CN113832115A

CN113832115A - Fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparation of antitumor drugs

Info

Publication number: CN113832115A
Application number: CN202110983048.9A
Authority: CN
Inventors: 居颂光; 葛彦
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2021-12-24

Abstract

The invention discloses a fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparing antitumor drugs. The fusion gene RIL-7 and CCL19 combined recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 is a gene sequence formed by inserting the fusion gene RIL-7, a chemotactic factor CCL19 and a regulatory element after the 81001bp position of a vaccinia virus maternal genome (the genome sequence takes GenBank: AY243312.1 as a reference); wherein the gene sequence inserted into the vaccinia virus maternal genome is shown in S1 or S1'. The invention can avoid potential adverse reactions such as cytokine storm and the like while promoting the anti-tumor immune response, promote immune cells such as T cells and the like to chemotact to a tumor microenvironment and further enhance the anti-tumor effect of the recombinant oncolytic virus.

Description

Fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparation of antitumor drugs

Technical Field

The invention belongs to the field of medicines, and particularly relates to fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparation of antitumor drugs.

Background

1. Immunotherapy is increasingly becoming an important approach for tumor therapy

Overall, tumor prevalence and mortality are currently high. The existing tumor treatment means comprise traditional medical treatment means such as operation, radiotherapy, chemotherapy, targeted therapy, traditional Chinese medicine and the like, but the curative effect of the traditional tumor treatment means needs to be improved urgently.

In the pathological process of tumor, the interaction between tumor cells and anti-tumor immunity jointly determines the occurrence and progression of tumor. The number and functional status of immune cells (especially the dominant military T cells of anti-tumor immunity) in the tumor microenvironment is one of the key elements affecting the tumor immunotherapy efficacy and patient outcome. According to the existence of tumor infiltrating T cells, the tumor is divided into cold tumor (namely, the interior and the periphery of the tumor tissue lack T cells and other immune cells) and hot tumor (namely, a large amount of immune cells are infiltrated in the tumor tissue, and the T cells and other immune cells are in an activated or exhausted state); according to the number and functions of immune cells in a tumor microenvironment, an immunoinflammatory type (namely 'hot tumor'), an immune-privileged type (namely that immune cells such as T cells and the like can only reach peripheral matrixes of tumor cells and can not migrate into tumor tissues) and an immune desert type (namely 'cold tumor') can be realized. If the tumor microenvironment is provided with immune cells such as T cells with sufficient quantity, powerful functions and lasting effect, the treatment effect of the tumor can be greatly improved.

Therefore, immunotherapy provides a new means for treating tumors and brings new hopes for tumor patients. The tumor immunotherapy can be divided into active immunotherapy, passive immunotherapy and composite artificial means on the basis according to the technical principle. Active immunotherapy refers to the means of initiating the body's active immune attack against tumor cells by providing tumor antigens, such as tumor vaccines. Passive immunotherapy refers to passive acquisition of anti-tumor ability of a patient by infusion of anti-tumor immune response products, such as adoptive infusion of cell therapy such as T cells and NK cells, and infusion of body fluid immunotherapy such as PD-1 antibody. Researchers have also constructed novel tumor therapeutic means by artificial means, such as the construction of chimeric antigen receptor T cells (CAR-T) by introducing a combination of genes encoding an antibody variable region that recognizes a tumor antigen and a first signal and a second signal activation region required for T cell activation into T cells.

2. The bottleneck of severely restricting the tumor immunotherapy effect is that each part of the tumor immune circulation is blocked

The achievement of anti-tumor immune effects in the body depends on a continuous tumor immune cycle. The tumor immune cycle includes: firstly, tumor cells are killed, and tumor antigens are released; identifying, absorbing and processing the tumor antigen by the antigen presenting cell; ③ the antigen presenting cells migrate to the adjacent lymphoid tissue organs; antigen presenting cell presents tumor antigen to antigen specific T cell, and activates, proliferates and differentiates to effect T cell; fifthly, migrating the effector T cells from the lymphoid tissue organ to the tumor tissue; sixthly, the effector T cells kill the tumor cells and release more tumor antigens. These steps can be repeated and amplified continuously to realize effective antitumor effect. Clinical tumor treatment or clinical experimental research shows that the immunotherapy technical means can improve the tumor treatment effect to different degrees but is severely restricted. For example, the total effective rate of PD-1 mab is about 10% to 30%, and further improvement of the therapeutic effect is still needed. Many immunotherapies are applied to tumor immune circulation in a single step, and are often dismissed. The innovation for improving the tumor immunotherapy effect in a breakthrough way is how to effectively maintain the continuity of tumor immune circulation.

3. Concept, efficacy and deficiencies of oncolytic viruses

Oncolytic Viruses (OV), also known as conditionally replicating viruses or selectively replicating viruses, selectively infect and replicate in tumor cells, while not replicating or killing in normal tissue cells. OV is proliferated in tumor cells, and is cracked and killed; the progeny virus particles are released to further infect surrounding tumor cells, and meanwhile, the OV killed tumor cells release tumor antigens to induce specific anti-tumor immunity. The established oncolytic virus therapy is a novel tumor therapy which utilizes the selective replication of virus in tumor cells, then kills the tumor cells and stimulates the body to generate specific anti-tumor immune effect. Compared with other tumor treatment methods, the oncolytic virus therapy has high replication efficiency, good killing effect and small toxic and side effects.

However, there are significant drawbacks to using oncolytic viruses alone. After some time of oncolytic virus entering the body as virus, the immune system exerts antiviral effect to eliminate it. In addition, the safety of the virus is also an important index for considering the potential of clinical application.

4. Advantages and disadvantages of vaccinia virus

Oncolytic viruses can be classified as RNA viruses and DNA viruses. Common RNA oncolytic viruses include: newcastle disease virus, measles virus, vesicular stomatitis virus, reovirus, and the like; common DNA oncolytic viruses include: adenovirus, herpesvirus, Vaccinia Virus (VV), and the like. Vaccinia Virus (VV) has great advantages for use as an oncolytic virus: the replication speed is high, and the tumor killing capability is strong; secondly, the safety is good, and unlike other DNA viruses, VV is only replicated in cytoplasm and cannot be integrated into a host genome, so that the safety is good in gene and genetic level; the genome is large, and a large-fragment exogenous gene can be inserted, so that the modification is facilitated; the four has two forms of IMV (intracellular mature virus) and EEV (extracellular enveloped virus), which are absolutely unique in the virus; the IMV is fast in replication and can be used as a main force for killing tumors, and the EEV has a protective envelope, so that viruses are not easy to be attacked by antibodies and can move in vivo for a long distance to infect distant tumors.

However, at the same time, Vaccinia Virus (VV) has disadvantages as a sole oncolytic virus: VV is eliminated by the antiviral immunity of the organism in vivo; VV can also be inhibited by the negative immune mechanism in the body of the patient; ③ VV-induced systemic and memory advantages of anti-tumor immunity are to be further enhanced.

Therefore, vaccinia virus needs to be modified and "armed" (i.e., recombined into the genome of vaccinia virus) with anti-tumor immunopotentiators so that it can express multiple immunomodulatory factors simultaneously when infecting tumor cells, increasing the anti-tumor effect of oncolytic viruses, thereby achieving more effective and durable anti-tumor efficacy.

5. Advantages and disadvantages of the cytokine IL-7 and advantages of the cell membrane anchored IL-7 constructed according to the present invention

Interleukin-7 (Interleukin-7, IL-7), is a soluble cytokine. The human IL-7 gene is located on chromosome 8, the total length of the coding region is 534bp, and the gene encodes 177 amino acid residues polypeptide, and the molecular weight is about 20 kDa. IL-7 is mainly expressed in epithelial cells and stromal cells of lymphoid organs such as thymus, lymph nodes, spleen, and bone marrow. The IL-7receptor (IL-7receptor, IL-7R) consists of IL-7R α (CD127) and the common cytokine receptor γ chain (CD 132). The human IL7R alpha gene is located on chromosome 5, encodes a polypeptide of 439 amino acid residues, and has a molecular weight of 49.5 kDa. IL-7R alpha is expressed in hematopoietic cells of the lymphoid lineage (including T cells, NK cells and B lymphocyte precursors), developing T cells and B cells, macrophages and the like.

IL-7/IL-7R participates in the regulation and control of cell proliferation, differentiation and apoptosis by activating signal channels such as PI3-K kinase, JAK-STAT and the like. Through the IL-7/IL-7R signaling pathway, T cell development, differentiation, proliferation and function can be regulated. IL-7 can promote thymocyte development, stimulate initial T cell proliferation, maintain T cell functional diversity, enhance T cell effector function, induce memory T cell formation and prolong memory T cell survival time, inhibit Treg cell function, and enhance anti-tumor immune response through multiple approaches.

The cell factor has the characteristics of high efficiency, multiple functions, networking property and the like on the immune system, is an important means for enhancing the anti-tumor immune curative effect, and has great clinical application prospect. However, systemic and large dose of cytokine often generates various toxic and side effects, and even faces the potential risks of 'cytokine storm', and the like, which becomes a bottleneck restricting clinical application of cytokine drugs. The use of natural IL-7 as a cytokine also faces the above-mentioned problems

If the biological activity of the IL-7 is maintained and the IL-7 is anchored on the cell membrane (namely the cell membrane anchoring type IL-7 is constructed) by the technical means, the secretion of wild type IL-7 to the outside of the cell can be avoided to generate the whole body effect, so that the safety is improved, the anchoring type IL-7 can be intensively expressed in a tumor microenvironment, so that the local action concentration is improved, and the anchoring type IL-7 acts on immune cells in the form of cell membrane molecules, so that the biological efficiency is improved.

6. Effect of chemokine CCL19

Chemokines are a class of soluble small molecule proteins that can chemotactic immune cells for directed movement. The chemokine CCL19 and its receptor CCR7 are expressed on dendritic cells, T cells and various tumor cells. The chemokine, CCL19, also known as MIP-3 β, is a member of the CC subfamily of chemokines. The human CCL19 gene is located in chromosome 9, its cDNA full length is 687bp, and its coded product is polypeptide chain of 228 amino acids. The CCL19 receptor CCR7 is expressed in mature DCs, naive T cells, Tregs, central memory T cells, naive B cells, and the like.

In recent years, the role of CCL19 and CCR7 in antitumor therapy has become a research hotspot and made significant progress. CCL19 promotes tumor immune circulation by inducing DC maturation and enhancing DC endocytosis. CCL19 also plays an important role in T cell lymph node homing, where upon stimulation by CCL19, the CCR 7receptor of T cells is internalized, triggering T cell adhesion and migration. Most of T cells such as naive T cells, central memory T cells and Tregs enter lymph nodes through the action of adhesion endothelial cells and the like. Therefore, the chemokine CCL19 can induce the directional migration of DC and T cells, etc. critical for the anti-tumor immune response to the expected action site.

However, the above applications all have some inevitable defects, such as the enhancement of the anti-tumor immune effect by the cytokine IL-7, but also have the potential risk of toxic side effects as the cytokine. The chemokine CCL19 plays a role in promoting the migration of immune cells to tumor tissues, but has a limited effect of directly promoting the function of anti-tumor immune cells. In addition, IL-7 and CCL 19-mediated anti-tumor immunity are easily inhibited by negative immunity in patients, and are bottlenecks that limit the curative effect of tumor immunotherapy.

The characteristic of oncolytic virus capable of selectively infecting and destroying tumor cells and further inducing anti-tumor immunity makes the oncolytic virus a new field of tumor immunotherapy, however, the oncolytic virus has the defect of being eliminated by organisms, and the anti-tumor immunity induced by the oncolytic virus has the common problem that the anti-tumor immune circulation is blocked.

Disclosure of Invention

In order to solve the problems, the invention provides a fusion gene RIL-7 combined with CCL19 recombinant oncolytic vaccinia virus and application thereof in preparing an antitumor medicament, wherein the fusion gene RIL-7 combined with CCL19 recombinant oncolytic vaccinia virus can overcome the common problem that the antitumor immunity induced by oncolytic virus is blocked in the anti-tumor immune circulation, and break through the bottleneck of tumor immunotherapy that the antitumor immune cells are not easy to enter tumor tissues and the antitumor immunity is easy to be inhibited by negative immunity in the body of a patient. The potential risk caused by the systemic effect of the cell factors is avoided while the anti-tumor immunity is promoted, the cell factors can play the treatment effect of the anti-tumor immunity in the tumor tissues (tumor microenvironment) in a centralized manner, and the anti-tumor efficacy is further enhanced.

In order to achieve the purpose, the technical scheme of the invention is as follows: the fusion gene RIL-7 and CCL19 combined recombinant oncolytic vaccinia virus with the code VV-RIL-7-CCL19 is the gene sequence of the fusion gene RIL-7, chemokine CCL19 and regulatory elements inserted after the 81001bp position of the vaccinia virus maternal genome with the genome sequence based on GenBank: AY 243312.1; wherein the gene sequence inserted into the vaccinia virus maternal genome is shown in S1 or S1'.

The recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 is constructed by simultaneously inserting a chemokine CCL19 gene sequence and a fusion gene RIL-7 into a maternal vaccinia virus genome and knocking out a Thymidine Kinase (TK) gene.

Wherein, the fusion gene RIL-7 is formed by connecting the following three gene segments: fragment 1 is the IL-7 gene sequence with the deletion stop code; fragment 2 is a connecting peptide gene sequence, and is an EA3K rigid connecting peptide sequence or a (G4S)3 flexible connecting peptide sequence; fragment 3 is the cell membrane anchoring gene sequence, the GPI anchoring sequence of CD16b with the start code removed; the base sequence of the fusion gene RIL-7 is shown in S2 or S2' according to the difference of the fragment 2 connecting peptide gene. The base sequence of the fusion gene RIL-7 is S2 due to the rigid connection peptide sequence of EA3K, and the base sequence of the fusion gene RIL-7 is S2' due to the flexible connection peptide sequence of (G4S) 3.

The gene information of the VV-RIL-7-CCL19 recombinant oncolytic virus is as follows.

1. The genomic sequence of the vaccinia virus female parent used for the construction of the VV-RIL-7-CCL19 recombinant oncolytic virus was based on GenBank: AY243312.1, on which the gene sequences of the vaccinia virus female parent mentioned in the following statements were based.

2. The fusion gene RIL-7 and CCL19 gene are subjected to gene recombination with the vaccinia virus female parent through a recombinant vector (PV-RIL-7-CCL 19). In the process, after the 81001bp position of the vaccinia virus maternal genome, namely the position between Thymidine Kinase (TK) genes, the RIL-7 gene, the CCL19 gene and related regulatory elements are inserted, so that not only is the complete TK gene damaged (TK is knocked out), but also the expression of RIL-7 and CCL19 is realized. Because the related regulatory elements comprise Yellow Fluorescent Protein (YFP) genes (loxp sequences are arranged on two sides of the genes) used for screening the recombinant viruses, the recombinant viruses are infected with 293T cells transfected by Cre genes, and the YFP is deleted, so that the recombinant vaccinia virus VV-RIL-7-CCL19 is obtained. The S1 fragment which is finally inserted into the gene sequence of the female vaccinia virus, namely the VV-RIL-7-CCL19 genome sequence is as follows (the underlined partial sequence is RIL-7 gene; the underlined and slant written partial sequence is CCL19 gene; both are in 3 'to 5' direction; and other sequences are regulatory elements such as promoter):

the S1 sequence adopts EA3K rigid connecting peptide sequence as connecting peptide gene, namely the base sequence of the fusion gene RIL-7 is S2; if (G4S)3 flexible connecting peptide sequences are adopted for connection to form RIL-7, namely the base sequence of the fusion gene RIL-7 is S2 ', the gene sequence of vaccinia virus, namely the S1' segment in the VV-RIL-7-CCL19 genome sequence is finally inserted as follows (the underlined partial sequence is RIL-7 gene, the underlined and obliquely written partial sequence is CCL19 gene, both are 3 'to 5' direction, and other sequences are regulatory elements such as promoter):

the invention also discloses construction of the fusion gene RIL-7, wherein the fusion gene RIL-7 is formed by connecting the following three gene segments: fragment 1 is the IL-7 gene sequence with the deletion stop code; fragment 2 is a linker peptide gene, and is an EA3K rigid linker peptide sequence, or a (G4S)3 flexible linker peptide sequence; fragment 3 is the cell membrane anchoring gene sequence, the GPI anchoring sequence of CD16b with the start code removed; the base sequence of the fusion gene RIL-7 is shown in S2 or S2' according to the difference of the fragment 2 connecting peptide gene. The base sequence of the fusion gene RIL-7 is S2 due to the rigid connection peptide sequence of EA3K, and the base sequence of the fusion gene RIL-7 is S2' due to the flexible connection peptide sequence of (G4S) 3.

When constructing the fusion gene RIL-7, for example, EA3K rigid connecting peptide is adopted, the gene sequence of the fusion gene RIL-7 (adopting EA3K rigid connecting peptide) is S2, which is as follows: wherein, the segment 1 is the gene sequence of IL-7 with deleted termination code and is marked by underlining; fragment 2 is EA3K rigid connecting peptide gene sequence and is indicated by italics; fragment 3 is the GPI anchor sequence of CD16b, indicated by italic underlining.

When the fusion gene RIL-7 is constructed, if (G4S)3 flexible connecting peptide is adopted, the base sequence of the fusion gene RIL-7 is S2', and the specific steps are as follows: wherein fragment 1 (the gene sequence of IL-7 deleted for the stop codon) is underlined; fragment 2 is a (G4S)3 flexible linker peptide sequence, shown in italics; fragment 3 (GPI anchor sequence of CD16 b) is italicized and underlined.

The fusion gene RIL-7 and CCL19 combined recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 established by the invention is recombinant oncolytic vaccinia virus with the chemokine CCL19 gene and the fusion gene RIL-7 inserted at the same time and Thymidine Kinase (TK) gene knocked out. The fusion gene RIL-7 is formed by splicing an interleukin-7 gene with a deleted termination code, a connecting peptide gene and a CD16b cell membrane anchoring sequence.

Wherein the cell membrane-anchored gene sequence is the GPI-anchor sequence of human CD16 b.

The VV-RIL-7-CCL19 is established by the following method:

1) construction of cell membrane anchoring fusion gene RIL-7: splicing the gene sequence of the wild type IL-7 with the deletion of the termination code, the EA3K rigid connecting peptide sequence or the (G4S)3 flexible connecting peptide sequence and the GPI anchor sequence of CD16b into a fusion gene by PCR, and naming the fusion gene as RIL-7;

2) the fusion gene RIL-7 is flanked by restriction sites: introducing SdaI and HpaI restriction sites into two wings of the fusion gene RIL-7 through PCR, and separating gene segments;

3) preparation of the cohesive end of the support PV-1: cutting the VV homologous recombinant vector PV-1 by using SdaI and HpaI, and separating a plasmid fragment;

4) the fusion gene RIL-7 is connected with the viscous end of the VV homologous recombinant vector PV-1: connecting the recovered products in step 2) and step 3);

5) competent cells were transformed and positive clones were screened: co-incubating the ligation product obtained in the step 4) with competent escherichia coli, performing amplification culture, extracting recombinant plasmids, and confirming that the fusion gene RIL-7 is correctly cloned into a homologous recombination vector through gene sequencing, wherein the vector is named as PV-RIL-7;

6) introducing SalI and SgsI into two wings of a CCL19 gene through PCR, recovering a PCR product, performing double enzyme digestion by using SalI and SgsI, and separating a gene fragment for later use;

7) preparation of the cohesive end of the vector PV-RIL-7: cutting another polyclonal insertion site of the VV homologous recombinant vector PV-RIL-7 by using SalI and SgsI, and separating a plasmid fragment for later use;

10) connection of CCL19 gene and PV-RIL-7 homologous recombination vector cohesive end: connecting the recovered products in the step 1) and the step 2) by using a kit;

11) transforming competent cells and screening positive clones, co-incubating the ligation product obtained in the step 10) with competent escherichia coli, extracting plasmids after culturing, and confirming that the CCL19 gene is correctly cloned into a homologous recombination vector by gene sequencing, wherein the vector is named as PV-RIL-7-CCL 19;

12) the fusion gene RIL-7 and the CCL19 gene are simultaneously recombined into the VV genome and the TK gene is deleted: PV-RIL-7-CCL19 undergoes homologous recombination with VV while it replicates in tumor cells; the fusion gene RIL-7, the CCL19 gene and the tracing Yellow Fluorescent Protein (YFP) gene sequence are simultaneously homologously recombined into the TK gene of the VV genome to form recombinant virus; at the same time, the TK gene is disrupted (i.e., the TK gene is deleted) due to the insertion of the foreign gene; screening the monoclonal recombinant VV to obtain high-purity recombinant VV;

13) infecting 293T cells expressing cre enzyme with the obtained high-purity recombinant VV, cutting YFP gene in a recombinant VV genome, screening monoclonal recombinant VV to obtain seeds containing recombinant virus, confirming fusion gene RIL-7 and CCL19 gene recombination into VV through gene sequencing analysis, and finally obtaining fusion gene RIL-7 combined with CCL19 gene recombinant oncolytic vaccinia virus which is named as VV-RIL-7-CCL 19.

The invention also provides application of the fusion gene RIL-7 and CCL19 recombinant oncolytic vaccinia virus in preparation of antitumor drugs, and the recombinant oncolytic vaccinia virus is obtained by inserting VV-RIL-7-CCL19, namely a chemotactic factor CCL19 gene sequence and the fusion gene RIL-7 into the 81001bp position of a vaccinia virus maternal genome and knocking out TK gene. The fusion gene RIL-7 is a fusion gene spliced by an interleukin-7 gene of a deleted termination code, a connecting peptide gene sequence and a human CD16b cell membrane anchoring gene sequence.

The fusion gene RIL-7 and CCL19 combined recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 can be independently used in preparation of antitumor drugs or combined with other antitumor drugs or means.

The fusion gene RIL-7 and CCL19 combined recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 can be used for preparing medicines or products for treating 'immunoinflammatory type' tumor tissues, 'immunodesert type' tumor tissues and 'immune privileged type' tumor tissues.

The fusion gene RIL-7 and CCL19 combined recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 can be used for preparing medicines and products for treating body surface tumors and deep tumors through direct injection and combined endoscopic injection;

the body surface tumor includes but is not limited to melanoma and other solid tumors existing in the body surface part;

the combined endoscope injection treatment comprises but is not limited to the intratumoral injection treatment of deep solid tumors, which is accurately carried out on the basis of clearly observing tumor bodies through a laparoscope, a cholangioscope, a thoracoscope, an enteroscope, a brain surgery endoscope, a neuroendoscope and the like.

The fusion gene RIL-7 and CCL19 recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 can be used for preparing medicaments and products for treating tumors in various clinical stages, wherein the tumors comprise but are not limited to pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophagus cancer, brain glioma, ovarian cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma and melanoma.

The fusion gene RIL-7 is formed by connecting the following three gene segments: fragment 1 is the IL-7 gene sequence with the deletion stop code; fragment 2 is a linker peptide gene, and is an EA3K rigid linker peptide sequence, or a (G4S)3 flexible linker peptide sequence; fragment 3 is the cell membrane anchoring gene sequence, the GPI anchoring sequence of CD16b with the start code removed; the base sequence of the fusion gene RIL-7 is shown in S2 or S2' according to the difference of the fragment 2 connecting peptide gene;

the fusion gene RIL-7 can be used for the transformation and preparation of CAR-T; and can also be used for the transformation and construction of other types of oncolytic viruses including adenovirus, herpes virus, parvovirus, Maraba virus, reovirus, coxsackievirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, Seneca valley virus, poliovirus, alphavirus, Zika virus and the like.

The invention constructs a fusion gene and further constructs recombinant oncolytic vaccinia virus. In order to anchor IL-7 with biological function to cell membrane and form stable expression product, through molecular design, the interleukin-7 gene deleted termination code, the connecting peptide gene sequence and the human CD16b cell membrane anchoring gene sequence deleted initiation code are spliced to construct a novel fusion gene, named RIL-7; then recombining RIL-7 into the genome of the vaccinia virus to construct a novel recombinant vaccinia virus containing RIL-7 gene sequence, which is named as VV-RIL-7; VV-RIL-7 retains the oncolytic properties of VV and can express a large amount of fusion protein coded by RIL-7 after infecting tumor cells. The fusion protein coded by RIL-7 not only retains the property of IL-7 for promoting anti-tumor immunity, but also becomes a membrane molecule due to the cell membrane anchoring structure, thereby avoiding potential risks caused by the systemic effect of wild type IL-7 as a soluble molecule, and also ensuring that IL-7 is limited in tumor tissues (tumor microenvironment) to play a role in enhancing the treatment effect of anti-tumor immunity.

The chemotactic factor CCL19 gene and the fusion gene RIL-7 are simultaneously recombined into VV to construct the fusion gene RIL-7 combined with CCL19 recombinant vaccinia virus which is named as VV-RIL-7-CCL 19. After the VV-RIL-7-CCL19 infects tumor cells, the tumor cells can play a role in dissolving tumors, can reach tumor tissues (tumor microenvironment) through expressing a series of anti-tumor immune cells such as CCL19, chemotactic DC, T cells and the like, and can also enhance anti-tumor immunity through expressing a large amount of RIL-7 coding fusion protein, so that the immune situation in the tumor tissues is reconstructed, and the anti-tumor efficacy is comprehensively enhanced.

As described in the background of the invention, if the recombinant oncolytic vaccinia virus carries wild-type IL-7 to infect tumor cells, the recombinant oncolytic vaccinia virus can enable the tumor cells to express and secrete a large amount of soluble IL-7, and the soluble IL-7 can reach the whole body to enhance the anti-tumor immune effect, but inevitably has the potential risk of toxic and side effects as a cytokine. In the invention, a wild type IL-7 gene is connected with a cell membrane anchoring gene sequence through rigid or flexible molecular connecting peptide to construct a fusion gene RIL-7, and the fusion gene RIL-7 is introduced into a homologous recombination vector to further perform homologous recombination with vaccinia virus to construct a recombinant oncolytic virus VV-RIL-7. Meanwhile, the TK gene of the vaccinia virus is deleted, so that the TK gene is safer and the selectivity of infecting tumor cells is further enhanced.

After VV-RIL-7-CCL19 infects tumor cells, it can express a large amount of fusion protein coded by RIL-7 while replicating. Since RIL-7 contains GPI anchor sequence, the fusion protein encoded by RIL-7 can be anchored to the cell membrane and localized to the tumor microenvironment to play a role. Thus, potential IL-7 systemic side effects can be avoided, local IL-7 high-concentration expression of tumor tissues can be realized, and stronger local anti-tumor efficacy is exerted. This is an advantage of the cell membrane anchored recombinant oncolytic vaccinia virus VV-RIL-7-CCL 19.

The recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 can play a role in direct oncolytic and starting anti-tumor immunity, and RIL-7 encoding protein can play a high-efficiency immunity enhancing role in local tumor tissues. However, in many cases, the number of immune cells in tumor tissues such as T cells is small, and there is a phenomenon called "cold tumor" or "desertification" of immune cells. The invention establishes a novel recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 containing a chemokine CCL19 gene sequence and a fusion gene RIL-7 by simultaneously recombining the chemokine CCL19 gene and the fusion gene RIL-7 into the vaccinia virus. After the VV-RIL-7-CCL19 infects tumor cells, CCL19 is synthesized while the large amount of CCL is replicated and secreted out of the cells, chemotactic immune cells enter tumor tissues, phenomena of 'cold tumor' and 'desertification' are changed, and the anti-tumor efficacy of the vaccinia virus and RIL-7 is further enhanced, which is the advantage of the recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 combining cell membrane anchoring type IL-7 with CCL 19.

The RIL-7 fusion gene and the encoding protein thereof not only retain the biological function of IL-7, but also are anchored on the cell surface to become a membrane molecule, and can be used for modifying other types of oncolytic viruses such as adenovirus, herpes virus, parvovirus, malaba virus, reovirus, coxsackievirus, newcastle disease virus, vesicular stomatitis virus, measles virus, senega virus, poliovirus, alphavirus, Zika virus and the like based on the requirements on the function and safety of IL-7 in addition to the modification of vaccinia virus by 'armed outfitting'; can also be used for transforming and constructing CAR-T.

The application of VV-RIL-7-CCL19 in preparing tumor drugs has the following situations and effects.

Application of VV-RIL-7-CCL19 in improving anti-tumor immune situation of tumor microenvironment

The interaction of tumor cells with anti-tumor immunity, together, determines the development and progression of tumors. The number and functional status of immune cells (especially T cells) in the tumor microenvironment is one of the key elements affecting tumor immunotherapy efficacy and patient outcome.

VV-RIL-7-CCL19 mainly dissolves local tumor tissues through the characteristics of oncolytic virus, starts immune response, establishes inflammatory environment of tumor immunity in local tumor, chemotaxis and recruits T cells and other immune cells into the tumor tissues through expressing CCL19, and enhances and maintains the anti-tumor functional state of the tumor tissues such as 'inflammatory environment' T cells and other immune cells through expressing RIL-7 encoding protein. Thus, VV-RIL-7-CCL19 has an effect on tumor tissue in a three-type immune state: based on the immune typing angle of the tumor microenvironment, the VV-RIL-7-CCL19 can be used for 'immunoinflammatory type' tumor tissues (namely 'hot tumors', existing T cells and other immune cells infiltrate), so that the 'hot tumors' are more 'hot', namely the number and the activity of immune cells in the tumor microenvironment are further enhanced; the VV-RIL-7-CCL19 can be used for 'immune desert type' tumor tissues (namely 'cold tumor', and the interior and the periphery of the tumor tissues lack T cells and other immune cells), so that the 'cold tumor' is converted into 'hot tumor', namely, the migration and infiltration of T cells and other immune cells to the tumor tissues are promoted, the anti-tumor activity of the tumor tissues is enhanced, and conditions are created for combining PD-1 monoclonal antibody and other immunotherapy; VV-RIL-7 can be used for immune-privileged tumor tissues (immune cells such as T cells exist at the periphery of the tumor tissues but cannot infiltrate into the tumor tissues), not only promotes the immune cells to break through various barriers and enter the tumor tissues, but also promotes the anti-tumor function of the immune cells.

VV-RIL-7-CCL19 for treatment of body and deep tumors by direct injection and combined endoscopic injection

Based on the application (administration) mode, the VV-RIL-7-CCL19 can be used for a series of solid tumors such as melanoma and the like existing on the body surface part through direct intratumoral injection; VV-RIL-7-CCL19 can be used for accurately injecting deep solid tumor into tumor body through laparoscope, choledochoscope, thoracoscope, enteroscope, brain surgery endoscope, neuroendoscope and the like on the basis of clearly observing tumor body, and can stimulate anti-tumor immune response while directly dissolving tumor cells.

VV-RIL-7-CCL19 for treating solid tumors such as colorectal cancer, pancreatic cancer and melanoma

Based on the tumor type, the VV-RIL-7-CCL19 takes the action principle of directly dissolving tumor and starting and enhancing immune cell efficiency such as T cells, so the VV-RIL-7-CCL19 can treat pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, brain glioma, ovarian cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, melanoma and the like in each clinical stage by the administration mode.

The invention well realizes the technical effects of constructing fusion genes and recombining the oncolytic virus, keeping the advantages of the original oncolytic virus and avoiding the weaknesses of the original oncolytic virus. The invention splices the interleukin-7 gene, the connecting sequence and the human CD16b cell membrane anchoring gene sequence deleted the termination code into a fusion gene RIL-7. Then the fusion gene RIL-7 is introduced into the vaccinia virus VV through homologous recombination to establish the recombinant oncolytic vaccinia virus VV-RIL-7. The recombinant oncolytic vaccinia virus VV-RIL-7 is applied to a medicine for tumor immunotherapy, when a tumor cell is infected and the effect of dissolving the tumor cell is obtained, RIL-7 encoding fusion protein anchored on a cell membrane is expressed, so that IL-7 is concentrated and limited to be expressed in a tumor microenvironment, potential adverse reactions such as cytokine storm and the like are avoided while anti-tumor immune response is promoted, and the bottleneck of tumor immunotherapy that anti-tumor immune cells are not easy to enter tumor tissues and anti-tumor immunity is easy to be inhibited by negative immunity in a patient body is broken through. Furthermore, the chemotactic factor CCL19 gene and the fusion gene RIL-7 are simultaneously recombined into the vaccinia virus to establish the recombinant oncolytic vaccinia virus VV-RIL-7-CCL 19. After VV-RIL-7-CCL19 infects tumor cells, CCL19 is synthesized while being replicated in a large quantity and secreted outside the cells, chemotactic immune cells to enter tumor tissues, change the phenomena of 'cold tumor' and 'desertification' and the like, and further enhance the anti-tumor efficacy of vaccinia virus and RIL-7 encoding protein. Overcomes the common problem that the anti-tumor immunity induced by the oncolytic virus is blocked in the anti-tumor immune circulation, and realizes the performance of promoting the anti-tumor immunity. Meanwhile, potential risks caused by systemic action of cytokines are avoided, the treatment effect of enhancing anti-tumor immunity is realized in a centralized manner in tumor tissues (tumor microenvironment), and the anti-tumor efficacy is further enhanced.

Drawings

FIG. 1 is a schematic diagram of the gene fragment introduced into the recombinant vector PV-1 and the structure of the PV-1 vector.

FIG. 2 is a graph showing the results of flow cytometry for detecting membrane-anchored IL-7 on the surface of MC-38 cells of intestinal cancer cells infected with recombinant viruses.

FIG. 3 is a graph showing the results of flow cytometry for detecting membrane-anchored IL-7 on the surface of melanoma cells B16-F10 after recombinant virus infection.

FIG. 4 shows the effect of MTT assay on the proliferative capacity of resting T cells after infection of MC-38 cells with recombinant oncolytic virus.

FIG. 5 shows the effect of MTT assay on the proliferative capacity of activated T cells after infection of MC-38 cells with recombinant oncolytic virus.

FIG. 6 shows the result of ELISA detection of CCL19 secretion from recombinant vaccinia virus infected tumor cells.

FIG. 7 shows the biological activity of CCL19 secreted by recombinant vaccinia virus infected tumor cells identified by the Transwell chemotaxis assay.

FIG. 8 is a graph showing the results of comparing the proliferation potency of various groups of recombinant oncolytic viruses in tumor cells by a virus titer assay.

FIG. 9 shows MTT assay for in vitro detection and comparison of oncolytic capacity of recombinant viruses.

FIG. 10 is the observation result of the efficacy of recombinant vaccinia virus VV-RIL-7 in treating abdominal tumor model of mouse intestinal cancer.

FIG. 11 is an observation of the efficacy of recombinant oncolytic viruses in treating tumors.

FIG. 12 shows a comparison of the antitumor efficacy of RIL-7 gene recombinant oncolytic viruses linked by two linking peptides.

FIG. 13 is a flow cytometry analysis of the number and proportion of T cells in the tumor microenvironment after treatment with recombinant oncolytic virus VV-RIL-7-CCL 19.

FIG. 14 shows the results of intracellular cytokine staining and flow cytometry to detect interferon-. gamma.production by T cells in the tumor microenvironment after treatment with recombinant oncolytic viruses VV-RIL-7-CCL19, etc.

FIG. 15 shows the results of flow cytometry for detecting the expression of PD-1 by T cells in the tumor microenvironment after treatment with recombinant oncolytic viruses VV-RIL-7-CCL19 and the like.

Detailed Description

The technical solution of the present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.

The invention is described in detail below with the aid of non-limiting examples. It will be apparent to those skilled in the art that certain changes and modifications may be made in the invention without departing from the spirit and principles of the invention. It is to be understood that such changes and modifications in parallel are intended to be included within the scope of the appended claims.

Example 1

1. Establishment of recombinant oncolytic vaccinia virus VV-RIL-7

1) Construction of IL-7 fusion Gene with cell Membrane anchoring sequence: the gene sequence of wild type IL-7 with the deletion of the termination code, the gene sequence of EA3K rigid peptide [ or (G4S)3 flexible connecting peptide ] and the GPI anchor gene sequence of human CD16b were linked and spliced by PCR to form a novel fusion gene, which was named RIL-7.

2) The fusion gene RIL-7 is flanked by restriction sites: SdaI and HpaI restriction enzyme sites are introduced into two wings of the fusion gene RIL-7 through PCR, after a recovery kit PCR product is adopted, SdaI and HpaI double enzyme digestion is used, gene segments are separated through gel electrophoresis, segments with expected sizes are cut, and the gene segments are separated through a DNA gel electrophoresis kit for later use.

3) Preparation of VV homologous recombination vector cohesive end: SdaI and HpaI are used for cutting VV homologous recombinant vector PV-1 (containing yellow fluorescent protein YFP gene sequence), gene segments are separated through gel electrophoresis, segments with expected sizes are cut, and plasmid segments are separated through a DNA gel electrophoresis kit for later use.

4) And (3) connecting the fusion gene RIL-7 with the viscous end of the VV homologous recombination vector: the recovered products in step 2) and step 3) were ligated using T4-Liganse kit for 2h at 16 ℃.

5) Competent cells were transformed and positive clones were screened: the ligation product and competent DH5 alpha colibacillus are incubated for 30min, then thermal shock is carried out for 90s at 42 ℃, 800 mu L precooled LB culture medium is quickly added, the mixture is cultured for 90min in a temperature-controlled shaking table at 37 ℃ and 125rpm, the mixture is coated on a semisolid LB culture plate containing 100 mu g/mL ampicillin, the mixture is cultured overnight at 37 ℃, a single colony is selected, recombinant plasmid is extracted after amplification culture, and gene sequencing is carried out to confirm that the fusion gene RIL-7 is correctly inserted into the homologous recombinant vector, which is named as PV-RIL-7.

6) The fusion gene RIL-7 is recombined into the genome of wild Vaccinia Virus (VV) to construct a novel recombinant oncolytic virus: the green monkey kidney cell CV-1 is inoculated to a 24-well plate and cultured until the abundance is about 80 percent. After incubating the liposome and the PV-RIL-7 plasmid for 20min, CV-1 cell culture supernatant was added simultaneously with VV. Thus, VV replicates in infected CV-1 cells and PV-RIL-7 undergoes homologous recombination with it. The fusion gene RIL-7 segment and the yellow fluorescent protein YFP gene sequence can be simultaneously homologously recombined into the VV genome, and the Thymidine Kinase (TK) gene is knocked out to form a recombinant virus.

7) Screening monoclonal recombinant VV: digesting CV-1 cells infected with VV and transfected with PV-RIL-7 plasmid in step 6) to prepare a single cell suspension. According to the characteristic that the recombinant virus contains YFP sequence and can express yellow fluorescent protein, through high-speed flow cytometry sorting (sorting), the CV-1 cell population with positive YFP is sorted. Adding YFP-positive CV-1 cells into CV-1 cells which are pre-inoculated in a 96-well plate according to 1 cell/hole by a limiting dilution method, culturing for 72h, selecting culture holes containing YFP-positive CV-1 cells with only single plaque under a fluorescence microscope, collecting the cells, and repeatedly freezing and thawing to obtain seeds containing recombinant viruses. The recombinant virus with the gene YFP deleted by cre enzyme is named VV-RIL-7 after gene sequencing confirmation, and subsequent function identification and analysis are carried out (remark: part of experiments which need YFP as an indication adopt the recombinant virus without YFP deletion).

8) Purity analysis of recombinant viruses: freezing and thawing the CV-1 cells containing the recombinant viruses obtained in the step 7), determining the virus titer (pfu) of the lysate according to a conventional virus titer detection method, adding CV-1 cells with the abundance of about 80% according to 0.1, 1 and 5MOI, and detecting the ratio of the virus plaque expression YFP after 72h, wherein the ratio is 100%, so that the available high-purity recombinant VV can be determined.

9) Gene sequencing analysis of VV-RIL-7: and (3) taking CV-1 cells infected by the recombinant virus VV-RIL-7, adopting a DNA purification kit to obtain DNA, and then performing gene sequencing to confirm that the correct fusion gene RIL-7 is recombined into VV. A VV-D (TK gene-knocked-out VV) infection group and the like were set as controls.

10) Expression and cell membrane localization analysis of fusion gene RIL-7 after VV-RIL-7 infects tumor cells: the recombinant virus VV-RIL-7 infects intestinal cancer cells MC-38, melanoma cells B16-F10 and the like according to the concentration of MOI of 1, 5 and the like, the cells infected by the virus are collected to prepare single cell suspension, and whether RIL-7 fusion protein is positioned on a cell membrane or not and the expression quantity of the RIL-7 fusion protein are analyzed at the protein level through anti-IL-7 monoclonal antibody staining and flow cytometry.

11) Comparing the tumoricidal effect of the recombinant virus VV-RIL-7 with that of VV-D (TK gene knockout VV): the recombinant virus VV-RIL-7 infects tumor cells such as MC-38 according to the concentration of MOI of 0, 0.1, 1, 5 and the like, and whether the recombinant virus retains the oncolytic capacity of the maternal virus is evaluated.

12) In vitro functional analysis of RIL-7 fusion protein expressed after VV-RIL-7 infected tumor cells: the recombinant virus VV-RIL-7 infects MC-38 cells according to the concentration of MOI of 1, 5 and the like, collects the cells infected by the virus to prepare single cell suspension, co-cultures the single cell suspension with PHA activated T cells, and detects the stimulation effect of RIL-7 fusion protein expressed in cell membranes on immune cells in vitro.

13) Analyzing the in vivo anti-tumor effect of VV-RIL-7 by adopting a tumor animal membrane type: inoculating MC-38 cells to the abdominal cavity of mouse to establish a late-stage systemic tumor-bearing mouse model, injecting recombinant oncolytic virus VV-RIL-7 to the abdominal cavity, and observing the survival period of the mouse. Cells such as MC-38 are inoculated to a mouse subcutaneous tissue to construct a tumor-bearing mouse model, recombinant oncolytic virus VV-RIL-7 is injected into a tumor, and the tumor growth and growth conditions are observed. And setting up an experimental control group. Remarking: because of species factors, in vivo experimental validation was performed using VV-RIL-7 constructed from the mouse IL-7 gene sequence.

2. Recombinant oncolytic vaccinia virus VV-RIL-7-CCL19

1) Introducing SalI and SgsI into two wings of a CCL19 gene through PCR, performing double enzyme digestion by using SalI and SgsI after adopting a recovery kit PCR product, separating a gene fragment by gel electrophoresis, cutting a fragment with an expected size by using a surgical blade, and separating the gene fragment by using a DNA gel electrophoresis kit for later use.

2) Preparation of VV homologous recombination vector cohesive end: the other polyclonal insertion site of VV homologous recombinant vector PV-RIL-7 is cut by SalI and SgsI, gene fragments are separated by gel electrophoresis, fragments with expected sizes are cut by a surgical blade, and plasmid fragments are separated by a DNA gel electrophoresis kit for later use.

3) Connection of CCL19 gene and PV-RIL-7 homologous recombination vector cohesive end: the recovered products from step 1) and step 2) were ligated using T4-Liganse kit for 2h at 16 ℃.

4) Competent cells were transformed and positive clones were screened: the ligation product and competent DH5 alpha colibacillus are incubated for 30min, then thermal shock is carried out for 90s at 42 ℃, 800 mu L precooled LB culture medium is quickly added, the mixture is cultured for 90min in a temperature-controlled shaking table at 37 ℃ and 125rpm, the mixture is coated on a semisolid LB culture plate containing 100 mu g/mL ampicillin, the mixture is cultured overnight at 37 ℃, a single colony is selected, recombinant plasmid is extracted after amplification culture, and gene sequencing is carried out to confirm that CCL19 gene is correctly cloned into a homologous recombinant vector, which is named as PV-RIL-7-CCL 19.

5) The fusion gene RIL-7 and the CCL19 gene are simultaneously recombined into the VV genome: the green monkey kidney cell CV-1 is inoculated to a 24-well plate and cultured until the abundance is about 80 percent. CV-1 cell culture supernatant was added simultaneously with wild-type vaccinia virus VV after incubating the liposomes with the PV-RIL-7-CCL9 plasmid for 20 min. Thus, VV replicates in infected CV-1 cells and PV-RIL-7-CCL19 undergoes homologous recombination with it. The fusion gene RIL-7 fragment, the CCL19 gene and the yellow fluorescent YFP sequence can be simultaneously homologously recombined into the VV genome to form recombinant viruses.

6) Screening monoclonal recombinant VV: digesting CV-1 cells infected with VV and transfected with PV-RIL-7-CCL9 plasmid in step 5) to prepare a single cell suspension. According to the characteristic that the recombinant virus contains YFP sequence and can express yellow fluorescent protein, through high-speed flow cytometry sorting (sorting), the CV-1 cell population with positive YFP is sorted. Adding YFP-positive CV-1 cells into CV-1 cells which are pre-inoculated in a 96-well plate according to 1 cell/hole by a limiting dilution method, culturing for 72h, selecting culture holes containing YFP-positive CV-1 cells with only single plaque under a fluorescence microscope, collecting the cells, and repeatedly freezing and thawing to obtain seeds containing recombinant viruses. The recombinant virus in which the YFP gene was deleted by cre enzyme was named VV-RIL-7-CCL19, and the subsequent confirmation and functional identification analysis was performed (note: part of experiments requiring YFP as an indicator used a recombinant virus in which YFP was not deleted).

7) Purity analysis of recombinant viruses: freezing and thawing the CV-1 cells containing the recombinant viruses obtained in the step 6), determining the virus titer (pfu) of the lysate according to a conventional virus titer detection method, adding CV-1 cells with the abundance of about 80% according to 0.1, 1 and 5MOI, and detecting the ratio of the virus plaque expression YFP after 72h, wherein the ratio is 100%, so that the available high-purity recombinant VV can be determined.

8) Gene sequencing analysis of VV-RIL-7-CCL 19: and (3) taking CV-1 cells infected by the recombinant virus VV-RIL-7-CCL19, obtaining DNA by adopting a DNA purification kit, and then performing gene sequencing to confirm that the correct fusion gene RIL-7 and CCL19 are recombined into VV. Various necessary recombinant virus-infected groups were set up as controls.

9) Expression and cell membrane localization analysis of fusion gene RIL-7 and expression analysis of CCL19 after VV-RIL-7-CCL19 infects tumor cells: infecting intestinal cancer cells MC-38, melanoma cells B16-F10 and the like with recombinant virus VV-RIL-7-CCL9 according to the concentration of MOI of 1, 5 and the like, collecting the virus infected cells to prepare single cell suspension, and analyzing whether RIL-7 fusion protein is positioned on cell membranes and the expression quantity thereof at the protein level by anti-IL-7 monoclonal antibody staining and flow cytometry; collecting the cell culture supernatant, and detecting the content of CCL19 protein by using an ELISA kit.

10) In vitro functional analysis of RIL-7 fusion protein expressed after VV-RIL-7-CCL19 infects tumor cells and CCL 19: the recombinant virus VV-RIL-7 infects MC-38 cells according to the concentration of MOI of 1, 5 and the like, collects the cells infected by the virus to prepare single cell suspension, co-cultures the single cell suspension with PHA activated T cells, and detects the stimulation effect of RIL-7 fusion protein expressed in cell membranes on immune cells in vitro. The cell supernatant is collected and placed in a Transwell lower chamber, a mouse spleen single cell suspension is placed in an upper chamber, and directional chemotaxis of CCL19 on immune cells in the supernatant is observed. A control group was set up.

11) The tumoricidal effect of the recombinant virus VV-RIL-7-CCL19 was evaluated: the recombinant virus VV-RIL-7-CCL9 infects tumor cells such as MC-38 according to the concentration of MOI of 0, 0.1, 1, 5 and the like, and whether the recombinant virus retains the oncolytic capacity of the maternal virus is evaluated.

12) Analyzing the in vivo anti-tumor effect of VV-RIL-7-CCL19 by adopting a tumor animal membrane type: inoculating MC-38 cells into mouse abdominal cavity to establish late-stage systemic tumor-bearing mouse model, injecting recombinant oncolytic virus VV-RIL-7-CCL19 into abdominal cavity, and observing survival period of mouse. Cells such as MC-38 and the like are inoculated to a mouse subcutaneous to construct a tumor-bearing mouse model, and the tumor growth and growth conditions are observed by injecting recombinant oncolytic virus VV-RIL-7-CCL19 into a tumor. And setting up an experimental control group. Remarking: because of species factors, VV-RIL-7-CCL19 constructed by using a mouse IL-7 gene sequence and a mouse CCL19 gene sequence is used for in vivo experimental verification.

FIG. 1 is a schematic diagram of the gene fragment introduced into the recombinant vector PV-1 and the structure of the PV-1 vector. As shown in FIG. 1, the TK 5 'and TK 3' sequences in the vector are homologous to portions of the vaccinia virus genome sequence, flanking the gene sequence of interest. The recombinant vector is constructed and introduced into host cells infected by wild type vaccinia virus, TK 5 'and TK 3' sequences can be subjected to homologous recombination with wild type vaccinia virus genes, and gene sequences between TK 5 'and TK 3' in the vector are introduced into vaccinia virus genomes to construct recombinant viruses.

Wherein, MCSn_(1、2...): a multiple cloning site, into which a target gene can be inserted; IRES: a ribosome entry site.

Example 2 testing of recombinant oncolytic Virus-infected tumor cells expressing the immune factors RIL-7 and CCL19

FIG. 2 is a graph showing the results of detecting the expression of cell membrane IL-7 by flow cytometry after MC-38 cells were infected with the recombinant oncolytic virus of fusion gene RIL-7. FIG. 2 shows the results of confirming whether or not the fusion proteins encoded by RIL-7 genes carried by recombinant oncolytic viruses VV-RIL-7 and VV-RIL-7-CCL19 are anchored to the cell membrane. After the MC-38 cells are infected by the VV-RIL-7 and VV-RIL-7-CCL19 recombinant oncolytic viruses, the expression of the cell membrane IL-7 is detected by adopting anti-IL-7 monoclonal antibody staining and flow cytometry. The results show that the surface of the MC38 cell membrane of the intestinal cancer cells infected by VV-RIL-7 and VV-RIL-7-CCL19 after the recombination of RIL-7 gene detects the existence of membrane-anchored IL-7, while the surface of MC38 cells infected by VV-D (TK gene knock-out VV), VV-IL-7 (recombinant into the gene coding soluble IL-7 and TK gene knock-out VV) and VV-CCL19 (recombinant into CCL19 and TK gene knock-out VV) has no membrane-anchored IL-7. This demonstrates that the expression of membrane-anchored IL-7 fusion protein on the surface of tumor cells can be achieved by infecting tumor cells after the fusion gene RIL-7 is inserted into recombinant viruses VV-RIL-7 and VV-RIL-7-CCL 19.

FIG. 3 is a graph showing the results of detecting the expression of cell membrane IL-7 by flow cytometry after B16-F10 cells were infected with the recombinant oncolytic virus of fusion gene RIL-7. Intestinal cancer cell strains MC-38 and melanoma cells B16-F10 in logarithmic growth phase are infected according to different virus multiplicity of infection (MOI) of 0, 0.1, 1 and 5, cells are digested after 24 hours to prepare single cell suspension, rabbit anti-IL-7 monoclonal antibodies and fluorescein labeled anti-rabbit Ig polyclonal markers are adopted, and the expression of IL-7 is analyzed by flow cytometry. This experiment was to verify the generality of the experimental results from fig. 2. Similar results are obtained by repeating the virus infection process in figure 2 by using melanoma cells B16-F10 as target cells, and the fact that the expression of membrane anchoring type IL-7 fusion protein on the surface of tumor cells can be realized after different tumor cells are infected by recombinant viruses VV-RIL-7 and VV-RIL-7-CCL19 is proved.

The results in FIG. 2 and FIG. 3 show that both recombinant vaccinia virus VV-RIL-7 and VV-RIL-7-CCL19 infected tumor cells harboring the fusion gene RIL-7 of IL-7 and GPI anchor region expressed membrane type IL-7 and the expression intensity increased with the increase of infection index (MOI). This result confirmed that the recombinant vaccinia virus constructed in the present invention can carry RIL-7 gene and express membrane type IL-7 after infecting tumor cells.

Wherein, YFP: yellow fluorescent protein for tracing virus infected cells;

VV-D: a TK gene knock-out vaccinia virus infected group;

VV-IL-7: VV-IL-7 recombinant vaccinia virus infected group, after infection, tumor cells can express wild type IL-7 gene;

VV-CCL 19: VV-CCL19 recombinant vaccinia virus infected group, wherein tumor cells can secrete chemotactic factor CCL19 after infection;

VV-RIL-7: VV-RIL-7 recombinant vaccinia virus infected group, after infection, tumor cells can express IL-7 and GPI anchor region fusion gene, and IL-7 and GPI anchor region fusion protein is anchored to tumor cell membrane;

VV-RIL-7-CCL 19: VV-RIL-7-CCL19 recombinant vaccinia virus infected group, after infection tumor cells expressed anchoring to the cell membrane of IL-7, at the same time secreted CCL 19.

Example 3 biological Activity assay

In order to identify whether a fusion gene RIL-7 expression product carried by the recombinant vaccinia virus has biological activity, T cells are obtained from spleen cells in the experiment and are divided into a static T cell group and a PHA activated T cell group which are respectively inoculated in a 96-well plate, meanwhile, MC-38 cells are infected by different recombinant vaccinia viruses according to MOI (molar equivalent of 1) for 24 hours, a single cell suspension is prepared by digestion, the static T cell group and the PHA activated T cell group are added in proportion after PBS (phosphate buffer solution) washing, and after 48 hours of incubation, the OD (optical density) value of each group is detected by adopting an MTT method. The results show that tumor cells infected by recombinant vaccinia virus carrying the fusion gene RIL-7 can effectively stimulate the proliferation of resting and activated T cells, and the biological effect of the expression product of the fusion gene RIL-7 is further proved by the blocking of the effect by an anti-IL-7 neutralizing antibody.

Wherein: PHA: a phytohemagglutinin for activating T cells;

t: a T cell;

a: anti-IL-7 neutralizing antibodies

MC 38-vv-D: VV-D infected MC-38 cells;

MC 38-VV-IL-7: VV-IL-7 recombinant vaccinia virus infected MC-38 cells;

MC38-VV-CCL 19: VV-CCL19 recombinant vaccinia virus infected MC-38 cells;

MC 38-VV-RIL-7: VV-RIL-7 recombinant vaccinia virus infected MC-38 cells;

MC38-VV-RIL-7-CCL 19: VV-RIL-7-CCL19 recombinant vaccinia virus infected MC-38 cells.

FIG. 4 shows the MTT assay to determine the effect of recombinant oncolytic virus on the proliferative capacity of resting T cells after infection of MC-38 cells. In order to detect whether a membrane anchoring type IL-7 molecule coded by an RIL-7 gene carried by recombinant oncolytic virus has biological activity on a static T cell by an in vitro experiment, MC-38 cells infected by VV-RIL-7 are pretreated by mitomycin and then are co-cultured with the static T cell, and the proliferation capacity of the T cell is detected by an MTT method. The results show that MC-38 cells after VV-RIL-7 infection can promote T cell proliferation, and the effect can be blocked by an anti-IL-7 neutralizing antibody, and the expressed membrane anchoring type IL-7 molecules after the fusion gene RIL-7 is recombined into VV are shown to have biological activity on T cells.

FIG. 5 shows MTT assay to determine the effect of recombinant oncolytic virus on the proliferative capacity of PHA-activated T cells after infection of MC-38 cells. In order to detect whether the membrane anchoring type IL-7 molecule coded by the RIL-7 gene carried by the recombinant oncolytic virus has biological activity on activated T cells through in vitro experiments, MC-38 cells infected by VV-RIL-7 are pretreated by mitomycin and then are co-cultured with PHA activated T cells, and the T cell proliferation capacity is detected by an MTT method. The results show that MC-38 cells after VV-RIL-7 infection can promote the proliferation of activated T cells, and the effect can also be blocked by an anti-IL-7 neutralizing antibody, and further show that the expressed membrane anchoring type IL-7 molecules after the fusion gene RIL-7 is recombined into VV have biological activity on the T cells.

Example 4 recombinant Gene expression assay

In order to confirm whether the CCL19 gene carried by the recombinant virus can be normally expressed, the MC-38 cells and the B16-F10 cells are respectively infected by the recombinant virus for 48 hours according to MOI (molar equivalent of identity) 1, and the secretion amount of CCL19 is detected by ELISA (enzyme-linked immuno sorbent assay) after collecting supernatant. The result shows that the recombinant oncolytic viruses VV-CCL19 and VV-RIL-7-CCL19 carrying the CCL19 gene can secrete high-level CCL19 after infecting host cells.

FIG. 6 shows the detection of CCL19 secretion from tumor cells infected by recombinant vaccinia virus carrying CCL19 gene, wherein a is the detection of CCL19 secretion from MC-38 tumor cells infected by recombinant vaccinia virus carrying CCL19 gene, and B is the detection of CCL19 secretion from B16-F10 tumor cells infected by recombinant vaccinia virus carrying CCL19 gene. In order to detect whether the CCL19 gene carried by the recombinant virus can realize expression after infecting tumor cells, after MC-38 cells (a) and B16-F10 cells (B) are infected by the recombinant viruses VV-CCL19 and VV-RIL-7-CCL19, the secretion amount of CCL19 in culture supernatant is detected by ELISA. The results show that the VV-CCL19 and VV-RIL-7-CCL19 can produce CCL19 and secrete to the outside of the cell after infecting the tumor cells, while the tumor cells do not produce CCL19 after infecting the recombinant virus VV in the control group.

Example 5 secreted chemokine bioactivity assay

In order to identify whether CCL19 generated after recombinant virus VV-RIL-7-CCL19 infects tumor cells has the biological activity of chemotactic immune cells, VV-RIL-7-CCL19 infects MC-38 cells, supernatant is collected, chemotactic effect of CCL19 in the supernatant on PBMCs is detected by a Transwell method, the supernatant is added into a lower chamber of a Transwell plate, and T cell suspension is added into an upper chamber for migration experiment. FIG. 7 shows the biological activity identification of CCL19 secreted by recombinant vaccinia virus infected tumor cells carrying CCL19 gene. The results show that the cell supernatants of MC-38 infected by VV-CCL19 and VV-RIL-7-CCL19 can chemotactic T cells, the chemotactic ability can be blocked by an anti-CCL 19 neutralizing antibody, and the cell supernatants of MC-38 infected by control viruses VV-D and VV-RIL-7 have almost no capacity of chemotactic T cells.

Example 6 comparison of the proliferation Capacity of groups of recombinant oncolytic viruses in host cells

In order to confirm the influence of gene recombination on the replication and proliferation capacity of the vaccinia virus in tumor cells, MC-38 cells and B16-F10 cells are respectively infected by each recombinant virus, the cells are respectively harvested at 24h, 48h and 72h after infection, green monkey kidney cells CV-1 are infected after repeated freeze thawing and dilution, and the virus yield is determined by a plaque lysis method. FIG. 8 shows a comparison of the proliferation potency of various groups of recombinant oncolytic viruses in host cells, wherein a is infected with MC-38 cells and B is infected with B16-F10 cells. The results show that the virus titers generated after the various groups of viruses infect tumor cells have no obvious difference, which indicates that the recombination of RIL-7 or the combination of RIL-7 and CCL19 genes has no influence on the self-proliferation and replication capacity of the viruses.

Example 7 in vitro experiments to compare the Effect of genetic recombination on the ability of vaccinia virus to kill tumor cells

In order to confirm whether the gene recombination influences the virus oncolytic effect or not, the recombinant viruses infect MC-38 cells and B16-F10 cells according to different MOIs (0, 0.1, 0.5 and 5), and the living cell ratio is detected by an MTT method after 48 hours. FIG. 9 shows the effect of in vitro experiments comparing gene recombination on the ability of vaccinia virus to kill tumor cells, where a is infected with MC-38 cells and B is infected with B16-F10 cells. The results show that the oncolytic capacities of various groups of viruses after infecting tumor cells have no obvious difference, which indicates that the recombinant RIL-7 or RIL-7 and CCL19 genes have no influence on the oncolytic capacity of the viruses, and the gene recombination does not influence the killing capacity of the viruses in vitro.

Example 8 therapeutic efficacy test of recombinant vaccinia virus VV-RIL-7 for treatment of tumor model of mouse intestinal cancer

The fusion gene RIL-7 is recombined into the vaccinia virus genome to construct a novel virus VV-RIL-7 recombinant vaccinia virus. In order to identify the curative effect of VV-RIL-7 on tumor treatment, an abdominal cavity model of intestinal cancer of a mouse (C57/BL6 strain mouse) is established by adopting tumor cells MC-38, and on the 9 th day of establishment of the tumor model, the abdominal cavity injection treatment is carried out by adopting recombinant virus VV-RIL-7, and the following control treatment groups are set up: VV-IL-7 (inserted with a gene coding soluble IL-7, VV-D (TK gene-knocked-out VV) and phosphate buffer solution treatment group (PBS) are treated by intraperitoneal injection, and the survival time of the mice is observed.A result of the observation of the curative effect of the recombinant vaccinia virus VV-RIL-7 on an abdominal tumor model of mice intestinal cancer is shown in figure 10a, compared with the PBS, the VV-D can effectively prolong the survival time of tumor-bearing mice, compared with the VV-D treatment group, the wild type IL-7 recombinant vaccinia virus (VV-IL-7) and RIL-7 recombinant vaccinia virus (VV-RIL-7) can both prolong the survival time of the tumor-bearing mice, and the curative effect of the VV-RIL-7 treatment group is better than that of the VV-IL-7.

To observe the anti-tumor immune memory effect (persistence of anti-tumor effect) after the above treatment, mice in the cured group (mice survived for more than 90 days) were taken, normal mice were set as controls, and MC-38 cells were subcutaneously injected into one flank of the mice (FIG. 10B), and B16-F10 cells were injected into the opposite flank (FIG. 10c), and the tumor major axis (L) and minor axis (R) were measured every other day, and the tumor volume was calculated according to the formula π/6 XL XR. The result shows that after the MC-38 tumor-bearing mouse model tumor is cured by the recombinant virus VV-RIL-7, the same tumor (MC-38) is adopted for inoculation, the tumor is not formed any more, and the tumor can be formed after other tumors (B16-F10) are inoculated; while the same number of MC-38 cells and B16-F10 cells were able to form tumors and continue to grow in the control C57/BL6 strain of mice. The results show that the mice in the cured group generate durable and specific anti-tumor immunological memory, while the mice in the control group have no anti-tumor capability, and the adoption of the recombinant oncolytic virus VV-RIL-7 can induce the organism to generate the specific anti-tumor immunological memory.

Therefore, the recombinant oncolytic virus VV-RIL-7 established by the invention can improve and trigger durable antitumor capability.

Example 9 detection of therapeutic efficacy of VV-RIL-7-CCL19 in treating tumors

In order to confirm the anti-tumor efficacy of the VV-RIL-7-CCL19 recombinant oncolytic virus established by the invention, an abdominal cavity model of intestinal cancer of a mouse (C57/BL6 strain mouse) is established by adopting tumor cells MC-38, and on the 9 th day of establishment of the tumor model, the recombinant virus VV-RIL-7-CCL19 is adopted for abdominal cavity injection treatment, and the following control groups are established: the VV-RIL-7, VV-CCL19, VV-D and PBS treatment groups were treated by intraperitoneal injection, and the survival time of the mice was observed. FIG. 11 shows the comparison of the therapeutic effects of VV-RIL-7-CCL19 on the tumors in the control groups, which indicates that the VV-D treated group can effectively inhibit the tumor growth compared with the PBS treated group (FIG. 11 a); compared with the VV-D treatment group, the VV-CCL19 treatment group and the VV-RIL-7 treatment group have more obvious tumor growth inhibition; the VV-RIL-7-CCL19 recombinant oncolytic virus can effectively inhibit the growth of tumors, and the curative effect of the recombinant oncolytic virus is obviously superior to that of PBS, VV-D and VV-RIL-7 treatment groups.

To confirm whether the therapeutic efficacy of the VV-RIL-7-CCL19 recombinant virus-cured mice of the present invention is maintained for a long time (i.e., whether effective anti-tumor immune memory is available), the mice in the cured group (mice survive for more than 90 days) were selected, and normal mice were set as controls, and then mice were injected subcutaneously into one flank of the flank (fig. 11B) and the other flank of the mice with B16-F10 cells (fig. 11 c), and the tumor growth was observed and compared. The results show that the same tumor (MC-38) can not be used for inoculation to form a tumor any more, and other tumors (B16-F10) can still be used for inoculation to form a tumor; while the same number of MC-38 cells and B16-F10 cells were able to form tumors and continue to grow in the control C57/BL6 strain of mice. The above results indicate that the mice in the cured group developed persistent and specific anti-tumor immune memory, while the mice in the control group had no anti-tumor ability. Therefore, the recombinant oncolytic virus VV-RIL-7-CCL19 established by the invention has more efficient and durable anti-tumor capacity.

Example 10 comparison of antitumor Effect of recombinant oncolytic vaccinia virus constructed from RIL-7 Gene comprising different linker genes

In the invention, the fusion gene RIL-7 is formed by connecting three gene segments: fragment 1 is the gene sequence of wild-type IL-7 deleted of the stop codon; fragment 2 is EA3K rigid linker peptide sequence or (G4S)3 flexible linker peptide sequence; fragment 3 is the GPI anchor sequence of CD16 b. In order to identify whether there is a difference in biological function of RIL-7 ligated using different fragments 2, RIL-7 constructed using EA3K rigid linker peptide sequence was further designated as RIL-7/EA3K, and the recombinant vaccinia virus thus created was designated as VV-RIL-7/EA 3K; RIL-7 composed of the (G4S)3 flexible linker peptide sequence was further designated as RIL-7/(G4S)3, and the recombinant vaccinia virus thus established was designated as VV-RIL-7/(G4S) 3.

For this purpose, the experiment was first conducted by immunofluorescence staining and flow cytometry analysis to detect that both VV-RIL-7/(G4S)3 and VV-RIL-7/EA3K produce membrane-anchored IL-7 after infecting tumor cells, and the results showed (FIG. 12a) that both VV-RIL-7/(G4S)3 and VV-RIL-7/EA3K produce membrane-anchored IL-7 after infecting MC-38 cells, and that the expression levels thereof increased with increasing MOI.

Furthermore, in order to verify and compare the antitumor efficacy of VV-RIL-7/(G4S)3 and VV-RIL-7/EA3K, in this experiment, mouse intestinal cancer cells MC-38(106 cells/spot) were inoculated subcutaneously into the buttocks of mice of the C57/BL6 strain, the tumor major axis (L) and minor axis (R) were measured at intervals, the tumor volume was calculated according to the formula pi/6 XLXRXR, and when the tumor volume reached about 200mm3, the cells were randomly grouped and injected intratumorally with recombinant viruses VV-RIL-7/EA3K, VV-RIL-7/(G4S)3, VV-D and PBS, respectively, and the tumor size was measured at intervals. As shown in FIG. 12b, VV-RIL-7/EA3K, VV-RIL-7/(G4S)3, VV-D were all effective in inhibiting tumor growth compared to PBS treated group; the efficacy of VV-RIL-7/EA3K and VV-RIL-7/(G4S)3 group in inhibiting tumor growth is obviously better than that of VV-D group; the two treatment groups of VV-RIL-7/EA3K and VV-RIL-7/(G4S)3 have obvious tumor growth inhibition, but have no difference. Thus, it was demonstrated by this experiment that both linker peptides have no significant effect on the biological function of RIL-7, and thus in other examples of the present application RIL-7 was in the form of RIL-7/EA3K, VV-RIL-7 and VV-RIL-7-CCL19 were in the form of RIL-7/EA3K in the construction of recombinant viruses involving RIL-7. Accordingly, RIL-7/EA3K and RIL-7/(G4S)3 are collectively referred to herein as RIL-7.

Example 11 analysis of the number and function of T cells in the tumor microenvironment following recombinant oncolytic virus treatment

According to the design idea, the recombinant virus VV-RIL-7-CCL19 is used as a complete oncolytic virus, except for the oncolytic effect of the virus, CCL19 expressed after the recombinant virus infects tumor cells can chemotact anti-tumor immune cells to a tumor microenvironment, and the expressed anchored IL-7 can promote the functions of the anti-tumor immune cells. In order to further verify the rationality of the design idea and the effectiveness of the related recombinant viruses, healthy C57/BL6 mice of 6-8 weeks are selected in the experiment, and intestinal cancer cell strains MC-38 (10) are injected into the abdominal cavity⁶Individual cell/mouse) to establish a tumor-bearing mouse with intestinal cancer; by 9 days after tumor inoculation, mice were randomly grouped and injected with VV-RIL-7-CCL19, VV-RIL-7, VV-CCL19, VV-D and PBS, respectively; after 5 days, mice are sacrificed, abdominal tumor tissues are collected, 1mL of digestive enzyme mixed liquor (containing 0.1mg/mL of hyaluronidase, 1mg/mL of collagenase and 30U/mL of deoxyribonuclease I) is added into every 100mg of tumor tissues, the mixture is digested for 2 hours at 37 ℃, after neutralization, the mixture is filtered through a 150-mesh screen, and finally, tumor tissue single cell suspension is obtained, and the number and the proportion of key anti-tumor immune components T cells in the tumor tissues and the levels of IFN-gamma and PD-1 molecules expressed by the T cells are analyzed by antibody staining and flow cytometry.

The results show that:

1. the flow cytometry analysis results shown in FIG. 13 demonstrate the effect of recombinant oncolytic virus VV-RIL-7-CCL19 and the like on the number and proportion of T cells in the tumor microenvironment after treatment. Roughly determining lymphocyte populations by adopting an FS-SS circle gate; the T cell population (i.e., CD45+ CD3+ population) was further analyzed for cell populations within the circle using the hematopoietic lineage derived marker molecule CD45 in combination with the T cell marker molecule CD 3; CD4+ and CD8+ T cell subsets in the T cell population were then analyzed using CD8 and CD4 index gates. By comprehensive analysis (product of the above 3-layer gate), it was found that: compared with the PBS treatment group, the proportion of T cells and the subgroup thereof in all the recombinant virus treatment groups in the unit mass of tumor tissues is increased, the VV-CCL19 and VV-RIL-7 treatment groups are superior to the VV-D treatment group, and the proportion of the VV-CCL19-RIL-7 treatment group is higher than that of all other treatment groups. These results support that CCL19 expressed by VV-CCL19-RIL-7 recruits T cells into tumor tissues and that RIL-7 locally expands T cells.

2. As shown in FIG. 14, the results of intracellular cytokine staining and flow cytometry analysis demonstrate the effect of recombinant oncolytic virus VV-RIL-7-CCL19 on the production of interferon-gamma by T cells in the tumor microenvironment after treatment. By analyzing the expression of IFN-gamma, a marker molecule for anti-tumor immunity, in CD8+ T cells after FS-SS and CD8 cycle gates are performed on the cells in the tumor tissues in sequence, the following results are found: compared with the PBS treatment group, the expression of IFN-gamma by CD8+ T cells of all the recombinant virus treatment groups is increased, and the increase of the VV-CCL19-RIL-7 treatment group is more obvious. The above results show that, after the recombinant oncolytic virus VV-RIL-7 and VV-CCL19-RIL-7 are used for treatment, not only the number of T cells is increased, but also the anti-tumor efficacy is improved, and especially the effect of the VV-CCL19-RIL-7 group is more ideal.

3. The flow cytometry analysis results shown in fig. 15 demonstrate the effect of recombinant oncolytic virus VV-RIL-7-CCL19 and the like on the expression of PD-1 by T cells in the tumor microenvironment after treatment. By analyzing the expression of the CD8+ T cells and CD4+ T cells surface immune checkpoint molecule PD-1 after sequential FS-SS and CD8/CD4 cycle gates on cells in tumor tissue, it was found that: the expression of PD-1 levels by CD8+ T cells and CD4+ T cells was down-regulated in the VV-RIL-7 and VV-RIL-7-CCL19 treated groups compared to the PBS, VV-D and VV-CCL19 treated groups. The above results illustrate that: RIL-7 expressed after the two recombinant viruses infect tumor cells plays a role in maintaining the functional state of T cells; in addition, the expression level of PD-1 by T cells in the treatment groups of VV-RIL-7 and VV-RIL-7-CCL19 is maintained at a higher level even though the expression level is down-regulated, which is the basis for the combined application with the anti-PD-1 monoclonal antibody.

The experiment shows that VV-RIL-7-CCL19 can obviously promote the infiltration of anti-tumor key component T cells to tumor tissues and maintain the anti-tumor functional state, and has the premise of being combined with anti-PD-1 monoclonal antibody for application.

According to the invention, a wild type IL-7 gene and a GPI-anchored protein (GPI-anchored protein) gene sequence are linked through rigid and flexible molecular connecting peptides to construct an IL-7-R-GPI fusion gene, a homologous recombination vector is introduced, homologous recombination is carried out with vaccinia virus, and meanwhile, a vaccinia virus TK gene is knocked out, so that the recombinant oncolytic virus VV-RIL-7 is safer, and thus, the recombinant oncolytic virus VV-RIL-7 is constructed. VV-RIL-7 has better safety because TK gene is deleted. After VV-RIL-7 infects tumor cells, it can express a large amount of recombinant IL-7(RIL-7) fusion protein while replicating. Since RIL-7 contains the GPI anchor sequence, it can be anchored to the cell membrane and is localized to function in the tumor microenvironment. Thus, potential IL-7 systemic side effects can be avoided, local IL-7 high-concentration expression of tumor tissues can be realized, and stronger local anti-tumor efficacy is exerted.

Furthermore, the chemotactic factor CCL19 gene and the IL-7-R-GPI fusion gene are simultaneously recombined into the vaccinia virus to establish the recombinant oncolytic vaccinia virus VV-RIL-7-CCL 19. After VV-RIL-7-CCL19 infects tumor cells, CCL19 is synthesized while being replicated in a large quantity and secreted outside the cells, chemotactic immune cells to enter tumor tissues, change the phenomena of 'cold tumor' and 'desertification' and the like, and further enhance the anti-tumor efficacy of vaccinia virus and RIL-7. Overcomes the common problem that the anti-tumor immunity induced by the oncolytic virus is blocked in the anti-tumor immune circulation, and realizes the performance of promoting the anti-tumor immunity. Meanwhile, potential risks caused by systemic action of cytokines are avoided, the treatment effect of enhancing anti-tumor immunity is realized in a centralized manner in tumor tissues (tumor microenvironment), and the anti-tumor efficacy is further enhanced.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.

Sequence listing

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Claims

1. The recombinant oncolytic vaccinia virus combining fusion gene RIL-7 and CCL19 is characterized in that the fusion gene RIL-7 and CCL19 are combined to prepare the recombinant oncolytic vaccinia virus, the code number VV-RIL-7-CCL19 is the gene sequence of the fusion gene RIL-7, chemokine CCL19 and regulatory elements inserted after the 81001bp position of the vaccinia virus maternal genome with the genome sequence based on the GenBank of AY 243312.1; wherein the gene sequence inserted into the vaccinia virus maternal genome is shown in S1 or S1'.

2. The recombinant oncolytic vaccinia virus of claim 1, wherein fusion gene RIL-7 in combination with CCL19 is formed by connecting the following three gene fragments: fragment 1 is the IL-7 gene sequence with the deletion stop code; fragment 2 is a linker peptide gene, and is an EA3K rigid linker peptide sequence, or a (G4S)3 flexible linker peptide sequence; fragment 3 is the cell membrane anchoring gene sequence, the GPI anchoring sequence of CD16b with the start code removed; the base sequence of the fusion gene RIL-7 is shown in S2 or S2' according to the difference of the fragment 2 connecting peptide gene.

3. The fusion gene RIL-7 in combination with CCL19 recombinant oncolytic vaccinia virus of claim 1, wherein the VV-RIL-7-CCL19 is established by:

3) preparation of vaccinia virus VV homologous recombinant vector PV-1 cohesive end: cutting the VV homologous recombinant vector PV-1 by using SdaI and HpaI, and separating a plasmid fragment;

4. The application of the recombinant oncolytic vaccinia virus with the fusion gene RIL-7 combined with CCL19 in preparation of antitumor drugs is characterized in that the recombinant oncolytic vaccinia virus VV-RIL-7-CCL19, namely the recombinant oncolytic vaccinia virus obtained by inserting a chemokine CCL19 gene sequence and a fusion gene RIL-7 sequence into the 81001bp position of a vaccinia virus maternal genome and knocking out a TK gene is disclosed, and the fusion gene RIL-7 is a fusion gene spliced by deleting an interleukin-7 gene, a connecting peptide gene sequence and a human CD16b cell membrane anchoring gene sequence of a termination code.

5. The use of the fusion gene RIL-7 in combination with CCL19 recombinant oncolytic vaccinia virus according to claim 4, wherein the fusion gene RIL-7 in combination with CCL19 recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 is used alone or in combination with other antineoplastic drugs or means in the preparation of antineoplastic drugs.

6. The use of the fusion gene RIL-7 in combination with CCL19 recombinant oncolytic vaccinia virus according to claim 4 for preparing an anti-tumor medicament, wherein the fusion gene RIL-7 in combination with CCL19 recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 can be used for preparing a medicament or product for treating "immunoinflammatory type" tumor tissue, "immunodesert type" tumor tissue, "immunoimmune privileged type" tumor tissue.

7. The use of the fusion gene RIL-7 in combination with CCL19 recombinant oncolytic vaccinia virus according to claim 4 for the preparation of antitumor drugs, wherein the fusion gene RIL-7 in combination with CCL19 recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 can be used for the preparation of drugs and products for treating superficial and deep tumors by direct injection and combined endoscopic injection;

the combined endoscope injection treatment includes but is not limited to the precise intratumoral injection treatment of the deep solid tumor on the basis of clearly observing the tumor body through a laparoscope, a choledochoscope, a thoracoscope, an enteroscope, a neuroendoscope and the like.

8. The use of the fusion gene RIL-7 in combination with CCL19 recombinant oncolytic vaccinia virus according to claim 4 for the preparation of antitumor drugs, wherein the fusion gene RIL-7 in combination with CCL19 recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 can be used for the preparation of drugs and products for the treatment of tumors in various clinical stages, including but not limited to pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, brain glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, melanoma.

9. The fusion gene RIL-7 is characterized in that the fusion gene RIL-7 is formed by connecting the following three gene segments: fragment 1 is the IL-7 gene sequence with the deletion stop code; fragment 2 is a linker peptide gene, and is an EA3K rigid linker peptide sequence, or a (G4S)3 flexible linker peptide sequence; fragment 3 is the cell membrane anchoring gene sequence, the GPI anchoring sequence of CD16b with the start code removed; the base sequence of the fusion gene RIL-7 is shown in S2 or S2' according to the difference of the fragment 2 connecting peptide gene;

the fusion gene RIL-7 can be used for the transformation and preparation of CAR-T; and can also be used for the transformation and construction of oncolytic viruses including but not limited to adenovirus, herpes virus, parvovirus, maraba virus, reovirus, coxsackievirus, newcastle disease virus, vesicular stomatitis virus, measles virus, senega valley virus, poliovirus, alphavirus, Zika virus and the like.