CN116948005A

CN116948005A - Novel cytokine delivered by virus and application thereof in tumor treatment

Info

Publication number: CN116948005A
Application number: CN202210400632.1A
Authority: CN
Inventors: 刘牧龙
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-04-16
Filing date: 2022-04-16
Publication date: 2023-10-27

Abstract

The invention relates to a novel cytokine delivered by virus and application thereof in tumor treatment, the cytokine delivered by virus is selected from an altered body of IL2, the altered body is selected from computer simulation design, the altered body of IL2 has the function of selectively activating dimeric receptors of IL2 Rbeta and yc, and simultaneously, the activity of natural IL2 serving as an IL2 Ralpha (CD 25) receptor agonist is lost. The invention provides a plurality of administration modes including local injection or systemic administration or intratumoral delivery, which can obviously inhibit the growth of malignant tumor, and simultaneously can effectively activate the autoimmune system of a patient, change the tumor microenvironment, activate the specific anti-tumor immune response of an organism, prevent the growth, diffusion and recurrence of tumor, achieve the purpose of eliminating or controlling tumor, relieve the immunosuppression in the tumor microenvironment, further enhance the systemic tumor killing activity of immune cells, and has obvious superiority in curative effect compared with the traditional therapy.

Description

Novel cytokine delivered by virus and application thereof in tumor treatment

Technical Field

The invention relates to oncology, virology and molecular cell biology, wherein non-integrated replicable negative-strand RNA viruses are used as vector systems for chimeric expression of super cytokines and are used in the field of cancer treatment, in particular to a strategy method for preparing novel therapeutic drugs for treating tumors or cancers, which comprises a method for treating different indications by adopting various administration modes by using replicative viral vectors modified by chimeric super cytokines.

Background

The onset and hazard of cancer is known, and cancer is the first cause of death for a long time. Especially, the lung cancer, liver cancer and intestinal cancer are rapidly increased, which is worth highly paying attention. Tumors are a worldwide public health problem.

Although the cure rate of several malignant tumors has been significantly improved, the outcome of patients with advanced solid tumors remains crudely unchanged during the last decades, which underscores the need for new therapies, and what has been put into clinical use today for solid tumors is antibodies to tumor immune checkpoints, such as antibody therapy, the key point of which is the effective production of monoclonal antibodies that antagonize immune checkpoint molecules per unit volume. In addition, the problem of drug resistance of antibody drugs is urgently solved, and the current research progress of tumor immunotherapy is focused on by various countries. A variety of immune-related tumor therapeutic strategies including T cell node inhibitors, oncolytic viruses, chimeric antigen receptor T cells, and the like are derived. It is well known that efficient immunotherapy needs to have several characteristics: inducing a durable clinical response; there is no typical resistance; inducing autoimmune-like toxicity.

In a plurality of immunotherapy modes at present, the capability of directly destroying tumor tissues of virus-mediated immunotherapy is strongest, a plurality of administration ways including intravenous administration are realized after a new generation of virus vectors are modified, the virus vectors can further reach the tumor local part through blood and lymphatic system circulation, the destruction of the inside of solid tumors is realized, the tumor immune response is generated, tumor antigens released by the tumor local part are subjected to antigen presentation, the adaptive immune response of an organism is activated, the infiltration of activated immune cells to the tumor local part is promoted, the killing of tumor cells is accelerated, and finally the aim of controlling or curing the solid tumors is achieved.

Although many drugs for various clinical indications are marketed, such drugs still suffer from the disadvantages of long administration period, drug resistance, high cost, etc. The invention describes in detail a novel recombinant cytokine delivery vector system for modifying tumor microenvironment by chimeric expression of a virus, specifically defined as an attenuated viral vector system for modifying tumor microenvironment having a polypeptide agonist that selectively activates dimeric receptors for IL2Rβ, yc without CD25/CD215 receptor activity. The agonist is named as IL-2 x 15 super cytokine, and the polypeptide sequence of the factor has about 100 amino acids, can bind IL2 Rbeta and yc with high intensity, but does not bind with CD25 (IL 2 Ralpha recognition receptor) and CD215 (IL 15 Ralpha recognition receptor), and has better thermostability, higher activity, lower immunogenicity and larger safety window than the natural IL 2. In the embodiment, the novel IL-2 x 15 super cytokine shows excellent pharmaceutical activity in animal models of metastatic non-small cell lung cancer, rectal cancer, breast cancer and the like.

The known IL2 and IL15 are two important cytokines, have important significance for T cell proliferation, production of cytotoxic T lymphocytes and stimulation of B cell synthesis of immunoglobulin and activation and survival of NK cells, and are hot targets for potential tumor treatment at present.

The cellular receptors for IL2 and IL15 are trimers, which share the IL2Rβ and yc receptors, but IL2Rα (CD 25) and IL15Rα (CD 215) exhibit cytokine-specific high affinities. IL2 is the earliest immunotherapy drug on the market, but because of the short half-life period and great toxicity and side effects of systemic administration, it is not much clinically applied, and only approved for tumor indications such as renal cancer, malignant black and the like. Although the current mechanism of human toxicity sources is not well understood, animal experiments have shown that IL 2-produced CD25 activity is the major source of toxicity. While traditional CD25 removal is mainly based on chemical modifications such as pegylation and point mutation, such modification has limited improvement in selectivity and sometimes affects activity, stability, etc. Fusion proteins, while extending half-life, may affect engineered cytokine tissue penetration because the molecules are too large. In addition, because the structure of the PEG modification or mutant is too close to that of natural IL2, if once the body produces neutralizing antibodies, endogenous IL2 is neutralized, continued resistance is further developed. IL2 x 15 supercytokines do not amplify tregs as much as IL2 because of no CD25 activity, and the CD8/Treg ratio is significantly higher than native IL2. This is important for tumor immunotherapy, because tregs are considered immunosuppressive T cells, while CD8 is tumor killing active.

The problems can be solved simultaneously by adopting a brand new strategy to modify the skeleton of the IL2 Rbeta and gamma c agonist. Endogenous signaling molecules are known to bind to many receptors, e.g., 14 known receptors for 5 HT. The body achieves selectivity by locally synthesizing and scavenging these signaling molecules, thus there are no potential toxicity issues with systemic administration. So these endogenous molecules are inherently less proprietary, but selective receptor subtype agonists or antagonists are the main source of drugs, 50% of which are GPCR-type ligands on the market before large-scale attack by macromolecular drugs and kinase inhibitors. These drug molecules, although similar in endogenous structure, can sometimes differ greatly, so that systemic administration is also sufficiently selective. In the present invention, the homology of the new IL-2 x 15 with the natural IL-2 is only about 20%.

The current mode of effectively breaking the barrier of solid tumors is mainly realized by a virus vector, the system can specifically express the exocrine super cytokine while finishing virus replication in tumor cells, release tumor antigens to activate immune cells to generate specific immune response against tumors, and simultaneously break the immune inhibition of tumor microenvironment, promote the elimination of killer T cells on tumor cells, and simultaneously promote organisms to generate systemic specific anti-tumor immune memory response, thereby effectively controlling the elimination of free or metastatic tumor cells. Some recombinant viruses modified by gene editing are used as a new tumor therapeutic preparation, and the anti-tumor immune response is initiated through two action mechanisms of killing tumor cells by the viruses and inducing systemic anti-tumor immune response. The specific molecular mechanism is not clear, and part of the existing research results show that the mechanism is a plurality of action factors such as the induction of cell death caused by the replication and proliferation of viruses in tumor cells, the interaction with antiviral elements of the tumor cells, the promotion of inherent spontaneous or specific antitumor immune response and the like, and the development of related research and development plans and clinical test projects are carried out in terms of the uniqueness of the modified viral vector system in great numbers by International biological medicine company and scientific research institutions.

The invention fills the problem that the domestic and foreign needs cannot be solved for a long time by designing and developing an attenuated virus vector system (AVS deimmunized inhibition virus vector system) capable of quickly and efficiently chimeric expressing human functional super cytokines. The invention inserts the nucleotide sequence of super cell factors such as IL2 x 15 into the modified virus expression vector through the means of gene editing, carries out gene recombination rescue in specific eukaryotic cells to obtain attenuated viruses of stably expressed chimeric super cell factors, screens and obtains AVS (M3) -rIL2 x 15 viruses capable of efficiently expressing novel recombinant cell factors in tumor tissues, and simultaneously utilizes the system to further evaluate the curative effect of the recombinant system in a solid tumor model, thereby providing a novel technical scheme and selection for developing treatment products of solid tumors.

The invention relates to an attenuated viral vector system chimeric novel recombinant cytokine and practical application thereof in solid tumors. The core structure gene position of the virus vector system can be embedded with one or more chimeric regions of coding super cytokines, and the super cytokines have the characteristics of effectively changing the immunosuppression effect in tumor microenvironment and recovering CD8 ⁺ T ability to kill tumor cells, the invention also relates to methods of treating tumors using one or more of such compositions in combination.

Cytokines and methods of use

Such fusion proteins comprising cytokines and their physiological receptors are sometimes also referred to as "hypercytokine" because of their high activity at lower doses than single cytokines and/or mixtures of cytokines with their soluble receptors.

Tumor immunotherapy

The concept of cancer therapeutic vaccines is based on the knowledge that acquired immunity can be elicited and activated to specifically recognize and kill tumor cells. Several vaccine studies have shown immune and clinical responses in selected patients (e.g., renal cell carcinoma patients) over the past 25 years (Kubler & vieneg 2006). With the discovery of tumor-associated antigens or Dendritic Cells (DCs) and advances in molecular biology and bioengineering (providing recombinant cytokines and gene delivery systems), several tumor vaccination strategies are proposed: tumor cell-based vaccines (e.g., BCG, corynebacterium parvum, or IFN) formed by mixing tumor cells with specific adjuvants; a genetically modified tumor vaccine based on tumor cells expressing genes encoding immunostimulatory factors; and DCs modified with tumor-derived RNAs that carry peptide/tumor lysates or are fused with tumor cells.

The inventors of the present application have studied a tumor therapeutic agent for mass production in advance. In research on tumor cells genetically modified to express supercytokines, therefore, the compositions of the present application comprising first and second allogeneic cell lines genetically modified to express the same or different supercytokines can be more suitable as tumor therapeutic drugs than drugs known in the art.

Disclosure of Invention

In a first aspect, the application relates to a composition comprising (1) one or more first cells modified to express a first ultrafine cytokine, and (2) one or more second cells modified to express a second ultrafine cytokine, wherein the one or more second cells are different from the one or more first cells.

In a second aspect, the present application relates to a composition for use in the first aspect of medicine.

In a third aspect, the present application relates to a pharmaceutical composition comprising the composition of the first or second aspect, further comprising a pharmaceutically acceptable diluent, carrier, excipient, filler, binder, lubricant, glidant, disintegrant, adsorbent and/or preservative.

In a fourth aspect, the present application relates to a composition of the first or second aspect or a pharmaceutical composition of the third aspect for use in the prevention or treatment of cancer.

In a fifth aspect, the present invention relates to the use of a composition according to the first or second aspect for the preparation of a pharmaceutical composition for the treatment or prophylaxis of cancer.

Various cytokines are known to have important potential application values in tumor immunotherapy, hematopoietic stem cell transplantation, immune vaccine preparation and the like. In the aspect of anti-tumor, animal experiments prove that the IL2 cytokine can partially delay the growth of tumors and possibly excite specific immune protection response to malignant melanoma, lymphoma, fibrosarcoma, liver tumor, ovarian cancer and other solid tumors or leukemia.

Firstly, a computer simulated modified body of IL2 (humanized IL 2) is synthesized into a full-length base sequence (363 bp) by adopting a gene synthesis technology, cloned to an expression vector pcDNA3.1, and expressed in eukaryotic cells, and the research of IL2 is mainly used for melanoma, renal cell carcinoma, prostate cancer and post-healing use of tumor patients after radiotherapy and chemotherapy. However, the eukaryotic expression yield is low, and the requirement of large-scale application cannot be met. The mutant is integrated into an AVS system, recombinant AVS virus is amplified in 293 cells, culture supernatant of recombinant factor IL2 x 15 expressed by the virus through cell secretion is filtered, fractogel EMD SO3-650 is separated, affinity chromatography column separation (crude IL2 x 15), concentration, superdex 200 separation and concentration are carried out, and finally recombinant IL2 x 15 pure product is obtained. This process is complex but of high purity, so there is a need to simplify the industrial production and preparation process of IL2 x 15.

In addition, there are many breakthroughs in the current foreign tumor immunotherapy. Therefore, in the invention, the rhabdovirus AVS (M3) is used for mediating the super cytokine to carry out the immunoregulation, thereby improving the anti-tumor immunity of the organism and achieving good curative effect for treating tumors. The combined gene therapy by selecting cytokines with synergistic immunoregulatory and anti-tumor effects is a promising development direction of tumor immune gene therapy. There are many studies on the combination of multiple cytokines for the treatment of malignancy, but no targeted treatment of malignancy has been reported to date for expression of IL2 x 15 in a viral vector.

Thus, based on the above-mentioned needs, the present invention provides nucleic acid sequences encoding human IL2 x 15 and methods of using the same to express human IL2 x 15 and methods of co-expressing IL2 x 15 and uses for treating different malignancies.

The present invention relates to a polypeptide as set forth in SEQ ID No.:2 and SEQ ID No.:2, and a polynucleotide sequence shown in seq id no.

The invention also relates to vectors, preferably BHK cytoskeletal vectors, containing said polynucleotide sequences.

The invention also relates to an attenuated rhabdovirus AVS positive recombinant containing the vector.

The invention further relates to IL2 x 15 expressed by said attenuated rhabdovirus AVS recombinant, i.e. SEQ ID No.:3, and a sequence of amino acids.

The invention further relates to a method for producing IL2 x 15 comprising the steps of:

1) Subcloning the polynucleotide sequence encoding IL2 x 15 onto a viral backbone vector, screening clones containing positive plasmids;

2) Transferring the positive framework plasmid into BHK cells, and inducing to express IL2 x 15 expressed in a secretion form;

3) Separating and purifying the secretion protein;

the method is characterized in that the polynucleotide sequence for encoding IL2 x 15 is SEQ ID NO.:2, and a polynucleotide sequence shown in seq id no.

The invention further relates to the use of IL2 x 15 for the manufacture of a medicament for stimulating proliferation of CAR-NK cells

The invention also relates to the use of IL2 x 15 in the preparation of an anti-malignant medicament.

The invention further relates to a polypeptide as set forth in SEQ ID No.:3 encoding IL2 x 15.

The invention further relates to a modified rhabdovirus AVS (M3) skeleton plasmid containing the polynucleotide sequence gene.

The invention further relates to recombinant rhabdovirus AVS (M3) containing said vector.

The invention further relates to the use of the IL2 x 15 and immunoadjuvant in combination expression for preparing an anti-malignant tumor medicament.

There are many cell culture systems that can be used in the present invention, e.g., prokaryotic, mammalian culture systems, all of which can be used to express SEQ ID No.:2 encoding IL2 x 15.

In accordance with the long-standing patent law, the specification includes the words "a" and "an" as used in the claims, including the word "one or more". Some embodiments of the invention may consist of or consist essentially of one or more of the elements, method steps and/or methods of the invention. It is contemplated that any of the methods or compositions described herein may be practiced with respect to any other method or composition described herein.

Detailed Description

Definition of the definition

Before describing the present invention in detail, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification, reference is made to several documents, each of which is cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, instructions for use, sequences submitted by GenBank accession numbers, etc.), whether previously or later, is incorporated herein by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The term "ultrafine cytokine" refers to a fusion protein comprising, consisting essentially of, or consisting of (a) a soluble portion of a cytokine receptor, and (b) a cytokine that can bind to the soluble portion of the cytokine receptor under physiological conditions, and an optional peptide linker located between the soluble cytokine receptor and the cytokine. In a preferred embodiment, the cytokine is GM-CSF, IL-6, IL-12, IL-15, anti-TGF, EPO, interferon, LIF, OSM, CNTF, CT-1. If the cytokine is located at the N-terminus relative to the cytokine receptor, it is preferred that the cytokine still contain its secretion signal, which will be cleaved off during protein maturation, i.e., the mature ultrafine cytokine protein will not contain the secretion signal. If the cytokine is located at the C-terminus relative to the cytokine receptor, it is preferred that the cytokine does not contain its secretion signal. The term "soluble cytokine receptor" refers to a soluble fragment of the cytokine receptor, such as lacking most or all of the transmembrane and cytoplasmic portions of the cytokine receptor, and includes most or all of the extracellular portion thereof; such as sIL-6R and sIL-12R. A receptor fragment is soluble if it has no or substantially no insertion onto the membrane of a mammalian cell (preferably a human cell) expressing the receptor fragment. If the cytokine receptor is located at the N-terminus relative to the cytokine, it is preferred that the cytokine receptor still contain its secretion signal, which will be cleaved off during protein maturation, i.e., the mature ultrafine cytokine protein will not contain the secretion signal. If the cytokine receptor is at the C-terminus relative to the cytokine, it is preferred that the cytokine receptor does not contain its secretion signal. As described above, the ultrafine cytokines optionally comprise a peptide linker between the cytokine receptor and the cytokine. Preferably, the peptide linker has low or no immunogenicity. More preferably, the peptide linker is non-immunogenic to humans. In a preferred embodiment, the soluble cytokine receptor is located in the amino-terminal portion of the ultrafine cytokine and the cytokine is located in the carboxy-terminal portion of the ultrafine cytokine.

The term "ultrafine cytokine activity" refers to the activity of a fusion protein. Although, based on the same molar amount, particularly preferred ultrafine cytokines have 100 to 1000-fold activity in the same assay as the cytokine or cytokine and cytokine receptor mixture (i.e., the unfused portion forms the supercytokine) upon which they are based, not every ultrafine cytokine exhibits such a significant increase, depending on the length of the cytokine and the portion of the soluble cytokine receptor involved and the length of the protein linker, if any. A large number of assays are known to be useful in assessing the activity of each cytokine, which forms the basis of the ultra-fine cytokines that can be used in the present invention. An activity of an ultrafine cytokine is considered to be present within the meaning of the invention if it is at least 10 times the activity of the naturally occurring cytokine on which it is based (in the same molar amount) or as a mixture of cytokine fraction and unfused fraction of soluble cytokine receptor. Preferably, at the same molar amount, the activity is at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold or 1000-fold that of the cytokine or combination of cytokine and soluble cytokine receptor on which it is based. Suitable assay systems include, for example, those described in Fischer M.et al (1997) for IL-6 ultrafine cytokines that induce BAF-3/cell proliferation.

The expression "at least 90% sequence identity" as used throughout the specification preferably means at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the respective reference polypeptide. When it is not specifically indicated which reference sequence is referred to calculate the percent sequence identity, then the percent sequence identity will be calculated with reference to the longer of the two sequences to be compared. If a reference sequence is indicated, the sequence identity is determined based on the full length sequence shown in SEQ ID. For example, a peptide sequence consisting of 21 amino acids hybridizes to SEQ ID NO:2 may exhibit a percent maximum sequence identity of 9.9% (21:212) and a sequence of 106 amino acids may exhibit a percent maximum sequence identity of 50% (106:212).

The similarity of nucleotide and amino acid sequences, i.e., percent sequence identity, can be determined by sequence alignment. Such an alignment may be performed by several algorithms well known in the art, preferably hmmalign (HMMER package, http:// HMMER. Wust. Edu /) or CLUSTAL algorithm (Thompson J.D. et al, 1994), with the preferred parameters used being default parameters set as indicated in http:// www.ebi.ac.uk/clustalw/index. Html#. The degree of sequence identity (sequence matching) can be calculated using, for example, BLAST, or BlastZ (or BlastX). Preferably, the sequence matching analysis is further supplemented by recognized homology profiling techniques such as Shuffle-lag an (Brudno m., 2003) or Markov random fields. When calculating the percentage of sequence identity in the context of the present invention, these percentages are calculated relative to the full length of the longer sequence, if not specifically indicated otherwise.

"peptide linker" in the context of the present invention refers to an amino acid sequence of 1-100 amino acids. In preferred embodiments, the minimum length of the peptide linker of the invention is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In further preferred embodiments, the peptide linker of the invention has a maximum length of 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids or less. In a particularly preferred embodiment, the above preferred minimum and maximum lengths of the peptide linker of the invention may be combined, provided that such combination is of arithmetic significance. In a further preferred embodiment, the peptide linker of the invention is non-immunogenic, and in a particularly preferred embodiment, the peptide linker is non-immunogenic to humans.

Specifically, the present invention provides:

a novel cytokine delivered by a virus, the cytokine delivered by the virus being selected from the group consisting of an altered body of IL2 selected from the group consisting of a computer-simulated design, the altered body of IL2 having a dimeric receptor that selectively activates IL2rβ, yc, while losing the activity of native IL2 as an agonist of IL2rα (CD 25) receptor.

Preferably, the virus is selected from the group consisting of RNA viruses selected from the Rhabdoviridae family, and the genus selected from the group consisting of attenuated strains screened for vesicular stomatitis.

Preferably, the attenuated strain is a three-locus nonsensical mutation produced by the viral vector encoding M, the amino acid sequence of the viral gene after a specific mutation being at least 90% identical to the amino acid sequence of SEQ ID NO. 1 and being aligned with the wild-type strain, the gene sequence of the attenuated strain having amino acid mutations at three specific positions of SEQ ID NO. 1 (M protein).

Preferably, the attenuated virus strain is AVS-M3, the M gene mutation sites of the AVS-M3 are respectively that the 21 st amino acid G of the M gene is mutated into the propylamino acid A, the 51 st amino acid M is mutated into the alanine A, the 221 th valine V is mutated into the phenylalanine F, and the 226 th serine S is mutated into the arginine R.

Preferably, the exogenous chimeric gene integration site of the novel cytokine on the virus is the spacer sequence of the envelope gene G and the polymerase gene L of the viral system.

Preferably, the exogenous chimeric gene is selected from the group consisting of humanized cytokines, said humanized cytokines having the property of altering the tumor microenvironment.

Preferably, the gene sequences of cytokines of IL2 and IL15 (IL 2 x 15), said IL2 x 15 cytokine is an active polypeptide which binds with high intensity to dimeric receptors of IL2rβ, yc but has NO binding capacity to the other recognition receptor IL2rα of IL2, the other recognition receptor CD215 of IL15, said engineered cytokine IL2 x 15 is SEQ ID NO:2, wherein the nucleotide sequence encoding IL2 x 15 is SEQ ID No.:1, and the novel cytokine delivered by the virus is expressed as AVS (M3) -IL2 x 15.

Preferably, AVS (M3) -IL2 x 15 is selected from vesicular stomatitis virus or maraba virus, or recombinant vesicular stomatitis virus or recombinant maraba virus that retains the biological activity of said vesicular stomatitis virus or maraba virus.

Preferably, the vesicular stomatitis virus is selected from the group consisting of the vesicular stomatitis virus indiana strain, the vesicular stomatitis virus new jersey strain.

Preferably, the recombinant vesicular stomatitis virus or recombinant genetically modified virus has potent oncolytic and/or attenuated activity relative to the natural wild-type virus.

Also provided is a method for preparing a cytokine composition for human use produced with a recombinant viral vector (AVS-EV), comprising the main steps of:

a) Subcloning the polynucleotide sequence encoding IL2 x 15 onto a backbone vector AVS (M3), screening cloned AVS (M3) -IL2 x 15 containing positive recombinant backbone plasmids;

b) The positive skeleton plasmid is transduced into BHK cells, and the induced expression is expressed in a secretory form

IL2 x 15 modification;

c) And separating and purifying the secreted protein.

Preferably, it has the activity of stimulating the proliferation of hematopoietic stem cells and activating immune cells in vitro, including NK/TCR-T, or as an anti-malignant pharmaceutical composition.

Preferably, the viral AVS-EV (M3) backbone may express a cytokine composition comprising one or more different expression cells modified to express a first ultrafine cytokine and one or more different expression cells modified to express a second ultrafine cytokine.

Preferably, the first and/or second ultrafine cytokines are fusion proteins comprising cytokine receptors and cytokines.

Preferably, the cytokine receptor is independently selected from (a) IL2R, IL15R, GMCSF-R, sIL-6R, sIL-11R, OSM-R, CNTF-R and CT-I-R; or (b) a polypeptide exhibiting at least 90% sequence identity to the polypeptide of (a); and wherein the cytokine is independently selected from (c) IL2, IL15, IL-6, IL-11, OSM, CNTF, and CT-I; or (d) a polypeptide exhibiting at least 90% sequence identity to the polypeptide of (c); wherein said cytokine is preferably an engineered version of the activity of an ultrafine cytokine.

Preferably, the virus is a vector of genome chimeric IL2 x 15, which has the characteristic of precisely targeting a tumor microenvironment system, and the chimeric vector of IL2 x 15 has the purpose of resisting malignant tumors.

Preferably, the virus further comprises one of attenuated strains with the same or similar characteristics in the genera vesicular virus, rabies virus, transient fever virus, non-virulent granoviruses and the like, and the attenuated virus is a stable genetic high-titer virus strain obtained by gene mutation screening libraries.

Preferably, the preparation method is obtained by recombinant rescue in an AVS vector system using a specifically genetically engineered cell line, which is a cell for industrial-grade production.

Preferably, the use of a therapeutically effective amount of a virus in treating a subject, comprising the step of delivering to the subject a therapeutically effective amount of the virus, wherein the subject is administered by one or more site-directed administration routes selected from the group consisting of intramuscular injection, intravenous injection, intratumoral administration, minimally invasive intervention of organ tissue, intelligent delivery by micro-robot, and the like.

Preferably, the cancer treatment is one or a combination of surgical treatment, radiation therapy, chemotherapy, immunotherapy, hormonal therapy.

Also provided is a composition of AVS (M3) -rIL2 x 15 attenuated virus in combination with an immune checkpoint inhibitor, the activity in the composition further comprising a combination with one or more additional active agents controlling or treating a tumor, said additional active agents comprising: clofibrate, choline, methionine, niacin or ursodeoxycholic acid.

Preferably, the composition further comprises a second oncolytic virus.

Preferably, the second oncolytic virus is selected from one or more comprising vaccinia virus, herpes virus, measles virus, newcastle disease virus, rhabdovirus, alphavirus, parvovirus, enterovirus strains.

Preferably, the second oncolytic viral drug is selected from attenuated oncolytic viruses.

Preferably, the use in the manufacture of a medicament for killing abnormally proliferative cells, inducing an immune response that promotes anti-tumour or eliminating micro-environmental immunosuppression of tumour tissue.

Preferably, the composition comprises a clinically administered dose of said AVS (M3) -ril2 x 15, said AVS (M3) -ril2 x 15 comprising 1 x 10 ⁸ PFU-1×10 ¹² A single administration dose of PFU, said immune checkpoint inhibitor comprising a single use dose of 1-50 mg/kg.

Preferably, AVS (M3) -ril2×15 comprises 1×10 ⁹ A single administration dose of PFU, the immune checkpoint inhibitor containing a single use dose of 10 mg/kg.

Preferably, the abnormally proliferative cell is contained in the patient.

Preferably, the aberrant proliferative cells are selected from tumor cells or tumor tissue-associated cells.

Preferably, the tumor cell is a cancer cell.

Preferably, the cancer cells are metastatic cancer cells.

Preferably, the use of the composition for the manufacture of a medicament for the treatment of a patient suffering from a tumor and/or cancer.

Preferably, the composition comprises a clinical dose ranging from 5 to 50mg/kg, said AVS (M3) -rIL2 x 15 comprising 1X 10 ⁸ PFU-1×10 ¹² Single administration dose of PFU.

Also provided is a method of inhibiting and/or killing cells of abnormal proliferation in a subject, the method comprising sequentially subjecting the subject to the steps of: 1) Administering AVS (M3) -ril2 x 15 to the subject, wherein said AVS (M3) -ril2 x 15 is capable of selectively replicating in tumor cells; 2) Administering an immune checkpoint inhibitor to said subject after administration of AVS (M3) -ril2 x 15 as described in step 1).

Preferably, AVS (M3) -ril2×15 is a clinically administered dose, said AVS (M3) -ril2×15 containing 1×10 ⁸ PFU-1×10 ¹² A single administration dose of PFU, said immune checkpoint inhibitor is a clinically administered dose, said immune checkpoint inhibitor is a single use dose comprising 5-50 mg/kg.

Preferably, AVS (M3) -rIL2 is administered intratumorally at a dose of every 100mm ³ Tumor correspondence 2×10 ⁷ A single administration dose of PFU, the immune checkpoint inhibitor containing a single use dose of 10 mg/kg.

Preferably, the dose of AVS (M3) -rIL2 x 15 is a clinical dose administered 1 time every 3 days, 3-7 times in succession; the administration dosage of the immune checkpoint inhibitor is 1 time every 2 days, and 3-5 times of continuous administration are carried out.

Preferably, AVS (M3) -ril2 x 15 recombinant, a composition or vaccine comprising the isolated recombinant AVS (M3) -ril2 x 15 recombinant is administered by a mode of administration comprising one or more of intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, intratumoral, subcutaneous or intranasal administration; the administration route of the administration mode comprises one or more of endoscope, intervention, minimally invasive and traditional operation; the immune checkpoint inhibitor is administered intravenously or intraperitoneally.

Preferably, the dysproliferating cells are selected from the group consisting of tumor and/or cancer cells.

Preferably, the method further comprises the step of administering a second anti-tumor therapy.

Preferably, the second anti-tumor therapy is selected from the group consisting of administration of a second oncolytic virus; the second oncolytic virus is selected from one or more of rhabdovirus, vaccinia virus, herpes virus, measles virus, newcastle disease virus, rhabdovirus AVS (M3), alphavirus, parvovirus, enterovirus strain.

Preferably, the second oncolytic virus is an attenuated oncolytic virus.

Preferably, the second oncolytic virus is an attenuated poxvirus.

Preferably, the tumor and/or cancer is selected from lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphatic cancer, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia.

Preferably, the second anti-tumor therapy is selected from one or more of chemotherapy, radiation therapy, immunotherapy, surgical therapy.

Also provided is a method of preparing recombinant viral AVS (M3) -ril2×15 that co-expresses IL2×15, comprising the steps of:

d) Subcloning the polynucleotide sequence encoding IL2 x 15 onto modified rhabdovirus pAVS (M3) skeleton vector, screening clone of positive skeleton plasmid containing IL2 x 15 gene;

e) Co-transfecting the positive framework plasmid with pP, pL, pN and pT7 genome plasmid pAVS-rIL2 x 15 in BHK21 cells by using a liposome transfection technology, and assembling virus particles in the cells to generate recombinant virus AVS-rIL2 x 15;

f) Collecting cell culture solution supernatant after 3 days, infecting freshly cultured Vero cells, and collecting the supernatant after 48-72 hours to obtain 1 st generation recombinant rhabdovirus AVS (M3) -rIL2 x 15;

293 cells were cultured in large amounts, 293 cells were infected with recombinant Rhabdoviral AVS (M3) -rIL 2X 15 expressed at high efficiency, and supernatants were collected after 1 day, and recombinant viral titers were determined in Vero cells by Karber method. The following drawings form a part of the present invention and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments.

Specifically, the medicine provides a plurality of administration modes including local injection or systemic administration or intratumoral delivery, which can obviously inhibit the growth of malignant tumor, and simultaneously can effectively activate the autoimmune system of a patient, change the tumor microenvironment, activate the specific anti-tumor immune response of the organism, prevent the growth, diffusion and recurrence of tumor, achieve the purpose of eliminating or controlling tumor, relieve the immunosuppressive effect in the tumor microenvironment, further enhance the systemic killing tumor activity of immune cells, and has obvious excellent curative effect compared with the traditional therapy.

Drawings

FIG. 1 is a schematic diagram of construction of eukaryotic expression vectors for the super cytokine IL 2X 15.

FIG. 2 shows the identification of IL 2X 15 super cytokine expression, FIG. 2A shows IL 2X 15 expression in cell lysates and cell culture supernatants, and FIG. 2B shows ELISA to detect IL 2X 15 concentration in culture supernatants.

Fig. 3. Proliferation of IL2 x 15 on T cells in vitro, fig. 3A shows proliferation of IL2 x 15 on CD 8T cells, and fig. 3B shows proliferation of IL2 x 15 on CD25 Treg in mice.

Fig. 4. Proliferation of il2 x 15 on NK natural killer cells in vitro.

Fig. 5.AVS system mediated novel recombinant cytokine AVS (M3) -ril2.15 attenuated strain, fig. 5A is a photograph 24 hours after AVS (M3) -ril2.15 infection of cells, fig. 5B is a comparison of AVS (M3) -ril2.15 replication growth curves with control AVS-3M.

FIG. 6 evaluation of the effects of AVS (M3) -rIL 2.15 attenuated strains in animal models of lung cancer, FIG. 6A is a method for establishing lung cancer models, FIG. 6B is the size of lung cancer tissues of mice treated in experimental and control groups, and FIG. 6C is the survival rate of mice.

FIG. 7 evaluation of the effect of AVS (M3) -rIL 2.15 attenuated strain in animal models of colon cancer, FIG. 7A is a method of colon cancer model establishment, FIG. 7B is the size of colon cancer tissue of mice treated in experimental and control groups, and FIG. 7C is the survival rate of mice.

FIG. 8 shows the evaluation of the effect of AVS (M3) -rIL 2.15 attenuated strain in animal models of breast cancer, FIG. 8A shows the method for establishing breast cancer model, FIG. 8B shows the size of breast cancer tissue of mice treated by experimental group and control group, and FIG. 8C shows the survival rate of mice.

Detailed Description

The present invention will be further described below. In the following paragraphs, various aspects of the invention will be described in more detail. Each aspect described may be combined with any other aspect unless clearly indicated to the contrary. In particular, any feature which is considered to be preferred or advantageous may be combined with any other feature which is considered to be preferred or advantageous.

In a first aspect, the present invention provides a composition comprising:

1) One or more first cells modified to express a first ultrafine cytokine,

2) One or more second cells modified to express a second ultrafine cytokine,

or consist essentially of, or consist of, the above substance, wherein the one or more second cells are different from the one or more first cells.

The second cell is different from the first cell if the first and second cells are derived from different cell lines, and/or if the second cell has a different genetic modification than the first cell (e.g., the first and second cells are modified to express different ultrafine cytokines). In a preferred embodiment, the first and second cells are derived from the tissue of two different individuals, respectively, preferably from two different persons. Preferably the two tissues (preferably tumour tissue) are of the same type. The term tissue as used herein refers to a solid tissue (e.g., skin, liver, brain, kidney, lung, stomach, colon, bladder or testis) and a moving population of cells (e.g., lymphocytes or stem cells). Although the cells may be autologous or allogeneic, it is particularly preferred that the first and/or second cells are allogeneic. The term "allogeneic" refers to a relationship between a cell and a patient receiving the cell. Cells from a particular individual are allogeneic to any other patient, however they are autologous to the particular individual. For the mass production of any cell-based vaccine, the allo-form is a prerequisite, since otherwise the cells must be isolated and cultured from the individual patient to be treated to produce each cell vaccine individually. Allogeneic cells also have other advantages, including the ease with which allogeneic cells induce a stronger immune response in a patient than autologous cells.

The terms "one or more first cells" and "one or more second cells" as used herein refer to individual cells, clonal populations of such cells, and a series of like cells. Thus, in a preferred embodiment, wherein the cells originate from a primary tissue (primary tumor), preferably a primary tumor, the cells need not be completely clonal, but need to consist of one, two, three or more clonal cell populations belonging to a particular cell and/or tumor type. In particular, tumor cells exhibit high genetic variability after passage, and it is therefore common that cells within a constructed cell line are not identical genetically. These cells are examples of a series of similar cells. Another example is primary tumor cells derived from a tumor, which have been passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times in vitro, which will result in the selection of a subset of proliferating cells, thus reducing the heterogeneity of the cell population, i.e. leading to a more similar series of cells. In one embodiment, wherein the first and/or second cells are derived from a primary tissue, preferably a primary tissue of the same type, in particular a tumor tissue, they are considered to be different if the first and second cells are derived from two different individuals, preferably from two different persons.

The term "modified to express" means that the gene encoding each of the ultra-fine cell factors is stably introduced into a cell capable of stably expressing the gene encoding the ultra-fine cell factor and then producing the corresponding ultra-fine cell factor.

Preferably, the gene encoding the ultrafine cytokine is introduced into an expression vector for mammalian cells, which vector generally comprises an origin of replication (necessary, see below), a promoter located before the gene to be expressed, optionally an inverted enhancer, and any necessary ribosome binding sites, RNA splice sites, polyadenylation sites and transcription termination sequences. Such expression vectors can then be used to modify cells to express various ultra-fine cytokines.

In a preferred embodiment, the expression vector of the invention comprises a plasmid; phagemid; a bacteriophage; a cosmid; artificial chromosomes, in particular artificial mammalian chromosomes or artificial yeast chromosomes; knocking out or knocking in the construct; viruses, in particular rhabdovirus AVS (M6), vaccinia virus, attenuated vaccinia virus, canary poxvirus, lentivirus (Chang and Gay, 2001), herpes viruses, in particular herpes simplex virus (HSV-1, carlezon, et al, 2000), baculovirus, retrovirus, adeno-associated virus (AAV, carter and samulki, 2000), rhinovirus, human Immunodeficiency Virus (HIV), filoviruses and engineered variants of the foregoing (see e.g. Kobinger et al, 2001); viral minibodies; "naked" DNA; a liposome; a virus-like particle; and nucleic acid coated particles, in particular gold spherical particles, or essentially consisting of, or consisting of, the above substances. Particularly preferred are viral vectors, such as Rhabdoviral AVS (M6) vectors, lentiviral vectors, baculovirus vectors or retroviral vectors (Lindemann et al, 1997 and Springer et al, 1998). Examples of plasmids that produce such recombinant viral vectors include pFastBac1 (Invitrogen Corp., carlsbad Calif.), pDCMUV (Wiznerowicz et al 1997) and pShuttle-CMV (Q-biological, carlsbad, california). In the case of rhabdovirus AVS (M6) as expression vector, the coding sequence may be ligated into rhabdovirus AVS (M6) transcription/translation control complexes, such as late promoters and triplex leader sequences. The ultrafine cytokine gene can be inserted into the Rhabdoviral AVS (M6) genome by means of in vitro or in vivo recombination. Recombinant viruses produced by insertion into non-essential regions of the viral genome (e.g., the E1, E3 or E4 regions) remain viable and express the corresponding ultrafine cytokines in infected cells. Preferably, the viral vector used is modified such that it loses replication capacity to prevent viral particles from being produced by the first and/or second cells modified to express the supercytokines.

In order to stably express the transferred gene, the expression vector must either have an origin of replication to enable it to replicate independently of the genome of the cell or must be integrated into the genome of the first and/or second cell. In the first case, the expression vector is maintained in the form of an episome. Suitable origins of replication may be derived from SV40 or other viruses (e.g., polyoma, rhabdovirus AVS (M6), CMV, VSV, BPV). In the latter case, if the expression vector is integrated into a genome such as a chromosome, it is not necessary to provide an origin of replication.

For direct expression of the supercytokines, the genes encoding them are operably linked to promoters and/or enhancers recognized by the cellular transcription machinery. Suitable promoters may be derived from mammalian cell genomes (e.g., metallothionein promoters) or mammalian viral genomes (e.g., rhabdovirus AVS (M6) late promoters, vaccinia virus 7.5K promoters, or cytomegalovirus promoters). The SV40 viral early and late promoters are particularly useful because both are readily available from viruses as fragments comprising the SV40 viral origin of replication. Shorter or longer SV40 fragments may also be used, provided that they comprise an about 250bp sequence from the HindIII site to the BglII site at the viral origin of replication. In addition, it may also be possible and may be desirable to use promoters or regulatory sequences typically associated with the cytokine or cytokine receptor of the polynucleotide on which the ultra-fine cytokine is encoded.

"operably linked" as used herein means incorporated into a genetic construct such that the expression control sequence is effective to control expression of the coding sequence of interest.

Specific initiation signals may also be required for efficient translation of the coding sequence for the ultra-fine cytokine. These signals include the ATG initiation codon and its adjacent sequences. Exogenous translational control signals (including the ATG initiation codon) may need to be provided in addition. Those of ordinary skill in the art can readily determine and provide the necessary signals. It is well known that the initiation codon must be in frame or in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons may be of various origins, both natural and synthetic. Expression efficiency can be increased by inserting appropriate transcription enhancing elements and transcription terminators. In eukaryotic expression, it is generally desirable to incorporate a suitable polyadenylation site (e.g., 5 '-AATAAA-3') in the transcriptional unit if not included in the original cloned segment. Typically, the poly A addition site is located about 30 to 2000 nucleotides "downstream" of the protein termination site prior to the transcription termination site.

As described above, in addition to using expression vectors containing viral origins of replication, the cells can be transformed using vectors and selectable markers under the control of appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After introduction of the exogenous DNA, the genetically engineered cells may be grown in the enrichment medium for 1-2 days and then transferred to the selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, allowing the cell to stably integrate the plasmid into its chromosome and grow to form a spot, which in turn forms a cell line by cloning and expansion.

Many selection systems can be used including, but not limited to, the herpes simplex virus thymidine kinase (tk) gene, the hypoxanthine guanine phosphoribosyl transferase (hgprt) gene, and the adenine phosphoribosyl transferase (aprt) gene in tk-cells, hgprt-cells, or aprt-cells, respectively. Antimetabolite resistance can also be used as a basis for selection against dihydrofolate reductase (dhfr) (conferring resistance against methotrexate), gpt (conferring resistance against mycophenolic acid), neomycin (neo) (conferring resistance against aminoglycoside G-418) and hygromycin (hygro) (conferring resistance against hygromycin).

In a preferred embodiment, the expression vector used to transform, transfect or infect the cell to be modified comprises a gene encoding a selectable marker as a transcript with a gene encoding a ultrafine cytokine. To confirm independent expression of the selectable marker and the ultra-fine cytokine, an Internal Ribosome Entry Site (IRES) was placed between these two coding sequences.

The cells comprised in the composition of the invention preferably multiply individually. Preferably they are propagated in vitro in one of two ways: as non-anchorage dependent cells grown in suspension throughout the culture or anchorage dependent cells that need to be attached to a solid substrate for propagation (i.e. the cells grow in a monolayer). Suitable growth conditions depend on the cell type and can be determined by the skilled person by routine experimentation.

In a preferred embodiment of the composition of the first aspect, the first and/or second ultra-fine cytokines are fusion proteins comprising, consisting essentially of or consisting of soluble cytokine receptors and cytokines. In preferred embodiments, the soluble cytokine receptor is independently selected from (a) sIL-6R, sIL-11R, sOSM-R, sCNTF-R and sCT-I-R, or (b) a polypeptide exhibiting at least 90% sequence identity to the polypeptide of (a); and the cytokine is independently selected from (c) IL-6, IL-11, OSM, CNTF, and CT-I, or (d) a polypeptide that exhibits at least 90% sequence identity to the polypeptide of (c); and optionally a peptide linker between the soluble cytokine receptor and the cytokine, wherein the resulting fusion protein has ultrafine cytokine activity. Preferably, the fusion protein is arranged N-terminal to C-terminal as follows: soluble cytokine receptor-optionally peptide linker-cytokine. To ensure secretion of the expressed ultrafine cytokines, the ultrafine cytokines comprise at least one natural or artificial secretion signal. Because all cytokines are secreted, they naturally contain such secretion signals. Similar signal transduction peptides are also found in cytokine receptors. Preferably, the secretion signal is located at the N-terminus of the fusion protein. The signal peptide will be cleaved off during processing and/or secretion of the ultra-fine cytokine.

An optional peptide linker;

or consist essentially of or consist of the above, wherein in a related assay of IL-2 activity, the fusion protein preferably has an ultrafine cytokine activity of SEQ ID NO:5, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more of the activity of the IL2-IL15 fusion protein shown in figure 5.

Preferred expression cassettes comprise super IL-2 x 15 and Neo selectable markers, both under the control of the CMV immediate early promoter. The expression cassette may be contained in a different vector, preferably a viral vector, such as a retroviral vector.

In a preferred embodiment of the first aspect, the second cells (preferably the allogeneic) are of a different Human Lymphocyte Antigen (HLA) type than the first cells (preferably allogeneic first cells). The HLA system is the name for the human Major Histocompatibility Complex (MHC). The gene group encoding this complex is located on chromosome 6 and encodes a cell surface antigen presenting protein and many other genes. Most HLA antigens are essential elements of immune function.

Because it is contemplated that the compositions of the present invention may further enhance the anti-tumor immune response, the compositions may further comprise adjuvants commonly used in vaccines to enhance the immune effect. Preferred adjuvants are selected from: unmethylated DNA, in particular unmethylated DNA comprising CpG dinucleotides (CpG motifs), in particular CpG ODN with Phosphorothioate (PTO) backbone (CpG PTO ODN) or CpG ODN with Phosphodiester (PO) backbone (CpG PO ODN); colloidal precipitation of aluminum hydroxide (alum); bacterial products from the outer membrane of gram-negative bacteria, in particular monophosphoryl lipid a (MPLA), lipopolysaccharide (LPS), muramyl dipeptide and derivatives thereof; synthetic lipopeptide derivatives, in particular Pam3Cys; lipoarabinomannan; peptidoglycan; a yeast polysaccharide; heat Shock Proteins (HSPs), particularly HSP70; dsRNA and synthetic derivatives thereof, in particular polyinosinic acid; polycationic peptides, in particular poly-L-arginine; paclitaxel; fibronectin; flagellin; imidazoquinolines; cytokines with adjuvant activity, in particular GM-CSF, interleukins- (IL-) 2, IL-6, IL-7, IL-18, types I and II, interferons, in particular interferon-gamma, TNF-alpha; an oil-in-water emulsifier, in particular MF59 consisting of squalene; higher purity derivatives of Tween 80, span 85 (sorbitan trioleate) and QS-21, quil A, nonionic block polymers, in particular Poloxamer 401, saponins and their derivatives, in particular immunostimulatory fragments of saponins; polyphosphazene; n- (2-deoxy-2-L-leucinylamino-beta-D-glucopyranosyl) -N-octadecyldodecanamide hydroacetate (BAY R1005), 25-dihydroxyvitamin D3 (calcitriol); DHEA; murametide [ MDP (Gln) -OMe ]; murapalmitine; polymers of lactic acid and/or glycolic acid; polymethyl methacrylate; sorbitan trioleate; squalane; stearoyl tyrosine; squalene; a multi-vitamin mineral tablet (theramide); oligopeptides, in particular peptides presented by MHC class II molecules, are synthesized. Particularly preferred adjuvants that may be included in the compositions of the present invention are selected from unmethylated DNA, in particular unmethylated DNA comprising CpG dinucleotides (CpG motifs), in particular CpG ODN with Phosphorothioate (PTO) backbone (CpG PTOODN) or CpG ODN with Phosphodiester (PO) backbone (CpG PO ODN), and synthetic lipopeptide derivatives, in particular Pam3Cys.

In a further aspect, the present invention relates to a composition of the present invention for use in medicine, the present invention relates to a pharmaceutical composition comprising a composition of the present invention, further comprising a pharmaceutically acceptable diluent, carrier, excipient, filler, binder, lubricant, glidant, disintegrant, adsorbent and/or preservative. Preferably, the pharmaceutical composition is formulated for parenteral administration, preferably in the form of a sterile aqueous solution which may also contain other substances, such as a sufficient amount of salt or glucose to render the solution isotonic with blood. The aqueous solution should be suitably buffered (preferably to a pH of 3-9) if necessary. A particularly preferred aqueous solution is Phosphate Buffered Saline (PBS).

In another aspect, the invention relates to a composition of the invention or a pharmaceutical composition of the invention for use in the treatment or prevention of cancer. Cancers that may be treated or prevented with the compositions of the present invention are preferably selected from: gastrointestinal cancer, colorectal cancer, liver cancer, pancreatic cancer, renal cancer, bladder cancer, prostate cancer, endometrial cancer, head and neck cancer, ovarian cancer, testicular cancer, prostate cancer, skin cancer, eye cancer, melanoma, dysplastic oral mucosa, invasive oral cancer, small cell and non-small cell lung cancer, hormone-dependent breast cancer, hormone-independent breast cancer, metastatic squamous cell carcinoma, neuromalignant tumors including neuroblastoma, glioma, astrocytoma, osteosarcoma, soft tissue sarcoma, hemangioma, endocrine tumors, hematological tumors including leukemia, lymphoma, other myeloproliferative and lymphoproliferative disorders, carcinoma in situ, proliferative disorders, adenoma, and fibroma. Particularly preferred are treatment or prevention of melanoma, pancreatic cancer and renal cancer.

In a further aspect, the present invention relates to the use of a composition of the invention for the preparation of a pharmaceutical composition for the treatment or prevention of cancer.

The compositions of the present invention may be used to treat and/or prevent a variety of different cancers, however, the cancers that the present invention may treat or prevent are preferably selected from: gastrointestinal cancer, colorectal cancer, liver cancer, pancreatic cancer, renal cancer, bladder cancer, prostate cancer, endometrial cancer, head and neck cancer, ovarian cancer, testicular cancer, prostate cancer, skin cancer, eye cancer, melanoma, dysplastic oral mucosa, invasive oral cancer, small cell and non-small cell lung cancer, hormone-dependent breast cancer, hormone-independent breast cancer, metastatic squamous cell carcinoma, neuromalignant tumors including neuroblastoma, glioma, astrocytoma, osteosarcoma, soft tissue sarcoma, hemangioma, endocrine tumors, hematological tumors including leukemia, lymphoma, other myeloproliferative and lymphoproliferative disorders, carcinoma in situ, proliferative disorders, adenoma, and fibroma. Particularly preferred are prevention or treatment of melanoma, pancreatic cancer and renal cancer. In particular, in the case of treatment and/or prophylaxis of cancer, it is envisaged that the patient is vaccinated with a "cancer vaccine" before any symptoms of the disease appear, i.e. is subjected to protective vaccination; or the patient is vaccinated after the symptoms of the disease, i.e. receiving a therapeutic vaccination.

Expression of at least one other cytokine, in particular IL6, by the first and/or second cell expressing the supercytokine, preferably superil-2, may provide an even stronger in vivo anti-tumor response in certain tumors, in particular melanoma and renal carcinoma, than cells expressing the supercytokine alone. Thus, in a preferred use, the use of a first and/or second cell expressing a supercytokine modified to express at least one other cytokine for the manufacture of a medicament for the prevention or treatment of a proliferative disease.

It is particularly preferred herein when the first and second cells are from the same type of tissue (preferably tumor tissue), but have a partially or completely different HLA type than the first and/or second cells.

The invention will be illustrated in more detail hereinafter by means of non-limiting examples:

description of the preferred embodiments

Embodiment 1 eukaryotic expression of IL2×15 super-cytokines

The DNA sequence of IL2 x 15 is synthesized by a total gene synthesis method, as shown in a sequence chart 2, 6 histidine His tags are designed at the N end of IL2 x 15, the subsequent detection and purification are facilitated, the DNA sequence encoding IL2 x 15 is cloned to a eukaryotic expression vector pcDNA3.1 through BamHI and xhoI cleavage sites, and a pcDNA-sIL2 x 15 eukaryotic expression plasmid is obtained, and the specific construction method is shown in the chart 1.

Further, we cultured 5X10 in 10cm dishes ⁶ HEK293T cells were transfected with 10ug of plasmid pcDNA-sIL 2X 15 using Invitrogen Lipofectamine2000 transfection reagent, and the cell supernatants were harvested 48 hours after transfection, concentrated, and the supercytokines were detected by western blot method using His-tagged antibodies and expressed in transfected cells and cell supernatants with molecular weights around 14kD as expected (FIG. 2A), while we also detected the supernatant with IL 2X 15 content of approximately 85ng/mL by anti-His ELISA kit (FIG. 2B). Example 2 functional identification of IL 2X 15 super-cytokines

To verify the biological function of IL2 x 15, we isolated mouse spleen-derived CD8T cells using a meitian-4 kit, then amplified and cultured with commercial IL-2, and we prepared IL2 x 15 by themselves, examined its effect on CD8T amplification, and found that 50ng IL-2 in mice was indeed effective for CD8T amplification after 5 days, but that the effective dose of IL-2 x 15 was five times higher than IL-2, about 10ng was able to stimulate the same number of CD8T cells, indicating that IL2 x 15 was indeed able to exert IL-2 function, promoting proliferation of CD8T cells (fig. 3A). Furthermore, we given mice with IL2 x 15,1,3 intravenously for 5 days, 10 ng/mouse; mice were sacrificed on day 7, cd25+ suppressor T cells CD25 tregs were detected in the spleen cells of the mice, and no significant difference was found between the administered CD25 tregs and the control group, indicating that IL2 x 15 had no stimulatory effect on cd25+ suppressor T cells (fig. 3B).

IL-15 is required for proliferation of NK92, a natural killer cell line of patients with malignant non-Hodgkin's lymphoma, and we next see the effect of IL2 x 15 on NK92 natural killer cell expansion. As shown in FIG. 4, NK cells grew well and proliferated in a complete medium (complete culture) containing IL-15 and IL-2 cytokines after 3 days; whereas cells grew singly and apoptosis occurred in minimal medium lacking IL-15 cytokines (basal medium), we basal medium supplemented with 10ng or 50ng IL-2 x 15 cells grown similarly to complete medium, and had clumped expansion occurred. Finally, counting the number of living cells, the approach of complete culture medium and the addition of IL-2 x 15 super cytokine, more than 90% of cells are living cells, and only 5.9% of cells in the culture medium are living cells (figure 4), and the partial result shows that IL2 x 15 can promote the expansion of NK cells.

Example 3 construction of vector System for novel Rhabdoviral chimeric exogenous super cytokines IL2 x 15 super cytokine AVS (M3) -rIL2 x 15

Expression vector systems of viral backbone origin used so far are used only for a very small number of cell types. In contrast, the AVS vector system of the present invention is a broad host cell source. Because the recombinant human adenovirus contains Glycoprotein (GP), the recombinant human adenovirus can enter host cells without specific receptor mediation, can infect almost all mammalian cells, simultaneously complete virus replication and realize high-efficiency expression of exogenous chimeric genes, and greatly improves the expression efficiency of exogenous chimeric antibody in vivo and in vitro.

In the design scheme, the AVS attenuated virus strain constructed by us is firstly constructed to be used as a vector skeleton for delivering IL-2 x 15. In a specific embodiment, the DNA sequence encoding IL-2 x 15 was inserted into the viral genome by molecular cloning between the viral G and L genes using a virus mutated in vesicular stomatitis virus VSV M genes M51R, V221F, S226R, through xhoI and NheI cleavage sites (fig. 5A). In BHK cells, we have rescued the novel attenuated rhabdovirus AVS (M3) -ril2.15 expressing IL-2.15 using the VSV virus reverse genetics system, and we found significant cytopathy 24 hours after infection of BHK cells with the virus (fig. 5B), while we also made a comparison of 72 hour replication growth curves of one virus in BHK cells for wild-type VSV, and found that our attenuated strain replication capacity was lower for wild-type VSV (fig. 5C).

Example 3 treatment of AVS (M3) -rIL 2. Times.15 on mouse non-small cell lung carcinoma

The method is characterized in that the effect evaluation of the AVS system related in the patent on the treatment of the solid tumor is utilized, the virus vector system is utilized to express the super cytokine with functions to bind the immune checkpoint antigen in tumor tissues, and the local immune suppression effect of the tumor is broken. Embodiments relate to therapeutic administration with antibodies expressed from specific purified recombinant non-segmented, negative-strand RNA viral vectors. The related super cytokine is specifically combined with immune checkpoint antigen IL2 x 15 molecules, so that the immune inhibition effect of tumor cells is blocked, and the killing activity of immune cells is enhanced.

The amino acid sequence of the related super cytokine IL2 x 15 is shown in SEQ ID NO. 2, and the super cytokine of the sIL2 x 15 has human-mouse homology, so that the treatment effect of AVS (M3) -rIL2 x 15 can be directly verified by using a non-humanized mouse tumor model.

Specific technical details are as follows: firstly, establishing a metastatic non-small cell lung cancer model, and subcutaneously inoculating 4.0 x 10≡5 (200 uL) LLC cells per CB7 BL/6. Tumor size was measured every 1 day and calculated as follows: m12 is M2/2 (M1: short diameter, M2: long diameter). After tumor volume of each group of mice grew over 50mm3, 10 was given at day8, day10 and day12, respectively ⁷ Intratumoral injection of PFU (20 ul) was treated with virus and changes in tumor volume were recorded by continuous observation (fig. 6A).

Pharmacodynamic performance test of AVS (M3) -ril2 x 15 immunotherapy as shown in fig. 6, the intratumoral injection therapy for 3 consecutive days, AVS (M3) -ril2 x 15, compared with control virus VSV-3M, effectively inhibited the tumor growth trend, greatly delayed the life cycle of mice, and by analyzing PBS untreated mice, tumor size was 175mm 3 at day 25 post-inoculation, VSV-3M therapy group size was about 510mm3, whereas mice received AVS (M3) -ril2 x 15 therapy group size was about 85mm3 (fig. 6B). By means of the mouse survival curve, it was found that about 50% of the mice tumors in the VSV-scFV-PDL1 treated group were reduced until they disappeared, and were completely relieved, the growth rate of the surviving mice tumors was effectively inhibited, and the final survival rate was 75% (6/8), which was greatly superior to that of the VSV-3M treated group (3/8; 37.5%), PBS untreated group (0%), as shown in FIG. 6C.

Example 4 treatment of AVS (M3) -rIL 2.times.15 on a mouse colon cancer model

Since our AVS (M3) -ril2×15 did not carry tumor-specific antigens, we speculate that AVS (M3) -ril2×15 also have therapeutic effects on other types of tumors. Referring to embodiment 3, we set up a colon cancer model in C57BL/6 mice, and subcutaneously inoculate 2.0 x 10≡5 (200 uL) MC38 cells per CB7 BL/6. Tumor size was measured every 1 day and calculated as follows: m12 is M2/2 (M1: short diameter, M2: long diameter). After tumor volume of each group of mice grew over 50mm3, 10 was given at day8, day10 and day12, respectively ⁷ Intratumoral injection of PFU (20 ul) was treated with virus and changes in tumor volume were recorded by continuous observation (fig. 7A).

Similar to the non-small cell lung cancer model, we have seen a good therapeutic effect of AVS (M3) -ril2×15 on the mouse colon cancer model, as shown in fig. 7B, PBS non-treated group with tumor volume size of 1300mm at 25 days ³ Whereas the tumor size of mice in the treatment group with AVS (M3) -rIL 2.multidot.15 was 100mm ³ The ratio is better than that of the control group AVS-3M 430mm ³ . The survival rate of the corresponding AVS (M3) -ril2.15 was 62.5%, which was superior to that of PBS and AVS-3M mice (fig. 7B and 7C).

Example 5 treatment of AVS (M3) -rIL 2.times.15 on mouse breast cancer model

Breast cancer is the tumor with the highest incidence in women, and in order to examine the effect of AVS (M3) -ril2×15 on breast cancer treatment, we established a mouse breast cancer model. 5.0 x 10≡5 (200 uL) 4T1 cells were inoculated subcutaneously per CB7 BL/6. Tumor size was measured every 1 day and calculated as follows: m12 is M2/2 (M1: short diameter, M2: long diameter). Treating tumor volume growth of each group of miceAfter 50mm3, 10 at day6, day8 and day10, respectively ⁷ Intratumoral injection of PFU (20 ul) was treated with virus and changes in tumor volume were recorded by continuous observation (fig. 8A).

We also seen that AVS (M3) -ril2×15 had good therapeutic effect on mouse breast cancer, as shown in fig. 8B, PBS non-treated group had a tumor volume size of 2200mm at 25 days ³ Whereas mice treated with AVS (M3) -rIL 2.multidot.15 had tumor sizes of 400mm ³ The following is superior to the AVS-3M 1100mm of the control group ³ (FIG. 7B), the survival rate of the corresponding AVS (M3) -rIL 2.15 was 57.1% (4/7), since AVS-3M 14.2% (1/7) (FIG. 7C).

Sequence listing

<110> Wang Xiaoxia

<120> a novel cytokine delivered by virus and its use in tumor treatment

<141> 2022-04-16

<160> 3

<170> SIPOSequenceListing 1.0

<210> 4

<211> 690

<212> DNA

<213> Adenovirus type 37

<400> 4

atgagttcct taaagaagat tctcggtctg aaggggaaag gtaagaaatc taagaaatta 60

gggatcgcac caccccctta tgaagaggac actagcatgg agtatgctcc gagcgctcca 120

attgacaaat cctattttgg agttgacgag agggacacct atgatccgaa tcaattaaga 180

tatgagaaat tcttctttac agtgaaaatg acggttagat ctaatcgtcc gttcagaaca 240

tactcagatg tggcagccgc tgtatcccat tgggatcaca tgtacatcgg aatggcaggg 300

aaacgtccct tctacaaaat cttggctttt ttgggttctt ctaatctaaa ggccactcca 360

gcggtattgg cagatcaagg tcaaccagag tatcacgctc actgcgaagg cagggcttat 420

ttgccacata ggatggggaa gacccctccc atgctcaatg taccagagca cttcagaaga 480

ccattcaata taggtcttta caagggaacg attgagctca caatgaccat ctacgatgat 540

gagtcactgg aagcagctcc tatgatctgg gatcatttca attcttccaa attttctgat 600

ttcagagaga aggccttaat gtttggcctg attgtcgaga aaaaggcatc tggagcgtgg 660

ttcctggact ctatccgcca cttcaaatga 690

<210> 2

<211> 366

<212> DNA

<213> Homo sapiens

<400> 2

atgggcagct cccaccatca ccatcaccac agcagcggac tggtgcccag aggaagccac 60

atgcccaaga agaagatcca gctccacgcc gagcacgccc tctacgatgc tctgatgatc 120

ctcaacatcg tgaagaccaa cagccctccc gccgaggaga agctggagga ctacgccttc 180

aacttcgagc tcattctgga ggagatcgcc agactgtttg agagcggcga ccagaaggat 240

gaggccgaga aggccaagag gatgaaggag tggatgaaga gaatcaagac cacagcctcc 300

gaggatgagc aagaggagat ggccaacgcc atcatcacca ttctgcagag ctggatcttc 360

agctga 366

<210> 3

<211> 121

<212> PRT

<213> Homo sapiens

<400> 3

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Ala Gly Ser His Met Pro Leu Leu Leu Ile Gly Leu His Ala Gly His

20 25 30

Ala Leu Thr Ala Ala Leu Met Ile Leu Ala Ile Val Leu Thr Ala Ser

35 40 45

Pro Pro Ala Gly Gly Leu Leu Gly Ala Thr Ala Pro Ala Pro Gly Leu

50 55 60

Ile Leu Gly Gly Ile Ala Ala Leu Pro Gly Ser Gly Ala Gly Leu Ala

65 70 75 80

Gly Ala Gly Leu Ala Leu Ala Met Leu Gly Thr Met Leu Ala Ile Leu

85 90 95

Thr Thr Ala Ser Gly Ala Gly Gly Gly Gly Met Ala Ala Ala Ile Ile

100 105 110

Thr Ile Leu Gly Ser Thr Ile Pro Ser

115 120

Claims

1. A novel cytokine delivered by a virus, wherein the cytokine delivered by the virus is selected from the group consisting of an altered form of IL2 selected from the group consisting of a computer-simulated design, and wherein the altered form of IL2 has the ability to selectively activate dimeric receptors for IL2rβ, yc while losing the activity of native IL2 as an agonist of IL2rα (CD 25) receptors.

2. The novel cytokine delivered by the virus of claim 1, wherein the virus is selected from the group consisting of RNA viruses selected from the family rhabdoviridae, and the genus of viruses is selected from the group consisting of attenuated strains screened for vesicular stomatitis.

3. The novel cytokine delivered by the virus of claim 2, wherein the attenuated strain is a three-locus nonsensical mutation of the viral vector encoding M, the amino acid sequence of the specifically mutated viral gene is at least 90% identical to the amino acid sequence of SEQ ID No. 1 and the gene sequence of the attenuated strain has amino acid mutations at three specific positions of SEQ ID No. 1 (M protein) in comparison to the wild-type strain.

4. The novel cytokine delivered by the virus of claim 3, wherein the attenuated strain is AVS-M3, the M gene mutation site of AVS-M3 is amino acid G at position 21 of the M gene mutated to propylamine a, amino acid M at position 51 mutated to alanine a, valine V at position 221 mutated to phenylalanine F, and serine S at position 226 mutated to arginine R, respectively.

5. The novel cytokine delivered by the virus of claim 4, wherein the novel cytokine has a foreign chimeric gene integration site on the virus that is a spacer sequence of envelope gene G and polymerase gene L of the viral system.

6. The novel cytokine delivered by the virus of claim 5, wherein the exogenous chimeric gene is selected from the group consisting of humanized cytokines, wherein the humanized cytokines have properties that alter the tumor microenvironment.

7. The novel cytokine delivered by the virus of claim 1, wherein the gene sequences of the cytokines IL2 and IL15 (IL 2 x 15), the IL2 x IL15 cytokine is an active polypeptide that binds with high intensity to dimeric receptors IL2rβ, yc but does not bind to both the other recognition receptor IL2rα of IL2 and the other recognition receptor CD215 of IL15, and the engineered cytokine IL2 x 15 is SEQ ID NO:2, wherein the nucleotide sequence encoding IL2 x 15 is SEQ ID No.:1, and the novel cytokine delivered by the virus is expressed as AVS (M3) -IL2 x 15.

8. The novel cytokine delivered by the virus of claim 7, wherein AVS (M3) -IL2 x 15 is selected from vesicular stomatitis virus or maraba virus, or recombinant vesicular stomatitis virus or recombinant maraba virus that retains the biological activity of the vesicular stomatitis virus or maraba virus.

9. The novel cytokine delivered by the virus of claim 8, wherein the vesicular stomatitis virus is selected from the group consisting of vesicular stomatitis virus indiana strain, vesicular stomatitis virus new jersey strain.

10. The novel viral-delivered cytokine according to claim 9, wherein the recombinant vesicular stomatitis virus or recombinant genetically modified virus has potent oncolytic and/or attenuated activity relative to the natural wild-type virus.

11. A method for preparing a cytokine composition for human use produced with a recombinant viral vector (AVS-EV), comprising the main steps of:

b) The positive skeleton plasmid is transduced into BHK cells, and IL2 x 15 modified body is expressed in a secretion form by induced expression;

c) And separating and purifying the secreted protein.

12. The method according to claim 11, wherein the composition has an activity for stimulating proliferation of hematopoietic stem cells and activating immune cells in vitro, and comprises NK/TCR-T or is used as an anti-malignant pharmaceutical composition.

13. The method of claim 12, wherein the viral AVS-EV (M3) backbone can express a cytokine composition comprising one or more different expressing cells modified to express a first ultrafine cytokine and one or more different expressing cells modified to express a second ultrafine cytokine.

14. The method of claim 13, wherein the first and/or second ultra-fine cytokines are fusion proteins comprising cytokine receptors and cytokines.

15. The method of claim 14, wherein the cytokine receptor is independently selected from the group consisting of (a) IL2R, IL, 15R, GMCSF-R, sIL-6R, sIL-11R, OSM-R, CNTF-R, and CT-I-R; or (b) a polypeptide exhibiting at least 90% sequence identity to the polypeptide of (a); and wherein the cytokine is independently selected from (c) IL2, IL15, IL-6, IL-11, OSM, CNTF, and CT-I; or (d) a polypeptide exhibiting at least 90% sequence identity to the polypeptide of (c); wherein said cytokine is preferably an engineered version of the activity of an ultrafine cytokine.

16. The method of claim 11, wherein the virus is a vector of genomic chimeric IL2 x 15, having the property of targeting precisely tumor microenvironment system, and wherein the chimeric vector of IL2 x 15 has anti-malignant use.

17. The method of claim 16, wherein the virus further comprises one of attenuated strains of the genus vesicular, rabies, transient fever, non-virulent rhabdovirus, and the like, having the same or similar traits, wherein the attenuated virus is a stable genetic high titer strain obtained by screening a library for genetic mutations.

18. The method of claim 11, wherein the method is obtained by recombinant rescue in an AVS vector system using a specific genetically engineered cell line that is a cell for industrial-scale production.

19. The use of a novel cytokine delivered by a virus according to claims 1-7, comprising the step of delivering a therapeutically effective amount of the virus to an individual in need of treatment thereof, wherein said individual is treated by one or more site-directed administration routes selected from the group consisting of intramuscular injection, intravenous injection, intratumoral administration, minimally invasive intervention in organ tissue, and intelligent delivery by a micro-robot.

20. The use of claim 19, wherein the cancer treatment is one or a combination of surgical treatment, radiation therapy, chemotherapy, immunotherapy, hormonal therapy.

21. A composition of AVS (M3) -ril2×15 attenuated virus in combination with an immune checkpoint inhibitor, wherein the activity in the composition further comprises a combination with one or more additional active agents that control or treat a tumor, the additional active agents comprising: clofibrate, choline, methionine, niacin or ursodeoxycholic acid.

22. The composition of claim 21, wherein the composition further comprises a second oncolytic virus.

23. The composition of claim 22, wherein the second oncolytic virus is selected from one or more comprising vaccinia virus, herpes virus, measles virus, newcastle disease virus, rhabdovirus, alphavirus, parvovirus, enterovirus strains.

24. The composition of claim 23, wherein the second oncolytic viral drug is selected from attenuated oncolytic viruses.

25. The use of a composition according to any one of claims 21 to 24, in the manufacture of a medicament for killing abnormally proliferative cells, inducing an anti-tumour immune response or eliminating micro-environmental immunosuppression of tumour tissue.

26. The use according to claim 25, wherein the composition comprises a clinically administered dose of AVS (M3) -ril2 x 15, said AVS (M3) -ril2 x 15 comprising 1 x 10 ⁸ PFU-1×10 ¹² A single administration dose of PFU, said immune checkpoint inhibitor comprising a single use dose of 1-50 mg/kg.

27. The use according to claim 26, wherein AVS (M3) -ril2 x 15 comprises 1 x 10 ⁹ A single administration dose of PFU, the immune checkpoint inhibitor containing a single use dose of 10 mg/kg.

28. The use of claim 25, wherein the abnormally proliferative cell is contained in the patient.

29. The use according to claim 28, wherein the abnormally proliferative cell is selected from a tumour cell or a tumour tissue-associated cell.

30. The use of claim 29, wherein the tumor cell is a cancer cell.

31. The use of claim 30, wherein the cancer cell is a metastatic cancer cell.

32. The use according to claim 25, wherein the composition is for the manufacture of a medicament for the treatment of a patient suffering from a tumor and/or cancer.

33. The use according to claim 25, wherein said composition comprises a clinical dose ranging from 5 to 50mg/kg, and said AVS (M3) -ril2 x 15 comprises 1 x 10 ⁸ PFU-1×10 ¹² Single administration dose of PFU.

34. A method of inhibiting and/or killing an abnormally proliferating cell in a subject, the method comprising sequentially performing the steps of: 1) Administering AVS (M3) -ril2 x 15 to the subject, wherein said AVS (M3) -ril2 x 15 is capable of selectively replicating in tumor cells; 2) Administering an immune checkpoint inhibitor to said subject after administration of AVS (M3) -ril2 x 15 as described in step 1).

35. The method of claim 34, wherein AVS (M3) -ril2 x 15 is administered at a clinical dose and wherein AVS (M3) -ril2 x 15 comprises 1 x 10 ⁸ PFU-1×10 ¹² A single administration dose of PFU, said immune checkpoint inhibitor is a clinically administered dose, said immune checkpoint inhibitor is a single use dose comprising 5-50 mg/kg.

36. The method of claim 34, wherein the AVS (M3) -rIL2 x 15 intratumorally administered dose is per 100mm ³ Tumor correspondence 2×10 ⁷ A single administration dose of PFU, the immune checkpoint inhibitor containing a single use dose of 10 mg/kg.

37. The method of claim 34 or 35 or 36, wherein the AVS (M3) -rIL2 x 15 is administered at a clinical dose of 1 every 3 days, 3-7 consecutive administrations; the administration dosage of the immune checkpoint inhibitor is 1 time every 2 days, and 3-5 times of continuous administration are carried out.

38. The method of claim 37, wherein the AVS (M3) -rIL2 x 15 recombinant, composition or vaccine comprising the isolated recombinant AVS (M3) -rIL2 x 15 recombinant is administered by a mode of administration comprising one or more of intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, intratumoral, subcutaneous, or intranasal administration; the administration route of the administration mode comprises one or more of endoscope, intervention, minimally invasive and traditional operation; the immune checkpoint inhibitor is administered intravenously or intraperitoneally.

39. The method of claim 38, wherein the abnormally proliferating cells are selected from cells of a tumor and/or cancer.

40. The method of claim 39, further comprising the step of administering a second anti-tumor therapy.

41. The method of claim 40, wherein the second anti-tumor therapy is selected from the group consisting of administering a second oncolytic virus; the second oncolytic virus is selected from one or more of rhabdovirus, vaccinia virus, herpes virus, measles virus, newcastle disease virus, rhabdovirus AVS (M3), alphavirus, parvovirus, enterovirus strain.

42. The method of claim 41, wherein the second oncolytic virus is an attenuated oncolytic virus.

43. The method of claim 42, wherein the second oncolytic virus is an attenuated poxvirus.

44. The method of claim 41, wherein the tumor and/or cancer is selected from lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphatic cancer, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia.

45. The method of claim 41, wherein the second anti-tumor therapy is selected from one or more of chemotherapy, radiation therapy, immunotherapy, surgical therapy.

46. A method for preparing recombinant viral AVS (M3) -ril2.15 co-expressing IL 2.15, comprising the steps of:

a) Subcloning the polynucleotide sequence encoding IL2 x 15 onto modified rhabdovirus pAVS (M3) skeleton vector, screening clone of positive skeleton plasmid containing IL2 x 15 gene;

b) Co-transfecting the positive framework plasmid with pP, pL, pN and pT7 genome plasmid pAVS-rIL2 x 15 in BHK21 cells by using a liposome transfection technology, and assembling virus particles in the cells to generate recombinant virus AVS-rIL2 x 15;

c) Collecting cell culture solution supernatant after 3 days, infecting freshly cultured Vero cells, and collecting the supernatant after 48-72 hours to obtain 1 st generation recombinant rhabdovirus AVS (M3) -rIL2 x 15;

293 cells were cultured in large amounts, 293 cells were infected with recombinant Rhabdoviral AVS (M3) -rIL 2X 15 expressed at high efficiency, and supernatants were collected after 1 day, and recombinant viral titers were determined in Vero cells by Karber method.