WO2019184459A1

WO2019184459A1 - Oncolytic virus vaccine and adoptive immune cell combination therapy

Info

Publication number: WO2019184459A1
Application number: PCT/CN2018/121292
Authority: WO
Inventors: 秦晓峰; 万永红; 沃尔什·斯科特; 吴飞; 陈岚
Original assignee: 苏州奥特铭医药科技有限公司; 鸿易医药科技有限公司
Priority date: 2018-03-27
Filing date: 2018-12-14
Publication date: 2019-10-03
Also published as: CN110305198A; CN110305198B

Abstract

The present disclosure relates to an oncolytic virus vaccine and an adoptive immune cell combination therapy. Specifically, the present disclosure relates to a modified matrix protein (M) of a recombinant oncolytic rhabdovirus, and an oncolytic virus and an oncolytic virus vaccine, as well as a combination therapy of the oncolytic virus vaccine and endogenous and adoptive immune cells. The present disclosure also provides an anti-tumor product, including independent administration of the oncolytic virus, the oncolytic virus vaccine, and multiple routes and combined administration therapy. The present disclosure further provides a combination therapy and strategy for treating cancers, and a method for preparing the oncolytic virus vaccine and the tumor antigen-specific central memory CD8⁺ T-cell population. The combination therapy provided by the present disclosure can develop the full potential of the adoptive immune cells and endogenous T-cells against various tumor-associated antigens, thus effectively eliminating the primary and secondary metastasis of primary tumors, preventing immune escape and long-term memory protection, and achieving the effect of radically curing tumors.

Description

Oncolytic virus vaccine and adoptive immune cell combination therapy

Technical field

The present disclosure relates to immunotherapeutic methods for treating cancer, including oncolytic virus therapy, oncolytic virus vaccines, and oncolytic virus vaccines in combination with T cells.

Background technique

Cancer and conventional cancer therapeutics currently present significant socioeconomic burdens in terms of emotional/physical pain, loss of life and increased health care costs. Conventional therapies show some beneficial clinical effects but are accompanied by toxic side effects that reduce the quality of life of patients [1]. There is a need for cancer therapies with more effective and lower toxic side effects in clinical treatment.

It is well understood that neoplastic malignancy can be effectively eliminated by the immune system, and immunotherapy is the most promising alternative to cancer therapy relative to other treatments [2].

For example, it is generally considered that immune cells such as T cells having specific recognition ability against tumor-associated antigens have the ability to destroy tumors, and as a result of natural tumor cell renewal or by administering a therapeutic vaccine, such a cancer patient can be induced. cell. Unfortunately, once infiltrated into tumor tissue, these cells are often proven to be inoperable, and they often exhibit a dysfunctional phenotype and lack of tumor-induced local immunosuppression to reduce or destroy tumors. The ability to fight against tumor cells [3-5]. Therefore, the immunosuppressive tumor microenvironment remains a significant obstacle to tumor therapy, which attenuates the immune response induced by conventional cancer immunotherapy.

Immunological checkpoint inhibitor-based therapies have been developed to attenuate inhibitory signaling to tumor infiltrating lymphocytes (TIL) and can activate endogenous anti-tumor responses [6,7]. Checkpoint inhibitors have shown efficacy in clinical trials, but no response has been observed in all treated subjects [8]. Immunological checkpoint inhibitor therapy relies on the activation of pre-existing TAA-specific T cells, but in some patients, there are not enough endogenous tumor antigen-specific T cells to cause tumors after treatment-induced reactivation. Complete regression [9,10].

At present, small molecule drugs, monoclonal antibodies, etc. have been developed for new treatment of tumors, but the cure rate is not high, and more research is needed. In addition, treatment with only a single drug may lead to drug resistance in tumor cells, so there is an urgent need to develop effective biological treatment methods. An oncolytic virus is a virus that has a replication ability by genetic modification. A highly diluted attenuated virus can utilize the inactivation or defect of a tumor suppressor gene in a tumor (target) cell to selectively replicate in a target cell. Eventually it leads to the lysis and death of tumor cells, which are only present in small amounts or unable to proliferate in normal cells. Tumor treatment with this virus is called oncolytic virus treatment. The oncolytic virus not only replicates itself in tumor cells, but also causes cell lysis and death; and the release of viral particles by dead cells produces a cascade effect that amplifies the cytolytic effect until the tumor cells are cleared. At the same time, rupture of tumor cells leads to the release of tumor antigens from tumor cells, thereby inducing a systemic anti-tumor immune response in vivo, which may enhance the cytolytic activity of the virus. After the oncolytic virus enters the tumor cells, it can gradually destroy the host cells due to self-replication, and then spread to the surrounding cells and enter other tumor cells. This repeated cycle can exert an effective anti-tumor effect.

A large number of reports have shown that in vitro, many viruses replicate and kill tumor cells in a variety of tumor cells, such as Sendai virus (Kinoh et al., 2004); Coxackie Virus (Shafren et al., 2004). ); herpes simplex virus (Mineta et al, 1995); parvovirus (Abschuetz et al, 2006); adenovirus (Heise et al, 2000); poliovirus (Gromeier et al, 2000); Newcastle disease virus; measles virus ( Grote et al., 2001); Reovirus (Coffey et al., 1998); Retrovirus (Logg et al., 2001); Vaccinia virus (Timiryasova et al., 1999) and Influenza virus (Bergmann et al., 2001)). In addition, these viruses have proven to be effective in treating tumor animal models. However, the safety of most live viruses is an important concern, so there is still a need to develop safer and more reliable ways to treat cancer.

With the advancement of RNA virus genetic technology, vesicular stomatitis virus vectors have been developed as an effective therapeutic preparation. The VSV (vesicular stomatitis virus) viral vector is a highly efficient oncolytic baculovirus vector with a very broad oncolytic range. According to the data, VSV vector can infect almost all tumor cells, and the oncolytic rate of VSV vector is more than 50% in vitro. In vivo experiments, VSV vector can significantly prolong the life of tumor-bearing animal model. The VSV vector has also been developed as an effective vaccine vector, and the VSV viral vector is used as a vaccine vector in the development of vaccines such as acquired immunodeficiency syndrome virus, influenza virus, hepatitis C virus and hepatitis B virus. Therefore, the vesicular stomatitis virus vector has a very good application prospect.

In the field of tumor gene therapy, viruses are often used as vectors for therapeutic genes. For safety reasons, it is common to control the replication of the virus in normal cells, so it is technically difficult to achieve 100% infection efficiency. Therefore, the use of a self-replicating virus (proliferative virus) to treat tumors (the treatment of oncolytic baculovirus) has attracted much attention and expectation. Oncolytic baculovirus treatment refers to the use of the virus to self-replicate to destroy the tumor host cells in the infected tumor cells, and to achieve the therapeutic purpose by utilizing the original direct killing effect of the virus. In addition, oncolytic baculovirus treatment differs from gene therapy mainly in that it replicates in tumor cells to produce an effect of killing tumor cells. The concept of oncolytic virus treatment has long existed. Some hundred years ago, people tried to treat tumors with wild-type or natural attenuated strains. With the development of genetic engineering technology, the research on virus for therapeutic development has developed rapidly. Now it has been developed to the second. Gene recombinant virus. The VSV-based genetic recombinant virus known in the prior art either has certain toxicity to normal somatic cells, resulting in a safety risk; or the oncolytic effect is poor, resulting in poor therapeutic effect on solid tumors.

At the same time, recent studies have demonstrated that central memory T cells (Tcm) of TAA-specific T cells represent a significant superiority to checkpoint inhibitor therapy for the treatment of malignant tumors. Reinfusion treatment is performed using tumor-reactive T cells isolated from cancer patients and grown ex vivo. Once the immunosuppressive power of the local microenvironment of the tumor tissue is removed, these immune cells are activated and proliferated to a higher number before being returned to the patient. Essentially, in vitro culture conditions can convert a small number of dysfunctional T cells with little or no killing activity to hundreds of millions of fully functional Ts that return to the tumor's local microenvironment and destroy tumor cells. cell. The lymphocytes used to prepare Tcm are derived from T lymphocytes (TIL) including peripheral blood mononuclear cells (PBMC) and tumor local infiltration. The preparation of Tcm is flexible and diverse, because not only can the activation of the antigen pathway be recognized by the TAA natural receptor, but also transgene transgenes encoding the TAA-specific T cell receptor (TCR) or chimeric antigen receptor (CAR). The way to modify lymphocytes in vitro is to obtain Tcm [12, 13].

At the same time, clinical evidence has shown good anti-tumor efficacy of Tcm in non-solid cancers such as B cell lymphoma. In fact, infusion therapy for effector T cells that specifically recognize human papillomavirus can also induce regression of HPV-positive therapeutic cervical cancer [14]. This strategy is currently extended to target non-viral tumor-associated antigens, but further improvements are needed to enhance the infiltration of local lymphocytes and to cure solid tumors. Previous studies involving Tcm have shown that the therapeutic potential of Tcm requires sufficient long-term infiltration of immune cells to invade tumor tissue in order to kill all malignant cells. Clinical evidence suggests that these needs can be achieved through high-dose adoptive therapy cells (billions) with a slightly differentiated phenotype and maintaining replication capacity [15]. However, in view of the serious conflicts between this and preparation, the production of high doses of T cells requires extensive in vitro expansion, resulting in loss of replication capacity of T cells due to excessive differentiation [16]. In fact, conventional Tcm therapy requires pre-radiotherapy or chemotherapy to remove autologous cleared lymphocyte populations before treatment with differentiated effector T cells, on the basis of which dozens of in vitro expansions can be allowed. Better input of billion immune cells [17,18]. In addition, IL2 therapy is required to maintain the persistence and activity of the infused cells [19].

Recent studies have shown that Tcm, a poorly differentiated immune cell, exhibits better persistence in adoptive infusion of cells and shows superiority in clinical outcomes [15,20]. A method for producing Tcm culture of TAA-specific T cells from PBMC samples has been disclosed [21], but this protocol requires a clinical grade cell sorter, which requires a large amount of preparation for enrichment of antigen-specific cells. Human resources (see US Patent Application Publication No. US 2015/0023938).

Summary of the invention

Problems to be solved by the invention

Based on the problems in the prior art, it is desirable to provide an attenuated strain of oncolytic baculovirus which is effective in reducing the toxicity of viruses in normal somatic cells. Preferably, it is desirable to provide an attenuated strain of oncolytic baculovirus which is capable of ensuring high selectivity for abnormal proliferating cells relative to normal cells and having a good oncolytic effect.

Therefore, there is still a need to develop a rhabdovirus-based recombinant oncolytic baculovirus which has both good safety and good oncolytic effect.

At the same time, it is also desirable to provide a method for treating or curing a tumor or cancer based on the aforementioned attenuated strain of oncolytic baculovirus.

Solution to solve the problem

Based on the problems in existing tumor immunotherapy techniques, the present disclosure provides an immunotherapeutic method for treating cancer comprising oncolytic virus alone, oncolytic virus vaccine, and using oncolytic virus vaccine and central memory T cells ( Tcm) combination therapy for solid tumors, which can completely cure solid tumors and produce a permanent protective effect.

In one embodiment, the present disclosure provides techniques and methods for producing an oncolytic virus vaccine and large scale amplification of antigen-specific central memory T cells for production of an oncolytic virus vaccine obtained in vivo and tumor antigen specific Sexual central memory T cells can elicit a cellular T cell immune response against specific tumor cells. In combination, this immunotherapy does not require the use of a cell sorter or pre-clears the endogenous T cells of the subject, which is significantly better than existing cellular immunotherapy. Further existing cell immunotherapy has been significantly improved.

In one embodiment, the present disclosure provides a method of treating cancer in a mammal (eg, a human), the method comprising: administering the oncolytic virus alone, the oncolytic virus vaccine, and the oncolytic virus vaccine in combination with adoptive T cells Three ways of therapy. In several embodiments, a combination therapy for treating cancer in a subject of a particular indication is provided, the combination therapy comprising: (i) tumor antigen-specific central memory T cells (Tcm) to the subject The adoptive cell infusion infusion, followed by (ii) the subject is vaccinated with a recombinant oncolytic virus (OV) vaccine, the oncolytic virus vaccine expression is consistent with the infused T cell stimulating antigen, thereby inducing autoimmune cells to the tumor Destroy and eliminate. In a preferred embodiment, T cells in Tcm are genetically modified to express one or more recombinant T cell receptors (TCRs) or chimeric antigen receptors (CARs) that specifically recognize tumor antigens. In other embodiments, the T cells in Tcm therapy are autologous T cells derived from the subject to be treated. Preferably, the combination therapy does not require a step of autoimmune cell clearance of the subject.

In one embodiment, the present disclosure relates to a modified matrix protein (M) of a recombinant oncolytic baculovirus, characterized in that the amino acid sequence encoding the modified matrix protein (M) is represented by SEQ ID NO: a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% identical to the amino acid sequence; and wherein the amino acid sequence is at position 51 compared to SEQ ID NO: The 221st position and the 226th position have amino acid substitutions at the same time.

In another embodiment, the amino acid sequence encoding the modified matrix protein (M) is at the 21st position, the 51st position, the 111th position, and the 221st position as compared to the amino acid sequence set forth in SEQ ID NO: 1. It also has amino acid substitutions.

In one embodiment, the present disclosure relates to a modified matrix protein (M), wherein the recombinant oncolytic baculovirus is selected from the group consisting of vesicular stomatitis virus; preferably, the recombinant oncolytic baculovirus is selected from the group consisting of vesicular Stomatitis virus MuddSummer strain.

In one embodiment, the present disclosure relates to a modified matrix protein (M), wherein the modified matrix protein (M) has a sequence encoding a modified matrix protein (M compared to SEQ ID NO: 1 The amino acid sequence has the following mutations: (i) the 21th glycine G mutation is glutamic acid E, (ii) the 51st methionine M mutation is alanine A, (iii) the 111th leucine The L mutation is phenylalanine F, and (iv) the 221th proline V mutation is phenylalanine F. Preferably, the sequence of the modified matrix protein (M) is the sequence shown in SEQ ID NO:

In another embodiment, the present disclosure relates to a modified matrix protein (M), wherein the sequence of the modified matrix protein (M) is compared to SEQ ID NO: 1, encoding a modified matrix protein ( The amino acid sequence of M) has the following mutations: (i) the 51th methionine M mutation is arginine R, (ii) the 221th valine V mutation is phenylalanine F, (iii) the 226th The glycine G mutation is arginine R. Preferably, the sequence of the modified matrix protein (M) is the sequence shown in SEQ ID NO: 5.

In one embodiment, the present disclosure relates to a recombinant oncolytic baculovirus, wherein the recombinant oncolytic baculovirus comprises a modified matrix protein (M), wherein the amino acid sequence of the modified matrix protein (M) is The amino acid sequence shown above; preferably, the recombinant oncolytic baculovirus is an attenuated oncolytic baculovirus.

In one embodiment, the present disclosure relates to a composition comprising an isolated recombinant oncolytic baculovirus having a nucleic acid fragment encoding a modified matrix protein (M), characterized in that The amino acid sequence of the modified matrix protein (M) is an amino acid sequence as shown above; preferably, the recombinant oncolytic baculovirus is an attenuated recombinant oncolytic baculovirus.

In another embodiment, the composition of the present disclosure further comprises a second oncolytic virus; preferably, the second oncolytic virus is selected from the group consisting of a rhabdovirus, a vaccinia virus, a herpes virus, a measles virus, and a Newcastle disease virus. Or one or more of an adenovirus, an alphavirus, a parvovirus, an enterovirus strain; more preferably, the second oncolytic virus is an attenuated oncolytic virus; most preferably, wherein the second dissolution The tumor virus is an attenuated rhabdovirus.

In another embodiment, the composition of the present disclosure further comprises a second anti-tumor preparation; preferably, the second anti-tumor preparation is an immunotherapeutic agent, a chemotherapeutic agent or a radiotherapeutic agent; more preferably, the The second anti-tumor preparation is selected from one or more of small molecules, macromolecules, cells, viral vectors, gene vectors, DNA, RNA, polypeptides, and nanocomposites.

In one embodiment, the present disclosure relates to a vaccine comprising a therapeutically effective amount of one or more recombinant oncolytic baculoviruses, wherein the one or more recombinant oncolytic baculoviruses comprise the aforementioned modifications Matrix protein (M).

In another embodiment, the vaccine of the present disclosure may further comprise a second oncolytic virus, or a second anti-tumor preparation.

In one embodiment, the disclosure relates to an isolated peptide encoded by an amino acid sequence selected from at least 80%, preferably at least 90%, more preferably comprising the amino acid sequence of SEQ ID NO: 1. At least 95%, most preferably at least 98% of the same sequence, and the amino acid sequence has an amino acid substitution at the 51st position, the 221st position, and the 226th position as compared to SEQ ID NO: 1.

In another embodiment, the amino acid sequence has an amino acid substitution at the 21st position, the 51st position, the 111th position, and the 221st position as compared to SEQ ID NO: 1.

In one embodiment, the disclosure relates to an amino acid sequence encoding the isolated peptide that simultaneously has the following mutations: (i) the 21th glycine G mutation to glutamate E, (ii) the 51st methionine M mutation For alanine A, (iii) the 111th leucine L is mutated to phenylalanine F, and (iv) the 221st valine V is mutated to phenylalanine F. Preferably, the amino acid sequence is the sequence set forth in SEQ ID NO: 3.

In another embodiment, the disclosure relates to an amino acid sequence encoding the isolated peptide that simultaneously has the following mutations: (i) the 51th methionine M mutation to arginine R, (ii) the 221st hydrazine ammonia The acid V mutation is phenylalanine F, and (iii) the 226th glycine G is mutated to arginine R. Preferably, the sequence of the modified matrix protein (M) is the sequence shown in SEQ ID NO: 5.

In one embodiment, the disclosure relates to an isolated nucleotide sequence for encoding the isolated peptide.

In one embodiment, the disclosure relates to a composition or vaccine comprising an isolated recombinant oncolytic baculovirus in the manufacture of a medicament for killing aberrant proliferative cells, inducing an anti-tumor immune response, or eliminating microenvironmental immunosuppression of tumor tissue Applications.

In another embodiment, in the above application of the present disclosure, the aberrant proliferative cells are included in a patient.

In another embodiment, in the above application of the present disclosure, the abnormally proliferating cells are selected from tumor cells or tumor tissue-associated cells; preferably, the tumor cells are cancer cells; more preferably, the cancer cells It is a metastatic cancer cell.

In one embodiment, the disclosure relates to the use of a composition or vaccine comprising an isolated recombinant oncolytic baculovirus for the manufacture of a medicament for treating a patient having a tumor.

In one embodiment, the present disclosure relates to a method of slowly and continuously killing aberrant proliferative cells comprising contacting the aberrant proliferative cells with a recombinant oncolytic baculovirus, a composition comprising the isolated recombinant oncolytic baculovirus, or a vaccine A step of.

In another embodiment, the present disclosure is directed to a method of slow and sustained killing of aberrant proliferative cells, the aberrant proliferative cells being included in a patient.

In another embodiment, the present disclosure relates to a method for slow and sustained killing of aberrant proliferating cells, wherein the aberrant proliferating cells are selected from tumor cells or tumor tissue-associated cells; preferably, the tumor cells are cancer cells; more preferably The cancer cells are metastatic cancer cells.

In another embodiment, the present disclosure is directed to a method of slow and sustained killing of aberrant proliferative cells, the recombinant oncolytic baculovirus, a composition comprising the isolated recombinant oncolytic baculovirus or a vaccine administered to a patient.

In another embodiment, the present disclosure relates to a method of slowly and continuously killing aberrant proliferative cells, the recombinant oncolytic baculovirus, a composition comprising the isolated recombinant oncolytic baculovirus or a vaccine comprising, by intraperitoneal, intravenous, Administration of one or more modes of intra-arterial, intramuscular, intradermal, intratumoral, subcutaneous or intranasal administration; preferably, the route of administration of the mode of administration includes endoscopy, cavities One or more of mirror, intervention, minimally invasive, and traditional surgery.

In another embodiment, the present disclosure is directed to a method of slow and sustained killing of aberrant proliferative cells, the method further comprising the step of administering a second anti-tumor therapy.

In another embodiment, the present disclosure relates to a method of slow and sustained killing of aberrant proliferative cells, wherein the second anti-tumor therapy is selected from the group consisting of administering a second oncolytic virus.

In another embodiment, the present disclosure relates to a method of slow and sustained killing of aberrant proliferative cells, wherein the second oncolytic virus is selected from the group consisting of a rhabdovirus, a vaccinia virus, a herpes virus, a measles virus, a Newcastle disease virus, a gland One or more of a virus, an alphavirus, a parvovirus, an enterovirus strain; more preferably, the second oncolytic virus is an attenuated oncolytic virus; most preferably, wherein the second oncolytic virus Attenuated rhabdovirus.

In another embodiment, the present disclosure relates to a method of slow and sustained killing of aberrant proliferative cells, wherein the second anti-tumor therapy is selected from one or more of chemotherapy, radiation therapy, immunotherapy, surgery therapy.

In one embodiment, the present disclosure relates to a method of inducing an immune response in a subject, the method comprising administering to the subject a recombinant oncolytic baculovirus, a composition comprising the isolated recombinant oncolytic baculovirus, or a vaccine One or more of them.

In one embodiment, the present disclosure relates to a method of inducing an anti-tumor immune response or ameliorating microenvironment immunosuppression of a tumor tissue comprising the step of attaching a tumor or tumor tissue to a recombinant oncolytic baculovirus comprising an isolated recombinant oncolytic baculovirus The step of contacting the composition or vaccine.

In one embodiment, a method for producing a tumor antigen-specific central memory CD8+ T cell is provided, the method comprising: a step of ex vivo immune cell culture, the step comprising: in the presence of a tumor antigen, dendritic, etc. Lymphocytes are cultured from PBMC or TIL in the presence of antigen presenting cells, IL21, IL15 and rapamycin and preferably in the absence of IL2. Preferably, CD25+ cells (including regulatory T cells and activated T and B cells) are removed from PBMC prior to culture. A tumor antigen, a tumor associated antigen (TAA), is a substance produced in tumor cells that elicits an immune response in a mammal. In some embodiments, the tumor antigen is an autoantigen. In other embodiments, the tumor antigen is a tumor-specific antigen that is unique to the tumor and is not expressed in normal cells or expressed in very low amounts in normal cells (eg, new antigen).

In a related embodiment, the tumor antigen has higher tissue-specific expression in cancer cells compared to normal cellular contents, non-limiting examples of which include tyrosinase, MART-1, gp100, TRP-1/ Proteins such as gp75 and TRP-2. In other embodiments, the tumor antigen is a tumor-specific and common antigen that is expressed in cancer and testis but not expressed in other normal tissues or expressed in very small amounts (testicular cancer antigen or CT antigen), Non-limiting examples include BAGE, CAMEL, MAGE-A1, and NY-ESO-1. In other embodiments, the tumor antigen is a tumor-specific and single antigen (new antigen) that is expressed only in tumor cells, non-limiting examples of which include CDK4, catenin, cysteine protease-8, MUM- 1. MUM-2, MUM-3, MART-2, OS-9, p14ARF, GAS7, GAPDH, SIRT2, GPNMB, SNRP116, RBAF600, SNRPD1, PRDX5, CLPP, PPP1R3B, EF2, TcmN4, ME1, NF-YC, HLA-A2, HSP70-2 and KIAA1440. In other embodiments, the tumor antigen is overexpressed in cancer cells as compared to normal cells. Examples of tumor-associated antigens include: oncofetal antigens such as alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA); surface glycoproteins such as CA 125; oncogenes such as Her2; melanoma Related antigens such as dopachrome tautomerase (DCT), GP100 and MART1; cancer-testis antigens such as MAGE protein and NY-ESO1; viral oncogenes such as HPV E6 and E7; usually limited to embryonic or extraembryonic tissues A protein that is ectopically expressed in a tumor, such as PLAC1. As will be understood by those of skill in the art, the present method can be used to select antigens based on the type of cancer being treated, as one or more antigens may be particularly suitable for the treatment of certain cancers. For example, for the treatment of melanoma, a melanoma-associated antigen such as DCT can be used.

In some preferred embodiments, to generate tumor antigen (eg, TAA)-specific CD8 ⁺ T cells, the method includes the step of, in the step, ex vivo cultured cells (eg, obtained from a subject) Autologous PBMCs or PBMCs having a phenotype compatible with the tissue of the subject are genetically modified to express one or more recombinant TCRs or CARs to confer tumor antigen specificity. The transduced cells are cultured ex vivo in the presence of tumor antigens, such as antigen-presenting cells such as dendritic cells, IL21, IL15, and rapamycin. For example, a classical CAR consists of two segments, and the corresponding extracellular segment is composed of a single-chain antibody variable region (scFV) that recognizes a tumor antigen, and the corresponding intracellular segment is activating a T cell domain. The recombinant TCR comprises an alpha chain and a beta chain, for example, an HLA-A2/peptide complex can be identified. The TCR/CAR expression gene can be transduced into a cell of interest by a vector carrying a selected TCR/CAR expressed transgene expression vector, such as a lentivirus or retroviral vector. Methods for introducing foreign genes into such vectors are well known to those skilled in the art. Typically, the vector is modified to express TCR/CAR. In this regard, the nucleic acid sequence encoding the selected TCR/CAR is cloned into the vector of choice using established recombinant techniques. In some embodiments, the TCR or CAR is specific for a member of the MART-1, gp100, NY-ESO-1, or MAGE family (eg, MAGE-A3). Superiority, tumor antigens (TAA) generated by the methods described herein are not required to be used prior to amplification of specific CD8 ⁺ T cells (eg, rapid amplification using anti-CD3 antibodies and anti-CD28 antibodies, and optionally IL2) The tetramer label and the clinical grade sorter were further enriched. Accordingly, in a preferred embodiment, a method for adoptive cell infusion is provided, the method comprising: (i) in the presence of antigenic delivery cells (APCs, such as autologous dendritic cells) loaded with tumor antigens, IL21 , in the case of IL15 and rapamycin, and preferably in the absence of IL2, culturing PBMC or TIL obtained from a subject having cancer, thereby producing a population of cells rich in tumor antigen-specific CD8 ⁺ T cells, (ii) amplifying tumor antigen-specific CD8 ⁺ T cells using an anti-CD3 antibody and an anti-CD28 antibody, and optionally IL2, and (iii) reintroducing the cells into the subject, wherein the method is not included in step (i) The step of enriching T cells, such as sorting CD8 ⁺ cells, is performed between (ii) and (ii).

The oncolytic virus expressing the tumor antigen is administered to the subject following the combination therapy of the tumor antigen-specific CD8 ⁺ T cells according to the combination therapy described herein. Adoptive therapy of tumor antigen-specific CD8 ⁺ T cells can be accomplished by any suitable method.

In some embodiments, the combination therapy comprises: (i) culturing TIL from a subject having a tumor ex vivo in the presence of antigen presenting cells (APCs) and IL21 loaded with a tumor antigen peptide. And reintroducing the cells in the expanded culture into the subject, and (ii) administering the oncolytic virus expressing the same tumor antigen to the subject.

In other embodiments, the combination therapy comprises: (i) ex vivo culture of TILS from a subject having a tumor in the presence of APC, IL21, IL15, and rapamycin loaded with a tumor antigen peptide, amplification The cells in the culture are reintroduced into the subject, and (ii) the oncolytic virus expressing the same tumor antigen is administered to the subject.

In other embodiments, the combination therapy comprises: (i) culturing the PBMC from the subject ex vivo in the presence of APC and IL21 loaded with the tumor antigen peptide, expanding the cells in the culture, and reintroducing them To the subject, and (ii) the oncolytic virus expressing the same tumor antigen is administered to the subject.

In other embodiments, the combination therapy comprises: (i) ex vivo culturing PBMC from a subject, in an expanded culture, in the presence of APC, IL21, IL15, and rapamycin loaded with a tumor antigen peptide Cells, and reintroducing them into a subject, and (ii) administering an oncolytic virus that expresses the same tumor antigen to the subject.

In a related embodiment, the PBMC are transduced with a recombinant TCR or CAR capable of specifically recognizing a tumor antigen prior to ex vivo culture.

In some embodiments, the oncolytic virus expressing the tumor antigen is administered to the mammal about 8 to 72 hours after treatment of the tumor antigen-specific CD8 ⁺ T cells. In a preferred embodiment, the oncolytic virus expressing the tumor antigen is administered to the subject about 12 to 48 hours, about 20 to 28 hours, or about 24 hours after the treatment of the tumor antigen-specific CD8 ⁺ T cells.

The tumor-expressing antigen administered according to the combination therapy described herein is any oncolytic virus having replication ability. In some preferred embodiments, the replicative oncolytic virus is a rhabdovirus such as VSV or Maraba Rhabdovirus, which preferably comprises one or more genetic modifications to increase the selectivity of the virus for cancer cells.

The replicative oncolytic virus vaccine can be administered by one or more of a plurality of routes. In some embodiments, the replicative oncolytic virus is a rhabdovirus and is administered to a mammal by an intravenous route. In other embodiments, the replicative oncolytic virus is a rhabdovirus RV and is administered to a mammal intravenously (IV), intramuscularly (IM), intraperitoneally (IP) or intratumor (IT). As will be understood by those skilled in the art, a replicating oncolytic virus (e.g., rhabdovirus or vaccinia virus) will be administered as a suitable carrier, e.g., physiological saline or other pharmaceutically suitable buffer.

In one embodiment, the present disclosure provides an anti-tumor preparation comprising (i) adoptive immune cells and (ii) a replicative oncolytic virus vaccine (OVV), wherein the replicating type The oncolytic virus is an oncolytic baculovirus or an oncolytic vaccinia virus. Alternatively, the replicative oncolytic virus expresses a tumor antigen.

In some embodiments, the adoptive immune cells are central memory T cells (Tcm), preferably, the central memory T cells (Tcm) are CD8 ⁺ T cell populations that produce tumor antigen specificity, more Preferably, at least 50%, 60% or 70% of said CD8 ⁺ T cells exhibit a central memory phenotype. Wherein, the CD8 ⁺ T cell population is obtained by ex vivo culture of the PBMC cleared by CD25. Wherein the tumor antigen is selected from the group consisting of alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), CA 125, Her2, dopachrome tautomerase (DCT), GP100, MART1, MAGE protein, NY - any one or combination of the group consisting of ESO1, HPV E6, and HPV E7. The tumor is melanoma, sarcoma, lymphoma, brain cancer (such as glioma), breast cancer, liver cancer, lung cancer, kidney cancer, pancreatic cancer, esophageal cancer, gastric cancer, colon cancer, colon Colorectal cancer, bladder cancer, prostate cancer, and leukemia. In other embodiments, the tumor is a solid tumor.

In some embodiments, the CD8 ⁺ T cell population is produced by the presence of antigen-presenting cells (APCs) loaded with tumor antigens, IL21, IL15, and rapamycin, and preferably in the absence of IL2. Obtaining peripheral blood mononuclear cells (PBMC), or tumor infiltrating lymphocytes (TIL), or intramedullary lymphocytes from a subject having cancer, and treating said peripheral blood mononuclear cells (PBMC), or tumors Invasive lymphocytes (TIL) or intramyelocytes are cultured in vitro. In other embodiments, the CD8 ⁺ T cell population is produced by obtaining PBMC from a subject having cancer by transducing a recombinant T cell receptor (TCR) or a chimeric antigen receptor to PBMC ( CAR) genetically modified it and cultured transduced PBMC in vitro. After PBMC or TIL in vitro culture of CD8 + T cells obtained, further comprising using an anti-CD3 antibody and anti-CD28 antibody, and optionally amplifying the IL2 vitro CD8 ⁺ T cells.

In some embodiments, in the anti-tumor preparation, the replicative oncolytic virus is a baculovirus, preferably a vesicular stomatitis virus (VSV). In other embodiments, the baculovirus is a recombinant or wild type Maraba virus, preferably a Maraba MG1 virus. In some other embodiments, the replicative oncolytic virus is a wild-type or recombinant vaccinia virus; wherein the vaccinia virus is a Copenhagen, Western Reserve, Lister or Wyeth strain; alternatively, the vaccinia virus lacks thymidine Kinase gene or deletion of vaccinia virus growth factor gene. The vaccinia virus is administered to the subject intratumorally, intraperitoneally, intravenously, intraarterially or intramuscularly

In some embodiments, the oncolytic virus vaccine is administered to a subject by intravascular administration, preferably by intravenous administration.

The present disclosure also provides the use of a composition for the preparation of a medicament for treating a tumor, the composition comprising (i) adoptive immune cells and (ii) a replicative oncolytic virus vaccine. Wherein the adoptive immune cell and the replicative oncolytic virus vaccine are the same as the corresponding components in the antitumor preparation described above in the present disclosure.

The present disclosure provides methods of treating cancer in a mammal (eg, a human), the method comprising: adoptive cell therapy and subsequent administration of an oncolytic virus vaccine. In several embodiments, a combination therapy for treating cancer in a subject in need thereof is provided, the combination therapy comprising: (i) tumor antigen-specific central memory T cells (Tcm) to the subject Adoptive cell therapy, followed by (ii) subjecting the subject to a recombinant oncolytic virus (OV) vaccine expressing the same antigen targeted by adoptive cell therapy T cells, thereby inducing cancer destruction and elimination. In a preferred embodiment, central memory T cells (Tcm) are genetically modified to express one or more recombinant T cell receptors (TCRs) or chimeric antigen receptors (CARs) specific for tumor antigens. In some embodiments, the central memory T cell (Tcm) is an autologous T cell derived from a subject to be treated. Preferably, the combination therapy does not include the step in which the subject is immunodepleted.

In one embodiment, a method for producing a tumor antigen-specific central memory CD8 ⁺ T cell is provided, the method comprising: a step of ex vivo cell culture, the step comprising: in the presence of a tumor antigen, such as a dendritic cell, In the case of antigen presenting cells, IL21, IL15 and rapamycin, and preferably in the absence of IL2, lymphocytes are cultured from PBMC or TIL. Preferably, CD25 ⁺ cells (regulatory T cells as well as activated T cells and B cells) are removed from PBMC prior to culture. The tumor antigen can be, for example, a tumor associated antigen (TAA), which is a substance produced in tumor cells that elicits an immune response in a mammal. In some embodiments, the tumor antigen is an autoantigen. In other embodiments, the tumor antigen is a tumor-specific antigen that is unique to the tumor and is not expressed in normal cells or expressed in very low amounts in normal cells (eg, new antigen).

In a related embodiment, the tumor antigen is a tissue-specific tumor antigen having higher expression in cancer cells as compared to normal cells, non-limiting examples of which include tyrosinase, MART-1, gp100, TRP- 1/gp75 and TRP-2 proteins. In other embodiments, the tumor antigen is a tumor-specific and common antigen that is expressed in cancer and testis but not expressed in other normal tissues or expressed in very small amounts (cancer-testis antigen or CT antigen), Non-limiting examples thereof include BAGE, CAMEL, MAGE-A1, and NY-ESO-1. In other embodiments, the tumor antigen is a tumor-specific and unique antigen (new antigen) that is expressed only in tumor cells, non-limiting examples of which include CDK4, catenin, cysteine protease-8, MUM -1, MUM-2, MUM-3, MART-2, OS-9, p14ARF, GAS7, GAPDH, SIRT2, GPNMB, SNRP116, RBAF600, SNRPD1, PRDX5, CLPP, PPP1R3B, EF2, TcmN4, ME1, NF-YC , HLA-A2, HSP70-2 and KIAA1440. In other embodiments, the tumor antigen is an overexpressed tumor antigen that is overexpressed in cancer cells as compared to normal cells. Examples of tumor-associated antigens include: oncofetal antigens such as alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA); surface glycoproteins such as CA 125; oncogenes such as Her2; melanoma Related antigens such as dopachrome tautomerase (DCT), GP100 and MART1; cancer-testis antigens such as MAGE protein and NY-ESO1; viral oncogenes such as HPV E6 and E7; usually limited to embryonic or extraembryonic tissues A protein that is ectopically expressed in a tumor, such as PLAC1. As will be understood by those skilled in the art, the method can be used to select antigens based on the type of cancer to be treated, as one or more antigens may be particularly suitable for the treatment of certain cancers. For example, for the treatment of melanoma, a melanoma-associated antigen such as DCT can be used.

In some preferred embodiments, to generate tumor antigen (eg, TAA)-specific CD8+ T cells, the method includes the step of, in the step, ex vivo cultured cells (eg, obtained from a subject) Autologous PBMCs or PBMCs having a phenotype compatible with the tissue of the subject are genetically modified to express one or more recombinant TCRs or CARs to confer tumor antigen specificity. Transduced cells are cultured ex vivo in the presence of tumor antigens, such as antigen presenting cells such as dendritic cells, IL21, IL15, and rapamycin. CAR is a fusion protein formed by an extracellular single-chain variable fragment (scFv) derived from an antibody having an antigen-recognizing portion and an intracellular T cell activation domain. The recombinant TCR comprises an alpha chain and a beta chain and, for example, can recognize an HLA-A2/peptide complex. The cells can be transduced with a vector such as a lentivirus or retroviral vector carrying a transgenic cassette that supports expression of the selected TCR/CAR. Methods for introducing a transgenic cassette into a vector are well known to those skilled in the art. Typically, the vector is modified to express TCR/CAR. In this regard, the nucleic acid sequence encoding the selected TCR/CAR is incorporated into the vector of choice using established recombinant techniques. In some embodiments, the TCR or CAR is specific for a member of the MART-1, gp100, NY-ESO-1, or MAGE family (eg, MAGE-A3). Advantageously, tumor antigen (eg, TAA)-specific CD8+ T cells produced by the methods described herein are not required to be used prior to amplification (eg, rapid amplification using anti-CD3 antibodies and anti-CD28 antibodies, and optionally IL2) Further enrichment of tetramer labeling and clinical grade sorters. Accordingly, in a preferred embodiment, a method for adoptive cell therapy is provided, the method comprising: (i) in the presence of antigen presenting cells loaded with a tumor antigen (APC, eg, autologous dendritic cells), IL21, IL15 And culturing PBMC or TIL obtained from a subject having cancer in the absence of ILPA and preferably in the absence of IL2, thereby producing a cell population enriched with tumor antigen-specific CD8+ T cells, (ii) Amplifying tumor antigen-specific CD8 ⁺ T cells using an anti-CD3 antibody and an anti-CD28 antibody, and optionally IL2, and (iii) reintroducing the cells into the subject, wherein the method is not included in steps (i) and Ii) a step of enriching T cells such as sorting tetramers + cells.

In a preferred embodiment, lymphocytes from PBMC or TIL are isolated in the presence of tumor antigens, such as antigen presenting cells such as dendritic cells, IL21, IL15 and rapamycin, and preferably in the absence of IL2. The body culture is from about 1 week to about 4 weeks, for example about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or any range therebetween, and then in the presence of IL21, IL15 and rapamycin, Ex vivo culture in the presence of tumor antigens and antigen presenting cells for about 1 week to about 2 weeks, about 1 week, or about 2 weeks. In a particularly preferred embodiment, lymphocytes from PBMC are cultured ex vivo in the presence of dendritic cells loaded with tumor antigen peptide, IL21, IL15 and rapamycin for about 2 weeks, and in the presence of IL21, IL15 In vitro and rapamycin in the absence of dendritic cells loaded with tumor antigen peptide for about 1 week. Advantageously, tumor antigen-specific CD8+ T cells produced according to the methods described herein can be introduced into a mammal without lymphocyte depletion and without the need to administer IL-2 to the subject. Accordingly, in some embodiments, a method for adoptive cell therapy is provided, the method comprising: (i) in the presence of a tumor antigen, such as an antigen presenting cell such as a dendritic cell, IL21, IL15, and rapamycin Lymphocytes from PBMC or TIL are cultured ex vivo, and (ii) the resulting tumor antigen (eg, TAA)-specific CD8+ T cells are administered to a mammal without destroying existing mammals by chemotherapy or radiology. Lymphocytes (lymphocyte depletion or lymphoablation and do not give IL-2 to the subject.

The oncolytic virus expressing the tumor antigen is administered to the subject following the combination therapy of the tumor antigen-specific CD8 ⁺ T cells according to the combination therapy described herein. The adoptive therapy of tumor antigen-specific CD8 ⁺ T cells can be accomplished by any suitable method, including the methods described herein and the contents thereof, as described in US Patent Application Publication No. US2015/0023938, which is incorporated herein by reference. method.

In some embodiments, the combination therapy comprises: (i) ex vivo culture of TILS, expanded culture from a subject having a tumor in the presence of antigen presenting cells (APC) and IL21 loaded with a tumor antigen peptide The cells in and reintroduced into the subject, and (ii) the oncolytic virus expressing the same tumor antigen is administered to the subject.

In other embodiments, the combination therapy comprises: (i) ex vivo culture of TILS from a subject having a tumor in the presence of APC, IL21, IL15 and rapamycin loaded with a tumor antigen peptide, amplification The cells in the culture are reintroduced into the subject, and (ii) the oncolytic virus expressing the same tumor antigen is administered to the subject.

In other embodiments, the combination therapy comprises: (i) ex vivo culturing PBMC from the subject in the presence of APC, IL21, IL15 and rapamycin loaded with the tumor antigen peptide, in the expanded culture Cells, and reintroducing them into a subject, and (ii) administering an oncolytic virus that expresses the same tumor antigen to the subject.

In a related embodiment, the PBMCs are transduced with recombinant TCR or CAR of a specific tumor antigen prior to ex vivo culture.

In some embodiments, the oncolytic virus expressing the tumor antigen is administered to the mammal about 8 to 72 hours after treatment of the tumor antigen-specific CD8+ T cells. In a preferred embodiment, the oncolytic virus expressing the tumor antigen is administered to the subject about 12 to 48 hours, about 20 to 28 hours, or about 24 hours after the treatment of the tumor antigen-specific CD8 ⁺ T cells.

Any replication competent oncolytic virus that expresses a tumor antigen can be administered according to the combination therapies described herein. In some preferred embodiments, the replication-oncolytic virus is a baculovirus such as vesicular stomatitis virus (VSV) or Maraba baculovirus, which preferably comprises one or more genetic modifications to increase the selectivity of the virus for cancer cells. .

The replicative oncolytic virus vaccine can be administered by one or more of a plurality of routes. In some embodiments, the replicative oncolytic virus is a baculovirus and is administered to a mammal by an intravenous route. In other embodiments, the replicative oncolytic virus is a vaccinia virus and is administered to a mammal intravenously (IV), intramuscularly (IM), intraperitoneally (IP) or intratumor (IT). As will be understood by those skilled in the art, replicative oncolytic viruses (e.g., baculovirus or vaccinia virus) will be administered in a suitable carrier, such as saline or other pharmaceutically suitable buffer.

In one embodiment of the present disclosure, in order to establish pre-existing immunity, the methods of the present disclosure comprise the step of vaccinating a mammal with a tumor antigen suitable for inducing an immune response against a target tumor cell. For example, the tumor antigen can be a tumor associated antigen (TAA), such as a substance produced in a tumor cell that elicits an immune response in a mammal. Examples of such antigens include oncofetal antigens (e.g., alpha-fetoprotein, AFP) and carcinoembryonic antigen (CEA), surface glycoproteins (e.g., CA 125), oncogenes (e.g., Her2), melanin. Tumor-associated antigens (eg, dopachrome tautomerase (DCT)), GP100 and MART1, cancer-testes antigen (eg, MAGE protein and NY-ESO1), viral oncogenes (eg, HPV E6 and E7) A protein (e.g., PLAC1) that is ectopically expressed in a tumor that is usually limited to embryonic tissue or extra-embryonic tissue. As will be appreciated by those skilled in the art, antigens can be selected depending on the type of cancer to be treated using the methods of the present disclosure, as one or more antigens may be particularly useful for treating certain cancers. For example, for the treatment of melanoma, a melanoma-associated antigen, such as DCT, can be used.

The antigen itself may be administered, or preferably by a vector, such as an adenovirus (Ad) vector, a poxvirus vector or a retroviral vector, a plasmid or a loaded antigen presenting cell, such as a dendritic cell. Methods for introducing an antigen into a vector are known to those skilled in the art. In general, the vector can be modified to express an antigen. In this aspect, a nucleic acid encoding a selected antigen is integrated into a selected vector using widely accepted recombinant techniques.

The antigen is administered to the mammal by any of several methods including, but not limited to, intravenous, intramuscular or intranasal. As will be appreciated by those skilled in the art, the antigen or antigen-doped carrier can be administered in a suitable vehicle such as saline or other suitable buffer. After vaccination with the selected tumor antigen, the mammal produces an immune response during the interval of the immune response, for example, for about 4 days and for several months, years, or possibly for life.

An immune response to the antigen is established, and vaccination with the antigen is carried out using widely accepted techniques. Thus, the selected antigen or vector expressing the antigen can be administered to the mammal in an amount sufficient to produce an immune response. As will be understood by those skilled in the art, the amount required to generate an immune response will vary depending on a number of factors including, for example, the selected antigen, the vector used to deliver the antigen, and the mammal to be treated, such as breed, age, size. Wait. In this regard, for example, adenoviral vectors administered to mice intramuscularly at least about 10 ⁷ PFU minimum amount sufficient to generate an immune response. For administration to a human, the corresponding amount should be sufficient to produce an immune response.

In another embodiment, the immune response to the antigen can occur naturally in the mammal without the need for a first vaccination step to induce an immune response. The naturally occurring immune response to an antigen can result from any prior exposure to the antigen.

Once an immune response is produced in a mammal during an appropriate interval of immune response, for example at least about 24 hours, preferably at least about 2-4 days or longer, such as at least about 1 week, the amount will be appropriate for the oncolytic virus therapy. The oncolytic virus expressing the tumor antigen is administered to the mammal, as will be understood by those skilled in the art, which may vary with the oncolytic virus selected and the mammal to be treated. For example, a minimum of 10 ⁸ PFU oncolytic VSV administered intravenously to a mouse is sufficient for oncolytic therapy. A corresponding amount may be sufficient for a person.

An oncolytic virus expressing a selected tumor antigen can be prepared by integrating a transgene encoding an antigen into a virus using standard recombinant techniques. For example, the transgene can be integrated into the viral genome, or the transgene integrated plasmid can be used to integrate the transgene into the virus. The methods of the present disclosure are not particularly limited to available oncolytic viruses, and may include any oncolytic virus capable of destroying a tumor while being suitable for administration to a mammal.

In one embodiment, the present disclosure describes an attenuated baculovirus that is produced by a reverse genetics operating system and is a novel recombinant system for gene tumor therapy development. The attenuated baculovirus quadruple mutant (RV-4Mut) has been produced and demonstrated to be safe and effective by systemic delivery in a variety of tumor models (tumor models with immune function).

In one embodiment, the attenuated quadruple mutant baculovirus (and/or other oncolytic agent) of the present disclosure can be used continuously without causing a host's strong immune response to the treatment of the virus. Based on this, the host can be treated with the same virus system multiple times in a certain period of time, prolonging the treatment time, further reducing the body's resistance to single drug, and thus improving the effect of tumor treatment. Embodiments of the present disclosure include compositions and methods related to baculovirus and their use as anti-tumor therapies. These baculoviruses have the property of killing tumor cells both in vivo and in vitro. In the present disclosure, the baculovirus may be a genetically engineered variant of an attenuated baculovirus or an attenuated baculovirus. The viruses described herein can be used in combination with other baculoviruses.

In one embodiment of the present disclosure, there is provided attenuated baculovirus and a composition comprising an attenuated baculovirus encoding an M protein of an attenuated baculovirus (ie, as set forth in SEQ ID NO: 1 The amino acid sequence) has at least or at most 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% (including all of these values) Range and percentage) of amino acid identity variant M proteins. The specificity of the M protein of the attenuated baculovirus described above means that the M protein of the attenuated baculovirus has a conserved mutation that normally maintains the function of the protein. A representative example of a conservative mutation is a conservative substitution. The conservative substitution is, for example, a mutation in which Phe, Trp, and Tyr are substituted with each other when the substitution site is an aromatic amino acid, and a substitution between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. a mutation; a mutation that replaces each other between Gln and Asn in the case of a polar amino acid; a mutation that replaces each other between Lys, Arg, and His in the case of a basic amino acid; in the case of an acidic amino acid A mutation that is substituted between Asp and Glu; and a mutation that is substituted between Ser and Thr in the case of an amino acid having a hydroxyl group. Specific examples of substitutions considered as conservative substitutions include substitution of Ala to Ser or Thr, substitution of Arg to Gln, His or Lys, substitution of Asn to Glu, Gln, Lys, His or Asp, and Asp orientation. Substitution of Asn, Glu or Gln, substitution of Cys to Ser or Ala, substitution of Gln to Asn, Glu, Lys, His, Asp or Arg, substitution of Glu to Gly, Asn, Gln, Lys or Asp, Gly to Pro Substitution, substitution of His to Asn, Lys, Gln, Arg or Tyr, substitution of Ile to Leu, Met, Val or Phe, substitution of Leu to Ile, Met, Val or Phe, Lys to Asn, Glu, Gln, His or Substitution of Arg, substitution of Met to Ile, Leu, Val or Phe, substitution of Phe to Trp, Tyr, Met, Ile or Leu, substitution of Ser to Thr or Ala, replacement of Thr to Ser or Ala, Trp to Phe or Substitution of Tyr, substitution of Tyr to His, Phe or Trp, and substitution of Val to Met, Ile or Leu. Further, the mutation of the M protein of the attenuated baculovirus further includes a naturally occurring mutation resulting from an individual difference in the baculovirus derived from the gene, a difference in strain, and species.

In some cases, for a single random mutation, although the individual single mutant may reduce the virulence of the virus to normal healthy cells, once the above-mentioned multiple sets of random mutations are combined, the virus is in the tumor cell It may become more toxic than the wild type virus. Therefore, the therapeutic index of the recombinant oncolytic baculovirus in the present disclosure is unexpectedly increased, and is an unexpected finding established in the process of large-scale screening of attenuated strains in vitro. When a plurality of single mutant attenuated strains undergo simultaneous multi-gene mutations, Some viruses lose their infectivity at the same time in tumor cells and normal cells, a small part of which is strong and cytotoxicity is strong. The present disclosure unexpectedly found that the four amino acid mutations of RV-4Mut did not rejuvenate the virus itself, while retaining the characteristics of killing tumors, although the time point of lysing tumor cells was found to be delayed at the cellular level in vitro, but the tumor was specifically killed. The complete retention of the properties, the most valuable is that RV-4Mut does not have any toxicity to normal cells, fully in line with biosafety requirements.

The methods and compositions of the present disclosure can include a second therapeutic virus, such as an oncolytic virus or a replication defective virus. Oncolytic usually refers to the ability to kill, dissolve or prevent the growth of tumor cells. Oncolytic virus refers to a certain degree of replication in tumor cells, resulting in tumor cell death, lysis (oncolytic) or tumor cell growth arrest, and usually has a slight toxic effect on non-tumor cells. The second virus includes, but is not limited to, rhabdovirus, vaccinia virus, herpes virus, measles virus, Newcastle disease virus, adenovirus, alphavirus, parvovirus, enterovirus strain, and the like.

Embodiments of the present disclosure include compositions and methods related to rhabdoviruses comprising heterologous N, P, M, G, and/or L proteins and their use as anti-tumor therapies. This rhabdovirus has tumor cell killing properties in vivo and in vitro. Thus, a VSV virus as described in the present disclosure can be further modified by combining heterologous N, P, M, G and/or L proteins. As used in the present disclosure, heterologous N, P, M, G, and/or L proteins include Rhabdovirus N, P, M, G, and/or L proteins.

The methods of the present disclosure can further comprise administering a second anti-tumor therapy, such as a second therapeutic virus. In certain aspects, the therapeutic virus can be an oncolytic virus, more particularly a VSV virus. In other aspects, the second anti-tumor therapy is a chemotherapeutic agent, a radiotherapeutic agent or an immunotherapeutic agent, surgery, or the like.

In another aspect, the composition is a pharmaceutically acceptable composition. The composition may also include a second anti-tumor agent, such as a chemotherapeutic agent, a radiotherapeutic agent, or an immunotherapeutic agent.

A further embodiment of the present disclosure relates to a method of killing a proliferating cell, the method comprising contacting the cell with an isolated oncolytic rhabdovirus composition of the present disclosure.

A further embodiment of the present disclosure relates to the treatment of a cancer patient comprising administering an effective amount of an oncolytic rhabdovirus composition of the present disclosure.

In certain aspects of the present disclosure, cells can be included in a patient, which can be proliferative, neoplastic, pro-cancerous, metastatic cells. Rhabdovirus can be administered to a patient having cells that are susceptible to being killed by at least one rhabdovirus or a therapeutic regimen or composition comprising a rhabdovirus. Administration of the therapeutic composition can be carried out using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rhabdoviruses or recombinant rhabdoviruses, either alone or in combination in various ways. , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Administration can be intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, subcutaneous or nasal. In certain aspects, the compositions are administered systemically, particularly by intravascular administration, including injection, infusion, and the like.

Effect of the invention

In one embodiment, the present disclosure provides attenuated oncolytic coronaviruses capable of effectively reducing the toxicity of a virus in normal somatic cells by genetic recombination techniques. Preferably, the attenuated lytic baculovirus strain provided by the present disclosure simultaneously ensures a high selectivity for abnormal proliferating cells relative to normal cells, and has an oncolytic baculovirus which infects tumor cells and thereby produces a good oncolytic effect. Attenuated strain. In another embodiment, the aforementioned attenuated strain has sustained replication expression, high titer, an immune response that stimulates the local microenvironment of the tumor, and is characterized by low toxicity to normal cells while maintaining high selectivity for infecting tumor cells.

In one embodiment, the tumor of the present disclosure has an effect of inducing an anti-tumor immune response and eliminating immunosuppression of tumor tissue microenvironment. In another embodiment, a therapeutic vaccine that continuously and stably expresses a tumor antigen is developed based on an oncolytic virus vector, which can be administered directly by various routes, preferably intravenously, based on the tumor vaccine, combined with adoptive T cell therapy, for a variety of refractory tumors, to achieve the elimination of solid tumors and control of metastases, while generating autoimmune memory protection response, overcoming the recurrence of tumor immunotherapy.

In one embodiment, the present disclosure provides a combination therapy method that allows simultaneous analysis of endogenous T cell regulated anti-tumor immunity by therapy. In addition to promoting T cell expansion and tumor infiltration, oncolytic virus vaccines can also be vaccinated endogenously against various tumor-associated antigen T cells. The T cells of the therapy contribute to the rapid destruction of large tumor masses, while the endogenous T cells simultaneously prevent the appearance of antigen-deficient variants. When the T cells of the therapy disappear shortly after the tumor has subsided, the endogenous T cells acquire long-term memory with an antigen-specific broad pool. Our findings suggest that this combination strategy can exploit the full potential of T-cell therapy and endogenous T-cells for a variety of tumor-associated antigens to eliminate primary tumors, prevent immune escape, and provide long-term protective memory.

In one embodiment, the present disclosure enhances the therapeutic efficiency of adoptive cell therapy by injecting a baculovirus vaccination virus oncolytic vaccine in advancing systemic expansion and tumor infiltration of adoptive therapy T cells.

In one embodiment, the anti-tumor preparations and combination treatment regimens provided by the present disclosure can result in complete and long-lasting tumor regression without pretreatment. Using this model, the contribution of the endogenous T cells of the therapy to the therapeutic effect was also monitored. The results of the trials show that pre-existing endogenous T-cells against various tumor-associated antigens are critical for obtaining durable regression and long-term immune memory by adoptive cell therapy to prevent and/or eliminate antigenic escape variants.

In one embodiment, the anti-tumor preparations and combination therapies provided by the present disclosure can induce the promotion of an anti-tumor immune response, anti-tumor immune cell-specific proliferation, anti-tumor immune cells infiltrating tumor tissue or eliminating tumor tissue microenvironment immunosuppression.

DRAWINGS

Embodiments of the present disclosure are shown in greater depth by the following description of the respective figures of the figures listed below.

Figure 1 shows the viral rescue of VSV attenuated oncolytic virus random mutation (RV-Mut) single-point multipoint mutation.

Figure 2 shows viral replication at different time points in different attenuated strains including tumor cell LLC and normal fibroblast MEF, including RV-4Mut.

Figure 3 shows the in vitro killing of various tumor-bearing cells with different viral loads, including RV-4Mut, against different tumor cells.

Figure 4 shows the sustained expression of foreign proteins in MEF and Vero cells of different oncolytic virus attenuated strains including RV-4Mut.

Figure 5 shows a comparison of the sustained replication expression ability of different oncolytic virus attenuated strains including RV-4Mut in Vero and human tumor cell A549.

Figure 6 shows a comparison of the ability of different oncolytic virus attenuated strains including RV-4Mut to stimulate interferon immunoreactivity in different cells.

The neurotoxicity of different attenuated strains of oncolytic virus including RV-4Mut shown in Figure 7 in different strains of mice, including mouse body weight and survival rate statistics.

Figure 8 shows the evaluation of the therapeutic effects of different oncolytic virus attenuated strains including RV-4Mut in a tumor model.

Figure 9 shows a schematic diagram of the establishment of a subcutaneous xenograft model for different tumor cells, including colon cancer (MC38), melanoma (B16), and lung cancer cells (LLC).

Figure 10 shows a comparison of the efficacy of intravenous administration of oncolytic virus RV and intratumoral administration in the treatment of lung cancer models.

Figure 11 shows the pharmacodynamic evaluation of intravenous administration of RVV (gp33) oncolytic virus vaccine in a colon cancer (MC38-gp33) tumor model.

Figure 12 shows the statistical effect of treatment of oncolytic virus vaccine (RVV-DCT) in combination with Tcm and melanoma tumor model (B16).

Figure 13 shows the statistical effect of the oncolytic virus vaccine (RVV-mERK) in combination with Tcm in a malignant osteosarcoma (CMS5) tumor model.

Figure 14 shows a comparison of the therapeutic effects of oncolytic virus vaccine (RVV-gp33) in combination with Tcm in colon cancer.

The oncolytic virus vaccine RVV-gp33 shown in Figure 15 was combined with Tcm to induce anti-tumor T cells and to eliminate malignant melanoma in the B16-gp33 tumor model.

Figure 16 shows that the combination of the oncolytic virus vaccine RVV-mERK and Tcm stimulates CD8 ⁺ T cells to infiltrate the tumor core region (CMS5 tumor tissue).

Figure 17 shows the combination of oncolytic virus vaccine and Tcm to control tumor recurrence by stimulating endogenous lymphocytes.

Figure 18 shows that the combination of the oncolytic virus vaccine RVV-gp33 and Tcm effectively controls tumor recurrence by maintaining endogenous T cells.

Figure 19 shows a comparison of Tcm cells with optimal anti-tumor effects in combination therapy under different ex vivo culture conditions (in combination with IL15, IL21 and rapamycin).

Specific implementation

definition

As will be understood by those skilled in the art, the definitions and embodiments described in this section and other sections are intended to apply to all embodiments of the application described herein, unless otherwise indicated.

The term "comprising" and its derivatives as used herein is intended to be an open-ended term that refers to the presence of the features, elements, components, groups, integers and/or steps, but not Exclude other undescribed functions, elements, components, groups, integers, and/or steps. The foregoing also applies to words having similar meanings such as the terms "including", "having" and their derivatives. The term "consisting of" and its derivatives, as used herein, is intended to be a closed term that specifies the presence of the features, elements, components, groups, integers and/or steps, but excludes the presence of other The existence, elements, components, groups, integers, and/or steps of the illustrated features. As used herein, the term "consisting essentially of" is intended to designate the presence of such features, elements, components, groups, integers and/or steps and does not substantially affect the features, elements, or components. Those of the basic and novel features of the group, integers and/or steps.

It is to be understood that "combination therapy" is the sequential administration of a tumor antigen-specific central memory T cell population produced according to the Tcm therapy described herein plus a replicative oncolytic virus expressing one or more of the same tumor antigens. The memory T cell population produced according to the Tcm therapy described herein and the oncolytic virus expressing the same tumor antigen are shown to exert synergy in the subject at certain time intervals. In a preferred embodiment, the memory T cell population and oncolytic virus can be within 1 to 72 hours (eg, within 1, 2, 3, 6, 12, 24, 48, or 72 hours) or at 4, 5, 6 Or administered within 7 days, or at 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, Give within 29, 30 or 31 days.

As used herein, terms of degree such as "substantially", "about" and "approximately" are intended to mean a reasonable amount of variation of the term that does not cause a significant change in the result. If the deviation does not negate the underlying meaning of the word being modified, the use of these degrees of terminology should be limited to a range of plus or minus five percent of the definition of the deviation from the modified term.

The singular forms "a", "the", and "the" For example, an embodiment comprising "a T cell" is understood to mean a component or a component of two or more than two other substances.

In embodiments comprising a "other" or "second" component, such as another or a second cytokine, the second component as used herein is chemically distinct from the other components or the first component. The "third" component is different from the other components, the first component, and the second component, and further enumerates similarities to the "other" component.

The term "and/or" as used herein means that the listed items are used alone or in combination. In fact, the term means "at least one" or "one or more" that is already present or in use in the listed items.

When used in conjunction with the term "comprising" in the claims and/or the specification, the words "a" or "an" may mean "the", but may also mean "one or more", One "and one or more than one".

The word "comprising," "having," "including," or "include," or "includes" or "includes" or "includes" or "includes" or "includes" or "includes" does not exclude additional, unquoted elements or methods. step.

Throughout the application, the term "about" means that a value includes the standard deviation of the error of the device or method used to determine the value.

The disclosure of the term "or" in the claims is intended to be merely a substitute and "and/or" unless the meaning is merely a substitute or substitute, and the term "or" in the claim means "and / or".

The terms "inhibiting," "reducing," or "preventing" or any variant of these terms, when used in the claims and/or specification, include any measurable reduction or complete inhibition to achieve a desired result (eg, tumor treatment). . Desirable outcomes include, but are not limited to, remission, reduction, slowing or eradication of cancer or proliferative disorders or cancer-related symptoms, as well as improved quality of life or prolonged life.

The vaccination methods of the present disclosure can be used to treat tumors in a mammal. Alternatively, the vaccination methods of the present disclosure can be used to treat cancer in a mammal. The term "cancer" as used in this disclosure includes any cancer including, but not limited to, melanoma, sarcoma, lymphoma, cancer (eg, brain cancer, breast cancer, liver cancer, gastric cancer, lung cancer, and colon cancer) and leukemia.

The term "mammal" refers to both humans as well as non-human mammals.

The methods of the present disclosure comprise administering to a mammal an oncolytic vector expressing a tumor antigen to which a mammal has pre-existing immunity. The term "pre-existing immunity" as used in the present disclosure is meant to include immunity induced by vaccination with an antigen as well as immunity naturally present in a mammal.

The term "RV virus" as used in this disclosure refers to an attenuated VSV oncolytic baculovirus. The term "RV-Mut" refers to the presence of a mutated oncolytic baculovirus as compared to a wild-type VSV oncolytic baculovirus. Illustratively, "RV-4Mut" refers to an oncolytic baculovirus having a four amino acid mutation compared to a wild type VSV oncolytic baculovirus; "RV-3Mut" refers to a wild type VSV oncolytic baculovirus In contrast, there are three amino acid mutant oncolytic baculoviruses.

Central memory T cell (Tcm)

Central memory T cells, also known as central memory T cells, are T cells with long-term memory that can be homaged to lymph nodes for antigen re-stimulation after naive T cells are activated by antigen. . Illustratively, the biomarker of Tcm can be selected from the double positive of CD62L and CD45RO, indicating that Tcm can pass through the lymphatic shield, return to the lymph nodes, and be in a state activated by the antigen.

Oncolytic rhabdovirus

The vesicular stomatitis virus (VSV) is a negative-strand RNA virus that infects most mammalian cells and expresses up to 60% of the total protein in the infected cells. In nature, VSV infects pigs, cattle and horses and causes varicella disease near the mouth and feet. Although it has been reported that humans are infected with VSV, VSV does not cause any serious symptoms in humans. VSV encodes five proteins, including nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), surface glycoprotein (G), and RNA-dependent RNA polymerase (L). Blocking host cell protein synthesis by VSV matrix protein (M) induces cell death.

Oncolytic virus vaccine

A replication-competent oncolytic virus that expresses a tumor antigen according to the present disclosure includes any naturally occurring (eg, from "field source") or modified replication competent oncolytic virus. In addition to expressing a tumor antigen, the oncolytic virus can be modified, for example, to increase the selectivity of the virus for cancer cells.

Oncolytic viruses having replication ability according to the present disclosure include, but are not limited to, oncolytic viruses that are members of the following families: myoviridae, siphoviridae, podpviridae, The phage family (teciviridae), the phage family (corticoviridae), the bacteriological bacteriophage (plasmaviridae), the lipophage family (lipothrixviridae), the microscopy phage family (fuselloviridae), the pocyiridae (poxyiridae), the iridescent virus family ( Iridoviridae), phycodnaviridae, baculoviridae, herpesviridae, adnoviridae, papovaviridae, polydnaviridae , inoviridae, microviridae, geminiviridae, circoviridae, parvoviridae, hepadnaviridae, retrovirus Retroviridae, cystoviridae, reoviridae, biRNAV (birnavi) Ridae), paramyxoviridae, rhabdoviridae, filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae , leviviridae, picaraviridae, sequiviridae, comoviridae, potyviridae, caliciviridae, stellate Virology (astroviridae), Nodaviridae, tetraviridae, tombusviridae, coronaviridae, glaviviridae, togaviridae And the genus Barnaviridae.

Specific examples of viral backbones for replication competent oncolytic virus vaccines for use in the practice of the present disclosure include rhabdovirus, adenovirus, retrovirus, reovirus, baculovirus, Newcastle disease virus (NDV), polyomavirus, Vaccinia virus (VacV), herpes simplex virus, picornavirus, coxsackie virus, and parvovirus.

In some preferred embodiments, the replication competent oncolytic virus that expresses a tumor antigen according to the present disclosure is an oncolytic rhabdovirus.

Prototype baculoviruses are rabies and vesicular stomatitis virus (VSV), the most studied viruses in the family of viruses. The baculovirus is a family of bullet-shaped viruses that have a non-segmented (-) sense RNA genome. The baculoviridae include, but are not limited to, Arajas virus, Chandipura virus (AF128868/gi: 4583436, AJ810083/gi: 57833891, AY871800/gi: 62861470, AY871799/gi: 62861468, AY871798/gi: 62861466 , AY871797/gi:62861464, AY871796/gi:62861462, AY871795/gi:62861460, AY871794/gi:62861459, AY871793/gi:62861457, AY871792/gi:62861455, AY871791/gi:62861453), Cocal virus (AF045556/gi: 2865658), Isfahan virus (AJ810084/gi: 57834038), Maraba virus (SEQ ID ON: 1-6 of U.S. Patent No. 8,481,023, incorporated herein by reference; HQ660076 .1), Carajas virus (SEQ ID NO: 7-12 of US Patent No. 8,481,023, which is incorporated herein by reference, AY335185/gi:33578037), Piry virus (D26175/gi:442480, Z15093/ Gi: 61405), Vesicular stomatitis Alagoas virus, BeAn 157575 virus, Boteke virus, Calchaqui virus, Eel virus American, Gray Dolce virus Gray Lodge virus), Julona virus (Ju Rona virus), Klamath virus, Kwatta virus, La Joya virus, Malpais Spring virus, Mount Elgon bat virus (DQ457103/gi: 91984805) , Perinet virus (AY854652/gi:71842381), Tupaia virus (NC_007020/gi:66508427), Bahia Grande virus (SEQ ID NO: 13-18 of U.S. Patent No. 8,481,023, incorporated herein by reference) , KM205018.1), Muir Springs virus (KM204990.1), Reed Ranch virus, Hart Park virus, Flanders virus (AF523199/gi: 25140635, AF523197/gi: 25140634, AF523196/gi: 25140633, AF523195/gi: 25140632, AF523194/gi: 25140631, AH012179/gi: 25140630), Kamese virus, Mosqueiro virus, Mossuril virus, Baru virus ( Barur virus), Fukuoka virus (AY854651/gi:71842379), Kern Canyon virus, Nkolbisson virus, Le Dantec virus (AY854650/gi:71842377), Keuraliba virus, Connecticu T virus, New Minto virus, Sawgrass virus, Chaco virus, Sena Madureira virus, Timbo virus, Almpiwar virus ) (AY854645/gi:71842367), Aruac virus, Bangoran virus, Bimbo virus, Bivens Arm virus, Blue crab virus, Charlieville virus Charleville virus), Coastal Plains virus, DakArK7292 virus, Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus (AY854643/gi:71842363), Joinjakaka virus ), Kannamangalam virus, Kolongo virus (DQ457100/gi: 91984799 nuclear protein (N) mRNA, partial cds); Koolpinyah virus, Kotonkon virus (DQ457099/ Gi: 91984797, AY854638/gi: 71842354); Landjia virus, Manitoba virus, Marco virus, Nasoule virus, Navarro virus, Ngaingan virus ( AY854649/gi:71842375), Oak-Vale virus (AY854670/gi:71842417), Obodhiang virus (DQ457098/gi|91984795), Oita virus (AB116386/gi:46020027) , Ouango virus, Parry Creek virus (AY854647/gi:71842371), Rio Grande cichlid virus, Sandjimba virus (DQ457102/gi|91984803), West Sigma virus (AH004209/gi: 1680545, AH004208/gi: 1680544, AH004206/gi: 1680542), Sripur virus, Sweetwater Branch virus, Tibrogargan virus (AY854646/gi: 71842369), Xiburema virus, Yatta virus (Yata virus), Rhode Island virus, Adelaide River virus (U10363/gi: 600151, AF234998/gi: 10443747, AF234534/gi: 9971785, AY854635/gi: 71842348), Berrimah virus (AY854636/gi: 71842350]), Kimberley virus (AY854637/gi: 71842352), or Bovine ephemeral fever virus (NC_002526/gi: 10086561). Any of these baculoviruses or variants thereof can be engineered to express tumor antigens for use in accordance with the present disclosure.

In some preferred embodiments, the oncolytic baculovirus expressing a tumor antigen of the present disclosure is a wild-type vesiculovirus or a recombinant vesicular virus, such as wild-type or recombinant VSV, Jindipula, Maraba or Carajas, Includes its variants. In other embodiments, the oncolytic baculovirus is a wild-type or recombinant non-vesicular virus, such as Muir Springs, Farmington or Grand Bahia virus, including variants thereof.

In a particularly preferred embodiment, the oncolytic virus of the present disclosure that expresses a tumor antigen is a wild-type Maraba strain baculovirus or variant thereof, which is optionally genetically modified, for example, to increase tumor selectivity.

Oncolytic virus vaccine and adoptive T cell combination therapy

Although the results of adoptive T cell therapy with central memory T cells (Tcm) are often associated with the function of vaccinated T cells, the role of endogenous T cells remains unclear. The success of checkpoint blockade therapy demonstrates the potential therapeutic value of pre-existing tumor-initiating T cells in cancer therapy. Given the results of checkpoint blockade therapy, we hypothesized that endogenous T cells contribute to long-term survival after Tcm. Here, we describe a method of combining Tcm with an oncolytic vaccine that allows simultaneous analysis of anti-tumor immunity mediated by metastatic and endogenous T cells. We found that in addition to promoting the expansion of metastatic T cells and tumor infiltration, oncolytic virus vaccines also promote tumor-priming host T cells. We determined that metastatic T cells help to rapidly destroy large tumor masses, while endogenous T cells simultaneously prevent the appearance of antigen-loss variants. Furthermore, although metastatic T cells disappear shortly after tumor regression, endogenous T cells acquire long-term memory with broad antigen specificity. Our results suggest that this combination strategy can take advantage of the full potential of Tcm and tumor-initiated host T cells to eliminate primary tumors, prevent immune escape, and provide long-term protective memory.

Tumor antigen

The oncolytic viruses of the present disclosure express the same antigen targeted by T cells produced by the Tcm method described herein. Suitable antigens that can be expressed by oncolytic viruses include, but are not limited to, carcinoembryonic antigens, such as alpha-fetoprotein (ALF) and carcinoembryonic antigen (CEA), surface glycoproteins (eg, CA 125), oncogenes such as Her2, melanoma-associated antigens For example, dopachrome tautomerase (DCT), GP100 and MART-1, cancer-testis antigens such as MAGE protein and NY-ESO-1, viral oncogenes such as HPV E6 and E7, and usually limited to embryos or extraembryo A protein that is ectopically expressed in a tumor of a tissue such as PLAC1 or a tumor-associated antigen. A "variant" of a tumor-associated antigen refers to a protein that: (a) includes at least one tumor-associated antigenic epitope from a tumor-associated antigenic protein, and (b) at least 70%, preferably at least 80%, of the tumor-associated antigenic protein. More preferably, at least 90% or at least 95% are the same. Van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B, in "Database of T cell-defined human tumor antigens: the 2013 update." Cancer Immun 2013 13:15 provides a database summarizing recognized epitopes, Available online at www.cancerimmunity.org/peptide.

In some embodiments, the oncolytic virus expresses MAGEA3, a human papillomavirus E6/E7 fusion protein, a human six transmembrane epithelial antigen of prostate protein, or a cancer testis antigen 1.

In other embodiments, the disclosure provides methods of identifying a tumor antigen comprising using a test composition comprising IL21, IL15 and rapamycin or consisting essentially of IL21, IL15 and rapamycin Isolated tumor material co-cultures PBMC and antigen presenting cells (eg, dendritic cells); isolates T cell populations from culture; clones individual T cells from T cell populations; and characterizes antigen specificity of T cell clones. In some embodiments, the tumor material comprises total RNA, lysed tumor cells, or apoptotic bodies. The Tcm methods described herein can be used to generate a population of central memory T cells specific for antigens recognized by the method, and in combination with oncolytic viruses engineered to express the same antigen to provide synergistic cancer therapy.

cancer

Cancers treated according to the combinations described herein include, but are not limited to, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, myeloblast promyelocytic leukemia, myelomonocytic leukemia, chronic leukemia, chronic Myeloid (granulocyte) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal B-cell lymphoma, polycythemia lymphoma, Hodgkin Disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumor, sarcoma, and cancer, fibrosarcoma, mucinous sarcoma , liposarcoma, chondrosarcoma, osteosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial cell tumor, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma , colon sarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, Cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, Embryonic cancer, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma , craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal cancer , basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer , eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasm, kidney cancer, laryngeal cancer, liver cancer, lung cancer (including small cell lung cancer, squamous non-small cell lung cancer and non-squamous non-small cell lung cancer), melanoma (including Therapeutic melanoma) Tumor; oral cancer (eg lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterus Cancer and urinary system cancer. In some preferred embodiments, the cancer to be treated is selected from the group consisting of non-small cell lung cancer (NSCLC), breast cancer (eg, hormone refractory breast cancer), head and neck cancer (eg, head and neck squamous cell carcinoma), therapy Colorectal cancer, hormone sensitive or hormone refractory prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, chondrosarcoma sarcoma and small cell lung cancer.

In one aspect, the subject treated with the combination is a human having cancer that is difficult to treat for treatment with one or more chemotherapeutic agents and/or for one or more The treatment of antibodies is difficult to treat.

Sequence table meaning

In the present disclosure, SEQ ID NO: 1 is the amino acid sequence of the original matrix protein (M) of the oncolytic baculovirus MuddSummer strain;

In the present disclosure, SEQ ID NO: 2 shows the nucleotide sequence of the original matrix protein (M) of the oncolytic baculovirus MuddSummer strain;

In the present disclosure, SEQ ID NO: 3 shows the amino acid sequence of the four mutant strain (RV-4Mut) modified matrix protein (M) of the oncolytic baculovirus MuddSummer strain;

In the present disclosure, SEQ ID NO: 4 is the nucleotide sequence of the four mutant strain (RV-4Mut) modified matrix protein (M) of the oncolytic baculovirus MuddSummer strain;

In the present disclosure, SEQ ID NO: 5 shows the amino acid sequence of the four mutant strain (RV-3Mut) modified matrix protein (M) of the oncolytic baculovirus MuddSummer strain;

In the present disclosure, SEQ ID NO: 6 shows the nucleotide sequence of the four mutant strain (RV-3Mut) modified matrix protein (M) of the oncolytic baculovirus MuddSummer strain.

Statement of excellence

The role of endogenous T cells in the clinical outcome of adoptive cell therapy is underestimated because of the use of lymphocyte depletion pretreatment. The maintenance and activation of endogenous cells by oncolytic viruses results in a more diverse anti-tumor response, thereby preventing tumor recurrence due to the growth of antigen-mutant cells.

Accumulating evidence suggests that host T cells can be activated spontaneously by recognizing tumor antigens, thereby responding to growing tumors. In particular, since the immune system recognizes the immunogenicity of a foreign new antigen, tumors containing a large number of mutations are more likely to activate endogenous T cells, thereby providing a target for T cell attack. The correlation between the mutation load and the results of immunological checkpoint blockade therapy emphasizes the clinical relevance of these findings, which block the presence of tumor-initiated T cell populations. However, in most cases, spontaneous T cell responses to mutant antigens are relatively inefficient and do not mediate tumor rejection. In fact, only a subset of patients show a persistent response to immune checkpoint blockade, a fact that not all patients have tumor-initiating T cells sufficient to eradicate tumors.

Adoptive T cell therapy (Tcm) with antigen-specific T cells is an excellent alternative to checkpoint inhibitor therapy for malignant tumors. Healthy tumor-specific T cells can grow in vitro and infuse large amounts into patients with advanced disease, thereby overcoming the problem of insufficient spontaneous T cell response in cancer patients. Importantly, clinical approaches to the development of functional tumor-specific T cells in vitro have been developed, and recent clinical success in leukemia, melanoma, neuroblastoma, and EBV-associated malignancies has demonstrated that Tcm is feasible in humans. And effective strategies.

However, the necessary step for the success of Tcm is to clear the host lymphocytes before infusion of T cells. This process is called preconditioning and is highly toxic to some patients. In addition, although pre-processing can create an advantageous environment.

Adoptive transfer of T cells (ie elimination of regulatory T cells and steady-state cytokine "sink"), which also removes pre-existing tumor-initiating T cells, making it difficult, if not impossible, to determine endogenous anti-tumor immunity The role and benefits in Tcm.

Oncolytic viruses that express tumor-associated antigens are effective in binding and expanding tumor-specific memory T cells while retaining their inherent ability to directly infect and attenuate tumors and reverse the immunosuppressive tumor microenvironment. In this part of the disclosure, we use a rhabdovirus-based oncolytic vaccine to drive systemic expansion and tumor infiltration of adoptively transferred T cells, thereby increasing the therapeutic effect of Tcm. This reasonable combination results in complete and long-lasting regression of the tumor without pretreatment. Using this model, we also monitored the contribution of metastatic and endogenous T cells to treatment outcomes. Our data suggest that pre-existing, tumor-primed host T cells are essential for preventing and/or eliminating antigen escape variants, achieving durable regression through Tcm, and long-term immune memory.

In some embodiments, the combination therapies described herein utilize adoptive therapy of tumor antigen-specific central memory T cells produced according to the methods described herein. Thus, it has surprisingly been found that in the presence of APC, tumor antigens and compositions comprising IL21, IL15 and rapamycin or consisting essentially of IL21, IL15 and rapamycin, T cells are cultured (eg undifferentiated) T cells) result in a tumor antigen-specific T cell population that is substantially enriched in T cells showing a central memory phenotype compared to IL21, IL15 or rapamycin alone, which can be expanded ex vivo and Introducing the patient, then administering the oncolytic virus expressing the same antigen to the patient to synergistically treat the cancer without the need for expensive cell sorting techniques, and without administering a lymphocyte clearance protocol to the patient prior to introduction of the T cell, and without administering IL2 to the patient . In some embodiments, IL21 and IL15 are present at a concentration of from about 1 ng/ml to about 20 ng/ml, preferably about 10 ng/ml. In a related embodiment, the rapamycin is present at a concentration of from about 10 ng/ml to about 30 ng/ml, preferably about 20 ng/ml.

For example, APC can be an autologous dendritic cell that can differentiate adherent PBMCs into dendrites by culturing adherent PBMCs with GM-CSF (eg, about 800 U/ml) and IL-4 (eg, about 500 U/ml) for about 5 days. Cell-like cells are obtained. TNFα (eg, about 10 ng/ml), IL-1b (eg, about 2 ng/ml), IL-6 (eg, about 1000 U/ml), PGE-2 (eg, about 1000 ng/ml), IL can be added by day 5. The resulting dendritic cells are stimulated by -4 (e.g., about 500 U/ml) and GM-CSF (e.g., about 800 U/ml), and then cultured for 2 days. On day 7, dendritic cells can be pulsed with 40 [mu]g/ml of peptide tumor antigen for about 2 hours and irradiated as APC loaded with tumor antigen peptide according to the methods described herein.

In some embodiments, culturing undifferentiated T cells in the presence of APC, tumor antigen and IL21, IL15 and rapamycin results in a tumor antigen-specific T cell population, at least 50%, at least 60%, at least 70 %, at least 80% or more shows the central memory phenotype. In one embodiment, the central memory T cells obtained according to the Tcm method described herein display a CD44+CD62L+CD127+ phenotype.

In another embodiment, a CD8+ human T cell population for adoptive cell therapy according to the present disclosure is obtained by the presence of IL21, IL15 and rapamycin, a tumor antigen expressed by cancer and antigen presenting cells. Tumor infiltrating lymphocytes obtained from human cancer subjects (eg, 1 to 4 weeks, preferably about 3 weeks), amplifying CD8+ human T cell populations with anti-CD3 and anti-CD28 antibodies, and optionally IL2, and cells The patient is reintroduced and subsequently (eg, 24 hours apart) administered a replicative oncolytic virus (eg, oncolytic baculovirus) engineered to express a transgene encoding the same tumor antigen.

The oncolytic baculovirus expressing a tumor antigen of the present disclosure is a VSV strain or a variant thereof, optionally genetically modified, for example, to increase tumor selectivity. In a particularly preferred embodiment, the VSV comprises a deletion of methionine at position 51 of the M protein, as described in Stojdl et al., Cancer Cell., 4(4): 263-75 (2003), Its contents are incorporated herein by reference.

The replicative oncolytic baculovirus expressing a tumor antigen of the present disclosure may be 10, 100, 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ after Tcm treatment according to the methods described herein. One or more doses of 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ or more viral particles (vp) or plaque forming units (pfu) are administered systemically to the subject, preferably by intravascular (intravenous) And/or intraarterial) administration, including injection and perfusion, and the like. In a preferred embodiment, the oncolytic baculovirus expressing the tumor antigen is intravascularly 10 ⁵ -10 ¹⁴ pfu, 10 ⁶ -10 ¹² pfu, 10 ⁸ -10 ¹⁴ pfu after Tcm treatment according to the methods described herein. Or one or more doses of 10 ⁸ -10 ¹² pfu are administered to the subject.

Replicative oncolytic vaccinia viruses that express tumor antigens can be engineered to lack one or more functional genes, thereby increasing the cancer selectivity of the virus. The replicative oncolytic vaccinia virus of the present disclosure expressing a tumor antigen can be administered to a subject locally or systemically, for example by intratumoral, intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, intracranial, subcutaneous or intranasal. Administration, after treatment with Tcm according to the methods described herein, in one or more doses of 1 x 10 ⁵ plaque forming units (pfu), 5 x 10 ⁵ pfu, at least 1 x 10 ⁶ pfu, 5 x 10 ⁶ or about 5 x 10 ⁶ pfu, 1 x 10 ⁷ , at least 1 x 10 ⁷ pfu, 1 x 10 ⁸ or about 1 x 10 ⁸ pfu, at least 1 x 10 ⁸ pfu, about or at least 5 x 10 ⁸ pfu, 1 ×10 ⁹ or at least 1 × 10 ⁹ pfu, 5 × 10 ⁹ or at least 5 × 10 ⁹ pfu, 1 × 10 ¹⁰ pfu or at least 1 × 10 ¹⁰ pfu, 5 × 10 ¹⁰ or at least 5 × 10 ¹⁰ pfu, 1 × 10 ¹¹ or at least 1 × 10 ¹¹ , 1 × 10 ¹² or at least 1 × 10 ¹² , 1 × 10 ¹³ or at least 1 × 10 ¹³ pfu. For example, the virus may be between about 10 ⁷ -10 ¹³ pfu, about 10 ⁸ -10 ¹³ pfu, about 10 ⁹ -10 ¹² pfu, between about 10 ⁸ -10 ¹² pfu, or about 10 ⁹ and 10 ¹⁰ The dose of pfu is administered.

A single dose of an oncolytic virus that is expected to express a tumor antigen refers to the amount administered to the subject or tumor during 0.1, 0.5, 1, 2, 5, 10, 15, 20 or 24 hours, including all values during the period. The dose can be spread over time or by a single injection. Typically, multiple doses are administered to the same general target area, such as in the vicinity of the tumor, or in the case of intravenous administration, the subject's bloodstream or a particular entry point in the lymphatic system. In certain aspects, the viral dose is delivered by an injection device that includes a needle that provides a plurality of ports in a single needle or a plurality of bifurcations that are coupled to the syringe, or a combination thereof. A single dose of oncolytic virus can be administered, or multiple doses can be administered over a treatment period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks. For example, the oncolytic virus can be administered every other day, every week, every other week, every three weeks for 1, 2, 3, 4, 5, 6 months or more.

As will be appreciated by those skilled in the art, oncolytic viruses that express tumor antigens are typically administered as part of a pharmaceutical composition with a pharmaceutically acceptable carrier such as saline or other suitable buffer. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, Suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Oncolytic virus specific embodiment

Other objects, features, and advantages of the present disclosure will be apparent from the description. It should be understood, however, that the particular embodiments of the invention, and the invention, Various changes and modifications will become apparent to those skilled in the art.

The reagents and consumables used in the present disclosure are as follows:

PBS (Hyclone SH30256.01), DMEM high glucose medium (Gibco C11995500), RPMI1640 (Gibco C22400500CP), diabody (Gibco 15140-122), fetal bovine serum (Gibco 10099141),

I Reduced Serum Medium (Gibco 31985-070), Lipofectamine LTX (Invitrogen 15338100), 96-well cell culture plate (Corning 3599), 6-well cell culture plate (Corning 3516), 0.22 um filter (Millipore SLGP033rb), DMSO (Macklin D806645) ), Thiazole Blue (Sigma M2128).

Establishment of cell lines and tumor models

All cells were maintained at 37 ° C in a humidified environment containing 5% CO ₂ . MC38-gp33 cells, which are engineered to express MC38 cells corresponding to the gene for the LCMV gp33 epitope, are maintained in 10% FBS, 2 mM L-glutamine, 5 ml sodium pyruvate, 5 ml non-essential amino acids, 5 ml vitamins Solution, 55 μM 2-mercaptoethanol, 100 U/mL penicillin and 100 ng/mL streptomycin in MEM/F11. Expression of the gp33 minigene was maintained with 800 μg/ml G418. All cell culture reagents were from Invitrogen (Invitrogen, Grand Island, NY).

Recurrent osteosarcoma CMS5 cells (referred to as "CMS5r") were obtained from the end point NRG mice and expanded in the same medium as used for CMS5. CMS5 cells and B16-gp33 were maintained in DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 ng/mL streptomycin until the cells confluent. Tumor cells were washed twice with PBS and resuspended at a concentration of 10 ⁶ cells/30 μL in PBS. Certification testing but not screened cells with PlasmoTest ^TM (InvivGen) years once (bi-yearly). The cells were grown for no more than 3 generations after thawing and before administration in vivo. Mice were challenged by intradermal injection and tumors were grown to an average volume of approximately 150 mm ³ prior to treatment initiation.

animal

C57BL/6 and BALB/c mice were purchased from Vitalliwa and housed in the Gima animal house. P14 mouse, a transgenic mouse strain carrying a TCR of gp33 peptide (B6.Cg-Tcratm1Mom Tg (TcrLCMV) 327Sdz). NRG mice (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ) were purchased from Vitalliwa and the mice were housed under ultra-clean conditions. Animal models of skin graft tumors, 6-8 weeks old BALB/c, C57BL/6 mice were inoculated subcutaneously with 2 x 10 ⁵ B16-gp33 or B16 cells or 10 ⁶ CMS5 cells. Tcm and subsequent OVV (oncolytic virus vaccine) vaccination were given 5 to 7 days after tumor transplantation (when the tumor reached a diameter of ~5 mm). Tumor growth was monitored daily and measured with a caliper every other day. Tumor volume is calculated as width x length x depth.

virus

The RV-gp33 vector is a recombinant vesicular stomatitis virus MuddSummer subtype, which expresses dominant CD8 ⁺ and CD4 ⁺ T cell epitopes of lymphocytic choriomeningitis virus glycoprotein (LCMV-gp33-41 and LCMV-, respectively). Gp61-80).

In one embodiment, the modified matrix protein (M) of the recombinant vesicular stomatitis virus MuddSummer subtype strain is selected from the 51st position, the 221st position, and the 226th position, and has an amino acid substitution. : The 51th methionine M was replaced by arginine R, the 221th valine V was replaced by phenylalanine F, and the 226th glycine G was replaced by arginine R.

In another embodiment, the modified matrix protein (M) of the recombinant vesicular stomatitis virus MuddSummer subtype strain is selected from the 21st position, the 51st position, the 111th position, and the 221st position, and has an amino acid substitution. The amino acid substitution method is: the 21th glycine G is replaced by glutamic acid E, the 51st methionine M is replaced by alanine A, the 111th leucine L is replaced by phenylalanine F, the 221st position. Proline V was replaced with phenylalanine F.

Synthesis of antigenic peptides

LC-2-GP H-2Db-restricted peptide (gp33-41; KAVYNFATM), DCT H-2Kb-restricted peptide (DCT180–188SVYDFFVWL), and mutant ERK H-2Kd-restricted peptide (Erk9M136–144; QYIHSANVL) Purchased from Wujiang Nearshore Protein Biotechnology Co., Ltd. Each peptide was dissolved in distilled water and stored at -20 °C.

In vitro Tcm culture and Tcm vaccination protocol

Splenocytes were isolated from TCR transgenic mice by conventional methods. Splenocytes (containing a large amount of APC) were seeded at a density of 3 million/mL in RPMI medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 55 μM 2-mercapto Ethanol, 100 U/mL penicillin, 100 ng/mL streptomycin, 10 ng/mL IL21, 10 ng/mL IL15, 20 ng/mL rapamycin, and 0.1 μg/mL homologous antigen peptide. Four days after the initial inoculation, the culture was expanded by 5 volumes using the initial medium and the peptide-free supplement. Cells were harvested after 3 days, washed and suspended in PBS for injection. Mice were treated with 200μL PBS in 10 ⁶ Tcm cells were injected intravenously (IV), after 24 hours, with PBS 200μL (unless otherwise stated) 2 × 10 ⁸ pfu in RV-gp33, RV-DCT, 5 × 10 ⁸ pfu of RV-erk9m was injected intravenously. A lymphocyte clearance treatment was performed, which indicated that 20 mg/kg cyclophosphamide (CPX) was injected via IP 3 days before Tcm cell infusion.

Flow staining of surface markers

The cells cultured in vitro were collected, precipitated and treated with Fc blocking. Cells were then stained for surface expression of CD8, CD44, CD62L and CD127. Data were acquired using LSRFortessa (BD Biosciences, Mississauga, ON, Canada) containing FACSDiva software and analyzed by FlowJo software (Tree Star, Ashland, OR).

Detection of antigen-specific T cell responses

Peripheral blood mononuclear cells were obtained from blood samples from the periorbital sinus. Red blood cells were lysed with ACK lysis buffer. Mononuclear cells were stimulated with gp33 peptide (1 μg/mL) for 5 hours at 37 ° C and antibiotic A (Golgi Plug, 1 μg/mL; BD Biosciences) was added within 4 hours after the incubation. Cells were treated with Fc blocking and stained for surface expression of CD8. The cells were then fixed, transfected (Cytofix/Cytoperm, BD Biosciences) and needle-stained intracellular interferon-gamma. Data were acquired using LSRFortessa (BD Biosciences) containing FACSDiva software and analyzed by FlowJo software (Tree Star, Ashland, OR).

Histopathology and immunohistochemical staining

The tissue was fixed in 10% formalin for 24 hours and then dehydrated in 70% ethanol. The fixed tissue was embedded in paraffin and cut into 5 μm sections. The sections were either stained with hematoxylin and eosin or by immunohistochemistry (IHC) to detect insulin expression. For IHC, sections were treated with 3% hydrogen peroxide for 15 minutes at room temperature and blocked with 5% BSA and 2% goat serum in PBS containing 0.2% Triton X-100 for 45 minutes, followed by antibiotic protein/biotin blocking kit ( Vector labs) processing. Slides were incubated overnight at 4 °C with rabbit anti-mouse insulin antibody (abcam; ab181547) followed by incubation with biotinylated rabbit antibody (Vector labs). Sections were developed using continuous processing with Vectastain ABC kit (Vector labs) and ImmPTcm AMEC Red Peroxidase Substrate Kit (Vector labs), then counterstained with Harris hematoxylin and sealed with Permount (Fisher) sheet.

In vitro T cell differentiation

Most of the splenocytes from the transgenic mice were isolated and cultured for 7 days in the presence of 100 ng/mL of ErkM or gp33 peptide or DCT antigen peptide. For Tcm differentiation, 10 ng/mL of IL-15, 10 ng/mL of IL-21, and 20 ng/mL of rapamycin were added to the culture.

Combination therapy administration method

When tumors reached an average volume of approximately 150 mm ³ , in vitro differentiated CD8+ transgenic T cells were intravenously injected (iv) into tumor-bearing mice at a dose of 10 ⁶ cells/200 μL PBS. After 24 hours, the mice were treated with different vaccines. The form of intravenous delivery of the RVV oncolytic virus vaccine (2 x 10 ⁸ pfu) was administered via the intravenous route.

T cell surface and intracellular staining

Monoclonal antibodies recognizing the following targets were used for flow cytometry: CD16/CD32 (Fc Block), CD4 (GK1.5), CD8 (53-6.7), IFN-γ (XMG1.2), Thy1.1 ( OX-7), CD62L (MEL-14), CD44 (IM-7) (Biolegend, San Diego, CA). Blood, spleen and bone marrow samples were taken and treated with ACK lysis buffer to remove red blood cells prior to surface staining. For intracellular staining, cells were stimulated with 1 μg/mL of ErkM peptide for 4 hours in the presence of brefeldin A to block cytokine secretion, followed by BD Cytofix/Cytoperm buffer (BD Biosciences). Fluorescence was measured using a BD LSR Fortessa flow cytometer (BD Biosciences) and data was analyzed using FlowJo (10th Edition) flow cytometry software (Tree Star, Ashland, OR).

In vivo clearance of T lymphocytes

Anti-mouse CD4, CD8 (produced by GK1.5 and 2.43 hybridomas, respectively, injected intraperitoneally (ip) at a dose of 250 μg/200 μL for 48 hours, purchased from the American Type Culture Collection (American Type Culture Collection) , ATCC)), or Thy1. antibody (30H12, from BioXcell), to achieve T cell clearance. The same pattern was used for treatment with the isotype control antibody (HRPN from BioXcell). For prolonged observation, the corresponding antibodies were injected intraperitoneally once every two weeks to maintain clearance. Cyclophosphamide (Sigma) was administered intraperitoneally at a dose of 3 mg/mouse 1 day prior to T cell therapy. The removal efficiency was monitored by FACS analysis of the PBMC at different time points during the process.

Histology

Sections from formalin-fixed paraffin-embedded tissues were immunohistochemically analyzed using a Leica Bond Rx automatic staining machine (Leica Biosystem). Slides were deparaffinized and pretreated with Leica Bond Epitope Repair Buffer #2 (Leica Biosystems) for 20 minutes prior to staining with rat anti-mouse CD8α antibody (1:1000 dilution; clone 4SM15 from Thermo Fisher Scientific). . The color was visualized using a Leica Bond Polymer Accurate Detection Kit (Leica Biosystems), and the main components were replaced with a rabbit anti-rat antibody (1:100, Vector labs).

Example 1: Viral rescue of an attenuated mutant strain (RV-Mut) constructed based on VSV rod-shaped oncolytic virus

Based on the efficient rescue system of VSV virus, different attenuated strain systems were constructed, and the single-point multi-point virus rescue was randomly mutated. Furthermore, it was confirmed whether the M protein of the VSV virus can be arbitrarily mutated, and virions can be obtained (that is, whether any mutation can be performed and the virions can be rescued).

The specific steps of the above virus rescue system construction are as follows:

1. BSR-T7 cells were plated in 6-well plates to make the cell volume reach 4×10 ⁵ cells/well. After 14-16 hours, the cells were added with vT7, and the virus was infected for 4 hours, then transfected.

2. The plasmid was diluted using opti-MEM medium. Among them, the total amount of the plasmid was 5 μg, and 7.5 μl of PLUS Reagent was further added. 10 μl of Lipofectamine LTX was diluted using medium.

3. Mix the LTX mixture with the DNA mixture in an equal volume and incubate at room temperature.

4. Replace the medium in the 6-well plate with Opti-MEM medium, add the mixture in step 3 to the 6-well plate of the cultured cells, and gently shake the 6-well plate to evenly distribute the 6-well plate. Inside.

5. After 6-8 hours of transfection, the transfection reagent was aspirated and fresh complete medium was added.

6. After 72 hours of culture, the cell supernatant was harvested and filtered using a 0.22 μm filter.

The virion is successfully rescued using conventional methods in the art. The test results of virus packaging success rate are shown in Figure 1.

The results in Figure 1 demonstrate that the 111 amino acid of the RV virus M gene can only be mutated from leucine to phenylalanine. Mutation to other amino acids severely affects the rescue of virions, and the 221th amino acid of the viral M protein can only be replaced. In the case of phenylalanine, if the amino acid at this position is replaced with alanine, no recombinant virus can be produced.

From the statistical list of the packaging success rate of the virus shown in Fig. 1, it is reflected that a mutation at a specific site of the pathogenic gene M can obtain an attenuated strain, and the mutation selection of the amino acid is not arbitrarily selected, but needs to follow a specific rule. Only specific virions can be made.

Example 2: Virus titer detection of different RV-Mut strains, viral replication of different attenuated strains including RV-4Mut in normal cell MEF and tumor cell LLC at different time points.

In the MEF/LLC cell culture medium, the following viruses were added: VSV-GFP-WT, RV-GFP-M51R, RV-GFP-M51R-V221F-G226R (RV-3Mut), RV-GFP-G21E-M51A-L111F -V221F (RV-4Mut) 200 PFU each, and the virus titer (TCID50) generated by the virus strain was detected.

The specific steps for detecting the titer of the above virus are as follows:

1. Add 3 mL of Vero (LLC/Hela) cell suspension to each well in a 6-well culture plate to make the cell volume reach 4 × 10 ⁵ /well, a total of 6 wells, and incubate at 37 ° C, 5% CO _{2 for} 16 h.

2. Two wells of virus RV-WT, RV-M51R, RV-M51R-V221F-G226R, and RV-G21E-M51A-L111F-V221F were added to each well, and two wells of normal cells were set. Cell supernatants were harvested at 100 h at 12 h, 24 h, 48 h, 72 h, 80 h, and 96 h.

3. 100 μl of Vero cell suspension was added to each well of a 96-well culture plate to make the cell volume reach 1 × 10 ⁴ /ml, and cultured at 37 ° C, 5% CO _{2 for} 16 h.

4. The supernatant harvested in step 2 was diluted 10 times in a 1.5 ml EP tube from 10 ^-1 to 10 ^-11 for a total of 11 titers.

5. Inoculate the diluted supernatant into a 96-well culture plate, inoculate a total of 8 wells per dilution, and inoculate 100 μl per well. A normal cell control group was set up.

After 6 and 48 hours, the fluorescence of each well was observed. If there was fluorescence, it was recorded that the well was infected.

7. Calculate TCID50 according to the Karber method.

The results of the titer of the above virus are shown in Fig. 2.

It can be seen from Fig. 2A-2B that the number of virions released by RV-4Mut in the in vitro tumor cells during early infection replication to the supernatant is much lower than that of the control virus, but when the infection time is extended to 5 days, the virus-infected tumor cells are released. The viral load is consistent with wild-type virus, demonstrating that RV-4Mut has the property of slowly infecting tumor cells.

From Fig. 2C-2D, it can be found that when the RV-4Mut attenuated strain infects normal fibroblasts, the viral load added to the original supernatant is 200 PFU, which is different in the detection time at the two time points of 24 h and 120 h. The virus titer of the attenuated strains showed that the number of virions in the RV-4Mut attenuated strain was lower than that in the original supernatant (significantly decreased in the supernatant of the infection for 120 h), while the control group showed different degrees of increase, indicating RV. The -4Mut strain does not have the ability to replicate in normal fibroblasts, while the wild and control strains RV-M51R and RV-3Mut still have strong replication-infecting ability (from Figure 2, and RV- The infection replication ability of the 4Mut attenuated strain was as high as three orders of magnitude difference compared to the wild virus strain and the control strains RV-M51R and RV-3Mut.

Example 3: Comparison of in vitro killing of RV-4Mut with different viral loads in various tumor cells

In vitro killing of different tumor cells by different viral load of RV-4Mut was detected by MTT assay.

The specific steps of the above detection method are as follows:

1. 100 μl of (LLC/Hela/MEF) cell suspension was added to each well of a 96-well culture plate to achieve a cell volume of 1 × 10 ⁴ cells/well, and cultured at 37 ° C, 5% CO _{2 for} 16 hours.

2. Dilute the virus RV-WT, RV-M51R, RV-M51R-V221F-G226R (RV-3Mut), RV-G21E-M51A-L111F-V221F (RV-4Mut) to MOI (multiplicity of infection) of 0.001 , 0.01, 0.1, 1.0, 4 wells were inoculated for each dilution gradient, 100 μl per well, and cultured at 37 ° C, 5% CO _{2 for} 40 h.

3. Discard the supernatant from the 96-well culture plate, add fresh medium, and add MTT solution at 20 μL/well. Incubate for 4 h at 37 ° C, 5% CO ₂ .

4. Centrifuge the 96-well plate, set the rotation speed at 2500 rpm/min, and centrifuge at room temperature for 5 minutes.

5. Using a 1 mL disposable sterile syringe, gently aspirate the supernatant.

6. Add DMSO to each well, 100 ul/well, and place at 37 ° C for 10 minutes.

7. Using a multi-function microplate reader, shake for 2 minutes, and measure the OD value of each well at a wavelength of 570 nm or 490 nm.

The results of the in vitro killing of the above different strains are shown in FIG.

As shown in Figures 3A-3D, when the viral infection complex number is 0.01, the lytic ability of RV-4Mut in tumor cells in Hell and Hela is slightly lower than that in the control group, and the viral infection number of RV-4Mut strain is increased. By the time of 0.1, the direct killing ability of tumor cells such as LLC or Hela was significantly improved, and there was no significant difference compared with the wild strain virus, indicating that RV-4Mut retains the unique pair of wild viruses despite the mutation of four sites. The invasiveness of tumor cells has a strong ability to continuously kill tumor cells.

At the same time, the same experiment was repeated on MEF cells, as shown in Figures 3E-3F. The experimental results showed that the RV-4Mut strain had no significant difference in toxic side effects on normal cells at the MOI of 0.1 or 0.01 compared with the PBS group.

The results of the MTT assay showed that the four mutant strain RV-4Mut had no significant toxic side effects on normal cells in vitro, and did not cause apoptosis and necrosis of normal cells. The wild strain and the control group RV-M51R virus had certain toxic and side effects on normal cells, and the wild strain had the strongest side effects. The RV-4Mut strain had the lowest toxic and side effects and the best safety among the three attenuated strains.

Example 4: Peripheral expression of foreign genes in different attenuated strains including different attenuated strains including RV-4Mut.

The expression of the exogenous gene GFP of different attenuated strains in different cells was detected by FACS flow cytometry.

The specific steps of the above detection are as follows:

1. Add 100 μl of Vero/MEF cell suspension to each well of a 48-well culture plate to bring the cell volume to 2 × 10 ⁴ cells/well, and incubate at 37 ° C, 5% CO _{2 for} 16 h.

2. Each well was added with virus RV-GFP-WT, RV-GFP-M51R, RV-GFP-M51R-V221F-G226R (RV-3Mut), RV-GFP-G21E-M51A-L111F-V221F (RV-4Mut). ) 100 PFU each, 21 wells per virus, and 12 wells in the blank control group.

3. Harvest cells at each time point (24h, 36h, 48h, 60h, 72h, 84h, 96h), harvest 3 cells of each cell in each virus, and harvest 1 cell of cells in the blank control group, all with 400ul PBS. The cells were resuspended, and 100 μL of the cell suspension was analyzed by a Life Launch Attune NxT-Next flow cytometer to count the total number of GFP-positive cells.

The detection results of the above flow detection are shown in FIG.

As shown in Fig. 4A, in the Vero cells (interferon deficiency), the proportion of GFP-positive cells continued to increase in the Vero cells (interferon deficiency). After 72 hours of infection, the positive cells infected by the RV-4Mut strain exceeded the wild. The proportion of the virus indicates that the RV-4Mut strain has stronger sustained replication and replication ability in cells with defective interferon expression ability than other candidate strains, and the replication time in Vero engineering cell line is better than other controls. The virus strain is extended, which is more in line with the requirements of industrial scale production.

In MEF cells, as shown in Figure 4B, after the initial viral load of the four viruses was increased by 200-fold, RV-4Mut was all viruses, and GFP-positive cells did not significantly increase with time migration, while MEF The interferon pathway of the cell is normal, and when the external pathogen is infected, the response is rapidly generated, and the result of Fig. 6B proves that RV-4Mut has higher safety than the control attenuated strain (viral infection replication in MEF cells) The underlying cause is that RV-4Mut is more sensitive to interferon.

Therefore, when the virus GMP is industrially produced, when the virus is amplified in Vero engineered cells (interferon deficiency), the RV-4Mut strain has the ability to efficiently and continuously express the foreign gene, compared with the control attenuated strain. With the natural advantage, in the same volume of the reaction tank system, it can produce virus products with higher titer.

Example 5: Expression of the same exogenous gene GFP chimeric in different attenuated strains in tumor cells (A549) and Vero cells

The expression of the same exogenous gene (GFP) chimeric by different attenuated strains in different tumor cells (Hela and A549) was detected by flow cytometry.

The specific steps of the above detection are the same as in the fourth embodiment.

The results of the above flow detection are shown in Figures 5A-5B.

From Fig. 5B, by the ratio of flow-staining living cells, the proportion of living cells of RV-4Mut is relatively high in A549 cells, which is conducive to virus replication in cells, and finally by making more virions. Expand the range of infected tumor cells. At the same time, in the continuous expression of exogenous genes in Vero cells (Fig. 5A), the ability of RV-4Mut to continuously express proteins was significantly higher than that of other control groups after 72 h of in vitro infection amplification. Further detection revealed RV-4mut. Excellent ability to express foreign proteins.

The above results indicate that RV-4Mut has the ability to continuously replicate in tumor cells, and the expression of the foreign gene is more persistent and superior in comparison with the control group.

Example 6: Expression levels of antiviral interferons of different attenuated strains of different cell lines

Different attenuated strains were infected with MEF or tumor cell line LLC in vitro, and the expression levels of antiviral interferons of different attenuated strains were detected by RT-PCR.

The specific steps of the above detection are as follows: total RNA is extracted from LL, MEF cells with TRIzol (Invitrogen), and reverse transcribed into cDNA using PrimeScript RT Reagent Kit with gDNA Eraser (Takara) reverse transcription kit, and used

480 SYBR Green I Master (Roche) dye for dyeing

The Ct value of each gene was detected on a 480 quantitative PCR machine. The expression levels of the target genes IFN-β and VSV-G relative to the housekeeping gene GAPDH were calculated by the ΔΔCt method.

The detection results of the above detection are shown in FIG. 6.

As shown in Fig. 6B and Fig. 6D, the four viruses infect the MEF and the tumor cell LLC, respectively. After the MEF was infected by the above four viruses, the mRNA level of IFN was changed as shown in Fig. 6B, and the interferon induced by RV-4Mut was shown. The ability is the lowest compared to the other two attenuated strains. At the same time, the viral self-replication ability of VSV-G mRNA level compared with the control group (Fig. 6A), RV-4Mut RNA level in MEF cells is very low, further supporting the safety of RV-4Mut virus, while Tumor cell LLC performed the same experiment and found that, contrary to the phenomenon in MEF cells, the attenuated strain of RV-4Mut stimulated the highest amount of interferon in LLC cells, and the virus replication ability in tumor cells was also the strongest compared with the control group. (Figure 6C).

The above experimental results demonstrate the high selective infectivity of RV-4Mut on tumor cells and the enhancement of the expression of interferon in tumor cells (the increase in the amount of interferon in tumors can significantly increase the ability of local immune cells to kill tumor cells). At the same time, the ability of the virus to replicate was not affected, and the interferon-inducing ability against the RV-4Mut in normal cells was not significantly enhanced, further demonstrating that RV-4Mut has continuous stimulation of local immune cells of the tumor and produces a sustained anti-tumor immune response.

Example 7: Verification of differences in neurotoxicity between RV-4Mut and other oncolytic virus attenuated strains

In order to verify the neurotoxicity difference between RV-4Mut and other strains, 6-week-old Balb/c mice were selected as experimental subjects, 10 mice in each group, and each group of mice was given 50 μl of 10 ⁸ PFU/mL. Virus dilution (the aforementioned viruses are VSV-GFP-WT (ie RV-GFP), RV-GFP-M51R, RV-GFP-M51R-V221F-G226R (RV-3Mut), RV-GFP-G21E-M51A- L111F-V221F (RV-4Mut)), nasal drops once every other day, a total of 2 nasal drops. It is known that infection of wild baculovirus through the nasal cavity may cause paralysis of the hind limbs of some experimental mice, and severe mice will die. The five groups of experimental mice are continuously recorded and counted, and the body weight changes after the nasal drop test, the hind limb paralysis of the mice In case, the mouse is smooth and has flu-like symptoms.

The specific statistical results of the above experiments are shown in Table 1 and Figure 7.

Table 1 Neurotoxicity identification of RV-4Mut in animal models

From Table 1 and Figure 7, in the RV-WT group, 10 mice were all ill, and the symptoms were severe, and the disease deteriorated rapidly. The mortality rate reached 60% (as shown in Figure 7B, 10 wild virus strains were shown). There were 6 cases of dead mice, and there was hind limb paralysis. The symptoms lasted for a long time, while the symptoms of the other 3 attenuated strains were slightly relieved, especially in the RV-4Mut group. The mice have mild symptoms and the RV-4Mut group has the fastest recovery ability. At the same time, there was no hind limb paralysis in the RV-4Mut group, and only slight hair clutter, as shown in Fig. 7A, the RV-4Mut group was the least obvious experimental group with weight loss, and PBS. The trend of the nasal drops was consistent.

The above experimental results demonstrate that the safety advantage of the attenuated strain of RV-4Mut in animal models is very significant, and the excess of RV-4Mut does not have any adverse effects on the survival of mice.

Example 8: Establishment of a mouse lung cancer model (LLC tumor cells) to verify the efficacy of the attenuated strain of RV-4Mut

Further, the mouse lung cancer model was established, and the performance of the attenuated strain was tested by local intratumoral administration to verify the efficacy of the attenuated strain of RV-4Mut.

The specific steps for establishing the above mouse lung cancer model are as follows.

1.0×10 ⁶ (200 μL) of LLC cells were inoculated subcutaneously per CB7BL/6 mouse. The tumor size was measured every other day and calculated as follows: 1/2 x M2 x M1 ² (M1: short diameter, M2: long diameter). After tumor volume growth of each group of mice exceeded 200 mm ³ , 10 ⁶ PFU (20 ul) of intratumoral injection of virus was administered on day 12, day 14 and day 16, respectively, and the changes in tumor volume were recorded continuously.

The results of the above experiment are shown in Fig. 8.

As shown in Fig. 8A, intratumoral injection of RV-4Mut for three consecutive days can effectively inhibit the growth of tumors, greatly delay the life cycle of mice, and independently treat each mouse to receive three different attenuated strains. It was found that RV-4Mut and RV-GFP-M51R in the control group can shrink 30% of the tumors of the mice until they disappear, and achieve complete remission. The growth rate of tumors in nearly 40% of mice is effectively inhibited and partially relieved.

It can be seen from Figure 8B that the number of lung metastases in the RV-4Mut-free treatment group was minimal. Further, through the effective survival record of the tumor-bearing mice after treatment, the survival rate of the experimental group was the highest, and nearly 40% of the mice maintained normal life state in the past 2 months, and the tumor of some tumor model mice. Gradually subsided and completely cured, further indicating that RV-4Mut attenuated strain has a significant therapeutic effect on solid tumors, and has good clinical application value.

Example 9: Establishment of a subcutaneous xenograft model of different tumor cells

In the present disclosure, it relates to murine colon cancer (MC38), melanoma (B16) mouse lung cancer model (LLC), and the specific tumor model is as follows: subcutaneously inoculation of tumor cells on day 0, and mice after 8-12 days The tumor volume was started near 100 mm ³ , and the oncolytic virus RV was administered intratumorally, three times a day, and the corresponding RVV tumor vaccine was administered intravenously and administered once in the tail vein. Continuous observation and recording of changes in tumor volume in mice.

As shown in Fig. 9, the RV-4Mut and other mutant strains in the case 8 can only be administered in multiple ways in the tumor, and the RVV oncolytic virus vaccine which further develops the RV virus skeleton can be controlled by controlling the growth of the tumor. A variety of treatments have been implemented, and the increase in the mode of intravenous administration reduces the complexity of clinical local intervention, while the RVV oncolytic virus vaccine only needs to be administered once, reducing the cost of administration and the cumbersome multiple administration. .

Example 10: Comparison of the efficacy of intravenous administration of oncolytic virus RV with intratumoral administration (lung cancer)

First, a mouse unilateral lung cancer model (LLC tumor cells) was established to verify the efficacy of intravenous and intratumoral administration of RV oncolytic virus. The procedure for establishing the mouse lung cancer model described above is as follows.

1.0 × 10 ⁶ (200 μL) LLC-t2 cells were inoculated subcutaneously per CB7BL/6 mouse. The tumor size was measured every other day and calculated as follows: 1/2 x M2 x M1 ² (M1: short diameter, M2: long diameter). After the tumor volume of each group of mice grew more than 200 mm ³ , 10 ⁶ PFU (20 ul) of intratumoral injection of virus was given on the 12th, 14th and 16th day after tumor formation, and another group of RV veins were given. The drug was also administered intravenously three times according to the above administration time, and the tumor volume changes of the mice in the three groups of different administration groups and the cancer cell metastasis of the lung tissues of the mice at the experimental termination point were observed.

As shown in Fig. 10A, the tumor control effect of the RV intratumor treatment group was significantly better than that of the intravenous administration group, and nearly 30% of the mouse lung cancer was effectively controlled, while the tumor volume of all the mice in the intravenous administration group eventually exceeded the control. Scope, further through the experimental termination point of the mouse lung tissue, fluorescent labeled tumor cells observed that the intratumoral administration of oncolytic virus RV on lung cancer metastasis was significantly better than the control group and intravenous administration group, directly proved to dissolve The tumor virus RV can only be administered intratumorally, and intravenous administration cannot achieve effective immunotherapy.

Example 11: Evaluation of the efficacy of intravenous administration of oncolytic virus vaccine RVV

The oncolytic virus vaccine RVV (gp33) immunotherapy shown in Fig. 11B can induce regression of MC38-gp33 tumors, and the effect thereof was induced to increase the number of endogenous tumor-specific T cells (Fig. 11A). Specific Protocols RV, RVV-gp33 (2 x 10 ⁸ pfu; intravenously administered) were administered to C57BL/6 mice inoculated with MC38-gp33 tumors. Tumor volume is monitored at the indicated time points, indicated by ³ mm. 11A is a detecting body source-specific CD8 ⁺ T the ability to express an interferon, RVV-gp33 administration on 5 days, the activity of endogenous specific T cells peaked at MC38 colon carcinoma mouse model, intravenous The oncolytic virus vaccine RVV (gp33) effectively inhibited tumor growth after administration, compared to the control group RV (no antigen gene expression), and even some tumors were controlled and eliminated, although some individuals had tumors. Relapsed after healing, directly indicates that the oncolytic virus vaccine can effectively control the growth of tumor by intravenous administration.

Example 12: Therapeutic effect of oncolytic virus vaccine (OVV) combined with Tcm on expression of a self-tumor model (DCT)

The B16 cell autologous tumor antigen DCT was cloned into the RVV tumor vaccine by genetic engineering, and the DCT antigen peptide-stimulated Tcm was prepared simultaneously. The specific method was further studied in C57BL/6 mice with reference to the ex vivo Tcm culture and Tcm vaccination protocol and the T cell differentiation protocol. Six days after inoculation of malignant melanoma tumors, 10 ⁶ / RVV (DCT) + Tcm tumor vaccine (2 × 10 ⁸ pfu), TCM alone, RVV vaccine alone or RV treatment, as shown in Figure 12, only oncolytic virus vaccine The combination with Tcm can effectively remove melanoma tumors, and the mice that have been cured do not relapse after half a year of observation.

Example 13: Therapeutic effect of oncolytic virus vaccine (RVV) combined with Tcm in malignant osteosarcoma tumors with tumor antigen mutations

Inspired by the unique ability of the Rhabdovirus Oncolytic Virus Vaccine (RVV) to simultaneously cause the peripheral expansion of existing central memory T cells (Tcm) and the rapid infiltration into the tumor, we explored the combined use of in vitro Adoptive T cell therapy therapy with differentiated tumor antigen-specific Tcm and oncolytic virus (RVV) vaccine to treat established solid tumors. Wild type BALB/c mice were intradermally inoculated with CMS5 cells, a fibrosarcoma cell derived from methyl cholestyrium expressing a novel antigenic determinant (ErkM136-144, QYIHSANVL) in the ERK2 mutant gene. ErkM136-144-specific CD8+ T cells from DUC18 transgenic mice were cultured and expanded in the presence of IL-15, IL-21 and rapamycin, which prompted the acquisition of a typical Tcm phenotype (CD62L ⁺ CD44 ⁺ ).

Test method: BALB / c mice 7 days after the inoculation CMS5 cells, adoptive infusion DUC18 memory T cells (10 ⁶ cells / mouse) at day 1 infusion of memory T cells, using specific virus vaccine (RVV-mERK) The mice were treated with a mouse administered with RV-ErkM, Tcm or PBS alone as a control. The tumor volume of the CMS5-inoculated mice was monitored at the indicated time after treatment for 0 days, indicating that the tumor volume unit was mm on the day of vaccination. ³ . Tumor volume 1000 mm ^{3 was} used as the endpoint of survival analysis.

Test Results: Six days after tumor inoculation, mice were treated with an intravenous injection of 10 ⁶ DUC18 Tcm, or 10 ⁸ pfu of RVV-ErkM, or DUC18 Tcm 24h followed by a combination of RVV-ErkM. Previous examples have demonstrated that intravenous administration is to achieve antigen presentation in the periphery to enhance Tcm and tumor targeting for oncogenesis, and is the best route of treatment by means of RVV oncolytic virus vaccine and induction of T cell infiltration. Figure 13 shows that neither Tcm alone nor RVV vaccine alone has a significant effect on tumor growth, whereas the combination of Tcm and RVV vaccines induces complete tumor regression and significantly prolongs survival. However, effector DUC18T cells (Tcm) or RVV- alone. Administration of ErkM alone failed to induce complete and sustained tumor regression in all treated mice, with a sustained regression induced by Tcm + RVV-ErkM treatment, with subsequent survival significantly prolonged compared to the Tcm treated group.

It was confirmed that antigen-specific RVV vaccination is required to amplify the therapeutic T cells and induce infiltration into tumor tissues. Consistent with this view, the use of non-oncolytic vaccines is less effective than RVV vaccines.

Example 14: Complete cure of colon cancer (MC38-gp33) tumor requires oncolytic RVV tumor vaccine and tumor-specific T cell combination therapy

Test method: C57 mice were inoculated with MC38-gp33 cells for 6 days, and then gp33 memory T cells were inoculated (10 ⁶ cells/cell). After infusion of memory T cells for 1 day, specific oncolytic virus vaccine (RVV- Gp33) The mice were treated with RVV-gp33, Tcm or PBS alone as a control, and the tumor volume of colon cancer mice was monitored at the time specified after treatment. 0 days indicates the day of vaccination, tumor The volume unit is mm ³ . Tumor volume 1000 mm ^{3 was} used as the endpoint of survival analysis.

Test results: After 6 days of tumor inoculation, mice were treated with a combination of 10 ⁶ Tcm, or 10 ⁸ pfu of RVV-gp33, or Tcm 24 h followed by a combination of viral vaccines. Figure 14 shows that neither Tcm alone nor RVV alone have a significant control of tumor growth (compared to the PBS treated group), whereas the combination of Tcm and RVV vaccines induced complete tumor regression and significantly prolonged survival, Despite the tumor recurrence in some mice after 25 days of administration, administration alone with RVV-gp33 failed to induce complete and sustained tumor regression in all treated mice, colon cancer tumors induced by Tcm+RVV-gp33 Regression, and subsequent survival was significantly longer than treatment with the single administration group.

Example 15: Oncolytic virus vaccine RVV combined with Tcm induces a large number of anti-tumor T cells in the B16-gp33 tumor model, effectively eliminating radical malignant melanoma

Tumors affect the expansion and survival of endogenous ErkM-reactive CD8+ T cells during combination therapy. To further understand how the RVV vaccine affects the progression of T cells, we also monitored T cell responses peripherally.

Test method: C57BL/6 mice inoculated with B16-gp33 tumors were administered with PBS, RVV-gp33 (2×10 ⁸ pfu; vein), P14TCM (10 ⁶ /mouse) or RVV-gp33+P14 Tcm. Tumor volume is monitored at specified time points, expressed in mm ^3. After administration indicated time points assessed gp33-specific CD8 + response and the percentage of CD8 T cells in the peripheral blood circulation ⁺ T cells (CD8 ⁺ IFN- [gamma] of the T cells were stimulated in vitro by a gp33 polypeptide). Blood was collected on the number of days after the treatment, and the frequency of gp33-specific CD8 ⁺ T cell reaction was evaluated (the results are shown in Fig. 15A). The therapeutic effect of RVV (gp33) in combination with Tcm in melanoma expressing viral antigen was evaluated (results are shown in Figure 15B).

Experimental results: As shown in Fig. 15A, RVV can induce a ratio of endogenous antigen-specific CD8 of less than 10%, while RVV-gp33+P14 Tcm combined treatment group induced more than 30%, while P14 Tcm treatment group almost The endogenous CD8 response could not be induced. Further, it can be seen from Fig. 15B that the therapeutic effect of the co-administered group is also the best, and almost all of the mouse tumors are controlled to finally achieve a healing effect, and the drug alone is administered. There was no cure in the group, and some of the tumors in the RVV-treated group were controlled, and the Tcm-administered group was not significantly effective. Further excellent healing results produced by the combination administration group were directly related to the induction of endogenous specific CD8+ T cells by the combination therapy.

Example 16: Tumor infiltration and localization of tumors in CD8 ⁺ T cells in mice treated with combination

To directly observe tumor infiltration of CD8 ⁺ T cells and their localization in tumors, we stained tumor tissue on day 5 post inoculation on Tcm or Tcm+RVV (ErkM) oncolytic virus vaccine. Microscopic images of CMS5 tumor tissue stained with anti-CD8 antibody showed T cell infiltration around the tumor core and tumor tissue induced by the indicated therapeutic approach. The left side is a low-magnification image of the entire tumor, and the right side is a high-magnification image of the tumor periphery (black box outline) and the tumor core (blue frame area). The scale lengths are 500 μm and 200 μm, respectively.

Test Results: Microscopic images of CMS5 tumor tissue stained with anti-CD8 antibody showed that the tumor core and peripheral T cells were relatively infiltrated under the induction of the indicated therapeutic method. On the right is a low-magnification image of the entire tumor, and on the right is a high-magnification image of the tumor periphery (black frame outline) and the tumor core (blue frame outline). The scale lengths are 500 μm and 200 μm, respectively. As shown in Figure 16, the distribution around the tumor of CD8 ⁺ T cells was evident after only Tcm therapy and Tcm in combination with RVV oncolytic virus vaccination, but a high density of deep penetration into tumor tissue was observed only after RVV enhancement. CD8 ⁺ T cells confirmed that RVV vaccine + Tcm has a clear advantage over conventional vaccines. Finally, 100% of long-term survivors (over 60 days) after Tcm+RVV-ErkM treatment rejected re-vaccination of CMS5 cells after 2 months of treatment discontinuation and showed significantly prolonged survival, indicating the formation of effective immune memory. The oncolytic virus vaccine combined with Tcm treatment promoted the infiltration of more metastatic CD8 ⁺ T cells into the core area of the tumor (Fig. 16).

Example 17: Endogenous T cells form long-term anti-tumor immunity in mammals after combination therapy

To determine the persistence and protective function of endogenous T cells, particularly those that recognize non-targeted tumor antigens, we decided to use an intradermally injected osteosarcoma model of CMS5 cells (ie, tumor autoantigen mutations) to demonstrate oncolytic Viral vaccine and T cell combination therapy to control tumor recurrence is through stimulation of endogenous lymphocytes.

NRG mice were subcutaneously inoculated with CMS5 cells, and 7 days later, DUC18 Tcm (10 ⁶ cells/cell) was adoptively transferred. One day after Tcm input, mice were vaccinated with RVV-ErkM vaccine (2 x 10 ⁸ pfu/head). Mice using RVV-ErkM, TCM or PBS alone served as controls. Tumor volume changes in tumor-bearing mice were recorded after the indicated treatment, and tumor volume was represented by mm ³ .

Test Results: To determine which lymphocyte populations were needed for protection against CMS5r, we performed CD8+, CD4+ or both T cell subtype deletion mice (NRG mice) in C57 tumor model mice prior to revaccination. . As shown in Figure 17, although the combination treatment group had the best effect in controlling tumor growth in all treatment groups, the tumors that inhibited growth all relapsed as the number of days after administration increased, eventually being the same as other treatments. Eventually lead to the death of the mouse. These results demonstrate endogenous T lymphocytes and are also critical for the formation of long-term protective immunity with broad antigen specificity.

Example 18: Maintenance of endogenous T lymphocyte administration can prevent tumor recurrence

C57BL/6 mice were intradermally inoculated with B16-gp33 tumors (6 days), treated with adoptive cells, 10 ⁶ gp33-specific Tcm. intravenously, and then intravenously injected with 5 x 10 ⁷ pfu RVV-gp33 tumor vaccine. mouse. The antibody (negative control and αThy1.2 experimental group) was administered 1 day before Tcm administration, and then administered once a week for three weeks, and CPX was injected once a day before Tcm. (A) Monitoring tumor volume and (B) mouse survival are shown. Data are representative of two independent experiments with n=5 per group. Treatment with CPX chemotherapy and alpha Thy1.2 antibody results in a decrease in the number of endogenous T cells.

We determined the role of endogenous T lymphocytes in achieving complete and long-lasting tumor regression by evaluating the combination platform for Tcm+RVV vaccination in different tumor models.

Test Results: As shown in A in Figure 18, complete and long-lasting tumor regression was achieved in mice receiving combination therapy, confirming the use of targeting different tumor antigens and/or introducing different oncolytic viral backbones. The effectiveness and flexibility of this joint platform. Interestingly, however, mice treated with anti-Thy1.2 antibody or CPX showed a significant reduction in survival (A and B in Figure 18) prior to combination therapy due to tumor recurrence after initial remission, which reinforces The importance of pre-existing host T lymphocytes may be broadened by the broad selection of immunotherapeutic tumors.

Example 19: Specific combinations of IL21, IL15 and rapamycin are necessary for the induction of differentiation of antigen-specific central memory CD8+ T cells with optimal anti-tumor effects in combination therapy.

As shown in Figure 19, the fold expansion (A) after 7 days of in vitro culture using the indicated combination of IL15, IL21 and rapamycin, and CD62L (B) of CD8+ T cells in P14 mice. And CD44 (C) expression levels. A vernier caliper was used to measure the tumor volume and the unit was expressed as mm ³ . The graphical representation of DG shows the results of tumor volume (DG) after infusion of P14 cells cultured in combination with the indicated IL15, IL21 and rapamycin in mice treated with the oncolytic virus vaccine RVV-gp33. A graph showing the maintenance of tumor size below 1000 mm ³ (tumor measurement endpoint cutoff) is shown in Figure DG.

The specific combination of rapamycin, IL15 and IL21 is essential for the in vitro culture, expansion and differentiation of antigen-specific central memory CD8+ T cells with optimal anti-tumor effects in combination therapy as described herein. Each and all of these components are essential for the production of such Tcm cells, and in the absence of one or more of these components, the cells produced have suboptimal T after in vivo infusion and OV inoculation. The cells expand and/or reduce the anti-tumor effect.

IL15 is required in culture protocols that drive CD8+ T cell expansion and central memory differentiation. Cells cultured in the absence of IL15 showed impaired expansion of ex vivo culture and reduced cell yield compared to cells grown in complete combination (IL15/IL21/Rapa in A in Figure 19) (19 L21/Rapa in A). In addition, the cells so cultured exhibited an altered bias towards the initial phenotype (reduced CD44 levels in C in Figure 19) rather than a central memory phenotype - as in B in Figure 19 and C in Figure 19 Illustrated by cells grown in complete combination. At the time of infusion and RVV-gp33 stimulation, cells grown in the absence of IL15 produced a slightly reduced antigen-specific CD8 T cell response and were unable to completely abolish the tumor (E in Figure 19).

IL21 plays an integral role in programming cultured cells into a good quality central memory phenotype with optimal antigen-specific cytolytic function. The cells cultured in the absence of IL21 (IL15/Rapa) have normal expression levels of CD62L (B in Figure 19) and CD44 (C in Figure 19), with ex vivo proliferation comparable to cells in fully combined culture. After infusion and stimulation with VSV-gp33, cells grown in the absence of IL21 produced in vivo responses comparable in size to cells in complete combination culture.

discuss

Strategies for selectively enhancing T cell reactivity against genetically defined neoantigens are currently under development [65, 66]. In the current study, the inventors explored a therapeutic platform that combines ex vivo expanded new antigen-specific T cells with an oncolytic virus vaccine (OVV). This strategy employs a "push-pull" mechanism in which OVV promotes T cell activation and peripheral expansion and then infiltrates T cells to the tumor site. Indeed, the inventors demonstrated that adoptive therapy of antigen-specific central memory T cells (Tcm) followed by oncolytic vaccines triggered intense T cell expansion, tumor infiltration, and complete tumor regression, revealing a Strong synergy. More importantly, there are no pretreatments (ie, whole body irradiation or lymphodepleting chemotherapy before cell therapy) and exogenous IL-2 (usually used in two other antigen-specific T cell environments). In the case of adjuvants, good efficacy is achieved [52, 53], emphasizing the transformational significance of the combination therapy of the present disclosure, which can provide a strong therapeutic experience for patients. In addition, most importantly, bypassing the pretreatment retains tumor-induced endogenous T cells, which not only complements T lymphocytes, thereby eliminating primary tumors and preventing the appearance of antigenic variants, but also forms immune Long-term memory of surveillance.

Despite the success in treating B-cell malignancies and melanoma, antigen-specific T cells have limited effects on most solid tumors [67,68]. Among them, a sufficient amount of T cells are prevented from infiltrating into the tumor and persist for a sufficient period of time to kill all malignant cells [69, 70]. Increasing the dose of cellular immunotherapy can increase its ability to acquire and kill solid tumors, but the large number of T cells produced ex vivo requires extensive amplification, which inevitably leads to excessive differentiation and replication aging of T cells [71,72]. ].

In addition, highly inhibitory microenvironments and heterogeneous antigenic landscapes associated with solid tumors often nullify T cells and promote antigen escape [73, 74]. Therefore, in order to achieve a sustained regression of solid tumors, antigen-specific T cells must be combined with other methods that can simultaneously stimulate T cell expansion, infiltrate T cells into tumors, overcome tumor-mediated immunosuppression, and broaden T cell-specific profiles. Combination. The inventors have previously demonstrated that oncolytic vaccines can effectively amplify tumor-specific Tcm while retaining their beneficial oncolytic properties, which led the inventors to hypothesize that OV vaccines may represent antigens due to vaccination and oncolytic functions of OV vaccines. An ideal platform for specific T cell combinations [55, 56]. Indeed, the inventors provided evidence in this study that both oncolytic viruses (no tumor antigens) and conventional vaccines (non-oncolytic) are not sufficient to synergize with antigen-specific T cells and disrupt the need for established solid tumors. Expansion of therapeutic T cells and tumor infiltration. For this reason, because of the effective implantation and proliferative capacity of Tcm, Tcm is superior to TEFF (effector T cells), which is consistent with the increasingly important concept in the field of antigen-specific T cells [71, 72].

The inventors conducted two additional observations that may have important implications for the current clinical practice and rational design of antigen-specific T cells. First, although tumor regression was achieved in immunodeficient mice following Tcm+OVV tumor vaccination, tumors recurred within 2 weeks. It is clear that recurrent tumor cells no longer contain epitope targets for therapeutic T cells. This result seems to support the view that targeting tumors with a population of T cells specific for a restricted set of antigens may result in selective derivation of antigen-negative tumor variants. However, persistent regression is consistently achieved in wild-type animals, suggesting that tumor heterogeneity and/or immune escape can be addressed by mobilizing endogenous T cells during Tcm, even targeting a single antigen.

The inventors observed that clearance of CD4 ⁺ or CD8 ⁺ T cells prior to Tcm+OVV treatment did not affect initial tumor regression, but resulted in recurrence, indicating a synergistic effect between the T cells of the therapy and endogenous T cells. This shows that therapeutic T cells play a major role in regulating tumor debulking (eradication of ErkM+ tumor cells), while elimination of escaped variants (ErkM-tumor cells) requires endogenous T cells. One possible mechanism for explaining the activation of endogenous T cells is epitope spreading, which involves the in vivo cross-presentation of tumor-derived antigens released in a wave of immune challenge to promote subsequent rounds of resistance against different antigens. The phenomenon of tumor T cells [75,76]. These consecutive events may be particularly effective in the context of the present disclosure due to intense tumor lysis and inflammation mediated by therapeutic T cells and oncolytic viruses. However, another possible mechanism is to release and/or amplify pre-existing tumor-primed T cells by Tcm+OVV treatment, thereby providing a broader repertoire to supplement the T cells of the therapy to completely eradicate all tumor cells. This latter possibility is supported by the following evidence: (1) the presence and enhancement of ErkM-specific CD8 ⁺ T cells can be detected as early as 2 days after Tcm+OVV; (2) 5 days after combination therapy, It also shows an increase in the response of T cells to non-targeted new epitopes, both of which are unlikely to be the result of epitope expansion requiring activation of undifferentiated T cells; and (3) clearance of CD4 ⁺ or CD8 6 days after treatment the fact ⁺ T cells did not result in recurrence showed that pre-existing but subsequently cleared early involvement with tumor cell-induced T cell and host T cell therapy. Nonetheless, Tcm+OVV is likely to re-exist existing antigen-specific T cells and induce new T cell responses via epitope diffusion in a continuous manner reflecting their relative importance in producing anti-tumor immunity [75, 77]. More work is needed in both cases to determine whether those unidentified antigens recognized by natural development or treatment-induced T cells are derived from tumor-specific mutations or autoantigens.

Secondly, it is believed that the long-term persistence of therapeutic T cells is important, and there is a positive correlation between tumor regression and the persistence of T cell clones for adoptive therapy [78,79]. Interestingly, however, the anti-ErkM CD8 ⁺ T cells of the disclosed therapeutics disappeared immediately after tumor regression, while endogenous ErkM-specific CD8 ⁺ T cells survived for a long time and were able to provide antigen-specific protective immunity. The inventors hypothesize that the unique observations in this disclosure are due to the introduction of oncolytic vaccines that not only accelerate the T cell response of the therapy and enhance their interaction with tumors, but are also involved in endogenous anti-tumor T cell responses. Indeed, the present disclosure demonstrates that therapeutic T cells dominate early expansion and tumor infiltration and are responsible for mediating initial tumor regression. Thus, therapeutic T cells undergo tumor-induced apoptosis, a phenomenon documented in previous studies [80, 81]. In contrast, endogenous T cell expansion did not peak until several days after tumor regression, indicating that most of the enhanced and/or therapeutically induced endogenous T cells did not undergo interaction with tumor cells, thus surviving to maintain resistance Tumor immunity. The fact that therapeutic T cells survive long-term in tumor-free mice demonstrates that the short-lived progression of therapeutic T cells in tumor-bearing mice may be the result of their interaction with tumor cells. The results of the present disclosure suggest that retention of endogenous tumor-reactive T cells during Tcm is critical to ensure disease elimination and long-term anti-tumor memory, which may be compromised by pre-treatment to increase survival of adoptive therapy cells [ 53].

Collectively, the data of the present disclosure support the involvement of tumor-primed host endogenous T lymphocytes in tumor localization during administration of an oncolytic virus vaccine, and thus minimize or even eliminate antigens caused by the introduction of a single selection pressure The risk of mutation. The fact that many patients contain CD4 ⁺ and/or CD8 ⁺ T cells that recognize different new epitopes derived from the patient's own tumors underscores the relevance of the findings of the present disclosure [82, 83]. In addition, clinical methods have been developed for the production of tumor-specific T cells from cancer patients, along with the established safety profiles of various oncolytic viruses, supporting the therapies of the present disclosure to be clinically highly transformable [40-42, 84,85].

Different embodiments of the present disclosure are illustrated by the above embodiments. Those skilled in the art can develop alternatives to the above-described methods, which are within the scope of the disclosure and the claims.

All publications, patents, and patent applications are hereby incorporated by reference in their entirety herein in their entirety in the the the the the the Where a term in this application is defined differently in the documents incorporated by reference herein, the definitions provided herein are used as the definition of the term.

references:

1. Minniti G, Sanctis V, Muni R, Filippone F, Bozzao A, Valeriani M, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma in elderly patients. J. Neurooncol. 2008 88: 97–103.

2. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature 2011 480: 480–9.

3. Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE, et al. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood. 2009 114: 1537–44.

4. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive Strategies that are Mediated by Tumor Cells. Annu. Rev. Immunol. 2007 25: 267–96.

5. Kim R, Emi M, Tanabe K, Arihiro K. Tumor-driven evolution of immunosuppressive networks during malignant progression. Cancer Res. 2006 66: 5527–36.

6. Mahoney KM, Freeman GJ, McDermott DF. The Next Immune-Checkpoint Inhibitors: PD-1/PD-L1 Blockade in Melanoma. Clin. Ther. 2015 37: 764-82.

7. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996 271: 1734–6.

8. Camacho LH. CTLA-4 blockade with ipilimumab: biology, safety, efficacy, and future considerations. Cancer Med. 2015 4: 661–72.

9. Zamarin D, Holmgaard RB, Subudhi SK, Park JS, Mansour M, Palese P, et al. Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy. Sci. Transl. Med. 2014 6: 226–32.

10. Winograd R, Byrne KT, Evans RA, Odorizzi PM, Meyer ARL, Bajor DL, et al. Induction of T-cell Immunity Overcomes Complete Resistance to PD-1 and CTLA-4 Blockade and Improves Survival in Pancreatic Carcinoma. Res. 2015 3: 399–411.

11. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 2012. 12: 269–81.

12. Rapoport AP, Stadtmauer EA, Binder-Scholl GK, Goloubeva O, Vogl DT, Lacey SF, et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat. Med 2015 21: 1–20.

13. Nakazawa Y, Huye LE, Salsman VS, Leen AM, Ahmed N, Rollins L, et al. PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor. Mol. Ther. 2011 19 : 2133–43.

14.

S, Draper LM, Langhan MM, Campbell TE, Kwong ML, Wunderlich JR, et al. Sustainable regression of metastatic cervical cancer after treatment with human papillomavirus-targeted tumor-infiltrating T cells. J. Clin. Oncol. 2015 33: 1543– 50.

15. Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR, Finkelstein SE, et al. Central memory self/tumor-reactive CD8+T cells confer superior antitumor immunity compared with effector memory T cells. Proc. Natl Acad. Sci. U. S. A. 2005 102: 9571–6.

16. Topalian SL, Muul LM, Solomon D, Rosenberg SA. Expansion of human tumor infiltrating lymphocytes for use in immunotherapy trials. J. Immunol. Methods 1987 102: 127–41.

17. Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, Kammula U, et al. Adoptive cell therapy for patients with metastatic melanoma: Evaluation of intensive myeloablative chemoradiation preparative regimens. J. Clin. Oncol. 2008; 26: 5233–9.

18. Ram R, Storb R, Sandmaier BM, Maloney DG, Woolfrey A, Flowers MED, et al. Non-myeloablative conditioning with allogeneic hematopoietic cell transplantation for the treatment of high-risk acute lymphoblastic leukemia. Haematologica. 2011 96: 1113– 20.

19. Topalian SL, Solomon D, Avis FP, Chang AE, Freerksen DL, Linehan WM, et al. Immunotherapy of patients with advanced cancer using tumor-infiltrating lymphocytes and recombinant interleukin-2: apilot study. J Clin Oncol 1988 6: 839 –53.

20. Chapuis AG, Thompson JA, Margolin KA, Rodmyre R, Lai IP, Dowdy K, et al. Transferred melanoma-specific CD8+T cells persist, mediate spectral regression, and acquire central memory phenotype. Proc. Natl. Acad. Sci . 2012 109: 4592–7.

21. Pollack SM, Jones RL, Farrar E a, Lai IP, Lee SM, Cao J, et al. Tetramer guided, cell sorter assisted production of clinical grade autologous NY-ESO-1 specific CD8 (+) T cells. J. Immunother. Cancer 2014 2: 36.

22. Zhang L, Bridle BW, Chen L, Pol J, Spaner D, Boudreau JE, et al. Delivery of viral-vectored vaccines by B cells represents a novel strategy to accelerate CD8 (+) T-cell recall responses. Blood. 2013 121: 2432–9.

23. Oldstone MB, Tishon A, Eddleston M, de la Torre JC, McKee T, Whitton JL. Vaccination to prevent persistent viral infection. J. Virol. 1993 67: 4372–8.

24. Pol JG, Zhang L, Bridle BW, Stephenson KB, Rességuier J, Hanson S, et al. Maraba virus as a potent oncolytic vaccine vector. Mol. Ther. 2014 22: 420–9.

25. Boudreau JE, Bridle BW, Stephenson KB, Jenkins KM, Brunellière J, Bramson JL, et al. Recombinant vesicular stomatitis virus transduction of dendritic cells enhances their ability to prime innate and adaptive antitumor immunity. Mol. Ther. 2009 17: 1465 –72.

26. Brun J, McManus D, Lefebvre C, Hu K, Falls T, Atkins H, et al. Identification of genetically modified Maraba virus as an oncolytic rhabdovirus. Mol. Ther. 2010 18: 1440–9.

27. Rochman Y, Spolski R, Leonard WJ. New insights into the regulation of T cells by gamma (c) family cytokines. Nat. Rev. Immunol. 2009 9: 480–90.

28. Crawley AM, Katz T, Parato K, Angel JB. IL-2 receptor gamma chain cytokines differentially regulate human CD8+CD127+and CD8+CD127-T cell division and susceptibility to apoptosis. Int. Immunol. 2009 21: 29– 42.

29. Dudley ME, Wunderlich JR, Shelton TE, Even J, Rosenberg SA. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J. Immunother. 2003 26:332–42.

30. Hinrichs CS, Spolski R, Paulos CM, Gattinoni L, Kerstann KW, Palmer DC, et al. IL-2 and IL-21 conferencing differentiation programs to CD8+T cells for adoptive immunotherapy. Blood 2008 111: 5326–33 .

31. Schluns KS,

L. Cytokine control of memory T-cell development and survival. Nat. Rev. Immunol. 2003 3: 269–79.

32. Zeng R, Spolski R, Finkelstein SE, Oh S, Kovanen PE, Hinrichs CS, et al. Synergy of IL-21 and IL-15 in regulating CD8+T cell expansion and function. J. Exp. Med. 2005 201 : 139–48.

33. Araki K, Turner AP, Shaffer VO, Gangappa S, Keller S a, Bachmann MF, et al. mTOR regulates memory CD8 T-cell differentiation. Nature. 2009 460: 108–12.

34. Rao RR, Li Q, Odunsi K, Shrikant PA. The mTOR Kinase Determines Effector versus Memory CD8+T Cell Fate by Regulating the Expression of Transcription Factors T-bet and Eomesodermin. Immunity. 2010 32: 67–78.

35. Li Q, Rao R, Vazzana J, Goedegebuure P, Odunsi K, Gillanders W, et al. Regulating mammalian target of rapamycin to tune vaccination-induced CD8 (+) T cell responses for tumor immunity. J. Immunol. 2012 188 : 3080–7.

36. Cameron BJ, Gerry AB, Dukes J, Harper J V, Kannan V, Bianchi FC, et al. Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells Sci. Transl. Med. 2013 5: 197ra103.

37. Chinnasamy N, Wargo J a, Yu Z, Rao M, Frankel TL, Riley JP, et al. ATCR targeting the HLA-A*0201-restricted epitope of MAGE-A3 recognizes multiple epitopes of the MAGE-A antigen superfamily in Several types of cancer. J. Immunol. 2011 186: 685–96.

38. Schumacher TN, Hacohen N. Neoantigens encoded in the cancer genome. Curr Opin Immunol 2016; 41: 98-103 doi 10.1016/j. coi. 2016.07.005.

39. Chabanon RM, Pedrero M, Lefebvre C, Marabelle A, Soria JC, Postel-Vinay S. Mutational Landscape and Sensitivity to Immune Checkpoint Blockers. Clin Cancer Res 2016; 22 (17) : 4309-21 doi 10.1158/1078-0432 . CCR-16-0903.

40. Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A, et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 2014; 371 (23) : 2189-99 Doi 10.1056/NEJMoa1406498.

41. Hugo W, Zaretsky JM, Sun L, Song C, Moreno BH, Hu-Lieskovan S, et al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. Cell 2016; 165 (1) : 35-44 doi 10.1016/j. cell. 2016.02.065.

42. McGranahan N, Furness AJ, Rosenthal R, Ramskov S, Lyngaa R, Saini SK, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016; 351 (6280) : 1463-9 doi 10.1126/ Science. aaf1490.

43. Houot R, Schultz LM, Marabelle A, Kohrt H. T-cell-based Immunotherapy: Adoptive Cell Transfer and Checkpoint Inhibition. Cancer Immunol Res 2015; 3 (10) : 1115-22 doi 10.1158/2326-6066. CIR- 15-0190.

44. Pollack SM, Jones RL, Farrar EA, Lai IP, Lee SM, Cao J, et al. Tetramer guided, cell sorter assisted production of clinical grade autologous NY-ESO-1 specific CD8 (+) T cells. J Immunother Cancer 2014; 2 (1) : 36 doi 10.1186/s40425-014-0036-y.

45. Kaiser AD, Assenmacher M, Schroder B, Meyer M, Orentas R, Bethke U, et al. Towards a commercial process for the manufacture of genetically modified T cells for therapy. Cancer Gene Ther 2015; 22 (2) : 72- 8 doi 10.1038/cgt. 2014.78.

46. Sadelain M, Riviere I, Riddell S. Therapeutic T cell engineering. Nature 2017; 545 (7655) : 423-31 doi 10.1038/nature22395.

47. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014; 371 (16) : 1507-17 doi 10.1056 /NEJMoa1407222.

48. Robbins PF, Kassim SH, Tran TL, Crystal JS, Morgan RA, Feldman SA, et al. Apilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell receptor: long-term follow-up and Correlation with response. Clin Cancer Res 2015; 21 (5) : 1019-27 doi 10.1158/1078-0432. CCR-14-2708.

49. Louis CU, Savoldo B, Dotti G, Pule M, Yvon E, Myers GD, et al. Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood 2011; 118 (23) : 6050-6 doi 10.1182/blood-2011-05-354449.

50. Bollard CM, Gottschalk S, Torrano V, Diouf O, Ku S, Hazrat Y, et al. Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane proteins. J Clin Oncol 2014; (8) : 798-808 doi 10.1200/JCO. 2013.51.5304.

51. Chapuis AG, Roberts IM, Thompson JA, Margolin KA, Bhatia S, Lee SM, et al. T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results In Long-Term Cell Persistence and Durable Tumor Regression. J Clin Oncol 2016 doi 10.1200/JCO. 2015.65.5142.

52. Ledford H. Safety concerns blight promising cancer therapy. Nature 2016; 538 (7624) : 150-1 doi 10.1038/538150a.

53. Muranski P, Boni A, Wrzesinski C, Citrin DE, Rosenberg SA, Childs R, et al. Increased intensity lymphodepletion and adoptive immunotherapy--how far can we go? Nat Clin Pract Oncol 2006; 3 (12) : 668-81 doi 10.1038/ncponc0666.

54. Wrzesinski C, Paulos CM, Kaiser A, Muranski P, Palmer DC, Gattinoni L, et al. Increased intensity lymphodepletion enhances tumor treatment efficacy of adoptively transferred tumor-specific T cells. J Immunother 2010; 33 (1) : 1- 7 doi 10.1097/CJI. 0b013e3181b88ffc.

55. Bridle BW, Stephenson KB, Boudreau JE, Koshy S, Kazdhan N, Pullenayegum E, et al. Potentiating cancer immunotherapy using an oncolytic virus. Mol Ther 2010; 18 (8) : 1430-9 doi 10.1038/mt. 2010.98.

56. Bridle BW, Nguyen A, Salem O, Zhang L, Koshy S, Clouthier D, et al. Privileged Antigen Presentation in Splenic B Cell Follicles Maximizes T Cell Responses in Prime-Boost Vaccination. J Immunol 2016; 196 (11) : 4587-95 doi 10.4049/jimmunol. 1600106.

57. Pol JG, Zhang L, Bridle BW, Stephenson KB, Resseguier J, Hanson S, et al. Maraba virus as a potent oncolytic vaccine vector. Mol Ther 2014; 22 (2) : 420-9 doi 10.1038/mt. 2013.249 .

58. Ikeda H, Ohta N, Furukawa K, Miyazaki H, Wang L, Kuribayashi K, et al. Mutated mitogen-activated protein kinase: atumor rejection antigen of mouse sarcoma. Proc Natl Acad Sci U S A 1997; 94 (12) : 6375-9.

59. Hanson HL, Donermeyer DL, Ikeda H, White JM, Shankaran V, Old LJ, et al. Eradication of established tumors by CD8+T cell adoptive immunotherapy. Immunity 2000; 13 (2) : 265-76.

60. Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR, Finkelstein SE, et al. Central memory self/tumor-reactive CD8+T cells confer superior antitumor immunity compared with effector memory T cells. Proc Natl Acad Sci U S A 2005; 102 (27) : 9571-6 doi 10.1073/pnas. 0503726102.

61. Cho HI, Jung SH, Sohn HJ, Celis E, Kim TG. An optimized peptide vaccine strategy capable of inducing multivalent CD8 (+) T cell responses with potent antitumor effects. Oncoimmunology 2015; 4 (11) : e1043504 doi 10.1080/ 2162402X. 2015.1043504.

62. North RJ. Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor depends on elimination of tumor-induced suppressor T cells. J Exp Med 1982; 155 (4) : 1063-74.

63. Salem ML, Kadima AN, El-Naggar SA, Rubinstein MP, Chen Y, Gillanders WE, et al. Defining the ability of cyclophosphamide preconditioning to enhance the antigen-specific CD8+T-cell response to peptide vaccination: creation of a J Immunother 2007; 30 (1) : 40-53 doi 10.1097/01. cji. 0000211311.28739. e3.

64. Duan F, Duitama J, Al Seesi S, Ayres CM, Corcelli SA, Pawashe AP, et al. Genomic and bioinformatic profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity. J Exp Med 2014; 211 (11) : 2231 -48 doi 10.1084/jem. 20141308.

65. Tran E, Robbins PF, Rosenberg SA. 'Final common pathway' of human cancer immunotherapy: targeting random somatic mutations. Nat Immunol 2017; 18 (3) : 255-62 doi 10.1038/ni. 3682.

66. Yarchoan M, Johnson BA, 3rd, Lutz ER, Laheru DA, Jaffee EM. Targeting neoantigens to augment antitumour immunity. Nat Rev Cancer 2017; 17 (4) : 209-22 doi 10.1038/nrc. 2016.154.

67. Fousek K, Ahmed N. The Evolution of T-cell Therapies for Solid Malignancies. Clin Cancer Res 2015; 21 (15) : 3384-92 doi 10.1158/1078-0432. CCR-14-2675.

68. Gilham DE, Debets R, Pule M, Hawkins RE, Abken H. CAR-T cells and solid tumors: tuning T cells to challenge an inveterate foe. Trends Mol Med 2012; 18 (7) : 377-84 doi 10.1016/ j. molmed. 2012.04.009.

69. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol 2012; 12 (4) : 269-81 doi 10.1038/nri3191.

70. Wu R, Forget MA, Chacon J, Bernatchez C, Haymaker C, Chen JQ, et al. Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic melanoma: current status and future outlook. Cancer J 2012; 18 ( 2) : 160-75 doi 10.1097/PPO. 0b013e31824d4465.

71. Busch DH, Frassle SP, Sommermeyer D, Buchholz VR, Riddell SR. Role of memory T cell subsets for adoptive immunotherapy. Semin Immunol 2016; 28 (1) : 28-34 doi 10.1016/j. smim. 2016.02.001.

72. Crompton JG, Sukumar M, Restifo NP. Uncoupling T-cell expansion from effector differentiation in cell-based immunotherapy. Immunol Rev 2014; 257 (1) : 264-76 doi 10.1111/imr. 12135.

73. Junttila MR, de Sauvage FJ. Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 2013; 501 (7467) : 346-54 doi 10.1038/nature12626.

74. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol 2013; 14 (10) : 1014-22 doi 10.1038/ni. 2703.

75. Corbiere V, Chapiro J, Stroobant V, Ma W, Lurquin C, Lethe B, et al. Antigen spreading contributes to MAGE vaccination-induced regression of melanoma metastases. Cancer Res 2011; 71 (4) : 1253-62 doi 10.1158 /0008-5472. CAN-10-2693.

76. Chapuis AG, Lee SM, Thompson JA, Roberts IM, Margolin KA, Bhatia S, et al. Combined IL-21-primed polyclonal CTL plus CTLA4 blockade controls refractory metastatic melanoma in a patient. J Exp Med 2016; 213 (7 ) : 1133-9 doi 10.1084/jem. 20152021.

77. Boon T, Coulie PG, Van den Eynde BJ, van der Bruggen P. Human T cell responses against melanoma. Annu Rev Immunol 2006; 24: 175-208 doi 10.1146/annurev. immunol. 24.021605.090733.

78. Chapuis AG, Desmarais C, Emerson R, Schmitt TM, Shibuya K, Lai I, et al. Tracking the Fate and Origin of Clinically Relevant Relevant Adoptively Transferred CD8 (+) T Cells In Vivo. Sci Immunol 2017; 2 (8) Doi 10.1126/sciimmunol. aal2568.79. Robbins PF, Dudley ME, Wunderlich J, El-Gamil M, Li YF, Zhou J, et al. cutting edge: persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy J Immunol 2004; 173 (12) : 7125-30.

80. Norian LA, Allen PM. Rapid maturation of effector T cells in tumors, but not lymphoid organs, during statistical regression. PLoS One 2007; 2 (9) : e821 doi 10.1371/journal. pone. 0000821.

81. Radoja S, Saio M, Frey AB. CD8+tumor-infiltrating lymphocytes are primed for Fas-mediated activation-induced cell death but are not apoptotic in situ. J Immunol 2001; 166 (10): 6074-83.

82. Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME, et al. Cancer immunotherapy based on mutation-specific CD4+T cells in a patient with epithelial cancer. Science 2014; 344 (6184) : 641 -5 doi 10.1126/science. 1251102.

83. Tran E, Robbins PF, Lu YC, Prickett TD, Gartner JJ, Jia L, et al. T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer. N Engl J Med 2016; 375 (23) : 2255-62 doi 10.1056 /NEJMoa1609279.

84. Breitbach CJ, Burke J, Jonker D, Stephenson J, Haas AR, Chow LQ, et al. Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature 2011; 477 (7362) : 99-102 doi 10.1038/nature10358.

85. LeBlanc AK, Naik S, Galyon GD, Jenks N, Steele M, Peng KW, et al. Safety studies on intravenous administration of oncolytic recombinant vesicular stomatitis virus in purpose-bred beagle dogs. Hum Gene Ther Clin Dev 2013; 24 ( 4) : 174-81 doi 10.1089/humc. 2013.165.

86. Oldstone MB et al. Vaccination to prevent persistent viral infection. . J. Virol. 1993; 67 (7) : 4372–8.

Claims

A modified matrix protein (M) of a recombinant oncolytic baculovirus, characterized in that the amino acid sequence encoding the modified matrix protein (M) has at least 80 compared to the amino acid sequence shown in SEQ ID NO: 1. %, preferably at least 90%, more preferably at least 95%, most preferably at least 98% identical; and wherein the amino acid sequence is compared to SEQ ID NO: 1, there is a substitution as shown in (i) or (ii) :

(i) the 21st position, the 51st position, the 111th position, and the 221st position have both amino acid substitutions;

(ii) The 51st position, the 221st position, and the 226th position have both amino acid substitutions.
The modified matrix protein (M) according to claim 1, wherein the recombinant oncolytic baculovirus is selected from the group consisting of vesicular stomatitis virus; preferably, the recombinant oncolytic baculovirus is selected from the group consisting of vesicular stomatitis virus Indiana More preferably, the recombinant oncolytic baculovirus is selected from the MuddSummer strain.
The modified matrix protein (M) according to any one of claims 1 to 2, wherein the modified matrix protein (M) has a sequence encoding a modified substrate as compared with SEQ ID NO: 1. The amino acid sequence of the protein (M) has a substitution as shown in (i) or (ii):

(i) The 21st position, the 51st position, the 111th position, and the 221st position have both amino acid substitutions, and the amino acid substitutions are:

(a) the 21th glycine G is replaced by glutamic acid E,

(b) the 51th methionine M is replaced by alanine A,

(c) the 111th leucine L is replaced by phenylalanine F,

(d) replacing the 221th proline V with phenylalanine F;

or

(ii) The 51st position, the 221st position, and the 226th position have both amino acid substitutions, and the amino acid substitutions are:

(a) the 51st methionine M is replaced by arginine R,

(b) Replacement of the 221th proline V with phenylalanine F,

(c) the 226th glycine G is replaced by arginine R;

Preferably, the sequence of the 21st position, the 51st position, the 111th position and the 221st position of the modified matrix protein (M) having an amino acid substitution is a sequence as shown in SEQ ID NO: 3;

The sequence in which the 51st position, the 221st position, and the 226th position of the modified matrix protein (M) have an amino acid substitution at the same time is the sequence shown in SEQ ID NO: 5.
A recombinant oncolytic baculovirus characterized in that the recombinant oncolytic baculovirus genome comprises a modified matrix protein (M), characterized in that the amino acid sequence of the modified matrix protein (M) is as claimed in claim 1. The amino acid sequence shown in any one of 3; preferably, the recombinant oncolytic baculovirus is an attenuated oncolytic baculovirus; more preferably, the recombinant oncolytic baculovirus is attenuated by attenuated delayed infection Tumor baculovirus.
A composition comprising an isolated recombinant oncolytic baculovirus having a nucleic acid fragment encoding a modified matrix protein (M), characterized in that the modified matrix protein The amino acid sequence of M) is the amino acid sequence according to any one of claims 1-3; preferably, the recombinant oncolytic baculovirus is an attenuated recombinant oncolytic baculovirus; more preferably, the recombinant lysate Tumor baculovirus is an oncolytic baculovirus with attenuated delayed infection.
The composition according to claim 5, further comprising a second oncolytic virus; preferably, the second oncolytic virus is selected from the group consisting of a rhabdovirus, a vaccinia virus, a herpes virus, a measles virus, and a Newcastle disease One or more of a virus, an adenovirus, an alphavirus, a parvovirus, an enterovirus strain; more preferably, the second oncolytic virus is an attenuated oncolytic virus; most preferably, wherein the second The oncolytic virus is an attenuated rhabdovirus.
The composition according to any one of claims 5 to 6, further comprising a second antitumor agent; preferably, the second antitumor agent is an immunotherapeutic agent, a chemotherapeutic agent or a radiotherapeutic agent; More preferably, the second anti-tumor preparation is selected from one or more of a small molecule, a macromolecule, a cell, a viral vector, a gene vector, a DNA, an RNA, a polypeptide, and a nanocomposite.
A vaccine composition, comprising: a therapeutically effective amount of one or more recombinant oncolytic baculoviruses, wherein the one or more recombinant oncolytic baculovirus genomes comprise a modified matrix The amino acid sequence of the protein (M), the modified matrix protein (M), which is an amino acid sequence according to any one of claims 1-3.
The vaccine composition according to claim 8, which may further comprise a second oncolytic virus as claimed in claim 6, or a second antitumor preparation as claimed in claim 7.
An isolated peptide encoded by an amino acid sequence selected from the group consisting of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% of the amino acid sequence of SEQ ID NO: 1. The same sequence, and the amino acid sequence is compared to SEQ ID NO: 1, there is a substitution as shown in (i) or (ii):

(i) The 21st position, the 51st position, the 111th position, and the 221st position have both amino acid substitutions, and the amino acid substitutions are:

(a) the 21th glycine G is replaced by glutamic acid E,

(b) the 51th methionine M is replaced by alanine A,

(c) the 111th leucine L is replaced by phenylalanine F,

(d) replacing the 221th proline V with phenylalanine F;

or

(ii) The 51st position, the 221st position, and the 226th position have both amino acid substitutions, and the amino acid substitutions are:

(a) the 51st methionine M is replaced by arginine R,

(b) Replacement of the 221th proline V with phenylalanine F,

(c) the 226th glycine G is replaced by arginine R;

Preferably, the sequence of the 21st position, the 51st position, the 111th position and the 221st position of the modified matrix protein (M) having an amino acid substitution is a sequence as shown in SEQ ID NO: 3;

The sequence in which the 51st position, the 221st position, and the 226th position of the modified matrix protein (M) have an amino acid substitution at the same time is the sequence shown in SEQ ID NO: 5.
A nucleotide sequence for encoding the isolated peptide of claim 10.
The use of the composition comprising the isolated recombinant oncolytic baculovirus or the vaccine according to any one of claims 8 to 9 in the preparation of a medicament according to any one of claims 5 to 7, which can kill abnormalities Proliferating cells, induction and promotion of anti-tumor immune response, anti-tumor immune cell-specific proliferation, anti-tumor immune cell homing invasive tumor tissue and/or elimination of tumor tissue microenvironment immunosuppression.
The use according to claim 11, wherein said abnormally proliferating cells are contained in a patient.
The use according to claim 12, wherein said abnormal proliferative cells are selected from tumor cells or tumor tissue-associated cells; preferably, said tumor cells are cancer cells; more preferably, said cancer cells are metastatic cancer cells .
Use of a composition comprising an isolated recombinant oncolytic baculovirus according to any one of claims 5-7 or a vaccine according to any one of claims 8-9 for the preparation of a medicament for treating a patient having a tumor .
A method for slow and sustained killing of aberrant proliferating cells, comprising the abnormally proliferating cells and the recombinant oncolytic baculovirus according to claim 4, comprising the isolated recombinant according to any one of claims 5-7 A composition of oncolytic baculovirus or a step of contacting a vaccine according to any one of claims 8-9.
The method of claim 16 wherein said abnormally proliferating cells are contained within a patient.
The method according to claim 16, wherein said abnormally proliferating cells are selected from tumor cells or tumor tissue-associated cells; preferably, said tumor cells are cancer cells; more preferably, said cancer cells are metastatic cancer cells .
The method of claim 16, wherein the recombinant oncolytic baculovirus, a composition comprising the isolated recombinant oncolytic baculovirus or a vaccine is administered to a patient.
The method according to claim 16, wherein said recombinant oncolytic baculovirus, composition or vaccine comprising the isolated recombinant oncolytic baculovirus comprises intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, intratumoral Administering one or more modes of administration, subcutaneous or intranasal administration; preferably, the route of administration of the mode of administration includes endoscopy, endoscopy, intervention, minimally invasive, conventional surgery One or more.
The method of claim 16 further comprising the step of administering a second anti-tumor therapy.
The method according to claim 21, wherein said second anti-tumor therapy is selected from the group consisting of administering a second oncolytic virus; preferably, said second oncolytic virus is selected from the group consisting of rhabdovirus, vaccinia virus, herpes virus, measles One or more of a virus, Newcastle disease virus, adenovirus, alphavirus, parvovirus, enterovirus strain; more preferably, the second oncolytic virus is an attenuated oncolytic virus; most preferably, wherein The second oncolytic virus is an attenuated rhabdovirus.
The method of claim 21, wherein the second anti-tumor therapy is selected from one or more of chemotherapy, radiation therapy, immunotherapy, surgery therapy.
A method of inducing an immune response in a subject, the method comprising administering to the subject a recombinant oncolytic baculovirus according to claim 4, comprising any one of claims 5-7 A composition of the isolated recombinant oncolytic baculovirus or one or more of the vaccines of any one of claims 8-9.
A method for inducing an anti-tumor immune response, anti-tumor immune cell-specific proliferation, anti-tumor immune cells infiltrating tumor tissue or eliminating micro-environmental immunosuppression of tumor tissue, comprising recombining tumor or tumor tissue with claim 4 An oncolytic baculovirus, a composition comprising an isolated recombinant oncolytic baculovirus according to any one of claims 5-7, or a step of contacting a vaccine according to any one of claims 8-9.
An antitumor composition comprising (i) a replicative oncolytic virus vaccine; (ii) adoptive immune cells.
The antitumor composition according to claim 26, wherein the replicative oncolytic virus is an oncolytic baculovirus or an oncolytic vaccinia virus; alternatively, the replicative oncolytic virus expresses a tumor antigen.
The antitumor composition according to claim 27, wherein the oncolytic baculovirus is the recombinant oncolytic baculovirus according to claim 4.
The antitumor composition according to any one of claims 26 to 28, wherein the adoptive immune cells are central memory T cells (Tcm), preferably, the central memory T cells (Tcm) are produced. A tumor antigen-specific CD8 + T cell population, more preferably, at least 50%, 60% or 70% of said CD8 + T cells exhibit a central memory cell-specific phenotype.
The antitumor composition according to any one of claims 26 to 29, wherein the tumor antigen is selected from the group consisting of NY-ESO1, cancer testis antigen, carcinoembryonic antigen (CEA), Composition of full-length protein or partially encoded peptides of alpha-fetoprotein (AFP), CA 125, Her2, dopachrome tautomerase (DCT), GP100, MART1, CMV pp65, EBV, HPV E6 and HPV E7 Any one or combination of the groups; alternatively, the cancer test antigen is selected from the group consisting of MAGE.
The antitumor composition according to any one of claims 26 to 30, wherein the tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, colon cancer, colorectal cancer ), pancreatic cancer, thyroid cancer, nasopharyngeal cancer, oral cancer, head and neck cancer, cholangiocarcinoma, prostate cancer, nest cancer, uterine cancer, cervical cancer, kidney cancer, bladder cancer, ureteral cancer, brain cancer (eg glioma) , melanoma, skin cancer, sarcoma, lymphoma, and leukemia; optionally, the tumor includes a primary, secondary or metastatic tumor.
The antitumor composition according to any one of claims 26 to 31, wherein the tumor is a solid tumor.
Use of a composition for the preparation of a medicament for treating a tumor, the composition comprising (i) adoptive immune cells and (ii) a replicative oncolytic virus vaccine.
The use according to claim 33, wherein the replicative oncolytic virus is an oncolytic baculovirus or an oncolytic vaccinia virus, and optionally, the replicative oncolytic virus expresses a tumor antigen.
The use according to claim 34, wherein the oncolytic baculovirus is the recombinant oncolytic baculovirus according to claim 4.
The use according to any one of claims 33 to 35, wherein the adoptive immune cell is a central memory T cell (Tcm), preferably, the central memory T cell (Tcm) is a tumor antigen specificity More preferably, at least 50%, 60% or 70% of the CD8+ T cells exhibit a central memory cell-specific phenotype.
The use according to any one of claims 33 to 36, wherein the oncolytic virus vaccine is administered to a subject by intravascular administration, preferably by intravenous administration to a subject.
The use according to any one of claims 33 to 37, wherein the vaccinia virus is selected from the group consisting of Wyeth, Western Reserve, Lister or Copenhagen; alternatively, the vaccinia virus lacks the thymidine kinase gene or the vaccinia virus growth factor gene.
The use according to any one of claims 33 to 38, wherein the vaccinia virus is administered to a patient intratumorally, intrathoracicly, intrapleurally, intraperitoneally, intraperitoneally, intracranically, intravenously, intraarterially or intramuscularly or Subject.
The use according to any one of claims 33 to 39, wherein the tumor antigen is selected from the group consisting of NY-ESO1, cancer testis antigen, carcinoembryonic antigen (CEA), alpha-fetoprotein ( AFP), CA 125, Her2, dopachrome tautomerase (DCT), GP100, MART1, CMV pp65, EBV, HPV E6, and HPV E7, etc. Any one or a combination thereof; alternatively, the cancer test antigen is selected from the group consisting of MAGE.
The use according to any one of claims 33 to 40, wherein the tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, colon cancer, colorectal cancer, pancreatic cancer , thyroid cancer, nasopharyngeal cancer, oral cancer, head and neck cancer, cholangiocarcinoma, prostate cancer, nest cancer, uterine cancer, cervical cancer, kidney cancer, bladder cancer, ureteral cancer, brain cancer (eg glioma), melanoma , skin cancer, sarcoma, lymphoma, and leukemia; optionally, the tumor includes a primary, secondary or metastatic tumor.
The use according to any of claims 33-41, wherein the tumor is a solid tumor.
A combination therapy for treating cancer, the method comprising (i) administering an adoptive immune cell to a subject having cancer; and (ii) subsequently administering a replicative oncolytic virus vaccine to a subject having cancer Tester.
The combination therapy according to claim 43, wherein said replicative oncolytic virus is an oncolytic baculovirus or an oncolytic vaccinia virus, and optionally, said replicative oncolytic virus expresses a tumor antigen.
The combination therapy method according to claim 44, wherein the oncolytic baculovirus is the recombinant oncolytic baculovirus according to claim 4.
The combination therapy method according to any one of claims 43 to 45, wherein the adoptive immune cell is a central memory T cell (Tcm), preferably, the central memory T cell (Tcm) is a tumor antigen producing A specific CD8 + T cell population, more preferably, at least 50%, 60% or 70% of said CD8 + T cells exhibit a central memory cell-specific phenotype.
The combination therapy method according to any one of claims 43 to 46, wherein the CD8 + T cell population is produced by the presence of antigen-presenting cells (APC) loaded with tumor antigens, IL21, IL15 and Repa And preferably obtain peripheral blood mononuclear cells (PBMC), or tumor infiltrating lymphocytes (TIL), or intramedullary lymphocytes from a subject having cancer in the absence of IL2, and Peripheral blood mononuclear cells (PBMC), or tumor infiltrating lymphocytes (TIL) or intramedullary lymphocytes are cultured in vitro.
The combination therapy method according to any one of claims 43 to 47, wherein the CD8+ T cell population is produced by obtaining PBMC from a subject having cancer by transducing a recombinant T cell receptor to PBMC (TCR) or chimeric antigen receptor (CAR) genetically modify it and culture the transduced PBMC ex vivo.
The combination therapy according to any one of claims 43 to 48, wherein the CD8+ T cell population is obtained by ex vivo culture of the PBMC cleared by CD25.
The combination therapy according to any one of claims 43 to 49, further comprising, after in vitro culture of PBMC or TIL, CD3+ T cells, further comprising using an anti-CD3 antibody and an anti-CD28 antibody, and optionally an IL2 in vitro amplification method. Said CD8 + T cells.
The combination therapy method according to any one of claims 43 to 50, wherein the oncolytic virus vaccine is administered to a subject by intravascular administration, preferably by intravenous administration.
The combination therapy according to any one of claims 43 to 51, wherein the tumor antigen is selected from the group consisting of NY-ESO1, cancer testis antigen, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA 125, Her2, dopachrome tautomerase (DCT), GP100, MART1, CMV pp65, EBV, HPV E6 and HPV E7 and other viral antigen full-length proteins or partially encoded peptides Any one or a combination thereof; alternatively, the cancer test antigen is selected from the group consisting of MAGE.
The combination therapy method according to any one of claims 43 to 52, wherein the tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, colon cancer, colorectal cancer, pancreas Cancer, thyroid cancer, nasopharyngeal cancer, oral cancer, head and neck cancer, cholangiocarcinoma, prostate cancer, nest cancer, uterine cancer, cervical cancer, kidney cancer, bladder cancer, ureteral cancer, brain cancer (eg glioma), melanin Tumor, skin cancer, sarcoma, lymphoma, and leukemia; optionally, the tumor includes a primary, secondary or metastatic tumor.
The combination therapy method according to any one of claims 43 to 53, wherein the tumor is a solid tumor.
The combination therapy according to any one of claims 43-54, wherein said IL21 and IL15 are present at a concentration of from about 5 ng/ml to about 15 ng/ml, said rapamycin being from 15 ng/ml to about A concentration of 25 ng/ml exists.
The combination therapy according to any one of claims 43 to 55, wherein said replicative oncolytic virus vaccine (OVV) is administered to the subject 1 to 72 hours after administration of said adoptive immune cells to the subject By.
A combination therapy according to any one of claims 43-56, wherein the subject does not undergo lymphocyte depletion prior to receiving adoptive immune cells.
The combination therapy method according to any one of claims 43 to 57, wherein the replication-type oncolytic virus vaccine is administered to a subject by multiple administrations.
The combination therapy method according to any one of claims 43 to 58, wherein the CD8 + T cell population is autologous to the subject.
The combination therapy method according to any one of claims 43 to 59, wherein the antigen presenting cell is a dendritic cell.