WO2019184459A1 - Oncolytic virus vaccine and adoptive immune cell combination therapy - Google Patents
Oncolytic virus vaccine and adoptive immune cell combination therapy Download PDFInfo
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- WO2019184459A1 WO2019184459A1 PCT/CN2018/121292 CN2018121292W WO2019184459A1 WO 2019184459 A1 WO2019184459 A1 WO 2019184459A1 CN 2018121292 W CN2018121292 W CN 2018121292W WO 2019184459 A1 WO2019184459 A1 WO 2019184459A1
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Definitions
- 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.
- 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].
- 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.
- T cells T cells having specific recognition ability against tumor-associated antigens
- a therapeutic vaccine such a cancer patient can be induced. cell.
- 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].
- 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.
- 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.
- 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.
- the oncolytic 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.
- 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)).
- 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.
- VSV vesicular stomatitis virus
- 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.
- 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.
- oncolytic baculovirus treatment differs from gene therapy mainly in that it replicates in tumor cells to produce an effect of killing tumor cells.
- 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.
- Tcm central memory T cells
- Tcm T lymphocytes
- PBMC peripheral blood mononuclear cells
- Tcm T lymphocytes
- PBMC peripheral blood mononuclear cells
- Tcm TAA-specific T cell receptor
- CAR chimeric antigen receptor
- Tcm tumor-associated antigens
- 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.
- an attenuated strain of oncolytic baculovirus which is effective in reducing the toxicity of viruses in normal somatic cells.
- 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.
- 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.
- 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.
- 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.
- a combination therapy for treating cancer in a subject of a particular indication 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.
- 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.
- the T cells in Tcm therapy are autologous T cells derived from the subject to be treated.
- the combination therapy does not require a step of autoimmune cell clearance of the subject.
- 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.
- 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.
- 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.
- M modified matrix protein
- 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.
- the sequence of the modified matrix protein (M) is the sequence shown in SEQ ID NO:
- 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.
- the sequence of the modified matrix protein (M) is the sequence shown in SEQ ID NO: 5.
- 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.
- M modified matrix protein
- the present disclosure relates to a composition
- 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.
- M modified matrix protein
- 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.
- 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
- 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.
- 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).
- M Matrix protein
- the vaccine of the present disclosure may further comprise a second oncolytic virus, or a second anti-tumor preparation.
- 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.
- 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.
- 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.
- the amino acid sequence is the sequence set forth in SEQ ID NO: 3.
- 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
- (iii) the 226th glycine G is mutated to arginine R.
- the sequence of the modified matrix protein (M) is the sequence shown in SEQ ID NO: 5.
- the disclosure relates to an isolated nucleotide sequence for encoding the isolated peptide.
- 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.
- the aberrant proliferative cells are included in a patient.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- the second oncolytic virus is an attenuated oncolytic virus; most preferably, wherein the second oncolytic virus Attenuated rhabdovirus.
- 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.
- 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.
- 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.
- a method for producing a tumor antigen-specific central memory CD8+ T cell 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.
- CD25+ cells including regulatory T cells and activated T and B cells
- a tumor antigen, a tumor associated antigen (TAA) is a substance produced in tumor cells that elicits an immune response in a mammal.
- the tumor antigen is an autoantigen.
- 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).
- 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.
- 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.
- 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.
- the tumor antigen is overexpressed in cancer cells as compared to normal cells.
- 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.
- 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
- 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.
- a melanoma-associated antigen such as DCT can be used.
- 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.
- 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.
- the vector is modified to express TCR/CAR.
- the nucleic acid sequence encoding the selected TCR/CAR is cloned into the vector of choice using established recombinant techniques.
- 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.
- a method for adoptive cell infusion 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.
- 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.
- APCs antigen presenting cells
- IL21 loaded with a tumor antigen peptide
- 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.
- 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.
- 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.
- the PBMC are transduced with a recombinant TCR or CAR capable of specifically recognizing a tumor antigen prior to ex vivo culture.
- 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.
- 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.
- the replicative oncolytic virus is a rhabdovirus and is administered to a mammal by an intravenous route.
- the replicative oncolytic virus is a rhabdovirus RV and is administered to a mammal intravenously (IV), intramuscularly (IM), intraperitoneally (IP) or intratumor (IT).
- a replicating oncolytic virus e.g., rhabdovirus or vaccinia virus
- a suitable carrier e.g., physiological saline or other pharmaceutically suitable buffer.
- 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
- OSV replicative oncolytic virus vaccine
- the oncolytic virus is an oncolytic baculovirus or an oncolytic vaccinia virus.
- the replicative oncolytic virus expresses a tumor antigen.
- 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.
- the CD8 + T cell population is obtained by ex vivo culture of the PBMC cleared by CD25.
- 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.
- the tumor is a solid tumor.
- 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.
- APCs antigen-presenting cells
- PBMC peripheral blood mononuclear cells
- TIL tumor infiltrating lymphocytes
- 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.
- 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.
- TCR recombinant T cell receptor
- CAR chimeric antigen receptor to PBMC
- the replicative oncolytic virus in the anti-tumor preparation, is a baculovirus, preferably a vesicular stomatitis virus (VSV).
- the baculovirus is a recombinant or wild type Maraba virus, preferably a Maraba MG1 virus.
- 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
- 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.
- 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.
- a combination therapy for treating cancer in a subject in need thereof 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.
- Tcm tumor antigen-specific central memory T cells
- OV oncolytic virus
- central memory T cells are genetically modified to express one or more recombinant T cell receptors (TCRs) or chimeric antigen receptors (CARs) specific for tumor antigens.
- TCRs recombinant T cell receptors
- CARs chimeric antigen receptors
- the central memory T cell (Tcm) is an autologous T cell derived from a subject to be treated.
- the combination therapy does not include the step in which the subject is immunodepleted.
- a method for producing a tumor antigen-specific central memory CD8 + T cell 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.
- a tumor antigen such as a dendritic cell
- lymphocytes are cultured from PBMC or TIL.
- CD25 + cells progulatory T cells as well as activated T cells and B cells
- 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.
- TAA tumor associated antigen
- the tumor antigen is an autoantigen.
- 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).
- 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.
- 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.
- 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.
- the tumor antigen is an overexpressed tumor antigen that is overexpressed in cancer cells as compared to normal cells.
- 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.
- 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
- 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.
- a melanoma-associated antigen such as DCT can be used.
- 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.
- the nucleic acid sequence encoding the selected TCR/CAR is incorporated into the vector of choice using established recombinant techniques.
- the TCR or CAR is specific for a member of the MART-1, gp100, NY-ESO-1, or MAGE family (eg, MAGE-A3).
- 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.
- a method for adoptive cell therapy 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.
- APC tumor antigen
- IL21 eg, autologous dendritic cells
- IL15 IL15
- TIL2 tumor antigen-specific CD8+ T cells
- 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.
- tumor antigens such as antigen presenting cells such as dendritic cells, IL21, IL15 and rapamycin
- 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.
- 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.
- 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.
- a method for adoptive cell therapy 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.
- 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
- the resulting tumor antigen eg, TAA
- 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.
- 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.
- APC antigen presenting cells
- IL21 loaded with a tumor antigen peptide
- the oncolytic virus expressing the same tumor antigen is administered to the subject.
- 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.
- 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.
- 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.
- the PBMCs are transduced with recombinant TCR or CAR of a specific tumor antigen prior to ex vivo culture.
- 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 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. .
- VSV vesicular stomatitis virus
- 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.
- the replicative oncolytic virus is a baculovirus and is administered to a mammal by an intravenous route.
- the replicative oncolytic virus is a vaccinia virus and is administered to a mammal intravenously (IV), intramuscularly (IM), intraperitoneally (IP) or intratumor (IT).
- IV intravenously
- IM intramuscularly
- IP intraperitoneally
- IT intratumor
- replicative oncolytic viruses e.g., baculovirus or vaccinia virus
- a suitable carrier such as saline or other pharmaceutically suitable buffer.
- 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.
- 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.
- TAA tumor associated antigen
- 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
- 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.
- 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.
- a melanoma-associated antigen such as DCT, can be used for the treatment of melanoma.
- 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.
- 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.
- the antigen is administered to the mammal by any of several methods including, but not limited to, intravenous, intramuscular or intranasal.
- the antigen or antigen-doped carrier can be administered in a suitable vehicle such as saline or other suitable buffer.
- the mammal 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.
- the selected antigen or vector expressing the antigen can be administered to the mammal in an amount sufficient to produce an immune response.
- 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.
- adenoviral vectors administered to mice intramuscularly at least about 10 7 PFU minimum amount sufficient to generate an immune response.
- the corresponding amount should be sufficient to produce an immune response.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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.
- 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.
- the mutation of the M protein of the attenuated baculovirus further includes
- 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.
- 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.
- 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.
- a VSV virus as described in the present disclosure can be further modified by combining heterologous N, P, M, G and/or L proteins.
- 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.
- a second anti-tumor therapy such as a second therapeutic virus.
- the therapeutic virus can be an oncolytic virus, more particularly a VSV virus.
- the second anti-tumor therapy is a chemotherapeutic agent, a radiotherapeutic agent or an immunotherapeutic agent, surgery, or the like.
- 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.
- 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.
- the compositions are administered systemically, particularly by intravascular administration, including injection, infusion, and the like.
- the present disclosure provides attenuated oncolytic coronaviruses capable of effectively reducing the toxicity of a virus in normal somatic cells by genetic recombination techniques.
- 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 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.
- the tumor of the present disclosure has an effect of inducing an anti-tumor immune response and eliminating immunosuppression of tumor tissue microenvironment.
- 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.
- the present disclosure provides a combination therapy method that allows simultaneous analysis of endogenous T cell regulated anti-tumor immunity by therapy.
- 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.
- the endogenous T cells acquire long-term memory with an antigen-specific broad pool.
- 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.
- the anti-tumor preparations and combination treatment regimens provided by the present disclosure can result in complete and long-lasting tumor regression without pretreatment.
- 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.
- 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.
- FIG 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.
- FIG. 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.
- 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).
- MC38 colon cancer
- B16 melanoma
- LLC lung cancer cells
- 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).
- RVV-DCT oncolytic virus vaccine
- 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).
- 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.
- “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.
- 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.
- 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 "about” means that a value includes the standard deviation of the error of the device or method used to determine the value.
- inhibiting 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.
- the vaccination methods of the present disclosure can be used to treat cancer in a mammal.
- 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.
- mamal 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.
- 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.
- RV virus refers to an attenuated VSV oncolytic baculovirus.
- RV-Mut refers to the presence of a mutated oncolytic baculovirus as compared to a wild-type VSV oncolytic baculovirus.
- 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 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.
- 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.
- VSV vesicular stomatitis virus
- N nucleocapsid protein
- P phosphoprotein
- M matrix protein
- G surface glycoprotein
- L RNA-dependent RNA polymerase
- a replication-competent oncolytic virus that expresses a tumor antigen includes any naturally occurring (eg, from "field source") or modified replication competent oncolytic virus.
- the oncolytic virus can be modified, for example, to increase the selectivity of the virus for cancer cells.
- Oncolytic viruses having replication ability 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 Retrovi
- 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) KM205018.1
- Muir Springs virus KM204990.1
- Reed Collins virus Hart Park virus
- Flanders virus AF523199/gi: 25140635, AF523197/gi: 25140634, AF523196/gi: 25140633, AF523195/gi: 25140632, AF523194/gi: 25140631, AH012179/gi: 25140630
- 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.
- the oncolytic baculovirus is a wild-type or recombinant non-vesicular virus, such as Muir Springs, Farmington or Grand Bahia virus, including variants thereof.
- 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.
- 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
- ALF alpha-fetoprotein
- CEA carcinoembryonic antigen
- surface glycoproteins eg, CA 125
- oncogenes such as Her2, melanoma-associated antigens
- DCT dopachrome tautomerase
- GP100 and MART-1 cancer-testis antigens
- 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 extraembr
- 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.
- 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.
- 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.
- 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.
- 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, endotheli
- 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.
- NSCLC non-small cell lung cancer
- breast cancer eg, hormone refractory breast cancer
- head and neck cancer eg, head and neck squamous cell carcinoma
- therapy ectal cancer
- hormone sensitive or hormone refractory prostate cancer eg, colorectal cancer
- ovarian cancer hepatocellular carcinoma
- renal cell carcinoma chondrosarcoma sarcoma
- small cell lung cancer small cell lung cancer
- 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.
- SEQ ID NO: 1 is the amino acid sequence of the original matrix protein (M) of the oncolytic baculovirus MuddSummer strain;
- SEQ ID NO: 2 shows the nucleotide sequence of the original matrix protein (M) of the oncolytic baculovirus MuddSummer strain
- 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;
- 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;
- 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;
- 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.
- 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.
- 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.
- 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.
- Tcm 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.
- Tcm 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
- Tcm Adoptive transfer of T cells
- 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.
- 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.
- 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.
- the combination therapies described herein utilize adoptive therapy of tumor antigen-specific central memory T cells produced according to the methods described herein.
- 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 .
- IL21 and IL15 are present at a concentration of from about 1 ng/ml to about 20 ng/ml, preferably about 10 ng/ml.
- the rapamycin is present at a concentration of from about 10 ng/ml to about 30 ng/ml, preferably about 20 ng/ml.
- 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. TNF ⁇ (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.
- GM-CSF eg, about 800 U/ml
- IL-4 eg, about 500 U/ml
- TNF ⁇ 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
- 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.
- 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.
- 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.
- the central memory T cells obtained according to the Tcm method described herein display a CD44+CD62L+CD127+ phenotype.
- a CD8+ human T cell population for adoptive cell therapy 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.
- a replicative oncolytic virus eg, oncolytic baculovirus
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- 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 ⁇ M 2-mercaptoethanol, 100 U/mL penicillin and 100 ng/mL streptomycin in MEM/F11. Expression of the gp33 minigene was maintained with 800 ⁇ g/ml G418. All cell culture reagents were from Invitrogen (Invitrogen, Grand Island, NY).
- CMS5r Recurrent osteosarcoma CMS5 cells
- 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 ⁇ L 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.
- mice 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
- 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.
- 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).
- 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
- the 226th glycine G was replaced by arginine R.
- 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.
- 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.
- 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 ⁇ M 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 ⁇ g/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.
- FBS fetal bovine serum
- 2-mercapto Ethanol 100 U/mL penicillin
- streptomycin 10 ng/mL IL21
- 10 ng/mL IL15 10 ng/mL IL15
- Tcm cells were harvested after 3 days, washed and suspended in PBS for injection. Mice were treated with 200 ⁇ L PBS in 10 6 Tcm cells were injected intravenously (IV), after 24 hours, with PBS 200 ⁇ L (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.
- CPX cyclophosphamide
- 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).
- 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 ⁇ g/mL) for 5 hours at 37 ° C and antibiotic A (Golgi Plug, 1 ⁇ g/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).
- 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 ⁇ m sections.
- the sections were either stained with hematoxylin and eosin or by immunohistochemistry (IHC) to detect insulin expression.
- IHC immunohistochemistry
- 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.
- 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.
- 10 ng/mL of IL-15, 10 ng/mL of IL-21, and 20 ng/mL of rapamycin were added to the culture.
- in vitro differentiated CD8+ transgenic T cells were intravenously injected (iv) into tumor-bearing mice at a dose of 10 6 cells/200 ⁇ L 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.
- Monoclonal antibodies recognizing the following targets were used for flow cytometry: CD16/CD32 (Fc Block), CD4 (GK1.5), CD8 (53-6.7), IFN- ⁇ (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.
- cells were stimulated with 1 ⁇ g/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).
- Anti-mouse CD4, CD8 (produced by GK1.5 and 2.43 hybridomas, respectively, injected intraperitoneally (ip) at a dose of 250 ⁇ g/200 ⁇ L 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.
- 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 CD8 ⁇ 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
- 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.
- the plasmid was diluted using opti-MEM medium. Among them, the total amount of the plasmid was 5 ⁇ g, and 7.5 ⁇ l of PLUS Reagent was further added. 10 ⁇ l of Lipofectamine LTX was diluted using medium.
- the transfection reagent was aspirated and fresh complete medium was added.
- the cell supernatant was harvested and filtered using a 0.22 ⁇ m 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.
- 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.
- VSV-GFP-WT VSV-GFP-WT
- RV-GFP-M51R RV-GFP-M51R-V221F-G226R
- RV-3Mut RV-GFP-G21E-M51A-L111F -V221F
- RV-4Mut 200 PFU each
- TCID50 virus titer
- RV-WT 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.
- Vero cell suspension 100 ⁇ l 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.
- 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.
- Fig. 2A-2B 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.
- 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
- RV-WT 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 ⁇ l per well, and cultured at 37 ° C, 5% CO 2 for 40 h.
- MOI multiplicity of infection
- 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.
- RV-GFP-WT virus
- RV-GFP-M51R virus
- RV-GFP-M51R-V221F-G226R RV-3Mut
- RV-GFP-G21E-M51A-L111F-V221F RV-4Mut.
- the proportion of GFP-positive cells continued to increase in the Vero cells (interferon deficiency).
- 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.
- 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.
- 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
- 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
- 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- ⁇ and VSV-G relative to the housekeeping gene GAPDH were calculated by the ⁇ Ct method.
- the four viruses infect the MEF and the tumor cell LLC, respectively.
- 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.
- the viral self-replication ability of VSV-G mRNA level compared with the control group (Fig.
- 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).
- Example 7 Verification of differences in neurotoxicity between RV-4Mut and other oncolytic virus attenuated strains
- 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.
- VSV-GFP-WT ie RV-GFP
- RV-GFP-M51R RV-GFP-M51R
- RV-GFP-M51R-V221F-G226R RV-3Mut
- RV-GFP-G21E-M51A- L111F-V221F RV-4Mut
- mice 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.
- Example 8 Establishment of a mouse lung cancer model (LLC tumor cells) to verify the efficacy of the attenuated strain of RV-4Mut
- mice 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.
- 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.
- Example 9 Establishment of a subcutaneous xenograft model of different tumor cells
- MC38 murine colon cancer
- B16 mouse lung cancer model
- LLC mouse lung cancer model
- 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.
- 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)
- 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.
- 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.
- 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.
- 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
- RVV oncolytic virus vaccine
- 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.
- 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
- 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)
- RVV-mERK specific virus vaccine
- 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.
- mice 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.
- 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.
- effector DUC18T cells Tcm
- RVV-ErkM effector DUC18T cells
- Tcm + RVV-ErkM treatment 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.
- 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.
- RVV- Gp33 specific oncolytic virus vaccine
- 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.
- 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.
- 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).
- 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.
- Fig. 15B 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
- Tcm or Tcm+RVV ErkM
- 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 ⁇ m and 200 ⁇ m, 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 ⁇ m and 200 ⁇ m, 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
- NRG mice were subcutaneously inoculated with CMS5 cells, and 7 days later, DUC18 Tcm (10 6 cells/cell) was adoptively transferred.
- DUC18 Tcm (10 6 cells/cell) was adoptively transferred.
- 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 ⁇ Thy1.2 experimental group
- A Monitoring tumor volume and
- mice receiving combination therapy 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.
- 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.
- rapamycin, IL15 and IL21 are 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).
- 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.
- 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 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.
- IL21 IL15/Rapa
- CD62L B in Figure 19
- CD44 CD44
- ex vivo proliferation comparable to cells in fully combined culture.
- VSV-gp33 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.
- pretreatments ie, whole body irradiation or lymphodepleting chemotherapy before cell therapy
- exogenous IL-2 usually used in two other antigen-specific T cell environments.
- 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.
- 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.
- 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]. ].
- 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.
- 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].
- 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.
- 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.
- 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].
- 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.
- 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.
- 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.
- therapeutic T cells dominate early expansion and tumor infiltration and are responsible for mediating initial tumor regression.
- therapeutic T cells undergo tumor-induced apoptosis, a phenomenon documented in previous studies [80, 81].
- 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].
- 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].
- 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].
- 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.
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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
本公开涉及用于治疗癌症的免疫治疗方法,包括溶瘤病毒治疗,溶瘤病毒疫苗以及溶瘤病毒疫苗与T细胞组合疗法。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.
癌症和常规的癌症治疗剂目前在情绪/身体痛苦、失去生命和增加医疗保健成本方面带来了显著的社会经济负担。常规的疗法显示出一些有益的临床效果但是伴随着产生降低患者的生活质量的毒副作用[1]。临床治疗中需要有更有效的且更低的毒副作用的癌症疗法。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.
充分理解的是,恶性肿瘤(neoplastic malignancy)可以被免疫系统有效地消除,并且相对于其它治疗方式,免疫疗法是癌症治疗最具前景的替代方式[2]。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].
例如通常认为,具有针对肿瘤相关抗原具有特异性识别能力的的T细胞等免疫细胞具有破坏肿瘤的能力,作为自然肿瘤细胞更新的结果或者通过给予治疗性疫苗,可以在癌症患者中诱导出这样的细胞。不幸的是,一旦浸润至肿瘤组织中,这些细胞通常被证明是不能发挥功能的,并且它们经常显示出功能障碍的表型并且由于肿瘤诱导的局部免疫抑制而导致缺乏对于减少或破坏肿瘤必要的对抗肿瘤细胞的能力[3-5]。因此,免疫抑制型肿瘤微环境仍然是肿瘤治疗的显著障碍,其会减弱常规的癌症免疫治疗诱导产生的免疫应答。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.
基于免疫检查点抑制剂的疗法已经开发用于减轻对肿瘤浸润性淋巴细胞(TIL)的抑制性信号传导并且可以激活内源性抗肿瘤应答[6,7]。检查点抑制剂已经在临床试验中显示出疗效,但是未在所有经治疗的受试者中观察到应答[8]。免疫检查点抑制剂疗法依赖于预先存在的TAA特异性T细胞的活化,但是在一些患者中,由于没有足够的内源性肿瘤抗原特异性T细胞,以致于在治疗诱导再活化后不能引起肿瘤的完全消退(tumor regression)[9,10]。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].
目前小分子药物、单克隆抗体等被开发应用于肿瘤的新型治疗,但治愈率不高,有待更多的研究。另外,仅用单一药物治疗可能会导致肿瘤细胞出现耐药性,因此急需开发有效的生物治疗方法。溶瘤病毒是一种通过遗传学改变而具有复制能力的病毒,经过高度稀释的减毒病毒能利用肿瘤(靶)细胞中抑癌基因的失活或缺陷,选择性地在靶细胞内复制,最终导致肿瘤细胞的溶解和死亡,而在正常细胞内它只是少量存在或不能增殖。利用这种病毒进行的肿瘤治疗称为溶瘤病毒治疗。溶瘤病毒不但自身在肿瘤细胞内复制,导致细胞溶解和死亡;而且通过死亡的细胞释放出病毒颗粒,产生一种级联效应,放大溶细胞效果,直至肿瘤细胞被清除。同时,肿瘤细胞的破裂会导致肿瘤抗原从肿瘤细胞中释放,从而诱导体内系统性的抗肿瘤免疫反应,这可能会增强病毒的溶细胞活性。溶瘤病毒进入肿瘤细胞后由于自我复制可陆续破坏宿主细胞,进而向周围扩散,进入其他肿瘤细胞。如此反复循环,可 发挥有效的抗肿瘤效果。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.
已有大量报道显示,在体外实验中,很多病毒会在多种肿瘤细胞中复制并杀死肿瘤细胞,例如仙台病毒(Kinoh等,2004);柯萨奇病毒(Coxackie Virus)(Shafren等,2004);单纯性疱疫病毒(Mineta等,1995);细小病毒(Abschuetz等,2006);腺病毒(Heise等,2000);脊髓灰质炎病毒(Gromeier等,2000);新城疫病毒;麻疹病毒(Grote等,2001);呼肠病毒(Coffey等,1998);逆转录病毒(Logg等,2001);痘苗病毒(Timiryasova等,1999)以及流感病毒(Bergmann等,2001))。此外,已证明,这些病毒对治疗肿瘤动物模型有效。但是,大多数活病毒的安全性是一个重要的关注点,因此仍有必要开发更加安全可靠的方法来治疗癌症。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.
随着RNA病毒遗传技术的进展,水疱性口炎病毒属载体已被开发成为一种有效的治疗制剂。VSV(水疱性口炎病毒)病毒载体是一种高效的溶瘤棒状病毒载体,具有非常广的溶瘤范围。根据资料报道,VSV载体几乎能够侵染溶解所有的肿瘤细胞,在体外实验中VSV载体的溶瘤率都在50%以上,在体内实验中VSV载体能够显著延长荷瘤动物模型的寿命。VSV载体也被开发成为一种有效的疫苗载体,VSV病毒载体作为疫苗载体应用到获得性免疫缺陷综合症病毒、流感病毒、丙型肝炎病毒和乙肝病毒等疫苗的研制过程中。因此水疱性口炎病毒载体具有非常好的应用前景。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.
在肿瘤基因治疗领域,常常利用病毒作为治疗基因的载体。出于安全考虑,通常会控制病毒在正常细胞体内复制,因此要达到100%的侵染效率在技术上很困难。于是,运用可自我复制的病毒(增殖型病毒)来治疗肿瘤(溶瘤棒状病毒治疗)备受瞩目和期待。溶瘤棒状病毒治疗是指在被感染的肿瘤细胞内,利用病毒自我复制达到破坏肿瘤宿主细胞,并利用病毒原有的直接杀伤细胞的功效达到治疗目的。另外,溶瘤棒状病毒治疗与基因治疗不同,主要依赖其在肿瘤细胞内复制而产生杀伤肿瘤细胞的效应。溶瘤病毒治疗的概念早已存在,100年前就有人尝试用野生型或自然弱毒株来治疗肿瘤,随着基因工程学技术的发展,病毒用于治疗的研究飞跃发展,现在已开发到第2代基因重组病毒。现有技术已知的基于VSV的基因重组病毒,要么存在一定的对于正常体细胞的毒性,导致存在安全性风险;要么溶瘤效果不佳,导致治疗实体瘤的效果不好。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.
同时,最新的研究证明TAA特异性T细胞的中心记忆性T细胞(Tcm)代表了对用于治疗恶性肿瘤的检查点抑制剂疗法的显著优异性。使用从癌症患者分离并且离体生长的肿瘤反应性T细胞来进行回输治疗。一旦将肿瘤组织局部微环境的免疫抑制力除去,使这些免疫细胞在回输至患者之前活化并且增殖至较高的数量。本质上,离体培养的条件下可以使少量的具有很少杀伤肿瘤细胞活性或者是没有杀伤活性的功能障碍T细胞转变为数以亿计具备返回肿瘤局部微环境并且破坏肿瘤细胞的全功能性T细胞。用于制备Tcm的淋巴细胞源自包括外周血单核细胞(PBMC)以及肿瘤局部浸润的T淋巴细胞(TIL)。Tcm的制备是灵活多样的,这是因为不仅可以通过TAA天然受体识别抗原通路的激活、也可以通过编码TAA特异性T细胞受体(TCR)或嵌合抗原受体(CAR)的转基因转导的方式来体外修饰淋巴细胞的途径获得Tcm[12,13]。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].
同时临床证据已经显示出Tcm在例如B细胞淋巴瘤等非实体癌症中的良好的抗肿瘤疗效。事实上,特异性识别人乳头瘤病毒的效应性T细胞的输注治疗也可以诱导HPV阳性的疗法性宫颈癌的消退[14]。目前将该策略扩展至靶向非病毒性肿瘤相关抗原,但是仍然需要进一步改进从而增强肿瘤局部淋巴细胞的浸润以及治愈实体瘤的目的。前期涉及到Tcm的研究工作已经表明,Tcm的治疗潜力需要足够的免疫细胞长期持久性浸润肿瘤组织从而才能够杀灭所有恶性肿瘤细胞。临床证据表明,可以通过具有微分化表型并维持复制能力的高剂量过继疗法细胞(数十亿)来实现这些需要[15]。然而,鉴于此目的与制备产生严重的冲突,生产制备高剂量的T细胞需要大量的体外扩增,而导致T细胞因过度分化而丧失复制能力[16]。实际上,常规的Tcm疗法使用已经分化的效应T细胞治疗前,需要对患者进行预先的放射治疗或化疗从而将自体的清除淋巴细胞群清除,在此基础上才能允许体外扩增的的数十亿免疫细胞的更好的输入[17,18]。此外,还需要IL2治疗来维持输注的细胞的持久性和活性[19]。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].
最近的研究已经表明,如Tcm,是一种低分化的免疫细胞,在细胞的过继性输注中展现了较好的持久性,在临床效果方面显示较好的优异性[15,20]。目前已经公开了用于从PBMC样品生产TAA特异性T细胞的Tcm培养的方法[21],但是该方案需要临床级细胞分选仪,对于富集抗原特异性细胞,所述的制备方案需要大量的人力物力(参见U.S.专利申请公开No.US2015/0023938)。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).
发明内容Summary of the invention
发明要解决的问题Problems to be solved by the invention
基于现有技术中存在的问题,需要提供一种能够有效降低病毒在正常体细胞中的毒性的溶瘤棒状病毒减毒株。优选的,需要提供一种能够保证相对于正常细胞,对于异常增生性细胞具有高选择性,并且具有良好溶瘤效果的溶瘤棒状病毒减毒株。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.
因此,仍需要开发一种既具有良好的安全性,又具有良好的溶瘤效果的基于弹状病毒的重组溶瘤棒状病毒。Therefore, there is still a need to develop a rhabdovirus-based recombinant oncolytic baculovirus which has both good safety and good oncolytic effect.
与此同时,同样需要提供一种基于前述溶瘤棒状病毒减毒株,用于治疗或者是治愈肿瘤或癌症的方法。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.
用于解决问题的方案Solution to solve the problem
基于现有肿瘤免疫治疗技术中存在的问题,本公开提供了一种治疗癌症的免疫治疗方法,包括溶瘤病毒单独给药,溶瘤病毒疫苗以及利用溶瘤病毒疫苗和中心记忆性T细胞(Tcm)联合疗法治疗实体瘤,该方法可以彻底治愈实体瘤,并且产生永久保护效果。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.
在一个实施方案中,本公开提供用于产生溶瘤病毒疫苗以及抗原特异性中心记忆性T细胞的规模化扩增的技术和方法,用于生产从体内获取得到溶瘤病毒疫苗以及肿瘤抗原特异性中心记忆性T细胞,可以引起特异性抗肿瘤细胞的细胞T细胞免疫应答反应。结合起来,这种免疫疗法不需要使用细胞分选仪或着预先清除受试者的内源性T细胞,明显优于已有的细胞免疫疗法。进一步现有的细胞免 疫疗法得到显著的改进。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.
在一个实施方案中,本公开提供了在哺乳动物(例如人类)中治疗癌症的方法,所述方法包括:溶瘤病毒单独给药,溶瘤病毒疫苗以及溶瘤病毒疫苗与过继性T细胞联合疗法三种方式。在数个实施方案中,提供了用于在特定适应症的受试者中治疗癌症的联合疗法,所述联合疗法包括:(i)肿瘤抗原特异性中心记忆T细胞(Tcm)向受试者的过继细胞输注输注,接着是(ii)受试者接种重组溶瘤病毒(OV)疫苗,该溶瘤病毒疫苗表达与中输注的T细胞刺激抗原一致,从而诱导自体免疫细胞对肿瘤破坏和消除。在优选的实施方案中,对Tcm中的T细胞进行遗传修饰从而表达特异识别肿瘤抗原的一种或多种重组T细胞受体(TCR)或嵌合抗原受体(CAR)。在另一些实施方案中,Tcm疗法中的T细胞为源自要治疗的受试者的自体T细胞。优选地,联合疗法无需对受试者进行自体免疫细胞清除的步骤。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.
在一个实施方案中,本公开涉及一种重组溶瘤棒状病毒的改性基质蛋白(M),其特征在于,编码所述改性基质蛋白(M)的氨基酸序列与SEQ ID NO:1所示的氨基酸序列相比,具有至少80%,优选至少90%,更优选至少95%,最优选至少98%相同的序列;并且,所述氨基酸序列和SEQ ID NO:1相比,在第51位置、第221位置、第226位置同时具有氨基酸替换。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.
在另一个实施方案中,编码所述改性基质蛋白(M)的氨基酸序列与SEQ ID NO:1所示的氨基酸序列相比,在第21位置、第51位置、第111位置和第221位置同时具有氨基酸替换。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.
在一个实施方案中,本公开涉及一种改性基质蛋白(M),其中,所述重组溶瘤棒状病毒选自水疱性口炎病毒;优选的,所述重组溶瘤棒状病毒选自水疱性口炎病毒MuddSummer株。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.
在一个实施方案中,本公开涉及一种改性基质蛋白(M),其中,所述的改性的基质蛋白(M)的序列和SEQ ID NO:1相比,编码改性基质蛋白(M)的氨基酸序列同时存在如下突变:(i)第21位甘氨酸G突变为谷氨酸E,(ii)第51甲硫氨酸M突变为丙氨酸A,(iii)第111位亮氨酸L突变为苯丙氨酸F,(iv)第221位缬氨酸V突变为苯丙氨酸F。优选的,所述改性基质蛋白(M)的序列为如SEQ ID NO:3所示的序列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:
在另一个实施方案中,本公开涉及一种改性基质蛋白(M),其中,所述的改性的基质蛋白(M)的序列和SEQ ID NO:1相比,编码改性基质蛋白(M)的氨基酸序列同时存在如下突变:(i)第51甲硫氨酸M突变为精氨酸R,(ii)第221位缬氨酸V突变为苯丙氨酸F,(iii)第226位甘氨酸G突变为精氨酸R。优选的,所述改性基质蛋白(M)的序列为如SEQ ID NO:5所示的序列。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.
在一个实施方案中,本公开涉及一种重组溶瘤棒状病毒,其中,所述重组溶瘤棒状病毒包含改性基质蛋白(M),其中,所述改性基质蛋白(M)的氨基酸序列为如上所示的氨基酸序列;优选的,所述重组溶瘤棒状病毒为减毒的溶瘤棒状病毒。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.
在一个实施方案中,本公开涉及一种包含分离的重组溶瘤棒状病毒的组合物,所述重组溶瘤棒状病毒具有核酸片段,所述核酸片段编码改性基质蛋白(M),其特征在于,所述改性基质蛋白(M)的氨基酸序列为如上所示的氨基酸序列;优选的,所述重组溶瘤棒状病毒为减毒的重组溶瘤棒状病毒。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.
在另一个实施方案中,本公开涉及的组合物进一步包含第二溶瘤病毒;优选的,所述第二溶瘤 病毒选自包含弹状病毒、牛痘病毒、疱疹病毒、麻疹病毒、新城疫病毒、腺病毒、甲病毒、细小病毒、肠道病毒株的一种或多种;更优选的,所述第二溶瘤病毒为减毒的溶瘤病毒;最优选的,其中所述第二溶瘤病毒为减毒的弹状病毒。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.
在另一个实施方案中,本公开涉及的组合物进一步包括第二抗肿瘤制剂;优选的,所述第二抗肿瘤制剂是免疫治疗剂、化学治疗剂或放射治疗剂;更优选的,所述第二抗肿瘤制剂选自小分子,大分子,细胞,病毒载体,基因载体,DNA,RNA,多肽,和纳米复合物中的一种或多种。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.
在一个实施方案中,本公开涉及一种疫苗,所述疫苗包含治疗有效量的一种或多种重组溶瘤棒状病毒,其中,所述一种或多种重组溶瘤棒状病毒包含前述改性基质蛋白(M)。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).
在另一个实施方案中,本公开涉及的疫苗还可以包含第二溶瘤病毒,或第二抗肿瘤制剂。In another embodiment, the vaccine of the present disclosure may further comprise a second oncolytic virus, or a second anti-tumor preparation.
在一个实施方案中,本公开涉及一种分离的肽,其由氨基酸序列编码而成,所述氨基酸序列选自包含与SEQ ID NO:1的氨基酸序列至少80%,优选至少90%,更优选至少95%,最优选至少98%相同的序列,并且,所述氨基酸序列和SEQ ID NO:1相比,在第51位置、第221位置、第226位置同时具有氨基酸替换。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.
在另一个实施方案中,所述氨基酸序列和SEQ ID NO:1相比,在第21位置、第51位置、第111位置和第221位置同时具有氨基酸替换。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.
在一个实施方案中,本公开涉及的编码所述分离的肽的氨基酸序列同时存在如下突变:(i)第21位甘氨酸G突变为谷氨酸E,(ii)第51甲硫氨酸M突变为丙氨酸A,(iii)第111位亮氨酸L突变为苯丙氨酸F,(iv)第221位缬氨酸V突变为苯丙氨酸F。优选的,所述氨基酸序列为如SEQ ID NO:3所示的序列。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.
在另一个实施方案中,本公开涉及的编码所述分离的肽的氨基酸序列同时存在如下突变:(i)第51甲硫氨酸M突变为精氨酸R,(ii)第221位缬氨酸V突变为苯丙氨酸F,(iii)第226位甘氨酸G突变为精氨酸R。优选的,所述改性基质蛋白(M)的序列为如SEQ ID NO:5所示的序列。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.
在一个实施方案中,本公开涉及一种用于编码所述的分离的肽的分离的核苷酸序列。In one embodiment, the disclosure relates to an isolated nucleotide sequence for encoding the isolated peptide.
在一个实施方案中,本公开涉及包含分离的重组溶瘤棒状病毒的组合物或疫苗在制备用于杀死异常增生性细胞、诱导促进抗肿瘤免疫反应或消除肿瘤组织微环境免疫抑制的药物中的应用。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.
在另一个实施方案中,本公开涉及的上述应用中,所述异常增生性细胞被包含在患者体内。In another embodiment, in the above application of the present disclosure, the aberrant proliferative cells are included in a patient.
在另一个实施方案中,本公开涉及的上述应用中,所述异常增生性细胞选自肿瘤细胞或肿瘤组织相关细胞;优选的,所述肿瘤细胞是癌细胞;更优选的,所述癌细胞是转移性癌细胞。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.
在一个实施方案中,本公开涉及包含分离的重组溶瘤棒状病毒的组合物或疫苗在制备治疗患有肿瘤的患者的药物中的应用。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.
在一个实施方案中,本公开涉及—种缓慢持续杀伤异常增生性细胞的方法,包括将所述异常增生性细胞与重组溶瘤棒状病毒、包含分离的重组溶瘤棒状病毒的组合物或疫苗接触的步骤。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.
在另一个实施方案中,本公开涉及的缓慢持续杀伤异常增生性细胞的方法,所述异常增生性细 胞被包含在患者体内。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.
在另一个实施方案中,本公开涉及的缓慢持续杀伤异常增生性细胞的方法,所述异常增生性细胞选自肿瘤细胞或肿瘤组织相关细胞;优选的,所述肿瘤细胞是癌细胞;更优选的,所述癌细胞是转移性癌细胞。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.
在另一个实施方案中,本公开涉及的缓慢持续杀伤异常增生性细胞的方法,所述重组溶瘤棒状病毒、包含分离的重组溶瘤棒状病毒的组合物或疫苗被施用至患者体内。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.
在另一个实施方案中,本公开涉及的缓慢持续杀伤异常增生性细胞的方法,所述重组溶瘤棒状病毒、包含分离的重组溶瘤棒状病毒的组合物或疫苗通过包括腹膜内、静脉内、动脉内、肌肉内、皮肤内、瘤内、皮下或鼻内给药中的一种或多种的给药方式而被施用;优选的,所述给药方式的给药途径包括内镜、腔镜、介入、微创、传统手术中的一种或多种。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.
在另一个实施方案中,本公开涉及的缓慢持续杀伤异常增生性细胞的方法,所述方法进ー步包括施用第二抗肿瘤疗法的步骤。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.
在另一个实施方案中,本公开涉及的缓慢持续杀伤异常增生性细胞的方法,其中所述第二抗肿瘤疗法选自施用第二溶瘤病毒。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.
在另一个实施方案中,本公开涉及的缓慢持续杀伤异常增生性细胞的方法,其中所述第二溶瘤病毒选自包含弹状病毒、牛痘病毒、疱疹病毒、麻疹病毒、新城疫病毒、腺病毒、甲病毒、细小病毒、肠道病毒株的一种或多种;更优选的,所述第二溶瘤病毒为减毒的溶瘤病毒;最优选的,其中所述第二溶瘤病毒为减毒的弹状病毒。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.
在另一个实施方案中,本公开涉及的缓慢持续杀伤异常增生性细胞的方法,其中所述第二抗肿瘤疗法选自化学疗法、放射疗法、免疫疗法、手术疗法中的一种或多种。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.
在一个实施方案中,本公开涉及—种在受试者中诱导免疫应答的方法,所述方法包含对受试者施用重组溶瘤棒状病毒、包含分离的重组溶瘤棒状病毒的组合物或疫苗中的一种或多种。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.
在一个实施方案中,本公开涉及—种诱导促进抗肿瘤免疫反应或消除肿瘤组织微环境免疫抑制的方法,包括将肿瘤或肿瘤组织与重组溶瘤棒状病毒、包含分离的重组溶瘤棒状病毒的组合物或疫苗接触的步骤。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.
在一个实施方案中,提供了用于产生肿瘤抗原特异性中心记忆CD8+T细胞的方法,所述方法包括:离体免疫细胞培养的步骤,所述步骤包括在存在肿瘤抗原、树突状等抗原递呈细胞、IL21、IL15和雷帕霉素的情况下并且优选在不存在IL2的情况下从PBMC或TIL中培养淋巴细胞。优选地,在培养之前从PBMC除去CD25+细胞(包括调节T细胞以及活化的T和B细胞)。肿瘤抗原即肿瘤相关抗原(TAA),其为在哺乳动物中引起免疫应答的在肿瘤细胞中产生的物质。在一些实施方案中,肿瘤抗原为自体抗原。在其它实施方案中,肿瘤抗原为肿瘤特异性抗原,其对于肿瘤是独特的并且在正常细胞中不表达或者在正常细胞中以非常低的量表达(例如新抗原)。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).
在相关的实施方案中,与正常细胞内容物相比肿瘤抗原在癌症细胞中具有更高的组织特异性表达,其非限制性实例包括酪氨酸酶、MART-1、gp100、TRP-1/gp75和TRP-2等蛋白。在其它实施方案中,肿瘤抗原为肿瘤特异性的且共同的抗原,其在癌症和睾丸中表达但是在其它正常组织中不表达或者以非常小的量表达(睾丸癌抗原或CT抗原),其非限制性实例包括BAGE、CAMEL、MAGE-A1和NY-ESO-1。在其它实施方案中,肿瘤抗原为肿瘤特异性的且单一抗原(新抗原),其仅在肿瘤细胞中表达,其非限制性实例包括CDK4、连环蛋白、半胱氨酸蛋白酶-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和KIAA1440。在其它实施方案中,与正常细胞相比肿瘤抗原在癌症细胞中过表达。肿瘤相关抗原的实例包括:癌胚抗原(oncofetal antigen),如甲胎蛋白(AFP)和癌胚抗原(carcinoembryonic antigen,CEA);表面糖蛋白,如CA 125;癌基因,如Her2;黑素瘤相关抗原,如多巴色素互变异构酶(DCT)、GP100和MART1;癌-睾丸抗原,如MAGE蛋白和NY-ESO1;病毒癌基因,如HPV E6和E7;通常限于胚胎或胚外组织的在肿瘤中异位表达的蛋白,如PLAC1。如本领域技术人员一般都会理解,可以使用本方法基于要治疗的癌症的种类来选择抗原,这是因为一种或多种抗原可能特别适用于某些癌症的治疗。例如,对于黑素瘤的治疗,可以使用例如DCT等黑素瘤相关抗原。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.
在一些优选的实施方案中,为了产生肿瘤抗原(例如TAA)特异性CD8
+T细胞,本方法包括如下步骤,在所述步骤中,对离体培养的细胞(例如,从受试者获得的自体PBMC或具有与受试者组织相容的表型的PBMC)进行遗传修饰从而表达一种或多种重组TCR或CAR以赋予肿瘤抗原特异性。在存在肿瘤抗原、例如树突细胞等抗原递呈细胞、IL21、IL15和雷帕霉素的情况下,离体培养转导的细胞。例如经典的CAR是由2段组成,对应的胞外段是由识别肿瘤抗原的单链抗体可变区域(scFV)构成,,胞内段是对应的融合蛋白是激活T细胞结构域。重组TCR包含α链和β链,例如可以识别HLA-A2/肽复合物。可以通过携带所选择的TCR/CAR表达的转基因表达载体:例如慢病毒或逆转录病毒载体等载体,将TCR/CAR表达基因转导到目的细胞。将外源基因导入至该类载体中的方法对于本领域技术人员是公知的。通常,对载体进行修饰从而表达TCR/CAR。在这一点上,使用已经确立的重组技术将编码所选择的TCR/CAR的核酸序列克隆至所选择的载体中。在一些实施方案中,TCR或CAR对于MART-1、gp100、NY-ESO-1、或MAGE家族的成员(例如MAGE-A3)是特异性的。优异性,进一步通过本文中所述的方法产生的肿瘤抗原(TAA)在扩增特异性CD8
+T细胞(例如使用抗CD3抗体和抗CD28抗体以及任选IL2的快速扩增)之前不需要使用四聚体标记和临床级分选仪进一步富集。因此,在优选的实施方案中,提供了用于过继细胞输注的方法,所述方法包括:(i)在存在荷载有肿瘤抗原的抗原递程细胞(APC,例如自体树突细胞)、IL21、IL15和雷帕霉素的情况下并且优选在不存在IL2的情况下培养从患有癌症的受试者获得的PBMC或TIL,从而产生富含肿瘤抗原特异性CD8
+T细胞的细胞群,(ii)使用抗CD3抗体和抗CD28抗体以及任选IL2扩增肿瘤抗原特异性CD8
+T细胞,和(iii)将细胞重 新导入至受试者,其中所述方法不包括在步骤(i)与(ii)之间进行富集T细胞如分选CD8
+细胞的步骤。
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).
根据本文中所述的联合疗法,在肿瘤抗原特异性CD8
+T细胞的过继疗法之后将表达肿瘤抗原的溶瘤病毒给予至受试者。肿瘤抗原特异性CD8
+T细胞的过继疗法可以通过任意合适的方法来完成。
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.
在一些实施方案中,联合疗法包括:(i)在存在荷载有肿瘤抗原肽的抗原递呈细胞(APC)和IL21的情况下离体培养来自患有肿瘤的受试者的TIL。并且将扩增培养物中的细胞重新导入至受试者中,和(ii)将表达相同的肿瘤抗原的溶瘤病毒给予至受试者。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.
在其它实施方案中,联合疗法包括:(i)在存在荷载有肿瘤抗原肽的APC、IL21、IL15和雷帕霉素的情况下离体培养来自患有肿瘤的受试者的TILS、扩增培养物中的细胞、并且将它们重新导入至受试者中,和(ii)将表达相同的肿瘤抗原的溶瘤病毒给予至受试者。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.
在其它实施方案中,联合疗法包括:(i)在存在荷载有肿瘤抗原肽的APC和IL21的情况下离体培养来自受试者的PBMC、扩增培养物中的细胞、并且将它们重新导入至受试者中,和(ii)将表达相同的肿瘤抗原的溶瘤病毒给予至受试者。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.
在其它实施方案中,联合疗法包括:(i)在存在荷载有肿瘤抗原肽的APC、IL21、IL15和雷帕霉素的情况下离体培养来自受试者的PBMC、扩增培养物中的细胞、并且将它们重新导入至受试者中,和(ii)将表达相同的肿瘤抗原的溶瘤病毒给予至受试者。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.
在相关的实施方案中,在离体培养之前,用能够特异性识别肿瘤抗原的重组TCR或CAR转导PBMC。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.
在一些实施方案中,在疗法肿瘤抗原特异性CD8
+T细胞之后约8至72小时,将表达肿瘤抗原的溶瘤病毒给予至哺乳动物。在优选的实施方案中,在疗法肿瘤抗原特异性CD8
+T细胞之后约12至48小时、约20至28小时、或约24小时,将表达肿瘤抗原的溶瘤病毒给予至受试者。
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.
根据本文中所述的联合疗法中给予的表达肿瘤抗原是具有复制能力的任意溶瘤病毒。在一些优选的实施方案中,复制型溶瘤病毒为例如VSV或Maraba弹状病毒等弹状病毒,其优选包含一种或多种遗传修饰从而增加病毒对癌症细胞的选择性。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.
可以通过多个途径中的一种或多种来给予复制型溶瘤病毒疫苗。在一些实施方案中,复制型溶瘤病毒为弹状病毒并且通过静脉内途径给予至哺乳动物。在其它实施方案中,复制型溶瘤病毒为弹状病毒RV,并且静脉内(IV)、肌内(IM)、腹腔内(IP)或瘤内(IT)给予至哺乳动物。如本领域技术人员将理解的,复制型溶瘤病毒(例如弹状病毒或痘苗病毒)将以例如生理盐水或其它药学上合适的缓冲液做为合适的载体给药。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.
在一个实施方案中,本公开提供了一种抗肿瘤用制品,所述抗肿瘤用制品包括(i)过继性免疫细胞和(ii)复制型溶瘤病毒疫苗(OVV),其中所述复制型溶瘤病毒为溶瘤棒状病毒或溶瘤痘苗病毒,可选的,所述复制型溶瘤病毒表达肿瘤抗原。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.
在一些实施方案中,所述的过继性免疫细胞为中心记忆性T细胞(Tcm),优选的,所述中心记 忆性T细胞(Tcm)为产生肿瘤抗原特异性的CD8
+T细胞群,更优选的,所述CD8
+T细胞中的至少50%、60%或70%显示中心记忆表型。其中,所述CD8
+T细胞群是通过对CD25清除的所述PBMC进行离体培养得到的。其中所述肿瘤抗原为选自由甲胎蛋白(AFP)、癌胚抗原(carcinoembryonic antigen,CEA)、CA 125、Her2、多巴色素互变异构酶(DCT)、GP100、MART1、MAGE蛋白、NY-ESO1、HPV E6和HPV E7组成的组中的任一项或其组合。其中所述肿瘤为黑素瘤、肉瘤、淋巴瘤、脑癌(例如胶质瘤)、乳腺癌、肝癌、肺癌、肾癌、胰腺癌、食管癌、胃癌、结肠癌(colon cancer)、结直肠癌(colorectal cancer)、膀胱癌、前列腺癌和白血病。在另外一些实施方案中,所述肿瘤为实体瘤。
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.
在一些实施方案中,所述CD8
+T细胞群的生产方法为:在存在加载有肿瘤抗原的抗原呈递程细胞(APC)、IL21、IL15和雷帕霉素且优选在不存在IL2的情况下,从患有癌症的受试者获得外周血单核细胞(PBMC)、或肿瘤浸润性淋巴细胞(TIL)、或骨髓内淋巴细胞,并对所述外周血单核细胞(PBMC)、或肿瘤浸润性淋巴细胞(TIL)或骨髓内淋巴细胞进行离体培养。在另一些实施方案中,所述CD8
+T细胞群的生产方法为:从患有癌症的受试者获得PBMC,通过对PBMC转导重组T细胞受体(TCR)或嵌合抗原受体(CAR)对其进行遗传修饰,并离体培养经转导的PBMC。在对PBMC或TIL离体培养得到CD8+T细胞后,还进一步包括使用抗CD3抗体和抗CD28抗体以及任选IL2体外扩增所述CD8
+T细胞。
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.
在一些实施方案中,在所述抗肿瘤用制品中,复制型溶瘤病毒为棒状病毒,优选为水疱性口炎病毒(VSV)。在另一些实施方案中,所述棒状病毒为重组或野生型Maraba病毒,优选为Maraba MG1病毒。在其他一些实施方案中,所述复制型溶瘤病毒为野生型或重组型痘苗病毒;其中所述痘苗病毒是Copenhagen、Western Reserve、Lister或Wyeth株;可选的,所述痘苗病毒缺失胸苷激酶基因或者缺失痘苗病毒生长因子基因。所述痘苗病毒通过瘤内、腹膜内、静脉内、动脉内或肌内给予受试者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
在一些实施方案中,所述溶瘤病毒疫苗通过血管内给予受试者,优选的通过静脉给予受试者。In some embodiments, the oncolytic virus vaccine is administered to a subject by intravascular administration, preferably by intravenous administration.
本公开还提供了一种组合物在制备治疗肿瘤的药物中的用途,所述组合物包括(i)过继性免疫细胞和(ii)复制型溶瘤病毒疫苗。其中所述过继性免疫细胞和复制型溶瘤病毒疫苗和本公开上文所述抗肿瘤用制品中对应的成分相同。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.
本公开提供了在哺乳动物(例如人类)中治疗癌症的方法,所述方法包括:过继细胞疗法和随后的溶瘤病毒疫苗的给予。在数个实施方案中,提供了用于在有需要的受试者中治疗癌症的联合疗法,所述联合疗法包括:(i)肿瘤抗原特异性中心记忆性T细胞(Tcm)向受试者的过继细胞疗法,接着是(ii)使受试者接种表达由过继细胞疗法T细胞靶向的相同的抗原的重组溶瘤病毒(OV)疫苗,从而诱导癌症破坏和消除。在优选的实施方案中,对中心记忆性T细胞(Tcm)进行遗传修饰从而表达特异于肿瘤抗原的一种或多种重组T细胞受体(TCR)或嵌合抗原受体(CAR)。在一些实施方案中,中心记忆性T细胞(Tcm)为源自要治疗的受试者的自体T细胞。优选地,联合疗法不包括其中使受试者免疫耗竭的步骤。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.
在一个实施方案中,提供了用于产生肿瘤抗原特异性中心记忆CD8
+T细胞的方法,所述方法包括:离体细胞培养的步骤,所述步骤包括在存在肿瘤抗原、例如树突细胞等抗原呈递细胞、IL21、IL15和雷帕霉素的情况下并且优选在不存在IL2的情况下从PBMC或TIL培养淋巴细胞。优选地,在培养之前从PBMC除去CD25
+细胞(调节T细胞以及活化的T细胞和B细胞)。肿瘤抗原可以例如为肿瘤相关抗原(TAA),其为在哺乳动物中引起免疫应答的在肿瘤细胞中产生的物质。在一些实施方案中,肿瘤抗原为自体抗原。在其它实施方案中,肿瘤抗原为肿瘤特异性抗原,其对于肿瘤是独特的并且在正常细胞中不表达或者在正常细胞中以非常低的量表达(例如新抗原)。
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).
在相关的实施方案中,肿瘤抗原为与正常细胞相比在癌症细胞中具有更高的表达的组织特异性肿瘤抗原,其非限制性实例包括酪氨酸酶、MART-1、gp100、TRP-1/gp75和TRP-2蛋白。在其它实施方案中,肿瘤抗原为肿瘤特异性的且共同的抗原,其在癌症和睾丸中表达但是在其它正常组织中不表达或者以非常小的量表达(癌-睾丸抗原或CT抗原),其非限制性实例包括BAGE、CAMEL、MAGE-A1和NY-ESO-1。在其它实施方案中,肿瘤抗原为肿瘤特异性的且独特的抗原(新抗原),其仅在肿瘤细胞中表达,其非限制性实例包括CDK4、连环蛋白、半胱氨酸蛋白酶-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和KIAA1440。在其它实施方案中,肿瘤抗原为与正常细胞相比在癌症细胞中过表达的过表达肿瘤抗原。肿瘤相关抗原的实例包括:癌胚抗原(oncofetal antigen),如甲胎蛋白(AFP)和癌胚抗原(carcinoembryonic antigen,CEA);表面糖蛋白,如CA 125;癌基因,如Her2;黑素瘤相关抗原,如多巴色素互变异构酶(DCT)、GP100和MART1;癌-睾丸抗原,如MAGE蛋白和NY-ESO1;病毒癌基因,如HPV E6和E7;通常限于胚胎或胚外组织的在肿瘤中异位表达的蛋白,如PLAC1。如本领域技术人员将理解的,可以使用本方法基于要治疗的癌症的种类来选择抗原,这是因为一种或多种抗原可能特别适用于某些癌症的治疗。例如,对于黑素瘤的治疗,可以使用例如DCT等黑素瘤相关抗原。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.
在一些优选的实施方案中,为了产生肿瘤抗原(例如TAA)特异性CD8+T细胞,本方法包括如下步骤,在所述步骤中,对离体培养的细胞(例如,从受试者获得的自体PBMC或具有与受试者组织相容的表型的PBMC)进行遗传修饰从而表达一种或多种重组TCR或CAR以赋予肿瘤抗原特异性。在存在肿瘤抗原、例如树突细胞等抗原呈递细胞、IL21、IL15和雷帕霉素的情况下,离体培养转导的细胞。CAR为由具有抗原识别部分的抗体衍生的细胞外单链可变片段(scFv)和细胞内T细胞激活结构域形成的融合蛋白。重组TCR包含α链和β链并且例如可以识别HLA-A2/肽复合物。可以用携带支持所选择的TCR/CAR的表达的转基因盒的例如慢病毒或逆转录病毒载体等载体转导细胞。将转基因盒导入至载体中的方法对于本领域技术人员是公知的。通常,对载体进行修饰从而表达TCR/CAR。在这一点上,使用已经确立的重组技术将编码所选择的TCR/CAR的核酸序列并入至所选择的载体中。在一些实施方案中,TCR或CAR对于MART-1、gp100、NY-ESO-1、或MAGE家族的成员(例如MAGE-A3)是特异 性的。有利地,通过本文中所述的方法产生的肿瘤抗原(例如TAA)特异性CD8+T细胞在扩增(例如使用抗CD3抗体和抗CD28抗体以及任选IL2的快速扩增)之前不需要使用四聚体标记和临床级分选仪的进一步富集。因此,在优选的实施方案中,提供了用于过继细胞疗法的方法,所述方法包括:(i)在存在加载有肿瘤抗原的抗原呈递细胞(APC,例如自体树突细胞)、IL21、IL15和雷帕霉素的情况下并且优选在不存在IL2的情况下培养从患有癌症的受试者获得的PBMC或TIL,从而产生富含肿瘤抗原特异性CD8+T细胞的细胞群,(ii)使用抗CD3抗体和抗CD28抗体以及任选IL2扩增肿瘤抗原特异性CD8
+T细胞,和(iii)将细胞重新导入至受试者,其中所述方法不包括在步骤(i)与(ii)之间进行富集T细胞如分选四聚体+细胞的步骤。
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.
在优选的实施方案中,将来自PBMC或TIL的淋巴细胞在存在肿瘤抗原、例如树突细胞等抗原呈递细胞、IL21、IL15和雷帕霉素的情况下和优选在不存在IL2的情况下离体培养约1周至约4周,例如约1周、约2周、约3周、约4周、或其间的任意范围,接着可以在存在IL21、IL15和雷帕霉素的情况下、在不存在肿瘤抗原和抗原呈递细胞的情况下离体培养约1周至约2周、约1周、或约2周。在特别优选的实施方案中,将来自PBMC的淋巴细胞在存在加载有肿瘤抗原肽的树突细胞、IL21、IL15和雷帕霉素的情况下离体培养约2周,并且在存在IL21、IL15和雷帕霉素的情况下和在不存在加载有肿瘤抗原肽的树突细胞的情况下离体培养约1周。有利地,可以将根据本文中所述的方法产生的肿瘤抗原特异性CD8+T细胞导入至哺乳动物中而不需要淋巴细胞耗竭(lymphodepletion)并且不需要将IL-2给予至受试者。因此,在一些实施方案中,提供了用于过继细胞疗法的方法,所述方法包括:(i)在存在肿瘤抗原、例如树突细胞等抗原呈递细胞、IL21、IL15和雷帕霉素的情况下离体培养来自PBMC或TIL的淋巴细胞,和(ii)将所得肿瘤抗原(例如TAA)特异性CD8+T细胞给予至哺乳动物而不通过化学治疗或放射学手段破坏哺乳动物中的现有的淋巴细胞(淋巴细胞耗竭或淋巴细胞消融(lymphoablation)并且不将IL-2给予至受试者。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.
根据本文中所述的联合疗法,在肿瘤抗原特异性CD8
+T细胞的过继疗法之后将表达肿瘤抗原的溶瘤病毒给予至受试者。肿瘤抗原特异性CD8
+T细胞的过继疗法可以通过任意合适的方法来完成,所述方法包括本文中所述的方法和其内容通过参考引入本文的U.S.专利申请公开No.US2015/0023938中记载的方法。
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.
在一些实施方案中,联合疗法包括:(i)在存在加载有肿瘤抗原肽的抗原呈递细胞(APC)和IL21的情况下离体培养来自患有肿瘤的受试者的TILS、扩增培养物中的细胞、并且将它们重新导入至受试者中,和(ii)将表达相同的肿瘤抗原的溶瘤病毒给予至受试者。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.
在其它实施方案中,联合疗法包括:(i)在存在加载有肿瘤抗原肽的APC、IL21、IL15和雷帕霉素的情况下离体培养来自患有肿瘤的受试者的TILS、扩增培养物中的细胞、并且将它们重新导入至受试者中,和(ii)将表达相同的肿瘤抗原的溶瘤病毒给予至受试者。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.
在其它实施方案中,联合疗法包括:(i)在存在加载有肿瘤抗原肽的APC和IL21的情况下离体培 养来自受试者的PBMC、扩增培养物中的细胞、并且将它们重新导入至受试者中,和(ii)将表达相同的肿瘤抗原的溶瘤病毒给予至受试者。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.
在其它实施方案中,联合疗法包括:(i)在存在加载有肿瘤抗原肽的APC、IL21、IL15和雷帕霉素的情况下离体培养来自受试者的PBMC、扩增培养物中的细胞、并且将它们重新导入至受试者中,和(ii)将表达相同的肿瘤抗原的溶瘤病毒给予至受试者。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.
在相关的实施方案中,在离体培养之前,用特异性肿瘤抗原的重组TCR或CAR转导PBMC。In a related embodiment, the PBMCs are transduced with recombinant TCR or CAR of a specific tumor antigen prior to ex vivo culture.
在一些实施方案中,在疗法肿瘤抗原特异性CD8+T细胞之后约8至72小时,将表达肿瘤抗原的溶瘤病毒给予至哺乳动物。在优选的实施方案中,在疗法肿瘤抗原特异性CD8
+T细胞之后约12至48小时、约20至28小时、或约24小时,将表达肿瘤抗原的溶瘤病毒给予至受试者。
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.
可以根据本文中所述的联合疗法给予表达肿瘤抗原的任意具有复制能力的溶瘤病毒。在一些优选的实施方案中,复制型溶瘤病毒为例如水疱性口炎病毒(VSV)或Maraba棒状病毒等棒状病毒,其优选包含一种或多种遗传修饰从而增加病毒对癌症细胞的选择性。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. .
可以通过多个途径中的一种或多种来给予复制型溶瘤病毒疫苗。在一些实施方案中,复制型溶瘤病毒为棒状病毒并且通过静脉内途径给予至哺乳动物。在其它实施方案中,复制型溶瘤病毒为痘苗病毒并且静脉内(IV)、肌内(IM)、腹腔内(IP)或瘤内(IT)给予至哺乳动物。如本领域技术人员将理解的,复制型溶瘤病毒(例如棒状病毒或痘苗病毒)将以例如盐水或其它药学上合适的缓冲液等合适的载体给药。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.
在本公开的一个实施方案中,为了建立预存免疫力,本公开的方法包括用适于诱导针对目标肿瘤细胞的免疫反应的肿瘤抗原给哺乳动物接种疫苗的步骤。例如,肿瘤抗原可以是肿瘤相关抗原(TAA),例如在引发哺乳动物的免疫应答的肿瘤细胞中产生的物质。这类抗原的实例包括癌胚抗原(oncofetal antigen)(例如甲胎蛋白,AFP)和癌胚抗原(carcinoembryonic antigen,CEA)、表面糖蛋白(例如CA 125)、癌基因(例如Her2)、黑素瘤相关抗原(例如多巴色素互变异构酶(DCT))、GP100和MART1、癌-睾丸抗原(cancer-testes antigen)(例如MAGE蛋白和NY-ESO1)、病毒癌基因(例如HPV E6和E7)、在通常限于胚胎组织或胚外组织的肿瘤中异位表达的蛋白质(例如PLAC1)。正如本领域技术人员应理解的一样,可根据待采用本公开的方法治疗的癌症的类型选择抗原,因为一种或多种抗原可能特别适用于治疗某些癌症。例如对于治疗黑素瘤,可以使用黑素瘤相关抗原,例如DCT。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.
可以给予抗原本身,或者优选通过载体给予抗原,例如腺病毒(Ad)载体、痘病毒载体或反转录病毒载体、质粒或荷载的抗原呈递细胞,例如树突细胞。将抗原引入载体的方法为本领域技术人员所知。一般而言,可对载体进行修饰以表达抗原。在这一方面,使用广为接受的重组技术,将编码选定抗原的核酸整合到选定载体上。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.
用以下若干方法的任一种将抗原给予哺乳动物,包括但不限于静脉内、肌内或鼻内。正如本领域技术人员应理解的一样,可在合适的溶媒(例如盐水或其它合适的缓冲液)中给予抗原或掺有抗原的 载体。在用选定肿瘤抗原接种后,在免疫应答间隔期内,例如约4天内并延长达数月、数年或可能终身,哺乳动物产生免疫应答。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.
建立对抗原的免疫应答,采用广为接受的技术,进行使用抗原的疫苗接种。因此,可以足以产生免疫应答的量将选定抗原或表达抗原的载体给予哺乳动物。正如本领域技术人员应理解的一样,产生免疫应答所需要的量将随多种因素而变化,包括例如选定抗原、递送抗原所使用的载体和待治疗的哺乳动物,例如品种、年龄、体型等。在这一方面,例如,肌内给予小鼠腺病毒载体至少约10
7PFU的最低量足以产生免疫应答。对于给予人,相应的量应足够产生免疫应答。
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.
在另一个实施方案中,对抗原的免疫应答可在哺乳动物内天然发生,不需要第一疫苗接种步骤以诱导免疫应答。对抗原的天然存在的免疫应答可由任何既往暴露于抗原而产生。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.
一旦在适当的免疫应答间隔期内在哺乳动物中产生免疫应答,例如至少约24小时,优选至少约2-4天或更久,例如至少约1周,则以适于溶瘤病毒疗法的量将表达肿瘤抗原的溶瘤病毒给予哺乳动物,正如本领域技术人员应理解的一样,这可随选定的溶瘤病毒和待治疗的哺乳动物而变化。例如,静脉内给予小鼠10
8PFU溶瘤VSV的最低量足以用于溶瘤疗法。相应的量可足以用于人。
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.
可通过采用标准重组技术,将编码抗原的转基因整合到病毒中来制备表达选定肿瘤抗原的溶瘤病毒。例如,可将转基因整合到病毒基因组,或者可采用整合转基因的质粒将转基因整合到病毒中。本公开的方法对于可利用的溶瘤病毒没有特别限制,可包括能够破坏肿瘤同时适于给予哺乳动物的任何溶瘤病毒。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.
在一个实施方案中,本公开所描述的是ー种用以反向遗传操作系统制造的减毒棒状病毒病毒,是一种全新的基因肿瘤治疗开发的重组系统。减毒棒状病毒四重突变株(RV-4Mut)已被制造出来,并且在多种肿瘤模型(具有免疫功能的肿瘤模型)中证实通过系统递送是安全和有效的。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).
在一个实施方案中,本公开的减毒四重突变棒状病毒(和/或其它溶瘤试剂)可被连续使用,而不会引起宿主针对治疗病毒的强烈免疫反应。基于此,在一定时间内可以对宿主进行多次相同病毒系统的治疗,延长治疗时间,进一步降低了机体对单一药物耐药性的产生,进而改善肿瘤治疗的效果。本公开的实施方案包括有关棒状病毒的组合物和方法以及它们作为抗肿瘤治疗的用途。这些棒状病毒在体内和体外都具备杀死肿瘤细胞的性质。在本公开中,棒状病毒可以是减毒棒状病毒或减毒棒状病毒的基因工程变异体。本申请所述的病毒可以与其它棒状病毒结合使用。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.
在本公开的一个实施方案中,包括减毒棒状病毒以及包含减毒棒状病毒的组合物,所述减毒棒状病毒编码与减毒棒状病毒的M蛋白(即如SEQ ID NO:1所示的氨基酸序列)具有至少或至多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%(包括这些数值之间所有范围和百分数)的氨基酸同一性的变异M蛋白。上述减毒棒状病毒的M蛋白具有特定百分数的同一性指的是,减毒棒状病毒的M蛋白存在可正常维持蛋白质的功能的保守突变。保守突变的代表性例子为保守置换。 保守置换是指,例如,在置换部位为芳香族氨基酸的情况下,在Phe、Trp、Tyr间相互置换的突变;在置换部位为疏水性氨基酸的情况下,在Leu、Ile、Val间相互置换的突变;在为极性氨基酸的情况下,在Gln、Asn间相互置换的突变;在为碱性氨基酸的情况下,在Lys、Arg、His间相互置换的突变;在为酸性氨基酸的情况下,在Asp、Glu间相互置换的突变;在为具有羟基的氨基酸的情况下,在Ser、Thr间相互置换的突变。作为被视作保守置换的置换,具体而言,可以举出Ala向Ser或Thr的置换、Arg向Gln、His或Lys的置换、Asn向Glu、Gln、Lys、His或Asp的置换、Asp向Asn、Glu或Gln的置换、Cys向Ser或Ala的置换、Gln向Asn、Glu、Lys、His、Asp或Arg的置换、Glu向Gly、Asn、Gln、Lys或Asp的置换、Gly向Pro的置换、His向Asn、Lys、Gln、Arg或Tyr的置换、Ile向Leu、Met、Val或Phe的置换、Leu向Ile、Met、Val或Phe的置换、Lys向Asn、Glu、Gln、His或Arg的置换、Met向Ile、Leu、Val或Phe的置换、Phe向Trp、Tyr、Met、Ile或Leu的置换、Ser向Thr或Ala的置换、Thr向Ser或Ala的置换、Trp向Phe或Tyr的置换、Tyr向His、Phe或Trp的置换、及Val向Met、Ile或Leu的置换。此外,上述减毒棒状病毒的M蛋白的同一性突变还包括起因于基因所来源的棒状病毒的个体差异、株、种的差异时等的天然产生的突变。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.
在某些情况下,对于单独的随机突变,尽管各自的单一突变株可能会减少病毒对正常健康细胞的毒性作用,但是,一旦将上述多组单独的随机突变相结合,病毒在肿瘤细胞中极有可能会变得比野生型病毒更加具有毒性。因此,本公开中的重组溶瘤棒状病毒的治疗指数出人意料地増加,是建立在体外大规模筛选减毒株过程中的意外发现,多种单一突变的减毒株进行多基因同时突变时,大部分病毒在肿瘤细胞及正常细胞中同时丧失感染力,少部分返强,细胞毒性变强。本公开意外地发现RV-4Mut的4个氨基酸突变并没有让病毒本身返强,同时继续保留了杀伤肿瘤的特性,尽管在体外细胞水平发现裂解肿瘤细胞的时间点延后,但特异性杀伤肿瘤的属性完整的保留,最难能可贵的是RV-4Mut对正常细胞没有任何毒性,完全符合生物安全要求。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.
本公开的方法和组合物可包括第二种治疗病毒,例如溶瘤病毒或复制缺陷型病毒。溶瘤通常指的是能够杀死、溶解或阻止肿瘤细胞生长。溶瘤病毒是指可在肿瘤细胞中一定程度上复制,导致肿瘤细胞死亡、溶解(溶瘤)或肿瘤细胞生长停止,并且通常对非肿瘤细胞具有微小的毒性作用。第二种病毒包括但不限于弹状病毒、牛痘病毒、疱疹病毒、麻疹病毒、新城疫病毒、腺病毒、甲病毒、细小病毒、肠道病毒株等。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.
本公开的实施方案包括与包含异源性N、P、M、G和/或L蛋白的弹状病毒有关的组合物和方法以及它们作为抗肿瘤疗法的用途。这种弹状病毒具有体内和体外肿瘤细胞杀伤性质。因此,如本公开所述的VSV病毒可通过联合异源性N、P、M、G和/或L蛋白而被进一步修饰。如本公开所使用的,异源性N、P、M、G和/或L蛋白包括弹状病毒N、P、M、G和/或L蛋白。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.
本公开的方法可进一步包括施用第二种抗肿瘤疗法,例如第二种治疗病毒。在某些方面,治疗病毒可为溶瘤病毒,更特别为VSV病毒。在其他方面,第二种抗肿瘤疗法为化学治疗剂、放射治疗剂 或免疫治疗剂、手术等。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.
另一方面,所述组合物是药学上可接受的组合物。所述组合物还可包括第二抗肿瘤制剂,例如化学治疗剂、放射治疗剂或免疫治疗剂。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.
本公开的另外的实施方案涉及杀死增生性细胞的方法,该方法包括将该细胞与本公开所述的分离的溶瘤弹状病毒组合物接触。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.
本公开另外的实施方案涉及癌症患者的治疗,包括施用有效量的本公开所述的溶瘤弹状病毒组合物。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.
在本公开的某些方面,可将细胞包括在患者体内,该细胞可为增生性的、瘤性的、前癌性的、转移性的细胞。弹状病毒可被施用至患者,该患者具有易被至少一种弹状病毒或包含弹状病毒的治疗方案或组合物杀死的细胞。治疗组合物的施用可利用1、2、3、4、5、6、7、8、9、10种或更多种弹状病毒或重组弹状病毒(单独或以各种方式组合)进行1、2、3、4、5、6、7、8、9、10次或更多次。施用可为腹膜内、静脉内、动脉内、肌肉内、皮内、皮下或鼻腔施用。在某些方面,组合物被系统性施用,特别是通过血管内施用,包括注射、灌注等。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.
发明的效果Effect of the invention
在一个实施方式中,本公开通过基因重组技术,提供了一种能够有效降低病毒在正常体细胞中的毒性的溶瘤棒状病毒减毒株。优选的,本公开提供的溶瘤棒状病毒减毒株同时保证相对于正常细胞,对于异常增生性细胞具有高选择性,并且具有侵染肿瘤细胞,进而产生良好的溶瘤效果的溶瘤棒状病毒减毒株。在另一个实施方式中,前述减毒株具有持续性复制表达,高滴度,刺激肿瘤局部微环境的免疫反应,以及在保持高选择性侵染肿瘤细胞的同时对正常细胞毒性低的特点。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.
在一个实施方式中,本公开记载的肿瘤具有诱导促进抗肿瘤免疫反应,消除肿瘤组织微环境免疫抑制的效果。在另一个实施方式中,基于溶瘤病毒载体开发出持续稳定表达肿瘤抗原的治疗性疫苗,可以直接通过多种途径给药,优选的是静脉给药,基于该肿瘤疫苗的基础上,联合过继T细胞疗法,针对多种难治性肿瘤,实现实体瘤的消除以及控制转移灶的同时,产生自体免疫记忆保护反应,克服了肿瘤免疫治疗的复发的难题。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.
在一个实施方式中,本公开提供的联合治疗方法,允许受疗法的内源性T细胞调节的抗肿瘤免疫的同时分析。溶瘤病毒疫苗除了促进疗法的T细胞扩张和肿瘤浸润之外,还能够接种内源性针对各类肿瘤相关抗原T细胞。其中疗法的T细胞对大肿瘤块的快速摧毁有所贡献,而内源性T细胞同时防止抗原缺失变体的出现。当疗法的T细胞在肿瘤消退后不久消失时,内源性T细胞会获取具有抗原特异性宽组库的长期记忆。我们的发现暗示了该联合策略可以开发过继T细胞疗法和内源性针对各类肿瘤相关抗原T细胞的全部潜能,以消除初生肿瘤、防止免疫逃脱和提供长期保护性记忆。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.
在一个实施方式中,本公开通过将棒状病毒疫苗接种病毒类的溶瘤疫苗在推进过继性疗法T细胞的全身扩张(systemic expansion)和肿瘤浸润中的用途,强化过继细胞疗法的治疗效率。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.
在一个实施方式中,本公开提供的抗肿瘤用制品和联合治疗方案可导致在没有预处理的情况下完全而持久的肿瘤消退。使用该模型,同时监控了疗法的内源性T细胞对治疗效果的贡献。试验结果显示了预先存在的内源性针对各类肿瘤相关抗原T细胞对于通过过继细胞疗法来防止和/或消除抗原逃脱性变体从而获得持久的消退和长期免疫记忆是至关重要的。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.
在一个实施方式中,本公开提供的抗肿瘤用制品和联合治疗方案可以诱导促进抗肿瘤免疫反应,抗肿瘤免疫细胞特异性增殖,抗肿瘤免疫细胞浸润肿瘤组织或消除肿瘤组织微环境免疫抑制。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.
通过对以下列出的它们各自的附图的以下描述更深入地示出本公开的实施方案。Embodiments of the present disclosure are shown in greater depth by the following description of the respective figures of the figures listed below.
图1所示的是VSV减毒溶瘤病毒随机突变(RV-Mut)单点多点突变的病毒拯救情况。Figure 1 shows the viral rescue of VSV attenuated oncolytic virus random mutation (RV-Mut) single-point multipoint mutation.
图2所示的是在肿瘤细胞LLC中和正常成纤维细胞MEF中,包括RV-4Mut在内的不同减毒株的不同时间点的病毒复制情况。Figure 2 shows viral replication at different time points in different attenuated strains including tumor cell LLC and normal fibroblast MEF, including RV-4Mut.
图3所示的是不同病毒载量的,包括RV-4Mut在内的不同溶瘤弹状病毒减毒株对多种肿瘤细胞的体外杀伤比较。Figure 3 shows the in vitro killing of various tumor-bearing cells with different viral loads, including RV-4Mut, against different tumor cells.
图4所示的是包括RV-4Mut在内的不同溶瘤病毒减毒株在MEF和Vero细胞中的外源蛋白持续表达能力情况。Figure 4 shows the sustained expression of foreign proteins in MEF and Vero cells of different oncolytic virus attenuated strains including RV-4Mut.
图5所示的是包括RV-4Mut在内的不同溶瘤病毒减毒株在Vero以及人源肿瘤细胞A549中的持续复制表达能力的比较情况。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.
图6所示的是包括RV-4Mut在内的不同溶瘤病毒减毒株在不同细胞中刺激诱导干扰素免疫反应能力的比较情况。Figure 6 shows a comparison of the ability of different oncolytic virus attenuated strains including RV-4Mut to stimulate interferon immunoreactivity in different cells.
图7所示的包括RV-4Mut在内的不同溶瘤病毒减毒株在不同品系小鼠中神经毒性的检测,包括小鼠体重以及生存率的统计。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.
图8所示的是包括RV-4Mut在内的不同溶瘤病毒减毒株在肿瘤模型中的治疗效果评价。Figure 8 shows the evaluation of the therapeutic effects of different oncolytic virus attenuated strains including RV-4Mut in a tumor model.
图9所示的是不同肿瘤细胞皮下移植瘤模型建立示意图,包括结肠癌(MC38),黑色素瘤(B16)以及肺癌细胞(LLC)。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).
图10所示的是溶瘤病毒RV静脉给药与瘤内给药在治疗肺癌模型的药效比较。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.
图11所示的是RVV(gp33)溶瘤病毒疫苗在结肠癌(MC38-gp33)肿瘤模型中静脉给药的药效评价。Figure 11 shows the pharmacodynamic evaluation of intravenous administration of RVV (gp33) oncolytic virus vaccine in a colon cancer (MC38-gp33) tumor model.
图12所示的是溶瘤病毒疫苗(RVV-DCT)联合Tcm在和黑色素肿瘤模型(B16)中的治疗效果统计。Figure 12 shows the statistical effect of treatment of oncolytic virus vaccine (RVV-DCT) in combination with Tcm and melanoma tumor model (B16).
图13所示的是溶瘤病毒疫苗(RVV-mERK)联合Tcm在恶性骨肉瘤(CMS5)肿瘤模型中的治疗效果统计。Figure 13 shows the statistical effect of the oncolytic virus vaccine (RVV-mERK) in combination with Tcm in a malignant osteosarcoma (CMS5) tumor model.
图14所示的是溶瘤病毒疫苗(RVV-gp33)联合Tcm在结肠癌中的治疗效果比较。Figure 14 shows a comparison of the therapeutic effects of oncolytic virus vaccine (RVV-gp33) in combination with Tcm in colon cancer.
图15所示的溶瘤病毒疫苗RVV-gp33与Tcm联合治疗在B16-gp33肿瘤模型中诱导抗肿瘤T细胞以及清除恶性黑色素瘤的情况。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.
图16所示的是溶瘤病毒疫苗RVV-mERK与Tcm联合治疗刺激CD8
+T细胞向肿瘤核心区域浸润(CMS5肿瘤组织)。
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).
图17所示的是溶瘤病毒疫苗与Tcm联合治疗通过刺激内源性淋巴细胞控制肿瘤的复发。Figure 17 shows the combination of oncolytic virus vaccine and Tcm to control tumor recurrence by stimulating endogenous lymphocytes.
图18所示的是溶瘤病毒疫苗RVV-gp33与Tcm联合治疗通过维持内源性T细胞有效控制肿瘤的复发。Figure 18 shows that the combination of the oncolytic virus vaccine RVV-gp33 and Tcm effectively controls tumor recurrence by maintaining endogenous T cells.
图19所示的是比较在不同的离体培养条件下(IL15、IL21和雷帕霉素的组合使用),从而产生在联合疗法中具有最佳抗肿瘤效果的Tcm细胞。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).
定义definition
如本领域技术人员都能理解的那样,除非另有说明,否则本节和其他节中描述的定义和实施方案旨在应用于本文所述申请的所有实施案例,。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.
在理解本申请的范围时,如本文所使用的术语“包含”及其衍生词旨在是开放式术语,其指定存在所述特征,元素,组分,组,整数和/或步骤,但不排除存在其他未说明的功能、元素、组分、组、整数和/或步骤。前述内容也适用于具有相似含义的词语,例如术语“包括”、“具有”及其衍生词。本文使用的术语“由......组成”及其衍生词旨在是封闭式术语,其指定存在所述特征、元素、组分、组、整数和/或步骤,但排除存在其他未说明的特征的存在、元素、组分、组、整数和/或步骤。如本文所用的,术语“基本上由......组成”旨在指定存在所述特征、元素、组分、组、整数和/或步骤以及不会实质上影响特征、元素、组分、组、整数和/或步骤的基本和新颖特征的那些。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.
应理解“组合疗法”是根据本文所述的Tcm疗法产生的肿瘤抗原特异性中心记忆性T细胞群加上表达一种或多种相同肿瘤抗原的复制性溶瘤病毒按照顺序给予受试者。根据本文所述的Tcm疗法产生的记忆性T细胞群和表达相同肿瘤抗原的溶瘤病毒在一定的时间间隔内给予受试者显示出协同作用。在优选的实施方案中,记忆T细胞群和溶瘤病毒可以在1至72小时内(例如在1、2、3、6、12、24、48或72小时内)或者在4、5、6或7天内给药,或在8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30或31天内给予。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.
本文使用的诸如“基本上”、“约”和“近似”的程度术语意为修饰术语的合理偏差量,这个改变不会导致结果产生显著变化。如果该偏差不会否定其修饰的词的根本含义的话,这些程度术语的使用应被限定为偏离修饰的术语的定义控制范围在正负百分之五。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.
如在本申请中所使用的,单数形式“一个/种(a/an)”和“所述(the)”包括复数指代,除非内容另 有明确说明。例如,包括“一个T细胞”的实施方案应理解为具有一种物质或两种及两种以上其他物质的组分。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.
在包含“另一”或“第二”组分,例如另一或第二细胞因子的实施方案中,如本文所用的第二组分在化学上不同于其他组分或第一组分。“第三”组分与其他组分、第一组分和第二组分不同,并且进一步列举的与“另一”组分不同的相似点。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.
本文所用的术语“和/或”是指所列出的项单独存在或组合使用。实际上,该术语意为所列项中已经存在或者在使用的“至少一个/种”或“一个/种或多个/种”。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.
当在权利要求和/或说明书中与术语“包含”联用时,词语“一(a)”或“一(an)”可以指“一个”,但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。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".
如在权利要求和说明书中所使用的,词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的,并不排除额外的、未引述的元件或方法步骤。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.
在整个申请文件中,术语“约”表示:一个值包括测定该值所使用的装置或方法的误差的标准偏差。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.
虽然所公开的内容支持术语“或”的定义仅为替代物以及“和/或”,但除非明确表示仅为替代物或替代物之间相互排斥外,权利要求中的术语“或”是指“和/或”。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".
当用于权利要求和/或说明书中时,术语“抑制”、“降低”或“防止”或这些术语的任何变形,包括为实现期望结果(例如肿瘤治疗)的任何可测量的减少或完全抑制。期望结果包括但不限于癌症或增生性病症或癌症相关症状的缓解、降低、减慢或根除,以及改善的生活质量或生命延长。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.
本公开的疫苗接种方法可用于治疗哺乳动物的肿瘤,可选的,本公开的疫苗接种方法可用于治疗哺乳动物的癌症。本公开使用的术语“癌症”包括任何癌症,包括但不限于黑素瘤、肉瘤、淋巴瘤、癌(例如脑癌、乳癌、肝癌、胃癌、肺癌和结肠癌)及白血病。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.
术语“哺乳动物”是指人以及非人类哺乳动物。The term "mammal" refers to both humans as well as non-human mammals.
本公开的所述方法包括将表达哺乳动物对之具有预存免疫力的肿瘤抗原的溶瘤载体给予哺乳动物。本公开使用的术语“预存免疫力”意指包括通过用抗原接种诱导的免疫力以及哺乳动物内天然存在的免疫力。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.
本公开中采用的术语“RV病毒”是指减毒的VSV溶瘤棒状病毒。术语“RV-Mut”是指和野生型的VSV溶瘤棒状病毒相比,存在突变的溶瘤棒状病毒。示例性的,“RV-4Mut”是指和野生型的VSV溶瘤棒状病毒相比,存在4个氨基酸突变的溶瘤棒状病毒;“RV-3Mut”是指和野生型的VSV溶瘤棒状病毒相比,存在3个氨基酸突变的溶瘤棒状病毒。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.
中心记忆性T细胞(Tcm)Central memory T cell (Tcm)
中心记忆性T细胞又被称为中央记忆型T细胞,是幼稚T细胞(Naive T Cell)经过抗原激活后,产生的具有长期记忆性的,并能够归巢到淋巴结接受抗原再刺激的T细胞。示例性的,Tcm的生物标 记可以选自CD62L和CD45RO双阳性,表明Tcm能够通过淋巴屏蔽,回归淋巴结,同时处于被抗原激活的状态。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.
溶瘤弹状病毒Oncolytic rhabdovirus
水疱性口炎病毒(VSV)是一种负链RNA病毒,其感染大部分哺乳动物细胞并在受感染细胞中表达高达总蛋白60%的病毒蛋白。在自然界中,VSV感染猪、牛和马,并在口和足附近导致水痘性疾病。虽然已有报道人感染VSV,但是VSV在人类中没有导致任何严重的症状。VSV编码5种蛋白,包括核壳蛋白(N)、磷蛋白(P)、基质蛋白(M)、表面糖蛋白(G)和RNA依赖性RNA聚合酶(L)。由VSV基质蛋白(M)阻断宿主细胞蛋白合成会诱导细胞死亡。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.
溶瘤病毒疫苗Oncolytic virus vaccine
根据本公开的表达肿瘤抗原的具有复制能力的(replication-competent)溶瘤病毒包括任何天然存在的(例如来自“野生来源(field source)”)或修饰的具有复制能力的溶瘤病毒。除了表达肿瘤抗原之外,可以例如修饰溶瘤病毒从而增加病毒对癌细胞的选择性。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.
根据本公开的具有复制能力的溶瘤病毒包括但不限于,是以下科的成员的溶瘤病毒:肌尾噬菌体科(myoviridae)、长尾噬菌体科(siphoviridae)、短尾噬菌体科(podpviridae)、复层噬菌体科(teciviridae)、覆盖噬菌体科(corticoviridae)、芽生噬菌体科(plasmaviridae)、脂毛噬菌体科(lipothrixviridae)、微小纺锤形噬菌体科(fuselloviridae)、痘病毒科(poxyiridae)、虹彩病毒科(iridoviridae)、藻类DNA病毒科(phycodnaviridae)、杆状病毒科(baculoviridae)、疱疹病毒科(herpesviridae)、腺病毒科(adnoviridae)、乳多空病毒科(papovaviridae)、多分体DNA病毒科(polydnaviridae)、丝状噬菌体科(inoviridae)、微小噬菌体科(microviridae)、双生病毒科(geminiviridae)、圆环病毒科(circoviridae)、细小病毒科(parvoviridae)、嗜肝DNA病毒科(hepadnaviridae)、逆转录病毒科(retroviridae)、囊状噬菌体科(cyctoviridae)、呼肠孤病毒科(reoviridae)、双RNA病毒科(birnaviridae)、副粘液病毒科(paramyxoviridae)、棒状病毒科(rhabdoviridae)、丝状病毒科(filoviridae)、正粘液病毒科(orthomyxoviridae)、布尼安病毒科(bunyaviridae)、沙粒病毒科(arenaviridae)、光滑噬菌体科(leviviridae)、小RNA病毒科(picornaviridae)、伴生病毒科(sequiviridae)、豇豆花叶病毒科(comoviridae)、马铃薯Y病毒科(potyviridae)、杯状病毒科(caliciviridae)、星状病毒科(astroviridae)、野田病毒科(nodaviridae)、四病毒科(tetraviridae)、番茄丛矮病毒科(tombusviridae)、冠状病毒科(coronaviridae)、黄病毒科(glaviviridae)、披膜病毒科(togaviridae)和杆状RNA病毒科(barnaviridae)。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.
用于本公开实践的具有复制能力的溶瘤病毒疫苗的病毒骨架具体实例包括弹状病毒、腺病毒、逆转录病毒、呼肠孤病毒、棒状病毒、新城疫病毒(NDV)、多瘤病毒、痘苗病毒(VacV)、单纯疱疹病毒、小核糖核酸病毒、柯萨奇病毒(coxsackie virus)和细小病毒。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.
在一些优选的实施方案中,根据本公开的表达肿瘤抗原的具有复制能力的溶瘤病毒是溶瘤弹状病毒。In some preferred embodiments, the replication competent oncolytic virus that expresses a tumor antigen according to the present disclosure is an oncolytic rhabdovirus.
原型棒状病毒是狂犬病和水泡性口炎病毒(vesicular stomatitis virus,VSV),它们该病毒科中研究最多的病毒。棒状病毒是子弹形病毒的科,其具有非节段化(-)有义RNA基因组。棒状病毒科包括但不限于:Arajas病毒、金迪普拉病毒(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)、科卡病毒(Cocal virus)(AF045556/gi:2865658)、伊斯法罕病毒(Isfahan virus)(AJ810084/gi:57834038)、Maraba病毒(美国专利号8,481,023的SEQ ID ON:1-6,其通过引用并入本文;HQ660076.1)、Carajas病毒(美国专利号8,481,023的SEQ ID NO:7-12,其通过引用并入本文,AY335185/gi:33578037)、皮理病毒(Piry virus)(D26175/gi:442480、Z15093/gi:61405)、水泡性口炎阿拉戈斯病毒(Vesicular stomatitis Alagoas virus)、BeAn 157575病毒、博特克病毒(Boteke virus)、Calchaqui病毒、美洲鳗病毒(Eel virus American)、格雷洛奇病毒(Gray Lodge virus)、朱罗纳病毒(Jurona virus)、克拉马斯病毒(Klamath virus)、克瓦塔病毒(Kwatta virus)、La Joya病毒、Malpais Spring病毒、芒特埃尔岗蝙蝠病毒(Mount Elgon bat virus)(DQ457103/gi:91984805)、Perinet病毒(AY854652/gi:71842381)、Tupaia病毒(NC_007020/gi:66508427)、大巴伊亚病毒(Bahia Grande virus)(美国专利号8,481,023的SEQ ID NO:13-18,其通过引用并入本文,KM205018.1)、Muir Springs病毒(KM204990.1)、Reed Ranch病毒、哈特公园病毒(Hart Park virus)、费兰杜病毒(Flanders virus)(AF523199/gi:25140635、AF523197/gi:25140634、AF523196/gi:25140633、AF523195/gi:25140632、AF523194/gi:25140631、AH012179/gi:25140630)、凯米斯病毒(Kamese virus)、Mosqueiro病毒、莫苏里病毒(Mossuril virus)、巴鲁病毒(Barur virus)、Fukuoka病毒(AY854651/gi:71842379)、克恩峡谷病毒(Kern Canyon virus)、恩科比逊病毒(Nkolbisson virus)、利丹特病毒(Le Dantec virus)(AY854650/gi:71842377)、Keuraliba病毒、Connecticut virus、新明托病毒(New Minto virus)、莎草病毒(Sawgrass virus)、查可病毒(Chaco virus)、Sena Madureira病毒、蒂姆博病毒(Timbo virus)、阿尔姆皮瓦病毒(Almpiwar virus)(AY854645/gi:71842367)、阿鲁卡病毒(Aruac virus)、班戈兰病毒(Bangoran virus)、卞博病毒(Bimbo virus)、Bivens Arm病毒、Blue crab病毒、查里维勒河病毒(Charleville virus)、Coastal Plains病毒、DakArK7292病毒、Entamoeba病毒、加巴病毒(Garba virus)、戈萨斯病毒(Gossas virus)、Humpty Doo病毒(AY854643/gi:71842363)、约英杰卡卡病毒(Joinjakaka virus)、肯纳曼格拉姆病毒(Kannamangalam virus)、科隆各病毒(Kolongo virus)(DQ457100/gi:91984799核蛋白(N)mRNA,部分序列信息(partial cds));Koolpinyah病毒、Kotonkon病毒(DQ457099/gi:91984797、AY854638/gi:71842354);兰德几亚病毒(Landjia virus)、Manitoba病毒、马可病毒(Marco virus)、Nasoule病毒、纳瓦鲁病毒(Navarro virus)、Ngaingan病毒(AY854649/gi:71842375)、Oak-Vale病毒(AY854670/gi:71842417)、奥博第安病毒(Obodhiang virus)(DQ457098/gi|91984795)、奥衣塔病毒(Oita virus)(AB116386/gi:46020027)、奥安戈病毒(Ouango virus)、帕里河病毒(Parry Creek virus)(AY854647/gi:71842371)、Rio Grande cichlid病毒、圣 德吉姆巴病毒(Sandjimba virus)(DQ457102/gi|91984803)、西格马病毒(Sigma virus)(AH004209/gi:1680545、AH004208/gi:1680544、AH004206/gi:1680542)、Sripur病毒、Sweetwater Branch病毒、Tibrogargan病毒(AY854646/gi:71842369)、Xiburema病毒、雅塔病毒(Yata virus)、罗得岛(Rhode Island)病毒、Adelaide River病毒(U10363/gi:600151、AF234998/gi:10443747、AF234534/gi:9971785、AY854635/gi:71842348)、Berrimah病毒(AY854636/gi:71842350])、金伯利病毒(Kimberley virus)(AY854637/gi:71842352)、或牛短暂热病毒(Bovine ephemeral fever virus)(NC_002526/gi:10086561)。任何这些棒状病毒或其变体可以工程化从而表达肿瘤抗原来根据本公开使用。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.
在一些优选的实施方案中,本公开的表达肿瘤抗原的溶瘤棒状病毒是野生型水泡性病毒(vesiculovirus)或重组水泡性病毒,例如野生型或重组VSV、金迪普拉,Maraba或Carajas,包括其变体。在其他实施方案中,溶瘤棒状病毒是野生型或重组非水泡性病毒,例如Muir Springs、Farmington或大巴伊亚病毒病毒,包括其变体。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.
在特别优选的实施方案中,本公开的表达肿瘤抗原的溶瘤病毒是野生型Maraba株棒状病毒或其变体,其任选地经遗传修饰,例如以提高肿瘤选择性。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.
溶瘤病毒疫苗和过继性T细胞联合疗法Oncolytic virus vaccine and adoptive T cell combination therapy
虽然采用中心记忆性T细胞(Tcm)的过继性T细胞疗法的结果通常与接种的T细胞的功能相关,但内源性T细胞的作用尚不清楚。检查点阻断疗法的成功证明了预先存在的肿瘤启动T细胞在癌症治疗中的潜在治疗价值。鉴于检查点阻断治疗的结果,我们假设内源性T细胞有助于Tcm后的长期存活。这里,我们描述了一种将Tcm与溶瘤疫苗结合的治疗方法,该方法允许同时分析由转移和内源性T细胞介导的抗肿瘤免疫。我们发现,溶瘤病毒疫苗除了促进转移T细胞的扩张和肿瘤浸润外,还能促进肿瘤启动的宿主T细胞。我们确定转移的T细胞有助于快速破坏大的肿瘤肿块,而内源性T细胞同时阻止了抗原丢失变异体的出现。此外,虽然转移T细胞在肿瘤消退后不久消失,但内源性T细胞获得了具有广泛抗原特异性的长期记忆。我们的研究结果提示,这种联合策略可以充分利用Tcm和肿瘤启动的宿主T细胞的全部潜力,以消除原发肿瘤,防止免疫逃逸,并提供长期的保护性记忆。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.
肿瘤抗原Tumor antigen
本公开的溶瘤病毒表达由本文所述的Tcm方法产生的T细胞靶向的相同抗原。可由溶瘤病毒表达的合适抗原包括但不限于癌胚抗原,例如甲胎蛋白(ALF)和癌胚抗原(CEA),表面糖蛋白(例如CA 125),癌基因例如Her2,黑素瘤相关抗原例如多巴色素互变异构酶(DCT),GP100和MART-1,癌-睾丸抗原例如MAGE蛋白和NY-ESO-1,病毒癌基因如HPV E6和E7,以及在通常限于胚胎或胚外组织的肿瘤中异位表达的蛋白质例如PLAC1或肿瘤相关抗原的变体。肿瘤相关抗原的“变体”是指如下蛋白质:(a)包括来自肿瘤相关抗原蛋白的至少一种肿瘤相关抗原表位,和(b)与肿瘤相关的抗原蛋白至少70%、优选至少80%、更优选至少90%或至少95%相同。Van der Bruggen P,Stroobant V,Vigneron N,Van den Eynde B在"Database of T cell-defined human tumor antigens:the 2013update."Cancer Immun 2013 13:15中提供了总结公认的抗原表位的数据库,其可在www.cancerimmunity.org/peptide在线获取。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.
在一些实施方案中,溶瘤病毒表达MAGEA3、人乳头瘤病毒E6/E7融合蛋白、前列腺蛋白的人六跨膜上皮抗原、或癌睾丸抗原1。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.
在其他实施方案中,本公开提供识别肿瘤抗原的方法,其包括在包含IL21、IL15和雷帕霉素或基本上由IL21、IL15和雷帕霉素组成的组合物存在下,用从受试者分离的肿瘤材料共培养PBMC和抗原呈递细胞(如树突状细胞);从培养物中分离T细胞群;克隆来自T细胞群的单个T细胞;和表征T细胞克隆的抗原特异性。在一些实施方案中,肿瘤材料包含总RNA、裂解的肿瘤细胞或凋亡小体。本文所述的Tcm方法可用于产生对通过该方法识别的抗原特异的中心记忆性T细胞群,并与经过工程改造以表达相同抗原的溶瘤病毒组合以提供协同癌症治疗。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.
癌症cancer
根据本文所述的组合来治疗的癌症包括但不限于,白血病、急性淋巴细胞白血病、急性髓细胞白血病、成髓细胞早幼粒细胞白血病、髓单核细胞单核细胞红白血病、慢性白血病、慢性髓细胞(粒细胞)白血病、慢性淋巴细胞白血病、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、伯基特淋巴瘤和边缘区B细胞淋巴瘤、真性红细胞增多症淋巴瘤、霍奇金病、非霍奇金病、多发性骨髓瘤、瓦尔登斯特伦巨球蛋白血症(Waldenstrom’s macroglobulinemia)、重链病(heavy chain disease)、实体瘤、肉瘤、以及癌、纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、成骨肉瘤、骨肉瘤、脊索瘤、血管肉瘤、内皮肉瘤、淋巴管肉瘤、淋巴管内皮细胞瘤、滑膜瘤、间皮瘤、尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠肉瘤、结肠直肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、囊腺癌、髓样癌、支气管肺癌、肾细胞癌、肝癌、胆管癌、绒毛膜癌、精原细胞瘤、胚胎癌、维耳姆斯瘤、宫颈癌、子宫癌、睾丸肿瘤、肺癌、小细胞肺癌、非小细胞肺癌、膀胱癌、上皮癌、神经胶质瘤、星形细胞瘤、成神经管细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突神经胶质瘤、血管瘤、神经母细胞瘤、视网膜母细胞瘤、鼻咽癌、食管癌、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和中枢神经系统(CNS)癌、宫颈癌、绒毛膜癌、结直肠癌、结缔组织癌、消化系统癌、子宫内膜癌、食道癌、眼癌、头颈癌、胃癌、上皮内赘生物、肾癌、喉癌、肝癌、肺癌(包括小细胞肺癌、鳞状非小细胞肺癌和非鳞状非小细胞肺癌))、黑色素瘤(包括疗法性黑色素瘤)、神经母细胞瘤;口腔癌(例如唇、舌、口和咽)、卵巢癌、胰腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌;呼吸系统癌、肉瘤、皮肤癌、胃癌、睾丸癌、甲状腺癌、子宫癌和泌尿系统癌。在一些优选的实施方案中、待治疗的癌症选自非小细胞肺癌(NSCLC)、乳腺癌(例如激素难治性疗法性乳腺癌)、头颈癌(例如头颈部鳞状细胞癌)、疗法性结肠直肠癌、激素敏感或激素难治性前列腺癌、结肠直肠癌、卵巢癌、肝细胞癌、肾细胞癌、软骨组织肉瘤和小细胞肺癌。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.
在一个方面中,用所述组合治疗的受试者是患有癌症的人,所述癌症对于用一种或多种化学治 疗剂的治疗是难以治疗的和/或对于用一种或多种抗体的治疗是难以治疗的。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.
序列表含义Sequence table meaning
在本公开中,SEQ ID NO:1所示的是溶瘤棒状病毒MuddSummer株的原始基质蛋白(M)的氨基酸序列;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;
在本公开中,SEQ ID NO:2所示的是溶瘤棒状病毒MuddSummer株的原始基质蛋白(M)的核苷酸序列;In the present disclosure, SEQ ID NO: 2 shows the nucleotide sequence of the original matrix protein (M) of the oncolytic baculovirus MuddSummer strain;
在本公开中,SEQ ID NO:3所示的是溶瘤棒状病毒MuddSummer株的四突变株(RV-4Mut)改性基质蛋白(M)的氨基酸序列;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;
在本公开中,SEQ ID NO:4所示的是溶瘤棒状病毒MuddSummer株的四突变株(RV-4Mut)改性基质蛋白(M)的核苷酸序列;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;
在本公开中,SEQ ID NO:5所示的是溶瘤棒状病毒MuddSummer株的四突变株(RV-3Mut)改性基质蛋白(M)的氨基酸序列;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;
在本公开中,SEQ ID NO:6所示的是溶瘤棒状病毒MuddSummer株的四突变株(RV-3Mut)改性基质蛋白(M)的核苷酸序列。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.
优异性陈述Statement of excellence
内源性T细胞在过继细胞治疗临床结果中的作用被低估,因为淋巴细胞耗竭性预处理的使用。溶瘤病毒对内源性细胞的维持和激活导致更加多样的抗肿瘤反应,从而防止由于抗原变异细胞的生长而导致的肿瘤复发。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.
积累的相关证据表明,宿主T细胞可以通过识别肿瘤抗原而自发地被激活,从而对生长中的肿瘤作出反应。特别地,由于免疫系统识别为外来的新抗原的免疫原性,含有大量突变的肿瘤更有可能激活内源性T细胞,从而提供T细胞攻击的目标。突变负荷与免疫检查点阻断治疗的结果之间的相关性强调了这些发现的临床相关性,免疫检查点阻断依赖于肿瘤启动的T细胞群的存在。然而,在大多数情况下,针对突变抗原的自发T细胞反应相对低效,不能介导肿瘤排斥反应。事实上,只有一部分患者对免疫检查点阻断表现出持久的反应,这一事实表明,并非所有患者都具有足以根除肿瘤的肿瘤启动T细胞。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.
采用抗原特异性T细胞的过继性T细胞治疗(Tcm)是治疗恶性肿瘤的检查点抑制剂疗法的极好替代方案。健康肿瘤特异性T细胞可在体外生长,大量输注到晚期疾病患者体内,从而克服癌症患者自发T细胞应答不足的问题。重要的是,已经发展了体外培养功能性肿瘤特异性T细胞)的临床方法,最近在白血病、黑色素瘤、神经母细胞瘤和EBV相关恶性肿瘤中的临床成功已经证明,Tcm在人类中是可行和有效的策略。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.
然而,Tcm成功的必要步骤是在输注T细胞之前使宿主淋巴细胞清除,这个过程称为预适应,对某些患者具有高度毒性。此外,虽然预处理可以创造一个有利的环境。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.
过继转移T细胞(即消除调节性T细胞和稳态细胞因子“汇”),它还去除了预先存在的肿瘤启动T细胞,使得很难(如果不是不可能的话)确定内源性抗肿瘤免疫在Tcm中的作用和益处。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.
表达肿瘤相关抗原的溶瘤病毒能有效地结合和扩增肿瘤特异性记忆T细胞,同时保留它们直接感染和削弱肿瘤并逆转免疫抑制性肿瘤微环境的固有能力。在这部分本公开中,我们使用弹状病毒为基础的溶瘤疫苗来驱动过继转移T细胞的全身扩张和肿瘤浸润,从而提高Tcm的治疗效果。这种合理的组合导致肿瘤在没有预处理的情况下完全和持久的消退。利用这个模型,我们同时监测了转移和内源性T细胞对治疗效果的贡献。我们的数据表明,预先存在的、肿瘤引发的宿主T细胞对于预防和/或消除抗原逃逸变异体、通过Tcm实现持久回归以及长期免疫记忆至关重要。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.
在一些实施方案中,本文所述的组合疗法利用根据本文所述方法产生的肿瘤抗原特异性中心记忆性T细胞的过继疗法。因此,令人惊讶地发现,在存在APC,肿瘤抗原和包含IL21、IL15和雷帕霉素或基本上由IL21、IL15和雷帕霉素组成的组合物的情况下培养T细胞(例如未分化T细胞)导致肿瘤抗原特异性T细胞群,其与单独的IL21、IL15或雷帕霉素相比,基本上富含显示中心记忆表型的T细胞,所述T细胞可以离体扩增并引入患者,然后给予患者表达相同抗原的溶瘤病毒以协同治疗癌症,而不需要昂贵的细胞分选技术,也不需要在引入T细胞之前对患者施用淋巴细胞清除方案,并且不需要给予患者IL2。在一些实施方案中,IL21和IL15以约1ng/ml至约20ng/ml、优选约10ng/ml的浓度存在。在相关的实施方案中,雷帕霉素以约10ng/ml至约30ng/ml、优选约20ng/ml的浓度存在。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.
例如,APC可以是自体树突细胞,其可以通过用GM-CSF(例如约800U/ml)和IL-4(例如约500U/ml)培养粘附的PBMC约5天从而将它们分化为树突状细胞来获得。可以通过在第5天添加TNFα(例如约10ng/ml)、IL-1b(例如约2ng/ml)、IL-6(例如约1000U/ml)、PGE-2(例如约1000ng/ml)、IL-4(例如约500U/ml)和GM-CSF(例如约800U/ml)来刺激所得的树突细胞,并接着培养2天。在第7天,可以将树突细胞用40μg/ml的肽肿瘤抗原脉冲约2小时,并根据本文所述的方法照射来用作装载肿瘤抗原肽的APC。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.
在一些实施方案中,在存在APC、肿瘤抗原和IL21、IL15和雷帕霉素的情况下培养未分化T细胞导致肿瘤抗原特异性T细胞群,其中的至少50%、至少60%、至少70%、至少80%或更多显示中心记忆表型。在一个实施方案中,根据本文描述的Tcm方法获得的中心记忆性T细胞显示CD44+CD62L+CD127+表型。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.
在另一实施方案中,用于根据本公开的过继细胞疗法的CD8+人T细胞群通过如下获得:在IL21、IL15和雷帕霉素(一种由癌症和抗原呈递细胞表达的肿瘤抗原)存在下培养从人类癌症受试者获得的肿瘤浸润淋巴细胞(例如1至4周,优选约3周),用抗CD3和抗CD28抗体以及任选的IL2扩增CD8+人T细胞群,并将细胞重新引入患者体内,随后(例如,间隔24小时后)给予工程化以表达编码相同肿瘤抗原的转基因的复制性溶瘤病毒(例如溶瘤棒状病毒)。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.
本公开的表达肿瘤抗原的溶瘤棒状病毒是VSV毒株或其变体,其任选地经遗传修饰,例如,以 提高肿瘤选择性。在特别优选的实施方案中,VSV包含在M蛋白的51位处的甲硫氨酸的删除,如在Stojdl等人,Cancer Cell.,4(4):263-75(2003)中描述的,其内容通过引用引入本文。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.
本公开的表达肿瘤抗原的复制性溶瘤棒状病毒可以在根据本文所述的方法的Tcm治疗后以10、100、10
3、10
4、10
5、10
6、10
7、10
8、10
9、10
10、10
11、10
12、10
13或更多病毒颗粒(vp)或噬斑形成单位(pfu)中的一种或多种剂量全身地给予受试者,优选通过血管内(静脉内和/或动脉内)给药,所述给药包括注射和灌注等。在优选的实施方案中,在根据本文所述的方法的Tcm治疗后将表达肿瘤抗原的溶瘤棒状病毒血管内以10
5-10
14pfu、10
6-10
12pfu、10
8-10
14pfu或10
8-10
12pfu中的一种或多种剂量给予受试者。
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.
表达肿瘤抗原的复制性溶瘤痘苗病毒可以工程化以缺乏一种或多种功能基因,从而增加病毒的癌症选择性。本公开的表达肿瘤抗原的复制性溶瘤痘苗病毒可局部或全身地给予受试者,例如通过瘤内、腹膜内、静脉内、动脉内、肌内、皮内、颅内、皮下或鼻内给药,在根据本文所述的方法的Tcm治疗后,以一个或多个剂量的1×10
5噬斑形成单位(pfu)、5×10
5pfu、至少1×10
6pfu、5×10
6或约5×10
6pfu、1×10
7、至少1×10
7pfu、1×10
8或约1×10
8pfu、至少1×10
8pfu、约或至少5×10
8pfu、1×10
9或至少1×10
9pfu、5×10
9或至少5×10
9pfu、1×10
10pfu或至少1×10
10pfu、5×10
10或至少5×10
10pfu、1×10
11或至少1×10
11、1×10
12或至少1×10
12、1×10
13或至少1×10
13pfu。例如,病毒可以以约10
7-10
13pfu之间、约10
8-10
13pfu、约10
9-10
12pfu之间、约10
8-10
12pfu之间、或约10
9和10
10pfu的剂量给予。
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.
预期表达肿瘤抗原的溶瘤病毒的单剂量是指在0.1、0.5、1、2、5、10、15、20或24小时期间给予受试者或肿瘤的量,包括期间所有的值。剂量可以随时间推移或通过单次注射来扩散。通常,将多剂量给予相同的一般靶区域,例如在肿瘤附近、或者在静脉内给药的情况下受试者的血流或淋巴系统中的特定进入点。在某些方面,病毒剂量通过注射装置递送,所述注射装置包括在单个针中提供多个端口的针或连接到注射器的多个分叉,或其组合。可以给予单剂量的溶瘤病毒,或者可以在包括1、2、3、4、5、6、7、8、9、10、11、12以上周的治疗期内给予多剂量。例如,溶瘤病毒可以每隔一天、每周、每隔一周、每隔三周施用一次,持续1、2、3、4、5、6以上个月。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.
如本领域技术人员所理解的,表达肿瘤抗原的溶瘤病毒通常作为药物组合物的一部分与药学上可接受的载体(例如盐水或其他合适的缓冲液)一起给予。如本文所用的,“载体”包括任何和所有溶剂、分散介质、运载体(vehicle)、包衣、稀释剂、抗细菌剂和抗真菌剂、等渗和吸收延迟剂、缓冲液、载体溶液、悬浮液、和胶体等。这些介质和药剂用于药物活性物质的用途是本领域熟知的。除非任何常规介质或试剂与活性成分不相容,否则考虑其在治疗组合物中的用途。补充的活性成分也可以掺入组合物中。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.
溶瘤病毒具体实施例Oncolytic virus specific embodiment
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是,应当理解的是,详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出,因为在阅读该详细说明后,在本公开的精神和范围内所作出的各种改变和修饰,对于本领域技术人员来说将变得显而易见。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.
本公开采用的试剂及耗材如下:The reagents and consumables used in the present disclosure are as follows:
PBS(Hyclone SH30256.01),DMEM高糖培养基(Gibco C11995500),RPMI1640(Gibco C22400500CP),双抗体(Gibco 15140-122),胎牛血清(Gibco 10099141),
I Reduced Serum Medium(Gibco 31985-070),Lipofectamine LTX(Invitrogen 15338100),96孔细胞培养板(Corning 3599),6孔细胞培养板(Corning 3516),0.22um滤器(Millipore SLGP033rb),DMSO(Macklin D806645),噻唑蓝(Sigma M2128)。
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).
细胞系和肿瘤模型的建立Establishment of cell lines and tumor models
将所有细胞维持在37℃、含5%CO
2的潮湿环境中。将MC38-gp33细胞(其是工程化为表达对应于LCMV gp33表位的基因的MC38细胞)维持在含有10%FBS、2mM L-谷氨酰胺、5ml丙酮酸钠、5ml非必需氨基酸、5ml维生素溶液、55μM 2-巯基乙醇、100U/mL青霉素和100ng/mL链霉素的MEM/F11中。用800μg/ml G418维持gp33微基因的表达。所有细胞培养试剂都来自Invitrogen(Invitrogen,Grand Island,NY)。
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).
复发性骨肉瘤CMS5细胞(称为“CMS5r”)获得自终点NRG小鼠并在与CMS5所用相同的培养基中扩增。将CMS5细胞和B16-gp33维持在含有10%FBS、2mM L-谷氨酰胺、100U/mL青霉素和100ng/mL链霉素的DMEM中,直至细胞汇合。用PBS洗涤肿瘤细胞两次并以PBS中10
6细胞/30μL的浓度重悬。不进行认证检测而是用PlasmoTest
TM(InvivGen)两年一次地(bi-yearly)筛选细胞。在解冻后、体内施用前使细胞生长不超过3代。经由皮内注射攻击小鼠并使肿瘤在治疗开始之前生长至约150mm
3的平均体积。
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.
动物animal
C57BL/6和BALB/c小鼠购自于维通利华公司,并饲养在吉玛动物房中。P14小鼠,携带有gp33肽的TCR的转基因小鼠品系(B6.Cg-Tcratm1Mom Tg(TcrLCMV)327Sdz)。NRG小鼠(NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ)购自于维通利华公司,并将小鼠在超净条件下饲养。皮肤移植肿瘤动物模型,选用6至8周龄的BALB/c、C57BL/6小鼠用2×10
5B16-gp33或B16细胞或10
6CMS5细胞皮下接种。在肿瘤移植后5至7天(当肿瘤达到~5mm直径时)给予Tcm和随后的OVV(溶瘤病毒疫苗)接种。每日监测肿瘤生长并每隔一天用卡尺测量。肿瘤体积以宽×长×深计算。
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.
病毒virus
RV-gp33载体为重组水泡性口炎病毒MuddSummer亚型株,其表达淋巴细胞脉络丛脑膜炎病毒糖蛋白的显性CD8
+和CD4
+T细胞表位(分别为LCMV-gp33-41和LCMV-gp61-80)。
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).
在一个技术方案中,所述重组水泡性口炎病毒MuddSummer亚型株的改性基质蛋白(M)选自第51位置、第221位置、第226位置同时具有氨基酸替换,所述氨基酸替换方式为:第51位甲硫氨酸M替换为精氨酸R,第221位缬氨酸V替换为苯丙氨酸F,第226位甘氨酸G替换为精氨酸R。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.
在另一个技术方案中,所述重组水泡性口炎病毒MuddSummer亚型株的改性基质蛋白(M)选自第 21位置、第51位置、第111位置和第221位置同时具有氨基酸替换,所述氨基酸替换方式为:第21位甘氨酸G替换为谷氨酸E,第51甲硫氨酸M替换为丙氨酸A,第111位亮氨酸L替换为苯丙氨酸F,第221位缬氨酸V替换为苯丙氨酸F。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.
抗原肽的合成Synthesis of antigenic peptides
LCMV-GP的H-2Db-限制肽(gp33-41;KAVYNFATM)、DCT的H-2Kb-限制肽(DCT180–188SVYDFFVWL)、和突变体ERK的H-2Kd-限制肽(Erk9M136–144;QYIHSANVL)购自吴江近岸蛋白生物科技有限公司。将各肽溶解在蒸馏水中并保存在-20℃。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.
离体Tcm培养和Tcm接种方案In vitro Tcm culture and Tcm vaccination protocol
通过常规方法从TCR转基因小鼠中分离脾细胞。将脾细胞(含有大量APC)以3百万/mL的密度接种在RPMI培养基中,所述RPMI培养基补充有10%胎牛血清(FBS)、2mM L-谷氨酰胺、55μM 2-巯基乙醇、100U/mL青霉素、100ng/mL链霉素、10ng/mL IL21、10ng/mL IL15、20ng/mL雷帕霉素和0.1μg/mL同源抗原肽。初始接种后四天,使用初始培养基和无肽的补充剂将培养物扩大5倍体积。3天后收集细胞,洗涤并悬浮于PBS中用于注射。小鼠用200μL PBS中的10
6Tcm细胞静脉注射(IV),24小时后,用200μL的PBS(除非另有说明)中的2×10
8pfu的RV-gp33、RV-DCT、5×10
8pfu的RV-erk9m静脉注射。进行淋巴细胞清除处理,其表示在Tcm细胞输注前3天经由IP注射20mg/kg环磷酰胺(CPX)。
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.
表面标志物的流式染色Flow staining of surface markers
收集离体培养的细胞、沉淀并用Fc阻断处理。然后针对CD8、CD44、CD62L和CD127的表面表达来染色细胞。数据使用含有FACSDiva软件的LSRFortessa(BD Biosciences,Mississauga,ON,Canada)获取并用FlowJo软件(Tree Star,Ashland,OR)分析。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).
抗原特异性T细胞反应的检测Detection of antigen-specific T cell responses
从眼眶周窦血样获得外周血单核细胞。用ACK裂解缓冲液裂解红细胞。37℃下用gp33肽(1μg/mL)刺激单核细胞5小时并在温育后4小时内添加抗菌素A(Golgi Plug,1μg/mL;BD Biosciences)。细胞用Fc阻断处理并针对CD8的表面表达进行染色。随后将细胞固定、穿膜(Cytofix/Cytoperm,BD Biosciences)并针染色细胞内干扰素-γ。数据使用含有FACSDiva软件的LSRFortessa(BD Biosciences)获取并用FlowJo软件(Tree Star,Ashland,OR)分析。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).
组织病理学和免疫组化染色Histopathology and immunohistochemical staining
将组织在10%福尔马林中固定24小时,然后在70%乙醇中脱水。将固定的组织包埋在石蜡中并切成5μm切片。切片或者用苏木精和伊红染色或者通过免疫组化(IHC)检测胰岛素表达。对于IHC,室温下用3%过氧化氢处理切片15分钟并用5%BSA和含有0.2%Triton X-100的PBS中的2%山羊血清封闭45分钟,随后用抗生素蛋白/生物素封闭试剂盒(Vector labs)处理。将载玻片在4℃下用兔抗小鼠胰岛素抗体(abcam;ab181547)过夜温育,接着用生物素标记的兔抗体(Vector labs)温育。切片使用利用Vectastain ABC试剂盒(Vector labs)和ImmPTcm AMEC红色过氧化物酶底物试剂盒(Vector labs)的连 续处理来显色,然后用Harris的苏木精复染并使用Permount(Fisher)封片。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.
体外T细胞分化In vitro T cell differentiation
将来自转基因小鼠的大部分脾细胞分离并在100ng/mL的ErkM或gp33肽或DCT抗原肽的存在下培养7天。对于Tcm分化,将10ng/mL的IL-15、10ng/mL的IL-21和20ng/mL的雷帕霉素添加至培养物。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.
联合治疗给药方法Combination therapy administration method
当肿瘤达到大约150mm
3的平均体积时,将体外分化的CD8+转基因T细胞以10
6细胞/200μL PBS的剂量静脉注射(i.v.)至荷瘤小鼠中。24小时后,将小鼠用不同疫苗处理。静脉注射递送RVV溶瘤病毒疫苗(2×10
8pfu)的形式经由静脉注射途径施用。
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细胞表面和细胞内染色T cell surface and intracellular staining
识别下列靶标的单克隆抗体用于流式细胞术测定: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)。采集血液、脾脏和骨髓样品并用ACK裂解缓冲液处理,从而在表面染色前除去红细胞。对于细胞内染色,在布雷菲德菌素A的存在下用1μg/mL的ErkM肽刺激细胞4小时以阻断细胞因子分泌,接着使用BD Cytofix/Cytoperm缓冲液(BD Biosciences)穿膜液。使用BD LSRFortessa流式细胞仪(BD Biosciences)检测荧光,并使用FlowJo(第10版)流式细胞术分析软件(Tree Star,Ashland,OR)分析数据。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).
T淋巴细胞的体内清除In vivo clearance of T lymphocytes
以250μg/200μL的剂量,间隔48小时,2次腹膜内(i.p.)注射抗小鼠CD4、CD8(分别由GK1.5和2.43杂交瘤产生,购自美国典型培养物保藏中心(American Type Culture Collection,ATCC)),或Thy1.抗体(30H12,来自BioXcell),实现T细胞的清除。用同型对照抗体(HRPN from BioXcell)以相同的模式进行处理。为了长时间观察,使用每两周一次地腹膜内注射相应的抗体来维持清除。在T细胞疗法前1天以3mg/小鼠的剂量腹腔内施用环磷酰胺(Sigma)。清除效率通过PBMC的FACS分析在处理过程中的不同时间点监测。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.
组织学Histology
使用Leica Bond Rx自动染色机(Leica Biosystem)对来自福尔马林固定的石蜡包埋的组织的切片进行免疫组化。将载玻片脱蜡,在用大鼠抗小鼠CD8α抗体(1:1000稀释;克隆4SM15,来自ThermoFisher Scientific)染色之前,用Leica Bond表位修复缓冲液#2(Leica Biosystems)预处理20分钟。使用Leica Bond高分子精确检测试剂盒(Leica Biosystems)显色,用兔抗大鼠抗体(1:100,Vector labs)替换之后的主要组分。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).
实施例1:基于VSV棒状溶瘤病毒构建的减毒突变株(RV-Mut)的病毒拯救情况Example 1: Viral rescue of an attenuated mutant strain (RV-Mut) constructed based on VSV rod-shaped oncolytic virus
基于VSV病毒高效的拯救系统,构建了不同的减毒株体系,随机突变单点多点的病毒拯救情况。进而确认是否可以任意对VSV病毒的M蛋白进行突变,均可以获得病毒粒子(即是否可以进行任意突变,病毒粒子均可以被拯救)。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).
上述病毒拯救系统构建的具体步骤如下:The specific steps of the above virus rescue system construction are as follows:
1、BSR-T7细胞铺6孔板,使细胞量达到4×10
5个/孔,铺板14-16h后加入vT7,病毒感染4h后,进行转染。
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、使用opti-MEM培养基对质粒进行稀释。其中,质粒总量为5μg,再加入7.5μl PLUS Reagent。使用培养基对10μl Lipofectamine LTX进行稀释。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、将LTX混合液与DNA混合液等体积混合,室温孵育。3. Mix the LTX mixture with the DNA mixture in an equal volume and incubate at room temperature.
4、将6孔板中的培养基换成Opti-MEM培养基,将步骤3中的混合液逐滴加入培养细胞的6孔板中,轻轻摇动6孔板,使均匀分布在6孔板内。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、转染6-8h后,吸去转染试剂,加入新鲜的完全培养基。5. After 6-8 hours of transfection, the transfection reagent was aspirated and fresh complete medium was added.
6、培养72h后收获细胞上清,使用0.22μm滤器进行过滤。6. After 72 hours of culture, the cell supernatant was harvested and filtered using a 0.22 μm filter.
利用本领域常规的方法对病毒粒子是否成功拯救进行检测。病毒包装成功率的检测结果如图1所示。The virion is successfully rescued using conventional methods in the art. The test results of virus packaging success rate are shown in Figure 1.
图1的结果证明,RV病毒M基因的111位氨基酸只能由亮氨酸突变为苯丙氨酸,突变为其它氨基酸严重影响到病毒粒子的拯救,病毒M蛋白的第221位氨基酸只能替换为苯丙氨酸,若该位置氨基酸替换为丙氨酸,制造不出重组的病毒。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.
从图1所示的病毒的包装成功率的统计列表中反映出,在致病基因M的特定位点突变能够获得减毒株,氨基酸的突变选择并不是任意选择,而是需要遵循特定的规律,才可以制造出特定的病毒粒子。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.
实施例2:不同RV-Mut病毒株的病毒滴度检测,正常细胞MEF和肿瘤细胞LLC中,包括RV-4Mut在内的不同减毒株在不同时间点的病毒复制情况。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.
在MEF/LLC细胞培养液中,分别加入如下病毒:VSV-GFP-WT、RV-GFP-M51R、RV-GFP-M51R-V221F-G226R(RV-3Mut)、RV-GFP-G21E-M51A-L111F-V221F(RV-4Mut)各200PFU,检测病毒株产生的病毒的滴度(TCID50)。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.
检测上述病毒的滴度的具体步骤如下:The specific steps for detecting the titer of the above virus are as follows:
1、在6孔培养板中每孔加入Vero(LLC/Hela)细胞悬液3mL,使细胞量达到4×10
5个/孔,共6个孔,37℃,5%CO
2培养16h。
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、每个孔分别加入病毒RV-WT、RV-M51R、RV-M51R-V221F-G226R、RV-G21E-M51A-L111F-V221F各200PFU,设正常细胞对照2个孔。在12h、24h、48h、72h、80h、96h各时间点收获细胞上清100μl。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、在96孔培养板中每孔加入Vero细胞悬液100μl,使细胞量达到1×10
4个/ml,37℃,5%CO
2培养16h。
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、在1.5ml EP管中将步骤2中收获的上清作连续10倍的稀释,从10
-1~10
-11,共11个滴度。
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、将稀释好的上清接种到96孔培养板中,每一稀释度接种一列共8孔,每孔接种100μl。设正 常细胞对照组一列。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.
6、48h后观察每孔细胞荧光情况,有荧光则记为此孔被感染。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、按Karber法计算TCID50。7. Calculate TCID50 according to the Karber method.
上述病毒的滴度的检测结果如图2所示。The results of the titer of the above virus are shown in Fig. 2.
从图2A-2B中可以发现,RV-4Mut在体外肿瘤细胞中在早期感染复制释放到上清中的病毒粒子数较对照病毒大大降低,但是当感染时间延长到5天时,病毒感染肿瘤细胞释放的病毒载量与野生病毒趋于一致,证明RV-4Mut具备缓慢感染肿瘤细胞的特性。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.
从图2C-2D中可以发现,RV-4Mut减毒株在感染正常的成纤维细胞时,原始上清中加入的病毒载量为200PFU,在24h和120h这2个时间点检测上清中不同减毒株的病毒滴度,发现RV-4Mut减毒株较原始上清中的病毒粒子数降低(120h的感染上清液中显著降低),而对照组都呈现了不同程度的上升,表明RV-4Mut株在正常的成纤维细胞中不具备复制能力,而野生病毒株及对照病毒株RV-M51R和RV-3Mut仍具备较强的感染复制能力(从图2中可以得出,与RV-4Mut减毒株的感染复制能力相比,野生病毒株及对照病毒株RV-M51R和RV-3Mut存在高达三个数量级的差异)。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.
实施例3:检测不同病毒载量的RV-4Mut在多种肿瘤细胞的体外杀伤的对比情况Example 3: Comparison of in vitro killing of RV-4Mut with different viral loads in various tumor cells
通过MTT检测的方法,检测不同病毒载量的RV-4Mut减毒株对不同肿瘤细胞的体外杀伤情况。In vitro killing of different tumor cells by different viral load of RV-4Mut was detected by MTT assay.
上述检测方法的具体步骤如下:The specific steps of the above detection method are as follows:
1、在96孔培养板中每孔加入(LLC/Hela/MEF)细胞悬液100μl,使细胞量达到1×10
4个/孔,37℃,5%CO
2培养16h。
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、分别将病毒RV-WT、RV-M51R、RV-M51R-V221F-G226R(RV-3Mut)、RV-G21E-M51A-L111F-V221F(RV-4Mut)稀释到MOI(感染复数)分别为0.001,0.01,0.1,1.0,每一稀释梯度接种4个孔,每孔100μl,37℃,5%CO
2培养40h。
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、弃去96孔培养板中的上清,加入新鲜培养基,加入MTT溶液,20μL/孔。37℃,5%CO
2培养4h。
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、将96孔板离心,设置转速2500rpm/分钟,室温离心5分钟。4. Centrifuge the 96-well plate, set the rotation speed at 2500 rpm/min, and centrifuge at room temperature for 5 minutes.
5、使用1mL一次性无菌注射器,轻轻吸掉上清。5. Using a 1 mL disposable sterile syringe, gently aspirate the supernatant.
6、向每孔中加入DMSO,100ul/孔,37℃放置10分钟。6. Add DMSO to each well, 100 ul/well, and place at 37 ° C for 10 minutes.
7、使用多功能酶标仪,震荡2分钟,在570nm或490nm波长下,测定各孔的OD值。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.
上述不同病毒株的体外杀伤情况的检测结果如图3所示。The results of the in vitro killing of the above different strains are shown in FIG.
如图3A-3D所示,在病毒感染复数为0.01时,LLC和Hela中RV-4Mut对肿瘤细胞的裂解能力与对照组病毒相比有轻微的下降,而当RV-4Mut株病毒感染复数提高到0.1时,无论对LLC或者Hela等肿瘤细胞的直接杀伤能力显著提高,与野生株病毒相比没有显著差异,表明RV-4Mut尽管有四个位点的突变,但是保留了野生病毒特有的对肿瘤细胞的嗜侵性,具备较强的持续杀伤肿瘤细胞的能力。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.
同时在MEF细胞上重复相同的实验,如图3E-3F所示,实验结果显示RV-4Mut株在MOI为0.1或者0.01时对正常细胞的毒副作用与PBS组相比不存在显著差异。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.
MTT实验的结果表明,四突变株RV-4Mut在体外对正常细胞的不存在显著的毒副作用,同时不会引起正常细胞的凋亡和坏死。而野生株和对照组RV-M51R病毒对正常细胞都存在一定的毒副作用,野生株毒副作用最强,RV-4Mut病毒株在三种减毒株里的毒副作用最低,安全性最好。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.
实施例4:不同减毒株嵌合的外源基因包括RV-4Mut在内的不同减毒株在细胞中的外源蛋白持续表达能力情况。Example 4: Peripheral expression of foreign genes in different attenuated strains including different attenuated strains including RV-4Mut.
通过FACS流式检测不同减毒株嵌合的外源基因GFP在不同细胞中的表达情况。The expression of the exogenous gene GFP of different attenuated strains in different cells was detected by FACS flow cytometry.
上述检测的具体步骤如下:The specific steps of the above detection are as follows:
1、在48孔培养板中每孔加入Vero/MEF)细胞悬液100μl,使细胞量达到2×10
4个/孔,37℃,5%CO
2培养16h。
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、每个孔分别加入病毒RV-GFP-WT、RV-GFP-M51R、RV-GFP-M51R-V221F-G226R(RV-3Mut)、RV-GFP-G21E-M51A-L111F-V221F(RV-4Mut)各100PFU,每种病毒21个孔,空白对照组12个孔。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、在各时间点(24h、36h、48h、60h、72h、84h、96h)分别收获细胞,每种病毒收获3个细胞孔的细胞,空白对照组收获1个孔的细胞,均用400ul PBS重悬细胞,取100μL细胞悬液用Life Launch Attune NxT–Next型流式细胞仪进行分析,统计GFP阳性细胞总数。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.
上述流式检测的检测结果如图4所示。The detection results of the above flow detection are shown in FIG.
如图4A所示,RV-4Mut病毒株在Vero细胞(干扰素缺陷)中随着时间的推移,GFP阳性的细胞比例持续升高,感染72h后,被RV-4Mut病毒株感染阳性细胞超过野生病毒对应的比例,表明RV-4Mut病毒株与其它候选病毒株相比,在干扰素表达能力缺陷的细胞中具备更强的持续复制扩增能力,在Vero工程细胞系中复制的时间较其它对照病毒株延长,更符合工业规模生产的要求。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.
而在MEF细胞中,如图4B所示,将4种病毒的初始病毒载量增加200倍后,RV-4Mut是所有病毒中,GFP阳性的细胞没有随着时间的迁移显著增加的,而MEF细胞的干扰素通路是正常的,当外界病原感染时,会快速产生应答,而图6B的结果证明RV-4Mut与对照减毒株相比具备更高的安全性(MEF细胞中病毒感染复制情况),潜在的原因是RV-4Mut对干扰素更敏感。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.
因此,当病毒GMP工业化生产时,在Vero工程化细胞(干扰素缺陷)中扩增病毒时,RV-4Mut病毒株具备了高效持续的表达外源基因的能力,与对照减毒株相比,具备了天然的优势,同等体积的反应罐体系中,可以生产制备更高滴度的病毒制品。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.
实施例5:不同减毒株嵌合的相同的外源基因GFP在肿瘤细胞(A549)和Vero细胞中的表达情况Example 5: Expression of the same exogenous gene GFP chimeric in different attenuated strains in tumor cells (A549) and Vero cells
通过流式检测不同减毒株嵌合的相同的外源基因(GFP)在不同肿瘤细胞中(Hela和A549)的表达情况。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.
上述检测的具体步骤和实施例4相同。The specific steps of the above detection are the same as in the fourth embodiment.
上述流式检测的检测结果如图5A-5B所示。The results of the above flow detection are shown in Figures 5A-5B.
从图5B来看,通过流式染色活细胞的比例,在A549细胞中,RV-4Mut的活细胞比例都是比较高的,有利于病毒在细胞内复制,最终通过制造更多的病毒粒子来扩大感染肿瘤细胞的范围。同时在Vero细胞中持续表达外源基因的检测中(图5A),在体外感染扩增72h后,RV-4Mut的外源持续表达蛋白的能力显著高于其它对照组,进一步检测发现RV-4mut具备极佳的表达外源蛋白的能力。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.
上述结果表明,RV-4Mut具备在肿瘤细胞中持续复制的能力,与对照组相比,外源基因的表达的持续性更好,优异性显著。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.
实施例6:不同的细胞系对不同减毒株病毒的抗病毒干扰素的表达量Example 6: Expression levels of antiviral interferons of different attenuated strains of different cell lines
将不同的减毒株体外感染MEF或者肿瘤细胞系LLC,通过RT-PCR实验技术,分别检测不同的细胞系对不同减毒株病毒的抗病毒干扰素的表达量。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.
上述检测的具体步骤如下:从LLC,MEF细胞中用TRIzol(Invitrogen)提取总RNA,利用PrimeScript RT Reagent Kit with gDNA Eraser(Takara)反转录试剂盒逆转录成cDNA,并用
480 SYBR Green I Master(Roche)染料进行染色在
480定量PCR仪上检测各个基因的Ct值。用ΔΔCt法来计算目的基因IFN-β、VSV-G相对于管家基因GAPDH的表达量。
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.
上述检测的检测结果如图6所示。The detection results of the above detection are shown in FIG. 6.
如图6B和图6D所示,四种病毒分别侵染MEF和肿瘤细胞LLC,MEF被上述四种病毒感染后,IFN的mRNA水平的变化情况如图6B所示,RV-4Mut的诱导干扰素的能力与另外两株减毒株相比最低。与此同时,病毒本身复制能力VSV-G的mRNA水平与对照组相比(图6A),RV-4Mut在MEF细胞中的RNA水平非常低,进一步佐证RV-4Mut病毒的安全性高,同时在肿瘤细胞LLC进行相同的实验发现,与MEF细胞中的现象截然相反,RV-4Mut减毒株刺激LLC细胞的干扰素的量最高,同时在肿瘤细胞中病毒复制能力与对照组相比也是最强的(图6C)。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).
上述实验结果证明了RV-4Mut的对肿瘤细胞的高选择性侵染,同时诱导肿瘤细胞干扰素表达能力的增强(肿瘤内干扰素量的增加会显著提高局部免疫细胞杀伤肿瘤细胞的能力),同时不影响病毒的复制能力,而正常细胞中针对该RV-4Mut的干扰素诱导能力并没有显著增强,进一步证明RV-4Mut具备连续刺激肿瘤局部免疫细胞并产生持续抗肿瘤的免疫应答。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.
实施例7:RV-4Mut与其它溶瘤病毒减毒株在神经毒性上的差异的验证Example 7: Verification of differences in neurotoxicity between RV-4Mut and other oncolytic virus attenuated strains
为了验证RV-4Mut与其它病毒株在神经毒性上的差异,选取6周龄的Balb/c小鼠作为实验对象,每组10只小鼠,每组小鼠滴鼻50μl的10
8PFU/mL的病毒稀释液(前述病毒分别为VSV-GFP-WT(即RV-GFP)、RV-GFP-M51R、RV-GFP-M51R-V221F-G226R(RV-3Mut)、RV-GFP-G21E-M51A-L111F-V221F(RV-4Mut)),隔天滴鼻一次,共滴鼻2次。已知通过鼻腔感染野生的棒状病毒,会使部分实验小鼠后肢神经麻痹,严重的小鼠会死亡,持续记录并统计5组实验小鼠,在滴鼻实验后的体重变化,小鼠后肢瘫痪情况,小鼠毛发顺滑程度及是否有流感样症状。
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.
上述实验的具体统计结果如表1和图7所示。The specific statistical results of the above experiments are shown in Table 1 and Figure 7.
表1 RV-4Mut在动物模型中的神经毒性鉴定Table 1 Neurotoxicity identification of RV-4Mut in animal models
从表1和图7来看,RV-WT滴鼻组小鼠,10只全部发病,同时症状严重,病情恶化迅速最终,死亡率达到60%(如图7B所示10只接种野生病毒株的小鼠死了6只),均出现了后肢麻痹瘫痪现象,病症持续时间较长,而另外3株减毒株的症状稍有减轻,尤其是RV-4Mut滴鼻组小鼠仅有5只小鼠有轻微症状,同时RV-4Mut组小鼠恢复能力也是最快的。与此同时,RV-4Mut组的小鼠均没有出现后肢瘫痪现象,只有轻微的毛发杂乱的现象,如图7A所述的一样,RV-4Mut组是体重下降最不明显的实验组,与PBS滴鼻组的变化趋势一致。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.
上述实验结果证明了RV-4Mut减毒株在动物模型中的安全性的优势是非常显著的,过量的RV-4Mut对小鼠的生存情况没有任何不良影响。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.
实施例8:建立小鼠肺癌模型(LLC肿瘤细胞),验证RV-4Mut减毒株的药效Example 8: Establishment of a mouse lung cancer model (LLC tumor cells) to verify the efficacy of the attenuated strain of RV-4Mut
进一步通过建立小鼠肺癌模型,通过局部瘤内给药对减毒株的性能进行测试,验证RV-4Mut减毒株的药效。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.
上述小鼠肺癌模型的建立具体步骤如下。The specific steps for establishing the above mouse lung cancer model are as follows.
按每只CB7BL/6小鼠皮下接种1.0×10
6(200μL)LLC细胞。每隔1天测量肿瘤大小,按如下公式计算:1/2×M2×M1
2(M1:短径,M2:长径)。待每组小鼠肿瘤体积生长超过200mm
3后,分别在第12天,第14天和第16天给予10
6PFU(20ul)的瘤内注射病毒进行治疗,连续观察记录肿瘤体积的变化情况。
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.
上述实验的结果如图8所示。The results of the above experiment are shown in Fig. 8.
如图8A所示,连续3次隔天的瘤内注射RV-4Mut治疗能有效抑制肿瘤的生长趋势,大大延缓小鼠的生命周期,通过独立分析每只小鼠接受三种不同减毒株治疗效果发现RV-4Mut与对照组RV-GFP-M51R均能将30%的小鼠肿瘤缩小直至消失,达到完全缓解,近40%的小鼠肿瘤的生长速度得 到有效抑制,部分缓解。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.
从图8B可以发现,RV-4Mut免治疗组的小鼠肺部转移的数目最少。进一步通过对荷瘤小鼠接受治疗后的有效生存期记录发现,实验组的存活率最高,接近40%的小鼠在近2个月内维持正常的生命状态,同时部分肿瘤模型小鼠的肿瘤逐渐消退,彻底治愈,进一步表明RV-4Mut减毒株给予实体瘤治疗效果显著,具备很好的临床应用价值。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.
实施例9:不同肿瘤细胞皮下移植瘤模型建立图Example 9: Establishment of a subcutaneous xenograft model of different tumor cells
在本公开中,涉及到鼠结肠癌(MC38),黑色素瘤(B16)鼠肺癌模型(LLC),具体肿瘤模型的方案如下:在第0天开始皮下接种肿瘤细胞,8-12天后待小鼠肿瘤体积接近100mm
3开始给药,溶瘤病毒RV是采用瘤内给药,间隔一天,共给药三次,而对应的RVV肿瘤疫苗是静脉给药,尾静脉给药一次。持续观察并记录小鼠肿瘤体积变化情况。
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.
如图9所示实施案例8中的RV-4Mut及其它突变株,只能采取瘤内多次给药的方式,才具备控制肿瘤的生长,进一步以RV病毒骨架开发的RVV溶瘤病毒疫苗,已经实现了多种治疗方式,静脉给药的方式的增加降低了临床局部介入给药的复杂度,同时RVV溶瘤病毒疫苗只需要给药一次,降低了给药成本及多次给药的繁琐。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. .
实施例10:溶瘤病毒RV静脉给药与瘤内给药药效比较(肺癌)Example 10: Comparison of the efficacy of intravenous administration of oncolytic virus RV with intratumoral administration (lung cancer)
首先建立小鼠单侧肺癌模型(LLC肿瘤细胞),验证RV溶瘤病毒静脉和瘤内给药的药效上述小鼠肺癌模型的建立步骤如下。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.
按每只CB7BL/6小鼠皮下接种1.0×10
6(200μL)LLC-t2细胞。每隔1天测量肿瘤大小,按如下公式计算:1/2×M2×M1
2(M1:短径,M2:长径)。待每组小鼠肿瘤体积生长超过200mm
3后,分别在成瘤后第12天,第14天和第16天给予10
6PFU(20ul)的瘤内注射病毒进行治疗,另外一组RV静脉给药也是按照上述给药时间,静脉给药3次,观察三组不同的给药组的小鼠肿瘤体积变化及实验终止点小鼠肺部组织的癌细胞转移情况。
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.
如图10A,RV瘤内给药治疗组的肿瘤控制效果显著优于静脉给药组,有近30%的小鼠肺癌得到有效控制,而静脉给药组所有的小鼠肿瘤体积最终都超出控制范围,进一步通过实验终止点取小鼠肺部组织,进行荧光标记肿瘤细胞观察发现,瘤内给药溶瘤病毒RV对肺癌的转移的作用显著优于对照组及静脉给药组,直接证明溶瘤病毒RV目前只能采取瘤内给药的方式,静脉给药无法实现有效的免疫治疗效果。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.
实施例11:溶瘤病毒疫苗RVV静脉给药药效评价Example 11: Evaluation of the efficacy of intravenous administration of oncolytic virus vaccine RVV
如图11B所示溶瘤病毒疫苗RVV(gp33)免疫治疗可以诱导MC38-gp33肿瘤的消退,其疗效的产生是诱导增强了内源性肿瘤特异性T细胞的数量(图11A)。具体实验方案对接种MC38-gp33肿瘤的C57BL/6小鼠给药RV、RVV-gp33(2×10
8pfu;静脉给药.)。在指定的时间点对肿瘤体积进行监测,用mm
3表示。图11A是检测机体内源特异性CD8
+T的表达干扰素的能力,RVV-gp33在给药5天后,内源的 特异性T细胞的活性达到峰值,在MC38结肠癌小鼠模型中,静脉给药后与对照组RV(没有抗原基因的表达)相比,溶瘤病毒疫苗RVV(gp33)有效抑制了肿瘤的生长,甚至部分小鼠的肿瘤得到了控制并消除,尽管有一些个体的肿瘤治愈后又复发了,直接表明,溶瘤病毒疫苗可以通过静脉给药的方式有效控制肿瘤的生长。
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.
实施例12:溶瘤病毒疫苗(OVV)联合Tcm对表达自身肿瘤模型(DCT)的治疗效果Example 12: Therapeutic effect of oncolytic virus vaccine (OVV) combined with Tcm on expression of a self-tumor model (DCT)
将B16细胞自身肿瘤抗原DCT通过基因工程克隆到RVV肿瘤疫苗上,同时制备DCT抗原肽刺激的Tcm,具体方法参照离体Tcm培养和Tcm接种方案以及T细胞分化方案,进一步在C57BL/6小鼠接种恶性黑色素肿瘤6天后,分别给予10
6/只RVV(DCT)+Tcm肿瘤疫苗(2×10
8pfu)、单独TCM、单独RVV疫苗或RV治疗,如图12所示,只有溶瘤病毒疫苗与Tcm联合治疗能够将黑色素肿瘤有效清除根治,同时通过长达半年的时间观察,治愈的小鼠没有再次复发。
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.
实施例13:溶瘤病毒疫苗(RVV)联合Tcm在肿瘤抗原突变的恶性骨肉瘤肿瘤中的治疗效果Example 13: Therapeutic effect of oncolytic virus vaccine (RVV) combined with Tcm in malignant osteosarcoma tumors with tumor antigen mutations
受到弹状病毒溶瘤病毒疫苗(RVV)同时引起外周中的已经存在的中心记忆性T细胞(Tcm)的有效扩增和快速浸润至肿瘤中的独特的能力的启发,我们探索了联合使用体外分化的肿瘤抗原特异性Tcm的过继T细胞疗法疗法与溶瘤病毒(RVV)疫苗来治疗建立的实体瘤。用CMS5细胞(一种源自ERK2突变基因中表达新抗原决定簇(ErkM136-144,QYIHSANVL)的甲基胆蒽诱导的纤维肉瘤细胞)皮内接种野生型BALB/c小鼠。在存在IL-15、IL-21和雷帕霉素的情况下培养和扩增来自DUC18转基因小鼠的ErkM136-144特异性CD8+T细胞,其促使获得典型的Tcm表型(CD62L
+CD44
+)。
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 + ).
试验方法:BALB/c小鼠接种CMS5细胞7天后,过继性输注DUC18记忆T细胞(10
6个细胞/只),在输注记忆T细胞1天后,利用特异性病毒疫苗(RVV-mERK)对小鼠进行治疗,以RV-ErkM,Tcm或PBS单独给药的小鼠作为对照,在治疗后指定的时间,监测接种CMS5小鼠的肿瘤体积0天表示接种疫苗当天,肿瘤体积单位为mm
3。肿瘤体积1000mm
3作为存活率分析终点。
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.
试验结果:肿瘤接种6天后,静脉注射10
6个DUC18 Tcm、或10
8pfu的RVV-ErkM、或DUC18 Tcm24h之后是RVV-ErkM的组合来治疗小鼠。先前的实施案例已经证明了静脉给药是实现在外周的抗原呈递从而加强用于溶瘤的Tcm和肿瘤靶向,通过借助RVV溶瘤病毒疫苗以及诱导T细胞浸润是治疗的最佳途径。图13中显示单独Tcm或单独RVV疫苗均不会对肿瘤生长具有显著的影响,而Tcm和RVV疫苗的组合诱导了完全肿瘤消退并且显著地延长存活然而,单独效应DUC18T细胞(Tcm)或者RVV-ErkM单独给药未能在所有经治疗的小鼠中诱导完全且持续的肿瘤消退,由Tcm+RVV-ErkM治疗诱导的持续消退,随后的存活相比于Tcm治疗组显著地延长。
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.
证实了需要抗原特异性RVV接种来扩增疗法的T细胞并且诱导浸润至肿瘤组织中。与该观点一致,用非溶瘤疫苗加强不及RVV疫苗有效。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.
实施例14:结肠癌(MC38-gp33)肿瘤的完全治愈需要溶瘤RVV肿瘤疫苗和肿瘤特异性T细胞联合治疗Example 14: Complete cure of colon cancer (MC38-gp33) tumor requires oncolytic RVV tumor vaccine and tumor-specific T cell combination therapy
试验方法:C57小鼠接种MC38-gp33细胞6天后,过继性输注gp33记忆T细胞(10
6个细胞/只),在输注记忆T细胞1天后,利用特异性溶瘤病毒疫苗(RVV-gp33)对小鼠进行治疗,以RVV-gp33,Tcm或PBS单独给药的小鼠作为对照,在治疗后指定的时间,监测接种结肠癌小鼠的肿瘤体积,0天表示接种疫苗当天,肿瘤体积单位为mm
3。肿瘤体积1000mm
3作为存活率分析终点。
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.
试验结果:肿瘤接种6天后,静脉注射10
6个Tcm、或10
8pfu的RVV-gp33、或Tcm 24h之后是病毒疫苗的组合来治疗小鼠。图14中显示单独Tcm或单独RVV疫苗均不会对肿瘤生长有显著的控制作用(与PBS治疗组相比),而Tcm和RVV疫苗的联合治疗组诱导了完全肿瘤消退并且显著地延长存活,尽管给药25天后有部分小鼠的肿瘤复发,但是与RVV-gp33单独给药未能在所有经治疗的小鼠中诱导完全且持续的肿瘤消退,由Tcm+RVV-gp33诱导的结肠癌肿瘤消退,并且随后的存活率相比单独给药组的治疗显著地延长。
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.
实施例15:溶瘤病毒疫苗RVV与Tcm联合治疗在B16-gp33肿瘤模型中诱导大量抗肿瘤T细胞,有效清除根治恶性黑色素瘤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
在联合治疗期间,肿瘤影响内源性ErkM-反应性CD8+T细胞的扩增和存活,为了进一步理解RVV疫苗如何影响机体T细胞的进程,我们还在外周监测了T细胞反应。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.
试验方法:对接种B16-gp33肿瘤的C57BL/6小鼠给药PBS,RVV-gp33(2×10
8pfu;静脉)、P14TCM(10
6/小鼠)或RVV-gp33+P14 Tcm。在指定时间点监测肿瘤体积,以mm
3表示。在给药后指定的时间点评估gp33特异性CD8+T细胞的应答和CD8
+T细胞在外周血循环中产生的百分比(该CD8
+T细胞产生的IFNγ由gp33多肽体外进行刺激)。在治疗后指定的天数采血,评估gp33特异性CD8
+T细胞反应频率(结果如图15A所示)。评价RVV(gp33)联合Tcm在表达病毒抗原的黑色素瘤中的治疗效果(结果如图15B所示)。
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).
实验结果:如图15A所示,RVV可以诱导机体内源性抗原特异性CD8的比例不到10%,而RVV-gp33+P14 Tcm联合治疗组诱导的比例超过30%,而P14 Tcm治疗组几乎不能诱导内源性的CD8的反应,进一步从图15B可以发现,联合给药组的治疗效果也是最好的,几乎所有的小鼠肿瘤得得到了控制,最终达到治愈的效果,而单独给药组没有治愈的情况,在RVV治疗组有部分小鼠的肿瘤得到控制,Tcm给药组也是没有显著疗效。进一步得到联合给药组产生的绝佳的治愈效果与该组合治疗诱导内源特异性的CD8+T细胞有直接关系。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.
实施例16:联合治疗小鼠中CD8
+T细胞的肿瘤浸润及在肿瘤中的定位
Example 16: Tumor infiltration and localization of tumors in CD8 + T cells in mice treated with combination
为了直接观察CD8
+T细胞的肿瘤浸润和它们在肿瘤中的定位,我们在Tcm或Tcm+RVV(ErkM)溶瘤病毒疫苗在接种之后第5天将肿瘤组织染色。用抗CD8抗体染色的CMS5肿瘤组织的显微图像显示,在指定的治疗方法诱导下,肿瘤核心和肿瘤组织外围的T细胞浸润情况。左侧为整个肿瘤的低放大图像,右侧为肿瘤外围(黑框勾画)和肿瘤核心(蓝框区域)的高倍放大图像。比例尺长度分别为500μm和200μm。
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.
试验结果:用抗CD8抗体染色的CMS5肿瘤组织的显微图像显示,在指定的治疗方法诱导下,肿瘤核心和外围的T细胞相对浸润。右侧为整个肿瘤的低放大图像,右侧为肿瘤外围(黑框勾画)和肿瘤核心(蓝框勾勒)的高倍放大图像。比例尺长度分别为500μm和200μm。如图16中所示,CD8
+T细胞的肿瘤周围分布在仅Tcm疗法和Tcm联合RVV溶瘤病毒疫苗接种之后是明显的,但是仅在RVV加强之后观察到深入渗透至肿瘤组织中的高密度的CD8
+T细胞,证实了RVV疫苗+Tcm较常规疫苗具有明显优势。最后,在Tcm+RVV-ErkM治疗之后的100%的长期存活者(60天以上)在停止治疗2个月之后拒绝CMS5细胞的再次接种接种并且显示显著延长的存活,表明形成了有效的免疫记忆,溶瘤病毒疫苗联合Tcm治疗促使更多转移性CD8
+T细胞向肿瘤核心区域的浸润(图16)。
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).
实施例17:内源性T细胞在联合治疗之后在哺乳动物中形成长期抗肿瘤免疫Example 17: Endogenous T cells form long-term anti-tumor immunity in mammals after combination therapy
为了确定内源性T细胞特别是识别非靶向肿瘤抗原的那些的存活(persistence)和保护功能,我们决定用CMS5细胞的皮内注射的骨肉瘤模型(即肿瘤自身抗原突变)来证明溶瘤病毒疫苗与T细胞联合治疗控制肿瘤复发是通过刺激内源性淋巴细胞。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.
对NRG小鼠皮下接种CMS5细胞,7天后,过继转移DUC18 Tcm(10
6个细胞/只)。Tcm输入后1天,小鼠接种RVV-ErkM疫苗(2×10
8pfu/只)。单独应用RVV-ErkM、TCM或PBS的小鼠作为对照。在指定的治疗后记录荷瘤小鼠的肿瘤体积变化情况,用mm
3代表肿瘤体积。
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 .
试验结果:为了确定需要哪种淋巴细胞群用于针对CMS5r的保护,我们在再次接种之前在C57肿瘤模型鼠进行了CD8+、CD4+或这两种T细胞亚型的缺失小鼠(NRG小鼠)。如图17所示,尽管联合治疗组在所有治疗组中起始控制肿瘤生长的效果最好,但是随着给药后天数的延长,被抑制生长的肿瘤全部复发,最终与其它治疗方式一样,最终导致小鼠的死亡。这些结果证明内源性T淋巴细胞且对于形成具有广泛抗原特异性的长期保护性免疫也是至关重要的。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.
实施例18:维持内源性T淋巴细胞的给药治疗方式可预防肿瘤复发Example 18: Maintenance of endogenous T lymphocyte administration can prevent tumor recurrence
C57BL/6小鼠真皮内接种B16-gp33肿瘤(6天),给予过继细胞治疗,10
6gp33-特异性Tcm.静脉给药,然后用5x 10
7pfu RVV-gp33肿瘤疫苗静脉注射荷瘤小鼠。在Tcm给药前1天给予抗体(阴性对照和αThy1.2实验组),后每周给药1次,共持续三周,在Tcm的前一天注射了一次CPX。(A)监测肿瘤体积和(B)小鼠存活情况予以显示。数据是两组独立实验的代表性结果,每组n=5。CPX化疗和αThy1.2抗体治疗导致机体内源性T细胞数量降低。
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.
我们通过在不同的肿瘤模型中评价Tcm+RVV接种的联合平台(combination platform)从而确定内源性T淋巴细胞在实现完全和持久的肿瘤消退方面的作用。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.
试验结果:如图18中的A中所示,在接受联合治疗的小鼠中实现了完全和持久的肿瘤消退,证实了用于靶向不同的肿瘤抗原和/或引入不同的溶瘤病毒骨架的该联合平台的效力和灵活性。然而,有趣的是,由于在初始消退之后的肿瘤复发,在联合治疗之前用抗Thy1.2抗体或CPX治疗的小鼠显示存活率的显著降低(图18中的A和B),这加强了先前存在的宿主T淋巴细胞的重要性,它们可能被拓 宽免疫治疗肿瘤的多样性选择。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.
实施例19:IL21、IL15和雷帕霉素的特定组合对于在联合疗法中具有最佳抗肿瘤作用的抗原特异性中心记忆CD8+T细胞的诱导分化是必须的。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.
如图19所示在使用指定的IL15、IL21和雷帕霉素的组合离体培养7天之后的扩增倍数(fold expansion)(A)以及P14小鼠中CD8+T细胞的CD62L(B)和CD44(C)表达水平。使用游标卡尺来测量肿瘤体积并且表示单位为mm
3。图示D-G展现出了在用溶瘤病毒疫苗RVV-gp33处理的小鼠,在输注指定的IL15、IL21和雷帕霉素的组合培养的P14细胞之后的肿瘤体积的结果(D-G)。图D-G中示出了将肿瘤大小维持在低于1000mm
3(肿瘤测量终点截止值)的统计图。
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.
雷帕霉素、IL15和IL21的特定组合对于本文所述的在联合疗法中具有最佳抗肿瘤作用的抗原特异性中心记忆CD8+T细胞的离体培养、扩增和分化是必须的。每个和所有这些组分对于产生这样的Tcm细胞都是必须的,且在没有这些组分中的一个或多个的情况下所产生的细胞在体内输注和OV接种后具有次佳的T细胞扩增和/或降低抗肿瘤作用。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在驱动CD8+T细胞扩增和中心记忆分化的培养方案中是必须的。与完全组合中生长的细胞(图19中的A中的IL15/IL21/Rapa)相比,在没有IL15的情况下培养的细胞显示离体培养物的扩增受损和细胞产率减少(19中的A中的L21/Rapa)。另外,如此培养的细胞展现了改变的偏向初始的表型(图19中的C中降低的CD44水平),而不是中心记忆表型-如图19中的B和图19中的C中的在完全组合中生长的细胞所例示的。在输注和RVV-gp33刺激时,在没有IL15的情况下生长的细胞产生大小略微减少的抗原特异性CD8T细胞反应并且不能完全消退肿瘤(图19中的E)。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在将培养的细胞编程为具有最佳抗原特异性细胞溶解功能的良好品质的中心记忆表型中起到不可或缺的作用。在没有IL21的情况下培养的细胞(IL15/Rapa)具有CD62L(图19中的B)和CD44(图19中的C)的正常表达水平,具有与完全组合培养的细胞可比的离体增殖。在用VSV-gp33输注和刺激后,在没有IL21的情况下生长的细胞产生了具有大小与完全组合培养的细胞可比的体内反应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.
讨论discuss
用于选择性地增强针对遗传定义的新抗原(neoantigen)的T细胞反应性的策略目前正在开发中[65,66]。在目前的研究中,发明人探索了一种治疗平台,其将离体扩增的新抗原特异性T细胞与溶瘤病毒疫苗(OVV)组合。该策略采用“推-拉(push-pull)”机制,其中OVV促进T细胞活化和外周扩增,然后将T细胞浸润至肿瘤位点。确实,发明人证明了抗原特异性中心记忆性T细胞(Tcm)的过继疗法之后是溶瘤疫苗引发强烈的T细胞扩增、肿瘤浸润和完全肿瘤消退,这揭示了这两种治疗方法之间的强大的协同作用。更重要的是,在没有预处理(即细胞疗法之前的全身照射或淋巴细胞清除化学疗法(lymphodepleting chemotherapy))和外源性IL-2(通常用于其他抗原特异性T细胞环境中的两种佐剂)的情况下实现了很好的效力[52,53],强调了本公开的组合治疗的转化意义,可以为患者提供较强的疗效体验。此外,最重要的是,绕过预处理保留了肿瘤引发的内源性T细胞,这不仅补充了T淋巴细胞从而 消除原发性肿瘤并防止抗原变体的出现,而且还形成了用于免疫监视的长期记忆库。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.
尽管在治疗B细胞恶性肿瘤和黑色素瘤方面取得了成功,但抗原特异性T细胞对多数实体肿瘤的作用有限[67,68]。其中,阻碍了足够量的T细胞渗入肿瘤和持续足够时长杀死所有恶性细胞[69,70]。增加细胞免疫疗法的剂量可以提高其获取和杀死实体瘤的能力,但离体产生大量T细胞需要大量扩增,所述扩增不可避免地导致T细胞的过度分化和复制衰老[71,72]。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]. ].
此外,与实体瘤相关的高度抑制性微环境和异质抗原景观(landscape)通常使T细胞无效并促进抗原逃逸[73,74]。因此,为了实现实体瘤的持续消退,抗原特异性T细胞必须与可同时刺激T细胞扩增、将T细胞浸润到肿瘤中、克服肿瘤介导的免疫抑制和拓宽T细胞特异性谱的其他方法相组合。发明人之前已经证明,溶瘤疫苗可以有效地扩增肿瘤特异性Tcm,同时保留其有益的溶瘤特性,这使发明人假设因为OV疫苗的疫苗接种和溶瘤功能,OV疫苗可能代表与抗原特异性T细胞组合的理想平台[55,56]。确实,发明人在该研究中提供的证据表明,溶瘤病毒(无肿瘤抗原)和常规疫苗(非溶瘤性)都不足以与抗原特异性T细胞协同作用,并且破坏已建立的实体瘤需要疗法的T细胞的扩增和肿瘤浸润。为此,因为Tcm的有效的植入和增殖能力,Tcm优于TEFF(效应T细胞),这与抗原特异性T细胞领域日益受到重视的概念一致[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].
发明人进行了可能对抗原特异性T细胞的当前临床实践和合理设计具有重要意义的两个额外的观察。首先,尽管在Tcm+OVV肿瘤疫苗接种后在免疫缺陷小鼠中实现肿瘤消退,但肿瘤在2周内再次发生。很明显,复发的肿瘤细胞不再含有疗法的T细胞的表位靶标。该结果似乎支持这样的观点,即靶向具有对于一组受限抗原特异的T细胞群的肿瘤可能导致抗原阴性肿瘤变体的选择性派生。然而,在野生型动物中始终实现持久的消退,这表明可以通过在Tcm期间动员内源性T细胞来解决肿瘤异质性和/或免疫逃逸,甚至靶向单一抗原。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.
发明人观察到Tcm+OVV治疗前清除CD4
+或CD8
+T细胞不影响初始肿瘤消退,但导致复发,这表明疗法的T细胞与内源性T细胞之间存在协同作用。这显示,疗法的T细胞在调节肿瘤减积(debulking)(根除ErkM+肿瘤细胞)中起主要作用,而消除逃逸的变体(ErkM-肿瘤细胞)需要内源性T细胞。解释内源性T细胞的活化的一种可能的机制是表位扩散(epitope spreading),即涉及体内交叉呈递在一波免疫攻击中释放的肿瘤衍生抗原从而促进后续轮次的针对不同抗原的抗肿瘤T细胞的现象[75,76]。由于由疗法的T细胞和溶瘤病毒介导的强烈的肿瘤溶解(tumor lysis)和炎症,这些连续事件在本公开的情况下可能特别有效。然而,另一可能的机制是通过Tcm+OVV治疗释放和/或扩增预先存在的肿瘤引发的T细胞,从而提供更广泛的库(repertoire)以补充疗法的T细胞来完全根除所有肿瘤细胞。这后一种可能性由以下几个证据支持:(1)早在Tcm+OVV后2天就能检测到ErkM-特异性CD8
+T细胞的存在和增强;(2)组合治疗后5天,还显示T细胞对非靶向的新表位的反应增加,这两者都不太可能是需要激活未分化T细胞的表位扩散的结果;和(3)治疗后6天清除CD4
+或CD8
+T细胞未导致复发的事实表明,预先存在但未随后诱导的宿主T细胞与疗法的T细胞一起参与肿瘤细胞的早期清除。尽管如此, Tcm+OVV很可能可以以反映其在产生抗肿瘤免疫过程中的相对重要性的连续方式重新促进已存在的抗原特异性T细胞并通过表位扩散诱导新的T细胞应答[75,77]。在两种情况下需要做更多的工作以确定由天然发育或治疗诱导的T细胞识别的那些未鉴定的抗原是否源自肿瘤特异性突变或自体抗原。
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.
其次,认为疗法的T细胞的长期持续性是重要的,并且据报道肿瘤消退与过继疗法的T细胞克隆的持续程度之间存在正相关性[78,79]。然而,有趣的是,本公开的疗法的抗ErkM CD8
+T细胞在肿瘤消退后立即消失,而内源性ErkM特异性CD8
+T细胞长期存活并且能够提供抗原特异性保护性免疫。发明人推测,本公开中的独特观察是由于引入了溶瘤疫苗,其不仅加速了疗法的T细胞反应并加强了它们与肿瘤的相互作用,而且还参与了内源性抗肿瘤T细胞反应。确实,本公开证明疗法的T细胞主导了早期扩增和肿瘤浸润,并且负责介导最初的肿瘤消退。因此,疗法的T细胞经历肿瘤诱导的细胞凋亡,这是先前研究记录的现象[80,81]。相比而言,内源性T细胞扩增直到肿瘤消退后几天才达到峰值,表明多数增强和/或治疗诱导的内源性T细胞未经历与肿瘤细胞的相互作用,因此存活从而维持抗肿瘤免疫力。疗法的T细胞在无肿瘤小鼠中长期存活的事实证实,荷瘤小鼠中疗法的T细胞的短寿进程可能是其与肿瘤细胞相互作用的结果。本公开的结果认为,在Tcm期间保留内源性肿瘤反应性T细胞对于确保疾病消除和长期抗肿瘤记忆是至关重要的,其可能通过预处理来试图增加过继疗法细胞的存活而受到损害[53]。
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].
总的来说,本公开的数据支持通过在溶瘤病毒疫苗给药期间使肿瘤引发的宿主的内源性T淋巴细胞参与到肿瘤局部,并因此最小化甚至消除由引入单一选择压力引起的抗原突变产生的风险。许多患者含有识别源自患者自身的肿瘤的不同的新表位的CD4
+和/或CD8
+T细胞这一事实强调了本公开研究结果的相关性[82,83]。此外,已经开发了从癌症患者产生肿瘤特异性T细胞的临床方法,其与各种溶瘤病毒的已确定的安全特性一起,支持本公开的疗法是临床上高度可转化的[40-42,84,85]。
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].
本公开的不同实施方案通过上述实施例示出。本领域技术人员可以开发出上述方法的替代方案,这些替代方案在本公开和所限定的权利要求的范围内。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.
所有出版物、专利和专利申请均以其全部内容通过参考引入本文,其引入程度与如同每个单独的出版物、专利或专利申请以其全部内容通过参考引入而被具体和单独地指出一样。在本申请中的术语在通过引用引入本文的文件中被不同地定义的情况下,本文所提供的定义用作该术语的定义。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.
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Claims (60)
- 一种重组溶瘤棒状病毒的改性基质蛋白(M),其特征在于,编码所述改性基质蛋白(M)的氨基酸序列与SEQ ID NO:1所示的氨基酸序列相比,具有至少80%,优选至少90%,更优选至少95%,最优选至少98%相同的序列;并且,所述氨基酸序列和SEQ ID NO:1相比,存在如(i)或(ii)所示的替换: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)第21位置、第51位置、第111位置和第221位置同时具有氨基酸替换;(i) the 21st position, the 51st position, the 111th position, and the 221st position have both amino acid substitutions;(ii)第51位置、第221位置、第226位置同时具有氨基酸替换。(ii) The 51st position, the 221st position, and the 226th position have both amino acid substitutions.
- 根据权利要求1所述的改性基质蛋白(M),其中,所述重组溶瘤棒状病毒选自水疱性口炎病毒;优选的,所述重组溶瘤棒状病毒选自水疱性口炎病毒印第安纳株;更优选的,所述重组溶瘤棒状病毒选自MuddSummer株。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.
- 根据权利要求1-2任一项所述的改性基质蛋白(M),其特征在于,所述的改性的基质蛋白(M)的序列和SEQ ID NO:1相比,编码改性基质蛋白(M)的氨基酸序列存在如(i)或(ii)所示的替换: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)第21位置、第51位置、第111位置和第221位置同时具有氨基酸替换,所述氨基酸替换方式为:(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)第21位甘氨酸G替换为谷氨酸E,(a) the 21th glycine G is replaced by glutamic acid E,(b)第51甲硫氨酸M替换为丙氨酸A,(b) the 51th methionine M is replaced by alanine A,(c)第111位亮氨酸L替换为苯丙氨酸F,(c) the 111th leucine L is replaced by phenylalanine F,(d)第221位缬氨酸V替换为苯丙氨酸F;(d) replacing the 221th proline V with phenylalanine F;或or(ii)第51位置、第221位置、第226位置同时具有氨基酸替换,所述氨基酸替换方式为:(ii) The 51st position, the 221st position, and the 226th position have both amino acid substitutions, and the amino acid substitutions are:(a)第51位甲硫氨酸M替换为精氨酸R,(a) the 51st methionine M is replaced by arginine R,(b)第221位缬氨酸V替换为苯丙氨酸F,(b) Replacement of the 221th proline V with phenylalanine F,(c)第226位甘氨酸G替换为精氨酸R;(c) the 226th glycine G is replaced by arginine R;优选的,所述改性基质蛋白(M)第21位置、第51位置、第111位置和第221位置同时具有氨基酸替换的序列为如SEQ ID NO:3所示的序列;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;所述改性基质蛋白(M)第51位置、第221位置、第226位置同时具有氨基酸替换的序列为如SEQ ID NO:5所示的序列。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.
- 一种重组溶瘤棒状病毒,其特征在于,所述重组溶瘤棒状病毒基因组包含改性基质蛋白(M),其特征在于,所述改性基质蛋白(M)的氨基酸序列为如权利要求1-3任一项中所示的氨基酸序列;优选的,所述重组溶瘤棒状病毒为减毒的溶瘤棒状病毒;更优选的,所述重组溶瘤棒状病毒为减毒延时感染的溶瘤棒状病毒。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.
- 一种包含分离的重组溶瘤棒状病毒的组合物,所述重组溶瘤棒状病毒基因组具有核酸片段,所述核酸片段编码改性基质蛋白(M),其特征在于,所述改性基质蛋白(M)的氨基酸序列为根据权利要 求1-3任一项中所示的氨基酸序列;优选的,所述重组溶瘤棒状病毒为减毒的重组溶瘤棒状病毒;更优选的,所述重组溶瘤棒状病毒为减毒延时感染的溶瘤棒状病毒。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.
- 根据权利要求5所述的组合物,所述组合物进一步包含第二溶瘤病毒;优选的,所述第二溶瘤病毒选自包含弹状病毒、牛痘病毒、疱疹病毒、麻疹病毒、新城疫病毒、腺病毒、甲病毒、细小病毒、肠道病毒株的一种或多种;更优选的,所述第二溶瘤病毒为减毒的溶瘤病毒;最优选的,其中所述第二溶瘤病毒为减毒的弹状病毒。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.
- 根据权利要求5-6任一项所述的组合物,所述组合物进一步包括第二抗肿瘤制剂;优选的,所述第二抗肿瘤制剂是免疫治疗剂、化学治疗剂或放射治疗剂;更优选的,所述第二抗肿瘤制剂选自小分子,大分子,细胞,病毒载体,基因载体,DNA,RNA,多肽,和纳米复合物中的一种或多种。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.
- 一种疫苗组合物,其特征在于,所述疫苗组合物包含治疗有效量的一种或多种重组溶瘤棒状病毒,其中,所述一种或多种重组溶瘤棒状病毒基因组包含改性基质蛋白(M),所述改性基质蛋白(M)的氨基酸序列为如权利要求1-3任一项所示的氨基酸序列。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.
- 根据权利要求8所述的疫苗组合物,其特征在于,其还可以包含如权利要求6中所述的第二溶瘤病毒,或如权利要求7中所述的第二抗肿瘤制剂。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.
- 一种分离的肽,其由氨基酸序列编码而成,所述氨基酸序列选自包含与SEQ ID NO:1的氨基酸序列至少80%,优选至少90%,更优选至少95%,最优选至少98%相同的序列,并且,所述氨基酸序列和SEQ ID NO:1相比,存在如(i)或(ii)所示的替换: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)第21位置、第51位置、第111位置和第221位置同时具有氨基酸替换,所述氨基酸替换方式为:(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)第21位甘氨酸G替换为谷氨酸E,(a) the 21th glycine G is replaced by glutamic acid E,(b)第51甲硫氨酸M替换为丙氨酸A,(b) the 51th methionine M is replaced by alanine A,(c)第111位亮氨酸L替换为苯丙氨酸F,(c) the 111th leucine L is replaced by phenylalanine F,(d)第221位缬氨酸V替换为苯丙氨酸F;(d) replacing the 221th proline V with phenylalanine F;或or(ii)第51位置、第221位置、第226位置同时具有氨基酸替换,所述氨基酸替换方式为:(ii) The 51st position, the 221st position, and the 226th position have both amino acid substitutions, and the amino acid substitutions are:(a)第51位甲硫氨酸M替换为精氨酸R,(a) the 51st methionine M is replaced by arginine R,(b)第221位缬氨酸V替换为苯丙氨酸F,(b) Replacement of the 221th proline V with phenylalanine F,(c)第226位甘氨酸G替换为精氨酸R;(c) the 226th glycine G is replaced by arginine R;优选的,所述改性基质蛋白(M)第21位置、第51位置、第111位置和第221位置同时具有氨基酸替换的序列为如SEQ ID NO:3所示的序列;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;所述改性基质蛋白(M)第51位置、第221位置、第226位置同时具有氨基酸替换的序列为如SEQ ID NO:5所示的序列。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.
- 一种用于编码权利要求10所述分离的肽的核苷酸序列。A nucleotide sequence for encoding the isolated peptide of claim 10.
- 根据权利要求5-7任一项所述的包含分离的重组溶瘤棒状病毒的组合物或权利要求8-9任一项所述的疫苗在制备药物中的应用,所述药物可以杀死异常增生性细胞、诱导促进抗肿瘤免疫反应、抗肿瘤免疫细胞特异性增殖、抗肿瘤免疫细胞归巢侵润肿瘤组织和/或消除肿瘤组织微环境免疫抑制。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.
- 根据权利要求11所述的应用,其中所述异常增生性细胞被包含在患者体内。The use according to claim 11, wherein said abnormally proliferating cells are contained in a patient.
- 根据权利要求12所述的应用,其中所述异常增生性细胞选自肿瘤细胞或肿瘤组织相关细胞;优选的,所述肿瘤细胞是癌细胞;更优选的,所述癌细胞是转移性癌细胞。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 .
- 根据权利要求5-7任一项所述的包含分离的重组溶瘤棒状病毒的组合物或根据权利要求8-9任一项所述的疫苗在制备治疗患有肿瘤的患者的药物中的应用。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 .
- —种缓慢持续杀伤异常增生性细胞的方法,包括将所述异常增生性细胞与根据权利要求4所述的重组溶瘤棒状病毒、根据权利要求5-7任一项所述的包含分离的重组溶瘤棒状病毒的组合物或根据权利要求8-9任一项所述的疫苗接触的步骤。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.
- 根据权利要求16所述的方法,其中所述异常增生性细胞被包含在患者体内。The method of claim 16 wherein said abnormally proliferating cells are contained within a patient.
- 根据权利要求16所述的方法,其中所述异常增生性细胞选自肿瘤细胞或肿瘤组织相关细胞;优选的,所述肿瘤细胞是癌细胞;更优选的,所述癌细胞是转移性癌细胞。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 .
- 根据权利要求16所述的方法,其中所述重组溶瘤棒状病毒、包含分离的重组溶瘤棒状病毒的组合物或疫苗被施用至患者体内。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.
- 根据权利要求16所述的方法,其中所述重组溶瘤棒状病毒、包含分离的重组溶瘤棒状病毒的组合物或疫苗通过包括腹膜内、静脉内、动脉内、肌肉内、皮肤内、瘤内、皮下或鼻内给药中的一种或多种的给药方式而被施用;优选的,所述给药方式的给药途径包括内镜、腔镜、介入、微创、传统手术中的一种或多种。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.
- 根据权利要求16所述的方法,所述方法进ー步包括施用第二抗肿瘤疗法的步骤。The method of claim 16 further comprising the step of administering a second anti-tumor therapy.
- 根据权利要求21所述的方法,其中所述第二抗肿瘤疗法选自施用第二溶瘤病毒;优选的,所述第二溶瘤病毒选自包含弹状病毒、牛痘病毒、疱疹病毒、麻疹病毒、新城疫病毒、腺病毒、甲病毒、细小病毒、肠道病毒株的一种或多种;更优选的,所述第二溶瘤病毒为减毒的溶瘤病毒;最优选的,其中所述第二溶瘤病毒为减毒的弹状病毒。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.
- 根据权利要求21所述的方法,其中所述第二抗肿瘤疗法选自化学疗法、放射疗法、免疫疗法、手术疗法中的一种或多种。The method of claim 21, wherein the second anti-tumor therapy is selected from one or more of chemotherapy, radiation therapy, immunotherapy, surgery therapy.
- 一种在受试者中诱导免疫应答的方法,其特征在于所述方法包含对受试者施用选自如权利要求4所述的重组溶瘤棒状病毒、权利要求5-7任一项所述包含分离的重组溶瘤棒状病毒的组合物或权利要求8-9任一项所述的疫苗中的一种或多种。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.
- 一种诱导促进抗肿瘤免疫反应,抗肿瘤免疫细胞特异性增殖,抗肿瘤免疫细胞浸润肿瘤组 织或消除肿瘤组织微环境免疫抑制的方法,包括将肿瘤或肿瘤组织与根据权利要求4所述的重组溶瘤棒状病毒、根据权利要求5-7任一项所述的包含分离的重组溶瘤棒状病毒的组合物或根据权利要求8-9任一项所述的疫苗接触的步骤。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.
- 一种抗肿瘤用组合物,所述抗肿瘤用制品包括(i)复制型溶瘤病毒疫苗;(ii)过继性免疫细胞。An antitumor composition comprising (i) a replicative oncolytic virus vaccine; (ii) adoptive immune cells.
- 根据权利要求26所述的抗肿瘤用组合物,其中所述的复制型溶瘤病毒为溶瘤棒状病毒或溶瘤痘苗病毒;可选的,所述复制型溶瘤病毒表达肿瘤抗原。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.
- 根据权利要求27所述的抗肿瘤用组合物,其中,所述溶瘤棒状病毒为如权利要求4所述的重组溶瘤棒状病毒。The antitumor composition according to claim 27, wherein the oncolytic baculovirus is the recombinant oncolytic baculovirus according to claim 4.
- 根据权利要求26-28任一项所述的抗肿瘤用组合物,其中所述过继性免疫细胞为中心记忆性T细胞(Tcm),优选的,所述中心记忆性T细胞(Tcm)为产生肿瘤抗原特异性的CD8 +T细胞群,更优选的,所述CD8 +T细胞中的至少50%、60%或70%显示中心记忆性细胞特有表型。 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.
- 根据权利要求26-29中任一项所述的抗肿瘤用组合物,其中所述肿瘤抗原为选自由NY-ESO1、癌睾抗原(cancer testis antigen)、癌胚抗原(carcinoembryonic antigen,CEA)、甲胎蛋白(AFP)、CA 125、Her2、多巴色素互变异构酶(DCT)、GP100、MART1、CMV pp65、EBV、HPV E6和HPV E7等病毒抗原全长蛋白或部分编码肽段组成的组中的任一项或其组合;可选的,所述癌睾抗原选自MAGE。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.
- 根据权利要求26-30中任一项所述的抗肿瘤用组合物,其中所述肿瘤选自肺癌、乳腺癌、肝癌、胃癌、食管癌、结肠癌(colon cancer)、结直肠癌(colorectal cancer)、胰腺癌、甲状腺癌、鼻咽癌、口腔癌、头颈癌、胆管癌、前列腺癌、巢癌、子宫癌、宫颈癌、肾癌、膀胱癌、输尿管癌、脑癌(例如胶质瘤)、黑素瘤、皮肤癌、肉瘤、淋巴瘤和白血病;可选的,所述肿瘤包括原发、继发或转移性肿瘤。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.
- 根据权利要求26-31中任一项所述的抗肿瘤用组合物,其中所述肿瘤为实体瘤。The antitumor composition according to any one of claims 26 to 31, wherein the tumor is a solid tumor.
- 一种组合物在制备治疗肿瘤的药物中的用途,所述组合物包括(i)过继性免疫细胞和(ii)复制型溶瘤病毒疫苗。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.
- 根据权利要求33所述的用途,其中所述的复制型溶瘤病毒为溶瘤棒状病毒或溶瘤痘苗病毒,可选的,所述复制型溶瘤病毒表达肿瘤抗原。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.
- 根据权利要求34所述的用途,其中,所述溶瘤棒状病毒为如权利要求4所述的重组溶瘤棒状病毒。The use according to claim 34, wherein the oncolytic baculovirus is the recombinant oncolytic baculovirus according to claim 4.
- 根据权利要求33-35任一项所述的用途,其中所述过继性免疫细胞为中心记忆性T细胞(Tcm),优选的,所述中心记忆性T细胞(Tcm)为产生肿瘤抗原特异性的CD8+T细胞群,更优选的,所述CD8+T细胞中的至少50%、60%或70%显示中心记忆性细胞特有表型。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.
- 根据权利要求33-36中任一项所述的用途,其中所述溶瘤病毒疫苗通过血管内给予受试者,优 选的通过静脉给予受试者。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.
- 根据权利要求33-37中任一项所述的用途,所述痘苗病毒选自Wyeth、Western Reserve、Lister或Copenhagen株;可选的,所述痘苗病毒缺失胸苷激酶基因或者缺失痘苗病毒生长因子基因。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.
- 根据权利要求33-38中任一项所述的用途,其中所述痘苗病毒通过瘤内、胸腔内、胸膜内、腹腔内、腹膜内、颅内、静脉内、动脉内或肌内给予患者或受试者。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.
- 根据权利要求33-39中任一项所述的用途,其中所述肿瘤抗原为选自由NY-ESO1、癌睾抗原(cancer testis antigen)、癌胚抗原(carcinoembryonic antigen,CEA)、甲胎蛋白(AFP)、CA 125、Her2、多巴色素互变异构酶(DCT)、GP100、MART1、CMV pp65、EBV、HPV E6和HPV E7等病毒抗原全长蛋白或部分编码肽段组成的组中的任一项或其组合;可选的,所述癌睾抗原选自MAGE。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.
- 根据权利要求33-40中任一项所述的用途,其中所述肿瘤选自肺癌、乳腺癌、肝癌、胃癌、食管癌、结肠癌(colon cancer)、结直肠癌(colorectal cancer)、胰腺癌、甲状腺癌、鼻咽癌、口腔癌、头颈癌、胆管癌、前列腺癌、巢癌、子宫癌、宫颈癌、肾癌、膀胱癌、输尿管癌、脑癌(例如胶质瘤)、黑素瘤、皮肤癌、肉瘤、淋巴瘤和白血病;可选的,所述肿瘤包括原发、继发或转移性肿瘤。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.
- 根据权利要求33-41中任一项所述的用途,其中所述肿瘤为实体瘤。The use according to any of claims 33-41, wherein the tumor is a solid tumor.
- 一种治疗癌症的联合治疗方法,所述方法包括(i)将过继性免疫细胞给予至患有癌症的受试者;和(ii)随后将复制型溶瘤病毒疫苗给予至患有癌症的受试者。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.
- 根据权利要求43所述的联合治疗方法,其中所述的复制型溶瘤病毒为溶瘤棒状病毒或溶瘤痘苗病毒,可选的,所述复制型溶瘤病毒表达肿瘤抗原。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.
- 根据权利要求44所述的联合治疗方法,其中所述溶瘤棒状病毒为如权利要求4所述的重组溶瘤棒状病毒。The combination therapy method according to claim 44, wherein the oncolytic baculovirus is the recombinant oncolytic baculovirus according to claim 4.
- 根据权利要求43-45任一项所述的联合治疗方法,其中所述过继性免疫细胞为中心记忆性T细胞(Tcm),优选的,所述中心记忆性T细胞(Tcm)为产生肿瘤抗原特异性的CD8 +T细胞群,更优选的,所述CD8 +T细胞中的至少50%、60%或70%显示中心记忆性细胞特有表型。 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.
- 根据权利要求43-46任一项所述的联合治疗方法,其中所述CD8 +T细胞群的生产方法为:在存在加载有肿瘤抗原的抗原呈递程细胞(APC)、IL21、IL15和雷帕霉素且优选在不存在IL2的情况下,从患有癌症的受试者获得外周血单核细胞(PBMC)、或肿瘤浸润性淋巴细胞(TIL)、或骨髓内淋巴细胞,并对所述外周血单核细胞(PBMC)、或肿瘤浸润性淋巴细胞(TIL)或骨髓内淋巴细胞进行离体培养。 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.
- 根据权利要求43-47任一项所述的联合治疗方法,其中所述CD8+T细胞群的生产方法为:从患有癌症的受试者获得PBMC,通过对PBMC转导重组T细胞受体(TCR)或嵌合抗原受体(CAR)对其进行遗传修饰,并离体培养经转导的PBMC。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.
- 根据权利要求43-48任一项所述的联合治疗方法,其中所述CD8+T细胞群是通过对CD25清除 的所述PBMC进行离体培养得到的。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.
- 根据权利要求43-49任一项所述的联合治疗方法,在对PBMC或TIL离体培养得到CD8+T细胞后,还进一步包括使用抗CD3抗体和抗CD28抗体以及任选IL2体外扩增所述CD8 +T细胞。 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.
- 根据权利要求43-50任一项所述的联合治疗方法,其中所述溶瘤病毒疫苗通过血管内给予受试者,优选的通过静脉给予受试者。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.
- 根据权利要求43-51任一项所述的联合治疗方法,其中所述肿瘤抗原为选自由NY-ESO1、癌睾抗原(cancer testis antigen)、癌胚抗原(carcinoembryonic antigen,CEA)、甲胎蛋白(AFP)、CA 125、Her2、多巴色素互变异构酶(DCT)、GP100、MART1、CMV pp65、EBV、HPV E6和HPV E7等病毒抗原全长蛋白或部分编码肽段组成的组中的任一项或其组合;可选的,所述癌睾抗原选自MAGE。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.
- 根据权利要求43-52任一项所述的联合治疗方法,其中所述肿瘤选自肺癌、乳腺癌、肝癌、胃癌、食管癌、结肠癌(colon cancer)、结直肠癌(colorectal cancer)、胰腺癌、甲状腺癌、鼻咽癌、口腔癌、头颈癌、胆管癌、前列腺癌、巢癌、子宫癌、宫颈癌、肾癌、膀胱癌、输尿管癌、脑癌(例如胶质瘤)、黑素瘤、皮肤癌、肉瘤、淋巴瘤和白血病;可选的,所述肿瘤包括原发、继发或转移性肿瘤。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.
- 根据权利要求43-53任一项所述的联合治疗方法,其中所述肿瘤为实体瘤。The combination therapy method according to any one of claims 43 to 53, wherein the tumor is a solid tumor.
- 根据权利要求43-54任一项所述的联合治疗方法,其中所述IL21和IL15是以约5ng/ml至约15ng/ml的浓度存在,所述雷帕霉素是以15ng/ml至约25ng/ml的浓度存在.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.
- 根据权利要求43-55任一项所述的联合治疗方法,将所述过继性免疫细胞给予至受试者后的1至72小时之后将所述复制型溶瘤病毒疫苗(OVV)给予受试者。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.
- 根据权利要求43-56任一项所述的联合治疗方法,所述受试者在接受过继性免疫细胞之前不经历淋巴细胞耗竭。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.
- 根据权利要求43-57任一项所述的联合治疗方法,所述复制型溶瘤病毒疫苗通过多次给予至受试者。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.
- 根据权利要求43-58任一项所述的联合治疗方法,所述CD8 +T细胞群是受试者自体的。 The combination therapy method according to any one of claims 43 to 58, wherein the CD8 + T cell population is autologous to the subject.
- 根据权利要求43-59任一项所述的联合治疗方法,所述抗原呈递细胞是树突细胞。The combination therapy method according to any one of claims 43 to 59, wherein the antigen presenting cell is a dendritic cell.
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