Preparation method and application of attenuated rhabdovirus
阅读说明：本技术 一种减毒棒状病毒的制备方法及应用 (Preparation method and application of attenuated rhabdovirus ) 是由 秦晓峰 吴飞 于 2018-08-10 设计创作，主要内容包括：本公开涉及一种减毒棒状病毒的制备方法及应用。具体来说,本公开提供了一种减毒病毒载体的制备方法,通过上述方法可以制备嵌合表达针对特定靶点的抗体的减毒RNA病毒重组表达载体系统,例如靶向肿瘤微环境的减毒病毒载体系统。前述载体系统可以稳定表达针对特定靶点的相应抗体。本公开的载体的宿主细胞来源广泛,极大的提高了外源嵌合的类抗体的体内外的表达效率；与此同时,载体系统本身的基因组简单且稳定,突变率低,能够快速高效嵌合表达人源的特异性抗体。并且,本公开的载体系统由于进行了基因突变改造,使得病毒的毒性降低,在表达抗体时对宿主的正常细胞不产生明显的损伤。(The present disclosure relates to a method for preparing attenuated rhabdovirus and its application. In particular, the present disclosure provides a method for preparing an attenuated viral vector by which an attenuated RNA viral recombinant expression vector system can be prepared that chimerically expresses antibodies directed against a specific target, such as an attenuated viral vector system targeted to a tumor microenvironment. The aforementioned vector system can stably express the corresponding antibody against a specific target. The vector disclosed by the invention has wide host cell sources, and the in vivo and in vitro expression efficiency of the exogenous chimeric antibody-like body is greatly improved; meanwhile, the genome of the vector system is simple and stable, the mutation rate is low, and the humanized specific antibody can be quickly and efficiently chimeric expressed. Moreover, the vector system disclosed by the invention is subjected to gene mutation modification, so that the toxicity of the virus is reduced, and the normal cells of a host are not obviously damaged when the antibody is expressed.)
1. A method for producing an attenuated viral vector, comprising the steps of:
(S1) mixing the nucleotide sequence of the gene encoding the vesicular stomatitis virus matrix protein, as set forth in SEQ ID NO: 1(M gene of VSV), with the first vector, and adding a transposase to perform a transposition reaction;
(S2) mixing the transposition product obtained in the step (S1) with competent bacteria, and transforming;
(S3) extracting the plasmid of the bacterium obtained by the step (S2) to obtain the gene encoding the vesicular stomatitis virus matrix protein after transposition;
(S4) recombining the gene obtained in the step (S3) into a second vector to obtain the attenuated viral vector;
wherein the sequence encoding the second vector comprises the genomic sequence of vesicular stomatitis virus;
wherein the first vector is selected from a vector having a transposition function;
optionally, the first vector comprises a sequence shown as SEQ ID NO. 2;
optionally, the second vector comprises a sequence as set forth in GENEBANK numbering EU 849003.1.
2. An attenuated virus vector obtained by the production method according to claim 1.
3. The attenuated viral vector of claim 2, wherein the sequence encoding the attenuated viral vector comprises an amino acid sequence set forth in SEQ ID NO:3, and (b) is the sequence shown in the specification.
4. The attenuated viral vector of claim 3, wherein the sequence encoding the attenuated viral vector comprises the sequence set forth in SEQ ID NO:4, or a sequence shown in the figure.
5. A cloning scaffold vector system, wherein the cloning scaffold vector system recombines the sequence as shown in SEQ ID No. 5 into the vector of claim 3; wherein, the insertion site of the sequence shown in SEQ ID NO. 5 into the vector as shown in claim 3 is shown in SEQ ID NO:4, position 4632 of the sequence shown in fig. 4.
6. The cloning scaffold vector system according to claim 5, wherein the sequence encoding the cloning scaffold vector system comprises the sequence shown in SEQ ID NO 6.
7. A method of producing an attenuated monoclonal virus strain comprising the steps of:
(S1) culturing the first cell to be transfected;
(S2) co-transfecting a plasmid comprising the sequence shown as SEQ ID NO:3, and a plasmid (pN) comprising the sequence shown as SEQ ID NO:7, a plasmid (pL) comprising the sequence shown as SEQ ID NO:8, a plasmid mixture of a plasmid (pP) comprising the sequence shown as SEQ ID NO:9, into the cells to be transfected in step (S1);
(S3) extracting the supernatant of the cell mixture obtained after the co-transfection in the step (S2), and transfecting the cell mixture into a cell of a second cell to be transfected;
(S4) culturing the second cell to be transfected after being transfected in the step (S3), and screening to obtain an attenuated monoclonal virus strain.
8. The method according to claim 7, wherein the weight ratio of the plasmid comprising the sequence shown as SEQ ID NO. 3, the plasmid (pN) comprising the sequence shown as SEQ ID NO. 7, the plasmid (pL) comprising the sequence shown as SEQ ID NO. 8 and the plasmid (pP) comprising the sequence shown as SEQ ID NO. 9 is 10:4:1: 5.
9. The method according to any one of claims 7 to 8, wherein the plasmid comprising the sequence shown as SEQ ID NO. 3 is a plasmid comprising the sequence shown as SEQ ID NO. 4; optionally, the second cell to be transfected is selected from Vero cells; the first cells to be transfected are selected from BSR-T7 cells.
10. The method according to claim 9, wherein the plasmid comprising the sequence shown as SEQ ID NO. 4 in step (S2) is selected from the group consisting of plasmids comprising the sequence shown as SEQ ID NO. 6.
11. The method according to claim 10, wherein the coding sequence of the plasmid comprising the sequence shown as SEQ ID NO. 6 in step (S2) further comprises the sequence shown as SEQ ID NO. 10 and the sequence shown as SEQ ID NO. 11.
12. The method according to claim 10, wherein the coding sequence of the plasmid comprising the sequence shown as SEQ ID NO. 6 in step (S2) further comprises the sequence shown as SEQ ID NO. 12.
13. The method of claim 12, wherein the 5' end comprising the sequence set forth in SEQ ID NO 12 further comprises a signal peptide sequence; the sequence of the signal peptide is selected from the sequences shown as SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 or SEQ ID NO. 16; preferably, the signal peptide sequence is selected from the group consisting of the sequences shown in SEQ ID NO. 15.
14. An attenuated monoclonal virus strain produced by the method of producing an attenuated monoclonal virus strain according to any one of claims 7 to 13.
15. A monoclonal antibody secreted by the attenuated monoclonal virus strain of claim 14.
16. A monoclonal antibody comprising a fragment encoded by the sequence shown as SEQ ID NO. 4, the sequence shown as SEQ ID NO. 10 and the coding sequence shown as SEQ ID NO. 11.
17. The monoclonal antibody according to claim 16, wherein the sequence comprising SEQ ID NO. 4 is selected from the group consisting of the sequences comprising SEQ ID NO. 6.
18. The monoclonal antibody of claim 17, wherein the coding sequence further comprises a sequence as set forth in SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 or SEQ ID No. 16; preferably, the coding sequence further comprises the sequence shown in SEQ ID NO. 15.
19. A pharmaceutical composition comprising the monoclonal antibody of any one of claims 15-18.
20. Use of the monoclonal antibody of any one of claims 15-18 or the pharmaceutical composition of claim 19 in the preparation of a medicament for killing hyperproliferative cells, inducing promotion of an anti-tumor immune response, or eliminating microenvironment immunosuppression of tumor tissue.
21. The use of claim 20, wherein the hyperproliferative cell is contained in a patient.
22. The use of claim 21, wherein the hyperproliferative cell is selected from the group consisting of a tumor cell or a tumor tissue-associated cell; preferably, the tumor cell is a cancer cell; more preferably, the cancer cell is a metastatic cancer cell.
23. A method of slow and sustained killing of a hyperproliferative cell comprising the step of contacting said hyperproliferative cell with the monoclonal antibody of any one of claims 15-18 or the pharmaceutical composition of claim 19.
24. The method of claim 23, wherein the hyperproliferative cell is contained in a patient.
25. The method of claim 23, wherein the hyperproliferative cell is selected from the group consisting of a tumor cell or a tumor tissue-associated cell; preferably, the tumor cell is a cancer cell; more preferably, the cancer cell is a metastatic cancer cell.
26. The method of claim 23, wherein the monoclonal antibody of any one of claims 15-18 or the pharmaceutical composition of claim 19 is administered to a patient.
27. The method of claim 23, wherein the monoclonal antibody of any one of claims 15-18 or the pharmaceutical composition of claim 19 is administered by a mode of administration comprising one or more of intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, intratumoral, subcutaneous, or intranasal administration; preferably, the administration route of the administration mode comprises one or more of endoscopy, intervention, minimally invasive surgery and traditional surgery.
28. The method of claim 23, further comprising the step of ー administering a second anti-tumor therapy.
29. The method of claim 28, wherein the second anti-tumor therapy is selected from one or more of chemotherapy, radiation therapy, immunotherapy, surgical therapy.
30. A polynucleotide comprising the sequence shown in SEQ ID NO. 6.
Technical Field
The present disclosure relates to the fields of oncology, virology and molecular cell biology. In particular, the present disclosure relates to a vector system using non-integrated, replicable negative-sense strand RNA viruses, a method of constructing the same, and uses of the vectors constructed by the above method.
Background
The range of options for cancer patient treatment options has changed tremendously over the past decade. With the knowledge of the growth and development of the related driver mutations (driver mutation) of tumors and the development of the research and development of the specific mutant targeting molecule inhibitors, a new tumor treatment field, namely precise oncology, ensues, and the theoretical basis of tumor immunotherapy is that the immune system has the capacity of recognizing tumor-related antigens and regulating and controlling the body to attack tumor cells (highly specific cytolysis). This biological process is very complex and is still under considerable basic research.
In the last 90 s of the century, several research groups have discovered tumor antigens that can be recognized by T lymphocytes in a major histocompatibility complex-dependent manner. In some cases, antigens are often referred to as viral proteins, mutated self-antigens (some of which are driver oncogenes), derepressed embryonic antigens, over-expressed differentiated or autologous normal proteins. Tumor cells produce and release antigens in a variety of ways, such as intracellular kinases, primary necrosis of tumor cells, and the body's response to radiation, chemotherapy, or targeted therapies. In addition to antigens, in the context of cellular stress, hypoxia, nutrient depletion, and trauma, dead tumor cells can also release a variety of immunogenic molecules that can bind to cell surface or intracellular receptors (e.g., toll-like receptors) to trigger an innate immune response. In addition, specific antigen presenting cells (e.g., dendritic cells) in the tumor microenvironment can phagocytose dead tumor cells and soluble antigens, CD8+ T cells. To achieve better differentiation, T cell activation also sets a secondary signal recognition system, a signaling pathway mediated by co-stimulatory molecules. Once activated in the presence of co-stimulatory molecules, T cells can migrate into the tumor microenvironment following a concentration gradient of local chemokines. After the T cell reaches the vicinity of the tumor cell, the T cell receptor can recognize the cognate antigen on the surface of the tumor cell via MHC class I-polypeptide complexes. T cells can release cytotoxic factors (e.g., granzyme B and perforins) that can regulate direct lysis of antigen-expressing tumor cells while producing a bystander effect on adjacent non-antigen-expressing tumor cells. There are a number of activated effector lymphocytes present in the microenvironment of tumors that generally have a better prognosis and respond better to immunotherapy, and despite the existence of the tumor-immune cycle, established tumors may escape immune detection and elimination by a variety of hosts, tumors, and immune mechanisms.
In addition, studies have reported that tumor microenvironments accumulate a large number of suppressive immune cells, such as regulatory CD4+ positive T cells, tumor-associated macrophages, myeloid suppressive cells, which can suppress the activity of activated effector T cells. The manner in which tumor cells die may determine which immune response is activated. For example, apoptosis of tumor cells may induce T cell tolerance, while necrosis or apoptosis (pyroptosis) of tumor cells, programmed cell death may induce an activated tumor-specific T cell response
Cancer has long been at the head of various causes of death. The world health organization has long predicted that malignancies will become the "first killer" in humans in the 21 st century, and cancer control has become a key point of global health strategy. Although China is a developing country, the disease spectrum has been changed, and China has become the first cancer major country in the world. In recent years, the morbidity and mortality of malignant tumors are more serious, the number of new cases is about 160 ten thousand, the death number is 130 ten thousand, and more than 200 ten thousand of patients with the existing diseases are. Moreover, most cancers are on the rise and are worth high attention.
Although the cure rate for several malignancies has improved significantly, the outcome for patients with advanced solid tumors has remained crudely maintained over the past several decades. Currently, antibodies of tumor immune checkpoints, such as antibodies of PD-1/PDL1 and CTLA4, have been put into clinical use in solid tumors, and the key point of these monoclonal antibodies that effectively antagonize immune checkpoint molecules lies in the efficient production per unit volume, and in addition, the problem of drug resistance faced by immune checkpoint antibodies is urgently to be solved, and the research progress of tumor immunotherapy is currently receiving attention from various countries. A variety of immune-related tumor treatment strategies including T cell node inhibitors, oncolytic viruses, chimeric antigen receptor T cells, and the like have been derived. It is well known that efficient immunotherapy requires several major features: inducing a lasting clinical response; there is no typical drug resistance; inducing autoimmunity-like toxicity. Clinical oncologists need to have a thorough understanding of the current clinical application of tumor-targeted therapy and tumor immunotherapy, only to provide high quality treatment regimens for cancer patients. The rationale for tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens, regulate the body's ability to attack tumor cells (highly specific cytolysis).
Some gene editing modified recombinant viruses are used as a new tumor treatment preparation, and the anti-tumor immune response is initiated through two action mechanisms of the killing action of the viruses on tumor cells and the induction of systemic anti-tumor immune response. However, the specific molecular mechanism is not clear, and some of the existing research results show that the mechanism is the combined action of various action factors such as the replication and proliferation of the virus in tumor cells to induce cell death, the interaction with the anti-virus elements of the tumor cells, and the promotion of the intrinsic spontaneous or specific anti-tumor immune response.
Vesicular Stomatitis Virus (VSV) is a negative strand RNA virus that infects most mammalian cells and expresses up to 60% of the total protein of the viral protein in the infected cells. In nature, VSV infects pigs, cattle and horses and causes varicella diseases near the mouth and feet. Although human infection with VSV has been reported, VSV does not cause any serious symptoms in humans. VSV encodes 5 proteins, including a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a surface glycoprotein (G), and an RNA-dependent RNA polymerase (L). Blocking host cell protein synthesis by VSV matrix protein (M) induces cell death.
However, the existing medicines in the prior art still have the defects of long medication period, drug resistance, high price and the like. Therefore, there is a need to provide new medicaments to overcome the above-mentioned drawbacks.
Disclosure of Invention
Problems to be solved by the invention
Based on the deficiency of the prior art, the present disclosure provides an attenuated RNA virus recombinant expression vector system for chimeric expression of an antibody against a specific target, in particular an attenuated virus vector system (AVTM system) targeting tumor microenvironment. The aforementioned vector system can stably express the corresponding antibody against a specific target.
At the same time, the disclosure also relates to a preparation method and application of the attenuated virus vector system.
Means for solving the problems
The technical scheme related to the disclosure is as follows.
(1) A method for producing an attenuated viral vector, comprising the steps of:
(S1) mixing the nucleotide sequence of the gene encoding the vesicular stomatitis virus matrix protein, as set forth in SEQ ID NO: 1(M gene of VSV), with the first vector, and adding a transposase to perform a transposition reaction;
(S2) mixing the transposition product obtained in the step (S1) with competent bacteria, and transforming;
(S3) extracting the plasmid of the bacterium obtained by the step (S2) to obtain the gene encoding the vesicular stomatitis virus matrix protein after transposition;
(S4) recombining the gene obtained in the step (S3) into a second vector to obtain the attenuated viral vector;
wherein the sequence encoding the second vector comprises the genomic sequence of vesicular stomatitis virus;
wherein the first vector is selected from a vector having a transposition function;
optionally, the first vector comprises a sequence shown as SEQ ID NO. 2;
optionally, the second vector comprises a sequence as set forth in GENEBANK numbering EU 849003.1.
(2) An attenuated virus vector obtained by the production method of (1).
(3) The attenuated viral vector of (2), wherein the sequence encoding the attenuated viral vector comprises the sequence set forth in SEQ ID NO:3, and (b) is the sequence shown in the specification.
(4) The attenuated viral vector of (3), wherein the sequence encoding the attenuated viral vector comprises the sequence set forth in SEQ ID NO:4, or a sequence shown in the figure.
(5) A cloning scaffold vector system, characterized in that the cloning scaffold vector system recombines the sequence shown as SEQ ID NO. 5 into the vector described in (3); wherein, the site of the sequence shown in SEQ ID NO. 5 inserted into the vector coded in (3) is shown in SEQ ID NO:4, position 4632 of the sequence shown in fig. 4.
(6) The cloning scaffold vector system according to (5), wherein the sequence encoding the cloning scaffold vector system comprises the sequence shown in SEQ ID NO. 6.
(7) A method of producing an attenuated monoclonal virus strain comprising the steps of:
(S1) culturing the first cell to be transfected;
(S2) co-transfecting a plasmid comprising the sequence shown as SEQ ID NO:3, and a plasmid (pN) comprising the sequence shown as SEQ ID NO:7, a plasmid (pL) comprising the sequence shown as SEQ ID NO:8, a plasmid mixture of a plasmid (pP) comprising the sequence shown as SEQ ID NO:9, into the cells to be transfected in step (S1);
(S3) extracting the supernatant of the cell mixture obtained after the co-transfection in the step (S2), and transfecting the cell mixture into a cell of a second cell to be transfected;
(S4) culturing the second cell to be transfected after being transfected in the step (S3), and screening to obtain an attenuated monoclonal virus strain.
(8) The method according to (7), wherein the weight ratio of the plasmid comprising the sequence shown by SEQ ID NO. 3, the plasmid (pN) comprising the sequence shown by SEQ ID NO. 7, the plasmid (pL) comprising the sequence shown by SEQ ID NO. 8 and the plasmid (pP) comprising the sequence shown by SEQ ID NO. 9 is 10:4:1: 5.
(9) The method according to any one of (7) to (8), wherein the plasmid comprising the sequence shown by SEQ ID NO. 3 is a plasmid comprising the sequence shown by SEQ ID NO. 4; optionally, the second cell to be transfected is selected from Vero cells; the first cell to be transfected is selected from BSR-T7 cells;
(10) the method according to (9), wherein the plasmid comprising the sequence shown as SEQ ID NO. 4 in step (S2) is selected from plasmids comprising the sequence shown as SEQ ID NO. 6.
(11) The method according to (10), wherein the coding sequence of the plasmid comprising the sequence shown as SEQ ID NO. 6 in the step (S2) further comprises the sequence shown as SEQ ID NO. 10 and the sequence shown as SEQ ID NO. 11.
(12) The method according to (10), wherein the coding sequence of the plasmid comprising the sequence shown as SEQ ID NO. 6 in step (S2) further comprises the sequence shown as SEQ ID NO. 12.
(13) The method according to (12), wherein the 5' end comprising the sequence shown as
(14) An attenuated monoclonal virus strain produced by the method for producing an attenuated monoclonal virus strain according to any one of (7) to (13).
(15) A monoclonal antibody secreted by the attenuated monoclonal virus strain of (14).
(16) A monoclonal antibody comprising a fragment encoded by the sequence shown as SEQ ID NO. 4, the sequence shown as SEQ ID NO. 10 and the coding sequence shown as SEQ ID NO. 11.
(17) The monoclonal antibody according to (16), wherein the sequence comprising SEQ ID NO. 4 is selected from the group consisting of the sequences comprising SEQ ID NO. 6.
(18) The monoclonal antibody according to (17), wherein the coding sequence further comprises a sequence shown as SEQ ID NO 13,
(19) A pharmaceutical composition comprising the monoclonal antibody according to any one of (15) to (18).
(20) Use of the monoclonal antibody according to any one of (15) to (18) or the pharmaceutical composition of (19) for the preparation of a medicament for killing an abnormally proliferative cell, inducing a promotion of an anti-tumor immune response, or eliminating microenvironment immunosuppression of tumor tissue.
(21) The use of (20), wherein the hyperproliferative cell is contained in a patient.
(22) The use of (21), wherein the hyperproliferative cell is selected from the group consisting of a tumor cell and a tumor tissue-associated cell; preferably, the tumor cell is a cancer cell; more preferably, the cancer cell is a metastatic cancer cell.
(23) A method for slow and sustained killing of an hyperproliferative cell comprising the step of contacting said hyperproliferative cell with the monoclonal antibody of any one of (15) to (18) or the pharmaceutical composition of (19).
(24) The method of (23), wherein the hyperproliferative cell is contained in a patient.
(25) The method of (23), wherein the hyperproliferative cell is selected from the group consisting of a tumor cell and a tumor tissue-associated cell; preferably, the tumor cell is a cancer cell; more preferably, the cancer cell is a metastatic cancer cell.
(26) The method of (23), wherein the monoclonal antibody of any one of (15) to (18) or the pharmaceutical composition of (19) is administered into a patient.
(27) The method of (23), wherein the monoclonal antibody of any one of (15) to (18) or the pharmaceutical composition of (19) is administered by a mode of administration comprising one or more of intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, intratumoral, subcutaneous, or intranasal administration; preferably, the administration route of the administration mode comprises one or more of endoscopy, intervention, minimally invasive surgery and traditional surgery.
(28) The method of (23), further comprising the step of ー administering a second anti-tumor therapy.
(29) The method of (28), wherein the second anti-tumor therapy is selected from one or more of chemotherapy, radiation therapy, immunotherapy, surgical therapy.
(30) A polynucleotide comprising the sequence shown in SEQ ID NO. 6.
ADVANTAGEOUS EFFECTS OF INVENTION
The host cells of the AVTM vector systems of the present disclosure are widely available. Because of the existence of the surface glycoprotein (G) of the virus system, the virus system can enter host cells without specific receptor mediation, can infect almost all cells of mammals, and can simultaneously complete virus replication and realize high-efficiency expression of exogenous chimeric genes, thereby greatly improving the in vitro and in vivo expression efficiency of the exogenous chimeric antibody.
The genome of a rhabdovirus corresponding to the AVTM attenuated vector system is single negative strand RNA, and the expression of a foreign gene of the system is very stable, the attenuated viral vector system does not generate genome integration in cells, the genome of the viral vector system is simple and stable, and the mutation rate is low.
The system can quickly and efficiently carry out chimeric expression on the human specific antibody, complete specific replication in the tumor cells and express the specific antibody of an exocrine antagonistic tumor cell. Meanwhile, when the tumor antigen is released to activate the specific immune response of immune cells to tumor, a large number of specific antibodies without immunosuppression are gathered in a local tumor microenvironment in a short time, so that the regional immunosuppression barrier is effectively broken, the specific killing activity of T cells is activated, the removal of the killing T cells to the tumor cells is promoted, and the systemic specific anti-tumor immune memory reaction of an organism is promoted.
The gene of the AVTM of the disclosure is subjected to gene mutation modification, so that the toxicity of the virus is reduced, and when the AVTM expresses the antibody at 37 ℃, the normal cells of a host cannot be obviously damaged, so that the infected cells can continuously express the secretory antibody for a period of time. In contrast, for traditional transfected myeloma cells, the cell lines used to express specific antibodies need to replicate to an order of magnitude higher before they can be used for large-scale production of antibodies.
In one embodiment, the present disclosure inserts a nucleotide sequence encoding an antibody of an immune checkpoint molecule into a modified viral expression vector by means of gene editing, and recombines in a specific eukaryotic cell, thereby obtaining an attenuated viral system stably expressing the chimeric antibody.
In one technical scheme, the AVTM-scFV vector system capable of efficiently expressing the single-chain antibody in tumor body tissues is obtained by screening through optimizing a secretion signal peptide sequence of the antibody, and meanwhile, the system is utilized to further evaluate the curative effect of the recombinant system in a solid tumor model, so that a new technical scheme and a new choice are provided for developing a therapeutic product of the solid tumor.
Drawings
FIG. 1A is a schematic diagram showing the specific process of screening a library of attenuated virus strains established by random base insertion of exogenous bases in a rhabdovirus vector system and screening an attenuated strain of a four-gene mutant strain RV-
FIG. 1B is a schematic diagram of a modified pRV-2MCS vector rhabdovirus core backbone, and the modified system can integrate two different exogenous genes (PDL1 antibody heavy chain and PDL1 antibody light chain genes) at the same time.
FIG. 2 is a schematic diagram showing the expression of 2 genes simultaneously from outside source in RV-2MCS vector system. Wherein, shown in part a of fig. 2 are schematic diagrams of a heavy chain and a light chain expressing PDL1 antibody, respectively; shown in part B of fig. 2 is a schematic representation of an RV-2MCS vector system simultaneously expressing both the heavy and light chains of PDL1 antibody; FIG. 2, section C, is a schematic representation of the RV-2MCS vector system simultaneously integrating the fluorescent expression of Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP) in Vero cells.
FIG. 3 is a schematic diagram showing codon preference optimization by a mock analysis of a vector system of a single-chain antibody AVTM
FIG. 4 shows a comparison of the superiority of the RV-G21E-M51A-L111F-V221F (RV-Mut4) four mutant vector system. Wherein, part A in FIG. 4 and part B in FIG. 4 are schematic diagrams showing the characteristic of long-term sustained expression of foreign proteins in vitro cells of the vector RV-
FIG. 5 is a schematic diagram showing the ability of PDL1 single-chain antibody to bind to two tumor cell surface molecules detected by FACS, wherein the exogenous single-chain antibody secreted by an engineered cell line Vero (RV-Mut4-scFV-PDL1 high-expression PDL1 single-chain antibody) is used, and the antibody in the supernatant is incubated with two kinds of cells LLC and MC38 stably expressing human PDL1 in vitro.
FIG. 6 is a schematic representation of the identification of the level of exocrine in eukaryotic cells of the PDL1 single-chain antibody mediated by three different signal peptide linkages in vitro at the cell line level, by IB (Western blot) experiments.
FIG. 7A is a schematic representation of the secretion of four single-chain antibodies PDL1 into the supernatant mediated by the AVTM vector system in engineered cell Vero.
FIG. 7B is a schematic diagram showing detection of antibody presence in serum and local tumor tissue (near tumor tissue subcutaneous injection and intratumoral injection) by single-chain antibody linked with
FIG. 8 is a schematic diagram showing the evaluation of therapeutic effects of AVTM-mediated single-chain antibodies in lung cancer. As shown in part a of fig. 8, there are three treatment groups, which are an AVTM system mediated expression single chain antibody group (RV-scFV-PDL1 attenuated strain), an RV-WT experimental control group, and a PBS blank control group, respectively, and an overall statistical graph of the treatment effect on solid tumors in an LLC-mediated non-small cell lung cancer model mouse animal model; wherein, in order to further not determine the difference between individuals of different treatment groups, the individuals of each group are separately counted; section B of figure 8 shows the individual statistics for the PBS treated group; FIG. 8, section C, is a schematic statistical representation of individuals from the RV-GFP-WT treated group; section D of FIG. 8 shows a statistical chart of individuals in the RV-scFV-PDL1 treatment group. Inoculating the non-small cell lung cancer tissue of model mouse in
FIG. 9 is a graph of the individual therapeutic effect of PBS, RV-GFP and RV-scFV-PDL1, on portions A-C, respectively, on a lung cancer model; part D of fig. 9 is a statistical graph of the treatment effects of the above three groups of drugs on
FIG. 10 shows the evaluation of the effect of RV-scFV-PDL1 attenuated strain on lung cancer metastasis animal models, the metastasis of the model mouse lung tissue metastasis model (LLC-JSP) treated by the experimental group and the control group under low power microscope (part A in FIG. 10) and the survival rate of the model mouse (part B in FIG. 10).
FIG. 11 shows the first set up of a colon cancer model mouse model expressing human-derived CD274Type (human-derived PDL1), RV-scFV-PDL1 and corresponding control group virus (RV-WT) were intratumorally administered 30.
Detailed Description
Definition of
In the claims and/or the description of the present disclosure, the words "a" or "an" or "the" may mean "one", but may also mean "one or more", "at least one", and "one or more than one".
As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Also, the terms "comprising," "having," "including," or "containing" are intended to be inclusive and mean that there may be additional, unrecited elements or method steps.
Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.
Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.
The terms "inhibit," "reduce," or "prevent," or any variation of these terms, as used in the claims and/or the specification, include any measurable reduction or complete inhibition to achieve a desired result (e.g., tumor treatment). Desirable results include, but are not limited to, alleviation, reduction, slowing, or eradication of cancer or a proliferative disorder or cancer-related symptoms, as well as improved quality of life or prolongation of life.
The vaccination methods of the present disclosure can be used to treat tumors in mammals, and optionally, the vaccination methods of the present disclosure can be used to treat cancer in mammals. The term "cancer" as used in this disclosure includes any cancer, including, but not limited to, melanoma, sarcoma, lymphoma, cancer (e.g., brain, breast, liver, stomach, lung, and colon), and leukemia.
The term "mammal" refers to humans as well as non-human mammals.
The methods of the present disclosure comprise administering to a mammal an oncolytic vector that expresses a tumor antigen to which the mammal has a pre-existing immunity. The term "pre-existing immunity" as used in this disclosure is meant to include immunity induced by vaccination with an antigen as well as immunity naturally occurring in mammals.
The term "RV virus" as used in this disclosure refers to an attenuated VSV oncolytic rhabdovirus. The term "RV-Mut" refers to an oncolytic rhabdovirus that has a mutation compared to a wild-type VSV oncolytic rhabdovirus. The term "RV-
The term "VSV" refers to vesicular stomatitis virus, which is one of the oncolytic rhabdoviruses. It encodes 5 proteins, including the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), surface glycoprotein (G) and RNA-dependent RNA polymerase (L).
The term "
The term "vaccine" in the present disclosure is an immune preparation for preventing diseases, which is prepared from pathogenic microorganisms (such as bacteria, etc.) and metabolites thereof by artificial attenuation, inactivation, or using transgenosis, etc.
The term "radiotherapeutic agent" in the present disclosure includes the use of drugs that cause DNA damage. Radiotherapy has been widely used in cancer and disease treatment and includes those commonly referred to as gamma rays, X-rays and/or the targeted delivery of radioisotopes to tumor cells.
The term "chemotherapeutic agent" in the present disclosure is a chemical compound useful for the treatment of cancer. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, photosensitizers, anti-estrogen and selective estrogen receptor modulators, anti-progestins, estrogen receptor downregulators, estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, anti-androgens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Chemotherapeutic agents useful in the treatment methods of the present disclosure include cytostatic and/or cytotoxic agents.
The term "immunotherapeutic agent" in the present disclosure includes "immunomodulators" and agents that promote or mediate antigen presentation that promotes a cell-mediated immune response. Among these, "immune modulators" include immune checkpoint modulators, such as immune checkpoint protein receptors and their ligands that mediate the inhibition of T cell-mediated cytotoxicity and are typically expressed by tumors or on anergic T cells in the tumor microenvironment and allow the tumor to evade immune attack. Inhibitors of the activity of immunosuppressive checkpoint protein receptors and their ligands can overcome the immunosuppressive tumor environment to allow cytotoxic T cell attack of the tumor. Examples of immune checkpoint proteins include, but are not limited to, PD-1, PD-L1, PDL2, CTLA4, LAG3, TIM3, TIGIT, and CD 103. Modulation (including inhibition) of the activity of such proteins may be accomplished by immune checkpoint modulators, which may include, for example, antibodies, aptamers, small molecules that target checkpoint proteins, and soluble forms of checkpoint receptor proteins, among others. PD-1 targeted inhibitors include the approved pharmaceutical agents pembrolizumab and nivolumab, while plepima (ipilimumab) is an approved CTLA-4 inhibitor. Antibodies specific for PD-L1, PD-L2, LAG3, TIM3, TIGIT, and CD103 are known and/or commercially available and can also be produced by those skilled in the art.
"methods in general Biology in the art" in the present disclosure can be referred to corresponding methods described in publications such as "Current Protocols in Molecular Biology, Wiley publication", "Molecular Cloning, A Laboratory Manual, Cold spring harbor Laboratory publication", and the like.
The specific meanings of the nucleotide/amino acid sequences referred to in the present disclosure are as follows.
SEQ ID NO:1 shows the nucleotide sequence of the M gene in the Core backbone of VSV (i.e., the M gene in pRV-Core vector).
SEQ ID NO. 2 shows the nucleotide sequence of the vector Entrancepson with transposition function.
SEQ ID NO. 4 shows the nucleotide sequence of the pRV-core Mut4 vector.
SEQ ID NO 7 shows the nucleotide sequence of a plasmid containing the N gene in the core skeleton of VSV.
10 shows the nucleotide sequence of the heavy chain portion of the PDL1 antibody.
Shown in SEQ ID NO 11 is the nucleotide sequence of the light chain portion of the PDL1 antibody.
12 shows the nucleotide sequence of the single-chain PDL1 single-chain antibody.
13 shows the amino acid sequence of signal peptide Sig1 secreting PDL1 single chain antibody.
SEQ ID NO. 14 shows the amino acid sequence of signal peptide Sig2 secreting PDL1 single chain antibody.
SEQ ID NO. 15 shows the amino acid sequence of signal peptide Sig3 secreting PDL1 single chain antibody.
16 shows the amino acid sequence of signal peptide Sig4 secreting PDL1 single chain antibody.