CN117797250A - Individuation mRNA composition, vector, mRNA vaccine and application thereof - Google Patents
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides an individuation mRNA composition, a carrier, an mRNA vaccine and application thereof, and belongs to the technical field of biological vaccines. Wherein the personalized mRNA composition comprises at least one neoantigen mRNA encoding a tumor neoantigen and at least one related antigen mRNA encoding a tumor-associated antigen. The invention combines the new antigen mRNA and the related antigen mRNA to obtain the personalized vaccine material with the characteristic of high tumor killing bioactivity, which not only increases the targeting killing capacity of tumor specific T cells, but also quickens the progress of activating the immune system, improves the immune response of human body and prevents the recurrence possibly occurring in the follow-up.
Description
The present invention claims priority to the chinese application, application number "2022116972578", application day 2022, 12/28, entitled "personalized mRNA composition, vector, mRNA vaccine and uses thereof", which is incorporated herein in its entirety as part of the present invention.
Technical Field
The invention belongs to the technical field of biological vaccines, and particularly relates to an individuation mRNA composition, a carrier, an mRNA vaccine and application thereof.
Background
Immunotherapy, as a third revolution, has been mainly aimed at "survival with cancer" by enhancing and restoring the ability of the patient's own immune system to recognize and kill cancer cells, or by giving the patient an external immune power to help them kill cancer cells. Compared with radiotherapy and targeted therapy, immunotherapy has the advantages of simple treatment means, attack on cancer cells only, no damage to normal cells, and the like. In immunotherapy, however, immunotherapy based on immune checkpoint inhibitors, cytotoxic T lymphocyte-associated protein 4 antibodies and the like has progressed rapidly, and certain results have been achieved in solid tumor immunotherapy in the advanced stage, but more than 50% of patients do not respond to ICIs therapy due to lack of tumor-specific lymphocyte infiltration and the like, and more than 20% of patients develop ≡3-grade immunotherapy-related adverse reactions. Therefore, exploring a novel immunization strategy to enhance tumor-specific lymphocyte infiltration, further enhancing ORR for immunotherapy of solid tumor patients, may be the future direction of accurate immunotherapy of solid tumors.
More than 95% of the mutations in tumors are unique and patient-specific (Weide et al 2008:J.Immunother.31, 180-188). Based on these mutations, they can be transcribed into mRNA, translated into mutant peptide fragments, and presented on the surface of tumor cells via the Golgi apparatus as MHC-polypeptide complexes with major histocompatibility complexes (Majorhistocompatibility complex, MHC), which can be specifically recognized by TCR, thereby inducing specific killing of tumor cells by the body. Compared with the traditional tumor-associated antigen (Tumor associated antigen, TAA), the immunotherapy taking the new antigen corresponding to mutation as a target spot has the advantages of strong specificity, no central tolerance and autoimmune problem, small side effect, capability of realizing individuation accurate immunotherapy and the like. For example, the personalized RNA vaccine for cancer disclosed in patent EP2012000006W utilizes neoantigens to immunize patients against targeted personalized tumor cancers. However, such personalized RNA vaccines targeting only neoantigens have limited actual tumor killing effect. Therefore, there is a need in the art to develop more vaccines with good tumor killing effect.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an individuation mRNA composition, a vector, an mRNA vaccine and application thereof. On one hand, the invention takes the new antigen mRNA as a target point, realizes high-precision killing of tumors, enhances the targeting killing capability of tumor-specific T cells, and on the other hand, increases related antigen mRNA, so that the new antigen mRNA and the new antigen mRNA synchronously enter an immune system, and can rapidly activate the immune system while further enhancing the targeting killing capability of the tumor-specific T cells, thereby realizing the purpose of comprehensively improving the treatment progress and effect. The specific contents are as follows:
in a first aspect, the invention provides a personalized mRNA composition. The mRNA composition includes: at least one neoantigen mRNA encoding a tumor neoantigen and at least one associated antigen mRNA encoding a tumor associated antigen.
In some embodiments, the nascent antigen transcription template corresponding to the nascent antigen mRNA is a highly immunogenic mutant sequence that is screened for peptide selection and mutation site selection in sequence.
In some embodiments, the mRNA composition further comprises: at least one costimulatory factor mRNA encoding a costimulatory factor for promoting an immune response elicited by the tumor neoantigen and the tumor-associated antigen.
In some embodiments, the vector of the mRNA composition is an antigen presenting cell.
In some embodiments, the co-stimulatory factor is any of IL-2, IL-7, IL-12, IL-15, CD40L, CD, CD27L, CD, CD28, CD275, CD278, CD134, CD137, CD154, GITR, HVEM, LFA-1, CD2, CD58, ICAM-1, TNFSF4, TNFSF5, TNFSF7, TNFSF9, TNFSF14, TNFSF 18.
In some embodiments, the tumor-associated antigen is derived from or not derived from the same individual as the tumor-associated antigen.
In some embodiments, the tumor-associated antigen is a highly expressed tumor-associated antigen.
In some embodiments, the tumor-associated antigen is any one of WT1, MSLN, FSHR.
In some embodiments, the RNA length of the nascent antigen mRNA is 600 to 800nt; and/or
The RNA length of the related antigen mRNA is 1500-2000 nt; and/or
The RNA length of the costimulatory factor mRNA is 1100-3000 nt.
For example, the RNA length of the nascent antigen mRNA may be any value or combination of 600nt, 650nt, 700nt, 750nt, 800 nt.
For example, the RNA length of the relevant antigen mRNA may be any value or combination of 1500nt, 1600nt, 1700nt, 1750nt, 1800nt, 1850nt, 1900nt, 2000 nt.
For example, the RNA length of the co-stimulatory factor mRNA may be any value or combination of 1100nt, 1300nt, 1500nt, 1650nt, 1800nt, 1900 nt.
In a second aspect, the invention provides a vector comprising the mRNA composition of the first aspect.
In some embodiments, the carrier is one or more of a lipid, a liposome, a lipid complex, a lipid nanoparticle, a polymeric nanoparticle, a cell, a mimetic nanoparticle, a nanotube, or a conjugate comprising the mRNA composition.
In a third aspect, the invention provides an mRNA vaccine. The mRNA vaccine comprises the mRNA composition of the first aspect, or comprises the vector of the second aspect.
In a fourth aspect, the present invention provides the use of an mRNA composition according to the first aspect or a vector according to the second aspect or an mRNA vaccine according to the third aspect.
In some embodiments, the application comprises: use in the preparation of specific T cells for cancer; or (b)
Use in the preparation of TCR-T cells for cancer; or (b)
Use in the preparation of a diagnostic agent for cancer; or (b)
Use in the treatment and/or prophylaxis of cancer.
In some embodiments, the cancer is any one of breast cancer, ovarian cancer, gastric cancer, liver cancer, prostate cancer, lung cancer, and colon cancer.
The invention has the beneficial effects that: the novel antigen mRNA and the related antigen mRNA are combined to obtain an individualized vaccine material with the characteristic of high tumor killing bioactivity, and the vaccine material is applied to corresponding tumor treatment, so that the tumor specific T cell targeting killing capacity can be increased, the progress of activating an immune system can be accelerated, the immune response of a human body can be improved, and the subsequent possible recurrence can be prevented.
Drawings
FIG. 1 shows the results of protein detection of CD40L mRNA expression in DC tumor vaccine-1 prepared in example 4 of the present invention;
FIG. 2 shows the results of protein detection of WT1 mRNA expression in DC tumor vaccine-1 prepared in example 4 of the present invention;
FIG. 3 shows Western blot analysis of tumor neoantigen mRNA-1 and tumor neoantigen mRNA-2 expression proteins in DC tumor vaccines prepared in each of example 4 and example 6 of the present invention;
FIG. 4 shows the relevant detection data of tumor-specific CD8+ T cells in example 8 of the present invention; wherein, panel A is the amount of IFN-gamma expressed by tumor specific CD8+ T cells and panel B is the amount of TNF alpha expressed by tumor specific CD8+ T cells;
FIG. 5 shows the experimental results of the tumor weights of each group in example 9 of the present invention;
FIG. 6 shows the experimental results of the tumor suppression rates of each group in example 9 of the present invention;
FIG. 7 shows the experimental results of the tumor suppression rates of each group in example 10 of the present invention;
FIG. 8 shows the experimental results of the tumor suppression rates of each group in example 11 of the present invention;
FIG. 9 shows the experimental results of the tumor suppression rates of each group in example 12 of the present invention;
FIG. 10 shows the experimental results of the tumor suppression rates of each group in example 13 of the present invention;
FIG. 11 shows the experimental results of the tumor suppression rates of each group in example 14 of the present invention;
FIG. 12 shows the experimental results of the tumor suppression rates of each group in example 15 of the present invention;
FIG. 13 shows the experimental results of the tumor suppression rates of each group in example 16 of the present invention;
FIG. 14 shows the experimental results of the tumor suppression rates of each group in example 17 of the present invention;
FIG. 15 shows the experimental results of the tumor suppression rates of each group in example 18 of the present invention;
FIG. 16 shows the experimental results of the tumor suppression rates of each group in example 19 of the present invention;
FIG. 17 shows the experimental results of the tumor suppression rates of each group in example 20 of the present invention;
FIG. 18 shows the experimental results of the tumor suppression rates of each group in example 21 of the present invention;
FIG. 19 shows the experimental results of the tumor suppression rates of each group in example 22 of the present invention;
FIG. 20 shows the experimental results of the tumor suppression rates of each group in example 23 of the present invention;
fig. 21 shows the experimental results of the tumor suppression rates of each group in example 24 of the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description. The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
Various aspects of the invention are described in detail in the following examples. The examples are not intended to limit the invention. Each embodiment may be applied to any aspect of the present invention. In this application, unless otherwise indicated, the use of "or" means "and/or".
In order to facilitate understanding of the inventive concepts of the present invention and for convenience of applicant's expression, the following specific examples illustrate ovarian cancer, but the present invention is not limited to this field of ovarian cancer. The specific contents are as follows:
in a first aspect, embodiments of the present invention provide a personalized mRNA composition. The mRNA composition comprises: at least one neoantigen mRNA encoding a tumor neoantigen and at least one associated antigen mRNA encoding a tumor associated antigen.
In particular, the neoantigen mRNA is selected from all neoantigens having high immunogenicity. The mutation site of the selected neoantigen is also selected to have a higher score by adding all peptide segment scores of the same mutation site. In actual practice, a plurality of neoantigen mRNAs may be selected according to the criteria, for example, 5 neoantigens corresponding to the mutation sites corresponding to the top 5.
The related antigen mRNA is a tumor related antigen with strong immunogenicity, and can be used for the immunotherapy of tumor with high expression of related antigen. Meanwhile, the vaccine based on the related antigen mRNA has safety and effectiveness in clinical immunotherapy of diseases such as ovarian cancer, acute myelogenous leukemia and the like. The tumor-associated antigen is taken as the tumor-associated antigen which is highly expressed by more than 70% of ovarian cancer patients, and the tumor-specific T cells generated by stimulation can be improved to target the ovarian cancer tumor cells by adding the antigen.
According to the embodiment, the novel antigen mRNA and the related antigen mRNA are combined to obtain the personalized vaccine material with the characteristic of high tumor killing bioactivity, on one hand, the novel antigen mRNA is taken as a main body, high-precision tumor killing is realized, the tumor-specific T cell targeting killing capacity is enhanced, and on the other hand, the related antigen mRNA is added to enable the novel antigen mRNA and the novel antigen mRNA to synchronously enter an immune system, so that the immune system can be rapidly activated while the tumor-specific T cell targeting killing capacity is further improved, and the aim of comprehensively improving the treatment progress and effect is fulfilled.
In addition, since the mRNA is used as the vaccine raw material, the mRNA is firstly translated into the polypeptide in the carrier after entering the human body, and then the polypeptide is combined with the presentation effect of the carrier to activate the immune system, the immune system formed by the mode has memory, and the immune response of the human body to the corresponding tumor can be improved, so that the possibility of preventing subsequent recurrence is achieved.
In some embodiments, the nascent antigen transcription template corresponding to the nascent antigen mRNA is a highly immunogenic mutant sequence that is screened for peptides and mutant sites in sequence.
In specific implementation, the normal tissue sequence of a patient is taken as a control sample, and is subjected to sequence comparison with the tumor sample of the patient, so that all tumor neoantigens are screened. Then, tumor neoantigens were scored according to the following ranking conditions, ranked from high to low according to polypeptide scores, and top 10 high immunogenic neoantigens were screened. And adding all peptide segment scores of the same mutation site, sequencing the mutation sites (namely further screening out mutation sequences with higher immunogenicity), and selecting mutation points with the top 5 ranks (the mutation sequences corresponding to the 5 mutation points are the screened 5 high-immunogenicity mutation sequences). Finally, the mutation sequences corresponding to the 5 mutation points are respectively synthesized into a neoantigen transcription template for subsequent preparation of neoantigen mRNA for encoding tumor neoantigen.
Wherein the ordering condition includes: transcriptome sequencing data has mutant sequence support, TPM expression >3, affinity <300nM, mutation frequency >0.1, and non-homologous peptide.
The mRNA composition provided by the embodiment can also consist of a plurality of mutation sequences with high immunogenicity, so that the transcribed nascent antigen mRNA has higher immunogenicity, thereby further enhancing the tumor-specific T cell targeting killing capability and improving the accurate killing of tumors.
In addition, to ensure that the screened tumor neoantigen mutations are not present in normal tissue, tumor sample mutation point authenticity can be again verified. The specific verification operation may be: after the sequence of the screened neoantigen is determined, the accuracy of the mutation site of the neoantigen is verified in tumor tissue genome DNA and blood genome DNA by utilizing PCR and sanger generation sequencing methods so as to ensure that the neoantigen mutation after screening does not exist in normal tissues.
In some embodiments, the mRNA composition further comprises: at least one costimulatory factor mRNA encoding a costimulatory factor for use in promoting an immune response elicited by a tumor neoantigen and a tumor-associated antigen.
Costimulatory factor mRNA increases the secretion of interleukins by a mature vector cell, which has the function of promoting the production of cytotoxic T lymphocytes (hereinafter CTLs), by binding to the corresponding proteins produced by the vector cell itself. The interleukin (e.g. IL-12) acts in inducing multifunctional effector/memory cytotoxic T lymphocytes and can be used as a marker for immune cell differentiation.
In some embodiments, the vector of the mRNA composition is an antigen presenting cell. In particular, the antigen presenting cells may be dendritic cells.
Dendritic cells (hereinafter abbreviated DCs) are antigen presenting cells (hereinafter abbreviated APCs), and the mechanism of their antitumor activity is as follows: (1) DCs can highly express MHC-class I and MHC-class II molecules, which bind to their processed tumor antigens captured to form peptide-MHC molecule complexes, which are presented to T cells, thereby initiating both MHC-class I restricted Cytotoxic T Lymphocyte (CTL) responses and MHC-class II restricted CD4+ Th1 responses. At the same time, DCs also provide a secondary signal necessary for T cell activation through their highly expressed costimulatory molecules (CD 80/B7-1, CD86/B7-2, CD40, etc.), initiating an immune response. (2) DCs and T cells can be combined to secrete a large amount of IL-12, IL-18 activates T cell proliferation, CTL generation is induced, th1 type immune response is dominant, and tumor elimination is facilitated; activation of perforin granzyme B and FasL/Fas mediated pathways enhances NK cytotoxicity; (3) DCs secrete chemokines (CCK) specifically chemotactic initiating T cells promote T cell aggregation, and the excitation of T cells is enhanced. Maintaining effector T cells in the tumor site for a long period of time may affect tumor angiogenesis by releasing certain anti-angiogenic substances (e.g., IL-12, IFN- γ) and pro-angiogenic factors. The CCK further activates DC in a positive feedback paracrine mode, and up-regulates the expression of IL-12, CD80 and CD 86; meanwhile, DC also presents antigen peptide directly to CD8+ T cells, and the CD8+ T cells are activated with the help of activated CD4+ T cells, so that the CD4+ and CD8+ T cells can further enhance the anti-tumor immune response of organisms through secreting cytokines or directly killing.
In some embodiments, the co-stimulatory factor is any of CD40L, CD, CD27L, CD, CD28, CD275, CD278, CD134, CD137, CD154, GITR, HVEM, LFA-1, CD2, CD58, ICAM-1, TNFSF4, TNFSF5, TNFSF7, TNFSF9, TNFSF14, TNFSF 18.
In this example, the action of co-stimulatory factors is illustrated by the example of CD40L, specifically: CD40L mRNA increases secretion of IL-12 by binding to CD40 produced by mature DCs themselves, and IL-12 is a key signal that promotes production of cytotoxic T lymphocytes (hereinafter CTLs).
In some embodiments, the tumor-neoantigen is derived or not derived from the same individual as the tumor-associated antigen. In particular, the tumor neoantigen is a personalized tumor neoantigen from a patient, for which there is a very high specificity. The tumor-associated antigen is not limited to a certain patient, and may be derived from the patient, or may be a known tumor-associated antigen pre-stored in an antigen library.
In some embodiments, the tumor-associated antigen is a highly expressed tumor-associated antigen.
In some embodiments, the tumor-associated antigen is any one of WT1, MSLN, FSHR.
In some embodiments, the neoantigen mRNA is prepared from a corresponding plasmid template, and the release criteria for the immunization material after preparation include: the content is 1-4 mug/mug, the ratio of A260/A280 is 1.50-4.00, the RNA length is 600-800 nt, the integrity is more than or equal to 80%, the ultralong shortening is less than 20%, the capping efficiency is more than or equal to 70%, the DNA residue is less than 4 ng/mug, and the protein (enzyme) residue is less than 10 ng/mug.
In particular, the content may be any value or combination of 1. Mu.g/. Mu.L, 1.5. Mu.g/. Mu.L, 2. Mu.g/. Mu.L, 3. Mu.g/. Mu.L, 4. Mu.g/. Mu.L; the a260/a280 ratio may be any value or combination of 1.50, 2.00, 2.50, 3.00, 3.50, 4.00; the RNA length may be any value or combination of 600nt, 650nt, 700nt, 750nt, 800 nt; the integrity may be any value or combination of 80%, 83%, 86%, 89%, 93%, 95%, 98%; the extra long truncations may be any value or combination of 5%, 8%, 10%, 11%, 14%, 17%, 19%; capping efficiency may be any value or combination of 70%, 73%, 77%, 80%, 85%, 89%, 93%, 95%, 99%; the DNA residue may be any value or combination of 1 μg/μg, 1.5 μg/μg, 2 μg/μg, 2.5 μg/μg, 3ng/μg, 3.5ng/μg, 3.9ng/μg; the protein (enzyme) residue may be any value or combination of 5 ng/. Mu.l, 5.7 ng/. Mu.l, 6.5 ng/. Mu.l, 7 ng/. Mu.l, 7.5 ng/. Mu.l, 8 ng/. Mu.l, 9 ng/. Mu.l, 9.8 ng/. Mu.l.
In some embodiments, the relevant antigen mRNA is prepared from the corresponding plasmid template, and the release criteria for the immunization material after preparation include: the content is 1-4 mug/mug, the ratio of A260/A280 is 1.50-4.00, the RNA length is 1500-2000 nt, the integrity is more than or equal to 80%, the ultralong shortening is less than 20%, the capping efficiency is more than or equal to 70%, the DNA residue is less than 4 ng/mug, and the protein residue is less than 10 ng/mug.
In particular, the content may be any value or combination of 1. Mu.g/. Mu.L, 1.5. Mu.g/. Mu.L, 2. Mu.g/. Mu.L, 3. Mu.g/. Mu.L, 4. Mu.g/. Mu.L; the a260/a280 ratio may be any value or combination of 1.50, 2.00, 2.50, 3.00, 3.50, 4.00; the RNA length may be any value or combination of 1500nt, 1600nt, 1700nt, 1750nt, 1800nt, 1850nt, 1900nt, 2000 nt; the integrity may be any value or combination of 80%, 83%, 86%, 89%, 93%, 95%, 98%; the extra long truncations may be any value or combination of 5%, 8%, 10%, 11%, 14%, 17%, 19%; capping efficiency may be any value or combination of 70%, 73%, 77%, 80%, 85%, 89%, 93%, 95%, 99%; the DNA residue may be any value or combination of 1 μg/μg, 1.5 μg/μg, 2 μg/μg, 2.5 μg/μg, 3ng/μg, 3.5ng/μg, 3.9ng/μg; the protein (enzyme) residue may be any value or combination of 5 ng/. Mu.l, 5.7 ng/. Mu.l, 6.5 ng/. Mu.l, 7 ng/. Mu.l, 7.5 ng/. Mu.l, 8 ng/. Mu.l, 9 ng/. Mu.l, 9.8 ng/. Mu.l.
In some embodiments, costimulatory factor mRNA is prepared from the corresponding plasmid template, and the release criteria for the immunization material after preparation include: the content is 1-4 mug/mug, the ratio of A260/A280 is 1.00-4.00, the RNA length is 1100-3000 nt, the integrity is more than or equal to 80%, the ultralong shortening is less than 20%, the capping efficiency is more than or equal to 70%, the DNA residue is less than 4 ng/mug, and the protein residue is less than 10 ng/mug.
In particular, the content may be any value or combination of 1. Mu.g/. Mu.L, 1.5. Mu.g/. Mu.L, 2. Mu.g/. Mu.L, 3. Mu.g/. Mu.L, 4. Mu.g/. Mu.L; the a260/a280 ratio may be any value or combination of 1.50, 2.00, 2.50, 3.00, 3.50, 4.00; the RNA length may be any value or combination of 1100nt, 1300nt, 1500nt, 1650nt, 1800nt, 1900 nt; the integrity may be any value or combination of 80%, 83%, 86%, 89%, 93%, 95%, 98%; the extra long truncations may be any value or combination of 5%, 8%, 10%, 11%, 14%, 17%, 19%; capping efficiency may be any value or combination of 70%, 73%, 77%, 80%, 85%, 89%, 93%, 95%, 99%; the DNA residue may be any value or combination of 1 μg/μg, 1.5 μg/μg, 2 μg/μg, 2.5 μg/μg, 3ng/μg, 3.5ng/μg, 3.9ng/μg; the protein (enzyme) residue may be any value or combination of 5 ng/. Mu.l, 5.7 ng/. Mu.l, 6.5 ng/. Mu.l, 7 ng/. Mu.l, 7.5 ng/. Mu.l, 8 ng/. Mu.l, 9 ng/. Mu.l, 9.8 ng/. Mu.l.
In the embodiment of the invention, tumor neoantigen mRNA, related antigen mRNA and costimulatory factor mRNA are jointly introduced into autologous dendritic cells to obtain the high-expression personalized mRNA vaccine. The vaccine synergistically enhances immune and clinical responses through a variety of mechanisms. The specific mechanism is as follows: 1) Autologous DC cells improve reduced DC numbers and systemic dysfunction in cancer patients; 2) Developing a complete tumor antigen library, i.e. a combination of neoantigens and related antigens, to increase tumor-specific T cell targeted killing; 3) The immune co-stimulators can activate innate immunity, overcome the immunosuppressive effect in the tumor microenvironment, promote the type I T helper cell (T h cell) response, improve the microenvironment in the cancer patient, and provide a more effective immunotherapy approach.
In a second aspect, embodiments of the present invention provide a method for preparing an mRNA composition according to the first aspect. The preparation method comprises the following steps:
step 1, preparing a neoantigen mRNA based on a neoantigen plasmid by in vitro transcription;
step 2, preparing related antigen mRNA by in vitro transcription based on related antigen plasmid;
step 3, preparing costimulatory factor mRNA based on costimulatory factor plasmid in vitro transcription.
In specific implementation, the specific operation of step 1 may be as follows:
First, a nascent antigen plasmid was prepared: the mutation sequence with high immunogenicity is synthesized into a new antigen transcription template, and the new antigen plasmid is prepared for preparing new antigen mRNA.
Then, in vitro transcription prepares neoantigen mRNA: the BbsI restriction enzyme is incubated with the nascent antigen plasmid (each 1. Mu.g of the nascent antigen plasmid is reacted with 0.1 to 2. Mu.L of Bbs I, such as with any one of 0.1. Mu.L, 0.4. Mu.L, 0.9. Mu.L, 1.4. Mu.L, 1.7. Mu.L and 2.0. Mu.L of Bbs I) at 32 to 39 ℃ (such as at any one of 32 ℃, 35 ℃, 37 ℃ and 39 ℃ or a combination thereof) for 0.5 to 2 hours (such as any one of 0.5 hours, 1.0 hours, 1.5 hours and 2 hours or a combination thereof) to complete linearization of the nascent antigen plasmid;
purifying the linear plasmid DNA by using a PCR product purification kit, and eluting the linear plasmid DNA by using nuclease-free water; sampling for linearization confirmation detection and DNA content and purity detection;
preparing an in vitro transcription system by taking linearized neoantigen plasmid as a template, carrying out reaction on each 11 mu g neoantigen linearized plasmid with 1-4 mu L T7 transcriptases (such as any one value or combination of 1 mu L, 2 mu L, 3 mu L and 4 mu L), 1-4 mu L100mM GTP (such as any one value or combination of 1 mu L, 2 mu L, 3 mu L and 4 mu L), 1-4 mu L100mM ATP (such as any one value or combination of 1 mu L, 2 mu L, 3 mu L and 4 mu L), 1-4 mu L100mM UTP (such as any one value or combination of 1 mu L, 2 mu L, 3 mu L and 4 mu L), 1-4 mu L100mM CTP (such as any one value or combination of 1 mu L, 2 mu L, 3 mu L and 4 mu L), 1-4 mu L100mM DTT (such as any one value or combination of 1 mu L, 2 mu L, 3 mu L and 4 mu L), incubating at 32-39deg.C (such as any one of 32deg.C, 35deg.C, 37deg.C and 39deg.C or a combination thereof) for 1-4 h (such as any one of 1h, 1.0h, 1.5h and 2h or a combination thereof), adding 0.5-3 μL/μg Dnase I after transcription is completed, mixing (such as any one of 0.5 μL/μg, 1.0 μL/μg, 1.5 μL/μg, 2.0 μL/μg, 2.5 μL/μg and 3 μL/μg or a combination thereof), incubating at 32-39deg.C (such as any one of 32deg.C, 35deg.C, 37 ℃ and 39deg.C or a combination thereof) for 5-20 min (such as 5min, 10min, any one of 15min and 20min or a combination thereof) to digest the DNA template;
Purifying the nascent antigen mRNA by using a PCR product purification kit, eluting with water without a nuclease, and detecting the purity and the content of the nascent antigen RNA;
the purified nascent antigen mRNA is incubated with 0.1 to 1. Mu.L of 10mM GTP (e.g., any one of 0.1. Mu.L, 0.3. Mu.L, 0.7. Mu.L, and 1. Mu.L or a combination thereof), 0.01 to 0.1. Mu.L of 20mM SAM (e.g., any one of 0.01. Mu.L, 0.03. Mu.L, 0.07. Mu.L, and 0.1. Mu.L or a combination thereof), 0.01 to 0.1. Mu.L of 2' -O-methyltransferase (e.g., any one of 0.01. Mu.L, 0.03. Mu.L, 0.1. Mu.L, and 0.1. Mu.L or a combination thereof), 0.01 to 0.1. Mu.L of capping enzyme (e.g., any one of 0.01. Mu.L, 0.03. Mu.L, 0.07. Mu.L, and 0.1. Mu.L or a combination thereof), at 32 to 39 ℃ (e.g., any one of 32 ℃, 35 ℃, 37 ℃ and 39 ℃) for 1 to 4 hours (e.g., 1.1 hour, 1.5 hours, 1 hour, 2 hours, or a combination thereof);
purifying with PCR product purifying kit, eluting with nuclease-free water, and detecting the purity, content, RNA length, integrity, ultra-long truncation, capping efficiency, DNA residue and protein (enzyme) residue of the new antigen mRNA;
the concentration is regulated to the regulated standard (2 mug/mug), and the mixture is sub-packaged according to 44 mug/branch (40 mug specification), stored for 24 hours at the temperature of between-90 ℃ and-70 ℃ (such as any one of-90 ℃, -85 ℃, -80 ℃, -75 ℃ and-70 ℃ or the combination range thereof), and then transferred to a liquid nitrogen tank for storage.
Among these, the release criteria for neoantigen mRNA include: 1-4 mug/mug, purity (expressed by OD (A260/A280) ratio) of 1.50-4.00, RNA length of 600-800 nt, integrity of more than or equal to 80%, ultra-long truncated less than 20%, capping efficiency of more than or equal to 70%, DNA residue of less than 4 ng/mug, and protein (enzyme) residue of less than 10 ng/mug.
In the specific implementation, the specific operation of step 2 is similar to that of step 1, and is not described herein, but only the release criteria of the two are slightly different. In step 2, the release criteria for the relevant antigen mRNA include: 1-4 mug/mug, purity (expressed by OD (A260/A280) ratio) of 1.50-4.00, RNA length of 1500-2000 nt, integrity of more than or equal to 80%, ultra-long truncated less than 20%, capping efficiency of more than or equal to 70%, DNA residue of less than 4 ng/mug, and protein (enzyme) residue of less than 10 ng/mug.
In the implementation, the specific operation of step 3 is similar to that of step 1, and is not described herein, but only the release criteria of the two are slightly different. In step 3, the release criteria for co-stimulatory factor mRNA include: 1-4 mug/mug, purity (expressed by OD (A260/A280) ratio) 1.50-4.00, RNA length 1100-3000 nt, integrity more than or equal to 80%, ultra-long truncated less than 20%, capping efficiency more than or equal to 70%, DNA residue less than 4 ng/mug, protein (enzyme) residue less than 10 ng/mug.
In a third aspect, embodiments of the present invention provide a carrier. The vector comprises the mRNA composition of the first aspect. In some embodiments, the carrier may be any one or more of a lipid, a liposome, a lipid complex, a lipid nanoparticle, a polymeric nanoparticle, a cell, a mimetic nanoparticle, a nanotube, or a conjugate comprising the mRNA composition of the first aspect described above. The cells may be antigen presenting cells (e.g. dendritic cells DC) or B cells
Specifically, as follows: the mRNA composition may be complexed with a polymer or lipid component, or the mRNA composition may be encapsulated in some aspect in a liposome, or the mRNA composition may be encapsulated in Lipid Nanoparticles (LNPs).
In this example, the use of LNPs is effective in delivering chemically modified or unmodified mRNA vaccines.
In this example, the liposomes are amphiphilic lipids that can form bilayers in an aqueous environment to encapsulate the RNA-containing water core. These lipids may have anionic, cationic or zwitterionic hydrophilic head groups. Liposomes can be formed from a single lipid or a mixture of lipids. The mixture may comprise (i) a mixture of anionic lipids (ii) a mixture of cationic lipids (iii) a mixture of zwitterionic lipids (iv) a mixture of anionic lipids and cationic lipids (v) a mixture of anionic lipids and zwitterionic lipids (vi) a mixture of zwitterionic lipids and cationic lipids or (vii) a mixture of anionic lipids, cationic lipids and zwitterionic lipids.
In this embodiment, polymer microparticles or nanoparticles may also be used to encapsulate or adsorb mRNA. These particles may be substantially non-toxic and biodegradable. The particles used to deliver mRNA may have the optimal size and zeta potential. For example, the diameter of the microparticles may be in the range of 0.02 μm to 8 μm. In the case of compositions having a population of micro-or nano-particles of different diameters, at least 80%,85%,90% or 95% of these particles desirably have a diameter in the range of 0.03-7 μm. The particles may also have a zeta potential of between 40 and 100mV in order to maximize adsorption of mRNA to the particles. Non-toxic and biodegradable polymers include, but are not limited to, poly (ahydroxycid), polyhydroxybutyric acid, polylactones (including polycaprolactone), polydioxanones, polyglutarones, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine derived polycarbonates, polyvinylpyrrolidone or polyesteramides, and combinations thereof.
In this example, autologous DC cells can improve DC reduction and systemic dysfunction in cancer patients. Meanwhile, the autologous DC cells are selected as vectors, and the characteristics of the relevant antigens and the neoantigens expressed by the DCs are utilized, so that the two antigens can be efficiently presented, and the T cells of tumor effect can be effectively stimulated and killed.
Furthermore, the personalized neoantigens in this example: selected from tumor neoantigens specific for each patient themselves. They are expressed only by tumor cells and thus can elicit a truly tumor-specific T cell response, thereby preventing "off-target" damage to non-tumor tissues. Meanwhile, neoantigens are neoepitopes derived from somatic mutations, which have the potential to bypass the central tolerance of T cells to self-epitopes, thereby inducing immune responses to tumors. Furthermore, these vaccine-enhanced neoantigen-specific T cell responses persist and offer the potential for post-treatment immune memory, which provides the possibility of long-term prevention of disease recurrence.
Tumor associated antigen (tumor associated antigen, TAA) in this example: refers to a class of antigen molecules that are present on either tumor cells or normal cells, and are commonly used in clinical tumor diagnosis. It is not specific to tumor cells, and normal cells can be synthesized in minute amounts, but are highly expressed when tumor cells proliferate. In solid tumor treatment, the TAA target is also the first choice of tumor targets. The antigen is highly expressed by using a DC vaccine, and the antigen is efficiently presented, so that the tumor effector T cells can be effectively stimulated and killed. The specific antigen of interest may specifically be WT1.
The co-stimulatory factors of this embodiment, such as CD40L, are described in detail with respect to CD 40L: DC secretion of IL-12 was induced by the CD40-CD40L co-stimulatory axis. IL-12 is an important cytokine in innate and adaptive immunity, and is capable of enhancing the immunity of helper T cell type 1 (Th 1), increasing the cytotoxicity of cytotoxic T lymphocytes, and inhibiting angiogenesis.
The main concept of the vector (such as DC vaccine) carrying the mRNA composition provided in this example is: because of the over-expression antigen and the neoantigen generated by mutation in cancer cells (such as ovarian cancer cells), the characteristics of the cancer cells are utilized, and the neoantigen and the related antigen are used as targets, so that the killing effect on tumors can be improved.
In a fourth aspect, an embodiment of the present invention provides a method for preparing the carrier according to the third aspect. The preparation method comprises the following steps:
step 4, loading the mRNA composition described in the first aspect into a carrier.
When the vector is a cell, the preparation method further comprises: step 5, inducing the vector loaded with the mRNA composition.
In specific implementation, DC is taken as an example to describe the details, and the specific operation may be as follows:
at 1X 10 6 ～1×10 8 personal/mL (e.g. 1X 10 6 personal/mL, 10X 10 6 Per mL, 40X 10 6 personal/mL, 70X 10 6 individual/mL and 100×10 6 Any one of the values of the individual/mL or a combination thereof) is resuspended in electrotransport fluid, and each electrotransport cup is added with 1X 10 5 ～5×10 7 (0.1-0.5 mL) mDC (e.g., 1X 10) 6 Per mL, 50X 10 6 Per mL, 100X 10 6 personal/mL, 200X 10 6 personal/mL, 300X 10 6 personal/mL, 400X 10 6 Individual/mL and 500X 10 6 Any one of the values per mL or a combination thereof), 1 part of the RNA mixture (the ratio of the mixture is 1X 10) 6 1 to 4. Mu.g of co-stimulatory factor CD40L mRNA, 1 to 4. Mu.g of over-expressed WT1 mRNA, 0.1 to 3. Mu.g of tumor neoantigen mRNA are added to the individual DC cells, as follows: the added CD40L mRNA was any one of 1. Mu.g, 2. Mu.g, 3. Mu.g and 4. Mu.g, the added overexpressed WT1 mRNA was any one of 1. Mu.g, 2. Mu.g, 3. Mu.g and 4. Mu.g, and the added tumor neoantigen mRNA was any one of 0.1. Mu.g, 0.5. Mu.g, 1. Mu.g, 1.5. Mu.g, 2.0. Mu.g, 2.5. Mu.g and 3. Mu.g), and co-electrotransformation (voltage value: 200 to 350V, such as any one of 200V, 250V, 300V and 350V or a combination range thereof) was performed by an electrotransformation instrument.
The electrotransport device was Gene Pulser Xcell electrotransport device manufactured by Bio-Rad corporation.
The neoantigen mRNA may be selected from 1 to 10 sequences, that is, 1 sequence of neoantigen mRNA, or 2 sequences, 4 sequences, 6 sequences or 10 sequences of neoantigen mRNA.
After electrical stimulation, the cells were stimulated at 1X 10 6 ～5×10 6 personal/mL (e.g. 1X 10 6 Per mL, 2X 10 6 3X 10 per mL 6 Per mL, 4X 10 6 Individual/mL and 5X 10 6 Any one value of the individual/mL) is transferred into AIM-V culture medium containing GM-CSF and IL-4, and is placed into a carbon dioxide incubator for continuous culture, and the time from the beginning of electric transfer is 2-6 h (such as any one value of 2h, 3h, 4h, 5h and 6h or the combination thereof)) After that, the cells were transferred from the culture bag, counted by centrifugation and the stock solution was harvested. The stock solution is the DC tumor vaccine.
In a fifth aspect, embodiments of the present invention provide an mRNA vaccine. The mRNA vaccine comprises the mRNA composition of the first aspect described above, or comprises the vector of the third aspect described above. As described herein, mRNA vaccines can be used to induce balanced immune responses, including cellular and/or humoral immunity, without many of the risks associated with DNA vaccination.
In a sixth aspect, embodiments of the present invention provide a use of the mRNA composition of the first aspect or the vector of the third aspect or the mRNA vaccine of the fifth aspect.
In some embodiments, the use may be in the treatment and/or prevention of cancer, or in the preparation of specific T cells for cancer, or in the preparation of TCR-T cells for cancer, or in the preparation of diagnostic reagents for cancer.
In some embodiments, the cancer may be any one of breast cancer, ovarian cancer, gastric cancer, liver cancer, prostate cancer, lung cancer, and colon cancer.
In order to better understand the in vitro evaluation method of antigen immunogenicity provided by the examples of the present invention, the following detailed description will be given by way of specific examples. The reagents and instruments used are all commercial products and can be purchased directly unless otherwise specified.
In the following examples, WT1 was used as the tumor-associated antigen, dendritic cells were used as the vector, and CD40L was used as the co-stimulatory factor.
The ovarian cancer patients referred to in this example were diagnosed in the Huaxi hospital with concurrent ovariectomy. Patient samples were collected and used with the approval of the ethics committee of the western medicine hospital, and informed of the patient or patient family, both parties signed informed consent. Tumor tissue samples were immediately placed in plastic bottles containing mRNA protection fluid after the samples were collected from the operating room and patient information was marked. Additional 5-8mL of patient peripheral blood was collected using EDTA tubes as a control sample.
After checking the integrity and total amount of the sample, sequencing analysis is performed, and the sequencing result is screened under the following conditions: transcriptome sequencing data has mutant sequence support, TPM expression >5, affinity <100nM, mutation frequency >0.1, and non-homologous peptide. If the above conditions are not met, the rest are ranked from high to low according to the polypeptide scores, the top 10 high-immunogenicity neoantigens are screened, all peptide segment scores of the same mutation site are added, the mutation sites are ranked, and the top 5 mutation sites are selected.
And (3) verifying the accuracy of the mutation site of the neoantigen in the extracted tumor tissue genome DNA and the genomic DNA of the peripheral blood of the patient by using a PCR and sanger generation sequencing method. Ensuring that the neoantigen mutations after screening are not present in normal tissue.
EXAMPLE 1 preparation of neoantigen mRNA
In this example, the nascent antigen transcription template is a mutated sequence with high immunogenicity that is selected, and then a third party is commissioned to prepare a nascent antigen plasmid based on the nascent antigen template.
In vitro transcription to prepare neoantigen mRNA: 0.1 mu L of BbsI restriction enzyme was mixed with 1 mu g of the neoantigen plasmid and incubated for 0.5h at 32℃to complete linearization of the neoantigen plasmid; purifying the linear plasmid DNA by using a PCR product purification kit, and eluting the linear plasmid DNA by using nuclease-free water; sampling for linearization confirmation detection and DNA content and purity detection; preparing an in vitro transcription system by taking a linearization neoantigen plasmid as a template, reacting each 11 mu g neoantigen linearization plasmid with 1 mu L T transcriptase, 1 mu L100 mM GTP, 1 mu L100 mM ATP, 1 mu L100 mM UTP, 1 mu L100 mM CTP and 1 mu L100 mM DTT, incubating for 1h at 32 ℃, adding 0.5 mu L/mu g Dnase I after transcription is completed, mixing uniformly, and incubating for 5min at 32 ℃ to digest the DNA template; purifying the nascent antigen mRNA by using a PCR product purification kit, eluting with water without a nuclease, and detecting the purity and the content of the nascent antigen RNA; the purified nascent antigen mRNA is reacted with 0.1 mu L of 10mM GTP, 0.01 mu L of 20mM SAM, 0.01 mu L of 2' -O-methyltransferase and 0.01 mu L of capping enzyme per 1 mu g, and then incubated for 1h at 32 ℃ for capping modification; purifying with PCR product purifying kit, eluting with nuclease-free water, and detecting the purity, content, RNA length, integrity, ultra-long truncation, capping efficiency, DNA residue and protein (enzyme) residue of the new antigen mRNA; the concentration was adjusted to a prescribed standard (2. Mu.g/. Mu.L), and the mixture was dispensed at 44. Mu.g/branch (40. Mu.g in specification) and stored at-70℃for 24 hours, and then transferred to a liquid nitrogen tank for storage.
Wherein, the release standard of the new antigen mRNA is as follows: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) greater than 2.0, RNA length 600nt, integrity > 90%, ultralong truncations < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl. .
In this example, two neoantigen mRNAs were prepared according to the procedure described above, and the specific sequences are as follows:
the neoantigen mRNA-1 (SEQ ID NO: 1) is shown in Table 1,
TABLE 1 mRNA sequence information for the neoantigen mRNA-1
| RNA name | Gene | mRNA sequences |
| HLA-A0201 | SH3BP5L | AACCUGAUGCAGAUCAGCGAGCAGAUU |
The neoantigen mRNA-2 (SEQ ID NO: 2) is shown in Table 2,
TABLE 2 mRNA sequence information for the neoantigen mRNA-2
| RNA name | Peptide fragment information | mRNA sequences |
| SPINT2-0201 | VLLAGLFVMV | GUGCUUCUGGCGGGGCUGUUCGUGAUGGUG |
Example 2 preparation of related antigen WT1 mRNA
The specific operation and conditions of this embodiment are similar to those of embodiment 1 described above, and will not be described here. While the release criteria differ only slightly from each other. In this example, the release criteria for the relevant antigen mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length of 1500nt, integrity > 90%, ultralong truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence (SEQ ID NO: 3) of the related antigen WT1 mRNA prepared in this example is as follows:
CUGGACUUCCUCUUGCUGCAGGACCCGGCUUCCACGUGUGUCCCGGAGCCGGCGUCUCAGCACACGCUCCGCUCCGGGCCUGGGUGCCUACAGCAGCCAGAGCAGCAGGGAGUCCGGGACCCGGGCGGCAUCUGGGCCAAGUUAGGCGCCGCCGAGGCCAGCGCUGAACGUCUCCAGGGCCGGAGGAGCCGCGGGGCGUCCGGGUCUGAGCCGCAGCAAAUGGGCUCCGACGUGCGGGACCUGAACGCGCUGCUGCCCGCCGUCCCCUCCCUGGGUGGCGGCGGCGGCUGUGCCCUGCCUGUGAGCGGCGCGGCGCAGUGGGCGCCGGUGCUGGACUUUGCGCCCCCGGGCGCUUCGGCUUACGGGUCGUUGGGCGGCCCCGCGCCGCCACCGGCUCCGCCGCCACCCCCGCCGCCGCCGCCUCACUCCUUCAUCAAACAGGAGCCGAGCUGGGGCGGCGCGGAGCCGCACGAGGAGCAGUGCCUGAGCGCCUUCACUGUCCACUUUUCCGGCCAGUUCACUGGCACAGCCGGAGCCUGUCGCUACGGGCCCUUCGGUCCUCCUCCGCCCAGCCAGGCGUCAUCCGGCCAGGCCAGGAUGUUUCCUAACGCGCCCUACCUGCCCAGCUGCCUCGAGAGCCAGCCCGCUAUUCGCAAUCAGGGUUACAGCACGGUCACCUUCGACGGGACGCCCAGCUACGGUCACACGCCCUCGCACCAUGCGGCGCAGUUCCCCAACCACUCAUUCAAGCAUGAGGAUCCCAUGGGCCAGCAGGGCUCGCUGGGUGAGCAGCAGUACUCGGUGCCGCCCCCGGUCUAUGGCUGCCACACCCCCACCGACAGCUGCACCGGCAGCCAGGCUUUGCUGCUGAGGACGCCCUACAGCAGUGACAAUUUAUACCAAAUGACAUCCCAGCUUGAAUGCAUGACCUGGAAUCAGAUGAACUUAGGAGCCACCUUAAAGGGAGUUGCUGCUGGGAGCUCCAGCUCAGUGAAAUGGACAGAAGGGCAGAGCAACCACAGCACAGGGUACGAGAGCGAUAACCACACAACGCCCAUCCUCUGCGGAGCCCAAUACAGAAUACACACGCACGGUGUCUUCAGAGGCAUUCAGGAUGUGCGACGUGUGCCUGGAGUAGCCCCGACUCUUGUACGGUCGGCAUCUGAGACCAGUGAGAAACGCCCCUUCAUGUGUGCUUACCCAGGCUGCAAUAAGAGAUAUUUUAAGCUGUCCCACUUACAGAUGCACAGCAGGAAGCACACUGGUGAGAAACCAUACCAGUGUGACUUCAAGGACUGUGAACGAAGGUUUUCUCGUUCAGACCAGCUCAAAAGACACCAAAGGAGACAUACAGGUGUGAAACCAUUCCAGUGUAAAACUUGUCAGCGAAAGUUCUCCCGGUCCGACCACCUGAAGACCCACACCAGGACUCAUACAGGUAAAACAAGUGAAAAGCCCUUCAGCUGUCGGUGGCCAAGUUGUCAGAAAAAGUUUGCCCGGUCAGAUGAAUUAGUCCGCCAUCACAACAUGCAUCAGAGAAACAUGACCAAACUCCAGCUGGCGCUUUGA
EXAMPLE 3 preparation of Co-stimulatory factor CD40L mRNA
The specific operation and conditions of this embodiment are similar to those of embodiment 1 described above, and will not be described here. While the release criteria differ only slightly from each other. In this example, the release criteria for co-stimulatory factor CD40L mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length of 1100nt, integrity > 90%, ultralong truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence (SEQ ID NO: 4) of the costimulatory factor CD40L mRNA prepared in this example was as follows:
AUGAUCGAAACAUACAACCAAACUUCUCCCCGAUCUGCGGCCACUGGACUGCCCAUCAGCAUGAAAAUUUUUAUGUAUUUACUUACUGUUUUUCUUAUCACCCAGAUGAUUGGGUCAGCACUUUUUGCUGUGUAUCUUCAUAGAAGGUUGGACAAGAUAGAAGAUGAAAGGAAUCUUCAUGAAGAUUUUGUAUUCAUGAAAACGAUACAGAGAUGCAACACAGGAGAAAGAUCCUUAUCCUUACUGAACUGUGAGGAGAUUAAAAGCCAGUUUGAAGGCUUUGUGAAGGAUAUAAUGUUAAACAAGGAGGAGACGAAGAAAGAAAACAGCUUUGAAAUGCAAAAAGGUGAUCAGAAUCCUCAAAUUGCGGCACAUGUCAUAAGUGAGGCCAGCAGUAAAACAACAUCUGUGUUACAGUGGGCUGAAAAAGGAUACUACACCAUGAGCAACAACUUGGUAACCCUGGAAAAUGGGAAACAGCUGACCGUUAAAAGACAAGGACUCUAUUAUAUCUAUGCCCAAGUCACCUUCUGUUCCAAUCGGGAAGCUUCGAGUCAAGCUCCAUUUAUAGCCAGCCUCUGCCUAAAGUCCCCCGGUAGAUUCGAGAGAAUCUUACUCAGAGCUGCAAAUACCCACAGUUCCGCCAAACCUUGCGGGCAACAAUCCAUUCACUUGGGAGGAGUAUUUGAAUUGCAACCAGGUGCUUCGGUGUUUGUCAAUGUGACUGAUCCAAGCCAAGUGAGCCAUGGCACUGGCUUCACGUCCUUUGGCUUACUCAAACUCUAA
EXAMPLE 4 preparation of DC tumor vaccine-1 loaded with mRNA composition
The related antigen WT1 mRNA, tumor neoantigen mRNA and co-stimulatory factor CD40L mRNA are loaded into mDC through an electrotransfection technology, and then the loaded mDC is induced to obtain mature DC cells loaded with the related antigen, tumor neoantigen and co-stimulatory factor. The specific operation is as follows:
at 1X 10 6 The concentration of each mL is resuspended in electrotransport liquid, and each electrotransport cup is added with 1X 10 6 Each mL of mDC was added with 1 part of RNA mixture (1X 10 was contained in the mixture) 6 Co-electrotransformation (voltage value 200V) was performed by an electrotransformation apparatus with DC cells, 1. Mu.g of CD40L mRNA, 1. Mu.g of WT1 mRNA, 0.1. Mu.g of tumor neoantigen mRNA-1. The electrotransport device was Gene Pulser Xcell electrotransport device manufactured by Bio-Rad corporation.
After electrical stimulation, the cells were stimulated at 1X 10 6 The concentration of each mL is transferred into AIM-V culture medium containing GM-CSF and IL-4, and the culture medium is placed into a carbon dioxide incubator for continuous culture, after 2 hours from the beginning time of electric rotation, the cells are transferred out of a culture bag, and the stock solution is obtained after centrifugal resuspension counting. The stock solution is DC tumor vaccine-1.
CD40L mRNA expression verification: successful production in DC cells was verified by detecting proteins expressed by effector CD40L RNA. The verification operation is as follows: washing cells in the harvested stock solution by using FACS buffer, centrifuging, and dying the cells by using Dead Cell Discriminator; cells were fixed with Fixation buffer and Stop Reagent Discriminator; washing the fixed cells with FACS buffer; then re-suspending the cells with 1 XPerm/wash buffer and dividing into a control group and a CD40L group; adding 2.5uL purified CD154,CD40L groups in the control group, and incubating for 30min at room temperature; then 20uL of APC-CD154 is added to the control group and the CD40L group respectively, and the mixture is incubated for 30 minutes at room temperature; after washing the cells, the amount of RNA expressed in the cells of the control group and CD40L group was measured by a flow cytometer, and the data was analyzed by FlowJo.
FIG. 1 shows the results of protein detection of CD40L mRNA expression in DC tumor vaccine-1 (S group) prepared in example 4 of the present invention. As shown in FIG. 1, the expression level of CD154 in the DC tumor vaccine-1 can reach 60.2-94.9%, the average expression intensity is 80.3%, while the expression level of CD154 in the DC cells of the control group C (non-electrotransferred CD40L mRNA) is below 10%, and the average expression intensity is only 2.3%. From this result, it was found that CD40L mRNA was successfully produced in DC cells.
WT1 mRNA expression validation: successful production in DC cells was verified by detecting proteins expressed by the over-expressed antigen (WT 1) RNA. The validation procedure of this example is similar to that of CD40L mRNA expression, except that: the WTIgroup is supplemented with purified WT1 antibody, the control group is not supplemented, and after incubation for 30min, the control group and the WTIgroup are supplemented with 1.25uL of fluorescent secondary antibody APC Rat anti-Mouse IgG1.
FIG. 2 shows the results of protein detection of WT1 mRNA expression in DC tumor vaccine-1 prepared in example 4 of the present invention. As shown in FIG. 2, the expression level of WT1 in DC tumor vaccine-1 can reach 65.1-92.4%, the average expression level is 76.7%, while the expression level of WT1 in DC cells in the control group (non-electrotransformed WT1 mRNA) is below 10%, the average expression intensity is only 3.97%, which indicates that the over-expressed antigen (WT 1) RNA is successfully generated in DC cells.
Tumor neogenesis antigen mRNA-1 expression verification: the protein formed by the translation expression of the nascent antigen RNA in the cells is directly detected by a western blotting method. The detection method disclosed by the patent CN111440228B is used for detection, and comprises the following specific operations: washing cells in the harvested stock solution with PBS and centrifuging; removing supernatant, adding lysate (volume ratio of IP cell lysate, PMSF and protease inhibitor Cocktail is 1:0.002:0.002), swirling, adding 5X loadling buffer vortex, mixing, instantly separating, heating at 75deg.C for 5min, and pulverizing with cell pulverizer to obtain protein solution; the protein solution was electrophoresed on a 12% SDS-PAGE and transferred onto PVDF membrane, then blocked with 5% skim milk for 1h at room temperature, washed 3 times with PBST for 5min each time, then added with primary antibody (5% milk), left overnight at 4℃and the washing steps repeated, added with secondary antibody diluent (5% skim milk mixed with murine antibody at 1:500000) and incubated for 1h at 4℃and finally analyzed for Western blotting using a chemiluminescent imaging analysis system.
FIG. 3 shows Western blot analysis of tumor neoantigen mRNA-1 and tumor neoantigen mRNA-2 expression proteins in DC tumor vaccines prepared in each of example 4 and example 6 of the present invention. As shown in FIG. 3, the tumor neoantigen mRNA-1 can express proteins in a DC tumor vaccine. The DC cells of the control group (non-electrotransfer tumor neoantigen mRNA-1) did not have any blots at the same positions, indicating that the control group had no corresponding protein expression.
EXAMPLE 5 preparation of DC tumor vaccine-2 loaded with mRNA composition
The preparation method and specific conditions of this example are similar to those of example 4, except that: the RNA mixture was 1X 10 6 The cells consisted of individual DC cells, 1. Mu.g of WT1 mRNA and 0.1. Mu.g of tumor neoantigen mRNA-1. The other contents are the same, and detailed description is omitted in this embodiment. The stock solution was named DC tumor vaccine-2.
The WT1 mRNA expression test and the tumor neoantigen mRNA-1 expression test in this example were performed in the same manner as in example 4, and therefore, the description thereof will not be repeated in this example.
EXAMPLE 6 preparation of DC tumor vaccine-3 loaded with mRNA composition
The preparation method and specific conditions of this example are similar to those of example 4, except that: the RNA mixture was 1X 10 6 The cells consisted of DC cells, 1. Mu.g of CD40L mRNA, 1. Mu.g of WT1 mRNA and 0.1. Mu.g of tumor neoantigen mRNA-2. The other contents are the same, and detailed description is omitted in this embodiment. The stock solution was named DC tumor vaccine-3.
The WT1 mRNA expression test and the WT1 mRNA expression test in this example are the same as those in example 4, and therefore, the description thereof will not be repeated in this example.
The procedure for verifying expression of tumor neoantigen mRNA-2 was the same as that in example 4, and will not be described in detail in this example. FIG. 3 shows Western blot analysis of tumor neoantigen mRNA-1 and tumor neoantigen mRNA-2 expression proteins in DC tumor vaccines prepared in each of example 4 and example 6 of the present invention. As shown in FIG. 3, tumor neoantigen mRNA-2 was able to express protein in DC tumor vaccine, whereas DC cells of the control group (non-electrotransformed tumor neoantigen mRNA-2) did not have any blots at the same position, indicating that no corresponding protein was expressed in the control group.
EXAMPLE 7 preparation of DC tumor vaccine-4 loaded with mRNA composition
The preparation method and specific conditions of this example are similar to those of example 4, except that: the RNA mixture was 1X 10 6 The individual DC cells, 1. Mu.g of WT1 mRNA and 0.1. Mu.g of tumor neoantigen mRNA-2. The other contents are the same, and detailed description is omitted in this embodiment. The stock solution was named DC tumor vaccine-4.
The WT1 mRNA expression test and the tumor neoantigen mRNA-2 expression test in this example were performed in the same manner as in example 6, and therefore, the description thereof will not be repeated in this example.
Example 8 in vitro evaluation of immunogenicity of DC tumor vaccines
The evaluation group includes: CD40L/WT 1/neoantigen-treated group, control group, neoantigen-treated group, WT 1-treated group, CD 40L-treated group.
Wherein, the CD40L/WT 1/neoantigen treatment group is: DC tumor vaccine-1 and CD8 prepared in example 4 above + Co-stimulated culture of T cells at a cell number ratio of 1:10 to activate T cells to give tumor specific CD8 + Cell suspension number 1 of T cells, CD40L/WT 1/tumor neoantigen group. WT 1/neoantigen treated group: DC tumor vaccine-2 and CD8 prepared in example 5 above + Co-stimulated culture of T cells at a cell number ratio of 1:10 to activate T cells to give tumor specific CD8 + Cell suspension number 1 of T cells, WT 1/tumor neoantigen group.
Wherein, the control group is: preparation of RNA mixture by using enzyme-free water instead of RNA mixturePreparing RNA-free mature DC, and then adopting the RNA-free mature DC and CD8 + T cell co-incubation stimulation culture to obtain CD8 + T cell suspension, i.e. blank. The neoantigen treatment group was: DC and CD8 loaded with tumor neoantigen mRNA-1 prepared by the steps + Co-stimulated culture of T cells at a cell number ratio of 1:10 to activate T cells to give tumor specific CD8 + Cell suspension number 2 of T cells, tumor neoantigen treated group. The WT1 treatment group was: DC and CD8 loaded with WT1 mRNA prepared by the steps + Co-stimulated culture of T cells at a cell number ratio of 1:10 to activate T cells to give tumor specific CD8 + Cell suspension No. 3 of T cells, WT1 treatment group. The CD40L treatment group was: DC and CD8 loaded with the CD40L mRNA prepared by the steps + Co-stimulated culture of T cells at a cell number ratio of 1:10 to activate T cells to give tumor specific CD8 + Cell suspension number 4 of T cells, CD40L treatment group.
All 6 groups were incubated in vitro for 10 days, with 3 corresponding rounds of co-stimulation of DC cells, each round being 3 days apart.
The specific evaluation method comprises the following steps: tumor-specific CD8 detected in vitro using ICS detection technique + T cell internal effector IFN-gamma and tnfα. FIG. 4 shows tumor-specific CD8 in an embodiment of the invention + Relevant detection data of T cells; wherein, panel A is tumor-specific CD8 + T cells express IFN-gamma amounts, panel B is tumor specific CD8 + T cells express an amount of tnfα.
As shown in FIG. 4, it is clear from the test data that CD8 was subjected to 3 rounds of antigen-loaded stimulation by DC tumor vaccine-1 and DC tumor vaccine-2 + Both IFN-gamma and TNF-alpha expression of the T cell specific markers are up-regulated, exhibiting a greater ability to kill tumor cells.
Furthermore, from the data of the CD40L/WT 1/neoantigen-treated group and the WT 1/neoantigen-treated group, it was found that CD8 was stimulated when CD40L mRNA was added to an mRNA composition consisting of WT1 mRNA and tumor neoantigen mRNA-1 + T cell specific markers IFN-gamma and TNF-gammaAlpha expression was upregulated to a higher level than CD8 stimulated by mRNA composition consisting of WT1 mRNA and tumor neoantigen mRNA-1 + T cell specific markers IFN-gamma and TNF-alpha expression are up-regulated to a high degree. That is, the addition of CD40L mRNA can further increase the immunogenicity of DC tumor vaccines.
Example 9 evaluation of in vivo efficacy of DC tumor vaccine
We selected ovarian cancer-derived cell line OVCAR8 (HLA-A-02:01) to simulate human tumor cells, inoculated immunodeficient mice subcutaneously to construct tumor-bearing model, and then acclimatized CD8 in vitro + T humanized mice, the immune system of humans was reconstituted, and the objective was to observe the immune response caused by repeated intravenous injection of DC tumor vaccine-3 injection into NOG-dKO mice, resulting in tumor growth inhibition.
Experimental animals: species of genus&Strain: MHC class I-and class II-Deficient NOG (NOG-dKO for short), NOG background is NOD/Shi-Prkdc scid Il2rγ tm1Sug /Jic。
The specific experimental process comprises the following steps:
the test was repeated with 60 female NOG-dKO mice divided into 3 donors, each of which was assigned 20 mice, and divided into 6 groups (as shown in table 3): group 1 administration of tumor-bearing, in vitro domesticated CD8 + T cells and PBS (tumor bearing control, labeled control) at equal volume DC to other groups; group 2 administration of tumor-bearing, in vitro domesticated CD8 + T cells and 1X 10 6 DC with concentration of CD40L mRNA loaded (CD 40L treated group, labeled group 2); group 3 administration of tumor-bearing, in vitro domesticated CD8 + T cells and 1X 10 6 DC with concentration of WT1 mRNA loading (WT 1 treated group, labeled group 3); group 4 administration of tumor-bearing, DC in vitro domesticated CD8 + T cells and 1X 10 6 DC loaded with neoantigen mRNA-2 at concentration (neoantigen treated group, labeled group 4); group 5 administration of tumor-bearing, in vitro domesticated CD8 + T cells and 1X 10 6 Concentration DC tumor vaccine-3 (antigen combination treatment group, labeled group 5); group 6 administration of tumor-bearing, DC in vitro domesticated CD8 + T cells and 1X 10 6 Concentration DC tumor vaccine-4 (combination treatment group, labeled group 6).
TABLE 3 in vivo efficacy evaluation Experimental procedure information for each group
Note that: in this example, DC and CD8 were used + T cells were derived from the same patient (i.e., doner), and the DC tumor vaccine-3 and DC tumor vaccine-4 were prepared in this example with only the RNA compositions being different, and all cells were derived from the same patient.
Each mouse in each group was first injected 1 x 10 by subcutaneous injection 5 Is a tumor cell of (a). After 3 to 10 days of tumor bearing, each mouse is injected with 1X 10 by tail vein injection 7 cell/sub-concentration in vitro domesticated CD8 + T cells, injected at a volume of 0.10mL. Administration of in vitro domesticated CD8 + After T cells, each mouse of group 2 was injected 1×10 by tail vein injection 6 cell/time of DC cells loaded with CD40L mRNA and injection volume of 0.10mL; each mouse of group 3, group 4 was injected 1 x 10 by tail vein injection, respectively 6 cells/time of DC cells loaded with WT1 mRNA and DC cells loaded with tumor neoantigen mRNA-2 were injected at a volume of 0.10mL; each mouse of group 5, group 6 was injected 1 x 10 by tail vein injection 6 DC tumor vaccine-4 and DC tumor vaccine-3 at cell/sub-concentration DC doses were injected at a volume of 0.10mL. Then, the administration was once every 6 days, and the administration was 4 times in total. All mice were euthanized and observed anatomically until the end of the experiment. The detection indicators for animals include live imaging of small animals and monitoring of tumor size by tumor weight.
Taking 1 donor as an example, animals were randomly divided into 4 groups for study based on body weight of animals measured prior to tumor loading, and specific groupings are shown in Table 4 below.
TABLE 4 evaluation of in vivo efficacy data information for each group
Fig. 5 shows the experimental results of the tumor weights of each group in example 9 of the present invention. As can be seen from the experimental results shown in fig. 5, the tumor shrinkage results are: control group < CD40L treatment group < WT1 treatment group < neoantigen treatment group < WT 1/neoantigen treatment group < CD40L/WT 1/neoantigen treatment group. Namely, the DC tumor vaccine-3 and the DC tumor vaccine-4 prepared by the application have obvious killing effect on tumors, and the killing effect is obviously higher than that of the single use of WT1mRNA or tumor neogenesis antigen mRNA-2. Further, as is clear from the experimental results of group 2, group 5 and group 6 shown in FIG. 5, the tumor weight corresponding to the addition of CD40L mRNA to the mRNA composition consisting of WT1mRNA and tumor neoantigen mRNA-2 was lower than the tumor weight corresponding to the mRNA composition consisting of WT1mRNA and tumor neoantigen mRNA-2. That is, the addition of CD40L mRNA can further increase the efficacy of the DC tumor vaccine in killing tumors.
Fig. 6 shows the experimental results of the tumor suppression rates of each group in example 9 of the present invention. In fig. 6, the tumor inhibition rate detection of the experimental endpoint after 6 doses is shown; wherein, the tumor inhibition rates of group 2, group 3, group 4, group 5 and group 6 are calculated by taking group 1 as a datum point. As shown in the experimental results of FIG. 6, both group 3 and group 4 had a certain tumor inhibition rate, but the tumor inhibition rate of group 5 was significantly higher than that of group 3 and group 4 because DC tumor vaccine-4 was injected. That is, the experimental data of this example demonstrate that the tumor neoantigen and related antigen combination has a more pronounced tumor inhibiting effect after acting on tumors than when used alone.
Also, as is clear from the experimental results of group 2, group 5 and group 6 shown in FIG. 6, similarly, the tumor suppression rate corresponding to the addition of CD40L mRNA to the mRNA composition consisting of WT1mRNA and tumor neoantigen mRNA-2 was significantly higher than that corresponding to the mRNA composition consisting of WT1mRNA and tumor neoantigen mRNA-2, and was significantly higher than that of CD40L alone. That is, the addition of CD40L mRNA can further enhance the efficacy of the DC tumor vaccine in killing tumors.
Meanwhile, the following specific examples are also made in order to verify that the compositions provided herein are equally applicable to other cancers.
EXAMPLE 10 in vivo efficacy of ovarian cancer humanization
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01OVCAR8 cells, were acclimatized in vitro by intravenous injection after 5 days, and then divided into 6 groups, group 1 being a control group, given an equal volume of PBS at the same time, group 2 being a co-stimulatory factor modifying DC treatment group, given CD40L DC, group 3 being a combination of WT1 and FSHR treatments given WT1 and FSHR DC, group 4 being a combination of neoantigen treatments given neoantigen DC, group 5 being a combination of antigen treatments given WT1 and FSHR/neoantigen DC, and group 6 being a combination treatment group given co-stimulatory factors/WT 1 and FSHR/neoantigen DC. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
Fig. 7 shows the experimental results of the tumor suppression rate of each group in example 10 of the present invention. As can be seen from fig. 7, the tumor inhibition rate was calculated based on the detected tumor size change value, and when a plurality of relevant antigens were combined (as shown in WT1 FSHR treatment group), the tumor inhibition rate was 36.1%, i.e., the plurality of relevant antigens were used in combination, which also had a certain tumor inhibition rate. Meanwhile, after the neoantigen is added into the related antigen mixed group, the tumor inhibition rate of the obtained WT1/FSHR neoantigen treatment group is 56.9 percent, and compared with 36.1 percent, the tumor inhibition rate is also obviously improved.
On the other hand, after the co-stimulatory factors are added into the antigen treatment group, the added co-stimulatory factors have an enhancement effect on the neoantigen and the related antigen, so that the inhibition effect of the neoantigen and the related antigen on tumors is greatly improved, and experimental data also show that the obtained tumor inhibition rate can reach 70.4 percent after the co-stimulatory factors are added, and is improved by 13.5 percent compared with 56.9 percent.
The neoantigen used in this example was the neoantigen mRNA-1 produced in example 1. The RNA sequence of FSHR (SEQ ID NO: 5) is as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCACCAUGGCCCUGCUCCUGGUCUCUUU
GCUGGCAUUCCUGAGCUUGGGCUCAGGAUGUCAUCAUCGGAUCUGUCACUGCUCUAACAGGGUUUUUCUCUGCCAAGAGAGCAAGG
UGACAGAGAUUCCUUCUGACCUCCCGAGGAAUGCCAUUGAACUGAGGUUUGUCCUCACCAAGCUUCGAGUCAUCCAAAAAGGUGCA
UUUUCAGGAUUUGGGGACCUGGAGAAAAUAGAGAUCUCUCAGAAUGAUGUCUUGGAGGUGAUAGAGGCAGAUGUGUUCUCCAACC
UUCCCAAAUUACAUGAAAUUAGAAUUGAAAAGGCCAACAACCUGCUCUACAUCAACCCUGAGGCCUUCCAGAACCUUCCCAACCUU
CAAUAUCUGUUAAUAUCCAACACAGGUAUUAAGCACCUUCCAGAUGUUCACAAGAUUCAUUCUCUCCAAAAAGUUUUACUUGACAU
UCAAGAUAACAUAAACAUCCACACAAUUGAAAGAAAUUCUUUCGUGGGGCUGAGCUUUGAAAGUGUGAUUCUAUGGCUGAAUAAG
AAUGGGAUUCAAGAAAUACACAACUGUGCAUUCAAUGGAACCCAACUAGAUGAGCUGAAUCUAAGCGAUAAUAAUAAUUUAGAAG
AAUUGCCUAAUGAUGUUUUCCACGGAGCCUCUGGACCAGUCAUUCUAGAUAUUUCAAGAACAAGGAUCCAUUCCCUGCCUAGCUAU
GGCUUAGAAAAUCUUAAGAAGCUGAGGGCCAGGUCGACUUACAACUUAAAAAAGCUGCCUACUCUGGAAAAGCUUGUCGCCCUCAU
GGAAGCCAGCCUCACCUAUCCCAGCCAUUGCUGUGCCUUUGCAAACUGGAGACGGCAAAUCUCUGAGCUUCAUCCAAUUUGCAACA
AAUCUAUUUUAAGGCAAGAAGUUGAUUAUAUGACUCAGGCUAGGGGUCAGAGAUCCUCUCUGGCAGAGGACAAUGAGUCCAGCUAC
AGCAGAGGAUUUGACAUGACGUACACUGAGUUUGACUAUGACUUAUGCAAUGAAGUGGUUGACGUGACCUGCUCCCCUAAGCCAGA
UGCAUUCAACCCAUGUGAAGAUAUCAUGGGGUACAACAUCCUCAGAGUCCUGAUAUGGUUUAUCAGCAUCCUGGCCAUCACUGGGA
ACAUCAUAGUGCUAGUGAUCCUAACUACCAGCCAAUAUAAACUCACAGUCCCCAGGUUCCUUAUGUGCAACCUGGCCUUUGCUGAU
CUCUGCAUUGGAAUCUACCUGCUGCUCAUUGCAUCAGUUGAUAUCCAUACCAAGAGCCAAUAUCACAACUAUGCCAUUGACUGGCA
AACUGGGGCAGGCUGUGAUGCUGCUGGCUUUUUCACUGUCUUUGCCAGUGAGCUGUCAGUCUACACUCUGACAGCUAUCACCUUGG
AAAGAUGGCAUACCAUCACGCAUGCCAUGCAGCUGGACUGCAAGGUGCAGCUCCGCCAUGCUGCCAGUGUCAUGGUGAUGGGCUGG
AUUUUUGCUUUUGCAGCUGCCCUCUUUCCCAUCUUUGGCAUCAGCAGCUACAUGAAGGUGAGCAUCUGCCUGCCCAUGGAUAUUGA
CAGCCCUUUGUCACAGCUGUAUGUCAUGUCCCUCCUUGUGCUCAAUGUCCUGGCCUUUGUGGUCAUCUGUGGCUGCUAUAUCCACA
UCUACCUCACAGUGCGGAACCCCAACAUCGUGUCCUCCUCUAGUGACACCAGGAUCGCCAAGCGCAUGGCCAUGCUCAUCUUCACUG
ACUUCCUCUGCAUGGCACCCAUUUCUUUCUUUGCCAUUUCUGCCUCCCUCAAGGUGCCCCUCAUCACUGUGUCCAAAGCAAAGAUUC
UGCUGGUUCUGUUUCACCCCAUCAACUCCUGUGCCAACCCCUUCCUCUAUGCCAUCUUUACCAAAAACUUUCGCAGAGAUUUCUUCA
UUCUGCUGAGCAAGUGUGGCUGCUAUGAAAUGCAAGCCCAAAUUUAUAGGACAGAAACUUCAUCCACUGUCCACAACACCCAUCCA
AGGAAUGGCCACUGCUCUUCAGCUCCCAGAGUCACCAGUGGUUCCACUUACAUACUUGUCCCUCUAAGUCAUUUAGCCCAAAACUA
AUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAU
GAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGCUAGCAGCUCGCUUUCUUGCUGUCCAAU
UUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUA
AUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
EXAMPLE 11 in vivo efficacy of humanized breast cancer
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01MDA-MB-231 cells, were acclimated in vitro by intravenous injection after 5 days, and were then divided into 6 groups, group 1 being a control group given an equal volume of PBS at the same time, group 2 being a co-stimulatory factor modified DC treatment group given CD40L DC, group 3 being a WT1 treatment group given WT1DC, group 4 being a neoantigen treatment group given neoantigen DC, group 5 being an antigen combination treatment group given WT 1/neoantigen DC, group 6 being a combination treatment group given co-stimulatory factor/WT 1/neoantigen DC. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
The tumor suppression rate was calculated based on the detected tumor size change values, and fig. 8 shows the experimental results of the tumor suppression rates of each group in example 11 of the present invention. As can be seen from fig. 8, the composition provided by the present invention is also suitable for breast cancer, and the tumor inhibition rate of the antigen combination treatment group is 61.3%, which is significantly improved compared with the related antigen treatment group and the neoantigen treatment group alone. On the other hand, after the co-stimulatory factors are added into the antigen combination treatment group, the added co-stimulatory factors have an enhancement effect on the neoantigen and the related antigen, so that the inhibition effect of the neoantigen and the related antigen on tumors is greatly improved, and experimental data also show that the tumor inhibition rate obtained after the co-stimulatory factors are added can reach 77.9 percent, and is improved by 16.6 percent compared with 61.3 percent.
In this example, WT1 was used as the mRNA prepared in example 2 above; the costimulatory factor used the mRNA prepared in example 3 above; the new antigen mRNA-3 of breast cancer is selected from the published RNA sequences in the database, and the specific sequences (SEQ ID NO: 6) are as follows:
UACAAACAGUCCCAGCACAUGACAGAAGUUGUACGCCAUUGCCCACAUCACGAGCGCUGCUCUGACAGCGAUGGC
EXAMPLE 12 in vivo efficacy of humanized gastric cancer
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01HGC-27 cells, were acclimated in vitro by intravenous injection after 5 days, and then were divided into 6 groups, group 1 was control group given equal volume of PBS at the same time, group 2 was co-stimulatory factor modified DC treatment group given CD40L DC, group 3 was WT1 treatment group given WT1 DC, group 4 was neoantigen treatment group given neoantigen DC, group 5 was antigen combination treatment group given WT 1/neoantigen DC, and group 6 was combination treatment group given co-stimulatory factor/WT 1/neoantigen DC. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
The tumor suppression rate was calculated based on the detected tumor size change values, and fig. 9 shows the experimental results of the tumor suppression rates of each group in example 12 of the present invention. As can be seen from fig. 9, the composition provided by the present invention is also suitable for gastric cancer, and the tumor inhibition rate of the antigen combination treatment group is 58.5%, which is obviously improved compared with the single relevant antigen treatment group and the new antigen treatment group. In addition, when the co-stimulatory factors are added into the antigen combination treatment group, the added co-stimulatory factors have an enhancement effect on the neoantigen and the related antigen, so that the inhibition effect on tumors by the neoantigen and the related antigen is improved, and experimental data also show that the obtained tumor inhibition rate can reach 63.4% after the co-stimulatory factors are added, and the tumor inhibition rate is improved by 4.9% compared with 58.5%. Although the magnitude of the elevation is not very high, it is also a significant effect on the treatment of disease.
In this example, WT1 was used as the mRNA prepared in example 2 above; the costimulatory factor used the mRNA prepared in example 3 above; the nascent antigen mRNA-4 of gastric cancer is selected from the published RNA sequences in the database, and the specific sequences (SEQ ID NO: 7) are as follows:
CACAUCGUGGAACAGAAGAACGGCAAGGAAAGAGUGCAAUCCUGUGGCACUUCCUGCAGAAAGAGGCCGAGCUG
EXAMPLE 13 in vivo efficacy of liver cancer humanization
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01HepG2 cells, were acclimated in vitro by intravenous injection after 5 days, and then were divided into 6 groups, group 1 was control group, while the same volume of PBS was administered, group 2 was co-stimulatory factor-modified DC treatment group, CD40L DC was administered, group 3 was WT1 DC treatment group, group 4 was neoantigen DC treatment group, group 5 was antigen combination treatment group, WT 1/neoantigen DC was administered, and group 6 was co-stimulatory factor/WT 1/neoantigen DC was administered. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
The tumor suppression rate was calculated based on the detected tumor size change values, and fig. 10 shows the experimental results of the tumor suppression rates of each group in example 13 of the present invention. As can be seen from fig. 10, the composition provided by the invention is also suitable for liver cancer, and the tumor inhibition rate of the antigen combination treatment group is 43.7%, which is obviously improved compared with the single relevant antigen treatment group and the new antigen treatment group. Similarly, it can be seen from the experimental data that the tumor inhibition rate obtained after adding the co-stimulatory factor to the antigen combination therapy group reaches 51.7%, which is 8% higher than 43.7%.
In this example, WT1 was used as the mRNA prepared in example 2 above; the costimulatory factor used the mRNA prepared in example 3 above; the new antigen mRNA-5 of liver cancer is selected from the published RNA sequences in the database, and the specific sequences (SEQ ID NO: 8) are as follows:
GCUUUAGUAAAUAUAAUGAGGACCUAUACUUACGAAAUUCUACUGUGGACCACAAGCAGAGUGCUGAAGGUGCUA
EXAMPLE 14 in vivo efficacy of prostate cancer humanization
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01PC-3 cells, were acclimated in vitro by intravenous injection for 5 days, and then divided into 6 groups, group 1 being a control group, while an equal volume of PBS was administered, group 2 being a co-stimulatory factor modified DC treatment group, CD40L DC, group 3 being a WT1 treatment group, WT1 DC, group 4 being a neoantigen treatment group, neoantigen DC, group 5 being an antigen combination treatment group, WT 1/neoantigen DC, and group 6 being a combination treatment group, co-stimulatory factor/WT 1/neoantigen DC. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
The tumor suppression rate was calculated based on the detected tumor size change values, and fig. 11 shows the experimental results of the tumor suppression rates of each group in example 14 of the present invention. As can be seen from fig. 11, the composition provided by the present invention is also suitable for prostate cancer, and the tumor inhibition rate of the antigen combination treatment group is 46.3%, which is significantly improved (about 30% improvement) compared with the related antigen treatment group and the neoantigen treatment group alone. On the other hand, it can also be seen from the experimental data that when the co-stimulatory factors are added to the antigen combination therapy group, the tumor inhibition rate is improved to 53.6% due to the enhancement of the neoantigens and the related antigens by the added co-stimulatory factors.
In this example, WT1 was used as the mRNA prepared in example 2 above; the costimulatory factor used the mRNA prepared in example 3 above; the novel antigen mRNA-6 of the prostate cancer is selected from the published RNA sequences in the database, and the specific sequences (SEQ ID NO: 9) are as follows:
UCCACACCCCCGCCCGGCACCCGCGUCCGCGCCAUGACCAUCUACAAGCAGUCACAGCACAUGACGGAGGUUGUG
EXAMPLE 15 humanized in vivo efficacy of Lung cancer
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01H1299 cells, were acclimated in vitro by intravenous injection after 5 days, and then divided into 6 groups, group 1 being a control group, while an equal volume of PBS was administered, group 2 being a co-stimulatory factor modified DC treatment group, CD40L DC, group 3 being MSLN DC, group 4 being neonatal antigen DC, group 5 being antigen combination treatment group, MSLN/neonatal antigen DC, group 6 being co-stimulatory factor/MSLN/neonatal antigen DC. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
The tumor suppression rate was calculated based on the detected tumor size change values, and fig. 12 shows the experimental results of the tumor suppression rates of each group in example 15 of the present invention. As can be seen from fig. 12, the composition provided by the present invention is equally applicable to lung cancer, and the tumor suppression rate of the antigen combination treatment group is 52.5%, which is significantly improved compared with the related antigen treatment group and the neoantigen treatment group alone. On the other hand, when the co-stimulatory factor is added to the antigen combination therapy group, the tumor inhibition rate is improved by about 10% and reaches 62.8% due to the enhancement effect of the added co-stimulatory factor on the neoantigen and the related antigens. This has a good reference value for tumour treatment.
In this example, the mRNA prepared in example 3 was used as the costimulatory factor; the lung cancer neoantigen mRNA-7 is selected from the published RNA sequences in the database, and the specific sequences (SEQ ID NO: 10) are as follows:
UUCACCUUUCUCUACUGGCAUUUGGAAGACCUCAAUGUAACUACAACCCUCUUUGGGGUCUGUUCAGUCCUGAGU
in this example, the relevant antigen MSLN was prepared by the method described in example 2 above, and the specific MSLN RNA sequence (SEQ ID NO: 11) was as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCACCAUGGCCUUGCCAACGGCUCGACC
CCUGUUGGGGUCCUGUGGGACCCCCGCCCUCGGCAGCCUCCUGUUCCUGCUCUUCAGCCUCGGAUGGGUGCAGCCCUCGAGGACCCU
GGCUGGAGAGACAGGGCAGGAGGCUGCGCCCCUGGACGGAGUCCUGGCCAACCCACCUAACAUUUCCAGCCUCUCCCCUCGCCAACU
CCUUGGCUUCCCGUGUGCGGAGGUGUCCGGCCUGAGCACGGAGCGUGUCCGGGAGCUGGCUGUGGCCUUGGCACAGAAGAAUGUCA
AGCUCUCAACAGAGCAGCUGCGCUGUCUGGCUCACCGGCUCUCUGAGCCCCCCGAGGACCUGGACGCCCUCCCAUUGGACCUGCUGC
UAUUCCUCAACCCAGAUGCGUUCUCGGGGCCCCAGGCCUGCACCCGUUUCUUCUCCCGCAUCACGAAGGCCAAUGUGGACCUGCUCC
CGAGGGGGGCUCCCGAGCGACAGCGGCUGCUGCCUGCGGCUCUGGCCUGCUGGGGUGUGCGGGGGUCUCUGCUGAGCGAGGCUGAU
GUGCGGGCUCUGGGAGGCCUGGCUUGCGACCUGCCUGGGCGCUUUGUGGCCGAGUCGGCCGAAGUGCUGCUACCCCGGCUGGUGAG
CUGCCCGGGACCCCUGGACCAGGACCAGCAGGAGGCAGCCAGGGCGGCUCUGCAGGGCGGGGGACCCCCCUACGGCCCCCCGUCGAC
AUGGUCUGUCUCCACGAUGGACGCUCUGCGGGGCCUGCUGCCCGUGCUGGGCCAGCCCAUCAUCCGCAGCAUCCCGCAGGGCAUCGU
GGCCGCGUGGCGGCAACGCUCCUCUCGGGACCCAUCCUGGCGGCAGCCUGAACGGACCAUCCUCCGGCCGCGGUUCCGGCGGGAAGU
GGAAAAGACAGCCUGUCCUUCAGGCAAGAAGGCCCGCGAGAUAGACGAGAGCCUCAUCUUCUACAAGAAGUGGGAGCUGGAAGCCU
GCGUGGAUGCGGCCCUGCUGGCCACCCAGAUGGACCGCGUGAACGCCAUCCCCUUCACCUACGAGCAGCUGGACGUCCUAAAGCAUA
AACUGGAUGAGCUCUACCCACAAGGUUACCCCGAGUCUGUGAUCCAGCACCUGGGCUACCUCUUCCUCAAGAUGAGCCCUGAGGAC
AUUCGCAAGUGGAAUGUGACGUCCCUGGAGACCCUGAAGGCUUUGCUUGAAGUCAACAAAGGGCACGAAAUGAGUCCUCAGGUGGC
CACCCUGAUCGACCGCUUUGUGAAGGGAAGGGGCCAGCUAGACAAAGACACCCUAGACACCCUGACCGCCUUCUACCCUGGGUACCU
GUGCUCCCUCAGCCCCGAGGAGCUGAGCUCCGUGCCCCCCAGCAGCAUCUGGGCGGUCAGGCCCCAGGACCUGGACACGUGUGACCC
AAGGCAGCUGGACGUCCUCUAUCCCAAGGCCCGCCUUGCUUUCCAGAACAUGAACGGGUCCGAAUACUUCGUGAAGAUCCAGUCCU
UCCUGGGUGGGGCCCCCACGGAGGAUUUGAAGGCGCUCAGUCAGCAGAAUGUGAGCAUGGACUUGGCCACGUUCAUGAAGCUGCGG
ACGGAUGCGGUGCUGCCGUUGACUGUGGCUGAGGUGCAGAAACUUCUGGGACCCCACGUGGAGGGCCUGAAGGCGGAGGAGCGGCA
CCGCCCGGUGCGGGACUGGAUCCUACGGCAGCGGCAGGACGACCUGGACACGCUGGGGCUGGGGCUACAGGGCGGCAUCCCCAACG
GCUACCUGGUCCUAGACCUCAGCAUGCAAGAGGCCCUCUCGGGGACGCCCUGCCUCCUAGGACCUGGACCUGUUCUCACCGUCCUGG
CACUGCUCCUAGCCUCCACCCUGGCCUAAUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAA
GUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCG
CUAGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGA
AGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAA
EXAMPLE 16 in vivo efficacy of humanized colon cancer
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01CT26 cells, were acclimated in vitro by intravenous injection for 5 days, and then divided into 6 groups, group 1 being a control group, while an equal volume of PBS was administered, group 2 being a co-stimulatory factor modified DC treatment group, CD40L DC, group 3 being MSLN DC, group 4 being neonatal antigen DC, group 5 being antigen combination treatment group, MSLN/neonatal antigen DC, group 6 being co-stimulatory factor/MSLN/neonatal antigen DC. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
The tumor suppression rate was calculated based on the detected tumor size change values, and fig. 13 shows the experimental results of the tumor suppression rates of each group in example 16 of the present invention. As can be seen from fig. 13, the composition provided by the present invention is also suitable for colon cancer, and the tumor inhibition rate of the antigen combination treatment group is 45.9%, which is significantly improved compared with the related antigen treatment group and the neoantigen treatment group alone. On the other hand, when the co-stimulatory factors are added into the antigen combination treatment group, the added co-stimulatory factors have an enhancement effect on the neoantigens and the related antigens, so that the inhibition effect of the neoantigens and the related antigens on tumors is greatly improved, and experimental data also show that the tumor inhibition rate obtained after the addition of the co-stimulatory factors is improved to 58%.
In this example, the specific sequence of MSLN is the same as in example 16; the costimulatory factor used the mRNA prepared in example 3 above; the new antigen mRNA-8 of colon cancer is selected from the published RNA sequences in the database, and the specific sequences (SEQ ID NO: 12) are as follows:
GAUAGAAACACCUUCAGACAUUCAGUGGUUGUUCCAUGCGAACCGCCUGAGGUGGGAUCCGAUUGUACGACAAUU
from the experimental data of examples 11 to 16 described above, it is understood that the combination of the neoantigen and the tumor-associated antigen has a superior therapeutic effect when used for tumor suppression in terms of the size of tumor disappearance. From this, the personalized mRNA composition provided by the present application has a remarkable effect in tumor treatment.
Further, the specific examples of the present invention further demonstrate that the addition of co-stimulatory factors can further promote the tumor-inhibiting effects of neoantigens and tumor-associated antigens. The specific contents are as follows:
the neoantigens and related antigens referred to in the following examples were the neoantigens and related antigens prepared directly using example 1 and example 2, respectively.
Example 17
Step 1, preparation of Co-stimulatory factor CD27L mRNA
The preparation method of this embodiment is similar to that of embodiment 3, and description thereof is omitted in this embodiment. While the release criteria differ only in part from each other. In this example, the release criteria for co-stimulatory factor CD27L mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length 1200nt, integrity > 90%, extra-long truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence of the costimulatory factor CD27L mRNA (SEQ ID NO: 13) prepared in this example was as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCACCAUGCCGGAGGAGGGUUCGGGCUGCUCGGUGCGGCGCAGGCCCUAUGGGUGCGUCCUGCGGGCUGCUUUGGUCCCAUUGGUCGCGGGCUUGGUGAUCUGCCUCGUGGUGUGCAUCCAGCGCUUCGCACAGGCUCAGCAGCAGCUGCCGCUCGAGUCACUUGGGUGGGACGUAGCUGAGCUGCAGCUGAAUCACACAGGACCUCAGCAGGACCCCAGGCUAUACUGGCAGGGGGGCCCAGCACUGGGCCGCUCCUUCCUGCAUGGACCAGAGCUGGACAAGGGGCAGCUACGUAUCCAUCGUGAUGGCAUCUACAUGGUACACAUCCAGGUGACGCUGGCCAUCUGUUCCUCCACGACGGCCUCCAGGCACCACCCCACCACCCUGGCCGUGGGAAUCUGCUCUCCCGCCUCCCGUAGCAUCAGCCUGCUGCGUCUCAGCUUCCACCAAGGUUGUACCAUUGCCUCCCAGCGCCUGACGCCCCUGGCCCGAGGGGACACACUCUGCACCAACCUCACUGGGACACUUUUGCCUUCCCGAAACACUGAUGAGACCUUCUUUGGAGUGCAGUGGGUGCGCCCCUGAUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGCUAGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
step 2, in vivo efficacy of ovarian cancer DC vaccine (CD 27L) humanization
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01OVCAR8 cells, were acclimatized in vitro by intravenous injection after 5 days, and then divided into 6 groups, group 1 being a control group given an equal volume of PBS at the same time, group 2 being an immunoadjuvant modified DC treatment group given CD27 LDC, group 3 being a tumor-associated antigen treatment group given WT1DC, group 4 being a neoantigen treatment group given neoantigen DC, group 5 being an antigen combination treatment group given tumor-associated antigen/neoantigen DC, group 6 being a combination treatment group given immunoadjuvant/tumor-associated antigen/neoantigen DC. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
FIG. 14 shows the experimental results of the tumor suppression rates of each group in example 17 of the present invention. As shown in fig. 14, when the co-stimulatory factor is CD27L, the effect of enhancing the associated antigen and neoantigen is also exhibited, and the tumor suppression rate is increased from 49.2% to 64.2%.
Example 18
Step 1, preparation of costimulatory factor GITR mRNA
The preparation method of this embodiment is similar to that of embodiment 3, and description thereof is omitted in this embodiment. While the release criteria differ only slightly from each other. In this example, the release criteria for the co-stimulatory factor GITR mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length 1300nt, integrity > 90%, ultralong truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence of the costimulatory factor GITR mRNA (SEQ ID NO: 14) prepared in this example was as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCACCAUGGCACAGCACGGGGCGAUGGGCGCGUUUCGGGCCCUGUGCGGCCUGGCGCUGCUGUGCGCGCUCAGCCUGGGUCAGCGCCCCACCGGGGGUCCCGGGUGCGGCCCUGGGCGCCUCCUGCUUGGGACGGGAACGGACGCGCGCUGCUGCCGGGUUCACACGACGCGCUGCUGCCGCGAUUACCCGGGCGAGGAGUGCUGUUCCGAGUGGGACUGCAUGUGUGUCCAGCCUGAAUUCCACUGCGGAGACCCUUGCUGCACGACCUGCCGGCACCACCCUUGUCCCCCAGGCCAGGGGGUACAGUCCCAGGGGAAAUUCAGUUUUGGCUUCCAGUGUAUCGACUGUGCCUCGGGGACCUUCUCCGGGGGCCACGAAGGCCACUGCAAACCUUGGACAGACUGCACCCAGUUCGGGUUUCUCACUGUGUUCCCUGGGAACAAGACCCACAACGCUGUGUGCGUCCCAGGGUCCCCGCCGGCAGAGCCGCUUGGGUGGCUGACCGUCGUCCUCCUGGCCGUGGCCGCCUGCGUCCUCCUCCUGACCUCGGCCCAGCUUGGACUGCACAUCUGGCAGCUGAGGAGUCAGUGCAUGUGGCCCCGAGAGACCCAGCUGCUGCUGGAGGUGCCGCCGUCGACCGAAGACGCCAGAAGCUGCCAGUUCCCCGAGGAAGAGCGGGGCGAGCGAUCGGCAGAGGAGAAGGGGCGGCUGGGAGACCUGUGGGUGUGAUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGCUAGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
step 2, in vivo efficacy of ovarian cancer DC vaccine (GITR) humanization
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01OVCAR8 cells, were acclimatized in vitro by intravenous injection after 5 days, and then divided into 6 groups, group 1 being a control group to which an equal volume of PBS was administered at the same time, group 2 being an immunoadjuvant modified DC treatment group to which GITR DC was administered, group 3 being a tumor-associated antigen treatment group to which WT1DC was administered, group 4 being a neoantigen treatment group to which neoantigen DC was administered, group 5 being an antigen combination treatment group to which tumor-associated antigen/neoantigen DC was administered, and group 6 being a combination treatment group to which immunoadjuvant/tumor-associated antigen/neoantigen DC was administered. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
Fig. 15 shows the experimental results of the tumor suppression rates of each group in example 18 of the present invention. As shown in FIG. 15, when the co-stimulatory factor was GITR, it also had the effect of enhancing both the associated antigen and the neoantigen, and the tumor suppression rate increased from 43.1% to 64.7% upon addition of GITR.
Example 19
Step 1, preparing costimulatory factor LFA-1mRNA
The preparation method of this embodiment is similar to that of embodiment 3, and description thereof is omitted in this embodiment. While the release criteria differ only slightly from each other. In this example, the release criteria for the co-stimulatory factor LFA-1mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length of 3000nt, integrity > 90%, ultralong truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence of the costimulatory factor LFA-1mRNA prepared in this example (SEQ ID NO: 15) is as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCACCAUGCUGGGCCUGCGCCCCCCACUGCUCGCCCUGGUGGGGCUGCUCUCCCUCGGGUGCGUCCUCUCUCAGGAGUGCACGAAGUUCAAGGUCAGCAGCUGCCGGGAAUGCAUCGAGUCGGGGCCCGGCUGCACCUGGUGCCAGAAGCUGAACUUCACAGGGCCGGGGGAUCCUGACUCCAUUCGCUGCGACACCCGGCCACAGCUGCUCAUGAGGGGCUGUGCGGCUGACGACAUCAUGGACCCCACAAGCCUCGCUGAAACCCAGGAAGACCACAAUGGGGGCCAGAAGCAGCUGUCCCCACAAAAAGUGACGCUUUACCUGCGACCAGGCCAGGCAGCAGCGUUCAACGUGACCUUCCGGCGGGCCAAGGGCUACCCCAUCGACCUGUACUAUCUGAUGGACCUCUCCUACUCCAUGCUUGAUGACCUCAGGAAUGUCAAGAAGCUAGGUGGCGACCUGCUCCGGGCCCUCAACGAGAUCACCGAGUCCGGCCGCAUUGGCUUCGGGUCCUUCGUGGACAAGACCGUGCUGCCGUUCGUGAACACGCACCCUGAUAAGCUGCGAAACCCAUGCCCCAACAAGGAGAAAGAGUGCCAGCCCCCGUUUGCCUUCAGGCACGUGCUGAAGCUGACCAACAACUCCAACCAGUUUCAGACCGAGGUCGGGAAGCAGCUGAUUUCCGGAAACCUGGAUGCACCCGAGGGUGGGCUGGACGCCAUGAUGCAGGUCGCCGCCUGCCCGGAGGAAAUCGGCUGGCGCAACGUCACGCGGCUGCUGGUGUUUGCCACUGAUGACGGCUUCCAUUUCGCGGGCGACGGGAAGCUGGGCGCCAUCCUGACCCCCAACGACGGCCGCUGUCACCUGGAGGACAACUUGUACAAGAGGAGCAACGAAUUCGACUACCCAUCGGUGGGCCAGCUGGCGCACAAGCUGGCUGAAAACAACAUCCAGCCCAUCUUCGCGGUGACCAGUAGGAUGGUGAAGACCUACGAGAAACUCACCGAGAUCAUCCCCAAGUCAGCCGUGGGGGAGCUGUCUGAGGACUCCAGCAAUGUGGUCCAUCUCAUUAAGAAUGCUUACAAUAAACUCUCCUCCAGGGUCUUCCUGGAUCACAACGCCCUCCCCGACACCCUGAAAGUCACCUACGACUCCUUCUGCAGCAAUGGAGUGACGCACAGGAACCAGCCCAGAGGUGACUGUGAUGGCGUGCAGAUCAAUGUCCCGAUCACCUUCCAGGUGAAGGUCACGGCCACAGAGUGCAUCCAGGAGCAGUCGUUUGUCAUCCGGGCGCUGGGCUUCACGGACAUAGUGACCGUGCAGGUUCUUCCCCAGUGUGAGUGCCGGUGCCGGGACCAGAGCAGAGACCGCAGCCUCUGCCAUGGCAAGGGCUUCUUGGAGUGCGGCAUCUGCAGGUGUGACACUGGCUACAUUGGGAAAAACUGUGAGUGCCAGACACAGGGCCGGAGCAGCCAGGAGCUGGAAGGAAGCUGCCGGAAGGACAACAACUCCAUCAUCUGCUCAGGGCUGGGGGACUGUGUCUGCGGGCAGUGCCUGUGCCACACCAGCGACGUCCCCGGCAAGCUGAUAUACGGGCAGUACUGCGAGUGUGACACCAUCAACUGUGAGCGCUACAACGGCCAGGUCUGCGGCGGCCCGGGGAGGGGGCUCUGCUUCUGCGGGAAGUGCCGCUGCCACCCGGGCUUUGAGGGCUCAGCGUGCCAGUGCGAGAGGACCACUGAGGGCUGCCUGAACCCGCGGCGUGUUGAGUGUAGUGGUCGUGGCCGGUGCCGCUGCAACGUAUGCGAGUGCCAUUCAGGCUACCAGCUGCCUCUGUGCCAGGAGUGCCCCGGCUGCCCCUCACCCUGUGGCAAGUACAUCUCCUGCGCCGAGUGCCUGAAGUUCGAAAAGGGCCCCUUUGGGAAGAACUGCAGCGCGGCGUGUCCGGGCCUGCAGCUGUCGAACAACCCCGUGAAGGGCAGGACCUGCAAGGAGAGGGACUCAGAGGGCUGCUGGGUGGCCUACACGCUGGAGCAGCAGGACGGGAUGGACCGCUACCUCAUCUAUGUGGAUGAGAGCCGAGAGUGUGUGGCAGGCCCCAACAUCGCCGCCAUCGUCGGGGGCACCGUGGCAGGCAUCGUGCUGAUCGGCAUUCUCCUGCUGGUCAUCUGGAAGGCUCUGAUCCACCUGAGCGACCUCCGGGAGUACAGGCGCUUUGAGAAGGAGAAGCUCAAGUCCCAGUGGAACAAUGAUAAUCCCCUUUUCAAGAGCGCCACCACGACGGUCAUGAACCCCAAGUUUGCUGAGAGUUAGUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGCUAGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
step 2, efficacy of ovarian cancer DC vaccine (LFA-1) humanized in vivo
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01OVCAR8 cells, were acclimatized in vitro by intravenous injection after 5 days, and then divided into 6 groups, group 1 being a control group, given an equal volume of PBS at the same time, group 2 being an immunoadjuvant modified DC treatment group, given LFA-1DC, group 3 being a tumor-associated antigen treatment group, given WT1DC, group 4 being a neoantigen treatment group, given neoantigen DC, group 5 being an antigen combination treatment group, given tumor-associated antigen/neoantigen DC, group 6 being a combination treatment group, given immunoadjuvant/tumor-associated antigen/neoantigen DC. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
FIG. 16 shows the experimental results of the tumor suppression rates of each group in example 19 of the present invention. As shown in FIG. 16, when the co-stimulatory factor is LFA-1, the effect of enhancing the related antigen and the neoantigen is also achieved, and after LFA-1 is added, the tumor inhibition rate is increased from 55.9% to 76.0%.
Example 20
Step 1, preparation of costimulatory factor ICAM-1mRNA
The preparation method of this embodiment is similar to that of embodiment 3, and description thereof is omitted in this embodiment. While the release criteria differ only slightly from each other. In this example, the release criteria for the costimulatory factor ICAM-1mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length 2100nt, integrity > 90%, extra-long truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence (SEQ ID NO: 16) of the costimulatory factor ICAM-1mRNA prepared in this example was as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCACCAUGGCUCCCAGCAGCCCCCGGCCCGCGCUGCCCGCACUCCUGGUCCUGCUCGGGGCUCUGUUCCCAGGACCUGGCAAUGCCCAGACAUCUGUGUCCCCCUCAAAAGUCAUCCUGCCCCGGGGAGGCUCCGUGCUGGUGACAUGCAGCACCUCCUGUGACCAGCCCAAGUUGUUGGGCAUAGAGACCCCGUUGCCUAAAAAGGAGUUGCUCCUGCCUGGGAACAACCGGAAGGUGUAUGAACUGAGCAAUGUGCAAGAAGAUAGCCAACCAAUGUGCUAUUCAAACUGCCCUGAUGGGCAGUCAACAGCUAAAACCUUCCUCACCGUGUACUGGACUCCAGAACGGGUGGAACUGGCACCCCUCCCCUCUUGGCAGCCAGUGGGCAAGAACCUUACCCUACGCUGCCAGGUGGAGGGUGGGGCACCCCGGGCCAACCUCACCGUGGUGCUGCUCCGUGGGGAGAAGGAGCUGAAACGGGAGCCAGCUGUGGGGGAGCCCGCUGAGGUCACGACCACGGUGCUGGUGAGGAGAGAUCACCAUGGAGCCAAUUUCUCGUGCCGCACUGAACUGGACCUGCGGCCCCAAGGGCUGGAGCUGUUUGAGAACACCUCGGCCCCCUACCAGCUCCAGACCUUUGUCCUGCCAGCGACUCCCCCACAACUUGUCAGCCCCCGGGUCCUAGAGGUGGACACGCAGGGGACCGUGGUCUGUUCCCUGGACGGGCUGUUCCCAGUCUCGGAGGCCCAGGUCCACCUGGCACUGGGGGACCAGAGGUUGAACCCCACAGUCACCUAUGGCAACGACUCCUUCUCGGCCAAGGCCUCAGUCAGUGUGACCGCAGAGGACGAGGGCACCCAGCGGCUGACGUGUGCAGUAAUACUGGGGAACCAGAGCCAGGAGACACUGCAGACAGUGACCAUCUACAGCUUUCCGGCGCCCAACGUGAUUCUGACGAAGCCAGAGGUCUCAGAAGGGACCGAGGUGACAGUGAAGUGUGAGGCCCACCCUAGAGCCAAGGUGACGCUGAAUGGGGUUCCAGCCCAGCCACUGGGCCCGAGGGCCCAGCUCCUGCUGAAGGCCACCCCAGAGGACAACGGGCGCAGCUUCUCCUGCUCUGCAACCCUGGAGGUGGCCGGCCAGCUUAUACACAAGAACCAGACCCGGGAGCUUCGUGUCCUGUCCCCCCGGUAUGAGAUUGUCAUCAUCACUGUGGUAGCAGCCGCAGUCAUAAUGGGCACUGCAGGCCUCAGCACGUACCUCUAUAACCGCCAGCGGAAGAUCAAGAAAUACAGACUACAACAGGCCCAAAAAGGGACCCCCAUGAAACCGAACACACAAGCCACGCCUCCCUGAUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGCUAGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
step 2, in vivo efficacy of ovarian cancer DC vaccine (ICAM-1) humanization
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01OVCAR8 cells, were acclimatized in vitro by intravenous injection after 5 days, and then divided into 6 groups, group 1 being a control group to which equal volume of PBS was administered at the same time, group 2 being an immunoadjuvant modified DC treatment group to ICAM-1DC, group 3 being a tumor-associated antigen treatment group to WT 1DC, group 4 being a neoantigen treatment group to neoantigen DC, group 5 being an antigen combination treatment group to tumor-associated antigen/neoantigen DC, and group 6 being a combination treatment group to which immunoadjuvant/tumor-associated antigen/neoantigen DC was administered. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
Fig. 17 shows the experimental results of the tumor suppression rates of each group in example 20 of the present invention. As shown in FIG. 17, when the co-stimulatory factor was ICAM-1, it also had the effect of enhancing the relevant antigen and neoantigen, and after ICAM-1 was added, the tumor suppression rate was increased from 53.5% to 69.4%.
Example 21
Step 1, preparation of costimulatory factor IL-2mRNA
The preparation method of this embodiment is similar to that of embodiment 3, and description thereof is omitted in this embodiment. While the release criteria differ only slightly from each other. In this example, the release criteria for the co-stimulatory factor IL-2mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length of 1000nt, integrity > 90%, ultralong truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence of the costimulatory factor IL-2mRNA (SEQ ID NO: 17) prepared in this example was as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCCCAUGUACAGGAUGCAACUCCUGUCUUGCAUUGCACUAAGUCUUGCACUUGUCACAAACAGUGCACCUACUUCAAGUUCUACAAAGAAAACACAGCUACAACUGGAGCAUUUACUGCUGGAUUUACAGAUGAUUUUGAAUGGAAUUAAUAAUUACAAGAAUCCCAAACUCACCAGGAUGCUCACAUUUAAGUUUUACAUGCCCAAGAAGGCCACAGAACUGAAACAUCUUCAGUGUCUAGAAGAAGAACUCAAACCUCUGGAGGAAGUGCUAAAUUUAGCUCAAAGCAAAAACUUUCACUUAAGACCCAGGGACUUAAUCAGCAAUAUCAACGUAAUAGUUCUGGAACUAAAGGGAUCUGAAACAACAUUCAUGUGUGAAUAUGCUGAUGAGACAGCAACCAUUGUAGAAUUUCUGAACAGAUGGAUUACCUUUUGUCAAAGCAUCAUCUCAACACUGACUUGAUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGCUAGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
step 2, ovarian cancer DC vaccine (IL-2) humanized in vivo efficacy
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01OVCAR8 cells, 5 days later, CD8 was acclimatized in vitro by intravenous injection + T cells were then divided into 6 groups, group 1 being a control group with equal volume of PBS, group 2 being an immunoadjuvant-modified DC treatment group with IL-2DC, group 3 being a tumor-associated antigen treatment group with WT1 DC, group 4 being a neoantigen treatment group with neoantigen DC, group 5 being an antigen combination treatment group with tumor-associated antigen/neoantigen DC, and group 6 being a combination treatment group with immunoadjuvant/tumor-associated antigen/neoantigen DC. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific procedure and the dosage of the agent are the same as those of example 9, in this example And will not be described in detail.
Fig. 18 shows the experimental results of the tumor suppression rates of each group in example 21 of the present invention. As shown in FIG. 18, when the co-stimulatory factor is IL-2, the effect of enhancing the relevant antigen and neoantigen is also achieved, and the tumor inhibition rate is increased from 51.4% to 64.8% after IL-2 is added.
Example 22
Step 1, preparation of costimulatory factor IL-7mRNA
The preparation method of this embodiment is similar to that of embodiment 3, and description thereof is omitted in this embodiment. While the release criteria differ only slightly from each other. In this example, the release criteria for the co-stimulatory factor IL-7mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length 1200nt, integrity > 90%, extra-long truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence of the costimulatory factor IL-7mRNA (SEQ ID NO: 18) prepared in this example was as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCACCAUGUUCCAUGUUUCUUUUAGGUAUAUCUUUGGACUUCCUCCCCUGAUCCUUGUUCUGUUGCCAGUAGCAUCAUCUGAUUGUGAUAUUGAAGGUAAAGAUGGCAAACAAUAUGAGAGUGUUCUAAUGGUCAGCAUCGAUCAAUUAUUGGACAGCAUGAAAGAAAUUGGUAGCAAUUGCCUGAAUAAUGAAUUUAACUUUUUUAAAAGACAUAUCUGUGAUGCUAAUAAGGAAGGUAUGUUUUUAUUCCGUGCUGCUCGCAAGUUGAGGCAAUUUCUUAAAAUGAAUAGCACUGGUGAUUUUGAUCUCCACUUAUUAAAAGUUUCAGAAGGCACAACAAUACUGUUGAACUGCACUGGCCAGGUUAAAGGAAGAAAACCAGCUGCCCUGGGUGAAGCCCAACCAACAAAGAGUUUGGAAGAAAAUAAAUCUUUAAAGGAACAGAAAAAACUGAAUGACUUGUGUUUCCUAAAGAGACUAUUACAAGAGAUAAAAACUUGUUGGAAUAAAAUUUUGAUGGGCACUAAAGAACACUAAUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGCUAGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
step 2, humanized in vivo efficacy of ovarian cancer DC vaccine (IL-7)
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01OVCAR8 cells, were acclimatized in vitro by intravenous injection after 5 days, and then divided into 6 groups, group 1 being a control group to which an equal volume of PBS was administered at the same time, group 2 being an immunoadjuvant modified DC treatment group to which IL-7DC was administered, group 3 being a tumor-associated antigen treatment group to which WT1DC was administered, group 4 being a neoantigen treatment group to which neoantigen DC was administered, group 5 being an antigen combination treatment group to which tumor-associated antigen/neoantigen DC was administered, and group 6 being a combination treatment group to which immunoadjuvant/tumor-associated antigen/neoantigen DC was administered. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
Fig. 19 shows the experimental results of the tumor suppression rates of each group in example 22 of the present invention. As shown in FIG. 19, when the co-stimulatory factor is IL-7, the effect of enhancing the relevant antigen and neoantigen is also achieved, and the tumor inhibition rate is increased from 51.4% to 69.7% after IL-7 is added.
Example 23
Step 1, preparation of costimulatory factor IL-12mRNA
The preparation method of this embodiment is similar to that of embodiment 3, and description thereof is omitted in this embodiment. While the release criteria differ only slightly from each other. In this example, the release criteria for the co-stimulatory factor IL-12mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length of 3000nt, integrity > 90%, ultralong truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence of the costimulatory factor IL-12mRNA (SEQ ID NO: 19) prepared in this example was as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCCUCGAGCACCAUGUGUCAUCAGCAACUGGUCAUUAGCUGGUUUUCCUUGGUGUUCCUGGCCUCACCUCUCGUUGCAAUUUGGGAACUGAAGAAAGACGUAUAUGUCGUGGAACUGGACUGGUAUCCCGAUGCCCCAGGGGAAAUGGUGGUCCUUACUUGCGACACCCCCGAAGAGGACGGCAUUACAUGGACCCUUGACCAGUCCAGUGAGGUCCUCGGGAGUGGAAAGACACUGACAAUUCAGGUUAAGGAGUUCGGCGAUGCCGGUCAGUACACCUGUCAUAAAGGGGGAGAGGUUCUGUCCCAUUCACUCCUGCUGCUGCACAAGAAGGAAGAUGGUAUCUGGUCCACGGACAUUUUGAAAGAUCAAAAGGAGCCCAAGAACAAAACUUUCCUGCGGUGUGAAGCCAAAAAUUACAGCGGGCGAUUUACUUGUUGGUGGCUCACCACUAUUAGUACAGAUCUCACCUUCAGCGUUAAGAGUUCUAGGGGCUCCAGUGAUCCGCAGGGAGUGACGUGUGGCGCUGCGACCCUGUCCGCUGAGCGAGUGAGGGGCGAUAAUAAGGAGUAUGAGUAUAGUGUGGAGUGUCAGGAGGAUUCUGCUUGCCCUGCUGCCGAGGAGUCCCUUCCUAUCGAAGUGAUGGUCGAUGCUGUACACAAACUGAAAUACGAGAAUUAUACUAGCUCCUUUUUCAUAAGGGACAUCAUAAAGCCAGAUCCCCCCAAGAACCUUCAGCUGAAACCGCUGAAAAAUUCACGCCAGGUGGAGGUAUCUUGGGAGUACCCAGACACAUGGAGCACACCUCAUAGUUACUUCAGCCUGACCUUCUGUGUCCAGGUCCAGGGGAAGUCUAAAAGGGAAAAAAAGGACAGAGUUUUCACCGACAAGACAUCCGCAACCGUAAUCUGUAGGAAAAACGCCUCAAUUUCCGUUAGAGCGCAAGAUAGGUAUUAUUCCUCUUCCUGGUCUGAAUGGGCCUCUGUACCCUGCAGCGGGGGUGGCGGUAGCGGUGGGGGAGGGAGCGGAGGUGGAGGCAGCCGAAACCUGCCUGUGGCCACACCUGAUCCUGGGAUGUUCCCUUGCUUGCACCACAGUCAGAAUCUGCUCAGGGCCGUUAGCAACAUGCUGCAGAAGGCGCGGCAGACACUUGAGUUCUACCCUUGCACAAGUGAAGAGAUCGAUCACGAAGAUAUCACAAAGGAUAAAACGUCUACUGUCGAGGCCUGCCUCCCCCUCGAACUCACAAAGAACGAGAGUUGCCUCAAUUCCCGGGAAACCAGCUUCAUCACUAAUGGGUCCUGCCUGGCAAGCAGAAAAACUUCUUUCAUGAUGGCAUUGUGUUUGAGUAGCAUCUAUGAGGACCUCAAAAUGUACCAAGUUGAAUUUAAGACUAUGAACGCUAAGCUCCUCAUGGACCCUAAGCGGCAGAUCUUCCUGGAUCAGAACAUGUUGGCUGUCAUCGACGAACUCAUGCAAGCCCUGAACUUUAAUUCCGAAACAGUUCCCCAGAAGUCCAGUCUGGAGGAACCCGAUUUCUACAAAACCAAAAUUAAGCUGUGUAUCUUGCUGCAUGCUUUCCGGAUCAGGGCCGUUACGAUCGAUCGGGUAAUGAGCUACCUGAAUGCCAGUUAAUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGCUAGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
step 2, in vivo efficacy of ovarian cancer DC vaccine (IL-12) humanization
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01OVCAR8 cells, were acclimatized in vitro by intravenous injection after 5 days, and then divided into 6 groups, group 1 being a control group to which an equal volume of PBS was administered at the same time, group 2 being an immunoadjuvant modified DC treatment group to which IL-12DC was administered, group 3 being a tumor-associated antigen treatment group to which WT1DC was administered, group 4 being a neoantigen treatment group to which neoantigen DC was administered, group 5 being an antigen combination treatment group to which tumor-associated antigen/neoantigen DC was administered, and group 6 being a combination treatment group to which immunoadjuvant/tumor-associated antigen/neoantigen DC was administered. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
FIG. 20 shows the experimental results of the tumor suppression rates of each group in example 23 of the present invention. As shown in FIG. 20, when the co-stimulatory factor is IL-12, the effect of enhancing the relevant antigen and neoantigen is also achieved, and the tumor inhibition rate is increased from 50.2% to 79.7% after IL-12 is added.
Example 24
Step 1, preparing costimulatory factor IL-15mRNA
The preparation method of this embodiment is similar to that of embodiment 3, and description thereof is omitted in this embodiment. While the release criteria differ only slightly from each other. In this example, the release criteria for the co-stimulatory factor IL-15mRNA were: 1 μg/μl, purity (expressed as OD (A260/A280) ratio) less than 2.0, RNA length of 1100nt, integrity > 90%, ultralong truncation < 10%, capping efficiency > 90%, DNA residue <4ng/μg, protein (enzyme) residue <10ng/μl.
The RNA sequence (SEQ ID NO: 20) of the costimulatory factor IL-15mRNA prepared in this example was as follows:
GGGAGACAAGCUUCCUGCAGGUCGACACCGGUGGAUCCCGGGUACCGAGCUCGAAUUCACCAUGAGAAUUUCGAAACCACAUUUGAGAAGUAUUUCCAUCCAGUGCUACUUGUGUUUACUUCUAAACAGUCAUUUUCUAACUGAAGCUGGCAUUCAUGUCUUCAUUUUGGGCUGUUUCAGUGCAGGGCUUCCUAAAACAGAAGCCAACUGGGUGAAUGUAAUAAGUGAUUUGAAAAAAAUUGAAGAUCUUAUUCAAUCUAUGCAUAUUGAUGCUACUUUAUAUACGGAAAGUGAUGUUCACCCCAGUUGCAAAGUAACAGCAAUGAAGUGCUUUCUCUUGGAGUUACAAGUUAUUUCACUUGAGUCCGGAGAUGCAAGUAUUCAUGAUACAGUAGAAAAUCUGAUCAUCCUAGCAAACAACAGUUUGUCUUCUAAUGGGAAUGUAACAGAAUCUGGAUGCAAAGAAUGUGAGGAACUGGAGGAAAAAAAUAUUAAAGAAUUUUUGCAGAGUUUUGUACAUAUUGUCCAAAUGUUCAUCAACACUUCUUAAUCUAGAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCGCUAGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCUGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
step 2, humanized in vivo efficacy of ovarian cancer DC vaccine (IL-15)
Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01OVCAR8 cells, were acclimatized in vitro by intravenous injection after 5 days, and then divided into 6 groups, group 1 being a control group to which an equal volume of PBS was administered at the same time, group 2 being an immunoadjuvant modified DC treatment group to which IL-15DC was administered, group 3 being a tumor-associated antigen treatment group to which WT1DC was administered, group 4 being a neoantigen treatment group to which neoantigen DC was administered, group 5 being an antigen combination treatment group to which tumor-associated antigen/neoantigen DC was administered, and group 6 being a combination treatment group to which immunoadjuvant/tumor-associated antigen/neoantigen DC was administered. The dosing was 1 time per week, 5 times post dosing and 1 week post dosing, with tumor size monitored by in vivo imaging of small animals. The specific operation and dosage of the agent are the same as those of the above-mentioned embodiment 9, and the description thereof will be omitted in this embodiment.
Fig. 21 shows the experimental results of the tumor suppression rates of each group in example 24 of the present invention. As shown in FIG. 21, when the co-stimulatory factor is IL-15, the effect of enhancing the relevant antigen and neoantigen is also achieved, and the tumor inhibition rate is increased from 60.8% to 76.0% after IL-15 is added.
From the experimental data of examples 17-24, it is known that, in the composition provided by the present invention, when the co-stimulatory factor is added to the antigen treatment group, the added co-stimulatory factor has an enhancement effect on both the neoantigen and the related antigen, so that the tumor inhibition rates are obviously improved, and the difference between the improved tumor inhibition rates is greater than the tumor inhibition rate generated when the co-stimulatory factor is used alone.
For the purposes of simplicity of explanation, the methodologies are shown as a series of acts, but one of ordinary skill in the art will recognize that the present invention is not limited by the order of acts described, as some acts may, in accordance with the present invention, occur in other orders and concurrently. Further, those skilled in the art will recognize that the embodiments described in the specification are all of the preferred embodiments, and that the acts and components referred to are not necessarily required by the present invention.
The mRNA compositions, vectors, mRNA vaccines and uses thereof provided by the present invention are described in detail above, and specific examples are used herein to illustrate the principles and embodiments of the present invention, the above examples being provided only to assist in understanding the methods of the present invention and the core ideas thereof; meanwhile, as those skilled in the art will have variations in the specific embodiments and application scope in accordance with the ideas of the present invention, the present description should not be construed as limiting the present invention in view of the above.
Claims (12)
1. An individualised mRNA composition, the mRNA composition comprising: at least one neoantigen mRNA encoding a tumor neoantigen and at least one associated antigen mRNA encoding a tumor associated antigen.
2. The mRNA composition of claim 1, wherein the nascent antigen transcription template corresponding to the nascent antigen mRNA is a highly immunogenic mutant sequence selected by peptide screening and mutation site screening in sequence.
3. The mRNA composition of claim 1 or 2, wherein the mRNA composition further comprises: at least one costimulatory factor mRNA encoding a costimulatory factor.
4. The mRNA composition of claim 3, wherein the co-stimulatory factor is any of IL-2, IL-7, IL-12, IL-15, CD40L, CD, CD27L, CD, CD28, CD275, CD278, CD134, CD137, CD154, GITR, HVEM, LFA-1, CD2, CD58, ICAM-1, TNFSF4, TNFSF5, TNFSF7, TNFSF9, TNFSF14, TNFSF 18.
5. The mRNA composition of claim 1, wherein the tumor-associated antigen is any one of WT1, MSLN, FSHR.
6. The mRNA composition of claim 3, wherein the RNA length of the neoantigen mRNA is 600-800 nt; and/or
The RNA length of the related antigen mRNA is 1500-2000 nt and/or
The RNA length of the costimulatory factor mRNA is 1100-3000 nt.
7. A vector comprising the mRNA composition of any one of claims 1-6.
8. The carrier of claim 10, wherein the carrier is one or more of a lipid, a liposome, a lipid complex, a lipid nanoparticle, a polymeric nanoparticle, a DC cell, a B cell, a mimetic nanoparticle, a nanotube, or a conjugate comprising the mRNA composition.
9. An mRNA vaccine comprising the mRNA composition of any one of claims 1-6, or comprising the vector of claim 7 or 8.
10. Use of an mRNA composition according to any one of claims 1 to 6 or a vector according to claim 7 or 8 or an mRNA vaccine according to claim 9.
11. The application according to claim 10, characterized in that it comprises: use in the preparation of specific T cells for cancer; or (b)
Use in the preparation of TCR-T cells for cancer; or (b)
Use in the preparation of a diagnostic agent for cancer.
12. The use according to claim 11, wherein the cancer is any one of breast cancer, ovarian cancer, gastric cancer, liver cancer, prostate cancer, lung cancer and colon cancer.
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