CN114681600A - Universal polypeptide vaccine and application thereof in preparation of medicine for treating/preventing pancreatic cancer - Google Patents
Universal polypeptide vaccine and application thereof in preparation of medicine for treating/preventing pancreatic cancer Download PDFInfo
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Abstract
The invention discloses a universal polypeptide vaccine, which comprises KRAS-G12R mutation; the universal polypeptide vaccine can be used for preparing medicines for treating/preventing cancers (pancreatic cancer). The universal polypeptide vaccine has a good immunotherapy effect on pancreatic cancer.
Description
Technical Field
The invention relates to a universal polypeptide vaccine suitable for treating and preventing pancreatic cancer.
Background
Pancreatic cancer (PAAD) is a highly malignant malignancy of the digestive tract that is difficult to diagnose and treat. Its morbidity and mortality has increased dramatically in recent years and is one of the worst-prognosis malignancies. Pancreatic cancer is the closest cancer species in new and dead cases compared to other malignancies, indirectly indicating the refractory nature of pancreatic cancer. On the other hand, it is clear from the statistical report that the survival rate of most malignant tumors steadily increases year by year with the general increase of the diagnosis and treatment level of tumors in recent years, but only the survival rate of pancreatic cancer and lung cancer increases very slowly, and the relative survival rate of pancreatic cancer in five years is only 8% even in the united states.
At present, the basic treatment principle of pancreatic cancer is still mainly surgical operation treatment and assisted by radiotherapy and chemotherapy. Pancreatic cancer is difficult to diagnose at an early stage, the surgical resection rate is low, the five-year survival rate after surgery is low, and the sensitivity to radiotherapy is low; the chemotherapy drug has no specificity to kill tumor cells, can kill a large number of normal cells while killing cancer cells, causes toxic and side effects of nausea, vomiting, alopecia, leukopenia and the like on a human body, and often causes patients to be difficult to tolerate; the strong toxic side effects make chemotherapy difficult to administer and over-chemotherapy may shorten the survival of the patient. While traditional therapies do not provide the best therapeutic benefit, targeted drug therapy is also an alternative, and currently, the common drugs approved by CFDA in china for pancreatic cancer include Gemcitabine, Capecitabine and Erlotinib. However, the targeted drug therapy also has the problem of large side effect, and the drug resistance of patients is easy to appear along with the lengthening of the medication time, so that the targeted drug therapy is not sensitive. Compared to the drawbacks of the above methods, immunotherapy shows bright prospects, but only a small fraction of people are effective. In the case of pembrolizumab, the effective rate is only 33% in melanoma and is mostly between 10-30% in other tumors.
Under the background, the attention of tumor vaccines is paid, and the method for killing cancer cells by inducing the immune response of organisms by using the tumor polypeptide vaccine is safer and more effective. The tumor polypeptide vaccine is an accurate medical strategy, can specifically target cancer cells, and theoretically can eliminate all tumors and metastasis thereof. In recent years, the research of tumor polypeptide vaccines has been favored due to the discovery of tumor-associated antigens and tumor-specific antigens, as well as the intensive research and understanding of tumor-induced immune responses and tumor evasion immune surveillance mechanisms. In 2008, Yang B et al induced a strong specific cytotoxic T lymphocyte reaction in mice by loading dendritic cells with KRAS G12V mutant polypeptide, and could specifically kill pancreatic cancer cells. In 2010, Synne Wed en et al found that a polypeptide sequence with 17 th amino acid and mutation at amino acid 12 th position 5-21 of KRAS amino acid sequence can cause strong immune response in pancreatic cancer patients and effectively prolong the survival period of pancreatic cancer patients. A plurality of researches show that the tumor polypeptide vaccine has special advantages and bright prospect in the aspect of tumor treatment and even cure.
The general design process of individualized polypeptide vaccine is to find out the specific somatic mutation of tumor tissue of patient based on the individual genetic gene structure and function difference of tumor patient, analyze the neoantigen produced by these mutations, prepare polypeptide vaccine capable of being identified specifically by patient's histocompatibility complex (MHC) through chemical synthesis process, inject into patient's body, activate specific T cell response and immune storm. However, if a set of vaccine sequences and treatment regimens must be prepared separately for each patient according to current individualized design methods, the universality of these sequences and regimens is extremely low. At present, the whole process from the collection of patient samples, the detection of genetic information and the preparation of polypeptide vaccines takes about 70 to 90 days. Meanwhile, the patient is not only exposed to uncomfortable suffering, but also misses the best treatment opportunity once the condition of the patient is worsened. Although some studies report multiple vaccine peptides that can be used for treating pancreatic cancer, these studies focus on the immune effect of one or more vaccine peptides, and do not consider the applicable range of vaccine peptides, and the results of the studies only cover a small fraction of patients.
In addition, in view of the current situations of difficult diagnosis, difficult treatment and high mortality of pancreatic cancer, effective prevention in advance is particularly important. At present, there is no effective preventive measure against pancreatic cancer. Therefore, aiming at the problems, a universal polypeptide vaccine with both curative and preventive properties on pancreatic cancer is developed.
The references involved are as follows:
balachandran VP, et al, identification of unique neoantigenic qualities in long-term combustors of pancreatic cancer [ J ] Nature,2017,551: 512-516 (Balachandran VP et al, unique neoantigenic properties found in long-term surviving pancreatic cancer patients. Nature,2017,551: 512-516);
(iii) Wood MA, et al, position-level distribution and reactive immunology of Cancer neoepitopes [ J ]. BMC Cancer,2018,18:414(Wood MA et al, population level distribution of tumor neoepitopes and immune prophylactics. BMC Cancer,2018,18: 414);
pinku M, et al, progression of Pancreatic Adenocra Is Significantly Impped with a Combination of Vaccine and COX-2Inhibition [ J ] Immunol, 2009,182(1) 216-224(Pinku M et al, the combined use of a Vaccine and a COX-2 inhibitor effectively inhibits the progression of Pancreatic cancer J Immunol, 2009,182(1) 216-224);
ribas A, et al, Association of pembrolizumab with tumor response and subvalvular amonts with advanced melanomas [ J ]. Jama,2016,315(15): 1600-;
yang B, et al, specific immune cell induced by dendritic cell pulsed with tissue K-ras peptide [ J ]. Zhonghua Yi Xue Za Zhi,2008,88(28): 1956-containing 1960 (Yang Bo et al, K-ras mutant polypeptide loaded dendritic cell induced anti-pancreatic cancer specific immunity. Chinese J.J., 2008,88(28): 1956-containing 1960);
zhou Z, et al.TSNAD an integrated software for cancer therapeutic evaluation and tumor-specific neoantigen detection [ J ]. R Soc Open Sci, 2017,4(4):170050(Zhou Z et al, TSNAD: comprehensive software for tumor somatic mutation and tumor-specific neoantigen detection: RSoc Open Sci, 2017,4(4): 170050);
hundal J, et al. pVAC-Seq: A Genome-shaped in silico aproach to identifying tumor neoantigens [ J ] Genome Med.,2016,8(1):11(Hundal J et al, pVACSeq: bioinformatics method for identifying tumor Genome neoantigens. Genome Med.,2016,8(1): 11);
wed n S, et al, gaudernack G et al, Long-term follow-up of tissues with reconstructed pancreatic cancer following vaccination against an aid mutation K-ras [ J ]. International Journal of cancer.2011,128(5):1120-1128(Wed n S et al, pancreatic cancer patients injected with vaccine against K-ras mutations after long-term follow-up surgical resection. International Journal of cancer.2011,128(5): 1120-1128).
Disclosure of Invention
The technical problem to be solved by the invention is to provide a general vaccine which can be effectively applied to the treatment and prevention of cancers (particularly pancreatic cancers). In order to solve the technical problem, the invention provides a universal polypeptide vaccine, which comprises at least one of the following 6 mutations: KRAS-G12R, KRAS-G12V, CDKN2A-H83Y, KRAS-Q61H, TP53-R248W, CDKN2AP 94L.
Note: specifically as described in table 1.
As an improvement of the universal polypeptide vaccine of the present invention: comprising at least one of the polypeptide sequences directed against said 6 mutations.
As a further improvement of the universal polypeptide vaccine of the present invention: consists of the 6 mutations described.
As a further improvement of the universal polypeptide vaccine of the present invention: consists of a polypeptide sequence directed against the 6 mutations.
As a further improvement of the universal polypeptide vaccine, the polypeptide sequences corresponding to the 6 mutations are as follows:
KRAS-G12R：TEYKLVVVGARGVGKSALTIQLIQNHK；
KRAS-G12V：TEYKLVVVGAVGVGKSALTIQLIQNHK；
CDKN2A-H83Y：AEPNCADPATLTRPVYDAAREGFLDTLKKK；
KRAS-Q61H：KKKTCLLDILDTAGHEEYSAMRDQYMRTGE；
TP53-R248W：KNYMCNSSCMGGMNWRPILTIITLEDSSGN；
CDKN2A-P94L：APRRGAQLRRPRHSHLTRARRCPGGLPGHA。
the invention also provides the application of the general polypeptide vaccine: is used for preparing medicaments for treating/preventing cancers.
As an improvement of the use of the universal polypeptide vaccine of the present invention: the cancer is pancreatic cancer.
The present invention is specifically shown in table 1 below.
TABLE 1
Note: underlined fonts indicate mutated amino acids.
The invention comprises the following steps:
the tumor genome map (TCGA) plan has obtained a great deal of information of tumor genome, transcriptome and the like by utilizing a high-throughput sequencing technology, and greatly promotes people to know the molecular mechanism of the tumor. At present, the TCGA database is the largest tumor database, containing data for 33 cancer types, more than 11000 cancer patients. In addition, the data quality of the TCGA database is high, and the background error is small, so that the result obtained by analyzing the large sample volume of the TCGA database is relatively consistent with the real situation in clinic and has statistical significance.
The invention extracts and downloads the tumor somatic mutation data (182 cases) and the tumor gene read counts data (178 cases) of all pancreatic cancer patients from the TCGA database. The Read counts data contain the information of the reads abundance of all genes in different patients; somatic mutation data includes information on chromosomes, locations, genes, functions, protein changes, mutant samples, and the like. The invention firstly filters the mutation function of somatic mutation data, and reserves the mutation of exon regions and variable shearing types which can change protein amino acid sequences.
Furthermore, the read counts data were used to calculate the expression level (rpkm) of all genes, and mutation sites where the genes were not expressed were filtered out. In addition, the invention also filters out mutation sites with the number of samples less than or equal to 2, because the sites are probably only generated randomly and are not high-incidence in pancreatic cancer. Meanwhile, the invention also comprehensively analyzes the mutant polypeptide generated at the mutant site and the corresponding affinity condition of the wild-type polypeptide and the high-frequency HLA by utilizing 3 types of affinity analysis software netMHC, netMHCpan and PickPocket, and reserves the site with strong immunogenicity of the mutant polypeptide and stronger affinity than that of the wild-type polypeptide.
Then, mutation information and corresponding sample information are stored by constructing a weighted bipartite graph, and the optimized Hungary algorithm is used for calculating the combination of 6 individual cell mutations in the invention, so that the highest proportion of patient population can be covered, and the mutation reaches 46.2% (84/182). In addition to the combinations of the present invention, any combination of mutation sites (but not all sites of the present invention) can cover a proportion of pancreatic cancer patients that is less than 46.2% (FIG. 1).
Finally, a corresponding vaccine polypeptide sequence is designed through an iNeo algorithm to be used as a pancreatic cancer polypeptide vaccine therapy. In the process of designing the vaccine polypeptide, the invention considers the factors influencing the amino acid synthesis efficiency such as antigen epitope distribution, the length and the hydrophobic rate of the polypeptide and the like, and the factors influencing the safety of the polypeptide such as the toxicity and the homology of the polypeptide. The vaccine polypeptide combination of the present invention consists of a polypeptide sequence comprising the following 6 mutations: KRAS-G12R; KRAS-G12V; CDKN 2A-H83Y; KRAS-Q61H; TP 53-R248W; CDKN2A-P94L, as shown in Table 1.
In the practical application process of treating pancreatic cancer, the 6 mutation sites in the tumor DNA of a patient are detected by a rapid Sanger sequencing method, and if the patient carries one or more mutations, the polypeptide vaccine can be directly used. The vaccine peptide is cut into a plurality of short polypeptides in vivo, some of the short polypeptides can be presented by HLA class I, some of the short polypeptides can be presented by HLA class II, theoretically, the short polypeptides can be presented by HLA of different types, and the vaccine peptide has a synergistic effect.
The invention has the following technical advantages:
1. the pancreatic cancer polypeptide vaccine combination provided by the invention consists of polypeptide sequences aiming at the following 6 mutations: KRAS-G12R; KRAS-G12V; CDKN 2A-H83Y; KRAS-Q61H; TP 53-R248W; CDKN2A-P94L, which covers about 46.2% (84/182) of the patients with pancreatic cancer in TCGA database, has universality and universality. In addition to the combinations of the present invention, any combination of mutation sites (not including all sites of the present invention) can cover less than 46.2% of the pancreatic cancer patient population (FIG. 1).
2. Compared with individually customized polypeptide vaccines, the pancreatic cancer polypeptide vaccine combination therapy provided by the invention can be directly applied to patients carrying mutation without the steps of genomic sequencing, data analysis, vaccine customization and the like, and only simple individual mutation site detection is needed, so that the time for the patients to wait for treatment is shortened from 70-90 days to 1-2 days, the patients can not miss the optimal treatment time window, and the medical cost can be greatly reduced.
3. The combined vaccine peptide provided by the invention has stronger immune effect than that of a single vaccine peptide. The short peptides of the vaccine peptides, which are cleaved by enzymes in vivo, can be delivered by HLA class I to stimulate the production of CD8+ cells, and can be delivered by HLA class II to stimulate the production of CD4+ cells. Cytokines secreted by CD4+ cells stimulate more CD8+ cells to produce and aggregate, thereby killing tumor cells more effectively. The synergistic interaction between CD4+ cells and CD8+ cells and HLA was able to generate a stronger immune response (as described in experiment 2).
4. Most of the current targeting drugs for cancer treatment target a single target, and drug resistance is easy to generate after long-term administration. The 6 polypeptide vaccines provided by the invention can simultaneously target 6 targets at most, and the multi-target treatment scheme can effectively reduce the drug resistance occurrence probability of the tumor, so that the treatment effect is better and more durable (as shown in experiments 3, 4 and 6).
5. The combined vaccine peptide provided by the invention can be used for treating pancreatic cancer and preventing the occurrence and recurrence of pancreatic cancer. When healthy persons without pancreatic cancer or patients with pancreatic cancer after surgery are injected with the combined vaccine of the present invention, specific killer T cells and memory T cells targeting the 6 mutations of the present invention are generated in vivo. If one or more of the 6 mutations are generated in the canceration process of normal cells, the existing T cells in the body can rapidly identify and eliminate the cells carrying the mutations, thereby effectively preventing the occurrence of pancreatic cancer. Theoretically, according to the proportion of the population covered by 6 mutations in the invention, the prevention rate of the polypeptide vaccine of the invention on pancreatic cancer can reach 46.2% (as shown in experiment 5).
In the present invention, the ratio of 6 mutations each in pancreatic cancer patients is shown in Table 2.
TABLE 2
| Mutations | Ratio of covered sample |
| KRAS-G12R | 15.38％ |
| KRAS-G12V | 22.53％ |
| CDKN2A-H83Y | 2.20％ |
| KRAS-Q61H | 3.30％ |
| TP53-R248W | 2.75％ |
| CDKN2A-P94L | 3.30％ |
With the 6 sites of the present invention, a maximum of 46.2% of patients can be covered by the 6 site combinations. The universal polypeptide vaccine has a good immunotherapy effect on pancreatic cancer.
Drawings
FIG. 1 is a graph of the ratio of different number of sites combined to cover a patient;
the abscissa represents the combination of sites with different numbers, the ordinate represents the proportion of covered patients, the thick black line in the middle of the boxplot represents the median, and the horizontal dotted line represents the coverage rate of 46.2%. Boxplots for each site combination represent the results of 1000 random draws.
FIG. 2 is the amino acid sequence of each polypeptide expression cassette;
wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.
FIG. 3 is an immunogenicity assay for polypeptides;
1 is a control group, 2 is a PHA group, 3-8 is a single polypeptide group with 6 polypeptides, 9 is a mixed group with 6 polypeptides, 10 is a polypeptide group corresponding to a non-selection site KRAS-G12D, a and b represent that 2 mice are randomly selected from each group, and each mouse has 3 repeats.
FIG. 4 is a polypeptide-induced cytotoxicity assay;
a is a cytotoxicity experimental graph induced by BxPC-3 (wild); b is experimental graph of cytotoxic killing induced by BxPC-3(mut 6); t represents the number of effector T cells, the number of target cells and the ordinate represents the cell lysis rate; peptide i (i ═ 1 … 6) represents 6 polypeptides in the present invention, and peptide pool represents a mixed group of 6 polypeptides.
FIG. 5 shows the effect of the polypeptide vaccine on the inhibition of pancreatic cancer in mice;
the abscissa represents the days after vaccine injection and the ordinate represents the relative tumor volume. peptide i (i ═ 1 … 6) represents 6 polypeptides in the present invention, and peptide pool represents a mixed group of 6 polypeptides.
FIG. 6 shows the effect of the polypeptide vaccine on pancreatic cancer in mice;
the abscissa represents the number of days after vaccine injection and the ordinate represents the tumor volume; peptide i (i ═ 1 … 6) represents 6 polypeptides in the present invention, and peptide pool represents a mixed group of 6 polypeptides.
FIG. 7 is a graph showing the effect of a single polypeptide vaccine on pancreatic cancer inhibition in mice;
the abscissa represents the number of days after vaccine injection and the ordinate represents the relative tumor volume; peptide 1 represents the polypeptide to which the mutation corresponds, peptide 3 represents the mutated unrelated polypeptide, and peptide pool represents the mixed group of 6 polypeptides.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
examples 1,
A universal polypeptide vaccine, aiming at the following 6 mutations: KRAS-G12R; KRAS-G12V; CDKN 2A-H83Y; KRAS-Q61H; TP 53-R248W; CDKN 2A-P94L. The polypeptide sequences for the 6 mutations are specifically shown in table 1.
Comparative examples 1,
The combination of 6 sites with "KRAS-G12D" instead of "KRAS-G12V" in example 1 covered 53.8% of patients at the maximum. The polypeptide sequence corresponding to KRAS-G12D is: TEYKLVVVGADGVGKSALTIQLIQNH, underlined font indicates the mutated amino acid.
But the immunization effect of comparative example 1 was far less than that of example 1; KRAS-G12D proved to be extremely weak in immunogenicity as demonstrated in experiment 2 below.
Comparative examples 2,
Of all candidate sites, … … 9 site combinations of 1 site, 2 site combinations and 3 site combinations were randomly drawn 1000 times, each drawing covering the patient ratio as shown in fig. 1. As can be seen from the figure, the combination of 6 sites already achieved 46.2% patient coverage, with the combination of more than 6 sites covering no more than a 46.2% proportion of patients.
Experiment 1: construction of a Stable transgenic cell line containing specific mutation sites
Purpose of the experiment: in order to verify the therapeutic and preventive effects of the polypeptide vaccine for pancreatic cancer of the present invention, it is necessary to construct a set of stable transgenic cell lines containing the specific mutation sites of the present invention.
a. Construction of mutant site eukaryotic expression plasmid
The mini-gene capable of expressing all 6 mutated vaccine polypeptides in the invention is obtained by an artificial synthesis method and consists of the following parts: a signal peptide part (lysome-associated membrane glycoprotein 1, LAMP1), 6 mutant polypeptides and MHC class I transfection domain (MITD), wherein the 6 mutant polypeptides are connected by flexible connecting peptide GGSGGGGSGG, GATATC (EcoR V) is introduced at the upstream and CTCGAG (Xho I) is introduced at the downstream after codon optimization of genes, and the mutant polypeptides are cloned into an eukaryotic expression vector pcDNA3.1-hygro (+), namely mut6-pcDNA3.1 (+). Meanwhile, the corresponding wild type polypeptide is synthesized to be used as a control and is named as wild6-pcDNA3.1 (+). In order to verify the specificity of the target, a mutant polypeptide containing only 1 mutant site-KRAS-G12R was designed and named mut1-pcDNA3.1 (+). All gene fragments were synthesized and constructed by Nanjing Kingsrei Biotech, Inc., and the amino acid sequences are shown in FIG. 2.
b. Construction of cell lines capable of stably expressing mutant Polypeptides
Human pancreatic cancer cell strain BxPC-3 with 2 x 105Perwell in 6-well plates, transfection was initiated when cells were 70-80% covered. Mu.g of mut6-pcDNA3.1(+) plasmid, 2.5. mu.g of wild6-pcDNA3.1(+) plasmid and 2.5. mu.g of mut1-pc DNA3.1(+) plasmid were diluted in 3 parts of 100. mu.l serum-free RPMI-1640 medium, 2.5. mu.l of PLUSTMReagens were added, incubated at room temperature for 5min and then incubated with 5. mu.l of LipofectamineTMOf LTXSerum-free RPMI-1640100. mu.l were mixed and incubated at room temperature for 30 min. The liposome plasmid complex is respectively dropped into 3 cells to be transfected containing 1000 mul serum-free RPMI-1640, gently shaken from front to back, kept stand for 6h, replaced by an RPMI-1640 culture medium containing 10% serum, and continuously cultured for 48h, and then replaced by a 10% serum culture medium containing 700 mug/mL G418 and 400 mug/mL hygromycin B for cell screening. And (3) resistance screening for 10-14 days, after the cells of the control group are completely dead, the cells of the transfection group grow in a large quantity, the cells are digested, and the cells are planted into a 96-well plate by adopting a limiting dilution method. Monoclonal cells were selected under the microscope and cultured in medium containing 700. mu.g/mL G418, 400. mu.g/mL hygromycin B, with the medium changed every other day. After further culturing for about 10 days, the monoclonal cells grow into a larger mass, digested, and transferred to a 24-well plate for culture. Meanwhile, pcDNA3.1(+) -Hygro empty vector plasmid is transfected to carry out resistance screening by the same method and is used as a control cell named BxPC-3 (containing pcDNA3.1 (+)). In the above-described method, the cell lines constructed using the mut6-pcDNA3.1(+) plasmid, the wild6-pcDNA3.1(+) plasmid and the mut1-pcDNA3.1(+) plasmid were designated mut6-BxPC-3 (containing mut6-pcDNA3.1(+)), wild6-BxPC-3 (containing mut6-pcDNA3.1(+)), and mut1-BxPC-3 (containing mut1-pcDNA3.1 (+)), respectively.
Experiment 2: determination of immunogenicity of polypeptides
Purpose of the experiment: an ELISpot experiment proves that 6 polypeptides in the invention can cause immune reaction in a humanized mouse, and the immune effect of the mixture of 6 polypeptides is stronger than that of a single polypeptide; furthermore, the unselected site KRAS-G12D in comparative example 1 hardly elicited an immune response.
The experimental method comprises the following steps:
to detect the immune response of the polypeptide, an IFN- γ enzyme linked immunosorbent (ELISpot) assay is performed. The detailed experimental procedure is as follows: 27 humanized mice B-NSG (CD34+) were selected at 8 weeks of age and randomized into 9 groups of 3 mice each. After one week of adaptation, the samples were divided into negative control group number 1, 6 single polypeptide group numbers 3-8 (total 6 groups), 6 mixed polypeptide group number 9, and polypeptide group number 10 corresponding to unselected site KRAS-G12D. CpG was used as adjuvant (0.2. mu.g/mouse), 50. mu.g of polypeptide was added to Freund's incomplete adjuvant (FIA, Sigma-Aldrich) 1:1, mixing and emulsifying for 30 minutes, and mixing PBS with Freund incomplete adjuvant 1:1 mixed emulsification for 30 minutes as a negative control, four times of subcutaneous immunization is carried out on the right chest of the back of the neck, the total dose is 0.5 mL/mouse, once every 1 week for three weeks, and 10 days after the third immunization, the spleen of the mouse is taken to prepare mouse lymphocyte suspension for ELISpot detection. The mouse lymphocyte suspensions numbered 3-9 were retained for experiment 3.
And in the ELISpot detection result, the polypeptide with positive IFN-gamma result is judged as the positive candidate polypeptide. Diluting mouse lymphocytes to a concentration of 1-2 x 106Laying on 24-well plate, dividing into control group (DMSO with the same concentration as polypeptide), 6 single polypeptide groups, 6 polypeptide mixed groups, and polypeptide group corresponding to KRAS-G12D in comparative example 1, PHA positive control group number 2(PHA lymphocyte from negative control group), totaling 10 groups, adding corresponding polypeptide (10 μ G/mL), pre-incubating for 72h, centrifuging to separate cells, and adjusting cell concentration to 2 x 106The color was developed on IFN-. gamma.Elispot plates according to the instructions of the IFN-. gamma.Elispot kit, and the number of spots generated was read using a CTL-ImmunoSpots5 series enzyme-linked spot analyzer. The positive result of IFN-gamma indicates that antigen specific T cells are generated, the polypeptide can cause the immune reaction of the organism, and the number of spots reflects the intensity of the immunity.
The experimental results are as follows:
the results are shown in FIG. 3, where 6 single polypeptide groups produced about 30-150 spots per million cells, indicating that 6 polypeptides alone caused an immune response. The KRAS-G12D corresponding to the polypeptide group in comparative example 1 was produced substantially without spots, indicating that the polypeptide hardly elicited immune responses; meanwhile, the number of spots induced by the 6 polypeptide mixed groups is more than 400 on average and is obviously higher than that of 6 single polypeptide groups, which indicates that the combination can cause stronger immune response.
Experiment 3: in vitro cell killing experiment
Purpose of the experiment: the killing effect of the 6 polypeptides on in vitro cell level can be proved, and the killing effect after the 6 polypeptides are mixed is stronger than that of a single polypeptide.
The experimental method comprises the following steps:
(1)5- (6) -Carboxy-fluorochein succinimidyl ester (CFSE) dye was purchased from Invitrogen. The operation steps are carried out according to the kit instructions. CFSE was used to label mut6-BxPC-3 (containing mut6-pcDNA3.1(+)) and wild6-BxPC-3 (containing mut6-pcDNA3.1(+)) target cells under sterile conditions as target cells for the experimental and control groups, respectively.
(2) Killing experiment
a. Preparation of effector cells:
the 7 mouse lymphocyte suspensions retained in experiment 2 were resuspended in RPMI1640 medium and counted by trypan blue staining.
b. Induction culture of effector cells CTL:
the 7 groups of lymphocytes were diluted to a concentration of (1-2) × 10, respectively6Each well was plated at 3mL in 6-well plates, and cultured in RPMI1640+ 10% FBS +1 XPenicillin (100. mu.g/mL) + streptomycin (100. mu.g/mL) +1 XPEM non-essential amino acids +1mM sodium sulfate +10mM HEPES buffer medium, and supplemented with 50U/mL rhIL 2. Adding corresponding antigen peptide of 10 mug/mL into each hole, culturing for 7 days, changing liquid half every 3 days, and supplementing corresponding antigen peptide and rhIL 2; after one week, the cells were resuspended and washed 2 times with PBS to prepare effector CTLs.
1) The mut6-BxPC-3 (containing mut6-pcDNA3.1(+)) target cells were mixed with 7 effector cells CTL at a cell number ratio of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at 200. mu.L per well volume to serve as experimental groups, each of which was provided with three parallel control wells.
2) The target cells of wild6-BxPC-3 (containing mut6-pcDNA3.1(+)) and 7 effector cells CTL were mixed according to the cell number ratio of 1:5, 1:10 and 1:20, and added into a U-shaped 96-well plate with 200. mu.L of each well volume as a control group, and three parallel control wells were provided for each control group.
3) The 96-well plate was incubated at 37 ℃ for 4 hours.
4) The supernatant was centrifuged off from the 96-well plate, the cell pellet was resuspended in 200. mu.L of precooled PBS, transferred to a flow-up tube, labeled with Propidium Iodide (PI) staining at a concentration of 1. mu.g/m L for 3min, and immediately subjected to flow-up detection.
The experimental results are as follows:
as shown in FIG. 4, the killing efficiency of the effector T cells induced by the experimental group (FIG. 4B) was varied from 40% to 80%, and was significantly higher than that of the control group (FIG. 4A), indicating that the mutant polypeptide group had a killing effect on the target cells. In the mutant polypeptide group, the killing effect of T cells is stronger and stronger along with the increase of the effective target ratio, compared with a single polypeptide, the killing efficiency of the mixed polypeptide is higher, and when the effective target ratio is 20:1, the killing efficiency of the mixed polypeptide on target cells reaches over 90 percent. The experiment shows that 6 mutant polypeptides and a mixed group thereof can effectively activate specific T cell immune response, and the killing efficiency of the 6 polypeptide mixed is obviously higher than that caused by a single polypeptide.
Experiment 4: evaluation of the efficacy of the vaccine of the present invention for tumor treatment in humanized mouse model
Purpose of the experiment: the 6 polypeptides are verified to be capable of exciting the killing of T cells to tumor cells in vivo, and the killing effect of the 6 polypeptide mixture is stronger than that of a single polypeptide.
The experimental process comprises the following steps:
24 humanized mice B-NSG (CD34+) with the age of 8 weeks are selected, after one week of adaptation, BxPC-3(mut6) cells in logarithmic growth phase are collected and prepared into 5 x 106The cells were suspended in 0.2mL of suspension and placed under the left forelimb axilla of the mice. Taking subcutaneous tumor nodules with the diameter of about 5mm as a tumor formation standard, forming tumors within 9-12 days, selecting mice without hemorrhage, necrosis and infection, randomly dividing the mice into 8 groups, 3 mice in each group, 6 single polypeptide groups, 6 polypeptide mixed groups and blank groups (PBS), immunizing the mice on the same day after grouping, adopting CpG as an adjuvant (0.2 mu g/mouse), 50 mu g of polypeptide in each group, and further mixing the mice with Freund's incomplete adjuvant (FIA, Sigma-Aldrich) 1:1, mixing and emulsifying for 30 minutes, mixing PBS with Freund's incomplete adjuvant 1:1 mixed emulsion for 30 minutes as negative control, four times in neck, back and right chest subcutaneous immunization, total dose 0.5 mL/piece, 1 week once, three times of immunization. On day 28, mice were sacrificed, and the tumor length and length were measured daily with a vernier caliper to calculate the tumor volume;
TV＝1/2×a×b2wherein a and b represent major diameters, respectivelyAnd a short path, and calculating a Relative Tumor Volume (RTV) from the measurement result, wherein RTV is Vt/V0. Where V0 is the tumor volume measured at caging (i.e., d0) and Vt is the tumor volume at each measurement, a relative tumor volume curve is plotted and the body weight of each group of animals is recorded.
The experimental results are as follows:
the growth of transplanted tumors in each group before grouping treatment is basically consistent, and the size of the transplanted tumors is not obviously different (P > 0.05).
As shown in figure 5, the growth of transplanted tumors in each group is not obviously different from that of a control group (P is more than 0.05) in size 1 week after immunization, and the tumor size inhibition degree of an experimental group is more and more obvious than that of the control group along with the increase of the immunization times and the prolongation of time, which indicates that 6 polypeptides can play a role in inhibiting the growth of tumors in mice. Meanwhile, from the second week, the inhibition efficiency of the 6 polypeptide mixed group is obviously improved (P is less than 0.01) compared with that of each polypeptide alone, which indicates that the synergy exists among the polypeptides. The tumor shrinkage condition occurs 2 weeks after the immunization, which indicates that the immune response induced by the vaccine has a killing effect on the tumor cells.
Experiment 5: evaluation of tumor prevention efficacy of vaccine of the present invention in humanized mouse model
Purpose of the experiment: the 6 polypeptides are verified to be safe and effective on the humanized mouse and can effectively prevent the occurrence of tumors.
The experimental process comprises the following steps:
24 humanized mice B-NSG (CD34+) with the age of 8 weeks were selected, randomly divided into 8 groups of 3 mice, and after one week of adaptation, 50. mu.g of each polypeptide was adjuvanted with CpG (0.2. mu.g/mouse), and Freund's adjuvant (FIA, Sigma-Aldrich) 1:1, mixing and emulsifying for 30 minutes, mixing PBS with Freund's incomplete adjuvant 1:1 mixed emulsification for 30 minutes as a negative control, four times of subcutaneous immunization at the neck, back and right chest with 0.5 mL/mouse total dose, one time for 1 week, three immunizations, and 6 single polypeptide groups, 6 polypeptide mixed groups and blank groups (PBS). BxPC-3(mut6) cells were collected at logarithmic growth phase 1 week after the third immunization and prepared at 5 x 106One cell/mL cell suspension, 0.2mL in mice left forelimb axilla。
Recording the occurrence condition of tumors of each group of animals every day, measuring the long diameter and the short diameter of the tumors by using a vernier caliper day after the tumors grow out, and calculating the volume of the tumors;
TV＝1/2×a×b2wherein a and b represent the major and minor diameters, respectively, and the body weight of each group of animals was recorded. Evaluation: four weeks after tumor inoculation, all mice were sacrificed, the experiment was terminated, and the tumor inhibition rate was calculated and evaluated.
The tumor inhibition rate (%) is (mean tumor volume in control group-mean tumor volume in experimental group)/mean tumor volume in control group x 100%.
Data processing: SPSS software is adopted for statistical analysis, and single-factor variance analysis is adopted for comparison, wherein P <0.05 is the difference and has statistical significance.
The experimental results are as follows:
as shown in FIG. 6, 10 days after tumor inoculation, the tumor volume of the control group began to increase slowly, and the tumor volume inhibition of each experimental group was significant. At day 16, there was still no tumor formation in each experimental group compared to the control group. The tumor size of each experimental group slowly increased with the time, but the growth rate was significantly lower than that of the control group. Meanwhile, the inhibition efficiency of the 6 polypeptide mixed group is obviously improved compared with that of each polypeptide alone (P <0.01) from 23 days after tumor inoculation, and the tumor bodies of the 6 polypeptide mixed group hardly grow at 28 days after tumor inoculation. The test proves that the 6 polypeptides and the mixture thereof can prevent the specific tumor occurrence, and the prevention effect of the mixture of the 6 polypeptides is obviously superior to the prevention effect caused by each polypeptide alone.
Experiment 6: evaluation of the efficacy of the vaccines of the present invention in the treatment of single site mutant tumors in a humanized mouse model.
Purpose of the experiment: to more realistically model the clinical situation in reality, i.e., most patients generally carry only 1 mutation of the present invention (e.g., 93% of 84 patients (78) covered by 6 mutations of the present invention in the patient population we analyzed, carry only 1 mutation), it was verified that the therapeutic effect of the 6 polypeptide mixture on single-mutation tumors was superior to the therapeutic effect of the corresponding single polypeptide.
The experimental process comprises the following steps:
selecting 8-week-old humanized mouse B-NSG (CD34+)15, after adapting for one week, collecting logarithmic growth phase BxPC-3(mut 1) cells, and preparing into 5 x 106One cell/mL cell suspension, seeded 0.2mL in the mouse left forelimb axilla. Taking subcutaneous tumor nodules with the diameter of about 5mm as a tumor formation standard, forming tumors within 9-12 days, selecting mice without hemorrhage, necrosis and infection for grouping, randomly dividing into 4 groups, 3 mice in each group, dividing into a polypeptide group 1 (related polypeptide), a polypeptide group 3 (unrelated polypeptide), a 6-polypeptide mixed group and a blank group (PBS), immunizing the mice on the same day after grouping, adopting CpG as an adjuvant (0.2 mu g/mouse), 50 mu g of polypeptide for each, and then mixing with Freund's adjuvant (FIA, Sigma-Aldrich) 1:1, mixing, emulsifying for 30 minutes, mixing PBS with Freund's incomplete adjuvant 1:1 mixed emulsion for 30 minutes as negative control, four times in neck, back and right chest subcutaneous immunization, total dose 0.5 mL/piece, 1 week once, three times of immunization. The mice were sacrificed on day 28, and the tumor major and minor diameters were measured daily with a vernier caliper, and the tumor volume was calculated;
TV＝1/2×a×b2where a and b represent the long and short diameters, respectively, and Relative Tumor Volume (RTV) is calculated from the measurement results, where RTV is Vt/V0. Where V0 is the tumor volume measured at caging (i.e., d0) and Vt is the tumor volume at each measurement, a relative tumor volume curve is plotted and the body weight of each group of animals is recorded.
The experimental results are as follows:
the growth of the 4 transplanted tumors before group treatment was essentially consistent with no significant difference in size (P > 0.05). As shown in figure 7, the size of the transplanted tumor growth of each group is not obviously different from that of the control group (P >0.05) 1 week after immunization, the tumor size of the mixed group of the polypeptide group 1 and the 6 polypeptides is more and more obviously inhibited along with the increase of the immunization times and the prolongation of the time, and the tumor growth of the control group and the irrelevant polypeptide group 3 is similar, thereby proving that the killer T cells induced by the polypeptide have specificity. From 21 days, the inhibition efficiency of the 6 polypeptide mixed group is obviously improved (P is less than 0.01) compared with that of the polypeptide group 1 alone, and the tumor is shrunk after 2 weeks of immunization. Indicating that the polypeptides have synergistic effect.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Sequence listing
<110> Hangzhou Nianjin Biotechnology Co., Ltd
<120> universal polypeptide vaccine and application thereof in preparation of drugs for treating/preventing pancreatic cancer
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Claims (2)
1. A universal polypeptide vaccine, which is characterized by comprising KRAS-G12V mutation;
the KRAS-G12V mutant polypeptide sequence is as follows: TEYKLVVVGAVGVGKSALTIQLIQNHK are provided.
2. The use of the universal polypeptide vaccine of claim 1, wherein: is used for preparing a medicament for treating/preventing pancreatic cancer.
Applications Claiming Priority (3)
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| CN201811224668 | 2018-10-19 | ||
| CN2018112246689 | 2018-10-19 | ||
| CN201811430768.7A CN109550044B (en) | 2018-10-19 | 2018-11-28 | Universal polypeptide vaccine and application thereof in preparation of medicine for treating/preventing pancreatic cancer |
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| CN201811430768.7A Division CN109550044B (en) | 2018-10-19 | 2018-11-28 | Universal polypeptide vaccine and application thereof in preparation of medicine for treating/preventing pancreatic cancer |
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| CN201811430768.7A Active CN109550044B (en) | 2018-10-19 | 2018-11-28 | Universal polypeptide vaccine and application thereof in preparation of medicine for treating/preventing pancreatic cancer |
| CN202111660405.4A Withdrawn CN114573688A (en) | 2018-10-19 | 2018-11-28 | Universal polypeptide vaccine and application thereof in preparation of medicine for treating/preventing pancreatic cancer |
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| CN202111660405.4A Withdrawn CN114573688A (en) | 2018-10-19 | 2018-11-28 | Universal polypeptide vaccine and application thereof in preparation of medicine for treating/preventing pancreatic cancer |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5961978A (en) * | 1991-02-26 | 1999-10-05 | Norsk Hydro, A.S. | Therapeutically useful peptides and peptide fragments |
| WO2000066153A1 (en) * | 1999-04-30 | 2000-11-09 | Norsk Hydro Asa | RAS ONCOGEN p21 PEPTIDE VACCINES |
| CN106414493A (en) * | 2014-05-06 | 2017-02-15 | 塔格瓦克斯公司 | Peptide vaccine comprising mutant RAS peptide and chemotherapeutic agent |
| WO2018112420A1 (en) * | 2016-12-15 | 2018-06-21 | The Regents Of The University Of California | Compositions and methods for treating cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MY146381A (en) * | 2004-12-22 | 2012-08-15 | Amgen Inc | Compositions and methods relating relating to anti-igf-1 receptor antibodies |
| CN1830489A (en) * | 2006-04-11 | 2006-09-13 | 中国药科大学 | Tumour polypeptide vaccine based on gastrin release peptide |
| CA2745376A1 (en) * | 2008-12-01 | 2010-06-10 | Lifespan Extension Llc | Methods and compositions for altering health, wellbeing, and lifespan |
| FI3892295T3 (en) * | 2011-05-24 | 2023-05-10 | BioNTech SE | Individualized vaccines for cancer |
| CN105132463B (en) * | 2015-08-27 | 2018-10-26 | 中国科学院昆明动物研究所 | A method of establishing pancreatic cancer models |
-
2018
- 2018-11-28 CN CN202111665972.9A patent/CN114681600A/en not_active Withdrawn
- 2018-11-28 CN CN201811430768.7A patent/CN109550044B/en active Active
- 2018-11-28 CN CN202111660405.4A patent/CN114573688A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5961978A (en) * | 1991-02-26 | 1999-10-05 | Norsk Hydro, A.S. | Therapeutically useful peptides and peptide fragments |
| WO2000066153A1 (en) * | 1999-04-30 | 2000-11-09 | Norsk Hydro Asa | RAS ONCOGEN p21 PEPTIDE VACCINES |
| CN106414493A (en) * | 2014-05-06 | 2017-02-15 | 塔格瓦克斯公司 | Peptide vaccine comprising mutant RAS peptide and chemotherapeutic agent |
| WO2018112420A1 (en) * | 2016-12-15 | 2018-06-21 | The Regents Of The University Of California | Compositions and methods for treating cancer |
Non-Patent Citations (1)
| Title |
|---|
| SYNNE WEDEN等: "Long-term follow-up of patients with resected pancreatic cancer following vaccination against mutant K-ras", 《INTERNATIONAL JOURNAL OF CANCER》 * |
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| CN109550044B (en) | 2022-05-03 |
| CN109550044A (en) | 2019-04-02 |
| CN114573688A (en) | 2022-06-03 |
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