CN116836231A - New antigen peptide of t (8; 21) AML and application thereof - Google Patents
New antigen peptide of t (8; 21) AML and application thereof Download PDFInfo
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Abstract
The invention provides a novel antigen peptide of t (8; 21) AML and application thereof, wherein the amino acid sequence of the novel antigen peptide is KVLHSSLVCK. The invention finds that AE fusion protein can generate the new antigen and can be presented by HLA-A.times.31:01 or HLA-A.times.11:01 through analysis. The invention also discovers that the novel antigen can promote CTL to effectively kill t (8; 21) AML cells, and shows that the DC-CTL cell immunotherapy with KVLHSSLVCK as a target has potential curative effect. The novel antigen peptide can be applied to the development of T (8; 21) AML pharmaceutical compositions, or can be applied to the preparation of more specific and effective polypeptide vaccines, or can be applied to the induction generation of specific cytotoxic T cells, and has good development and application prospects in the field of T (8; 21) AML specific immunotherapy.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a novel antigen peptide of t (8; 21) AML and application thereof.
Background
Leukemia is one of ten major malignant tumors worldwide, with the incidence of acute myeloid leukemia (acute myelocytic leukemia, AML) being 2-4/10 ten thousand people, accounting for 80% -90% of adult acute leukemia. AML has complex pathogenesis, high mortality and high treatment cost, and brings about a heavy economic burden to society and families of patients. Chromosomal translocation is the most common genetic abnormality in acute leukemia, where t (8; 21) (q 22; q 22) occurs most frequently in AML, with t (8; 21) AML accounting for 10% -15% of all AML, and more so up to 40% -60% in M2-type AML. the induction remission rate of t (8; 21) AML is 85% -90%, 10% -15% of patients have primary drug resistance, 30% -50% of the patients who obtain remission have relapse, namely more than half of the patients finally become refractory/recurrent t (8; 21) AML, the re-induction remission rate of the patients is improved, and the total survival time is prolonged, so that the serious problem to be solved in clinical urgent need is solved.
The personalized immune cell therapy based on the tumor neoantigen (Neo-anti) provides a new idea for tumor immunotherapy and becomes a research hotspot. Tumor neoantigens are tumor-specific antigens that are formed by point mutations or fusion of genes during tumorigenesis, progression, and can be specifically recognized by T cells. The tumor neoantigen specific T cells have no cross immune reaction to normal tissues while killing tumor cells, and can not cause fatal toxicity of important organs (such as heart, liver, lung and the like). Cytotoxic T lymphocytes (Cytotoxic T lymphocytes, CTLs) are the most dominant immune effector cells that naturally occur in vivo to kill tumor cells, and neoantigens typically stimulate polyclonal CTL production in vivo. Immunotherapeutic strategies targeting neoantigens typically collect patient autologous lymphocytes that are either infused back into the body after in vitro expansion of CTLs by neoantigen stimulation or induced in vivo neoantigen-specific CTL expansion with a vaccine.
In recent years, most of immunotherapy clinical studies targeting tumor neoantigens have focused on the treatment of solid tumors, and a very successful case has been reported in 2014 Science journal, and researchers have excised the lung metastases of a patient with advanced cholangiocarcinoma to obtain tumor infiltrating T cells, and further isolate T cells capable of specifically recognizing the neoantigens generated by mutations in tumor ERBB2IP genes, and reinject the neoantigen-specific T cells amplified in vitro twice in order to completely relieve the patient. In a basic study of non-t (8; 21) AML, researchers found that the presence of a CBFB-MYH11 fusion gene in t (16; 16) AML cells can encode the generation of a corresponding fusion protein, and that a neoantigen peptide REEMEVHEL derived from the fusion protein can be presented by HLA-B40:01 and induce neoantigen-specific CTL to kill AML cells; studies by Dyantha et al showed that NPM1 gene produced NPM1c protein due to frameshift mutation, and that neoantigens WQWRKSL, CLAVEEVSL and AVEEVSLRK derived from NPM1c protein could stimulate specific CTL amplification in vitro, thereby recognizing and killing AML cells. It can be seen that cellular immunotherapy targeting tumor neoantigens has potentially considerable efficacy in AML treatment of non-t (8:21) AML. However, no studies have been reported using neoantigen-specific CTL to treat t (8:21) AML. Therefore, further development of neoantigenic peptides targeting t (8:21) AML tumor neoantigens has positive significance for improving the re-induction remission rate of refractory/recurrent t (8:21) AML patients and prolonging the total survival thereof.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, the present invention aims to provide a novel antigen peptide of t (8:21) AML and application thereof, aiming at solving the problem of lack of novel antigen peptide of targeting t (8:21) AML tumor neoantigen.
The technical scheme of the invention is as follows:
a novel antigen peptide of t (8; 21) AML, wherein the amino acid sequence of the novel antigen peptide is shown as SEQ ID NO. 1.
The novel antigenic peptide of t (8; 21) AML, wherein the novel antigenic peptide is presented by HLA-A.times.31:01 or HLA-A.times.11:01.
The novel antigenic peptide of T (8; 21) AML, wherein the novel antigenic peptide induces a cellular immune response in CD8+ T cells upon presentation by HLA-A.times.31:01 or HLA-A.times.11:01.
A method for developing a novel antigen peptide of t (8; 21) AML, comprising the steps of:
providing transcriptome high throughput second generation sequencing data for t (8; 21) AML cells;
analyzing fusion mutation of leukemia related fusion genes according to the transcriptome high-throughput second-generation sequencing data, and predicting to obtain candidate fusion new antigens;
and comparing the candidate fusion neoantigen with a normal protein through a tumor specific antigen prediction screening platform, and screening to obtain the neoantigen peptide of the t (8; 21) AML.
The development method of the novel antigen peptide of the t (8; 21) AML, wherein the amino acid sequence of the novel antigen peptide is shown as SEQ ID NO. 1.
A pharmaceutical composition, wherein the pharmaceutical composition comprises a neoantigenic peptide as defined in any one of the preceding claims.
The pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient, and the pharmaceutical form of the pharmaceutical composition can be vaccine.
Use of a neoantigenic peptide of T (8; 21) AML, wherein the neoantigenic peptide as described in any one of the above is used for the preparation of a vaccine specific for the T (8; 21) AML antigen, or for the induction of cytotoxic T cells specific for the neoantigenic peptide, or for the preparation of antigen presenting cells sensitized with the neoantigenic peptide.
Use of a neoantigenic peptide of T (8; 21) AML, wherein the neoantigenic peptide as described in any one of the above is used in the development of TCR-T, CAR-T or CAR-NK products.
Use of a neoantigenic peptide of t (8; 21) AML, wherein the neoantigenic peptide as described in any one of the above is used in the development of a diagnostic kit for t (8; 21) AML.
The beneficial effects are that: the invention provides a novel antigen peptide of t (8; 21) AML and application thereof. The present invention, by analyzing the RNA-seq data of t (8:21) AML cell lines and t (8:21) AML patient primary AML cells, finds that AE fusion proteins can produce neoantigen KVLHSSLVCK and can be presented by HLA-A.times.31:01 or HLA-A.times.11:01. By synthesizing KVLHSSLVCK peptide fragments and using an antigenic peptide-DC-CTL culture system, the invention discovers that a novel antigen KVLHSSLVCK generated by AE fusion protein can promote CTL to effectively kill t (8; 21) AML cells, and shows that DC-CTL cell immunotherapy with KVLHSSLVCK as a target has potential curative effects on t (8; 21) AML. The method of the invention firstly identifies that AE fusion protein can generate new antigen through data analysis and experimental verification, and AE fusion protein exists in all t (8; 21) AML patients. Thus, DC-CTL therapies targeting the novel antigenic peptides produced by AE can cover all t (8; 21) AML patients. Therefore, the novel antigen peptide can be applied to the development of T (8; 21) AML pharmaceutical compositions, or the preparation of more specific and effective polypeptide vaccines, or the induction of specific cytotoxic T cells, and has good development and application prospects in the field of T (8; 21) AML specific immunotherapy.
Drawings
FIG. 1 is a schematic diagram showing the identification result of a tumor neoantigen produced by AE in the example of the present invention.
FIG. 2 is a schematic representation of the results of killing t (8; 21) AML cells in vitro by neoantigen-specific CTLs in the examples of the present invention.
Detailed Description
The invention provides a novel antigen peptide of t (8; 21) AML and application thereof, and the invention is further described in detail below for the purpose, technical scheme and effect of the invention to be clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The embodiment of the invention provides a novel antigen peptide of t (8; 21) AML, wherein the amino acid sequence of the novel antigen peptide is shown as SEQ ID NO. 1.
SEQ.ID NO.1：KVLHSSLVCK
the most prominent feature in t (8; 21) AML is the presence of AE (AML 1-ETO, leukemia associated fusion gene) fusion proteins, the production of which is the first hit by t (8; 21) AML, which occurs throughout the development and progression of AML. By analyzing the RNA-seq data of t (8; 21) AML cell lines and t (8; 21) AML patient primary AML cells, the present examples found that AE fusion proteins produced novel antigen KVLHSSLVCK, which was presented by HLA-A.times.31:01 and HLA-A.times.11:01, respectively, and HLA-A.times.11:01 was the genotype with highest incidence in Chinese population, about 1/4 Chinese humans were positive for this type. By synthesizing KVLHSSLVCK peptide fragments and using an antigenic peptide-DC-CTL culture system, KVLHSSLVCK generated by AE fusion protein is found to promote CTL to effectively kill fusion protein cells, and the embodiment of the invention shows that DC-CTL cell immunotherapy with KVLHSSLVCK as a target has potential curative effect on fusion protein therapy.
In some embodiments, the neoantigenic peptide is presented by HLA-A x 31:01 or HLA-A x 11:01.
In some embodiments, the neoantigenic peptide induces a cd8+ T cell to generate a cellular immune response following presentation by HLA-A x 31:01 or HLA-A x 11:01.
According to the embodiment of the invention, KVLHSSLVCK polypeptide is synthesized by a chemical synthesis mode, and the synthesized antigen peptide is dissolved by DMSO. Collecting peripheral blood of healthy donors positive for HLA-A.multidot.31:01/11:01, isolating mononuclear cells (PBMC), isolating cultured DCs in vitro, and adding synthesized neoantigenic peptides to stimulate DCs, finally allowing presented antigenic peptides to activate antigen-recognizable CD8+ T cells for maturation, and obtaining a polyclonal CTL killer cell population by expansion. As a result, the novel antigenic peptides provided by the invention can obviously induce the expansion of CD8+ T cells.
The embodiment of the invention also provides a development method of the new antigen peptide of t (8; 21) AML, which comprises the following steps:
s10, providing transcriptome high-throughput second-generation sequencing data of t (8; 21) AML cells;
s20, analyzing fusion mutation of leukemia related fusion genes according to the transcriptome high-throughput second-generation sequencing data, and predicting to obtain candidate fusion new antigens;
s30, comparing the candidate fusion neoantigen with normal proteins through a tumor specific antigen prediction screening platform, and screening to obtain the neoantigen peptide of the t (8; 21) AML.
In some embodiments, the specific steps of the opening method include:
s100, providing transcriptome high throughput second generation sequencing (Next Generation Sequencing, NGS) data of t (8; 21) AML cells and t (8; 21) AML patient primary tumor cells;
s200, filtering non-target sequences and low-quality sequences of NGS data by SOAP-nuke software, comparing reference genome sequences by STAR software, and detecting fusion mutation by adopting easy fuse software;
s300, translating the fusion mutation into a fusion protein sequence by using an in-house process, cutting the fusion mutation into candidate fusion new antigens with the length of 8-11 amino acids, comparing the candidate fusion new antigens with normal proteins (Uniprot database), and filtering out the candidate fusion new antigens with the normal protein sequence;
s400, a tumor specific antigen prediction screening platform GIANT is adopted to predict the candidate fusion neoantigen and HLA types (HLA-A11:01, HLA-A02:01 and HLA-A24:02) of Chinese high frequency and the corresponding HLA types of the t (8; 21) AML cells, and the t (8; 21) AML cell AE fusion neoantigen peptide is obtained by screening.
In some embodiments, the amino acid sequence of the neoantigenic peptide is as set forth in seq id No. 1: KVLHSSLVCK
The embodiment of the invention also provides a pharmaceutical composition, which comprises the neoantigenic peptide KVLHSSLVCK.
In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient.
In some embodiments, the pharmaceutical form of the pharmaceutical composition may be a vaccine.
The embodiment of the invention also provides application of the novel antigen peptide of T (8; 21) AML, namely application of the novel antigen peptide KVLHSSLVCK in preparing a vaccine specific to T (8; 21) AML antigen, or application in inducing generation of cytotoxic T cells specific to the novel antigen peptide, or application in preparing antigen presenting cells sensitized by the novel antigen peptide, but is not limited to the application.
The embodiment of the invention also provides application of the novel antigen peptide of T (8; 21) AML, namely application of the novel antigen peptide KVLHSSLVCK to development of TCR-T, CAR-T or CAR-NK products targeting the novel antigen peptide, but the novel antigen peptide is not limited to the development.
The embodiment of the invention also provides application of the novel antigen peptide of the t (8; 21) AML, and the novel antigen peptide KVLHSSLVCK is applied to development of a t (8; 21) AML diagnostic kit.
Whether AE can produce a neoantigen is unknown in the art, and no study involving AE neoantigen has been published at present. The invention finds AE new antigen KVLHSSLVCK through works such as sequencing, bioinformatics analysis and synthesis verification, and proves that the AE new antigen KVLHSSLVCK has immunogenicity, can stimulate new antigen specific CTL amplification in vitro, and has potential of being applied to treatment of t (8; 21) AML by specific CTL taking the AE new antigen as a target point. According to the invention, a KVLHSSLVCK peptide fragment is synthesized, and an antigen peptide-DC-CTL culture system is used, so that a new antigen KVLHSSLVCK generated by the AE fusion protein can promote CTL to effectively kill t (8; 21) AML cells, and the DC-CTL cell immunotherapy with KVLHSSLVCK as a target point is proved to have potential curative effects on t (8; 21) AML. The method of the invention firstly identifies that AE fusion protein can generate new antigen through data analysis and experimental verification, and AE fusion protein exists in all t (8; 21) AML patients. Thus, DC-CTL therapies targeting the novel antigenic peptides produced by AE can cover all t (8; 21) AML patients. The novel antigen peptide can be applied to the development of T (8; 21) AML pharmaceutical compositions, or can be applied to the preparation of more specific and effective polypeptide vaccines, or can be applied to the induction generation of specific cytotoxic T cells, and has good development and application prospects in the field of T (8; 21) AML specific immunotherapy.
The novel antigenic peptide of t (8; 21) AML and its use according to the invention are further illustrated by the following examples:
the reagents, methods and apparatus employed in the present invention are those conventional in the art unless otherwise indicated.
EXAMPLE 1 analysis of AE-generated tumor neoantigen
Fusion mutations were analyzed and predictive screening of fusion neoantigens by transcriptome high throughput second generation sequencing (Next Generation Sequencing, NGS) of t (8; 21) AML cells (cell line SNKO-1) and t (8; 21) AML patient primary tumor cells.
Firstly, filtering non-target sequences and low-quality sequences by SOAP-nuke software in NGS data, comparing reference genome sequences by STAR software, and then carrying out fusion mutation detection by adopting easy fuse software; then using an in-house process to translate the fusion mutation into a fusion protein sequence, cutting the fusion mutation into candidate fusion new antigens with the length of 8-11 amino acids, comparing the candidate fusion new antigens with normal proteins (Uniprot database), and filtering out the candidate fusion new antigens with the normal protein sequence; finally, the present example will employ an international leading tumor-specific antigen predictive screening platform GIANT developed based on AI technology to predict candidate fusion neoantigens with the corresponding HLA types of the Chinese high frequency HLA types (HLA-A11:01, HLA-A02:01 and HLA-A24:02) and t (8; 21) AML cell lines, and screen to obtain the sequence of t (8; 21) AML cell line AE fusion neoantigens. By bioinformatic analysis of tumor neoantigen, it was found that AE fusion proteins in both t (8; 21) AML cells (cell lines) and t (8; 21) AML patient primary tumor cells produced neoantigen KVLHSSLVCK and could be presented by HLA-A11:01 (as shown in Table 1)
TABLE 1 analysis of tumor neoantigens by AE
As can be seen from the above table, through the bioinformatics analysis of the new antigen of primary tumor cells of t (8; 21) AML cell lines SKNO-1 and t (8; 21) AML patients, AE fusion proteins were found to produce new antigen KVLHSSLVCK with stronger immunogenicity, affinity and presentation ability.
EXAMPLE 2 identification of AE-generated tumor neoantigens
The KVLHSSLVCK polypeptide was synthesized by chemical synthesis, and the synthesized antigenic peptide was dissolved in DMSO. Collecting peripheral blood of healthy donors positive for HLA-A 31:01/11:01, isolating mononuclear cells (PBMC), isolating cultured DCs in vitro, and adding the synthesized corresponding antigen peptide to stimulate DCs, finally enabling the presented antigen peptide to activate CD8+ T cells capable of recognizing antigens to mature, and obtaining a polyclonal CTL killer cell population through amplification, wherein the specific steps are as follows:
a. collecting peripheral blood of healthy volunteers according to HLA-A ligand corresponding to the screened new antigen or target cells, and separating and purifying PBMC by using a Ficoll density gradient centrifugation method;
b. culturing cells in a lymphocyte serum-free medium for 2 hours by an adherence separation method to obtain adherence mononuclear cells and suspension cells (80% are T cells);
c. sorting the suspension Cell fraction using human Naive cd8+ T Cell isolation kit (130-093-244, miltenyibiotec) to obtain Naive cd8+ T cells, and cryopreserving;
d. adherent monocytes were cultured for 6 days via a culture system containing 10ng/ml IL-4, 1,600IU/ml GM-CSF cytokine, and were mediated to differentiate into Mo-DC. On day 5 of differentiation, 10ug/ml of the pool of neoantigenic peptides generated by the corresponding AE was added to the medium to form DCs loaded with the corresponding polypeptide. As a control, no peptide pool loaded DCs were set. On day 6 of differentiation, addition of 500U/ml TNF alpha and 1000U/ml IFN gamma to polypeptide-loaded DCs and control cells promoted DC maturation;
e. resuscitating the Naive cd8+ T cells frozen in step c, activating with IFN- γ, and mixing with antigen peptide-loaded DC cells;
f. culturing the DC and T mixed cells with a culture system containing 20U/ml IL-2, 10ng/ml IL-7 and 10ng/ml IL-15 for 7-8 days;
g. the activation of T cells was determined by flow-through detection of CD137 and CD28 expression, the total amount of T cells was counted to determine whether or not expansion of T cells occurred, the IFN-gamma level of the culture broth was detected by Elisa, and the production of CTL was confirmed by combining the results of multiple indicators.
Finally, the present invention found that the neoantigenic peptides significantly induced expansion of cd8+ T cells (as in fig. 1A-B), and the flow-through results showed that activated cd8+ T cells (cd137+) accounted for about 45.86% of the total (fig. 1C), and the levels of infγ in the culture supernatants after mixing the effector and target cells were examined, and found that the infγ content of the added antigenic peptide group was significantly up-regulated compared to the control group (fig. 1D). Wherein A is the proportion of the AE new antigen KVLHSSLVCK added group flow detection CD8+T cells; b is cell count to detect the expansion condition of CD8+ T cells; c is the proportion of activation in the CD8+ T cells of the set KVLHSSLVCK added AE neoantigen; d is the content of IFN gamma in the detection culture solution of Elisa.
EXAMPLE 3 in vitro killing of t (8; 21) AML cells by neoantigen-specific CTLs
Taking t (8; 21) AML cell line SKNO-1 as a target cell, taking the obtained CTL as an effector cell, and setting the effective target ratio to be 5:1 and 1:1 in sequence. Using CytoToxThe non-radioactive cytotoxicity detection kit (G1780, promega) adopts an LDH method to detect the killing level of CTL on target cells, calculates the killing efficiency of the CTL, and shows that KVLHSSLVCK groups can generate very good specific killing (shown in figure 2).
In summary, the present invention provides a novel antigenic peptide of t (8; 21) AML and uses thereof. The present invention, by analyzing the RNA-seq data of t (8:21) AML cell lines and t (8:21) AML patient primary AML cells, finds that AE fusion proteins can produce neoantigen KVLHSSLVCK and can be presented by HLA-A.times.31:01 or HLA-A.times.11:01. By synthesizing KVLHSSLVCK peptide fragments and using an antigenic peptide-DC-CTL culture system, the invention discovers that a novel antigen KVLHSSLVCK generated by AE fusion protein can promote CTL to effectively kill t (8; 21) AML cells, and shows that DC-CTL cell immunotherapy with KVLHSSLVCK as a target has potential curative effects on t (8; 21) AML. The method of the invention firstly identifies that AE fusion protein can generate new antigen through data analysis and experimental verification, and AE fusion protein exists in all t (8; 21) AML patients. Thus, DC-CTL therapies targeting the novel antigenic peptides produced by AE can cover all t (8; 21) AML patients. Therefore, the novel antigen peptide can be applied to the development of T (8; 21) AML pharmaceutical compositions, or the preparation of more specific and effective polypeptide vaccines, or the induction of specific cytotoxic T cells, and has good development and application prospects in the field of T (8; 21) AML specific immunotherapy.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (10)
1. A novel antigen peptide of t (8; 21) AML, which is characterized in that the amino acid sequence of the novel antigen peptide is shown as SEQ ID NO. 1.
2. The neoantigenic peptide of t (8; 21) AML according to claim 1, wherein said neoantigenic peptide is presented by HLA-A x 31:01 or HLA-A x 11:01.
3. The neoantigenic peptide of T (8; 21) AML according to claim 2, which induces a cd8+ T cell to generate a cellular immune response after being presented by HLA-A x 31:01 or HLA-A x 11:01.
4. A method for developing a novel antigen peptide of t (8; 21) AML, comprising the steps of:
providing transcriptome high throughput second generation sequencing data for t (8; 21) AML cells;
analyzing fusion mutation of leukemia related fusion genes according to the transcriptome high-throughput second-generation sequencing data, and predicting to obtain candidate fusion new antigens;
and comparing the candidate fusion neoantigen with a normal protein through a tumor specific antigen prediction screening platform, and screening to obtain the neoantigen peptide of the t (8; 21) AML.
5. The method for developing a neoantigenic peptide for t (8; 21) AML according to claim 4, wherein the amino acid sequence of said neoantigenic peptide is as shown in SEQ ID NO. 1.
6. A pharmaceutical composition comprising the neoantigenic peptide of any one of claims 1-3.
7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient, which may be in a vaccine in pharmaceutically acceptable form.
8. Use of a neoantigenic peptide of T (8; 21) AML, characterized in that it is used for the preparation of a vaccine specific for T (8; 21) AML antigens, or for the induction of cytotoxic T cells specific for said neoantigenic peptide, or for the preparation of antigen presenting cells sensitized by said neoantigenic peptide, according to any one of claims 1 to 3.
9. Use of a neoantigenic peptide of T (8; 21) AML, characterized in that it is applied to the development of TCR-T, CAR-T or CAR-NK products according to any one of claims 1 to 3.
10. Use of a neoantigenic peptide of t (8; 21) AML, characterized in that it is applied to the development of a diagnostic kit for t (8; 21) AML according to any one of claims 1 to 3.
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