CN112301088A - Method for screening neoantigen or neoantigen coding sequence - Google Patents
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Abstract
The invention discloses a method for screening neoantigens or neoantigen coding sequences, which comprises the steps of inserting a neoantigen coding sequence to be screened into an original expression vector to obtain a recombinant expression vector, then transfecting immature DC cells, culturing the transfected DC cells to ensure that the DC cells are mature, expressing corresponding neoantigen epitope peptides on the surface of the mature DC cells, presenting the neoantigen epitope peptides to T cells by the mature DC cells, inducing the T cells to be activated into effector T cells, then co-culturing the effector T cells and tumor cells, and screening effector T cells with good tumor cell killing effect, thereby screening neoantigens with good immunogenicity, wherein the corresponding coding sequence is the screened neoantigen coding sequence with good immunogenicity. The method for screening the neoantigen or the neoantigen coding sequence can quickly screen the neoantigen with strong immunogenicity, and the corresponding wild type peptide has no immunogenicity, so that the neoantigen is the optimal choice of the vaccine.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for screening a neoantigen or a neoantigen coding sequence.
Background
The close relationship between tumorigenesis and gene mutation has been confirmed by many studies. Point mutations, fragment deletions and insertions of the relevant genes, which lead to codon synonymity, missense, termination or frameshifting, result in a change in the protein sequence or loss of the relevant function. Those abnormal proteins which are produced by mutation of tumor cells and which are capable of activating the immune system are called tumor neoantigens. Existing animal and clinical studies have shown that T cells activated by tumor neoantigens are capable of specifically killing tumor cells and producing an immunological memory effect, thereby partially or completely regressing tumors.
In theory, tumor antigens can be processed and presented by both "endogenous" and "exogenous" pathways. Endogenous antigen can be cleaved by proteasome in cells to form I type neoantigen epitope peptide which is short peptide with 8-11 amino acids length, then the short peptide is transported to endoplasmic reticulum by antigen processing related transporter, and is combined with MHC I (major histocompatibility complex I) molecules of endoplasmic reticulum to form stable complex pMHC (peptide-MHC), finally reaches the surface of cell membrane through Golgi apparatus, and is supplied to CD8+T cell recognition. Different from endogenous antigens, exogenous antigens enter acidic endosomes through endocytosis and are degraded into polypeptides with the length of 10-18 amino acids, namely II type neoantigen epitope peptides, polypeptides and peptides: the MHC II molecule complex competes for binding, expelling weakly bound peptides, which are subsequently transported to the cell surface by the MHC II molecule for CD4+T cell recognition. Meanwhile, the process of MHC molecule antigen presentation also has the condition of cross presentation, also called as non-classical antigen presentation path, namely that the polypeptide formed by degrading endogenous antigen is possible to be presented by MHC class II molecule, and the polypeptide formed by degrading exogenous antigen is possible to be presented by MHC class II moleculePolypeptides may also be presented by MHC class I molecules.
The screening process of the neoantigens mainly comprises sequencing, software prediction, ELISpot verification and the like, wherein a high-throughput sequencing technology is a main means for obtaining genome mutation information, and no software or experimental method can help us to quickly screen out the neoantigens which can be presented by tumor cells and have immunogenicity from sequencing data 100% accurately at present. Most of the existing prediction software for screening tumor neoantigens only has higher accuracy in predicting the affinity of the neoantigens and MHC class I molecules, but has a plurality of defects in predicting other aspects such as the rule that the antigens are sheared by proteasomes in cells, the affinity of the antigens and MHC class II, the affinity of pMHC and TAP, and the like. Therefore, although the conventional bioinformatics prediction method is rapid and high-throughput, the algorithm still needs to be continuously verified and perfected, and the accuracy of prediction in other aspects is improved. Meanwhile, other screening methods and steps are also needed to be matched, and the accuracy of the neonatal antigen screening is further improved.
Another classical method for screening neoantigens is to detect the affinity between tumor neoantigens and HLA tetramers in vitro by flow assay to determine the specificity of tumor neoantigens, and based on visual observation of the affinity, the results are accurate, but the cost is high and the time is long. Yet another commonly used means of validating neoantigens is to plate PBMC or mouse lymphocytes in vitro, stimulate these cells with a stimulator (a polypeptide containing the amino acid sequence of the neoantigen, i.e., a neoantigen peptide), and count the spots either directly under the microscope or by an ELISpot assay system, with 1 spot representing 1 active cell, thereby calculating the frequency of cells secreting the protein or cytokine. However, the ELISpot experiment can only verify the IFN-gamma secretion capability of the T cell activated by the polypeptide, and cannot accurately reflect whether the neoantigen peptide can activate the T cell to recognize and kill the tumor cell. In addition, a further verification means is to synthesize a neoantigen peptide, and then add the neoantigen peptide to the culture of DC to allow the antigen to be expressedThe DC presents the new antigen epitope peptide to activate the tumor specific T cell, so that the tumor is specifically killed. However, the neoantigenic peptides used in this validation method are almost all short peptides, and neither can their degradation rate in culture systems be evaluated, nor can they be validated by killing experiments for efficient presentation and activation of T cells. In addition, the verification needs to use specific HLA typed cells, namely, the one-to-one killing verification of the primary tumor cells and the corresponding T cells can not be realized. When the synthesized new antigen long peptide is used for screening new antigen, after the antigen presenting cell phagocytoses the long peptide, the antigen presenting cell presents the new antigen epitope peptide through MHC II (major histocompatibility complex) pathway, and CD4 is activated+A T cell; in the immunotherapy of tumor, tumor cells are mostly somatic cells, and peptide-MHC class I complex is presented on the cell surface, so that the screening of neoantigens by using long peptides has limitations.
These factors affect the accuracy of neoantigen screening, often leading to the failure to see widespread tumor regression in cancer patients receiving neoantigen vaccine therapy. In order to further improve the accuracy of screening neoantigens and activate the recognition of tumor cells by the body more effectively, we hope to combine the coding nucleotide sequences of multiple neoantigens together by means of gene design to form an artificial gene, i.e. a short gene tandem sequence (TMG), which can code a neoantigen containing a large amount of neoantigen epitope peptides. By transferring the artificial genes into antigen presenting cells (such as DC cells) and inducing the expression mode thereof, a large amount of new antigen epitope peptide is simultaneously presented to CD8 by MHC class I molecules+T cells induce the ability of the T cells to kill cells carrying the corresponding neoantigens, and then the neoantigens with good immunogenicity are screened by detecting the killing effect.
The killing of the target cells by the lymphocytes can be realized through the ways of secretion of cytokines, release of perforin and granzyme, apoptosis of target cells mediated by Fasl and the like, so that the killing of the target cells by the activated lymphocytes is detected, and compared with an ELISpot experiment for singly detecting IFN-gamma secreted by the lymphocytes, the immunogenicity of the neoantigen is more intuitively and accurately expressed.
Disclosure of Invention
Aiming at the defects in the prior art, the application provides a method for screening the neoantigen or the neoantigen coding sequence, the neoantigen is screened through an in vitro cell killing experiment, the neoantigen with strong immunogenicity is found as a candidate site of the vaccine by combining the experiment on the basis of software prediction, and the effectiveness of the tumor neoantigen vaccine for activating anti-tumor immune response is improved.
A method of screening for a neoantigen or a neoantigen coding sequence comprising the steps of:
(1) inserting the coding sequence of the new antigen to be screened into the original expression vector to obtain a recombinant expression vector,
(2) transfecting the immature DC cells with the recombinant expression vector obtained in the step (1),
(3) culturing the transfected DC cells to ensure that the DC cells are mature, expressing corresponding neoantigen epitope peptide on the surface of the mature DC cells,
(4) presenting the newborn epitope peptide to T cells by the DC cells matured in the step (3), inducing the T cells to be activated into effector T cells,
(5) co-culturing the effector T cells and the tumor cells in the step (4), and screening the effector T cells with good effect of killing the tumor cells, thereby screening the neoantigen with good immunogenicity, wherein the corresponding coding sequence is the screened neoantigen coding sequence with good immunogenicity.
The invention also provides a method for screening the neoantigen coding sequence, the neoantigen with good immunogenicity is screened according to the method for screening the neoantigen, and the corresponding coding sequence is the screened neoantigen coding sequence with good immunogenicity.
The new antigen coding sequence is used as gene sequence for coding new antigen with new antigen epitope. The neoantigen to be screened may be a short peptide of 8-11 amino acids (i.e., a neoepitope peptide) or a long peptide of longer amino acid length, such as a long peptide of 25 amino acids.
Preferably, 1-50 coding sequences of the neoantigen are inserted into the original expression vector in the step (1).
More preferably, 1-10 coding sequences of the neoantigen are inserted into the original expression vector in the step (1).
Preferably, a plurality of neoantigen coding sequences are inserted into the original expression vector in the step (1), and the neoantigen coding sequences and the head and tail neoantigen coding sequences are connected with the original expression vector through flexible linker coding sequences. Flexible linkers are typically amino acids with short side chains, such as glycine, serine, which are commonly used. The flexible linker coding sequence is used for being expressed together with the neoantigen coding sequence, so that the neoantigens in the expressed product are separated by the flexible linker, and the mutual influence is reduced.
The process of intracellular presentation of neoantigens by MHC molecules requires degradation by the proteasome into short fragments of peptide fragments, binding to the MHC molecules in the endoplasmic reticulum, and presentation to the cell surface via golgi apparatus. The flexible Linker (Linker) can separate different neoantigens, each neoantigen can be cut separately as much as possible during proteasome cutting, and the condition that the short peptide obtained by cutting contains amino acids of two adjacent neoantigens is reduced.
More preferably, the upstream end of the first neoantigen coding sequence is connected with a starting flexible linker coding sequence, an intermediate flexible linker coding sequence is connected between two adjacent neoantigen coding sequences, and the downstream end of the last neoantigen coding sequence is connected with a last flexible linker coding sequence, wherein the amino acid sequence coded by the starting flexible linker coding sequence is GGSGGGSGG, the amino acid sequence coded by the intermediate flexible linker coding sequence is GGSGGGGSGG, and the amino acid sequence coded by the last flexible linker coding sequence is GGSLGGGGSG.
Preferably, a plurality of new antigen coding sequences are inserted into the original expression vector, the recombinant expression vector corresponding to the new antigen with better immunogenicity is screened out, then the immunogenicity of the encoded new antigen is verified respectively aiming at the plurality of new antigen coding sequences in the recombinant expression vector, and the new antigen with good immunogenicity is screened out.
Preferably, the activated effector T cells of step (4) are expanded. And amplifying the activated effector T cells to obtain a large number of effector T cells so as to facilitate the next screening process.
Preferably, the original expression vector in step (1) has a kozak sequence in front of the site where the neoantigen coding sequence is inserted, and the base sequence of the kozak sequence is shown in SEQ ID No. 1. The kozak sequence can be combined with a translation initiation factor to mediate the translation initiation of mRNA with a 5' cap structure, so that the function of improving the gene expression level is achieved.
More preferably, the original expression vector in step (1) further has a ubiquitin protein coding gene sequence between the kozak sequence and the site where the neoantigen coding sequence is inserted, and the base sequence of the ubiquitin protein coding gene sequence is shown in SEQ ID No. 4. Ubiquitin protein coding gene sequence correspondingly codes ubiquitin protein (ubiquitin), and the ubiquitin protein can mediate tumor neoantigen to be degraded by protease.
Preferably, the original expression vector in step (1) further has a signal peptide coding sequence between the kozak sequence and the ubiquitin protein coding gene sequence, and the base sequence of the signal peptide coding sequence is shown in SEQ ID No. 2. The signal peptide (signal peptide) is correspondingly coded by the signal peptide coding sequence, so that the neoantigen can be mediated to enter the endoplasmic reticulum, and the probability of combination of the neoantigen epitope peptide and the MHC I molecule in the endoplasmic reticulum is improved.
For the recombinant expression vectors of the present application, the original expression vector can be used as long as it can be used to express the neoantigen. The general experiment is carried out in mammalian cells, so the original vector needs to be selected from vectors suitable for expression in mammalian cells, for example, the original expression vector can be pVAX1 plasmid, pcDNA3.1 plasmid, etc.
Experimental research shows that the kozak sequence is arranged in the original expression vector in front of the insertion site of the neoantigen coding sequence, so that the presentation amount of a compound of the neoantigen and MHC I (peptide-MHC I compound) and a compound of the neoantigen and MHC II (peptide-MHC II compound) on the cell surface of BMDCs can be improved. Compared with an uninserted original expression vector, the expression vector can improve the presenting amount of a compound of a neoantigen and MHC I (peptide-MHC I compound) and a compound of the neoantigen and MHC II (peptide-MHC II compound) on the cell surface of BMDCs. The insertion of a kozak sequence, a signal peptide coding sequence and a ubiquitin protein coding gene sequence can maximize the presentation amount of a composition (peptide-MHC I composition) of a neoantigen and MHC I on the surface of BMDCs cells; however, in this combination, the increased amount of complex of neoantigen and MHC II presented on the cell surface of BMDCs may be effective, but not effective.
The method for screening the neoantigen comprises the steps of transfecting immature DC cells with recombinant expression plasmids inserted with neoantigen coding sequences, and presenting neoantigen epitope peptides after the DC cells are mature so as to activate T cells.
The invention relates to a method for screening neoantigens or neoantigen coding sequences, which comprises the steps of calculating neoantigens with high affinity, large mutation ratio, non-functionality and non-toxicity and presented by MHC I molecules by using a letter analysis platform algorithm continuously optimized by verified experimental data from the source, transfecting immature DC cells by constructing optimized recombinant expression plasmids, converting the unknown property of exogenously added neoantigens into endogenous addition expression, processing and presenting the neoantigens in the DC cells, activating corresponding T cells, carrying out a killing experiment with tumor cells of the same patient or animal sources, and screening the neoantigens with high affinity, large mutation ratio, non-functionality and non-toxicity and presented by the MHC I molecules and capable of activating T cells with specific tumor cell killing functions. Therefore, the neoantigen with strong immunogenicity can be obtained by rapid screening, and the corresponding wild-type peptide has no immunogenicity, and the neoantigen is the optimal choice for the neoantigen tumor vaccine. Correspondingly, the screened new antigen coding sequence can be applied to different forms of tumor vaccines such as polypeptide vaccines, mRNA vaccines, DNA vaccines and cell vaccines.
Drawings
FIG. 1 is a schematic diagram showing the structure of a recombinant expression vector for screening a neoantigen after insertion of a neoantigen coding sequence, wherein a is pVAX1-kozak-sgub-peptide, sgub indicates: signal peptide-ubiquitin; peptide represents: a neoantigen coding sequence; panel b shows pVAX 1-peptide.
FIG. 2 is a diagram of the immunization principle for screening neoantigens.
FIG. 3 is a diagram showing the gene structure design and detection results of a neoantigen, wherein, a is the gene design for screening the neoantigen, and M, KM, KUM and KSUM respectively represent the Mutation, kozak-ubiquitin-Mutation, and kozak-signal peptide-ubiquitin-Mutation; panel b shows the detection of peptide-MHC I complex on the surface of gene-transfected BMDC cells; panel c shows the detection of peptide-MHC II complex on the surface of gene-transfected BMDC cells.
FIG. 4 is a graph showing the results of primary screening of neoantigens, in which TMG1-5 encodes 10 neoantigens, TMG6 encodes 9 neoantigens, and the data of the graph is mean. + -. s.d.
Fig. 5 is a graph showing the results of screening for neoantigens, in which a: 10 neo-antigens mut1, mut2, mut3, mut4, mut5, mut6, mut7, mut8, mut9 and mut10 which constitute TMG2 induce the killing efficiency of T cells on B16F10 cells; and (b) figure: 10 neo-antigens mut11, mut12, mut13, mut14, mut15, mut16, mut17, mut18, mut19 and mut20 which constitute TMG3 induce the killing efficiency of T cells on B16F10 cells; and (c) figure: 10 neo-antigens mut21, mut22, mut23, mut24, mut25, mut26, mut27, mut28, mut29 and mut30 which constitute TMG3 induce the killing efficiency of T cells on B16F10 cells; FIG. d: wild-type antigenic peptides wt6, wt7, wt8, wt11, wt16, wt17, wt18, wt26, wt27 and wt28 induced the killing efficiency of T cells on B16F10 cells.
FIG. 6 is a graph showing the results of flow-testing neoantigens for their promotion of proliferation of CD4+ T and CD8+ T cells.
FIG. 7 is a graph showing the results of primary screening of neoantigens, in which TMG7-12 encodes 10 neoantigens, respectively, and the data of the curve is mean. + -. s.d.
Fig. 8 is a graph showing the results of screening for neoantigens, in which a: 10 neoantigens mut61, mut62, mut63, mut64, mut65, mut66, mut67, mut68, mut69 and mut70 which form TMG7 induce the killing efficiency of T cells on tumor cells; and (b) figure: 10 neoantigens mut71, mut72, mut73, mut74, mut75, mut76, mut77, mut78, mut79 and mut80 which form TMG8 induce the killing efficiency of T cells on tumor cells; and (c) figure: wild-type antigenic peptides wt61, wt62, wt63, wt64, wt65, wt66, wt67, wt68, wt69, wt70, wt71, wt76 and wt79 induce the killing efficiency of T cells to tumor cells.
Detailed Description
Example 1
(1) Gene design and synthesis for screening neoantigens
First, we designed genes for screening neoantigens, and added kozak, signal peptide and ubiquitin genes before the DNA sequence encoding neoantigens (neoantigen encoding sequence), respectively. Wherein the kozak sequence can be combined with a translation initiation factor to mediate the translation initiation of mRNA with a 5' cap structure, thereby playing a role in improving the gene expression level. As the assembly of the polypeptide and the MHC I-class molecule occurs in the endoplasmic reticulum, in order to improve the collision probability of the polypeptide and the MHC I-class molecule, a signal peptide and a ubiquitin gene are added in front of a neoantigen coding sequence to promote the amino acid sequence obtained by transcription and translation to be cut by a proteasome near the endoplasmic reticulum, and the short peptide obtained after cutting can be transported to the surface of a cell membrane by the MHC I-class molecule. signal peptide mediates the new antigen to enter endoplasmic reticulum, and the probability of combining the antigen and MHC I molecules in the endoplasmic reticulum is improved; ubiquitin mediates degradation of tumor neoantigens by proteases. We loaded these different genetic elements on the pVAX1 plasmid (fig. 1a), while we loaded the neoantigen-encoding gene only on the pVAX1 plasmid (fig. 1b) for subsequent cell transfection.
The group pVAX1-mutation (pVAX1-M) indicates that only the neoantigen-encoding sequence was inserted into the original vector, wherein mutation indicates the neoantigen-encoding sequence (abbreviated as M, the same below).
The pVAX1-Kozak-mutation (pVAX1-KM) group shows that the coding sequence of the neoantigen is inserted after the insertion of the Kozak base sequence in the original vector.
The pVAX1-Kozak-Ubiquitin-mutation (pVAX1-KUM) group shows that the coding sequence of the neoantigen is inserted after the Kozak base sequence and the Ubiquitin base sequence are inserted into the original vector.
The pVAX1-Kozak-signal peptide-Ubiquitin-mutation (pVAX1-KSUM) group shows that a Kozak base sequence, a signal peptide base sequence, and a Ubiquitin base sequence were inserted into the original vector, and then a neoantigen coding sequence was inserted.
Kozak base sequence: GCCGCCACC are provided.
Signal peptide amino acid sequence: RVTAPRTLILLLSGALALTETWAGSM are provided.
Signal peptide base sequence:
CGGGTCACGGCGCCCCGAACCCTCATCTTGCTGCTCTCGGGGGCCCTGGCCCTGACCGAGACCTGGGCGGGCTCCATG。
ubiquitin amino acid sequence:
QIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRG。
ubiquitin base sequence:
CAGATCTTCGTGAAGACCCTGACCGGCAAGACCATCACCCTAGAGGTGGAGCCCAGTGACACCATCGAGAACGTGAAGGCCAAGATCCAGGATAAAGAGGGCATCCCCCCTGACCAGCAGAGGCTGATCTTTGCCGGCAAGCAGCTGGAAGATGGCCGCACCCTCTCTGATTACAACATCCAGAAGGAGTCAACCCTGCACCTGGTCCTTCGCCTGAGAGGTGGC。
a base sequence of kozak-signal peptide-ubiquitin-mutation6(KSUM 6):
GCTAGCGCCGCCACCATGCGGGTCACGGCGCCCCGAACCCTCATCTTGCTGCTCTCGGGGGCCCTGGCCCTGACCGAGACCTGGGCGGGCTCCATGCAGATCTTCGTGAAGACCCTGACCGGCAAGACCATCACCCTAGAGGTGGAGCCCAGTGACACCATCGAGAACGTGAAGGCCAAGATCCAGGATAAAGAGGGCATCCCCCCTGACCAGCAGAGGCTGATCTTTGCCGGCAAGCAGCTGGAAGATGGCCGCACCCTCTCTGATTACAACATCCAGAAGGAGTCAACCCTGCACCTGGTCCTTCGCCTGAGAGGTGGCCACTGTCACTGGAACGATCTGGCCGTGATCCCTGCCGGCGTGGTGCACAACTGGGACTTCGAGCCCAGGAAGGTGTAGAAGCTT。
a base sequence of kozak-signal peptide-ubiquitin-mutation7(KSUM 7):
GCTAGCGCCGCCACCATGCGGGTCACGGCGCCCCGAACCCTCATCTTGCTGCTCTCGGGGGCCCTGGCCCTGACCGAGACCTGGGCGGGCTCCATGCAGATCTTCGTGAAGACCCTGACCGGCAAGACCATCACCCTAGAGGTGGAGCCCAGTGACACCATCGAGAACGTGAAGGCCAAGATCCAGGATAAAGAGGGCATCCCCCCTGACCAGCAGAGGCTGATCTTTGCCGGCAAGCAGCTGGAAGATGGCCGCACCCTCTCTGATTACAACATCCAGAAGGAGTCAACCCTGCACCTGGTCCTTCGCCTGAGAGGTGGCGACCCTTTCCCAAACCTGAACCCCGCCCCTGCCCCTCCCCTGGCTTGTAACCTGACCCTGGAGGACTTCTACGGCTAGAAGCTT。
a base sequence of kozak-signal peptide-ubiquitin-mutation8(KSUM 8):
GCTAGCGCCGCCACCATGCGGGTCACGGCGCCCCGAACCCTCATCTTGCTGCTCTCGGGGGCCCTGGCCCTGACCGAGACCTGGGCGGGCTCCATGCAGATCTTCGTGAAGACCCTGACCGGCAAGACCATCACCCTAGAGGTGGAGCCCAGTGACACCATCGAGAACGTGAAGGCCAAGATCCAGGATAAAGAGGGCATCCCCCCTGACCAGCAGAGGCTGATCTTTGCCGGCAAGCAGCTGGAAGATGGCCGCACCCTCTCTGATTACAACATCCAGAAGGAGTCAACCCTGCACCTGGTCCTTCGCCTGAGAGGTGGCCAGGGCTACCACCAGCTGTGTCACACACCTCACATCGGCAGCAGCGTGATCGATAGCGACGAGAAGTGGCTGTGTTAGAAGCTT。
wherein, the mutation6, mutation7 and mutation8 are three coding sequences of the neoantigen to be screened.
(2) Flow measurement of MHC I and MHC II related polypeptide amount presented on BMDC cell surface
Transfecting a plasmid loaded with a neoantigen encoding gene in a BMDC cell, collecting the BMDC cell presenting the neoantigen after 24 hours, and centrifuging at 1800rpm at 4 ℃ for 5 min. After each centrifugation, ensuring that cell precipitation can be seen by naked eyes, and centrifuging again if the cell precipitation can not be seen;
b. removing supernatant by suction, adding PBS and mixing, centrifuging at 1800rpm at 4 deg.C for 5min, repeating for 2 times;
c. removing supernatant, adding anti-mouse peptide-MHC I/II antibody diluted by PBS, mixing, and incubating at 4 deg.C for 30 min;
d. adding PBS and mixing, centrifuging at 1800rpm at 4 deg.C for 5min, repeating for 2 times;
e. removing the supernatant by suction, adding a Cy 3-goat anti-mouse IgG antibody diluted by PBS into each tube, uniformly mixing, and incubating for 30min at 4 ℃;
f. adding PBS and mixing, centrifuging at 1800rpm at 4 deg.C for 5min, repeating for 2 times;
g. absorbing and removing supernatant, adding 500 mu L PBS into each tube, mixing uniformly, transferring into a flow tube, keeping at 4 ℃ in a dark place until the tube is mounted;
h. and detecting the sample on a flow type computer.
The specific principle that pVAX1 plasmid carrying corresponding neoantigen genes is transfected in immature DC cells, the neoantigen epitope peptide is presented to T cells after the DC cells are matured for 24 hours, the T cells are induced to be activated into effector T cells, and when the effector T cells and tumor cells are cultured together, if the neoantigen epitope peptide which can be recognized by the T cells is presented on the surface of the tumor cells, the tumor cells can be killed by the T cells is shown in figure 2.
As shown in FIG. 3, the peptide-MHC I complexes of the pVAX1-kozak-signal peptide-ubiquitin-mutation (pVAX1-KSUM) group presented the highest amount on the cell surface of BMDCs relative to the other two gene designs, whereas the peptide-MHC II complexes of this group presented relatively less on the cell surface of BMDCs, but higher than the pVAX1-mutation (pVAX1-M) group. The amount of peptide-MHC II complexes presented on the cell surface of BMDCs was the greatest in the pVAX1-kozak-mutation (pVAX1-KM) group relative to the other three gene designs, whereas the amount of peptide-MHC I complexes presented on the cell surface of BMDCs was relatively small but higher than in the pVAX1-mutation (pVAX1-M) group.
Example 2
(1) Gene design and synthesis of short gene tandem sequence (TMG)
We inoculated mice subcutaneously on the right side with 2X 105B16F10 cells, mouse tail vein inoculation 1X 105Mouse subcutaneous melanoma tissue and lung metastatic melanoma tissue were obtained from individual B16F10 cells, respectively. We sequenced the whole exome and transcriptome of subcutaneous melanoma, lung metastatic melanoma and B16F10 cells. The FastQC software filters the poor quality data in the raw data, the BWA software compares the sequencing data with the mouse reference gene mm10 downloaded on the ensembl, and the mutect1, strelska, varscan and sniper in the GATK software analyze the somatic mutations. In addition, we also analyzed copy number mutations by CNVkit, FREEC and PyLOH software. The low quality mutations in the mutation sites were removed and the remaining mutations served as somatic mutation sites for subsequent analysis. Screening RNA reads greater than 5, Variant Allele Frequency (VAF)>0.1 of the neoantigen, PSSMHCpan, netMHC, netMHCpan and pickpocket software predict the affinity of the neoantigen to MHC class I molecules (H-2Kb, H-2Db), IEDB, netMHCII and netMHCIIpan software predict the avidity of the neoantigen to MHC class II molecules (H-2Ab)And, IEDB predicts the immunogenicity of neoantigens. According to the results predicted by the software, 59 neoantigens were synthesized in 6 TMGs (tables 1-6), respectively, the start linker (start linker sequence) of each TMG: GGSGGGSGG, midle linker (intermediate linker sequence): GGSGGGGSGG, end linker (end linker): GGSLGGGGSG, designing genes according to the preference of mouse-derived codons, synthesizing the gene sequence by Nanjing Kingsler Biotech Co., Ltd, and loading the gene sequence on pcDNA3.1 plasmid. Wherein TMG1, TMG2, TMG3, TMG4 and TMG5 encode 10 neoantigens, TMG6 encodes 9 neoantigens, each neoantigen comprises 25 amino acids, and the mutation site is located in the middle of the neoantigen.
TABLE 1 neoantigens of composition TMG1
TABLE 2 neoantigens of composition TMG2
| Gene | Transcript | Mutation site | Peptide fragment initiation | Peptide termination |
| Ap5b1 | ENSMUST00000096318 | K624T | 612 | 636 |
| Def8 | ENSMUST00000065534 | R255G | 243 | 267 |
| Hccs | ENSMUST00000033717 | R199T | 187 | 211 |
| Lrp10 | ENSMUST00000022782 | T190P | 178 | 202 |
| Mtf2 | ENSMUST00000081567 | D139G | 127 | 151 |
| Nacc1 | ENSMUST00000001975 | A434G | 422 | 446 |
| Nfrkb | ENSMUST00000086167 | S900R | 888 | 912 |
| Piga | ENSMUST00000033754 | K88N | 77 | 101 |
| Ttll12 | ENSMUST00000016901 | Q394R | 382 | 406 |
| Wdr82 | ENSMUST00000020490 | I221L | 209 | 233 |
TABLE 3 neoantigens of composition TMG3
TABLE 4 neoantigens of composition TMG4
| Gene | Transcript | Mutation site | Peptide fragment initiation | Peptide termination |
| Acot2 | ENSMUST00000021649 | L278R | 266 | 290 |
| B3galt6 | ENSMUST00000052185 | R228L | 216 | 240 |
| Ddx23 | ENSMUST00000003450 | V602A | 590 | 614 |
| Hipk1 | ENSMUST00000029438 | E413G | 401 | 425 |
| Kif24 | ENSMUST00000108055 | S594I | 582 | 606 |
| Klhl26 | ENSMUST00000066597 | E487A | 475 | 499 |
| Mta1 | ENSMUST00000009099 | P547L | 535 | 559 |
| Osbpl3 | ENSMUST00000114468 | F877L | 865 | 886 |
| Wdr5b | ENSMUST00000042203 | A311T | 299 | 323 |
| Zscan26 | ENSMUST00000032820 | K290T | 278 | 302 |
TABLE 5 neoantigens of composition TMG5
| Gene | Transcript | Mutation site | Peptide fragment initiation | Peptide termination |
| Ddx19b | ENSMUST00000040241 | K176T | 164 | 188 |
| Dmxl1 | ENSMUST00000041772 | N2558S | 2546 | 2570 |
| Dock9 | ENSMUST00000040700 | V1877M | 1865 | 1889 |
| Fam207a | ENSMUST00000045454 | S168I | 156 | 180 |
| Itsn2 | ENSMUST00000062580 | S1551R | 1539 | 1563 |
| Jmy | ENSMUST00000065537 | S860C | 848 | 872 |
| Smarcb1 | ENSMUST00000000925 | P45S | 33 | 57 |
| Tbc1d4 | ENSMUST00000162617 | V1112G | 1100 | 1124 |
| Tmem39b | ENSMUST00000102588 | A131P | 119 | 143 |
| Wdr13 | ENSMUST00000033506 | S460I | 448 | 472 |
TABLE 6 neoantigens of composition TMG6
| Gene | Transcript | Mutation site | Peptide fragment initiation | Peptide termination |
| Brca2 | ENSMUST00000044620 | K1997N | 1986 | 2010 |
| Dcaf15 | ENSMUST00000041367 | W111G | 99 | 123 |
| Ddit4l | ENSMUST00000053855 | G163A | 151 | 175 |
| Fzd7 | ENSMUST00000114246 | G304A | 292 | 316 |
| Il3ra | ENSMUST00000090591 | R310Q | 298 | 322 |
| Lyst | ENSMUST00000110559 | Q3359H | 3348 | 3372 |
| Mast4 | ENSMUST00000167058 | K1447T | 1435 | 1459 |
| Mvk | ENSMUST00000112239 | G321W | 309 | 333 |
| Rassf7 | ENSMUST00000046890 | S90R | 79 | 103 |
(2) Screening of candidate tumor neoantigen TMG
Transfecting BMDC (dendritic cells derived from mouse bone marrow) with pcDNA3.1 plasmid encoding TMG, centrifuging after 12h to remove supernatant, and suspending the cells by complete culture medium with RPIM-1640 containing 10% Gibco serum;
b. mixing spleen lymphocytes and BMDC cells according to the ratio of 10: 1, and culturing for 12 h;
B16F10 cells at 5X 10 per well3Plating in 96-well plate for overnight culture;
d. activated spleen lymphocytes are plated in a 96-well plate according to different proportions and are cultured for 8 hours together with B16F10 cells;
e. removing culture medium in the hole, and washing for 3 times by PBS;
f. add 100. mu.L serum-free RPIM-1640 medium containing MTT (1mg/mL) to each well;
g.4h later, removing culture medium from each well, adding 100 mu L DMSO, shaking at 37 ℃ for 10-20min to fully dissolve formazan, reading a plate by using a microplate reader, calculating the killing rate of immune cells to tumor cells,
percent killing ═ percent (control mean a value-experimental mean a value)/control mean a value × 100%.
The genes coding for 10 neoantigens are linked in a TMG through a linker (start linker): GGSGGGSGG, midle linker): GGSGGGGSGG and end linker): GGSLGGGGSG to carry out the immunogenicity screening of the neoantigens, which can help us to improve the efficiency of the screening and eliminate the neoantigens without immunogenicity. From the results of the first screening of neoantigens (fig. 4), the killing of B16F10 cells by TMG 1-activated spleen lymphocytes increased with the increase of the effective target ratio of activated spleen lymphocytes to B16F10, and TMG 6-activated spleen lymphocytes also showed a similar trend. However, TMG 1-activated spleen lymphocytes were less toxic to B16F10 cells than TMG2, TMG3 and TMG 4-activated spleen lymphocytes when the effective target ratio was less than 25: 1. When the effective target ratio is less than 25: 1, the killing ratio of the spleen lymphocytes activated by TMG2, TMG3 and TMG4 to the B16F10 cells ranges from 14.1% to 29.5%, which is significantly higher than the spleen lymphocytes activated by the TMG1, TMG5, TMG6 and PBS groups.
Example 3
After determining the TMG sequence with stronger immunogenicity, we further screened the neoantigens encoded on the TMG one by one, and loaded the neoantigen encoding genes, i.e. mutation genes, on the pVAX1-KSU plasmid respectively, wherein the mutation genes encode the neoantigens with 25 amino acid lengths, and the mutation sites are usually in the middle of the sequence.
(1) Gene construction for screening candidate neoantigens and corresponding wild-type antigenic peptides
Based on the existing kozak-signal peptide-ubiquitin (KSU) gene, a gene encoding a neoantigen (mutation, M) or a wild-type antigen peptide (wild type, WT) is added to the 3' end of the gene by PCR. We packaged these different genetic elements on the pVAX1 plasmid for subsequent cell transfection.
(2) Screening of candidate tumor neoantigens
Transfecting pVAX1 plasmid loaded with a neoantigen encoding gene in BMDC cells (mouse bone marrow-derived dendritic cells), centrifuging after 12h to remove supernatant, and suspending the cells by complete culture medium with RPIM-1640 containing 10% Gibco serum;
b. mixing spleen lymphocytes and BMDC cells according to the ratio of 10: 1, and culturing for 12 h;
B16F10 cells at 5X 10 per well3Plating in 96-well plate for overnight culture;
d. activated spleen lymphocytes are plated in a 96-well plate according to different proportions and are cultured for 8 hours together with B16F10 cells;
e. removing culture medium in the hole, and washing for 3 times by PBS;
f. add 100. mu.L serum-free RPIM-1640 medium containing MTT (1mg/mL) to each well;
g.4h later, removing culture medium from each well, adding 100 mu L DMSO, shaking at 37 ℃ for 10-20min to fully dissolve formazan, reading a plate by using a microplate reader, calculating the killing rate of immune cells to tumor cells,
percent killing ═ percent (control mean a value-experimental mean a value)/control mean a value × 100%.
(3) Flow detection of proliferation of spleen lymphocytes induced by candidate neoantigen
a. BMDC cells presenting the neoantigen were collected and T cells were stimulated and centrifuged at 1800rpm at 4 ℃ for 5 min. After each centrifugation, ensuring that cell precipitation can be seen by naked eyes, and centrifuging again if the cell precipitation can not be seen;
b. removing supernatant by suction, adding PBS and mixing, centrifuging at 1800rpm at 4 deg.C for 5min, repeating for 2 times;
c. removing supernatant by suction, adding FITC-CD4/CD8a staining antibody diluted by PBS, mixing uniformly, and incubating for 30min at 4 ℃ in the dark;
d. adding PBS and mixing, centrifuging at 1800rpm at 4 deg.C for 5min, repeating for 2 times;
e. absorbing and removing supernatant, adding 500 mu L PBS into each tube, mixing uniformly, transferring into a flow tube, keeping at 4 ℃ in a dark place until the tube is mounted;
f. and detecting the sample on a flow type computer.
Based on the previous TMG screening, 30 new antigens coded by TMG2, TMG3 and TMG4 were individually subjected to immunogenicity verification, and the results are shown in FIG. 5. mut1-10 is 10 neoantigens that make up TMG2, where the spleen lymphocytes activated by mut6, mut7 and mut8 had a stronger cytotoxic effect on B16F10 cells than the other 7 neoantigens. mut11-20 is the 10 neoantigens that make up TMG3, with spleen lymphocytes activated by mut11, mut16, mut17 and mut18 having a stronger cytotoxic effect on B16F10 cells than the other 6 neoantigens. mut21-30 is the 10 neoantigens that make up TMG4, of which mut26, mut27 and mut28 activated spleen lymphocytes had a stronger cytotoxic effect on B16F10 cells than the other 7 neoantigens. Therefore, by screening the neoantigens one by one, we obtained the 10 neoantigens with stronger immunogenicity: mut6, mut7, mut8, mut11, mut16, mut17, mut18, mut26, mut27, and mut 28.
Furthermore, we also performed immunogenicity validation on their corresponding wild-type antigenic peptides, as shown in fig. 5(d) and table 7. The results show that the 10 wild-type antigenic peptides of wt6, wt7, wt8, wt11, wt16, wt17, wt18, wt26, wt27 and wt28 have only weak cytotoxic effect on B16F10 cells and some or even no cytotoxic effect on B16F10 cells, such as wt17 and wt18, compared with the corresponding neoantigen and PBS group.
TABLE 7 comparison of killing rates of T cells induced by the selected neoantigen and its wild-type antigen peptide on B16F10 cells
Flow assay results of 10 neoantigens activating spleen-derived T cells are shown in FIG. 6, and only transfection reagents
Treated BMDC cell-activated T cells served as control group, PHA-treated BMDC cell-activated T cells served as positive control. Compared with the control group, 10 new antigens obtained by experimental screening can activate CD4+T and CD8+T cells proliferate to different extents, CD4+The more T cells proliferate, CD8+The higher the rate of T cell proliferation.
Example 4
(1) Gene design and synthesis of human-derived short gene tandem sequence (TMG)
The ethical lot number of the experiment: 2017KY 017; the ethical committee of the medical science of people hospitals in Zhejiang province.
We took patient tumor tissue and PBMCs for whole exome and transcriptome sequencing. WES sequenced fastq data was quality controlled using Fastqc and fastqsta and data filtered using trimmatic-0.36. The filtered high quality data was aligned to hg38(GRCh38) version of the human reference genome using bwa to generate bam files and sequence alignment optimization, repeated reads labeling, alignment quality correction and indel local re-alignment using software Picard. And on the other hand, inputting the filtered fastq file into an iNeo-HLA module for HLA typing identification of the sample. Samples were then analyzed for point mutations and indel mutations using Mutec 1 v 1.1.7, Strelka v 1.0.11, and Varscan v2.4.1, and high quality mutations were retained after mutation quality evaluation using iNeo-Mut. After prediction of neoantigens based on amino acid changes caused by mutations, the affinity of neoantigens to the identified HLA typing was predicted using iino _ Pred and NetMHCIIPan.
RNA-Seq generated fastq data quality control was also performed using Fastqc and Fastqstat and data filtering was performed using Trimmomatic-0.36. The filtered data were aligned using STAR to hg38(GRCh38) version of the human reference genome to generate bam files, which were then similarly post-aligned with Picard. And (4) after the comparison is finished, calculating the expression quantity by using htseq-count, and simultaneously, quantifying HLA by using a quantification program in iNeo-HLA according to the typing identification result of WES.
The epitope result with affinity is sorted out according to the above results, then we analysis generates key information and RNA-Seq analysis generates key information to be summarized, and according to the results predicted by software, 60 new antigens are respectively synthesized in 6 TMGs (as shown in tables 8-13, wherein part of the mutant peptide fragments with public transcript information gives the start and stop positions of the mutant peptide fragment, and the mutant peptide fragments without public transcript information gives the specific mutant peptide fragment sequence), and each TMG includes but is not limited to start linker (start connecting sequence): GGSGGGSGG, midle linker (intermediate linker sequence): GGSGGGGSGG, end linker (end linker): GGSLGGGGSG, designing gene according to the preference of human codon, synthesizing the gene sequence by Nanjing Kingsler Biotech Co., Ltd, and loading the gene sequence on pcDNA3.1 plasmid. Wherein TMG7, TMG8, TMG9, TMG10, TMG11 and TMG12 encode 10 neoantigens, each of which comprises 15-30 amino acids, and the mutation site is located in the middle of the sequence.
TABLE 8 neoantigens of composition TMG7
| Gene | Transcript | Mutation site | Mutant peptide fragments | Peptide fragment initiation | Peptide termination |
| SMAP2 | - | p.E120fs | KWKRGSEPVPEKKIG | - | - |
| ULK4 | - | p.K593fs | KGELIYLVATQEEKKKEP | - | - |
| RBMX | ENST00000570135 | p.G23V | - | 16 | 32 |
| CNOT3 | - | p.S242fs | LDLEDIPQALVATSPSQPQPHGKK | - | - |
| RNPC3 | - | p.E130fs | FSFKFMPQVYVPTTFQHNPSKH | - | - |
| ANKRD12 | - | p.E721fs | TEDLFLNMEHESLTLEKKIKIGKKHQR | - | - |
| AKR1C1 | - | p.A245fs | KPNSPVLLEDPVLCALAKKAQANPSP | - | - |
| AKR1C1 | - | p.A245fs | KAQANPSPDCPALPATAWG | - | - |
| MKL1 | ENST00000355630 | p.M847I | - | 833 | 859 |
| DYSF | ENST00000258104 | p.V891L | - | 887 | 903 |
TABLE 9 neoantigens of composition TMG8
TABLE 10 Novacntigens of composition TMG9
TABLE 11 neoantigens of composition TMG10
| Gene | Transcript | Mutation site | Mutant peptide fragments | Peptide fragment initiation | Peptide termination |
| STOML2 | ENST00000356493 | p.L12R | - | -2 | 26 |
| GLUD2 | ENST00000328078 | p.R300G | - | 291 | 312 |
| PHRF1 | ENST00000264555 | p.P592A | - | 587 | 607 |
| USP31 | ENST00000219689 | p.K898M | - | 884 | 912 |
| DIAPH2 | ENST00000373054 | p.H175L | - | 167 | 182 |
| TTR | ENST00000237014 | p.V114L | - | 105 | 129 |
| LAMP1 | ENST00000332556 | p.L179I | - | 165 | 192 |
| ITPR1 | ENST00000354582 | p.T2348P | - | 2336 | 2365 |
| ANKRD9 | ENST00000286918 | p.S19A | - | 10 | 28 |
| DPP7 | ENST00000371579 | p.F53V | - | 41 | 68 |
TABLE 12 Novacntigens of composition TMG11
TABLE 13 neoantigens of composition TMG12
| Gene | Transcript | Mutation site | Mutant peptide fragments | Peptide fragment initiation | Peptide termination |
| CYP3A7-CYP3A51P | ENST00000620220 | p.R478S | - | 467 | 494 |
| MCPH1 | ENST00000344683 | p.Q562P | - | 555 | 570 |
| NBPF12 | ENST00000611443 | p.V943L | - | 928 | 950 |
| HYKK | ENST00000388988 | p.D57E | - | 43 | 72 |
| HEATR5A | ENST00000543095 | p.K125N | - | 113 | 138 |
| SUSD3 | ENST00000375472 | p.S135C | - | 132 | 151 |
| MTCH2 | ENST00000302503 | p.R287Q | - | 279 | 295 |
| PIK3R5 | ENST00000447110 | p.S836P | - | 833 | 853 |
| SAMD9 | ENST00000379958 | p.P929L | - | 917 | 943 |
(2) Screening of candidate tumor neoantigen TMG
a. Attaching PBMC of the patient to the wall for 2 hours under the condition of no plasma culture medium, taking supernatant without attaching to the wall as T cells, and continuously culturing the T cells;
DC cell: scraping off the adherent cells, counting, re-plating according to the counting density, and transfecting the DC cells with pcDNA3.1 plasmid for encoding TMG;
DC cells plus TNF-a + IL-1 β + IL-6+ PGE2 to stimulate maturation;
c.1/2 DC: t is as follows 1: 10 for 5-7 days;
d.1/2 DC: t is as follows 1: 10 for 5-7 days;
e. counting and survival rate of a T cell culture bottle, and harvesting T cells;
f. treating Tumor tissue of a patient into single cell suspension according to the instruction of a Tumor Dissociation Kit human of the Kit, and culturing when the Tumor cell grows to 1 × 107At a rate of 5X 10 per hole3Plating in 96-well plate for overnight culture;
g. laying the activated T cells into a 96-well plate according to different proportions, and co-culturing single cells of tumor tissues of a patient for 8 hours;
h. removing culture medium in the hole, and washing for 3 times by PBS;
f. add 100. mu.L serum-free RPIM-1640 medium containing MTT (1mg/mL) to each well;
g.4h later, removing culture medium from each well, adding 100 mu L DMSO, shaking at 37 ℃ for 10-20min to fully dissolve formazan, reading a plate by using a microplate reader, calculating the killing rate of immune cells to tumor cells,
percent killing ═ percent (control mean a value-experimental mean a value)/control mean a value × 100%.
The genes coding for 10 neoantigens are linked in a TMG through a linker (start linker): GGSGGGSGG, middle linker): GGSGGGGSGG and end linker): GGSLGGGGSG, so as to carry out the immunogenicity screening of the neoantigens, which can help us to improve the efficiency of the screening and eliminate the neoantigens without immunogenicity. From the results of the first screening of neoantigens (figure), the killing of tumor cells by TMG 7-activated tumor-specific T cells increases with the increase of the effective target ratio of the activated tumor-specific cells to the tumor cells, and TMG 8-activated T cells also show a similar trend, and when the effective target ratio is less than 15:1, the toxicity of TMG 8-activated T cells to tumor cells is not as good as that of TMG7, but better than that of TMG9, TMG10, TMG11 and TMG 12. The killing ratio of the TMG 7-activated T cells ranges from 33% to 67%, which is obviously higher than that of the negative control group of other groups and the PBS group, when the effective target ratio is more than 25: the killing effect at 1 is not increased further but decreased, and the result is shown in fig. 7.
Example 5
After a TMG sequence with stronger immunogenicity is determined, the neoantigens coded on the TMG are further screened one by one, and the neoantigen coding genes, namely the mutation genes are respectively loaded on pVAX1-KSU plasmids, wherein the mutation genes code the neoantigens with the length of 15-30 amino acids, and the mutation sites are in partial middle positions of the sequences.
(1) Gene construction for screening candidate neoantigens and corresponding wild-type antigenic peptides
Based on the existing kozak-signal peptide-ubiquitin (KSU) gene, a gene encoding a neoantigen (mutation, M) or a wild-type antigen peptide (wild type, WT) is added to the 3' end of the gene by PCR. We packaged these different genetic elements on the pVAX1 plasmid for subsequent cell transfection.
(2) Screening of candidate tumor neoantigens
a. Attaching PBMC of the patient to the wall for 2 hours under the condition of no plasma culture medium, taking supernatant without attaching to the wall as T cells, and continuously culturing the T cells;
DC cell: scraping off the adherent cells, counting, re-plating according to the counting density, and transfecting the DC cells with pcDNA3.1 plasmid for encoding TMG;
DC cells plus TNF-a + IL-1 β + IL-6+ PGE2 to stimulate maturation;
c.1/2 DC: t is as follows 1: 10 for 5-7 days;
d.1/2 DC: t is as follows 1: 10 for 5-7 days;
e. counting and survival rate of a T cell culture bottle, and harvesting T cells;
f. treating Tumor tissue of a patient into single cell suspension according to the instruction of a Tumor Dissociation Kit human of the Kit, and culturing when the Tumor cell grows to 1 × 107At a rate of 5X 10 per hole3Plating in 96-well plate for overnight culture;
g. laying the activated T cells into a 96-well plate according to different proportions, and co-culturing single cells of tumor tissues of a patient for 8 hours;
h. removing culture medium in the hole, and washing for 3 times by PBS;
f. add 100. mu.L serum-free RPIM-1640 medium containing MTT (1mg/mL) to each well;
g.4h later, removing culture medium from each well, adding 100 mu L DMSO, shaking at 37 ℃ for 10-20min to fully dissolve formazan, reading a plate by using a microplate reader, calculating the killing rate of immune cells to tumor cells,
percent killing ═ percent (control mean a value-experimental mean a value)/control mean a value × 100%.
(3) Flow detection of proliferation of spleen lymphocytes induced by candidate neoantigen
a. T cells presenting the neoantigen were collected and centrifuged at 1800rpm at 4 ℃ for 5 min. After each centrifugation, ensuring that cell precipitation can be seen by naked eyes, and centrifuging again if the cell precipitation can not be seen;
b. removing supernatant by suction, adding PBS and mixing, centrifuging at 1800rpm at 4 deg.C for 5min, repeating for 2 times;
c. removing supernatant by suction, adding FITC-CD4/CD8a staining antibody diluted by PBS, mixing uniformly, and incubating for 30min at 4 ℃ in the dark;
d. adding PBS and mixing, centrifuging at 1800rpm at 4 deg.C for 5min, repeating for 2 times;
e. absorbing and removing supernatant, adding 500 mu L PBS into each tube, mixing uniformly, transferring into a flow tube, keeping at 4 ℃ in a dark place until the tube is mounted;
f. and detecting the sample on a flow type computer.
Based on the previous TMG screening, 20 neoantigens coded by TMG7 and TMG8 were individually subjected to immunogenicity validation, and the results are shown in FIG. 8. Mut61-70 is the 10 neoantigens that make up TMG 7. Wherein, compared with the other 4 neoantigens, the T cells activated by mut65, mut66, mut67, mut68, mut69 and mut70 have stronger cytotoxic effects on tumor cells. Of these mut68 is able to provoke the strongest cytotoxic effect.
Mut71-80 is 10 neoantigens that make up TMG8, wherein the T cells activated by Mut71, Mut76 and Mut79 have a certain cytotoxic effect on tumor cells compared to the other 7 neoantigens. While none of the other neoantigen-activated T cells showed a cytotoxic effect on tumor cells.
TABLE 14 comparison of killing rates of tumor cells induced by the neoantigens and their corresponding wild-type antigenic peptides
In addition, we also performed immunogenicity validation on their corresponding wild-type antigenic peptides, as shown in fig. 8 and table 14. The results showed that the 7 wild-type antigenic peptide-activated T cells, wt65, wt66, wt68, wt69, wt70, wt71 and wt79, had very weak cytotoxic effect on tumor cells. The other 2 wild-type antigen peptides wt67 and wt76 activated T cells did not even have a toxic effect on tumor cells. The specific contents of the encoding genes corresponding to the 10 neoantigens (table 14) we screened experimentally can be found in tables 8 and 9.
Sequence listing
<110> Hangzhou Nianjin Biotechnology Co., Ltd
<120> a method for screening neoantigen peptide
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Ala Leu Thr Glu Thr Trp Ala Gly Ser Met
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aggctgatct ttgccggcaa gcagctggaa gatggccgca ccctctctga ttacaacatc 180
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Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln
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Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser
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Thr Leu His Leu Val Leu Arg Leu Arg Gly
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gccctggccc tgaccgagac ctgggcgggc tccatgcaga tcttcgtgaa gaccctgacc 120
ggcaagacca tcaccctaga ggtggagccc agtgacacca tcgagaacgt gaaggccaag 180
atccaggata aagagggcat cccccctgac cagcagaggc tgatctttgc cggcaagcag 240
ctggaagatg gccgcaccct ctctgattac aacatccaga aggagtcaac cctgcacctg 300
gtccttcgcc tgagaggtgg ccactgtcac tggaacgatc tggccgtgat ccctgccggc 360
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<213> Artificial Sequence (Artificial Sequence)
<400> 7
gctagcgccg ccaccatgcg ggtcacggcg ccccgaaccc tcatcttgct gctctcgggg 60
gccctggccc tgaccgagac ctgggcgggc tccatgcaga tcttcgtgaa gaccctgacc 120
ggcaagacca tcaccctaga ggtggagccc agtgacacca tcgagaacgt gaaggccaag 180
atccaggata aagagggcat cccccctgac cagcagaggc tgatctttgc cggcaagcag 240
ctggaagatg gccgcaccct ctctgattac aacatccaga aggagtcaac cctgcacctg 300
gtccttcgcc tgagaggtgg cgaccctttc ccaaacctga accccgcccc tgcccctccc 360
ctggcttgta acctgaccct ggaggacttc tacggctaga agctt 405
<210> 8
<211> 405
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gctagcgccg ccaccatgcg ggtcacggcg ccccgaaccc tcatcttgct gctctcgggg 60
gccctggccc tgaccgagac ctgggcgggc tccatgcaga tcttcgtgaa gaccctgacc 120
ggcaagacca tcaccctaga ggtggagccc agtgacacca tcgagaacgt gaaggccaag 180
atccaggata aagagggcat cccccctgac cagcagaggc tgatctttgc cggcaagcag 240
ctggaagatg gccgcaccct ctctgattac aacatccaga aggagtcaac cctgcacctg 300
gtccttcgcc tgagaggtgg ccagggctac caccagctgt gtcacacacc tcacatcggc 360
agcagcgtga tcgatagcga cgagaagtgg ctgtgttaga agctt 405
<210> 9
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gly Gly Ser Gly Gly Gly Ser Gly Gly
1 5
<210> 10
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
1 5 10
<210> 11
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly
1 5 10
<210> 12
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Lys Trp Lys Arg Gly Ser Glu Pro Val Pro Glu Lys Lys Ile Gly
1 5 10 15
<210> 13
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Lys Gly Glu Leu Ile Tyr Leu Val Ala Thr Gln Glu Glu Lys Lys Lys
1 5 10 15
Glu Pro
<210> 14
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Leu Asp Leu Glu Asp Ile Pro Gln Ala Leu Val Ala Thr Ser Pro Ser
1 5 10 15
Gln Pro Gln Pro His Gly Lys Lys
20
<210> 15
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Phe Ser Phe Lys Phe Met Pro Gln Val Tyr Val Pro Thr Thr Phe Gln
1 5 10 15
His Asn Pro Ser Lys His
20
<210> 16
<211> 27
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Thr Glu Asp Leu Phe Leu Asn Met Glu His Glu Ser Leu Thr Leu Glu
1 5 10 15
Lys Lys Ile Lys Ile Gly Lys Lys His Gln Arg
20 25
<210> 17
<211> 26
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Lys Pro Asn Ser Pro Val Leu Leu Glu Asp Pro Val Leu Cys Ala Leu
1 5 10 15
Ala Lys Lys Ala Gln Ala Asn Pro Ser Pro
20 25
<210> 18
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Lys Ala Gln Ala Asn Pro Ser Pro Asp Cys Pro Ala Leu Pro Ala Thr
1 5 10 15
Ala Trp Gly
<210> 19
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Gly Gln Glu Pro Leu Ser His Pro Ala Leu Cys Ser Gly Ala Ser Arg
1 5 10 15
Trp Gly Cys Ala Gly
20
<210> 20
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Lys Val Asp Arg Glu Arg Ala Arg Gln Gly Thr Gly Ser Gly Pro Arg
1 5 10 15
Val
<210> 21
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Asp Pro Val Glu Asp Asp Lys Glu Lys Lys Arg Thr Trp Leu Phe Asn
1 5 10 15
Ser Arg Lys
<210> 22
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Lys Gln Pro Gly Glu Thr Asn Gly Glu Lys Lys Lys Cys Val Arg Tyr
1 5 10 15
Ile Gln Gly Glu Gly Ser
20
<210> 23
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Phe Gly Tyr Tyr Thr Pro Gln Gln Arg Ser Glu Thr Leu Ser Lys Lys
1 5 10 15
Lys Arg Lys Lys Glu Asn Arg Ser Gln Glu Trp Arg Pro Lys
20 25 30
Claims (10)
1. A method of screening for neoantigens comprising the steps of:
(1) inserting the coding sequence of the new antigen to be screened into the original expression vector to obtain a recombinant expression vector,
(2) transfecting the immature DC cells with the recombinant expression vector obtained in the step (1),
(3) culturing the transfected DC cells to ensure that the DC cells are mature, expressing corresponding neoantigen epitope peptide on the surface of the mature DC cells,
(4) presenting the newborn epitope peptide to T cells by the DC cells matured in the step (3), inducing the T cells to be activated into effector T cells,
(5) co-culturing the effector T cells and the tumor cells in the step (4), and screening the effector T cells with good effect of killing the tumor cells, thereby screening the neoantigens with good immunogenicity.
2. A method for screening the coding sequence of a neoantigen, characterized in that the neoantigen with good immunogenicity is screened according to the method for screening the neoantigen of claim 1, and the corresponding coding sequence is the screened coding sequence of the neoantigen with good immunogenicity.
3. The method according to claim 1 or 2, wherein step (1) comprises inserting between 1 and 50 neoantigen encoding sequences into the original expression vector.
4. The method according to claim 1 or 2, wherein a plurality of neoantigen coding sequences are inserted into the original expression vector in step (1), and each neoantigen coding sequence and the head and tail neoantigen coding sequences are connected with the original expression vector through flexible linker coding sequences.
5. The method of claim 4, wherein the upstream end of the first neoantigen coding sequence is linked to a starting flexible linker coding sequence, an intermediate flexible linker coding sequence is linked between two adjacent neoantigen coding sequences, and the downstream end of the last neoantigen coding sequence is linked to a last flexible linker coding sequence, wherein the amino acid sequence encoded by the starting flexible linker coding sequence is GGSGGGSGG, the amino acid sequence encoded by the intermediate flexible linker coding sequence is GGSGGGGSGG, and the amino acid sequence encoded by the last flexible linker coding sequence is GGSLGGGGSG.
6. The method according to claim 1 or 2, wherein a plurality of neoantigen coding sequences are inserted into the original expression vector, the recombinant expression vector corresponding to the neoantigen with better immunogenicity is screened out, then the immunogenicity of the encoded neoantigen is verified respectively aiming at the plurality of neoantigen coding sequences in the recombinant expression vector, and the neoantigen with good immunogenicity is screened out.
7. The method according to claim 1 or 2, wherein the activated effector T cells are expanded in step (4).
8. The method according to claim 1 or 2, wherein the original expression vector in step (1) has a kozak sequence before the site of insertion of the neoantigen-encoding sequence, and the base sequence of the kozak sequence is shown in SEQ ID No. 1.
9. The method according to claim 8, wherein the original expression vector in step (1) further has a ubiquitin protein-encoding gene sequence between the kozak sequence and the site where the neoantigen-encoding sequence is inserted, and the base sequence of the ubiquitin protein-encoding gene sequence is shown in SEQ ID No. 4.
10. The method according to claim 9, wherein the original expression vector in step (1) further has a signal peptide coding sequence between the kozak sequence and the ubiquitin protein coding gene sequence, and the base sequence of the signal peptide coding sequence is shown in SEQ ID No. 2.
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