CN112301088B - 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 the neoantigen coding sequences to be screened into an original expression vector to obtain a recombinant expression vector, transfecting immature DC cells, culturing the transfected DC cells to ensure that the DC cells are mature, expressing corresponding neoantigen epitope peptides on the surfaces 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, co-culturing the effector T cells and tumor cells, and screening the effector T cells with good tumor cell killing effect, so that the neoantigens with good immunogenicity are screened out, wherein the corresponding coding sequences are the screened neoantigen coding sequences 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, 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 the endoplasmic reticulum to form stable complex pMHC (peptide-MHC), and finally reaches the surface of cell membrane through Golgi apparatus to be 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 complex competes for binding, expelling weakly bound peptides, which are then transported to the cell surface by the MHC II molecules for CD4 supply + T cell recognition. Meanwhile, cross presentation also exists in the process of presenting antigens by MHC molecules, which is also called as a non-classical antigen presentation path, namely, polypeptides formed by degrading endogenous antigens are likely to be presented by MHC class II molecules, and polypeptides formed by degrading exogenous antigens are also likely to 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 current common bioinformatics method prediction method is fast 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 capability of the polypeptide for activating T cells to secrete IFN-gamma, and cannot accurately reflect whether the neoantigen peptide can activate the T cells to recognize and kill the tumor cells. In addition, a further verification means is to synthesize neoantigen peptide, add the neoantigen peptide into the culture of DC, so that the DC presents the neoantigen epitope peptide to activate tumor specific T cells, and 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 to the verificationThe killing verification of primary tumor cells and corresponding T cells in one-to-one correspondence can not be realized by using specific HLA typed cells. When the synthesized new antigen long peptide is used for screening new antigen, after the antigen presenting cell phagocytoses the long peptide, the new antigen epitope peptide is presented through MHC II path, 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 presented to CD8 by MHC class I molecules at the same time + 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 neoantigens or neoantigen-encoding sequences 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, step (1) inserts 1-50 coding sequences of the neoantigen into the original expression vector.
More preferably, step (1) inserts 1 to 10 coding sequences of the neoantigen into the original expression vector.
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 joint 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 new antigen coding sequence is connected with a starting flexible joint coding sequence, an intermediate flexible joint coding sequence is connected between two adjacent new antigen coding sequences, and the downstream end of the last new antigen coding sequence is connected with an end flexible joint coding sequence, wherein the amino acid sequence coded by the starting flexible joint coding sequence is GGSGGGSGG, the amino acid sequence coded by the intermediate flexible joint coding sequence is GGSGGGGSGG, and the amino acid sequence coded by the end flexible joint 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 before the site of insertion of the neoantigen coding sequence, 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 surface of BMDCs (BMDCs) cells can be improved. Compared with an uninserted original expression vector, the expression vector can improve the presentation 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 by the recombinant expression plasmid inserted with a neoantigen coding sequence, presenting neoantigen epitope peptide after the DC cells are mature, and activating 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 rapidly screened, and the corresponding wild-type peptide has no immunogenicity, so that 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 selecting a neoantigen after insertion of a neoantigen coding sequence, wherein a is pVAX1-kozak-sgub-peptide, sgub: signal peptide-ubiquitin; peptide represents: a neoantigen coding sequence; panel b shows pVAX1-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 curve is mean. + -. S.d.
Fig. 5 is a graph showing the results of screening for neoantigens, in which a: the 10 neoantigens mut1, mut2, mut3, mut4, mut5, mut6, mut7, mut8, mut9 and mut10 which form the TMG2 induce the killing efficiency of the T cells on the B16F10 cells; and (b) figure: the 10 neoantigens mut11, mut12, mut13, mut14, mut15, mut16, mut17, mut18, mut19, mut20 that make up TMG3 induce the killing efficiency of T cells on B16F10 cells; and (c) figure: the 10 neoantigens mut21, mut22, mut23, mut24, mut25, mut26, mut27, mut28, mut29, mut30 that make up 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, wt28 induced killing efficiency of T cells against B16F10 cells.
FIG. 6 is a graph showing the results of flow-based detection of proliferation of neoantigen-promoted 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: the 10 neoantigens mut61, mut62, mut63, mut64, mut65, mut66, mut67, mut68, mut69, mut70 which constitute TMG7 induce the killing efficiency of T cells on tumor cells; and (b) figure: the 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, wt79 induced the killing efficiency of T cells against 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). 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. 1 a), while we loaded the neoantigen-encoding gene only on the pVAX1 plasmid (fig. 1 b) for subsequent cell transfection.
The pVAX1-mutation (pVAX 1-M) group indicates that only the coding sequence of the neoantigen is inserted into the original vector, wherein mutation indicates the coding sequence of the neoantigen (abbreviated as M, the same below).
The pVAX1-Kozak-mutation (pVAX 1-KM) group shows that the coding sequence of the neoantigen is inserted after the insertion of the Kozak base sequence into the original vector.
The pVAX1-Kozak-Ubiquitin-mutation (pVAX 1-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 (pVAX 1-KSUM) group shows that a Kozak base sequence, a signal peptide base sequence, and a Ubiquitin base sequence are inserted into an original vector, and then a neoantigen coding sequence is inserted.
Kozak base sequence: GCCGCCACC.
Signal peptide amino acid sequence: RVTAPRTLILLLSGALALTETWAGSM.
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 new antigen 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 5min. After each centrifugation, ensuring that cell precipitates can be seen by naked eyes, and centrifuging again if the cell precipitates cannot 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 30min;
d. adding PBS and mixing, centrifuging at 1800rpm at 4 deg.C for 5min, repeating for 2 times;
e. the supernatant was discarded by aspiration, cy 3-goat anti-mouse IgG antibody diluted with PBS was added to each tube, mixed well and incubated at 4 ℃ for 30min;
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 away from light and storing at 4 ℃ until loading;
h. and detecting the sample on a flow type computer.
The specific principle is shown in figure 2 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.
As shown in FIG. 3, the amount of peptide-MHC I complexes in the pVAX1-kozak-signal peptide-ubiquitin-mutation (pVAX 1-KSUM) group presented on the cell surface of BMDCs was the highest relative to the other two groups of gene designs, while the amount of peptide-MHC II complexes presented on the cell surface of BMDCs was relatively low but higher than that in the pVAX1-mutation (pVAX 1-M) group. The amount of peptide-MHC II complexes presented on the cell surface of BMDCs was the highest in the pVAX1-kozak-mutation (pVAX 1-KM) group compared to the other three gene designs, whereas the amount of peptide-MHC I complexes presented on the cell surface of BMDCs was relatively low but higher than in the pVAX1-mutation (pVAX 1-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 10 5 B16F10 cells, mouse tail vein inoculation 1X 10 5 Mouse 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 mutation. 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-2 Db), IEDB, netMHCII and netMHCIIpan software predict the affinity of the neoantigen to MHC class II molecules (H-2 Ab), IEDB predicts the immunogenicity of the neoantigen. 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, middle linker (intermediate linker sequence): GGSGGGGSGG, end linker (end joining sequence): GGSLGGGGSG, according to murine codon preferenceThe gene design is carried out, the gene sequence is entrusted to Nanjing King Shirui biological science and technology limited company for synthesis, and the gene sequence is loaded on pcDNA3.1 plasmid. Wherein TMG1, TMG2, TMG3, TMG4 and TMG5 encode 10 neoantigens, TMG6 encodes 9 neoantigens, each neoantigen contains 25 amino acids, and the mutation site is located in the middle position of the neoantigen.
TABLE 1 neoantigens constituting TMG1
TABLE 2 neoantigens constituting 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 constituting 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 constituting 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
Transfection of BMDC cells (mouse bone marrow-derived dendritic cells) with pcDNA3.1 plasmid encoding TMG, 12h after centrifugation to remove supernatant, resuspension of cells in 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 12h;
B16F10 cells at 5X 10 per well 3 Plating in 96-well plate for overnight culture;
d. the activated spleen lymphocytes are plated in a 96-well plate according to different proportions and are cultured with B16F10 cells for 8 hours;
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 (1 mg/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 "= (control mean a value-experimental mean a value)/control mean a value x 100%.
The genes coding 10 neoantigens are linked in TMG through linker (start linker): GGSGGGSGG, middle linker): GGSGGGGSGG and end linker (end linker): GGSLGGGGSG to carry out the immunogenicity screening of the neoantigens, which can help us to improve the efficiency of screening and eliminate the neoantigens without immunogenicity. From the results of the first screening of neoantigens (FIG. 4), it is seen that the killing of B16F10 cells by TMG 1-activated spleen lymphocytes is increased with the increase of the effective target ratio of activated spleen lymphocytes to B16F10, and TMG 6-activated spleen lymphocytes also show a similar trend. However, when the effective target ratio is less than 25:1, TMG 1-activated spleen lymphocytes are less toxic to B16F10 cells than TMG2, TMG3 and TMG 4-activated spleen lymphocytes. When the effective target ratio is less than 25:1, the killing ratio of TMG2, TMG3 and TMG4 activated spleen lymphocytes to B16F10 cells ranges from 14.1% to 29.5%, which is significantly higher than the TMG1, TMG5, TMG6 and PBS group activated spleen lymphocytes.
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. the 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
A gene encoding a neo-antigen (mutation, M) or a wild-type antigen peptide (wild type, WT) was added to the 3' end of an existing kozak-signal peptide-ubiquitin (KSU) gene by PCR. We packaged these different genetic elements on the pVAX1 plasmid for subsequent cell transfection.
(2) Screening of candidate tumor neoantigens
Transfection of pVAX1 plasmid loaded with the neoantigen-encoding gene in BMDC cells (mouse bone marrow-derived dendritic cells), centrifugation 12h after supernatant, resuspension of cells in 10% Gibco serum-containing RPIM-1640 complete medium;
b. mixing spleen lymphocytes and BMDC cells according to the ratio of 10: 1, and culturing for 12h;
B16F10 cells by 5X 10 per well 3 Plating in 96-well plate for overnight culture;
d. the activated spleen lymphocytes are plated in a 96-well plate according to different proportions and are cultured with B16F10 cells for 8 hours;
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 (1 mg/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 "= (mean a value of control-mean a value of experimental)/mean a value of control × 100%.
(3) Flow detection of candidate neoantigen for inducing proliferation of spleen lymphocytes
a. BMDC cells presenting the neoantigen were collected and T cells were stimulated and centrifuged at 1800rpm at 4 ℃ for 5min. After each centrifugation, ensuring that cell precipitates can be seen by naked eyes, and centrifuging again if the cell precipitates cannot 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 a dark place;
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 step of TMG screening, we performed immunogenicity validation on 30 neoantigens encoded by TMG2, TMG3 and TMG4 one by one, and the results are shown in fig. 5.mut1-10 are 10 neoantigens that make up TMG2, with spleen lymphocytes activated by mut6, mut7 and mut8 having a stronger cytotoxic effect on B16F10 cells than the other 7 neoantigens. mut11-20 is 10 neoantigens that make up TMG3, where spleen lymphocytes activated by mut11, mut16, mut17 and mut18 have a stronger cytotoxic effect on B16F10 cells than the other 6 neoantigens. mut21-30 are 10 neoantigens that make up TMG4, with spleen lymphocytes activated by mut26, mut27 and mut28 having 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 mut28.
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 10 wild-type antigenic peptides, wt6, wt7, wt8, wt11, wt16, wt17, wt18, wt26, wt27 and wt28, activated spleen lymphocytes have only weak cytotoxic effect on B16F10 cells, and some have no or even no cytotoxic effect on B16F10 cells, such as wt17 and wt18, compared to their corresponding neoantigen and PBS group.
TABLE 7 comparison of killing rates of T cells induced by the obtained neoantigen and its wild-type antigenic 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 neoantigens obtained by experimental screening can activate CD4 + T and CD8 + T cells proliferate to different degrees, 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: 2017KY017; 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 using bwa to the hg38 (GRCh 38) version of the human reference genome to generate a bam file 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 Mutect1 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 the hg38 (GRCh 38) version of the human reference genome to generate bam files, which were then post-aligned also 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, middle linker (intermediate linker sequence): GGSGGGGSGG, end linker (end joining sequence): GGSLGGGGSG, gene design is carried out according to the preference of the humanized codon, and the gene sequence is entrusted to Nanjing King Shirui Biotech Co., ltd for synthesis and loaded on pcDNA3.1 plasmid. Wherein TMG7, TMG8, TMG9, TMG10, TMG11 and TMG12 encode 10 neoantigens, each neoantigen comprises 15-30 amino acids, and the mutation site is located in partial middle position 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 neoantigens 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 neoantigens constituting TMG11
TABLE 13 neoantigens constituting 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 are mature stimulated by adding TNF-a + IL-1 beta + IL-6+ PGE2;
c.1/2DC: t is as follows 1:10 for 5-7 days;
d.1/2DC: t is as follows 1:10 for 5-7 days;
e. counting and increasing the 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 × 10 7 At a rate of 5X 10 per hole 3 Plating in 96-well plate for overnight culture;
g. the activated T cells are plated in a 96-well plate according to different proportions, and the tumor tissue single cells of the patient are co-cultured 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 (1 mg/mL) to each well;
g.4h later, removing the culture medium from each well, adding 100 mu L DMSO, shaking at 37 ℃ for 10-20min to fully dissolve formazan, reading a plate by an enzyme-linked immunosorbent assay (ELISA) instrument, calculating the killing rate of the immune cells to the tumor cells,
percent killing "= (control mean a value-experimental mean a value)/control mean a value x 100%.
The genes coding 10 neoantigens are linked in TMG through linker (start linker): GGSGGGSGG, middle linker): GGSGGGGSGG and end linker (end linker): GGSLGGGGSG) to carry out the immunogenicity screening of the neoantigens, which can help us to improve the efficiency of 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 increasing effective target ratio of activated tumor-specific cells to tumor cells, and TMG 8-activated T cells also show a similar trend, and when the effective target ratio is less than 15. The killing ratio of the TMG7 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 determining a TMG sequence with stronger immunogenicity, we further screen neoantigens coded on the TMG one by one, and respectively load neoantigen coding genes, namely mutation genes, on pVAX1-KSU plasmids, wherein the mutation genes code the neoantigens with the length of 15-30 amino acids, and mutation sites are in partial middle positions 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) was added to the 3' end of the KSU by PCR. We packaged these different genetic elements on the pVAX1 plasmid for subsequent cell transfection.
(2) Screening of candidate tumor neoantigens
a. The PBMC of the patient adheres to the wall for 2 hours under the condition of no plasma culture medium, the supernatant without adhering to the wall is T cells, and the T cells are continuously cultured;
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 beta + IL-6+ PGE2 stimulate maturation;
c.1/2DC: t is as follows 1:10 for 5-7 days;
d.1/2DC: t is as follows: 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 × 10 7 At a ratio of 5X 10 per hole 3 Plating in 96-well plate for overnight culture;
g. the activated T cells are plated in a 96-well plate according to different proportions, and the tumor tissue single cells of the patient are co-cultured 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 (1 mg/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 "= (control mean a value-experimental mean a value)/control mean a value x 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 5min. 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 a dark place;
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, we performed immunogenicity validation of 20 neoantigens encoded by TMG7 and TMG8 one by one, and the results are shown in fig. 8.Mut61-70 are the 10 neoantigens that make up TMG 7. Among them, mut65, mut66, mut67, mut68, mut69 and mut70 activated T cells had a stronger cytotoxic effect on tumor cells than the other 4 neoantigens. Of which mut68 is capable of stimulating the strongest cytotoxic effect.
Mut71-80 is 10 neoantigens that make up TMG8, where Mut71, mut76 and Mut79 activated T cells have a 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 T cells induced by the neoantigen obtained by screening and the corresponding wild-type antigenic peptide
In addition, we also performed immunogenicity validation on their corresponding wild-type antigenic peptides, as shown in fig. 8 and table 14. The results show that the 7 wild-type antigen peptide-activated T cells of wt65, wt66, wt68, wt69, wt70, wt71 and wt79 have 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 cytotoxic effect on tumor cells. The specific contents of the genes encoding 10 neoantigens (Table 14) obtained by experimental screening can be found in tables 8 and 9.
Sequence listing
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ctggaagatg gccgcaccct ctctgattac aacatccaga aggagtcaac cctgcacctg 300
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<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 (5)
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,
inserting a plurality of neoantigen coding sequences into an original expression vector, connecting the neoantigen coding sequences and the head and tail neoantigen coding sequences with the original expression vector through flexible joint coding sequences,
the original expression vector in the step (1) has a kozak sequence in front of the site where the neoantigen coding sequence is inserted, the base sequence of the kozak sequence is shown as SEQ ID NO.1,
the original expression vector in the step (1) is also provided with a ubiquitin protein coding gene sequence between the kozak sequence and the inserted site of the new antigen coding sequence, the base sequence of the ubiquitin protein coding gene sequence is shown as SEQ ID NO.4,
the original expression vector in the step (1) is also provided with a signal peptide coding sequence between the kozak sequence and the ubiquitin protein coding gene sequence, the base sequence of the signal peptide coding sequence is shown as SEQ ID NO.2,
(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,
expanding the activated effector T cells in the step (4),
(5) Co-culturing the effector T cells and the tumor cells in the step (4), and screening the effector T cells with good tumor cell killing effect, thereby screening out the neoantigen 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) inserts 1 to 50 neoantigen encoding sequences into the original expression vector.
4. The method according to claim 1 or 2, wherein 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 an end 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 end flexible linker coding sequence is GGSLGGGGSG.
5. 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.
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