Method for preparing personalized cancer vaccine

A method for preparing a personalized cancer vaccine, comprising the following steps:

• (a) providing a first sample sequencing data set A1 and a first control sequencing data set R1 corresponding to a subject;
• and/or providing a second sample sequencing data set A2 and a second control sequencing data set R2 corresponding to a subject,
• wherein the first sample data set A1 and the first control sequencing data set R1 are obtained by a method including the following steps:
• t1) providing a first sample, and the first sample is a sample containing a CTC cell and a normal body fluid cell;
• t2) performing CTC cell enrichment treatment on the first sample, thereby obtaining an enriched first sample, wherein in the enriched first sample, the CTC cell abundance C1≥5% and the normal body fluid cell abundance C2≤95%, based on the total number of all cells in the enriched sample, and the ratio of the CTC cell abundance C1 to the normal body fluid cell abundance C2 is recorded as B1 (i.e., B1=C1/C2);
• t3) extracting DNA and/or RNA from the enriched first sample, thereby obtaining a first nucleic acid sample, wherein the first nucleic acid sample includes a nucleic acid sample from a CTC cell and a nucleic acid from a normal body fluid cell;
• and
• t4) sequencing the first nucleic acid sample, wherein the nucleic acid sample from a normal body fluid cell in the first nucleic acid sample is used as a control for the nucleic acid sample from a CTC cell, thereby obtaining the first sample sequencing data set A1 and the first control sequencing data set R1, wherein the first sample sequencing data set A1 corresponds to the sequencing data set of a CTC cell, and the first control sequencing data set R1 corresponds to the sequencing data set of a normal body fluid cell;
• wherein the second sample data set A2 and the second control sequencing data set R2 are obtained by a method including the following steps:
• w1) providing a second sample, and the second sample is a sample containing a circulating tumor DNA (ctDNA) and a circulating tumor RNA (ctRNA) and other free DNA (cfDNA) and free RNA (cfRNA);
• w2). enriching the second sample to obtain an enriched second nucleic acid sample;
• wherein, the enriched second nucleic acid sample includes ctDNA and ctRNA from a CTC cell and cfDNA and cfRNA from a normal body fluid cell, wherein based on the total weight of all nucleic acids, the content of ctDNA and ctRNA L1≥5%, while the content of cfDNA and cfRNA from a normal cell L2≤95%, and the ratio of the content L1 to L2 is recorded as B2 (i.e., B2=L1/L2);
• w3). sequencing the second nucleic acid sample, wherein the cfDNA and cfRNA from a normal cell in the second nucleic acid sample are used as a control for ctDNA and ctRNA from a CTC cell to obtain a second sample sequencing data set A2 and a second control sequencing data set R2, wherein the second sample sequencing data set A2 corresponds to the sequencing data set of a CTC cell, and the second control sequencing data set R2 corresponds to the sequencing data set of a normal body fluid cell;
• (b). performing sequence alignment treatment on the first sample sequencing data set A1 and the first control sequencing data set R1, or the second sample sequencing data set A2 and the second control sequencing data set R2, respectively, thereby obtaining a first candidate data set S1 or a second candidate data set S2;
• wherein any sequence element in the first candidate data set S1 is an element present in the A1 but not present in the R1;
• and any sequence element in the second candidate data set S2 is an element present in the A2 but not present in the R2;
• (c). performing an HLA type I or II receptor affinity prediction analysis on any sequence element in the first candidate data set S1 and/or the second candidate data set S2 to obtain a primarily selected sequence element, the primarily selected sequence element is a sequence element that binds tightly to the HLA type I or II receptor (IC50≤500 nm, preferably, 100 nm);
• (d). based on the primarily selected sequence element, synthesizing a DNA, RNA, short peptide chain corresponding to the primarily selected sequence element;
• (e). using the synthesized DNA, RNA, and short peptide chain to perform an in vitro T-cell receptor (TCR) binding test and CD8 + T cell and/or CD4 + T helper cell activation test to obtain 10-30 secondarily selected sequence elements, wherein the secondly selected sequence elements can bind to TCR and activate CD8 + T cells and/or CD4 + T helper cells;
• (f) based on the secondarily selected sequence elements, synthesizing DNA, RNA and peptide chains corresponding to the secondarily selected sequence elements;
• (g). mixing the DNA, RNA, and peptide chains synthesized in the previous step with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition, which is a personalized cancer vaccine.