WO2020101037A1 - Order-made medical core system - Google Patents

Abstract

Provided is a cell production method comprising: a step of generating a database of peptides specific to cancer cells from a cancer patient, the peptides being identified on the basis of information on the expression of gene products in the cancer cells and normal cells, wherein the database is specific to the cancer patient and includes the amino acid sequences of the peptides, information on the expression levels of proteins including the amino acid sequences, and information on the bonding ability of the peptides with respect to the human leukocyte antigen type of the cancer patient; a step of extracting an antigen peptide from the database on the basis of the expression level information and the information on the bonding ability; and a step of synthesizing the antigen peptide according to amino acid sequence information included in the database, and bringing the synthesized antigen peptide into contact with immature dendritic cells from the cancer patient to obtain mature dendritic cells that present the antigen peptide. The step of obtaining the mature dendritic cells includes: an inducing step for inducing the immature dendritic cells from mononuclear cells, or monocytes isolated from mononuclear cells, from the cancer patient; and a maturation step for bringing the induced immature dendritic cells into contact with a maturation inducer to mature the immature dendritic cells. In the maturation step, the antigen peptide is brought into contact with the immature dendritic cells 12 hours after bringing the maturation inducer into contact therewith.

Description

Made-to-order medical backbone system

The present invention relates to a method for producing cells effective for personalized medicine, and particularly to a personalized medical backbone system including the manufacturing method.

━ Three types of surgery, chemotherapy, and radiation therapy are said to be the three major therapies for cancer. Treatments using these are often accompanied by side effects and burdens. Therefore, immune cell therapy is drawing attention as a new treatment method for cancer. In immune cell therapy, cancer cells are treated by artificially proliferating immune cells capable of killing cancer cells and killing the cancer cells using the immune cells. Specifically, a method is used in which the action of immune cells collected from a cancer patient to attack the cancer cells is strengthened, the number is increased, and the cells are returned to the cancer patient.

Immune cell therapy using cancer patient's own cells has the advantage of less side effects and burden compared to the three major therapies. Immune cell therapy can be divided into several types depending on the type of cells returned to the patient. One is a method in which cells collected from a cancer patient are differentiated into dendritic cells that have a function of transmitting a marker of a cancer cell targeted for attack to T cells, and then returned to the body of the cancer patient. The other is to collect T cells from a cancer patient and contact them with dendritic cells that serve as markers for the cancer cells to be attacked, making them antigen-specific killer T lymphocytes. Is a method of artificially culturing and returning it to the body of a cancer patient. Other than this, there are methods called alpha / beta T cell therapy and gamma delta T cell therapy in which T cells are collected from a cancer patient and activated to be returned to the body of the cancer patient.

However, in immune cell therapy, it is not easy to make dendritic cells and T cells remember the characteristics of cancer cells, and it is not easy to efficiently produce immune cells that attack cancer cells. If it is possible to efficiently produce immune cells that attack cancer cells according to the condition of each cancer patient, the burden on the cancer patient will be reduced and effective personalized medicine (custom-made medicine) Medical care) can be provided more widely.

The present invention has been made in view of the above problems, starting from a cancer cell sample of the cancer patient itself, administering mature dendritic cells specialized for the cancer patient, patient-centered personalized medicine It is aimed to build a new system, which should be called a cycle, and aims to provide a bespoke medical backbone system capable of efficiently producing cells useful for cancer patients. To do.

The present inventors have conducted intensive studies to solve the above problems, using an antigen peptide selected by a predetermined method, by sensitizing dendritic cells, found that the above problems can be solved, The present invention has been completed.

That is, the present invention is as follows.
[1]
A database of peptides specific to the cancer cells, which is specified based on expression information of gene products in cancer cells and normal cells derived from cancer patients, wherein the amino acid sequences of the peptides and the expression of proteins containing the amino acid sequences Creating a database specific to the cancer patient, including amount information and information regarding the binding strength of the peptide to the human leukocyte antigen type of the cancer patient, and
A step of extracting an antigen peptide from the database based on the expression amount information and the information on the binding force;
Synthesizing the antigenic peptide according to the amino acid sequence information contained in the database, contacting the synthesized antigenic peptide with immature dendritic cells derived from the cancer patient to obtain mature dendritic cells presenting the antigenic peptide, Have
The step of obtaining the mature dendritic cells, from the mononuclear cells from the cancer patient or monocytes separated from mononuclear cells, an induction step of inducing the immature dendritic cells,
A maturation step of contacting a maturation inducer to the induced immature dendritic cells to mature the immature dendritic cells,
In the maturation step, 12 hours after the contact with the maturation inducer, the antigen peptide is contacted with the immature dendritic cell,
Method for producing cells.
[2]
In the step of creating the database,
Expression information of a gene product in a cancer cell derived from a cancer patient, which is obtained by RNA sequence analysis, and expression information of a gene product in a normal cell of a tissue to which the cancer cell belongs are compared, and the gene product is expressed only in the cancer cell. Specifying a protein and specifying a peptide specific to the cancer cell from the amino acid sequence of the protein,
The method for producing cells according to [1].
[3]
In the step of creating the database,
Obtained by exome analysis, the gene information in cancer cells derived from a cancer patient and the gene information in normal cells of the tissue to which the cancer cells belong are compared to identify a protein expressed only in the cancer cells, and the protein Identifying a peptide specific to the cancer cell from the amino acid sequence of
The method for producing cells according to [1] or [2].
[4]
In the step of creating the database,
The amino acid sequence of the peptide specific to the cancer cells, and based on the human leukocyte antigen type of the cancer patient, predict the binding strength of the peptide to the human leukocyte antigen type of the cancer patient,
The method for producing a cell according to any one of [1] to [3].
[5]
The database is
When there are multiple human leukocyte antigen types of the cancer patient, including information on the binding ability of the peptide to each of the human leukocyte antigen type,
The method for producing the cell according to any one of [1] to [4].
[6]
In the step of creating the database,
Obtaining information on the expression of a gene product in the cancer cell derived from the cancer patient, based on a body fluid sample containing DNA or a DNA fragment flowing out from the cancer cell,
The method for producing cells according to any one of [1] to [5].
[7]
In the step of obtaining the mature dendritic cells,
Using the immature dendritic cells derived from mononuclear cells from the patient,
The method for producing a cell according to any one of [1] to [6].
[8]
A step of contacting the mature dendritic cell with a T cell derived from a cancer patient to obtain an antigen-specific killer T lymphocyte,
The method for producing a cell according to any one of [1] to [7].
[9]
Further comprising the step of introducing a T cell receptor gene that recognizes the antigenic peptide to obtain an antigen-specific receptor gene-introduced T cell,
The method for producing cells according to any one of [1] to [8].
[10]
In the step of obtaining the mature dendritic cells,
The binding power of the human leukocyte antigen type of the plurality of antigen peptides and the cancer patient was evaluated in vitro using TAP gene-deficient cells, and each of the plurality of antigen peptides having high binding power to the human leukocyte antigen type was Contacting the immature dendritic cells from the cancer patient to obtain the mature dendritic cells,
The method for producing a cell according to any one of [1] to [9].
[11]
Improve the accuracy of binding power in epitope analysis by evaluating the binding power of the multiple antigenic peptides and the human leukocyte antigen type of the cancer patient in vitro using TAP gene-deficient cells, and feeding back the evaluation results. And the process of
A step of extracting an antigen peptide from the database based on the expression amount information and information on the binding strength with improved evaluation accuracy,
The method for producing a cell according to any one of [1] to [10].
[12]
In the information processing device,
A database of peptides specific to the cancer cells, which is specified based on expression information of gene products in cancer cells and normal cells derived from cancer patients, wherein the amino acid sequences of the peptides and the expression of proteins containing the amino acid sequences Creating a database unique to the cancer patient, including amount information and information regarding the binding strength of the peptide to the human leukocyte antigen type of the cancer patient, and
Executing the step of extracting an antigen peptide from the database based on the expression amount information and the information on the binding strength,
program.

According to the present invention, it is possible to provide a method for efficiently producing cells useful for personalized medical treatment of cancer patients.

An example of a flow chart of the method for producing cells of the present embodiment is shown. An example of a database created in the cell manufacturing method of the present embodiment is shown. In immature dendritic cells (Monocyte-derived DC) derived from monocytes isolated from human PBMC (peripheral blood mononuclear cells) and in immature dendritic cells induced without separating monocytes (Adherent PBMC-derived DC) The results of measuring the Purity (CD80 positive rate) and phenotypic latency (HLA-DR positive rate) by flow cytometry are shown. The detection result of the tetramer assay of the peptide specific CD8 positive cell when changing the pulse concentration and pulse time of a peptide is shown. An example of the hardware constitutions of the information processing apparatus in this embodiment is shown. An example of the block diagram which shows the functional structure of the information processing apparatus in this embodiment is shown.

The present invention relates to a bespoke medical backbone system centered on a patient, starting from a cancer cell sample of a cancer patient itself, in a series of cycles of administering mature dendritic cells specialized for the cancer patient, It is intended to provide a method for efficiently producing cells useful for cancer patients. Hereinafter, an embodiment of the present invention (hereinafter referred to as “the present embodiment”) will be described in detail, but the present invention is not limited to this, and various modifications can be made without departing from the gist thereof. Is.

[Method for producing cells]
The method for producing cells of the present embodiment is a database of peptides specific to cancer cells, which is specified based on the expression information of gene products in cancer cells and normal cells derived from cancer patients, wherein the amino acid sequences of the peptides are: A step of creating a cancer patient-specific database containing information on the expression level of a protein containing the amino acid sequence and information on the binding ability of the peptide to the human leukocyte antigen type of the cancer patient, and information on the expression level information and the binding ability. Based on the step of extracting the antigen peptide from the database, the antigen peptide is synthesized according to the amino acid sequence information contained in the database, and the synthesized antigen peptide is brought into contact with immature dendritic cells derived from a cancer patient to present the antigen peptide. To obtain mature dendritic cells.

According to such an embodiment, among the peptides specific to cancer cells of cancer patients, peptides that can be easily presented to dendritic cells and can give mature dendritic cells excellent in the attacking ability of cancer cells can be easily identified. Can be specified. This makes it possible to more efficiently obtain mature dendritic cells that effectively attack cancer cells of cancer patients. As a result, the use of mature dendritic cells that present an antigenic peptide as a drug (hereinafter, also referred to as “cancer vaccine”) used for the treatment or prevention of cancer by using immunity to cancer antigen in personalized medicine. Can be promoted.

In the present specification, the “gene product” is a generic term for a functional RNA transcribed from a gene and a protein translated from the mRNA of the transcript.

In the present specification, the “gene product expression information” is information on a gene product expressed in cells or tissues, and includes, for example, information obtained by RNA sequence analysis, exome analysis, or proteome analysis.

“Expression” as used herein includes the process by which a polypeptide is produced from a polynucleotide such as DNA. The process may involve transcription of the gene into mRNA and translation of this mRNA into a polypeptide. Depending on the context in which the term is used, "expression" may refer to the production of RNA, protein or both.

In the present specification, “human leukocyte type antigen type” means a human major histocompatibility complex (MHC) including leukocyte antigens.

In the present specification, the “database” means one having a data structure in which a peptide specific to a cancer cell and information regarding its expression and the like are linked.

In the present specification, the "cancer cell-specific peptide" is a peptide that is not expressed in normal cells and consists of a partial amino acid sequence of a cancer antigen protein (hereinafter, also referred to as "cancer antigen peptide"). means. This cancer cell-specific peptide is also called neoantigen. In addition, the "cancer antigen" refers to a protein that is specifically expressed in cancer cells but not in normal cells.

As used herein, the term “treatment of cancer” refers to inhibition (delay or arrest) of cancer development, reduction of tumor size (delay or arrest), inhibition of tumor metastasis (delay or arrest), inhibition of tumor growth (delay). Or cessation) and alleviation of one or more symptoms associated with cancer.

The subject to which the mature dendritic cells presenting the antigenic peptide according to the present invention are administered can be mammals including humans, and for example, humans, mice, rats, rabbits, cats, dogs, monkeys, sheep, horses, cows. Etc., but is not limited to these. The administration subject is typically a human.

The term “mature dendritic cell that presents an antigen peptide” as used herein means a dendritic cell that presents a complex of a cancer antigen peptide and an MHC class I molecule on the surface in vitro. Cancer antigen peptides are presented on the cell surface upon binding to major histocompatibility complex (MHC) class I molecules of antigen presenting cells.

As used herein, the term “T cell” refers to a CD8-positive T cell that has no cytotoxic activity before being stimulated by a complex of an antigen-presenting cell with an MHC class I molecule and an antigen peptide. Say.

As used herein, the term "antigen-specific killer T lymphocyte" refers to a complex of MHC class I molecule and cancer antigen peptide by administering to a subject a cancer vaccine containing mature dendritic cells that present the antigen peptide. Refers to activated T cells. Antigen-specific killer T lymphocytes, also called cytotoxic T cells (CTL), destroy cells expressing the same cancer antigen.

As used herein, the term "transcriptome" refers to the complete set of transcribed RNA molecules, including mRNAs, rRNAs, tRNAs and other non-coding RNAs produced in one cell or population of cells at a given time. Say. The term can be used for the sum of a set of transcripts in a given organism, or for a specific subset of transcripts present in a particular cell type. Unlike the genome, which is loosely fixed in a given cell line (excluding mutations), the transcriptome can fluctuate with external environmental conditions. Since it contains all mRNA transcripts in the cell, the transcriptome reflects genes that are actively expressed at any given time, with the exception of mRNA degradation events such as transcriptional decay.

Each step of the manufacturing method of this embodiment will be described below with reference to the drawings. FIG. 1 shows an example of a flowchart of the cell manufacturing method in the present embodiment. FIG. 2 shows an example of the data structure of the database. It should be noted that the processing procedure described below is merely an example, and each processing may be modified as much as possible within the scope of the technical idea of the present invention, and steps may be omitted, replaced, and added as appropriate. is there.

[Database creation process]
First, in the method for producing cells of the present embodiment, information on peptides specific to cancer cells is collected as a base information for selecting an antigen peptide specific to cancer cells of a cancer patient, and a database is created.

FIG. 2 shows an example of a data structure (table 200) included in the antigen peptide database. In this table 200, a peptide name 201 specific to a cancer cell, its amino acid sequence 202, information 203 on the expression amount of a protein containing the amino acid sequence, and information on the binding power of the peptide to the human leukocyte antigen type of a cancer patient. And 204 are stored in association with each other. It should be noted that the database may record the peptide evaluation 205 based on expression level information, binding strength, and the like.

The cancer cell-specific peptide name 201 is an ID (identifier) for identifying each peptide.

The amino acid sequence 202 is information indicating the amino acid sequence of a peptide specific to cancer cells identified by exome analysis. Based on this information, RNA sequence analysis can be used to evaluate the actual expression level of the amino acid sequence, epitope prediction can be used to evaluate the binding ability of peptides to the human leukocyte antigen type of cancer patients, and peptide synthesis can be performed. To do. The amino acid sequence also contains length information for the peptide. The peptide length information can be used to judge the binding to HLA. As an example, peptides A-1 to A-3 shown in FIG. 2 are all obtained from the same position of the same protein A except that they have different lengths. Is also obtained from the same position of the same protein A.

The protein expression amount information 203 including the amino acid sequence is information on the expression amount of the protein specified by the RNA sequence analysis, and is information indicating the actual expression amount of the cancer cell-specific peptide specified by the exome analysis. .. Peptides useful as antigens are extracted based on this information.

The information 204 on the avidity of the peptide for the human leukocyte antigen type is obtained by epitope prediction based on the information on one or more human leukocyte antigen types of cancer patients determined by HLA typing and the amino acid sequence of each peptide. This is information that evaluates these binding forces. Peptides useful as antigens are extracted based on this information. Information on one or more human leukocyte-type antigen types of a cancer patient determined by HLA typing can be shown by an allyl notation or the like.

Peptide evaluation 205 based on expression level information, binding strength, etc. is an index showing the usefulness of a peptide specific to cancer cells as an antigen. As such an index, for example, an index calculated from the expression level information 203 and the binding force information 204, such as the product or sum of the expression level information 203 multiplied by a predetermined coefficient and the binding force information 204 multiplied by a predetermined coefficient. Is mentioned.

The database creation step includes, for example, step S101 of preparing a sample, step S102 of identifying a peptide specific to a cancer cell, step S103 of identifying a human leukocyte antigen type of a cancer patient, and each peptide specific to a cancer cell. And step S104 of evaluating the binding strength with the human leukocyte-type antigen type. The order of these steps is not limited as long as a database as shown in FIG. 2 can be created.

(S101: Step of preparing sample)
The samples of cancer cells and normal cells used in creating the database are preferably derived from the same cancer patient. Cancer cells and normal cells can be directly collected from tissues containing cancer cells by a surgical technique. Further, in the present embodiment, it is not necessary to analyze the collected cancer cells themselves as a method for obtaining the expression information of the gene product in the cancer cells derived from the cancer patient. As a method for obtaining information on expression of gene products in cancer cells without analyzing the cancer cells themselves, instead of the cancer cells, a body fluid sample such as blood containing DNA or DNA fragments that have flowed out from the cancer cells is obtained. A method (liquid biopsy method) for obtaining expression information of a gene product in cancer cells derived from a cancer patient based on this body fluid sample can be adopted.

(S102: Step of identifying a peptide specific to cancer cells)
Exome analysis, RNA sequence analysis, etc. can be used as a method of identifying a peptide specific to a cancer cell. As one example, exome analysis is performed on each sample of cancer cells and normal cells to identify gene mutations specific to cancer cells (amino acid sequences specific to cancer cells). Then, the expression level of the protein having the gene mutation identified by the exome analysis is identified by the RNA sequence analysis. Thereby, the peptide specifically expressed in cancer cells can be confirmed, and its amino acid sequence and the information on the expression level of the protein containing the amino acid sequence can be obtained.

(Exome analysis)
As a method of identifying a peptide specific to a cancer cell, for example, exome analysis can be used. Exome analysis is a method for efficiently detecting mutations (SNV (SNP) / InDel) on exons by comprehensively analyzing only exon sequences in the entire genome. An exon is a base sequence that remains in mRNA, and is a part that has information such as protein synthesis. Exons are functionally important because they are regions translated into proteins, and it is presumed that most of the inherited diseases are caused by mutations in exon regions.

By performing exome analysis on both normal cells and cancer cells, it is possible to obtain genetic information on cancer cells derived from a cancer patient and gene information on normal cells of the tissue to which the cancer cells belong. By comparing the genetic information of normal cells and cancer cells, the protein expressed only in cancer cells can be specified. Then, from the amino acid sequence of the protein expressed only in cancer cells, a peptide specific to cancer cells which is not present in normal cells can be identified. Since exons account for 1 to 1.5% of the whole genome, exome analysis enables efficient analysis and identification of disease-related genes at a lower cost than whole genome sequence analysis.

As an analysis method, for example, a method of enriching an exon region (encoding DNA) using a known exome enrichment kit, performing a sequencing for examining a nucleotide sequence, and analyzing the determined nucleotide sequence can be mentioned.

By exome analysis, for example, it is possible to identify the presence or absence of a gene mutation and, if there is a mutation, identify the location. It is also possible to predict the effect of the gene mutation on the disease. In this prediction, a table (DB) that associates a disease with a gene mutation associated with the disease may be referred to.

(RNA sequence analysis)
RNA sequence analysis is a method of analyzing the base sequence of transcribed RNA and its transcription amount in a sample extracted from cells. By performing RNA sequence analysis on both normal cells and cancer cells, the expression information of the gene product in the cancer cells derived from the cancer patient and the expression information of the gene product in the normal cells of the tissue to which the cancer cells belong are obtained. be able to. By comparing the expression information of the gene products of normal cells and cancer cells, the protein expressed only in cancer cells can be specified. Then, from the amino acid sequence of the protein expressed only in cancer cells, a peptide specific to cancer cells which is not present in normal cells can be identified. Further, it is possible to obtain information on the expression level of a gene product together with the amino acid sequence of the protein.

As one embodiment, an exome analysis is performed on each of cancer cell and normal cell samples, and when the protein having the identified gene mutation is identified by exome analysis, RNA sequence analysis is performed on the cancer cell sample. It can also be done only. This makes it possible to obtain whether or not a protein having a mutation specified by exome analysis is expressed in cancer cells, and information on the expression level thereof. By identifying the gene mutation by exome analysis prior to RNA sequence analysis, it becomes possible to specify the protein to be targeted by RNA sequence analysis. As a result, it is possible to reduce the overall processing time and improve the analysis accuracy, as compared with the case where the gene mutation is specified only by the RNA sequence analysis.

Also, RNA sequence analysis makes it possible to analyze RNA (transcriptome) existing in cells under specific conditions. The comparison of expression information of gene products of normal cells and cancer cells may be performed based on data obtained by monitoring transcriptome change.

Note that RNA to be subjected to RNA sequence analysis includes mRNA, rRNA, or tRNA, or Total RNA, which is a mixture thereof.

Information relating to the peptide specifically expressed in cancer cells, the amino acid sequence thereof, and the expression level information of the protein containing the amino acid sequence, which is obtained by the exome analysis and / or RNA sequence analysis, should be recorded in a database as appropriate. You can

(S103: Step of identifying human leukocyte type antigen type of cancer patient)
The human leukocyte antigen type of a cancer patient can be determined by, for example, HLA (Human Leucocyte Antigen) typing. HLA typing refers to specifying the type of HLA (human leukocyte antigen), which is the major human histocompatibility serotype. By inoculating a peptide that is a cancer cell-specific peptide and is compatible with the HLA type of a cancer patient, dendritic cells with enhanced anti-cancer effect can be produced. Moreover, the cells obtained by the production method of the present embodiment are cells derived from the cancer patient himself, and the HLA types are completely the same.

The HLA molecule is partially embedded in the cell membrane, and the molecule is divided into the extracellular domain, cell transmembrane domain, and intracellular domain. The extracellular domain is further divided into a position far from the cell membrane (distal domain) and a proximal domain close to the membrane, and each has a different function. The distal domain has a structure involved in selective acceptance of antigenic peptides and presentation of antigens to T cells. The proximal domain has a structure involved in specific binding to molecules in T cells; CD4 and CD8. HLA contributes to the control of innate immunity and the presentation of antigens to T cells in adaptive immunity. The immune system is complicatedly configured to eliminate various non-self, and HLA is involved as the self to obtain non-self information.

There are two types of HLA, class I and class II, and HLA class I (HLA-A, B, C, etc.) is a cancer antigen to T cells in a form in which 9 endogenous amino acids are buried in a groove. Present the peptide. The T cells presented with the cancer antigen peptide become antigen-specific killer T lymphocytes and destroy the cancer cells. In addition, HLA class II (HLA-DR, DQ, DP, etc.) has a structure in which about 15 extraneous amino acids taken in by phagocytosis etc. are buried in the groove, and helper T cells receive cancer antigen peptides. Present. Helper T cells do not have cytotoxic activity by themselves, but release non-self amino acids and release Th1 and Th2 cytokines. The Th1 cytokine promotes the activation of antigen-specific killer T lymphocytes, and the Th2 cytokine promotes the immunoglobulin production of B cells.

The HLA typing is not particularly limited, but for example, it can be performed by the SBT method (Sequencing Based Typing) and the SSP method (sequence specific primers) that are HLA genotyping methods by DNA typing. As an example, in the SBT method, DNA is amplified by the PCR method, and the HLA genotype is determined by comparing the base sequence of the amplified product with the base sequence information of existing alleles. In addition, the genetic information obtained by whole exome analysis can also be used for HLA typing.

The notation of HLA typing recorded in the database can conform to the HLA allele notation. In this notation, following the gene name, the first section that describes the number corresponding to the HLA plateau (HLA specificity), the second section that describes groups with different amino acid sequences (non-synonymous substitution), and within the exon The third section that indicates alleles (alleles) that are distinguished by base substitutions that do not change the amino acid sequence (synonymous substitutions), and the alleles that are filled in by base substitutions other than exons (for example, differences in introns) It is marked from the 4th area that does.

(S104: Step of evaluating the binding power to human leukocyte type antigen type for each peptide specific to cancer cells)
The avidity of the cancer cell-specific peptide with the human leukocyte-type antigen type can be determined by, for example, epitope prediction. Epitope prediction is a prediction of a part (epitope) of an antigen recognized and bound by an antibody. For example, by predicting the binding strength of the peptide to the human leukocyte antigen type of the cancer patient based on the amino acid sequence of the peptide specific to the cancer cell and the human leukocyte antigen type of the cancer patient by epitope prediction, It is possible to improve the production efficiency of mature dendritic cells that present the antibody.

Note that if there are multiple human leukocyte antigen types of cancer patients, the database preferably contains the results of evaluating the binding ability of the cancer cell-specific peptide for each human leukocyte antigen type.

[S105: Extraction Step]
In the extraction step, an antigen peptide suitable for cell production is extracted from the database based on the expression amount information and the information on the binding strength. The number of peptides to be extracted is not particularly limited, but may be, for example, about 5 to 10 kinds. This makes it possible to reduce the subsequent in vivo and in vitro operation load. In addition, the extraction operation can be performed in silico, that is, on the data. Examples of the extraction method include a method in which a score serving as an evaluation index for extraction is attached to an antigen peptide in a database, and an antigen peptide suitable for cell production is extracted based on the score. Extraction of antigen peptides can be performed by selecting peptides in descending order of score.

Examples of the score include, for example, the product (the following formula) or the sum of the expression amount information multiplied by a predetermined coefficient and the binding force multiplied by the predetermined coefficient. By using the scores calculated in this way, we evaluate peptides that are relatively abundantly expressed in cancer cells (proteins) and that are easily taken up by dendritic cells to produce mature dendritic cells. It can be effectively used as a product. Note that the score calculation formula is not limited to the following. The score can be recorded in the peptide rating 205.
Score = (coefficient × expression amount information) × (coefficient × cohesion)

Also, the binding strength may be adjusted during extraction. For example, peptides A-1 to A-3 shown in FIG. 2 are all obtained from the same position of the same protein A except that the lengths are different. When the difference in the binding strength to HLA changes by changing the length of the peptides, such as peptides A-1 to A-3, the peptide with the maximum binding strength to the target HLA is extracted. can do. More specifically, in selecting an antigen peptide for HLA represented by A * 02: 01: ..., the binding amounts of peptides A-1 to A-3 containing gene mutation sites are compared and considered, The one with the highest bond strength can be selected.

[S106: Peptide Synthesis]
In the peptide synthesis step, the antigen peptide is synthesized by a known method based on the amino acid sequence information of the extracted antigen peptide as described above.

[S107: Peptide binding assay]
The binding strength of the synthesized antigen peptide may be evaluated prior to the cell production process. As an example, the binding force (affinity) between the antigenic peptide and HLA can be examined using TAP gene-deficient cells. Specifically, the antigen peptide is supplied to cells expressing HLA to which no peptide is bound. When the affinity between the antigenic peptide and HLA is high, HLA is stabilized on the cell surface. It should be noted that HLA to which no peptide is bound is constantly expressed from within the TAP gene-deficient cell and continues to be expressed on the cell surface. Therefore, cells to which an antigen peptide having a high affinity for HLA is added will present a large amount of the antigen peptide on the surface.

In this way, TAP gene-deficient cells to which the antigen peptide is added are cultured for a certain period of time, and fluorescence intensity is measured by flow cytometry using anti-HLA alternation. In addition, as a control, TAP gene-deficient cells to which an antigen peptide is added or TAP gene-deficient cells to which a peptide that does not bind is added are prepared, and fluorescence intensity is measured. TAP gene-deficient cells to which the antigen peptide was added by comparing the flow cytometry evaluation of TAP gene-deficient cells to which the antigen peptide was added with the evaluation of flow cytometry of TAP gene-deficient cells to which the antigen peptide was not added It can be evaluated that the higher the fluorescence intensity of is, the higher the binding force (affinity) between the antigen peptide and HLA. The accuracy of epitope analysis and the like may be improved by feeding back and learning the result of this operation.

[S108: Cell manufacturing process]
In the cell production step, the extracted antigenic peptide is used to obtain mature dendritic cells that present the antigenic peptide. Specifically, the antigen peptide extracted as described above is synthesized according to the amino acid sequence information contained in the database, and the synthesized antigen peptide is contacted with immature dendritic cells derived from a cancer patient (pulse), Obtaining mature dendritic cells displaying the peptide.

As the immature dendritic cells, it is preferable to use those derived from patient-derived mononuclear cells, such as by adding granulocyte-monocyte colony stimulating factor (GM-CSF) or interleukin. More preferably, those derived from monocytes are used. Thereby, the production efficiency of mature dendritic cells can be improved.

Figure 3 shows immature dendritic cells derived from monocytes isolated from human PBMC (peripheral blood mononuclear cells) (Monocyte-derived DC) and immature dendritic cells induced without separating monocytes (Adherent PBMC-). shows the results of flow cytometry of Purity (CD80 positive rate) and phenotypic latency (HLA-DR positive rate) of each immature dendritic cell.

As shown in Fig. 3, with both cytokine combinations of GM-CSF / IL-4 and GM-CSF / IFNa, monocytes were separated from peripheral blood mononuclear cells and immature dendritic cells were induced from the separated monocytes. It was found that the obtained immature dendritic cells had higher Purity and phenotypic potency.

Further, as one of the methods for efficiently obtaining mature dendritic cells that present an antigen peptide, it is preferable to contact (pulse) the antigen peptide with immature dendritic cells derived from a cancer patient in the latter half of the maturation stage. Specifically, an induction step of inducing immature dendritic cells from monocytes isolated from cancer patients or monocytes separated from the mononuclear cells, and contacting a maturation inducer with the induced immature dendritic cells. Then, when performing a maturation step of maturing immature dendritic cells, it is preferable to contact (pulse) the antigen peptide in the maturation step. Particularly, it is preferable that the immature dendritic cells are contacted (pulsed) with the antigen peptide in the latter half of the maturation step, for example, 12 hours after the contact with the maturation inducer. Although not particularly limited, the maturation step can be completed 24 hours after adding the maturation inducer. The timing of contact with the antigen peptide may be 15 hours, 18 hours, or 21 hours after the addition of the maturation inducer.

By this, mature dendritic cells that present the antigen peptide can be efficiently obtained, and the induction efficiency of peptide-specific CD8-positive cells can be improved. The reason for this is not particularly limited, but it is considered that the process of presenting to mature dendritic cells is different between a short antigen peptide such as 12 mer or less and a long antigen peptide such as 13 mer or more. When a short antigen peptide is previously synthesized and brought into contact as in the present embodiment, the antigen peptide is incorporated into the immature dendritic cell during maturation by contacting the antigen peptide immediately or simultaneously with the addition of a maturation inducer. This is probably because (phagocytosis) is finely processed and the target antigenic peptide is not presented at the HLA binding site of the resulting mature cells.

On the other hand, when synthesizing and contacting a short antigen peptide in advance as in the present embodiment, unlike a general phagocytosis process, the binding site of HLA existing on the surface of a mature immature dendritic cell is It is conceivable that the antigen peptide is physically bound and antigen presentation is performed. Therefore, in the present embodiment, after a certain period of time has passed since the addition of the maturation inducer, the immature dendritic cells can be contacted with the antigen peptide to efficiently obtain mature dendritic cells that present the antigen peptide. It is considered possible.

The length of the amino acid sequence of the antigen peptide is preferably 6 to 12 mer, more preferably 8 to 11 mer. When the length of the amino acid sequence of the antigen peptide is within the above range, mature dendritic cells presenting the antigen peptide can be obtained more efficiently. Further, as the antigen peptide to be pulsed, a mixture of peptides having an amino acid sequence length within the above range and having the same gene mutation site and different amino acid sequence lengths at portions other than the gene mutation site may be used. .. For example, the peptides A-1 to A-3 shown in FIG. 2 may be mixed and used. This makes it possible to obtain mature dendritic cells that present the antigenic peptide having the highest binding strength to HLA.

Monocytes were separated from human PBMCs, immature DCs were induced with GM-CSF / IFNa, and EBV-derived peptide (9 mer) was added (pulsed) at the same time as the initiation of maturation, followed by a 24-hour maturation process. The obtained mature dendritic cells and the EBV-derived peptide (9-mer) were added (pulsed) 22 hours after the start of maturation, and the peptide pulse time was set to 2 hours to perform a total of 24 hours of maturation process. The obtained mature dendritic cells were obtained. The resulting two types of mature dendritic cells were collected, frozen, and then thawed on the day of the experiment and cocultured with CD14-negative cells (T cells) for 11 days. Then, the cultured cells were collected, and the peptide-specific CD8-positive cells were detected by FACS by the tetramer assay. The result is shown in FIG.

As shown in Fig. 4, regardless of the contacted peptide concentration (3, uM, or 20, ug / ml), the contact time was 24 hours for mature dendritic cells when the contact time was 2 hours. It was found that the induction efficiency of peptide-specific CD8-positive cells was higher than that of mature dendritic cells in each case. It is considered that this is because the pulsed peptide was a short chain, so that the peptide was physically bound (ridden) to the binding site of HLA by a short pulse and the antigen was presented. On the other hand, when the contact time is set to 24 hours, mature dendritic cells in the process take up the peptide in the dendritic cell and are finely processed, and the target antigen peptide is presented on HLA of the dendritic cell. Probably because it was not done.

The maturation inducer is not particularly limited, but examples include Lipopolysaccharide, TNFα, and CD40L.

Also, it is preferable to produce mature dendritic cells for each antigenic peptide and mix the obtained multiple types of mature dendritic cells to prepare one vaccine. As a result, a more effective mature dendritic cell vaccine can be obtained.

[S109: Efficacy Evaluation]
Efficacy evaluation after administering the mature dendritic cells obtained as described above to a cancer patient can be performed as follows. For example, blood collected from a cancer patient is analyzed for a certain period (1 to 2 weeks) after administration of a mature dendritic cell vaccine, and the administered mature dendritic cells induce killer T lymphocytes specific to an antigen peptide. Check whether or not. In this evaluation, for example, when a vaccine containing 10 types of mature dendritic cells is used, it can be confirmed that killer T lymphocytes are induced in 3 types of them.

Based on such confirmation results, information such as induction efficiency of killer T lymphocytes, survival rate, and relationship of cancer tumor shrinkage may be stored in another database.

[S110: Step of obtaining antigen-specific killer T lymphocytes]
After producing the antigen-specific killer T lymphocytes outside the body of the cancer patient using the mature dendritic cells produced as described above, the produced antigen-specific killer T lymphocytes are returned to the body of the cancer patient. Good. Specifically, the antigen-specific killer T lymphocytes can be obtained by contacting the mature dendritic cells obtained as described above with T cells derived from a cancer patient. The thus-obtained antigen-specific killer T lymphocytes destroy the cancer cells expressing the antigen peptide, that is, the peptide specifically expressed by the cancer cells.

Alternatively, the mature dendritic cells produced as described above may be directly returned to the cancer patient's body to produce antigen-specific killer T lymphocytes in the cancer patient's body.

[Step of obtaining antigen-specific receptor gene-transferred T cell]
Furthermore, a T cell into which a gene for a T cell receptor that recognizes the above-identified antigen peptide is introduced (hereinafter also referred to as "antigen-specific receptor gene-introduced T cell") is produced, and the produced antigen is produced. The specific receptor gene-transfected T cells may be returned to the body of a cancer patient. Specifically, by cloning the gene of the T cell receptor specific for the antigenic peptide specified as described above, and introducing the cloned gene, an antigen-specific receptor gene-introduced T cell is obtained. Obtainable.

As an example, a method of identifying and extracting a gene for a T cell receptor from the antigen-specific killer T lymphocyte obtained as described above, cloning the gene, and introducing the cloned gene into another T cell is described. Conceivable. Thereby, an antigen-specific receptor gene-transferred T cell can be obtained. The T cell receptor obtained from an antigen-specific killer T lymphocyte has excellent binding properties to the above-mentioned antigenic peptide, and the T cell into which this is introduced is specific to a protein specifically expressed in cancer cells. It will have sex.

Prior to the production of antigen-specific receptor gene-transferred T cells, the above-mentioned mature dendritic cells or antigen-specific killer T lymphocytes may be administered to a cancer patient to confirm the effect. In this confirmation method, a body fluid sample such as blood containing DNA or a DNA fragment flowing out from a cancer cell is obtained, and based on this body fluid sample, expression information of a gene product in cancer cells derived from a cancer patient is obtained ( Liquid biopsy method) can be adopted.

In addition, when the cloned T cell receptor gene is obtained once, and the antigenic peptide recognized by the T cell receptor is extracted as described above, the cloned gene is directly expressed. It is also possible to produce an antigen-specific receptor gene-transfected T cell in which is introduced into another T cell and to administer this to a cancer patient. As a result, an antigen-specific receptor gene-introduced T cell can be efficiently produced without performing the step of identifying and extracting the T cell receptor gene again.

As a result of applying the liquid biopsy method to a cancer patient administered with the above-mentioned mature dendritic cells or antigen-specific killer T lymphocytes, when the cancer cells do not tend to decrease, the antigen-specific receptor gene transfer It is preferable to start the production of T cells.

〔how to use〕
As described above, by returning antigen-loaded dendritic cells into the same cancer patient's body by, for example, subcutaneous injection, the restored dendritic cells migrate to lymph nodes and become lymph nodes in the body. Activate the sphere. As a result, cells useful for cancer patients will be generated in a series of personalized medical care cycles centered on the patient, starting with the cancer cell sample of the cancer patient itself and administering mature dendritic cells specialized for the cancer patient. A method for efficiently manufacturing can be provided. In particular, according to the present invention, it becomes possible to efficiently produce immune cells that attack cancer cells according to the state of each cancer patient, reduce the burden on cancer patients, and achieve effective personalization. It becomes possible to provide medical care (made-to-order medical care) more widely.

(Information processing method related to extraction of antigen peptide from creation of database)
The database can be created by using the information processing device 300A. In creating the database, the information processing device 300A may be directly or indirectly connected to one or more measuring devices 300B for measuring information stored in the database. Examples of such a measuring device 300B include devices used for RNA sequence analysis, exome analysis, and HLA typing. It should be noted that the term "connection" refers to a state in which information can be directly or indirectly transmitted and received, and includes a case where information can be transmitted and received via a recording medium as well as a case where the information is connected via a network. In the following example, a mode via a network is described, but the embodiment is not limited to this.

The information processing apparatus 300A includes a processor 301, a memory 302, a storage 303, an input / output interface (input / output I / F) 304, and a communication interface (communication I / F) 305. Each component of the wear is connected to each other via a bus B, for example.

The processor 301 executes a function and / or a method realized by a code or an instruction included in a program stored in the storage 303. The processor 301 includes, for example, a central processing unit (CPU), etc., and is formed by a logic circuit or a dedicated circuit formed in an integrated circuit (IC (Integrated Circuit) chip, LSI (Large Scale Integration)) or the like, in each of the embodiments. Processing can be realized.

The memory 302 temporarily stores the program loaded from the storage 303 and provides a work area for the processor 301. The memory 302 also temporarily stores various data generated while the processor 301 is executing the program. The memory 302 includes, for example, a RAM (Random Access Memory) and a ROM (Read Only Memory).

The storage 303 stores a program. The storage 303 includes, for example, a HDD (Hard Disk Drive), an SSD (Solid State Drive), a flash memory, and the like.

The input / output I / F 304 includes an input device that inputs various operations on the information processing device 300A and an output device that outputs a processing result processed by the information processing device 300A. In the input / output I / F 304, an input device and an output device may be integrated, or an input device and an output device may be separated.

The input device is realized by any of all types of devices that can receive an input from a user and transmit information related to the input to the processor 301, or a combination thereof. The input device includes, for example, a hardware key such as a touch panel, a touch display, and a keyboard, a pointing device such as a mouse, a camera (operation input via an image), and a microphone (operation input by voice).

The output device is realized by any of all types of devices capable of outputting the processing result processed by the processor 301, or a combination thereof. In the case of outputting the processing result as a video and / or a moving image, the output device may be any one or all of the devices capable of displaying the display data according to the display data written in the frame buffer. It is realized by combination. The output device is, for example, a touch panel, a touch display, a monitor (for example, a liquid crystal display, an OELD (Organic Electroluminescence Display), etc.), a head mounted display (HDM: Head Mounted Display), projection mapping, a hologram, in the air (in a vacuum, A device capable of displaying images and text information, a speaker (voice output), a printer, and the like. Note that these output devices may be capable of displaying display data in 3D.

The communication I / F 305 sends and receives various data via the network. The communication may be executed by wire or wireless, and any communication protocol may be used as long as mutual communication can be executed. The communication I / F 305 has a function of performing communication with another information processing device via the network. The communication I / F 305 transmits various data to another information processing device according to an instruction from the processor 301. Further, the communication I / F 305 receives various data transmitted from another information processing device and transfers the data to the processor 301.

(Functional configuration of information processing device)
FIG. 6 shows an example of a block diagram showing a functional configuration of the information processing apparatus 300A for creating a database. The information processing device 300A is an example of an information processing device that creates a database, and includes an input / output I / F 411, a communication I / F 412, a control unit 420, and a storage unit 430. The input / output I / F 411 corresponds to the input / output I / F 304 in FIG. 5, and the communication I / F 412 corresponds to the communication I / F 305 in FIG. The storage unit 430 is realized using the memory 302 and / or the storage 303.

The functional unit 430 disclosed in FIG. 6 is realized by cooperation of the processor 301, the memory 302, the storage 303, the input / output I / F 304, and the communication I / F 305 included in the information processing device 300A. That is, the processor 301 of the information processing apparatus 300A illustrated in FIG. 5 expands various programs (control program, arithmetic program, etc.) stored in the storage 303 in the memory 302 (for example, RAM). Then, the processor 301 interprets and executes various programs expanded in the memory 302 to control each hardware component, thereby realizing the functional configuration described below.

Each function realized by the information processing device 300A may be realized by the processor 301 such as a general-purpose CPU, or a part or all of the function may be realized by one or a plurality of dedicated processors 301. Good. Furthermore, the functional configuration realized by the information processing apparatus 300 may be appropriately omitted, replaced, or added depending on the embodiment or the example.

The control unit 420 has a transmission / reception unit 421, a DB creation unit 422, an evaluation unit 423, and an output unit 424. The transmission / reception unit 421, the DB creation unit 422, the evaluation unit 423, and the output unit 424 are realized by the processor 301 reading and executing the program 432 stored in the storage unit 430. Further, the storage unit 430 stores an antigen peptide DB 431 and a program 432. The functional configuration realized by the information processing apparatus 300A may be appropriately omitted, replaced, or added depending on the embodiment or the example. The storage unit 430 will be described below, and then the control unit 420 will be described. ：

The antigen peptide DB 431 is a database that collects information on peptides specific to cancer cells, and one example is a database having the data structure shown in FIG. 2 above. The database is created for each patient.

The transmission / reception unit 421 receives information stored in the antigen peptide DB 431 from a measurement device 300B such as a device used for RNA sequence analysis, exome analysis, HLA typing, or transmits information necessary for analysis to these measurement devices 300B. It is a functional unit that performs processing. Each information received by the transmitting / receiving unit 421 is passed to the DB creating unit 422.

The DB creating unit 422 collects information on peptides specific to cancer cells as a base information for selecting an antigen peptide specific to cancer cells of a cancer patient, and creates a database. More specifically, a table is created based on each information passed from the transmitting / receiving unit 421, and an antigen peptide database for each cancer patient is created. The database for each patient created by the DB creation unit 422 is stored in the antigen peptide DB 431.

The evaluation unit 423 performs a process of attaching a score as an evaluation index to each antigen peptide stored in the database created by the DB creation unit 422. As described above, as the score, a parameter calculated by the product (the following formula) or the sum of the expression amount information multiplied by a predetermined coefficient and the binding force multiplied by a predetermined coefficient can be added. The score is stored in the antigen peptide DB 431 in association with each antigen peptide.

The output unit 424 extracts an antigen peptide suitable for cell production based on the score. The extraction method is not particularly limited, and examples thereof include a method of extracting the top 5 to 10 types of antigen peptides having a high score, and a method of extracting an antigen peptide having a score of a predetermined threshold value or more.

(Processing flow by the information processing device)
The process of creating a database will be described below. The processing procedure described below is merely an example, and each processing may be modified as much as possible within the scope of the technical idea of the present disclosure.

First, the transmission / reception unit 421 receives the gene information regarding the protein having the identified gene mutation from the exome analysis device 300B. Then, the DB creation unit 422, which has received the gene information from the transmission / reception unit 421, specifies a plurality of types of peptide sequences including the gene mutation site and stores them in the antigen peptide DB 431. Here, examples of a plurality of types of peptide sequences containing a gene mutation site include peptides A-1 to A-3 shown in FIG. Peptides A-1 to A-3 were obtained from the same position of the same protein A except that the lengths of the amino acid sequences other than the gene mutation site were different. Therefore, the binding force to HLA is different. Based on the gene information received from the transmission / reception unit 421, the DB creation unit 422 includes peptides having the same gene mutation site and different amino acid sequence lengths other than the gene mutation site (for example, peptides A-1 to A-3). It is possible to select an antigen peptide having the highest binding strength to HLA by preparing and storing

Then, the transmitting / receiving unit 421 transmits the gene information of the peptide stored in the antigen peptide DB 431 by the DB creating unit 422 in this manner to the RNA sequence analysis device 300B. Based on the received gene information, the RNA sequence analysis device 300B analyzes the presence / absence and the expression amount of the gene product (mRNA) having the gene information. Then, the transmitting / receiving unit 421 receives the analysis result from the RNA sequence analysis device 300B. Then, the DB creation unit 422, which has received the information regarding the presence / absence and the expression amount of the gene product (mRNA) from the transmission / reception unit 421, stores the information in the antigen peptide DB 431 in association with each already stored peptide.

In parallel with the above, the transmission / reception unit 421 receives information regarding the HLA type from the HLA typing analysis device 300B. Upon this reception, the transmitting / receiving unit 421 may transmit the gene information (all exome analysis results) of the peptides stored in the antigen peptide DB 431 by the DB creating unit 422 to the HLA typing analysis device 300B. The information on the HLA type received by the transmitting / receiving unit 421 from the HLA typing analysis device 300B is stored in the antigen peptide DB 431 by the DB creating unit 422.

Then, the transmission / reception unit 421 transmits the information about the HLA type and the gene information of the peptide stored in the antigen peptide DB 431 to the epitope analysis device 300B, and the information about the binding force between the HLA and the antigen peptide is transmitted from the epitope analysis device 300B. To receive. The transmitting / receiving unit 421 stores the received information on the binding force in the antigen peptide DB 431 in association with each of the already stored peptides. In this case, from the viewpoint of reducing the processing load of the epitope analysis, the gene information transmitted to the epitope analysis device 300B may be one in which an expression amount above a certain level (threshold value) is recognized in the RNA sequence analysis.

Then, the evaluation unit 423 performs a process of assigning a score as an evaluation index to each antigen peptide stored in the database created by the DB creation unit 422 as described above. Peptides that contain the same gene mutation site and differ in the length of the amino acid sequences other than the gene mutation site have the same expression level as proteins and different binding strengths. Therefore, by this scoring process, the peptide sequence having a certain gene mutation site and having the highest binding force can be selected. Finally, the output unit 424 extracts an antigen peptide suitable for cell production based on the score.

Further, the information processing device 300A may be connected to the peptide synthesis device 300C or the cell culture device 300D. As a result, the antigen peptide extracted as described above can be synthesized by the peptide synthesizer 300C. Further, when the cell culture device 300D uses the synthesized antigen peptide to perform cell culture of dendritic cells and the like, it is possible to store information about the culture result using the antigen peptide as a key.

INDUSTRIAL APPLICABILITY The present invention has industrial applicability as a method for producing cells effective for personalized medicine.

Claims (12)

1. A database of peptides specific to the cancer cells, which is specified based on the expression information of gene products in cancer cells and normal cells derived from cancer patients, wherein the amino acid sequences of the peptides and the expression of proteins containing the amino acid sequences Creating a database specific to the cancer patient, including amount information and information regarding the binding strength of the peptide to the human leukocyte antigen type of the cancer patient, and
   A step of extracting an antigen peptide from the database based on the expression amount information and the information on the binding force;
   Synthesizing the antigen peptide according to the amino acid sequence information contained in the database, contacting the synthesized antigen peptide with immature dendritic cells derived from the cancer patient, to obtain mature dendritic cells presenting the antigen peptide, Have
   The step of obtaining the mature dendritic cells, from the mononuclear cells from the cancer patient or monocytes separated from mononuclear cells, an induction step of inducing the immature dendritic cells,
   A maturation step of contacting a maturation inducer to the induced immature dendritic cells to mature the immature dendritic cells,
   In the maturation step, 12 hours after the contact with the maturation inducer, the antigen peptide is contacted with the immature dendritic cell,
   Method for producing cells.
2. In the step of creating the database,
   Expression information of a gene product in a cancer cell derived from a cancer patient, which is obtained by RNA sequence analysis, and expression information of a gene product in a normal cell of a tissue to which the cancer cell belongs are compared, and the gene product is expressed only in the cancer cell. Specifying a protein and specifying a peptide specific to the cancer cell from the amino acid sequence of the protein,
   The method for producing the cell according to claim 1.
3. In the step of creating the database,
   The gene information in cancer cells derived from cancer patients obtained by exome analysis and the gene information in normal cells of the tissue to which the cancer cells belong are compared to identify a protein expressed only in the cancer cells, and the protein is expressed. Identifying a peptide specific to the cancer cell from the amino acid sequence of
   The method for producing the cell according to claim 1 or 2.
4. In the step of creating the database,
   The amino acid sequence of the peptide specific to the cancer cells, and based on the human leukocyte antigen type of the cancer patient, predict the binding strength of the peptide to the human leukocyte antigen type of the cancer patient,
   The method for producing the cell according to any one of claims 1 to 3.
5. The database is
   When there are multiple human leukocyte antigen types of the cancer patient, including information on the binding ability of the peptide to each of the human leukocyte antigen type,
   The method for producing the cell according to any one of claims 1 to 4.
6. In the step of creating the database,
   Obtaining information on the expression of a gene product in the cancer cell derived from the cancer patient, based on a body fluid sample containing DNA or a DNA fragment flowing out from the cancer cell,
   The method for producing a cell according to any one of claims 1 to 5.
7. In the step of obtaining the mature dendritic cells,
   Using the immature dendritic cells derived from mononuclear cells from the patient,
   The method for producing the cell according to any one of claims 1 to 6.
8. Further comprising the step of contacting the mature dendritic cell with a T cell derived from a cancer patient to obtain an antigen-specific killer T lymphocyte,
   The method for producing the cell according to any one of claims 1 to 7.
9. Further comprising the step of introducing a T cell receptor gene that recognizes the antigenic peptide to obtain an antigen-specific receptor gene-introduced T cell,
   The method for producing the cell according to any one of claims 1 to 8.
10. In the step of obtaining the mature dendritic cells,
    The binding power of the human leukocyte antigen type of the plurality of antigen peptides and the cancer patient was evaluated in vitro using TAP gene-deficient cells, and each of the plurality of antigen peptides having high binding power to the human leukocyte antigen type was Contacting the immature dendritic cells from the cancer patient to obtain the mature dendritic cells,
    The method for producing the cell according to any one of claims 1 to 9.
11. Improve the accuracy of binding power in epitope analysis by evaluating the binding power of the multiple antigenic peptides and the human leukocyte antigen type of the cancer patient in vitro using TAP gene-deficient cells, and feeding back the evaluation results. And the process of
    A step of extracting an antigen peptide from the database based on the expression amount information and the information on the binding strength with improved evaluation accuracy,
    The method for producing the cell according to any one of claims 1 to 10.
12. In the information processing device,
    A database of peptides specific to the cancer cells, which is specified based on the expression information of gene products in cancer cells and normal cells derived from cancer patients, wherein the amino acid sequences of the peptides and the expression of proteins containing the amino acid sequences Creating a database specific to the cancer patient, including amount information and information regarding the binding strength of the peptide to the human leukocyte antigen type of the cancer patient, and
    Executing the step of extracting an antigen peptide from the database based on the expression amount information and the information on the binding strength,
    program.
    

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