T cell receptor and b cell receptor repertoire analysis system, and use of same in treatment and diagnosis

Abstract

The repertoire of the variable region of T cell receptors (TCR) or B cell receptors (BCR) is quantitatively analyzed using non-biased gene sequence analysis. The present invention provides the following: a method for quantitatively analyzing the repertoire of the variable region of the T cell receptors (TCR) or B cell receptors (BCR) of a subject by using a database, wherein the method includes (1) a step for providing a nucleic acid sample containing the nucleic acid sequence of T cell receptors (TCR) or B cell receptors (BCR) amplified in a non-biased manner from the subject; (2) a step for determining the nucleic acid sequence contained in the nucleic acid sample; and (3) a step for calculating the frequency of appearance of each gene or combination thereof on the basis of the determined nucleic acid sequence and deriving the TCR or BCR repertoire of the subject.

1 . A method of quantitatively analyzing a repertoire of a variable region of a T cell receptor (TCR) or a B cell receptor (BCR) of a subject by using a database, wherein the method comprises the steps of:
 (1) providing a nucleic acid sample comprising a nucleic acid sequence of the T cell receptor (TCR) or the B cell receptor (BCR) which is amplified from the subject in an unbiased manner;   (2) determining the nucleic acid sequence comprised in the nucleic acid sample; and   (3) calculating a frequency of appearance of each gene or a combination thereof based on the determined nucleic acid sequence to derive a TCR or BCR repertoire of the subject, wherein the nucleic acid sample comprises nucleic acid sequences of a plurality of types of T cell receptors (TCR) or B cell receptors (BCR) and the step (2) determines the nucleic acid sequence by a single sequencing performed with a common adaptor primer.   
     
     
         2 . The method of  claim 1 , wherein the step (1) comprises the following steps:
 (1-1) synthesizing a complementary DNA by using an RNA sample derived from a target cell as a template;   (1-2) synthesizing a double stranded complementary DNA by using the complementary DNA as a template;   (1-3) synthesizing an adaptor-added double stranded complementary DNA by adding a common adaptor primer sequence to the double stranded complementary DNA;   (1-4) performing a first PCR amplification reaction by using the adaptor-added double stranded complementary DNA, a common adaptor primer consisting of the common adaptor primer sequence, and a first TCR or BCR C region specific primer,
 wherein the first TCR or BCR C region specific primer is designed to comprise a sequence that is sufficiently specific to a C region of interest of the TCR or BCR and not homologous with other genetic sequences, and comprise a mismatching base between subtypes downstream when amplified; 
   (1-5) performing a second PCR amplification reaction by using a PCR amplicon of (1-4), the common adaptor primer, and a second TCR or BCR C region specific primer,
 wherein the second TCR or BCR C region specific primer is designed to have a sequence that is a complete match with the TCR or BCR C region in a sequence downstream the sequence of the first TCR or BCR C region specific primer, but comprise a sequence that is not homologous with other genetic sequences, and comprise a mismatching base between subtypes downstream when amplified; and 
   (1-6) performing a third PCR amplification reaction by using a PCR amplicon of (1-5), an added common adaptor primer in which a nucleic acid sequence of the common adaptor primer comprises a first additional adaptor nucleic acid sequence, and an adaptor-added third TCR or BCR C region specific primer in which a second additional adaptor nucleic acid sequence is added to a third TCR or BCR C region specific sequence,
 wherein the third TCR or BCR C region specific primer is designed to have a sequence that is a complete match with the TCR or BCR C region in a sequence downstream to the sequence of the second TCR or BCR C region specific primer, but comprise a sequence that is not homologous with other genetic sequences, and comprise a mismatching base between subtypes downstream when amplified. 
   
     
     
         3 . The method of  claim 1 , wherein the single sequencing is characterized in that at least one of the sequences used as a primer in amplification from the nucleic acid sample into a sample for sequencing has the same sequence as a nucleic acid sequence encoding a C region or a complementary strand thereof. 
     
     
         4 . The method of  claim 1 , wherein the unbiased amplification is not V region specific amplification. 
     
     
         5 . The method of  claim 1 , wherein the repertoire is the repertoire of a variable region of a BCR, and the nucleic acid sequence is a BCR nucleic acid sequence. 
     
     
         6 . The method of  claim 1 , wherein (3) derivation of the TCR or BCR repertoire is accomplished by a method comprising the following steps:
 (3-1) providing a reference database for each gene region comprising at least one of a V region, a D region, a J region and optionally a C region;   (3-2) providing an input sequence set which is optionally trimmed and optionally extracted to have a suitable length;   (3-3) searching for homology of the input sequence set with the reference database for the each gene region and recording an alignment with an approximate reference allele and/or a sequence of the reference allele;   (3-4) assigning the V region and the J region for the input sequence set and extracting a nucleic acid sequence of the D region based on a result of assigning;   (3-5) translating the nucleic acid sequence of the D region into an amino acid sequence and classifying the D region by utilizing the amino acid sequence; and   (3-6) calculating a frequency of appearance for each of the V region, the D region, and the J region and optionally the C region or a frequency of appearance of a combination thereof based on the classifying in (3-5) to derive the TCR or BCR repertoire.   
     
     
         7 . A system for quantitatively analyzing a repertoire of a variable region of a T cell receptor (TCR) or a B cell receptor (BCR) of a subject by using a database, wherein the system comprises:
 (1) a kit for providing a nucleic acid sample comprising a nucleic acid sequence of the T cell receptor (TCR) or the B cell receptor (BCR) which is amplified from the subject in an unbiased manner;   (2) an apparatus for determining the nucleic acid sequence comprised in the nucleic acid sample; and   (3) an apparatus for calculating a frequency of appearance of each gene or a combination thereof based on the determined nucleic acid sequence to derive a TCR or BCR repertoire of the subject,   wherein the nucleic acid sample comprises nucleic acid sequences of a plurality of types of T cell receptors (TCR) or B cell receptors (BCR) and the step (2) determines the nucleic acid sequence by a single sequencing performed with a common adaptor primer.   
     
     
         8 . The system of  claim 7 , wherein the repertoire is the repertoire of a variable region of a BCR, and the nucleic acid sequence is a BCR nucleic acid sequence. 
     
     
         9 . The system of  claim 7 , wherein the kit comprises the following:
 (1-1) means for synthesizing a complementary DNA by using an RNA sample derived from a target cell as a template;   (1-2) means for synthesizing a double stranded complementary DNA by using the complementary DNA as a template;   (1-3) means for synthesizing an adaptor-added double stranded complementary DNA by adding a common adaptor primer sequence to the double stranded complementary DNA;   (1-4) means for performing a first PCR amplification reaction by using the adaptor-added double stranded complementary DNA, a common adaptor primer consisting of the common adaptor primer sequence, and a first TCR or BCR C region specific primer,
 wherein the first TCR or BCR C region specific primer is designed to comprise a sequence that is sufficiently specific to a C region of interest of the TCR or BCR and not homologous with other genetic sequences, and comprise a mismatching base between subtypes downstream when amplified; 
   (1-5) means for performing a second PCR amplification reaction by using a PCR amplicon of (1-4), the common adaptor primer, and a second TCR or BCR C region specific primer,
 wherein the second TCR or BCR C region specific primer is designed to have a sequence that is a complete match with the TCR or BCR C region in a sequence downstream the sequence of the first TCR or BCR C region specific primer, but comprise a sequence that is not homologous with other genetic sequences, and comprise a mismatching base between subtypes downstream when amplified; and 
   (1-6) means for performing a third PCR amplification reaction by using a PCR amplicon of (1-5), an added common adaptor primer in which a nucleic acid sequence of the common adaptor primer comprises a first additional adaptor nucleic acid sequence, and an adaptor-added third TCR or BCR C region specific primer in which a second additional adaptor nucleic acid sequence is added to a third TCR or BCR C region specific sequence,
 wherein the third TCR or BCR C region specific primer is designed to have a sequence that is a complete match with the TCR or BCR C region in a sequence downstream to the sequence of the second TCR or BCR C region specific primer, but comprise a sequence that is not homologous with other genetic sequences, and comprise a mismatching base between subtypes downstream when amplified. 
   
     
     
         10 . The system of  claim 7 , wherein (3) an apparatus for deriving the TCR or BCR repertoire comprises the following:
 (3-1) means for providing a reference database for each gene region comprising at least one of a V region, a D region, a J region and optionally a C region;   (3-2) means for providing an input sequence set which is optionally trimmed and optionally extracted to have a suitable length;   (3-3) means for searching for homology of the input sequence set with the reference database for the each gene region and recording an alignment with an approximate reference allele and/or a sequence of the reference allele;   (3-4) means for assigning the V region and the J region for the input sequence set and extracting a nucleic acid sequence of the D region based on a result of assigning;   (3-5) means for translating the nucleic acid sequence of the D region into an amino acid sequence and classifying the D region by utilizing the amino acid sequence; and   (3-6) means for calculating a frequency of appearance for each of the V region, the D region, and the J region and optionally the C region or a frequency of appearance of a combination thereof based on the classifying in (3-5) to derive the TCR or BCR repertoire.   
     
     
         11 . A method of preparing a composition for use in a cancer idiotype peptide sensitization immune cell therapeutic method to a subject, the method comprising:
 (1) analyzing a T cell receptor (TCR) or B cell receptor (BCR) repertoire of the subject by the method of  claim 1 ;   (2) determining a TCR or BCR derived from a cancer cell of the subject, wherein the cancer is leukemia such as Acute T-lymphoblastic leukemia, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, adult T-cell leukemia, T-cell large granular lymphocyte leukemia, and malignant lymphoma, based on a result of the analysis, wherein the determining is done by selecting a high ranking sequence in a frequency of presence ranking of a TCR or BCR gene derived from the cancer cell of the subject as the TCR or BCR derived from the cancer cell;   (3) determining an amino acid sequence of a candidate HLA test peptide based on the determined TCR or BCR derived from cancer, wherein the determining is performed based on a score calculated by using an HLA binding peptide prediction algorithm; and   (4) synthesizing the determined peptide.   
     
     
         12 . A method of preparing an isolated cancer specific TCR gene by an in vitro antigen stimulation, comprising:
 (A) mixing an antigen peptide or antigen protein derived from a subject or the determined peptide of  claim 11  or a lymphocyte derived from the subject, an inactivated cancer cell derived from the subject, and a T lymphocyte derived from the subject and culturing the mixture to produce a tumor specific T cell;   (B) analyzing a TCR of the tumor specific T cell by the method of  claim 1 ; and   (C) isolating a desired tumor specific T cell based on a result of the analyzing.   
     
     
         13 . An in vitro method of preparing a T lymphocyte introduced with a tumor specific TCR gene for use in a cell processing therapeutic method, comprising:
 A) providing a T lymphocyte collected from a patient;   B) analyzing TCRs based on the method of  claim 1  after applying an antigen stimulation to the T lymphocyte, wherein the antigen stimulation is applied by an antigen peptide or antigen protein derived from the subject, an inactivated cancer cell derived from the subject, or an idiotype peptide derived from tumor;   C) selecting an optimal TCR and an optimal antigen in the analyzed TCRs; and   D) producing a tumor specific a and 13 TCR expression viral vector of a TCR gene of the optimal TCR.   
     
     
         14 . The method of  claim 2 , wherein the first, second and third TCR or BCR C region specific primers are each independently a primer for TCR or BCR repertoire analysis, the primer being selected to be a sequence that is a complete match with each isotype C region of IgM, IgG, IgA, IgD or IgE, and for a BCR, a complete match with subtypes for IgG and IgA, and not homologous with other sequences comprised in the database, and comprise a mismatching base between subtypes downstream of the primer, and
 wherein the common adaptor primer sequence is designed such that the sequence has a base length suitable for amplification, is unlikely to have homodimer and intramolecular hairpin structures, and is able to stably form a double strand, and designed not to be highly homologous with all BCR genetic sequences in the database and to have the same level of Tm as the C region specific primer.   
     
     
         15 . The method of  claim 2 , wherein the first TCR or BCR C region specific primer has the following structure: CM1 (SEQ ID NO: 5), CA1 (SEQ ID NO: 8), CG1 (SEQ ID NO: 11), CD1 (SEQ ID NO: 14), CE1 (SEQ ID NO: 17), CA1 (SEQ ID NO: 35) or CB1 (SEQ ID NO: 37). 
     
     
         16 . The method of  claim 2 , wherein the second TCR or BCR C region specific primer has the following structure: CM2 (SEQ ID NO: 6), CA2 (SEQ ID NO: 9), CG2 (SEQ ID NO: 12), CD2 (SEQ ID NO: 15), CE2 (SEQ ID NO: 18), CA2 (SEQ ID NO: 35), or CB2 (SEQ ID NO: 37). 
     
     
         17 . The method of  claim 2 , wherein each of the TCR or BCR C region specific primers is provided in a set compatible with all TCR or BCR subclasses. 
     
     
         18 . The method of  claim 13 , wherein the antigen stimulation is applied with the antigen peptide or antigen protein derived from the subject. 
     
     
         19 . The method of  claim 13 , wherein the antigen stimulation is applied with the inactivated cancer cell derived from the subject. 
     
     
         20 . The method of  claim 13 , wherein the step C) comprises selecting an antigen that is highly expressed in cancer tissue of the subject. 
     
     
         21 . The method of  claim 13 , wherein the step C) comprises selecting an antigen which most strongly activates a T cell in an antigen specific lymphocyte stimulation test.