Method and kit for determining whether a subject shows an immune response

Patent metadata

Field	Value
Publication number	US-10550430-B2
Application number	US-201415031415-A
Country	US
Kind code	B2
Filing date	Oct 24, 2014
Priority date	Oct 25, 2013
Publication date	Feb 4, 2020
Grant date	Feb 4, 2020

Abstract

The present invention relates to a kit and a method for determining whether a subject shows an immune response against an antigen.

First claim

The invention claimed is:

1. A method for detecting and quantifying individual HLA isotypes and individual T cell receptor α- and β-chains from a single biological sample comprising the steps of:
(a) obtaining a single biological sample from a human subject;
(b) determining, from the single biological sample, a nucleotide sequence of mRNA encoding HLA-A, HLA-B and HLA-C and a nucleotide sequence of mRNA encoding T cell receptor α- and β-chains transcribed from recombined T cell receptor loci,
wherein step (b) comprises:
(i) generating, from mRNA encoding HLA-A, HLA-B, or HLA-C, a first cDNA strand, the first cDNA strand having a 3′ poly (C) sequence comprising at least 3 C nucleotides at the 3′ end of the cDNA strand, which poly (C) sequence serves as a target sequence for a template switching oligonucleotide, wherein the template switching oligonucleotide comprises at least 3 G nucleotides and serves as a matrix for the elongation of the first cDNA strand at the 3′ end of the poly (C) sequence and introduces a primer binding site at the 3′ end of the first cDNA strand, and
(ii) reproducing the cDNA by polymerase chain reaction, with a forward primer which is capable of hybridizing to the primer binding site; and

(c) quantifying the individual HLA isotypes and individual T cell receptor chains from the single biological sample.

2. The method of claim 1 , wherein step (b)(i) comprises reverse transcription of mRNAs encoding HLA-A, HLA-B and HLA-C with a primer hybridizing to a target sequence within exon 3 or exon 4 of HLA-I and a reverse transcriptase that has terminal transferase activity.

3. The method of claim 2 , wherein the primer hybridizing to a target sequence within exon 3 or exon 4 of HLA-I comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 29.

4. A method of treating the subject by adoptive T cell therapy, comprising administering to the subject a T cell, said T cell having individual T cell receptor α- and β-chains identified and quantified by a method as defined in claim 1 , wherein said subject has or is suspected of having a disease or condition characterized by an antigen recognized by said T cell.

5. The method of claim 1 , wherein the single biological sample is obtained from the subject after the subject has been exposed to a tumor-associated antigen.

6. The method of claim 5 , wherein the tumor-associated antigen is expressed in at least tissue from which the sample has been obtained.

7. A method for detecting and quantifying individual HLA isotypes and individual T cell receptor α- and β-chains from a single biological sample comprising the steps of:
(a) obtaining a single biological sample from a human subject;
(b) determining, from the single biological sample, a nucleotide sequence of mRNA encoding HLA-A, HLA-B and HLA-C and a nucleotide sequence of mRNA encoding T cell receptor α- and β-chains transcribed from recombined T cell receptor loci,
wherein step (b) comprises:
(i) generating, from mRNA encoding a TCRα chain or TCRβ chain, a first cDNA strand, the first cDNA strand having a 3′ poly (C) sequence comprising at least 3 C nucleotides at the 3′ end of the cDNA strand, which poly (C) sequence serves as a target sequence for a template switching oligonucleotide, wherein the template switching oligonucleotide comprises at least 3 G nucleotides and serves as a matrix for the elongation of the first cDNA strand at the 3′ end of the poly (C) sequence and introduces a primer binding site at the 3′ end of the first cDNA strand, and
(ii) reproducing the cDNA by polymerase chain reaction, with a forward primer which is capable of hybridizing to the primer binding site.

8. The method of claim 7 , wherein step (b)(i) comprises reverse transcription of mRNAs encoding T cell receptor chains with a primer hybridizing to a target sequence within a constant region of the respective T cell receptor chain and a reverse transcriptase that has terminal transferase activity.

9. The method of claim 8 , wherein the primer hybridizing to a target sequence within a constant region of the respective T cell receptor chain comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

10. A method of treating the subject by adoptive T cell therapy, comprising administering to the subject a T cell, said T cell having individual T cell receptor α- and β-chains identified and quantified by a method as defined in claim 7 , wherein said subject has or is suspected of having a disease or condition characterized by an antigen recognized by said T cell.

11. The method of claim 7 , wherein the single biological sample is obtained from the subject after the subject has been exposed to a tumor-associated antigen.

12. The method of claim 11 , wherein the tumor-associated antigen is expressed in at least tissue from which the sample has been obtained.

13. A method for detecting and quantifying individual HLA isotypes and individual T cell receptor α- and β-chains from a single biological sample comprising the steps of:
(a) obtaining a single biological sample from a human subject;
(b) determining, from the single biological sample, a nucleotide sequence of mRNA encoding HLA-A, HLA-B and HLA-C and a nucleotide sequence of mRNA encoding T cell receptor α- and β-chains transcribed from recombined T cell receptor loci,
wherein step (b) comprises:
(i) generating, from mRNA encoding HLA-A, HLA-B, or HLA-C, an HLA-first cDNA strand, the HLA-first cDNA strand having a 3′ poly (C) sequence comprising at least 3 C nucleotides at the 3′ end of the HLA-first cDNA strand, which poly (C) sequence serves as a target sequence for a template switching oligonucleotide, wherein the template switching oligonucleotide comprises at least 3 G nucleotides and serves as a matrix for the elongation of the HLA-first cDNA strand at the 3′ end of the poly (C) sequence and introduces a primer binding site at the 3′ end of the HLA-first cDNA strand,
(ii) generating, from mRNA encoding a TCRα chain or TCRβ chain, a TCR-first cDNA strand, the TCR-first cDNA strand having a 3′ poly (C) sequence comprising at least 3 C nucleotides at the 3′ end of the TCR-first cDNA strand, which poly (C) sequence serves as a target sequence for a template switching oligonucleotide, wherein the template switching oligonucleotide comprises at least 3 G nucleotides and serves as a matrix for the elongation of the TCR-first cDNA strand at the 3′ end of the poly (C) sequence and introduces a primer binding site at the 3′ end of the TCR-first cDNA strand, and
(iii) reproducing the HLA-cDNA and TCR-cDNA by polymerase chain reaction, with a primer which is capable of hybridizing to the respective primer binding sites.

14. The method of claim 13 , wherein step (b)(i) comprises reverse transcription of mRNAs encoding HLA-A, HLA-B and HLA-C with a primer hybridizing to a target sequence within exon 3 or exon 4 of HLA-I and a reverse transcriptase that has terminal transferase activity.

15. The method of claim 13 , wherein step (b)(ii) comprises reverse transcription of mRNAs encoding T cell receptor chains with a primer hybridizing to a target sequence within a constant region of the respective T cell receptor chain and a reverse transcriptase that has terminal transferase activity.

16. The method of claim 14 , wherein the primer hybridizing to a target sequence within exon 3 or exon 4 of HLA-I comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 29.

17. The method of claim 15 , wherein the primer hybridizing to a target sequence within a constant region of the respective T cell receptor chain comprises the nucleo

Assignees

• Biontech Ag
• Tron—Translationale Onkologie An Der Univ Der Johannes Gutenberg—Universitaet Mainz Ge
• Biontech Diagnostics Gmbh
• Tron—Translationale Onkologie An Der Univ Der Johannes Gutenberg—Universitat Mainz Gem

Inventors

• Sahin Ugur
• Schäfer Michael
• Faryna Marta Magdalena
• Omokoko Tana
• Hebich Lisa
• Simon Petra
• Reichardt Annett
• Öz Özlem

Classifications

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6881 Primary

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/112

  Disease subtyping, staging or classification · CPC title