US 12,297,427 B2
Cloning and expression system for T-cell receptors
Paul Thomas, Memphis, TN (US); Xizhi Guo, Memphis, TN (US); and Pradyot Dash, Memphis, TN (US)
Assigned to ST. JUDE CHILDREN'S RESEARCH HOSPITAL, INC., Memphis, TN (US)
Appl. No. 15/780,938
Filed by St. Jude Children's Research Hospital, Inc., Memphis, TN (US)
PCT Filed Dec. 2, 2016, PCT No. PCT/US2016/064735
§ 371(c)(1), (2) Date Jun. 1, 2018,
PCT Pub. No. WO2017/096239, PCT Pub. Date Jun. 8, 2017.
Claims priority of provisional application 62/263,318, filed on Dec. 4, 2015.
Prior Publication US 2019/0040381 A1, Feb. 7, 2019
Int. Cl. C40B 50/06 (2006.01); C07K 14/725 (2006.01); C12N 15/10 (2006.01); C12N 15/66 (2006.01)
CPC C12N 15/1031 (2013.01) [C07K 14/7051 (2013.01); C12N 15/10 (2013.01); C12N 15/1003 (2013.01); C12N 15/66 (2013.01); C12N 2310/11 (2013.01); C40B 50/06 (2013.01)] 11 Claims
OG exemplary drawing
 
1. A method for cloning a T cell receptor (TCR) from a single T cell, wherein said method comprises:
(a) performing RT-PCR with a primer mixture on a single T cell to simultaneously obtain both α-chain and β-chain TCR DNA sequences or both γ-chain and δ-chain TCR DNA sequences, wherein said TCR DNA sequences comprise a partial variable (V) region, CDR3 region, and a partial constant (C) region, wherein the primer mixture comprises:
(i) sense primers comprising T-cell receptor alpha variable (TRAV) sequences and/or T cell receptor beta variable (TRBV) sequences and antisense primers comprising T-cell receptor alpha constant (TRAC) sequences and/or T-cell receptor beta constant (TRBC) sequences, wherein the TRAV and/or TRBV sense primers and the TRAC and/or TRBC antisense primers are selected from the primers comprising SEQ ID NO: 27-36, 167-184, and 187-315, or
(ii) sense primers comprising T-cell receptor gamma variable (TRGV) sequences and/or T cell receptor delta variable (TRDV) sequences and antisense primers comprising T-cell receptor gamma constant (TRGC) sequences and/or T-cell receptor delta constant (TRDC) sequences, wherein the TRGV and/or TRDV sense primers and the TRGC and/or TRDC antisense primers are selected from the primers having SEQ ID NO: 1-38 and 147-186,
(b) optionally sequencing the RT-PCR product obtained in step (a), and
(c) cloning the paired αβ or γδ full length TCR CDR3 DNA sequences obtained in step (a) into a corresponding TCRαβ or TCRγδ library.