TY - JOUR
T1 - Isolation of T-cell receptors specifically reactive with mutated tumor-associated antigens from tumor-infiltrating lymphocytes based on CD137 expression
AU - Parkhurst, Maria
AU - Gros, Alena
AU - Pasetto, Anna
AU - Prickett, Todd
AU - Crystal, Jessica S.
AU - Robbins, Paul
AU - Rosenberg, Steven A.
N1 - Publisher Copyright:
©2016 AACR.
PY - 2017/5/15
Y1 - 2017/5/15
N2 - Purpose: The adoptive transfer of lymphocytes genetically modified to express tumor reactive T-cell receptors (TCR) can mediate tumor regression. Some tumor-infiltrating lymphocytes (TIL) recognize somatic mutations expressed only in the patient's tumors, and evidence suggests that clinically effective TILs target tumor-specific neoantigens. Here we attempted to isolate neoantigen-reactive TCRs as a prelude to the treatment of patients with autologous T cells genetically modified to express such TCRs. Experimental Design: Mutations expressed by tumors were identified using whole-exome and RNA sequencing. Tandem minigene (TMG) constructs encoding 12-24 mutated gene products were synthesized, each encoding the mutated amino acid flanked by 12 amino acids of the normal protein sequence. TILs were cultured with autologous dendritic cells (DC) transfected with in vitro transcribed (IVT) mRNAs encoding TMGs and were evaluated for IFNg secretion and CD137 expression. Neoantigen-reactive T cells were enriched from TILs by sorting for CD137+ CD8+ T cells and expanded in vitro. Dominant TCR a and b chains were identified in the enriched populations using a combination of 5′ rapid amplification of cDNA ends, deep sequencing of genomic DNA, PairSeq analysis, and single-cell RT-PCR analysis. Human PBL retrovirally transduced to express the TCRs were evaluated for recognition of relevant neoantigens. Results: We identified 27 TCRs from 6 patients that recognized 14 neoantigens expressed by autologous tumor cells. Conclusions: This strategy provides the means to generate T cells expressing neoantigen-reactive TCRs for use in future adoptive cell transfer immunotherapy trials for patients with cancer.
AB - Purpose: The adoptive transfer of lymphocytes genetically modified to express tumor reactive T-cell receptors (TCR) can mediate tumor regression. Some tumor-infiltrating lymphocytes (TIL) recognize somatic mutations expressed only in the patient's tumors, and evidence suggests that clinically effective TILs target tumor-specific neoantigens. Here we attempted to isolate neoantigen-reactive TCRs as a prelude to the treatment of patients with autologous T cells genetically modified to express such TCRs. Experimental Design: Mutations expressed by tumors were identified using whole-exome and RNA sequencing. Tandem minigene (TMG) constructs encoding 12-24 mutated gene products were synthesized, each encoding the mutated amino acid flanked by 12 amino acids of the normal protein sequence. TILs were cultured with autologous dendritic cells (DC) transfected with in vitro transcribed (IVT) mRNAs encoding TMGs and were evaluated for IFNg secretion and CD137 expression. Neoantigen-reactive T cells were enriched from TILs by sorting for CD137+ CD8+ T cells and expanded in vitro. Dominant TCR a and b chains were identified in the enriched populations using a combination of 5′ rapid amplification of cDNA ends, deep sequencing of genomic DNA, PairSeq analysis, and single-cell RT-PCR analysis. Human PBL retrovirally transduced to express the TCRs were evaluated for recognition of relevant neoantigens. Results: We identified 27 TCRs from 6 patients that recognized 14 neoantigens expressed by autologous tumor cells. Conclusions: This strategy provides the means to generate T cells expressing neoantigen-reactive TCRs for use in future adoptive cell transfer immunotherapy trials for patients with cancer.
UR - http://www.scopus.com/inward/record.url?scp=85020385407&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85020385407&partnerID=8YFLogxK
U2 - 10.1158/1078-0432.CCR-16-2680
DO - 10.1158/1078-0432.CCR-16-2680
M3 - Article
C2 - 27827318
AN - SCOPUS:85020385407
SN - 1078-0432
VL - 23
SP - 2491
EP - 2505
JO - Clinical Cancer Research
JF - Clinical Cancer Research
IS - 10
ER -