Isolation of T-Cell Receptors Specifically Reactive with Mutated Tumor-Associated Antigens from Tumor-Infiltrating Lymphocytes Based on CD137 Expression.
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 within vitrotranscribed (IVT) mRNAs encoding TMGs and were evaluated for IFNγ secretion and CD137 expression. Neoantigen-reactive T cells were enriched from TILs by sorting for CD137+CD8+T cells and expandedin vitroDominant TCR α and β 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.Clin Cancer Res; 23(10); 2491-505. ©2016 AACR.
NIH/NCI Surgery Branch, Bethesda, Maryland. Maria_Parkhurst@nih.gov.
Maria
M
Parkhurst
NIH/NCI Surgery Branch, Bethesda, Maryland.
Alena
A
Gros
NIH/NCI Surgery Branch, Bethesda, Maryland.
Anna
A
Pasetto
NIH/NCI Surgery Branch, Bethesda, Maryland.
Todd
T
Prickett
NIH/NCI Surgery Branch, Bethesda, Maryland.
Jessica S
JS
Crystal
NIH/NCI Surgery Branch, Bethesda, Maryland.
Paul
P
Robbins
NIH/NCI Surgery Branch, Bethesda, Maryland.
Steven A
SA
Rosenberg
D019264:Adoptive Transfer / Q000379:methods; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology; D059472:Exome / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics / Q000276:immunology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016219:Immunotherapy, Adoptive; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology*; D008545:Melanoma / Q000235:genetics / Q000276:immunology / Q000628:therapy*; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000276:immunology
D000951:Antigens, Neoplasm; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9