Isolation of T-Cell Receptors Specifically Reactive with Mutated Tumor-Associated Antigens from Tumor-Infiltrating Lymphocytes Based on CD137 Expression

Abstract

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 IFNγ 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 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.

Figure 1.

Isolation of mutation reactive TCRs from TILs based on CD137 expression after in vitro stimulation. A and B, Treatment TIL from patients 3466 (A) and 3713 (B) were cocultured overnight with autologous DCs electroporated with IVT RNAs encoding TMG constructs previously identified as being recognized: TMG1 (containing mutated COL18A1) for patient 3466 and TMG2 (containing mutated SRPX) for patient 3713. CD3⁺ CD8⁺ CD137⁺ cells were sorted by FACS and expanded in vitro. C and D, The resulting T-cell populations were cocultured overnight with autologous DCs electroporated with IVT RNAs encoding the TMGs, and CD137 expression on CD3⁺CD8⁺ T cells was evaluated by FACS. E, TCR sequences in cDNA from the enriched populations were determined by 5⁻ RACE. F and G,TCRs were cloned into MSGV1 retroviral vectors and used to transduce autologous PBL. Transduction efficiencies were measured by staining cells with an anti-murine TCRβ-constant region antibody. As both the mutated COL18A1 and SRPX epitopes were previously identified as being HLA-A* 0201 restricted, the transduced T-cell populations were evaluated for recognition of peptide-pulsed T2 cells based on IFNgγ secretion.

Figure 2.

Isolation of a single mutated KIF16B-reactive TCR from a tumor biopsy fragment from patient 3784. A, TIL fragment F6 from patient 3784 was cocultured overnight with autologous DCs electroporated with IVT RNA encoding TMG5, and recognition was evaluated on the basis of IFNγ ELISPOT (■) and CD137 expression by FACS (). B, CD3⁺ CD8⁺ CD137⁺ cells were sorted by FACS and expanded in vitro, and the resulting T-cell population was again cocultured overnight with autologous DCs electroporated with IVT RNA encoding TMG5. Recognition was again evaluated on the basis of IFNγ ELISPOT and CD137 expression. C, Recognition of individual 25 amino acid peptides encoded by TMG5 by the enriched T-cell population was evaluated on the basis of IFNγ ELISPOT and CD137 expression after overnight coculture with autologous peptide-pulsed DCs (10 mg/mL pulsed for ~20 hours prior to coculture). D, TCR α and β chain sequences from the enriched population were determined by genomic DNA deep sequencing (Adaptive Biotechnologies), and frequencies (%) of productively rearranged sequences were calculated. E, The dominant TCR was cloned into an MSGV1 retroviral vector and used to transduce PBLs. Transduction efficiency was measured by staining cells with an anti-murine TCRβ constant region antibody. Recognition of the mutated KIF16B 25 mer as well as a shorter 11 mer predicted to bind to HLA-B*0702 was evaluated by TCR-transduced T cells based on IFNγ secretion after overnight coculture with peptide pulsed autologous or HLA-matched allogeneic DCs (10 mg/mL pulsed for ~20 hours prior to coculture for the 25 mer; 10⁻⁶–10 μg/mL pulsed for ~1.5 hours prior to coculture for the 11 mer). Recognition of IFNγ treated (10 ng/mL 24 hours prior to coculture) autologous and allogeneic melanoma cells lines was also evaluated on the basis of IFNγ secretion after overnight coculture.

Figure 3.

Isolation of multiple mutated TFDP2- reactive TCRs from a tumor biopsy fragment from patient 3716. A, TIL fragment F3 from patient 3716 was cocultured overnight with autologous DCs electroporated with IVT RNA encoding TMG3, and recognition was evaluated on the basis of IFNγ ELISPOT and CD137 expression. B, CD3⁺ CD8⁺ CD137⁺ cells were sorted by FACS and expanded in vitro, and the resulting T-cell population was again cocultured overnight with autologous DCs electroporated with IVT RNA encoding TMG3. Recognition was again evaluated on the basis of IFNγ ELISPOT and CD137 expression. C, TCR α and β chain sequences in genomic DNA from the enriched populations were determined by deep sequencing, and frequencies (%) of productively rearranged sequences were calculated. These were then compared to TCR α/β pair sequences identified in cDNA from FrTu digests using PairSeq analysis. The dominant TCR α/β chain pair identified by deep sequencing (TCR 1) was cloned into an MSGV1 retroviral vector as were the three TCR α/β chain pairs identified via PairSeq analysis (indicated by *). D, Retroviral vectors were used to transduce PBL, and transduction efficiencies were measured by staining cells with an anti-murine TCRβ constant region antibody. Recognition of TMG3 and a mutated TFDP2 25 amino acid peptide encoded by TMG3 were evaluated by the TCR- transduced T cells based on IFNγ secretion. In addition, we identified a minimal 9 amino acid HLA-B*1501- restricted epitope from TFDP2 encoded by TMG3. To evaluate the functional avidities of the reactive TCRs, recognition of titrated amounts of this minimal epitope pulsed onto HLA-matched DCs (~1.5 hours prior to coculture) was evaluated on the basis of IFNγ secretion after overnight coculture.

Figure 4.

Isolation of multiple mutated KIAA1279 reactive TCRs from a tumor biopsy from patient 3903. A, TIL fragment F1 from patient 3903 was cocultured overnight with autologous DCs electroporated with IVT RNA encoding TMG9, and recognition was evaluated on the basis of IFNγ ELISPOT and CD137 expression. B, CD3⁺ CD8⁺ CD137⁺ cells were sorted by FACS and expanded in vitro, and the resulting T-cell population was again cocultured overnight with autologous DCs electroporated with IVT RNA encoding TMG9. Recognition was again evaluated based on IFNγ ELISPOT and CD137 expression. C, TCR sequences in genomic DNA from the enriched populations were determined by deep sequencing, and frequencies (%) of productively rearranged sequences were calculated. These were then compared with TCR α/γ pair sequences identified in cDNA from FrTu digests using PairSeq analysis. Five different TCR α/γ chain pairs were cloned into MSGV1 retroviral vectors, including 2 identified using PairSeq analysis (indicated by *). D, Retroviral vectors were used to transduce PBL, and transduction efficiencies were measured by staining cells with an anti-murine TCRγ constant region antibody. Recognition of TMG9 was evaluated by the TCR-transduced T cells based on IFNγ secretion after overnight coculture with electroporated autologous DCs. E, We identified a minimal 8 amino acid HLA-B*3801 restricted epitope from KIAA1279 encoded by TMG9. To evaluate the functional avidities of the reactive TCRs, recognition of titrated amounts of this minimal epitope pulsed onto autologous DCs (~1.5 hours prior to coculture) was evaluated on the basis of IFNγ secretion after overnight coculture.

Figure 5.

Isolation of multiple mutated XPNPEP1-and UGGT2-reactive TCRs from a tumor biopsy fragment from patient 3678. A, TIL fragment F4 from patient 3678 was cocultured overnight with autologous DCs electroporated with IVT RNA encoding TMG9, and recognition was evaluated on the basis of IFNγ ELISPOT and CD137 expression. B, CD3⁺ CD8⁺ CD137⁺ cells were sorted by FACS and expanded in vitro, and the resulting T-cell population was again cocultured overnight with autologous DCs electroporated with IVT RNA encoding TMG9. Recognition was again evaluated on the basis of IFNγ ELISPOT and CD137 expression. C, TCR sequences in genomic DNA from the enriched populations were determined by deep sequencing, and frequencies (%) of productively rearranged sequences were calculated. In addition, single CD8⁺ T cells from the enriched population were sorted, and RT-PCR was conducted to identify TCR α and γ chain pairs. Four different TCR α/γ chain pairs were cloned into MSGV1 retroviral vectors based on single-cell RT-PCR analysis (indicated by *). D, These retroviruses were used to transduce PBL, and transduction efficiencies were measured by staining cells with an anti-murine TCRβ constant region antibody. Recognition of TMG9 and two mutated 25 amino acid peptides encoded by TMG9, UGGT2 and XPNPEP1, were evaluated by the TCR-transduced T cells based on IFNγ secretion after overnight coculture with peptide pulsed autologous DCs. In addition, we identified an HLA-A*0301-restricted 11 mer from XPNPEP1 and an HLA-A*0201 restricted 9 mer from UGGT2, both of which were encoded by TMG9. To evaluate the functional avidities of the reactive TCRs, recognition of titrated amounts of these minimal epitopes pulsed onto HLA-matched DCs (~1.5 hours prior to coculture) was evaluated on the basis of IFNγ secretion after overnight coculture.