Tumor- and Neoantigen-Reactive T-cell Receptors Can Be Identified Based on Their Frequency in Fresh Tumor

Abstract

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8(+)PD-1(+) T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734-43. ©2016 AACR.

Fig. 1. Strategy overview and TIL characterization

(A) Schematic representation of the multi-step process used to identify tumor reactive TCRs. 1) TCRB deep sequencing on bulk TIL and sorted CD8^+/− TIL and CD8+PD-1^+/− TIL populations is used to determine the subset with evidence of clonal expansion and to identify the TCRB sequences of the most dominant clonotypes within that subset. 2) The most dominant TCRB clonotypes in CD8⁺PD-1⁺ TIL are paired with TCRA chains identified by single cell RT-PCR and pairSEQ. 3) TCRA-TCRB pairs are cloned into expression vectors and engineered into T cells. 4) Engineered T cells are tested for tumor reactivity against tumor cell lines, shared tumor antigens and mutated neoantigens. (B) Unique productive TCRB clonotypes sequences are plotted for bulk TIL and sorted TIL subsets. Productive sequences do not contain stop codons or out of frame shifts so they are likely to be functional. These unique sequences represent a single, unique clonotype independent of its frequency in the samples. Wilcoxon matched-pairs signed rank test was applied (n = 10). For samples 1913 and 3922 the CD8⁻ subset was not available. (C) Total reads of TCRB clonotypes sequences are plotted for melanoma bulk TIL and sorted TIL subsets. Wilcoxon matched-pairs signed rank test was applied (n = 10). For samples 1913 and 3922 the CD8- subset was not available.

Fig. 2. Tumor-specific target recognition assay for reconstructed TCR pairs for patient 3998

(A) CD137 up-regulation on CD8⁺mTCRB⁺ cells is shown after co-culture with autologous tumor cell line for 8 of the TCR pairs reconstructed within the top 10 CD8⁺PD-1⁺ TIL for this tumor sample: the 1^st, 2^nd, 3^rd (in combination with 2 TCRAs), 4^th, 6^th, 7^th and 8^th most frequent TCRBs. Values are reported as mean ± SEM, the assay was done in duplicate. (B) CD137 up-regulation on CD8⁺mTCRB⁺ cells is shown after co-culture with autologous B cells transfected with tandem minigenes (TMG-1 to 7) encoding for 115 non-synonymous mutations for 6 of the TCR pairs reconstructed (3998-1, 3998-2, 3998-4, 3998-6, 3998-7, 3998-8). Values are reported as mean ± SEM, the assay was done in duplicate. (C) CD137 up-regulation is inhibited by pan MHC-I antibody. MHC-I restricted DMF5 TCR is reported as positive control and MHC-II restricted TCR MAGE-A3 is reported as negative control. * = greater than 50% inhibition. Values are reported as mean ± SEM, the assay was done in duplicate. (D) Murine TCRB expression and CD137 up-regulation are shown for reconstructed TCR pair 3998-8 after co-culture with, unpulsed autologous B cells, autologous B cells pulsed with 1 μg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, and 0.1 ng/ml of the mutated MAGEA6 peptide (KVDPIGHVY) and wild type MAGEA6 peptide (EVDPIGHVY) respectively.

Fig. 3. Summary of tumor and mutation reactivity for reconstructed TCR pairs

For every sample analyzed the graph represents the TCRB frequency of the top 10 CD8⁺PD-1⁺ clonotypes and color-coded their reactivity against autologous TC lines, shared melanoma/melanocyte and cancer-germline antigens, and tumor-specific mutations. All patients, except 3678, had a corresponding autologous TC line used for testing the TCR pairs. In 11 of 12 patients up to 5 tumor-reactive TCRs were found in the 5 most frequently expressed TCRs and this included recognition of mutated neoantigens in 5 of the patients. In 2 patients reactivity against MART-1 (3922-1) and NY-ESO-1 (3998-5) was also found. The most frequent TCR clonotype was found to be tumor reactive for 7 patients.