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.