Quantitative RT-PCR analysis of expression of mutated KRAS mRNA in pancreatic tumor lines. (A) cDNAs from five KRAS G12V-positive tumor lines and two KRAS G12V-negative tumor lines were synthesized for expression analysis. Mutation status was verified by sequencing of genomic DNA shown in . Primers specific for KRAS gene regardless of mutation status (designated as “Reference”), or for mutated KRAS G12V (designated as “G12V”) were used in the analysis. Results are presented relative to ACTB mRNA (encoding β-actin). (B) Similar analysis of KRAS expression of five KRAS G12D-positive tumor lines and three KRAS G12D-negative tumor lines.

Murine T cells reactive to KRAS G12V or KRAS G12D generated from HLA-A*11:01 transgenic mice by in vivo peptide immunization. (A) IFNγ production of murine T cells from splenocytes or draining lymph node lymphocytes (LN) from peptide-immunized HLA-A*11:01 transgenic mice. Spleen and LN from immunized mice were harvested, and stimulated with different concentrations (1, 0.1 or 0.01μM) of KRAS G12V_7-16 peptide once in vitro. 7 days after in vitro stimulation, T cells were co-cultured with COS7 stably transduced with HLA-A*11:01 (COS7/A11) and KRAS minigenes encoding the 23 N-terminal amino acids of wild type KRAS (WT), mutation variants KRAS G12D and KRAS G12V, and 3 HLA-A*11:01–transduced pancreatic tumor lines carrying KRAS G12V mutations. After overnight incubation, supernatants were harvested and IFNγ production was measured. (B) IFNγ production of murine T cells from splenocytes or LN from HLA-A*11:01 transgenic mice immunized three times with KRAS G12D_7-16 peptide. Spleen and LN from immunized mice were harvested, and stimulated with different concentrations (1, 0.1 or 0.01μM) of KRAS G12D_7-16 peptide once in vitro. Seven days after in vitro stimulation, T cells were cocultured with COS7/A11 transduced with KRAS minigenes, and four HLA-A*11:01–positive pancreatic tumor lines carrying KRAS G12D mutations. After overnight incubation, the supernatant was harvested and IFNγ production was measured.

Characteristics of HLA-A*11:01–restricted KRAS G12V-specific murine TCRs. (A) Expression of human PBL cotransduced with candidate TCR α and β chains. Two oligoclonal α chains and three oligoclonal β chains were identified from murine KRAS G12V-reactive splenocytes (1 μM) by 5′RACE (). All of them were constructed to retroviral vector, pMSGV1, separately. Allogeneic PBLs were stimulated with anti-CD3 (50 ng/ml) for 2 days and cotransduced twice with retroviruses encoding oligoclonal TCR α and β chains at 0.5 × 10⁶ cells per well in a 24-well plate. Three days after transduction, T cells transduced with all six possible TCR pairs were labeled with antibodies to CD3, CD8, and mouse TCRβ, and analyzed on a FACS Canto II. Data was gated on the live CD3⁺ population. (B) Reactivity of PBL cotransduced with oligoclonal TCR α and β chains. Anti-CD3 stimulated human PBL cotransduced with six pairs of α and β chains were cocultured with COS7/A11 transduced with WT, G12D, or G12V minigenes, or pulsed with KRAS wildtype_7-16 (WT_7-16), KRAS G12D_7-16, and KRAS G12V_7-16 10-mer peptides. (C) Affinity comparison of two KRAS G12V-reactive TCRs. Anti-CD3 stimulated human PBL were transduced with retroviruses encoding either TRAV3-3*01/BV4*01 or TRAV19*01/BV13-1*02 TCR as described above. Three days after transduction, TCR-transduced cells were cocultured with COS7/A11 pulsed with 1:10 serial diluted peptides starting from 10⁻⁶ M. (D) Both KRAS G12V-reactive TCRs were HLA-A*11:01–restricted. T cells transduced with either TRAV3-3*01/BV4*01 or TRAV19*01/BV13-1*02 were cocultured with KRAS G12V-positive pancreatic tumor lines transduced with HLA-A*11:01 and their parental HLA-A*11:01–negative tumor lines. (E) Both TCRs were KRAS G12V specific. T cells transduced with either TCR were cocultured with a panel of HLA-A*11:01–positive pancreatic tumor lines with or without the KRAS G12V mutation. (F) Correlation between mutated KRAS expression and IFNγ production by T cells transduced with TRAV3-3*01/BV4*01 and tested against a panel of pancreatic tumor lines with or without G12V mutation (R² = 0.68, P = 0.02). (G) TRAV3-3*01/BV4*01 had CD8 coreceptor–independent reactivity. CD8 or CD4 enrichment was performed on T cells transduced with retrovirus encoding TRAV3-3*01/BV4*01, and then cocultured with COS7/A11 KRAS transfectants and pancreatic tumor lines. From (B) to (G), all functional analysis was done by assessing IFNγ production from the coculture supernatant after overnight incubation. (H) TRAV3-3*01/BV4*01 proliferated upon antigen-specific stimulation. T cells transduced with TRAV3-3*01/BV4*01 were labeled with CFSE, cocultured with various targets for 3 days, and further labeled with antibodies to human CD3 and murine TCRβ, and then analyzed on a FACS Canto II. Data was gated on the live CD3⁺ population. (I) Antigen-specific degranulation of TRAV3-3*01/BV4*01. T cells transduced with TRAV3-3*01/BV4*01 were cocultured with various targets in the presence of anti-CD107a-FITC for 2 hr, labeled with antibodies to human CD3 and to murine TCRβ, and then analyzed on FACS Canto II. Data was gated on live CD3⁺CD8⁺ populations.

Characteristics of HLA-A*11:01–restricted KRAS G12D-reactive murine TCR, TRAV4-4*01/BV12-2*01. (A) Affinity of the KRAS G12D-reactive TCR. Anti-CD3 stimulated human allogeneic PBL were transduced with retrovirus encoding TRAV4-4*01/BV12-2*01. Three days after transduction, TCR-transduced cells were co-cultured with COS7/A11 pulsed with 1:10 serial diluted peptides. (B) TRAV4-4*01/BV12-2*01 was HLA-A*11:01–restricted. TCR-transduced T cells were cocultured with KRAS G12D-positive pancreatic tumor lines with or without HLA-A*11:01 expression. (C) TRAV4-4*01/BV12-2*01 was KRAS G12D specific. TCR-transduced T cells were cocultured with a panel of HLA-A*11:01 expressing pancreatic tumor lines with or without KRAS G12D mutation. (D) Reactivity of KRAS G12D-specific TCR against PANC-1. TCR-transduced T cells were cocultured with PANC-1, PANC-1 pulsed with 10-mer peptides, or PANC-1 transduced to overexpress HLA-A*11:01. (E) Reactivity of KRAS G12D-specific TCR against IFNγ treated pancreatic tumor lines. Pancreatic tumor lines were pre-treated with IFNγ (10ng/ml) for 48hrs, and then cocultured with TCR-transduced T cells. From (A) to (E), supernatant of cocultures were harvested and IFNγ production was assessed. (F) T cells transduced with TRAV4-4*01/BV12-2*01 proliferated upon antigen-specific stimulation. T cells transduced with TRAV4-4*01/BV12-2*01 were labeled with CFSE, cocultured with various targets. Three days after coculture, T cells were labeled with antibodies to human CD3 and to murine TCRβ, and then analyzed on a FACS Canto II. Data was gated on the live CD3⁺ population. (G) Antigen-specific degranulation of TRAV4-4*01/BV12-2*01–transduced T cells. T cells transduced with TRAV4-4*01/BV12-2*01 were cocultured with various targets in the presence of anti-CD107a-FITC for 4 hr, labeled with antibodies to human CD3 and to murine TCRβ, and then analyzed on FACS Canto II. Data was gated on live CD3⁺CD8⁺ populations.

Adoptive cell transfer of TRAV4-4*01/BV12-2*01-transduced cells to NSG mice. (A) Treatment efficacy of TRAV4-4*01/BV12-2*01. Pancreatic tumor line, FA6-2/A11, was injected into NSG mice subcutaneously, and 10 days after inoculation, 1 × 10⁷ T cells transduced with TRAV4-4*01/BV12-2*01 were injected intravenously, following by daily intraperitoneal IL2 injection for 3 days. Mice given no treatment, untransduced T cells, or mock-transduced T cells, served as controls. Serial tumor measurements were obtained, and tumor area calculated. Control groups had 5 mice and the treatment group10 mice. Center Bar = Mean; Error Bars = SEM. (B) Kaplan-Meier analysis of survival in tumor-bearing mice receiving adoptive transferred T cells transduced with TRAV4-4*01/BV12-2*01 versus controls; (TCR-tranduced T cells versus mock tranduced T cells; P <0.0001). “ACT” represents “adoptive cell transfer”. (C) HLA-A11 expression of tumors from treated mice. Tumors from mice which were treated with TRAV4-4*01/BV12-2*01–transduced cells were labeled with antibody to HLA-A11 and analyzed on FACS CantoII; Open: isotype control, Shaded: HLA-A11. FA6-2/A11 was used as the positive control. (D) Presence of transferred cells in treated mice. Spleens and tumors from 4 mice treated as described above were labeled with antibodies to human CD3, human CD8 and to mouse TCR β and analyzed on FACS CantoII. Data was gated on live CD3⁺ T cells.