T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer

Abstract

We identified a polyclonal CD8+ T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer. We observed objective regression of all seven lung metastases after the infusion of approximately 1.11×10¹¹ HLA-C*08:02-restricted tumor-infiltrating lymphocytes that were composed of four different T-cell clonotypes that specifically targeted KRAS G12D. However, one of these lesions had progressed on evaluation 9 months after therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histocompatibility complex (MHC) molecule. The loss of expression of this molecule provided a direct mechanism of tumor immune evasion. Thus, the infusion of CD8+ cells targeting mutant KRAS mediated effective antitumor immunotherapy against a cancer that expressed mutant KRAS G12D and HLA-C*08:02.

Figure 1. Adoptive Transfer of KRAS G12D–Specific T Cells

Panel A shows the flow cytometric analysis of the effector function of tumor-infiltrating lymphocytes in the infusion product with the use of intracellular cytokine staining (including interferon-γ [IFN-γ], tumor necrosis factor [TNF], and interleukin-2 [IL-2]), and cell-surface mobilization of the degranulation marker CD107a after 6-hour coculture with autologous dendritic cells incubated overnight with wild-type (WT) KRAS or KRAS G12D peptides consisting of 24 amino acids. Data are gated on CD8+ T cells. Pie charts represent the percentages of CD8+ cells that expressed the indicated number of effector functions. Panel B shows contrast-enhanced computed tomographic scans of the chest of Patient 4095 before and approximately 6 weeks and 9 months after the infusion of 1.48×10¹¹ tumor-infiltrating lymphocytes; at least 75% of these cells were specific for mutant KRAS G12D. Arrows highlight lung lesions before and after therapy. Shown are four of seven lesions; the remaining three lesions (not shown) had completely regressed at 9 months.

Figure 2. In Vivo Persistence and Reactivity Profile of KRAS G12D–Specific T-Cell Clones in the Infusion Product

Panel A shows the results of deep sequencing of the variable region of the T-cell receptor (TCR) beta chain to measure the frequency of each of the four identified KRAS G12D–reactive T-cell clones in the infusion product (Rx1), in three metastatic lung samples before cell transfer (designated Tu-1, Tu-2A, and Tu-2B), in the one progressing lesion after cell therapy (designated Tu-Pro), and in the peripheral blood of Patient 4095 before and at various times after cell therapy. T-cell receptors are identified according to their gene names (TRBV5-6 and TRBV10-02, with A, B, and C denoting different T-cell receptor clonotypes that share the same T-cell receptor beta variable gene family). The numbers in parentheses indicate the rank of the T-cell receptor sequence in the given sample. A circle with an X and ND denote not detected (<0.0002%). Autologous peripheral-blood T cells were genetically engineered to express each of the indicated KRAS G12D–reactive T-cell receptors that were present in the infusion product. Panel B shows the expression of the T-cell activation marker 4-1BB on T cells engineered with the indicated T-cell receptor after overnight coculture with autologous peripheral-blood mononuclear cells that were incubated with titrated amounts of wild-type KRAS or G12D mutant peptides consisting of either 9 or 10 amino acids (9mer or 10mer). Panel C shows interferon-γ (IFN-γ) production (left) and 4-1BB expression (right) of T cells that were genetically engineered with the indicated T-cell receptor after overnight coculture with two KRAS G12D–positive pancreatic-cancer cell lines not expressing the HLA-C*08:02 allele (mock) or expressing the HLA-C*08:02 allele with the use of enzyme-linked immunosorbent spot (ELISPOT) assay and flow cytometry, respectively. MDA denotes MDA Panc48 cell line, and HP HPAC cell line. Flow cytometry data are gated on CD8+ KRAS G12D–reactive cells. Values of more than 500 on the ELISPOT assay were not considered to be accurate.

Figure 3. Analysis of the Frequency of HLA-C Alleles in Tumor Samples

Shown is the frequency of the two HLA-C alleles in a representative metastatic lesion resected from Patient 4095 before cell therapy (Tu-1) and in the progressing lesion (Tu-Pro). The Tu-1 and Tu-Pro samples were estimated to contain 53% and 34% tumor, respectively, and thus contained some normal cells that also contributed to the calculation of the HLA-C allele frequency. As shown, the progressing lesion harbored cells with a genetic loss of the HLA-C*08:02 allele, which provided a direct mechanism of immune evasion by the tumor, since the infused KRAS G12D–reactive T cells require this molecule for direct tumor recognition. The I bars represent standard errors. The P value for the comparison of the allele frequency of a metastatic lesion before therapy and the progressing lesion after therapy was calculated with the use of the Wilcoxon matched-pairs signed-rank test.