Systematic discovery and validation of T cell targets directed against oncogenic KRAS mutations

Abstract

Oncogenic mutations in KRAS can be recognized by T cells on specific class I human leukocyte antigen (HLA-I) molecules, leading to tumor control. To date, the discovery of T cell targets from KRAS mutations has relied on occasional T cell responses in patient samples or the use of transgenic mice. To overcome these limitations, we have developed a systematic target discovery and validation pipeline. We evaluate the presentation of mutant KRAS peptides on individual HLA-I molecules using targeted mass spectrometry and identify 13 unpublished KRAS^G12C/D/R/V mutation/HLA-I pairs and nine previously described pairs. We assess immunogenicity, generating T cell responses to nearly all targets. Using cytotoxicity assays, we demonstrate that KRAS-specific T cells and T cell receptors specifically recognize endogenous KRAS mutations. The discovery and validation of T cell targets from KRAS mutations demonstrate the potential for this pipeline to aid the development of immunotherapies for important cancer targets.

Keywords: HLA; PRM; RAS; T cell; TCR; cancer; immunotherapy; mass spectrometry; neoantigen.

Graphical abstract

Figure 1

Establishment of a pipeline to discover and validate KRAS neoantigens Overview of the cell-based systematic pipeline established to discover and validate KRAS neoantigens using targeted MS (top) and an in vitro T cell stimulation protocol (middle). A375 cells were engineered to simultaneously express four KRAS^G12C/D/R/V mutations and an HLA-I molecule of interest. After purifying HLA-I peptides, targeted MS was used to detect individual KRAS neoantigens (lines with a colored box) in the presence of endogenous peptides that are also presented by the transduced HLA-I molecules (gray lines). An in vitro stimulation protocol was used to elicit naive T cell responses against the detected KRAS neoantigens. For a subset of the detected neoantigens (dashed arrows represent expanded pipeline), a cytotoxicity assay was performed to show that induced T cells can kill tumor cell lines that naturally express the KRAS^G12V mutation. Cytotoxicity assays were also used to demonstrate that cloned TCRs can kill tumor cells that naturally present the KRAS^G12V neoantigen.

Figure 2

Representative targeted MS data Targeted MS was used to detect the 10mer KRAS^G12V HLA-A3 superfamily neoantigen VVVGAVGVGK in (A) A375 cells engineered to express both the KRAS^G12V mutation and HLA-A∗11:01, (B) SW620 cells that naturally express the KRAS^G12V mutation but were engineered to express HLA-A∗11:01, and (C) NCI-H441 cells that naturally express both the KRAS^G12V mutation and HLA-A∗03:01. The top panels specify the KRAS mutation and HLA-I molecule present in each sample. The middle panels contain the mass spectra for the peptide detected in each sample (top), and the mass spectra for the heavy isotope-labeled synthetic peptide (bottom) used to confirm the detections. The bottom panels show mass error in parts per million (ppm) of the a-ions (purple), b-ions (blue), and y-ions (red) for the endogenous (top) and heavy isotope-labeled synthetic (bottom) mass spectra. Data from the same heavy isotope-labeled synthetic peptide was used in this figure. See also Data S2.

Figure 3

Evaluation of KRAS neoantigen immunogenicity in healthy donors (A) Flow cytometry plots of pHLA staining of KRAS neoantigens after naive T cell inductions in healthy donors with the appropriate HLA-I molecules. Multimer-positive populations are circled in red. Naive T cell inductions and multimer analysis were performed for all neoantigens detected by MS, except for KRAS^G12C on HLA-A∗68:01. See also Figure S3. (B) Cytotoxicity assay evaluating the specificity of multiple independent T cell cultures stimulated against KRAS^G12V on HLA-A∗11:01. T cell cultures with identifiable multimer-positive populations (left) were co-cultured with A375 cells transduced to express HLA-A∗11:01 and loaded with increasing amounts of the KRAS^G12V/HLA-A∗11:01 neoantigens (red dots) or a single concentration of the matching wild-type peptides (blue dots). Data are presented as mean ± SD. (C–E) Cytotoxicity assay evaluating the ability of a representative T cell culture to recognize endogenously processed and presented KRAS^G12V neoantigens. (C) Total HLA-I expression was evaluated on SW620 cells transduced to express HLA-A∗11:01 (red) compared with non-transduced SW620 cells (blue) or isotype control-stained cells (orange). See also Figure S1. (D and E) Cytotoxicity assays used to evaluate the ability of representative T cell cultures to recognize endogenously processed and presented KRAS^G12V neoantigens where (D) T cells were co-cultured with SW620^A∗11:01 cells and annexin V was measured over time (red) compared with SW620^A∗11:01 cells alone (blue) and (E) T cells were co-cultured with NCI-H441 cells, and annexin V was measured over time (red) compared with NCI-H441 cells alone (blue). Data are presented as mean ± SD and are representative of two independent experiments. See also Figure S4.

Figure 4

Characterization of KRAS-specific TCRs on common alleles (A–D) TCRs isolated from healthy donor T cells stimulated against different KRAS neoantigens were transduced into Jurkat^CD8 cells. TCR avidity was evaluated by titration of each cognate peptide presented on A375 cells transduced to express the cognate HLA-I molecule. TCR stimulation was assessed by IL-2 secretion or CD69 expression, and half-maximal effective concentration was calculated. (A) TCR specific for KRAS^G12V on HLA-A∗11:01. (B) TCR specific for KRAS^G12V on HLA-A∗03:01. (C) TCR specific for KRAS^G12D on HLA-A∗11:01. (D) TCR specific for KRAS^G12D on HLA-A∗03:01. Data are presented as mean ± SD and are representative of at least two independent experiments. See also Figure S5. (E) The TCR from (A) was transduced into PBMCs and used in a cytotoxicity assay against SW620^A∗11:01 across a range of specific effector to target (E:T) ratios (red). The ratio was calculated using the number of TCR-transduced CD8+ T cells in the well. As a control, an equivalent number of PBMCs transduced with an irrelevant TCR was co-cultured with SW620^A11:01 cells (blue). Both target cell growth (top) and target cell death (bottom) were measured. Data are presented as mean ± SD and are representative of three independent experiments. (F) The TCR from (B) was transduced into PBMCs and used in a cytotoxicity assay against SW620^A∗03:01 (red), and cell death of the target cell was measured. As a negative control, PBMCs transduced with the TCR from (A) were co-cultured with SW620^A∗03:01 (blue). Data are presented as mean ± SD and are representative of two independent experiments. (G) The TCR from (B) was transduced into PBMCs and used in a cytotoxicity assay against NCI-H441 (red), and cell death of the target cell was measured. As a negative control, PBMCs transduced with the TCR from (A) were co-cultured with NCI-H441 (blue). Data are presented as mean ± SD and are representative of two independent experiments.