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Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for i

Issuing time:2021-07-22 11:42


Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαβ isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein.


The RAS family of GTPases (KRAS, NRAS, HRAS) is mutated in approximately 20% of all human malignancies1,2. The vast majority of RAS genomic alterations are the result of missense mutations at codon positions G12, G13, or Q61 that involve the RAS protein GTP-binding domain, resulting in the activation of downstream effector substrates ERK and PI3-K leading to dysregulated cell growth and survival3. Among cancers in which KRAS mutations predominate, including pancreatic ductal adenocarcinomas (PDA), lung adenocarcinomas (LAC), and colorectal carcinomas (CRC), over 75% of amino acid substitutions occur at the G12 codon position4. The high frequency of G12 amino acid substitutions makes this codon position an ideal drug target; however, no small molecule inhibitors of G12 variants have been successfully developed aside from KRAS G12C that has only recently demonstrated clinical promise5,6.

Somatic gene mutations within cancer cells may be translated into peptides that are processed and presented on the surface of tumor cells7,8. These mutated peptides can serve as foreign epitopes, or neoantigens, that may be recognized by αβ T cells of the host immune system. Neoantigen-specific T cell responses have been well documented in patients for whom immune checkpoint blockade therapy has been successful, and they are believed to be the key mediators of anti-tumor activity9. Neoantigen-specific T cells can be isolated from the peripheral blood or tumor tissue of antigen-exposed cancer patients10, induced and expanded from the peripheral blood of cancer patients following neoantigen vaccination11, and generated in vitro utilizing the naive T-cell receptor (TCR) repertoire of healthy donors12. These observations have garnered interest in the development of neoantigen-targeted cancer vaccines and adoptive T cell therapies.

Neoantigen-based treatment strategies are inherently highly personalized as somatic tumor mutations are rarely shared between patients13,14. The high prevalence and conserved mutational profile of KRAS affords a unique opportunity to develop a neoantigen-targeted therapy with broad generalizability. Mutant KRAS (mKRAS) has been previously studied as a potential target of cancer vaccines, and clinical studies have demonstrated the successful generation of CD4+ and CD8+ αβ T cell responses with reactivity against allogeneic or autologous mKRAS tumor cell lines15,16,17,18. More recently, mKRAS-specific T cells have been isolated and characterized from the peripheral blood of patients with mKRAS epithelial cancers19,20, and they have been induced in vaccinated HLA-transgenic mice21. The therapeutic potential of targeting mKRAS as a cancer neoantigen was highlighted in a case report demonstrating clinical benefit in a patient with KRAS G12D metastatic CRC following the adoptive transfer of KRAS G12D-specific T cells restricted to HLA-C*08:0222. However, mKRAS as an immunological target remains poorly characterized, and there is a dearth of evidence regarding tumor cell processing and presentation of mKRAS-derived epitopes to guide the development of targeted immunotherapies.

In this work, we aim to validate mKRAS as an immunological target. Utilizing computational epitope prediction, biochemical assays, and proteomic analysis, we predict and identify high-affinity and/or high-stability mKRAS G12 peptides to HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02, and we confirm these epitopes as constituents of their corresponding HLA class I ligandome. The induction of mKRAS-reactive T cells from healthy donors by select peptides both confirms peptide immunogenicity and enables the isolation of mKRAS-specific TCRs directed against the following three epitopes: G12V/HLA-A*03:01, G12V/HLA-A*11:01, and G12R/HLA-B*07:02. Expression of these mKRAS-specific TCRαβ pairs in a NFAT-inducible reporter T cell line or TCRαβnull primary CD8+ T cells serves as sensitive probes to characterize the presentation of mKRAS G12 epitopes by tumor cell lines. TCR-redirected T cells display cytotoxic function against mKRAS tumor cell lines of various histologies without reactivity to wild-type (WT) KRAS peptides, thereby authenticating mKRAS G12V and G12R peptide ligands as bona fide neoantigens. Finally, the adoptive transfer of TCR redirected CD8+ human T cells specific for G12V/HLA-A*03:01 or G12V/HLA-A*11:01 leads to tumor eradication and prolongs survival in a mouse xenograft model of metastatic lung cancer. These findings provide further validation and strengthen the nomination of mKRAS as an immunological target.


Identification and characterization of mKRAS neoepitopes

Although predictive peptide-HLA (p-HLA) binding affinity and stability are important criteria for neoantigen nomination23,24,25, only a small percentage of the human proteome is presented by HLA molecules26. Therefore, only a fraction of candidate neoantigens are constituents of the tumor HLA ligandome27, and predicting high-affinity/stability p-HLA complexes is not indicative of intracellular processing and presentation28. To improve the identification of bona fide mKRAS neoepitopes, we employed a comprehensive multi-omic approach consisting of (1) computational epitope prediction, (2) biochemical assessment of p-HLA binding / stability, (3) proteomic validation of antigen processing and presentation, and (4) characterization of immunogenicity (Supplementary Fig. 1).

We utilized the neoantigen prediction tool antigen.garnish24 to perform a comprehensive identification of candidate HLA class I allele-restricted mKRAS G12 epitopes with recurrent amino acid substitutions (G12C, G12D, G12R, G12V) embedded within the 24 amino acid sequence derived from the WT KRAS protein. Epitopes derived from these 24-mer amino acid sequences were evaluated for predicted binding to HLA class I alleles to nominate HLA-A, HLA-B, and HLA-C nonamer (9-mer) and/or decamer (10-mer) mKRAS peptides (Supplementary Data 1, candidates <500 nM affinity). Predicted epitopes restricted to HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02 were selected for further study given the high prevalence of these HLA class I alleles within the USA population (Fig. 1a). Peptides encoding these candidate epitopes are designated by their KRAS protein amino acid positions followed by the G12 amino acid substitution (Fig. 1b). The predicted HLA class I affinities and p-HLA complex stabilities of candidate epitopes are presented in Supplementary Tables 1 and 2.

Fig. 1: Identification, characterization, and validation of candidate mKRAS epitopes.

a Heat maps displaying candidate WT and mKRAS epitopes with predicted HLA class I binding affinity (red) to the indicated alleles as determined by antigen.garnish, USA population HLA class I allele frequencies (blue) are as reported in the Allele Frequency Net Database, and mKRAS G12 variant frequencies (green) in pancreatic adenocarcinoma (PDA; n = 836), colorectal cancer (CRC; n= 866), and lung adenocarcinoma (LAC; n = 516) patients are as reported in the ICGC Data Portal. bGraphic depiction of the KRAS 24-mer containing candidate epitopes restricted by alleles shown in (a). The G12 codon is indicated in red. Epitopes are identified by colored lines, and amino acid positions as well as HLA class I restricting alleles are indicated. c Peptide affinity measurements of mKRAS epitopes to HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02. p-HLA affinities were assessed by fluorescence polarization competitive peptide-binding assays30. High peptide affinity is classified as a log10[IC50] < 3.7 nM and is indicated by the dashed line. d p-HLA complex stability measurements of mKRAS epitopes to HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02. p-HLA stability measurements were determined by scintillation proximity assay of β2-microglobulin dissociation29. For each HLA class I allele tested, β2-microglobulin association with MHC heavy chain in the absence of peptide served as negative controls. Displayed p-HLA affinity and stability values represent the mean of two independent tests. For affinity and stability assays, validated T cell epitopes for each allele were used as positive controls (open circles) and are listed in Supplementary Table 3. Error bars in panel (d) specify mean values ± SD of positive controls. mKRAS peptides with high measured binding affinity or stability are indicated as red circles. eSummary chart of MS/MS detected HLA class I restricted mKRAS epitopes processed and presented by TMC-engineered monoallelic cell lines (red). Validated viral epitopes (NLV, ILR, IVT, and TPR) encoded by the TMC construct were used as positive controls for epitope processing and presentation and are listed in Supplementary Table 3. Source data are provided as a Source Datafile.

We experimentally assessed p-HLA binding affinity and complex stability of candidate peptides as these two properties improve the performance of T cell epitope identification29. Furthermore, p-HLA complex stability positively correlates with immunogenicity12. To evaluate p-HLA binding affinity, we employed competitive peptide-binding assays using a FITC-labeled reference peptide and purified soluble HLA class I molecules to quantitate the binding capacity of non-labeled mKRAS peptide candidates. Peptide affinities are reported as concentrations of unlabeled peptide that result in 50% binding inhibition of FITC-labeled reference peptide (Supplementary Fig. 2 and Supplementary Table 1)30. To evaluate p-HLA complex stability, dissociation assays were performed where the stability of p-HLA complexes is expressed as the half-life of beta-2-microglobulin dissociation from the HLA heavy chain (Supplementary Table 2)29. Both assays incorporated validated HLA class I-restricted T cell epitopes as positive controls (Supplementary Table 3). Affinity binding values determined in competitive assays are reported as log10[IC50(nM)] and IC50(nM) and are defined according to a previously reported scale detailed in Supplementary Tables 1 and 3. For HLA-A*02:01 epitopes, only peptide 5-14V exhibited strong class I binding affinity (log10[IC50] = 3.469 nM) (Fig. 1c). However, all p-HLA-A*02:01 class I complexes exhibited short half-lives (t1/2 < 1 h) compared to control peptides (t1/2 1.3–10.1 h, Fig. 1d). For HLA-A*03:01 epitopes, peptides 7-16R, 8-16V, and 7-16V exhibited strong binding affinity (log10[IC50] ≤ 3.589 nM) (Fig. 1c) and formed p-HLA complexes with equivalent stability to positive controls (t1/2 > 1 h). All 9-mer (8-16C-V) and 10-mer (7-16C-V) peptides bound with strong affinity to HLA-A*11:01 (log10[IC50] ≤ 3.121 nM) (Fig. 1c), and 10-mer peptides formed more stable p-HLA-A*11:01 complexes compared to 9-mer peptides (t1/2 6–12 h vs 0.9–9.2 h) (Fig. 1d). For HLA-B*07:02 epitopes, peptides 10-19C, 10-19D, and 10-19R bound with high affinity (log10[IC50] ≤ 3.460 nM) (Fig. 1c), but only 10-19C and 10-19R exhibited p-HLA complex stability equivalent to positive control peptides (t1/2 > 1 h) (Fig. 1d). A comparison of predicted and experimentally determined p-HLA affinities showed a strong correlation for 8-16C-V and 7-16C-V peptides restricted by HLA-A*03:01 and HLA-A*11:01 (R2 > 0.9). However, no correlation was observed for either 5-14C-V peptides restricted by HLA-A*02:01 (R2 = 0.1033) or 10-19C-V peptides restricted by HLA-B*07:02 (R2 = 0.6945) (Supplementary Fig. 3 and Supplementary Table 1). A similar analysis of predicted and experimentally determined p-HLA stabilities (Supplementary Table 2) showed no correlation among p-HLA complexes studied. Experimental stabilities for all mKRAS/HLA-A*11:01 complexes yielded higher binding affinities than were predicted. In summary, we experimentally identified 8 mKRAS G12 epitopes restricted to HLA-A*03:01 (n = 3), HLA-A*11:01 (n = 3), or HLA-B*07:02 (n = 2) that display high affinity and formed high-stability p-HLA complexes equivalent to positive control peptides.

Validation of mKRAS antigen processing and presentation

To validate epitope processing and presentation, we performed targeted mass spectrometry (MS) on peptides isolated from single HLA class I allele/tandem minigene construct (TMC)-expressing cell lines. HLA class I negative cell lines were engineered to express a GFP-tagged HLA class I/β2-microglobulin single-chain dimer (HLA-SCD) construct and a mCherry-tagged TMC (Supplementary Fig. 4a). Use of monoallelic cells lines eliminates the ambiguity that arises from the coexpression of multiple HLA class I alleles31. The TMC consisted of nine minigenes encoding WT and mKRAS long 25-mer peptides (G12C, G12D, G12R, G12V) as well as validated viral epitopes restricted by HLA-A*02:01 (NLV), HLA-A*03:01 (ILR), HLA-A*11:01 (IVT), and HLA-B*07:02 (TPR) as positive controls (Supplementary Fig. 4b and Supplementary Table 3). TMC-expressing monoallelic cell lines were phenotyped for reporter and HLA class I expression (Supplementary Fig. 4c–e) then subjected to HLA class I immunoprecipitation and peptide elution. Peptide sequence identities were determined by quadrupole—orbitrap tandem MS using data-dependent acquisition (DDA) and targeted parallel reaction monitoring (PRM)32. Synthetic mKRAS peptides served as “beacons” to zero-in on TMC-encoded peptides that were naturally loaded onto HLA class I molecules. MS/MS fragmentation pattern comparison of eluted and synthetic peptides ensured accurate assignment of AA sequence identity of mKRAS epitopes (Fig. 1e and Supplementary Figs 57). In this manner, peptides 7-16D, 8-16V, 7-16V, and 8-16R were eluted in the context of HLA-A*03:01 (Supplementary Fig. 5), and peptides 8-16D, 7-16D, 8-16V, 7-16V, and 8-16R were eluted in the context HLA-A*11:01 (Supplementary Fig. 6). Among these peptides, only 8-16V and 7-16V form p-HLA complexes with HLA-A*03:01 and HLA-A*11:01 with comparable stability to positive controls. Lastly, peptides 10-19D and 10-19R were also identified in the context of HLA-B*07:02 (Supplementary Fig. 7). To further support the identity assignments between eluted and synthetic peptides, we determined precursor ion similarity via isotopic dot product (idotp, value >0.9) and dot product (dotp, value >0.5) measurements33. No mKRAS G12 peptides presented by HLA-A*02:01 were observed despite the detection of the validated NLV viral epitope (Supplementary Fig. 8). In addition, no cysteine-containing epitopes were detected. Free cysteines are prone to post-translational modifications and are underrepresented in MS-based HLA-peptide datasets34,35. Future analysis accounting for post-translation modifications will be performed to address the processing of mKRAS cysteine-containing peptides. Importantly, neither 8-16C nor 7-16C in the context of HLA-A*03:01 was observed by Wang et al.36 suggesting these peptides are not part of this HLA class I peptidome. In sum, we identified 11 processed and presented mKRAS epitopes restricted to HLA-A*03:01 (n = 4), HLA-A*11:01 (n = 5), or HLA-B*07:02 (n = 2) class I alleles (Fig. 1e).

mKRAS peptide immunogenicity and mKRAS-specific TCRs isolation

To evaluate the immunogenicity of selected mKRAS peptides, CD8+ T cells isolated from healthy donors were cocultured with autologous monocyte-derived mature dendritic cells (mDC) pulsed with candidate epitopes. Following two stimulations, cultures were screened for CD8+ T cell responses by IFN-γ ELISPOT assay (Fig. 2a). No responses were observed to peptides 5-14C-V among HLA-A*02:01+ donors. Responses were observed to peptide 7-16V in HLA-A*03:01+ (n = 1/4) and HLA-A*11:01+ (n = 4/4) donors, and responses to 7-16C (n = 4/4) and 7-16D (n = 1/4) were also noted in HLA-A*11:01+ donors. Responses to peptide 10-19R (n = 2/5) in HLA-B*07:02+ donors were also observed.

Fig. 2: Assessment of mKRAS neoantigen immunogenicity and identification of mKRAS-specific TCR sequences.

a IFN-γ ELISPOT assay results following in vitro expansion of healthy donor purified CD8+ T cells stimulated with mKRAS peptide-pulsed autologous mDC. IFN-γ ELISPOT Spot Forming Cell (SFC) values are substrated from media controls for each subject. The data presented are the pooled results from single experiments of individual donors represented by symbols indicated in the figure legend. Red symbols denote donors with positive responses. b p-HLA multimer analysis demonstrating positive 7-16V-specific HLA-A*03:01-restricted CD8+ T cell response isolated from donor D429. c p-HLA multimer analysis demonstrating positive 7-16V-specific HLA-A*11:01-restricted CD8+ T cell response isolated from donor D500. d p-HLA multimer analysis demonstrating positive 10-19R-specific HLA-B*07:02-restricted CD8+ T cell response isolated from donor D516. Cells were stained with p-HLA multimer and anti-CD8 Ab. p-HLA multimer staining of respective donor CD8+ T cells stimulated in the absence of peptide (non-specific) were used as staining controls. A total of 1 × 106 live events were collected gating on FSC/SSC/CD8+ cells. Data presented in panels (bd) are representative of two independent tests. Source data are provided as a Source Data file.

Given the high sensitivity of TCRs for p-HLA complexes, we sought to isolate TCRs directed against 7-16V and 10-19R epitopes in order to characterize their expression by tumor cell lines and validate these epitopes as neoantigens. To isolate TCRα/β pairs, cell cultures containing CD8+ T cell responses to 7-16V/HLA-A*03:01, 7-16V/HLA-A*11:01, and 10-19R/HLA-B*07:02 were expanded using peptide-pulsed artificial antigen presenting cells (APC). Antigen specificity was confirmed by p-HLA multimer staining (Fig. 2b–d), and multimer+cells were flow cytometrically sorted to >99% purity. TCRα/β sequences were determined by next-generation DNA and RNA sequencing37. A total of five TCRα/β pairs were identified, three of which were selected for further studies: TCRA3V (7-16V/HLA-A*03:01), TCRA11V (7-16V/HLA-A*11:01), and TCRB7R (10-19R/HLA-B*07:02) (Table 1).

Table 1 Summary table of mKRAS-TCRs.

We initially characterized the expression and function of these mKRAS-TCRs using a cell reporter system developed in our laboratory designated JASP90 reporter cells. The JASP90reporter cells are derived from CD8+/TCRαβ null Jurkat E6.1 cells engineered to express NFAT-inducible eGFP as a readout for TCR signaling. mKRAS-TCRα/β chain pairs were cloned into lentiviral vectors and expressed in JASP90 reporter cells. Custom p-HLA multimer staining was performed to validate TCRA3V, TCRA11V, and TCRB7R expression as well as confirm antigen specificity and HLA restriction (Fig. 3a).

Fig. 3: Validation and functional characterization of mKRAS-specific TCRs.

a FACS histogram plots demonstrating p-HLA multimer staining following lentiviral engineering of JASP90 reporter cells with TCRA3V, TCRA11V, and TCRB7R constructs (red) vs TCRαβnull controls (black). b JASP90 reporter assay to evaluate antigen recognition. JASP90 reporter cells expressing TCRA3V, TCRA11V, or TCRB7R were cocultured with HLA-matched monoallelic K562 cell lines pulsed with titrated concentrations of WT KRAS or mKRAS peptides. TCR signaling is indicated by eGFP expression upon NFAT activation. TCR avidity was determined as the mean peptide concentration required to achieve 50% NFAT activation of TCR-engineered JASP90 reporter cells. Specific Activity (%) = (%GFPTest – %GFPMin) / (%GFPMax – %GFPMin) x 100. Representative experiments of three independent evaluations are shown. Source data are provided as a Source Data file.

To further assess antigen specificity and measure TCR avidity, TCR-engineered JASP90reporter cells were cocultured with HLA class I matched monoallelic K562 cells pulsed with WT or mKRAS synthetic peptides. TCRA3V, TCRA11V, and TCRB7R-expressing JASP90reporter cells demonstrated specific reactivity to mKRAS cognate epitopes without reactivity to WT KRAS peptide-pulsed or unpulsed monoallelic K562 cells (Fig. 3b). Using titrated peptide concentrations, the functional avidities of each TCR were determined based on the mean concentration required to achieve 50% NFAT activation (Fig. 3b). The avidities of TCRA3V, TCRA11V, and TCRB7R were determined to be 1.64, 0.15, and 3.38 nM, respectively. Furthermore, TCRA3V and TCRB7R cells exhibited no cross-reactivity to other mKRAS G12 epitopes, while TCRA11V cells demonstrated cross-reactivity to peptide 7-16C, albeit with decreased avidity as compared to the cognate peptide. These TCRs constituted high-affinity probes for the characterization of naturally processed and presented mKRAS epitopes by human tumor cells.

mKRAS-specific TCRs confer lytic activity against human tumor cells

To probe for p-HLA complexes on tumor cell lines, the anti-tumor activity of mKRAS TCRs was evaluated using engineered TCRαβnull primary CD8+ T cells. To this end, healthy donor primary CD8+ T cells were gene edited using CRISPR/Cas9 and single guide RNAs (sgRNAs) to knock out endogenous TCRα and β expression (Supplementary Fig. 9a–c) to eliminate TCRαβ mispairing and enhance receptor cell surface expression38,39. Engineered TCR expression was assessed by cell surface CD3 and TCRαβ staining (Supplementary Fig. 9e). Secondary expansion of mKRAS-TCR T cells using peptide-pulsed artificial APCs led to enrichment of antigen-specific CD8+ T cells (Supplementary Fig. 9f), yielding populations with high (60–90%) expression of TCRA3V, TCRA11V, or TCRB7R as assessed by p-HLA multimer staining (Fig. 4a). In 4 h 51Cr-release assays, TCRA3V, TCRA11V, and TCRB7R-expressing CD8+ T cells kill HLA class I matched K562 cells pulsed with exogenous cognate mKRAS peptide but not WT KRAS peptide (Fig. 4b). In addition, recognition of processed and presented mKRAS antigen by TCRA3V, TCRA11V, and TCRB7R-expressing CD8+ T cells was demonstrated using TMC-expressing K562 cells as targets (Fig. 4b). Lack of recognition of processed and presented WT KRAS was demonstrated using a WT KRAS-expressing cell line (Fig. 4b, c). Altogether, peptide-pulsing experiments validate antigen specificity while TMC experiments, provide support for proteomic results (Fig. 1e), and validate presentation of cell surface mKRAS p-HLA complexes.

Fig. 4: mKRAS TCRαβ gene transfer confers HLA-restricted lytic activity against human tumor cells.

a FACS plot demonstrating p-HLA multimer binding to TCRA3V-, TCRA11V-, and TCRB7R-engineered TCRαβnull primary CD8+ T cells gated on viable/CD3+ population. The following p-HLA multimers were used as staining controls: gp17-25/A3, gp17-25/A11, NY60-72/B7 (Supplementary Table 3). b 4 h 51Cr-release cytotoxicity assay demonstrating TCRA3V, TCRA11V, and TCRB7 cell tumoricidal activity against TMC-expressing (red), mKRAS peptide-pulsed (blue), or WT KRAS peptide-pulsed (black) monoallelic K562 target cells. Percent-specific lysis of triplicates is shown for each data point. Data are presented as mean ± SD. p < 0.05 or p < 0.01 for all E:T ratios ≥0.3:1 comparing TMC-expressing or mKRAS peptide-pulsed to WT KRAS peptide-pulsed K562 targets, respectively; one-way ANOVA followed by post-hoc pair-wise Student’s t-test with multiple comparison adjustment. c, d. 4 h 51Cr-release cytotoxicity assay demonstrating TCRA3V and TCRA11V tumoricidal activity against cancer cell lines harboring WT KRAS (BxPC-3) or endogenous KRAS G12V mutation either non-modified (open circles, non-HLA matched) or engineered to express the restricting HLA allele (closed circles, HLA-matched). Each color represents a different cell line as identified in the figure legend. No significant cytotoxicity was observed against HLA matched vs non-matched BxPC-3 cell lines. Data are presented as mean values ± SD (n = 3 biologically independent samples). p < 0.05 for all E:T ratio ≥1:1 comparing HLA matched vs non-matched cell lines harboring endogenous KRAS G12V mutation; one-way ANOVA followed by post-hoc pair-wise Student’s t-test with multiple comparison adjustment. Source data are provided as a Source Data file.

In order to evaluate the recognition of p-HLA complexes on the surface of cancer cells by TCRs, we characterized the expression of HLA class I alleles in a panel of mKRAS G12 cancer cell lines of various tissue origins. Cell lines with reported KRAS WT (TPM 23), G12V (TPM 56-308), and G12R (TPM 16-130) expression, as well as cell lines expressing target HLA class I alleles but alternative KRAS mutations were selected for further study (Supplementary Table 4). Aside from G12V/HLA-A*03:01, no cell lines expressing endogenous G12V/HLA-A*11:01 or G12R/HLA-B*07:02 were identified. When applicable, KRAS G12V+ cell lines were modified to express either HLA-A*03:01 or HLA-A*11:01, and KRAS G12R+ lines were modified to express HLA-B*07:02. As shown in Fig. 4c, d, TCRA3V and TCRA11V-expressing CD8+ T cells exhibited specific cytotoxicity, irrespective of tissue of origin, against all HLA-A*03:01 or HLA-A*11:01-matched KRAS G12V+ cancer cell lines, respectively. Of note, neither TCR conferred cytotoxic activity against HLA matched BxPC-3 tumor cells, a KRAS WT pancreatic adenocarcinoma cell line, validating the specificity of each TCR for the cognate G12V peptide. These observations were replicated and expanded upon using the JASP90reporter cell system. Upon co-culture with mKRAS/HLA-matched cell lines, TCRA3V-, TCRA11V-, and TCRB7R-JASP90 cells demonstrated NFAT-induced GFP expression indicative of TCR sensing of p-HLA complexes presented on the surface of tumor cells (Supplementary Fig. 10a–c). No NFAT-induced GFP expression was observed in JASP90 reporter cells cultured with HLA matched KRAS WT-expressing BxPC-3 cells. The graded pattern of NFAT activation induced in JASP90 reporter cells may suggest varied surface expression of p-HLA complexes and/or differing patterns of coreceptor expression by cancer cells. Altogether, these results strongly support the hypothesis that mKRAS epitopes are processed and presented by tumor cells in the context of HLA-A*03:01, HLA-A*11:01 and HLA-B*07:02 at sufficient levels to activate recognition by mKRAS-TCR-engineered T cells.

Quantitation and recognition of mKRAS peptides presented by human tumor cells

To gain further insights into the processing and presentation of 8-16V and 7-16V peptides on the surface of cancer cell lines, we performed targeted MS and absolute peptide quantitation to enumerate p-HLA complexes on the surface of CORL23 lung tumor cells engineered to express HLA-A*03:01 (CORL23-A3) or HLA-A*11:01 (CORL23-A11)40. CORL23 was chosen for further studies given its ability to recapitulate a model of metastatic lung cancer (see below)41. In the context of HLA-A*03:01, we detected 8 complexes of 8-16V and 27 complexes of 7-16V per cell as compared to 43 complexes of 8-16V and 78 complexes of 7-16V per cell in the context of HLA-A*11:01 (Fig. 5a and Supplementary Fig. 11). This result confirms processing and presentation data obtained using TMC-engineered HLA class I monoallelic cell lines (Supplementary Figs 5 and 6) and suggests that KRAS G12V+ tumors present both 9-mer (8-16V) and 10-mer (7-16V) epitopes in the context of HLA-A*03:01 and HLA-A*11:01. Although these cells were genetically engineered to express the restricting HLA class I allele, HLA-A*03:01 and HLA-A*11:01 cell surface levels were noted to be within the range of expression comparable to other non-engineered HLA-A*03:01+ and HLA-A*11:01+mKRAS tumor cells lines (Supplementary Fig. 12a, b). Importantly, enumerated p-HLA complexes are within the ranges reported for other mKRAS/HLA complexes expressed by non-engineered cancer cell lines36.

Fig. 5: Quantitation and recognition of KRAS G12V peptide ligands presented by human tumor cell lines.

a Absolute quantification of cell surface expression of 8-16V/HLA and 7-16V/HLA complexes by CORL23-A3 and CORL23-A11 cells. Data are representative of two independent experiments. bCellular impedance to determine the kinetics of tumor cell death upon TCRA3V lytic recognition of CORL23-A3 vs TCR Ctrl (TCRA11V). Normalized cell index values over time for E:T ratios of 10:1, 3:1, and 1:1 are shown compared to tumor cells cultured in media alone. Data for cells exposed to triton-X to represent maximal impedance loss are displayed. Data are presented as mean values ± SD. p < 0.001 at 150 h for all E:T ratios for the TCRA3V group compared to media; one-way ANOVA followed by post-hoc pair-wise Student’s t-test with multiple comparison adjustment. Data are representative of two independent experiments. c Cellular impedance data following TCRA11V lytic recognition of CORL23-A11 vs TCR Ctrl (TCRA3V). Data are presented as mean values ± SD. p < 0.0001 at 150 h for all E:T ratios for the TCRA11V group compared to media; one-way ANOVA followed by post-hoc pair-wise Student’s t-test with multiple comparison adjustment. Data are representative of two independent experiments. d Comparative kinetics of the time to reach 50% cell death (KT50) of CORL23-A3 and CORL23-A11 cells upon TCRA3V and TCRA11V recognition, respectively, at E:T ratios of 10:1, 3:1, and 1:1. Data are presented as mean values with independent replicates displayed. *p < 0.05, **p < 0.01, ***p < 0.001; multiple t-test analysis. Data are representative of two independent experiments. e 4 h 51Cr-release cytotoxicity assay demonstrating TCRA3V tumoricidal activity against NCI-H441 cells (G12V+/HLA-A*03:01+) compared to TCR Ctrl (TCRA11V) cells. Data are presented as mean values ± SD. p < 0.01 for all E:T ratio ≥ 1:1; one-tailed Student’s t-test. Data are representative of two independent experiments. fCellular impedance data following TCRA3V lytic recognition of NCI-H441 vs TCR Ctrl (TCRA11V). Data are presented as mean values ± SD. p < 0.0001 at 130 h for all E:T ratios for the TCRA3V group compared to media; one-way ANOVA followed by post-hoc pair-wise Student’s t-test with multiple comparison adjustment. Data are representative of two independent experiments. Source data are provided as a Source Data file.

The cytotoxic activity of TCRA3V- and TCRA11V-engineered CD8+ T cells against CORL23 tumor cell lines was then characterized via live cell imaging and cellular impedance. These complimentary measurements of cell death allow for visualization of the loss of GFP-labeled tumor cells and the accumulation of Annexin V-CF594 dye (Supplementary Fig. 13 and Supplementary Movie 1), as well as cell shrinkage and fragmentation measurements over a 6-day period. TCRA3V cells promoted rapid apoptotic cell death of CORL23-A3 tumor cells at a 10:1 E:T ratio compared to control T cells, with slower killing kinetics at lower E:T ratios of 3:1 and 1:1 (Fig. 5b). TCRA11V cells promoted rapid cell death of CORL23-A11 cells at all E:T ratios (Fig. 5c). The kinetics to reach 50% cytolysis (KT50) was significantly faster for TCRA11V cells compared to TCRA3V cells at all E:T ratios (Fig. 5d), a finding consistent with the higher avidity of TCRA11V (Fig. 3b) and the higher number of 7-16V complexes detected in the context of HLA-A*11:01 expressed on the surface of CORL23-A11 cells.

We next evaluated the sensitivity of mKRAS TCRs to recognize p-HLA complexes in a non-engineered cell line. We identified the LAC cell line NCI-H441 as being both KRAS G12V+ and HLA-A*03:01+ (Supplementary Table 4), highlighting it as a potential target of TCRA3V. We confirmed this cell line retained HLA class I expression and expressed low levels of HLA-A*03:01 (Supplementary Fig. 12a). In both 4 h 51Cr-release and cellular impedance assays, TCRA3V cells exhibited specific lysis against parental (non-engineered) NCI-H441 cells, while TCRA11V cells, which recognizes the identical 7-16V epitope but restricted by HLA-A*11:01, exhibited no cytotoxic activity (Fig. 5e, f). Together, we conclude that TCRA3V and TCRA11V can “sense” low numbers of p-HLA complexes per cell and represent valuable reagents to immunologically target mKRAS.

In vivo anti-tumor activity of mKRAS-specific TCR+ CD8+ T cells

CORL23-A3 and CORL23-A11 tumor cells engineered to express click beetle red (CBR) luciferase were infused into NOD/scid/γcnull (NSG) mice via tail vein injection, and the establishment of pulmonary tumors was confirmed by bioluminescence imaging. Four days post tumor inoculation, mice were treated with TCRA3V- or TCRA11V-engineered CD8+ T cells. As controls, tumor-bearing mice received either no treatment (Mock) or TCRαβnull(TCR KO) CD8+ T cells. Mice treated with TCRA3V- or TCRA11V- engineered CD8+ T experienced complete eradication of CORL23-A3 (Fig. 6a, c) or CORL23-A11 (Fig. 6b, d) tumors, respectively, relative to control groups (p < 0.01). The treatment effect was long-lasting with no evidence of tumor outgrowth by day 39 in mice treated with mKRAS-TCR therapy, while control mice experienced persistent tumor growth. Consequently, mice treated with mKRAS-TCR therapy experienced prolonged survival relative to control groups (p < 0.0002, Fig. 6e, f). Altogether, these results demonstrate that the transfer of T cells engineered with high avidity mKRAS TCRs promotes tumor rejection and prolongs survival.

Fig. 6: Adoptive transfer of mKRAS-TCR T cells leads to in vivo eradication of KRAS G12V+ tumor cells in a xenograft model of metastatic lung cancer.

CORL23-A3 or CORL23-A11 tumors expressing CBR luciferase were engrafted into NSG mice via intravenous tail vein injection. Mice were left untreated (Mock) or treated with TCR αβ null (TCR KO) or mKRAS-TCR engineered CD8+ T cells 4 days after tumor engraftment. Tumor burden was assessed by bioluminescence imaging before and after treatment, and overall survival was monitored over time. Representative bioluminescence imaging prior to and following treatment of NSG mice bearing a CORL23-A3 pulmonary tumors treated with TCRA3V T cells and b CORL23-A11 pulmonary tumors treated with TCRA11V cells compared to control groups. Total Flux quantification over time of c CORL23-A3 and d CORL23-A11 tumor-bearing mice as shown in (a) and (b). Colored lines represent mean Total Flux values over time with individual data points corresponding to treatment groups presented as indicated in the figure legend. *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA followed by Tukey’s HSD post-test comparing Mock and TCRA3V or TCRA11V treated mice. No statistical difference was observed between Mock and TCR KO treated mice. Kaplan–Meier analysis of overall survival of e CORL23-A3 and f CORL23-A11 tumor-bearing mice. Colored lines correspond to treatment groups as indicated in the figure legend. p values as indicated; log-rank testing comparing Mock and TCRA3V or TCRA11V treated mice. No statistical difference was observed between Mock and TCR KO treated mice. Number of mice in representative experiment is as follows: CORL23-A3 Cohort—Mock (n = 6), TCR KO (n = 6), TCRA3V (n = 10). CORL23-A11 Cohort—Mock (n = 6), TCR KO (n = 6), TCRA11V (n = 12). Source data are provided as a Source Data file.


Epitope discovery is an essential step in designing immunotherapies such as cancer vaccines, bi-specific engagers, and TCR therapies. The ubiquity of mKRAS along with its role as a recurrent clonal driver makes this oncoprotein an ideal target42,43,44. To identify mKRAS epitopes, we undertook a comprehensive multi-omics approach (e.g., bioinformatic, biochemical, proteomic and immunological) to address the intricate processes involved in the generation of immunogenic peptides. By subjecting each candidate peptide to a series of assays that interrogate critical properties of antigen processing and presentation, we have identified a curated, albeit partial set of mKRAS peptides that represent candidate epitopes. Using this approach, we identified three epitopes (7-16V/HLA-A*03:01, 7-16V/HLA-A*11:01, and 10-19R/HLA-B*07:02) that fulfilled criteria of strong measured p-HLA binding affinity, high complex stability, and proteomically validated antigen processing and presentation. Furthermore, we determined that these epitopes were immunogenic allowing for the identification of mKRAS TCR sequences that were used as sensitive probes to validate epitope presentation by human cancer cell lines. Direct quantification of mKRAS p-HLA complexes on CORL23 cells highlights the sensitivity of mKRAS TCRs to recognize low abundance p-HLA complexes and eliminate mKRAS tumors cells in a xenograft mouse model of metastatic cancer.

Most mKRAS epitopes described herein have not been previously reported as constituents of the HLA peptidome. These epitopes include the following: HLA-A*03:01 – 8-16V, 8-16R, and 7-16V, HLA-A*11:01 – 8-16D, 8-16R, 8-16V, 7-16D, and 7-16V, HLA-B*07:02 – 10-19D and 10-19R. These epitopes exhibit high affinity for the restricting HLA class I allele and relatively high complex stability as each forms p-HLA complexes with t1/2 > 0.5 h. In agreement with our data, the processing and presentation of the 7-16D peptide in the context of HLA-A*03:01 has also recently been reported by Wang et al.36 despite exhibiting low measured affinity and complex stability. Our study and those of others36 using cell lines expressing mKRAS have failed to identify 5-14 mKRAS peptides as constituents of the HLA-A*02:01 peptidome. Furthermore, biochemical assessment of 5-14C-V peptide-binding and stability properties for HLA-A*02:01 suggest that these ligands fail to form stable p-HLA complexes, and only the 5-14V peptide demonstrated high HLA class I affinity in competitive binding assays. By contrast, Mishto et al.45 reported mKRAS linear (5-14V) and spliced (5-6/8-14V, lacking the V at position 7 of the canonical peptide) peptides as products of in vitro proteasome digests and have demonstrated these peptides to have high affinity for HLA-A*02:01. Given the high prevalence of HLA-A*02:01 in the population, further studies are needed to evaluate the discordant results between processing of the 5-14V mKRAS peptide by cell lines and in vitro proteasome digest assays.

To investigate the immunogenicity of mKRAS epitopes, immune responses were “outsourced” from healthy donors as naive TCR repertoires may recognize tumor neoantigens12. In addition to 7-16V and 10-19R epitopes described, we also identified CD8+ T cell responses to 7-16C and 7-16D in HLA-A*11:01+ donors. Of these epitopes, only 7-16D- and 7-16V-specific TCRs isolated from immunized transgenic HLA-A*11:01 mice have been previously reported21. We did not observe immune responses directed against 5-14C-V in HLA-A*02:01+ donors despite previous studies reporting HLA-A2-restricted 5-14D and 5-14V CD8+ T cell responses17,46. Given that these studies were reported over 15 years ago, it is likely high-resolution HLA typing was not performed, and it is possible responding patients expressed less prevalent HLA-A2 sub-alleles (e.g., HLA-A*02:03 or HLA-A*02:06) that may exhibit higher binding affinity to 5-14V and 5-14D peptides relative to HLA-A*02:01. Clearly, this issue related to mKRAS epitopes restricted by HLA-A*02:01 requires further investigation.

The availability of TCRs directed against 7-16V/HLA-A*03:01 (TCRA3V), 7-16V/HLA-A*11:01 (TCRA11V), and 10-19R/HLA-B*07:02 (TCRB7R) permitted their use as sensitive probes demonstrating the presence of these p-HLA complexes on the cell surface of various cancer cell lines from multiple histologies. Our observation that NCI-H441 can be recognized by TCRA3V-engineered CD8+ human T cells is noteworthy given this KRAS G12V mutant cell line naturally expresses the HLA-A*03:01 restricting allele. Further insights were gained by the quantification of p-HLA complexes on CORL23 cell lines demonstrating expression of both 9-mer and 10-mer peptides in the context of HLA-A*03:01 and HLA-A*11:01. As TCRA3V and TCRA11V recognize the same 7-16V peptide, we hypothesized differences in TCR avidity as well as p-HLA abundance by CORL23 lines may contribute to the distinct kinetics of in vitro killing. Indeed, higher expression of 7-16V/A*11:01 complexes (78 complexes/cell) relative to 7-16V/A*03:01 (27 complexes/cell) correlates with the more rapid elimination of CORL23-A11 relative to CORL23-A3 tumor cells. Importantly, the adoptive T cell transfer of either TCRA3V or TCRA11V engineered CD8+ human cells efficiently eliminated CORL23 tumor cells in vivo and proved curative for a majority of treated mice.

In summary, epitope identification and validation strategies reliant on a contemporary toolbox of bioinformatic, biochemical, genomic, and immunological assays have recently been employed by several groups in the development of personalized melanoma vaccines targeting passenger mutations11,47,48. Here, we have employed a similar strategy to characterize epitopes presented by high prevalence HLA class I alleles targeting a recurrent clonal driver—mKRAS. Our findings provide further evidence highlighting this oncogenic protein as a clinical target of immune-based therapies.

Article classification: Biological abstract
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