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the presentation of mutant KRAS peptides on individual HLA-I molecules using targeted mass spectrometry and identify 13 unpublished KRASG12C/D/R/V mutation/HLA-I pairs and nine previously described pairs.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.
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•A pipeline is developed to evaluate whether T cells can recognize cancer targets•Mass spectrometry is used to identify distinct KRAS mutation/HLA-I pairs•Nearly all KRAS mutation/HLA-I pairs tested can lead to a T cell response•TCRs lead to cytotoxicity of tumor cells with the cognate KRAS mutation/HLA-I pair
We created a pipeline for the systematic discovery and validation of T cell targets for the development of targeted cancer immunotherapies. This pipeline addresses the two critical aspects of a T cell target: presentation on an HLA molecule and immunogenicity. Applying our methodologies to the comprehensive analysis of common KRAS mutations, we demonstrated the therapeutic potential of our pipeline to increase patient populations that can be reached with T cell-based therapies.
T cell-based therapies for cancer rely on targets that are both presented on specific HLA molecules and are immunogenic. Choi et al. establish a systematic pipeline to evaluate both aspects sensitively and apply it to common KRAS mutations to broadly explore T cell targets from this oncogene.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
BackgroundMultiple myeloma (MM) is a rare cancer that leads to malignant plasma cells. There are efforts to uncover how MM develops from precursor disease states, such as monoclonal gammopathy of ...undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Characterizing the evolution of MM allows for earlier prognosis and intervention. Limited availability of tumor (CD138+) cells from patients poses challenges in studying MM stages. Immunopeptidomics typically requires a minimum of 50–100 million cells, exceeding what can typically be obtained from blood cancers. We optimized our HLA immunopurification protocol and paired it with ultra-sensitive mass spectrometry to enable analysis of sample inputs as low as 1 million cells. With this sensitive low input workflow, we characterized the immunopeptidomes of n=35 MM patients, across different stages of the disease.MethodsWe optimized our low input HLA immunopurification protocol by reducing lysis buffer volume, antibody concentration, protein G bead volume, bead wash volume, and desalting sorbent.1 2 Using HLA-I/II-expressing A375 cells, we refined the protocol with a range of cell inputs (0.5, 1, 2, 5, and 10 million cells). To confirm the method utility, we profiled a cohort of CD138+ MM samples (≤2 million cells) from a range of disease stages (MGUS; n=7, SMM; n=9, newly diagnosed multiple myeloma: NDMM; n=10, and relapsed/refractory multiple myeloma: RDMM; n=9). We used the Bruker trapped ion mobility spectrometry time of flight (timsTOF) single cell proteomics (SCP) instrument for low input HLA-I/II immunopeptidomics, interpreting spectra with Spectrum Mill against a database containing a reference human proteome and ribosome-sequencing-derived novel or unannotated open reading frames (nuORFs).3 ResultsOptimized low input immunopurification and ultra-sensitive mass spectrometry achieved ~700 HLA-I and ~650 HLA-II peptides on average across the cohort. In some samples, we identified up to ~4000 HLA-I and ~2000 HLA-II peptides. Given the depth, we found HLA-I nuORF-derived peptides, TAAs (e.g., BCMA, MMSET), and CTAs (e.g., MAGE family).ConclusionsHighly sensitive, low input immunopeptidome analysis enables the discovery of HLA peptides presented by primary MM tumor cells. We detected nuORF-derived peptides in the HLA-I and HLA-II immunopeptidome from MM samples for the first time. This optimized workflow and the resulting data may yield disease state biomarkers and immunotherapeutic targets.ReferencesAbelin JG, Bergstrom EJ, Rivera KD, Taylor HB, Klaeger S, Xu C, Verzani EK, Jackson White C, Woldemichael HB, Virshup M, Olive ME, Maynard M, Vartany SA, Allen JD, Phulphagar K, Harry Kane M, Rachimi S, Mani DR, Gillette MA, Satpathy S, Clauser KR, Udeshi ND, Carr SA. Workflow enabling deepscale immunopeptidome, proteome, ubiquitylome, phosphoproteome, and acetylome analyses of sample-limited tissues. Nat Commun. 2023 Apr 3;14(1):1851. doi: 10.1038/s41467-023-37547-0. PMID: 37012232; PMCID: PMC10070353.Phulphagar KM, Ctortecka C, Jacome ASV, Klaeger S, Verzani EK, Hernandez GM, Udeshi ND, Clauser KR, Abelin JG, Carr SA. Sensitive, high-throughput HLA-I and HLA-II immunopeptidomics using parallel accumulation-serial fragmentation mass spectrometry. Mol Cell Proteomics. 2023 May 2:100563. doi: 10.1016/j.mcpro.2023.100563. Epub ahead of print. PMID: 37142057.Ouspenskaia T, Law T, Clauser KR, Klaeger S, Sarkizova S, Aguet F, Li B, Christian E, Knisbacher BA, Le PM, Hartigan CR, Keshishian H, Apffel A, Oliveira G, Zhang W, Chen S, Chow YT, Ji Z, Jungreis I, Shukla SA, Justesen S, Bachireddy P, Kellis M, Getz G, Hacohen N, Keskin DB, Carr SA, Wu CJ, Regev A. Unannotated proteins expand the MHC-I-restricted immunopeptidome in cancer. Nat Biotechnol. 2022 Feb;40(2):209–217. doi: 10.1038/s41587-021-01021-3. Epub 2021 Oct 18. PMID: 34663921.
BackgroundAberrant translation of the non-coding genome in cancer can generate novel peptides capable of presentation by major histocompatibility complex class I (MHC-I; HLA-I in humans) and these ...non-canonical peptide sources can broaden the landscape of potentially targetable antigens in low-to-intermediate mutational burden malignancies. While emerging evidence suggests that translation of unannotated open reading frames (uORFs) can give rise to MHC class I-associated peptides (MAPs) across a range of malignancies, it is currently unknown to what extent these translation products are truly cancer-restricted and how effectively the resulting non-canonical MAPs (ncMAPs) can elicit a T cell response.MethodsWe leveraged twelve pancreatic cancer (PDAC) patient-derived organoids (PDOs) to purify the malignant compartment from low tumor cellularity tumor specimens. We developed a cutting-edge proteogenomics pipeline, coupled with high-depth immunopeptidomics to identify pancreatic cancer MAPs derived from somatic mutations, retained introns, and uORFs. To investigate the cancer-specificity of ncMAPs, we developed a translation-centric analysis pipeline that examines translation of uORFs encoding ncMAPs across a range of healthy tissues, including healthy thymus. To evaluate for immunogenicity, we employed a highly sensitive ex vivo platform to prime and expand ncMAP-specific cytotoxic T lymphocytes (CTLs) and evaluate cytolytic potential.ResultsWe demonstrate that ncMAPs are abundant and predominate over mutation-derived peptides in the pancreatic cancer immunopeptidome, establishing a novel class of recurrent cancer-restricted epitopes available for immune recognition. We observed widespread translation and MHC-I presentation of numerous ncMAPs across many healthy tissues, highlighting the importance of our translation-centric approach to assess cancer-restriction. Excitingly, we nominated over 500 ncMAPs that exhibit cancer-specific translation patterns. Approximately 30% of ncMAPs exhibited bona fide cancer-restricted translation patterns, and a substantial subset of these were shared among patients. We next interrogated immunogenicity using a highly sensitive ex vivo vaccination platform and demonstrated that the majority of cancer-restricted ncMAPs evaluated were highly immunogenic. Remarkably, the proportion of ncMAPs harboring immunogenic potential was substantially higher than mutation-derived neoepitopes and tumor-associated antigens, underscoring their therapeutic potential relative to traditional immunotherapy targets.ConclusionsThese findings demonstrate that aberrant translation in pancreatic cancer can give rise to recurrent cancer-restricted ncMAPs capable of recognition by cytotoxic T lymphocytes. Collectively, our findings furnish a novel set of recurrent, cancer-restricted immunotherapy targets not subject to central tolerance. We believe these findings will prompt translation-centric investigations in other solid tumors. We envision that these novel antigens will augment ongoing efforts to treat pancreatic cancer patients with vaccines and cell-based therapies.Ethics ApprovalInformed consent was obtained from patients at least 18 years old with pancreatic cancer under Dana-Farber/Harvard Cancer Center Institutional Review Board (IRB)-approved protocols 11–104, 17–000, 03–189, and/or 14–408 for tissue collection, molecular analysis, and organoid generation.
Abstract
Response rates to immunotherapies in pancreatic ductal adenocarcinoma (PDAC) patients are low. However, cases with long-term survival have been correlated with both T cell infiltration and ...quality of neoantigens (i.e., epitopes from mutations presented by Human Leukocyte Antigen (HLA) molecules). It is known that mutations in the KRAS oncogene, which occur in the majority of PDAC patients, can be recognized by T cells as neoantigens on specific HLA molecules and lead to tumor control. KRAS neoantigens may thus be an attractive therapeutic target in a substantial fraction of PDAC patients. In this study, we systematically evaluated a broad range of KRAS neoantigens using an integrated discovery and validation pipeline. To date, the discovery and validation of KRAS neoantigens has mainly relied on the detection of stochastic T cell responses in patient samples or the use of transgenic mouse models. As such, the reported coverage of common HLA alleles is relatively limited. To overcome these issues, we established a systematic pipeline that addresses two fundamental principles: (1) the presentation of the neoantigen on cells with the mutation and (2) ability of the neoantigen to elicit T cell responses. The presentation of potential neoantigens was evaluated by applying a targeted mass spectrometry approach to detect peptides eluted from specific class I HLA molecules isolated from cells harboring a KRAS mutation of interest. T cell responses were elicited to MS-observed peptides using a proprietary in vitro stimulation protocol, after which the T cells can further be used in cytotoxicity assays to orthogonally confirm neoantigen presentation. We substantiated our pipeline using KRAS codon 12 neoantigens reported in the literature (e.g., G12D on HLA-C*08:02), then evaluated novel neoantigens predicted to be presented by common class I HLA molecules in the U.S. population. Through this pipeline, we have increased the number of validated KRAS neoantigens and thus the targetable patient population expected to harbor at least one KRAS neoantigen. A more comprehensive understanding of KRAS neoantigens will help drive focused therapies, such as the development of tumor-specific T cell receptor (TCR) therapies or neoantigen vaccines in appropriate patient populations in PDAC or other indications.
Citation Format: Jaewon Choi, Scott P Goulding, Brandon P. Conn, Christopher D. McGann, Jared L. Dietze, Jessica Kohler, Divya R. Lenkala, Antoine Boudot, Daniel A. Rothenberg, Paul J. Turcott, John S. Srouji, Michael S. Rooney, Marit M. van Buuren, Richard B. Gaynor, Jennifer G. Abelin, Terri A. Addona, Vikram R. Juneja. Systematic discovery of KRAS neoantigens abstract. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-015.
Abstract
Translation of the ostensibly non-coding genome in cancer can generate novel peptides capable of presentation by major histocompatibility complex class I (MHC-I, HLA-I in humans). These ...non-canonical peptide sources can broaden the landscape of potentially targetable antigens in low-to-intermediate mutational burden malignancies, typified by pancreatic ductal adenocarcinoma (PDA). However, non-canonical MHC-I-associated peptides (ncMAPs) have yet to be explored in pancreatic cancer. We purified the malignant compartment from low tumor cellularity PDA specimens using patient-derived organoids (PDOs) and developed a personalized proteogenomics pipeline coupled with high-depth immunopeptidomics to deeply characterize the repertoire of HLA:peptide targets presented specifically on malignant cells. We demonstrate that ncMAPs are abundant on PDA and predominate over mutation-derived neoepitopes in the detectable immunopeptidome. While emerging evidence in other tumor types has implicated dysregulated translation of non-canonical open reading frames (ncORFs), it is currently unknown to what extent these translation products are truly cancer-restricted and how effectively the resulting ncMAPs can elicit a cytolytic T lymphocyte (CTL) response. To investigate the cancer-specificity of non-canonical peptides, we developed a translation-centric analysis pipeline that examines ncORF expression across a range of healthy tissues, including healthy thymus. We provide evidence that ~30% of ncMAPs exhibit cancer-restricted translation patterns, and a substantial subset of these are shared among pancreatic cancer patients with the appropriate HLA. To investigate immunogenicity, we employed a highly sensitive ex vivo platform to prime and expand antigen-specific T cells. We demonstrate that PDA-restricted ncMAPs are highly immunogenic, on par with or even exceeding the immunogenicity observed with mutation-derived peptides or tumor-associated antigens assayed using the same platform. These findings uncover a critical role for dysregulated translation in pancreatic cancer as a potential source for recurrent cancer-restricted epitopes capable of recognition by cytotoxic T cells. We envision that this novel class of antigens will accelerate ongoing efforts to treat pancreatic cancer patients with vaccines and cell-based therapies.
Citation Format: Zackery A. Ely, Zachary J. Kulstad, Eva K. Verzani, Jennifer G. Abelin, Sudarsana Addepalli, Karl R. Clauser, Marta Casarrubios, Kevin S. Kapner, Miles P. Agus, Connor J. Hennessey, Sine R. Hadrup, Susan Klaeger, Jennifer Su, Alex M. Jaeger, Brian M. Wolpin, Srivatsan Raghavan, Philip D. Greenberg, Andrew J. Aguirre, Steven A. Carr, Tyler Jacks, William A. Freed-Pastor. Non-canonical MHC class I-associated antigens in pancreatic cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A038.
Abstract
Pancreatic adenocarcinoma (PDAC) is among the most lethal cancer types and has been largely recalcitrant to traditional immunotherapy. A large subset of PDAC tumors is computationally ...predicted to harbor potentially immunogenic peptides for MHC class I (MHC-I) presentation, but the nature, expression, and immunogenicity of these peptides has yet to be determined. By investigating the PDAC immunopeptidome, we can uncover and exploit novel immune-based targets for PDAC and render it vulnerable to immunotherapy. Prior efforts to study the immunopeptidome in PDAC have largely focused on profiling MHC-associated peptides (MAPs) from bulk tumor samples. This approach is severely limited by the contribution of MAPs from the non-malignant compartments, which constitutes most of the cellular mass in PDAC. We can overcome this limitation by using patient-derived organoids (PDOs) to expand a pure cancer cell population for MHC-I immunoprecipitation, followed by LC/MS-MS. We applied this approach and detected 17,000-20,500 unique MAPs per sample, a dramatic increase in depth and resolution over prior efforts. To ascertain which MAPs may be PDAC-restricted, we first analyzed bulk RNA-sequencing data from the Genotype-Tissue Expression Project (767 patients, 30 tissues) to generate a set of genes that are functionally undetectable (Q90 <1 TPM) in healthy somatic tissues. We cross-referenced this list with our set of PDO MAPs, yielding 143 PDAC-restricted MAPs. To further expand our search space, we implemented a tiered tissue-based filter to relax TPM cutoffs in less essential tissues (ex. prostate) while maintaining strict cutoffs in essential tissues (ex. brain), generating 85 additional MAPs. Both approaches uncovered cancer-restricted MAPs present in most PDO lines, which may represent shared therapeutic targets. While somatic mutations are a well-established source of tumor-specific neoantigens, these have yet to be investigated with immunopeptidomics in human PDAC. Despite harboring an intermediate mutational burden, we detected at least one mutation-derived neoepitope in most PDOs, a detection frequency much closer to the detection frequency of cancers with high mutational burden than previously expected. Additionally, non-canonical peptide sources, including retained introns (RI) and novel unannotated open reading frames (nuORF), may represent a source of cancer-restricted MAPs. Interestingly, we detect nuORF- and RI-derived MAPs (n=298) in all PDOs, suggesting that these too may be therapeutically relevant in PDAC. To assess the immunogenicity of candidate MAPs, future studies will utilize established protocols for priming and expanding MAP-specific human T cells with autologous DCs. Functional evaluation of MAP-specific T cells will help prioritize strategies for vaccination, and the generation of T cell receptor sequences for adoptive cell therapy. Collectively, these data deepen our understanding of the PDAC immunopeptidome and provide a novel set of targets for immunotherapy in PDAC.
Citation Format: Zackery A. Ely, William A. Freed-Pastor, Zachary J. Kulstad, Jennifer G. Abelin, Eva Verzani, Kevin S. Kapner, Susan Klaeger, Karl R. Clauser, Miles Agus, Alex M. Jaeger, Nimisha B. Pattada, Arjun Bhutkar, Andrew J. Aguirre, Steven A. Carr, Tyler Jacks. Broadening the repertoire of PDAC-specific targets for immune-based therapy through high-resolution immunopeptidomics abstract. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C014.
Abstract
Background: There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Memory CD8+ T cell infiltration is now well established as a key prognostic indicator in ...CRC, and it is known that these tumor infiltrating lymphocytes (TILs) are specifically targeting and killing tumor cells. However, the epitopes that these TILs use to identify cancer cells have not been determined. This has limited the use of immunotherapies in CRC, despite their efficacy in other cancer types. Recently, phosphopeptides have emerged as strong candidates for tumor-specific epitopes, since dysregulation of signaling in cancers leads to aberrant protein phosphorylation. Here, we identify CRC-associated phosphopeptides and assess the tumor-resident immunity against these novel epitopes.
Methods: We compared tumor and healthy tissue from CRC patients, to identify tumor-specific MHC class-I associated phosphopeptides. The tissues were lysed, the MHC class-I complexes affinity purified, and the bound peptides eluted. Phosphopeptides were enriched using immobilized metal affinity chromatography, and characterized using mass spectrometry. TILs, from the same tumors, were extracted and expanded, and their responses to the phosphopeptides assessed using multiplexed intracellular cytokine staining. Cytolytic activity was observed by staining for surface mobilization of CD107a. Healthy donor responses were quantified using interferon-γ ELISpot, and functionality assessed using a europium release killing assay.
Results: We have identified 125 tumor-associated MHC class-I phosphopeptides from CRC, with different HLA-restrictions. There were, on average, 3.5 times more different phosphopeptides identified on cancer than healthy tissues, at 6.7-fold higher levels. Many of these novel epitopes are attributable to signaling events in well-defined cancer pathways and are therefore markers of malignancy. Through analysis of TIL's cytokine responses to these phosphopeptides, we have established that they are playing a key role in tumor-resident immunity. There were multifunctional TILs present in primary and metastatic tumors that recognized and killed in response to these phosphopeptides. Up to 0.7% of expanded TILs targeted each phosphopeptide, comparable with responses seen to viral epitopes. Thus, immunity to tumor-associated phosphopeptides represents a biological strategy for distinguishing tumor from healthy tissue.
Furthermore, we have shown that healthy donors have pre-existing, memory T cell responses to many (58%) of these CRC-associated phosphopeptides. These phosphopeptide-specific T cells are readily expanded ex vivo and can kill CRC cell lines. Therefore, MHC class-I associated phosphopeptides are ideal immunotherapeutic targets, as immunity must spare healthy tissue.
Conclusion: The identification of this novel class of MHC class-I epitopes in CRC offers new hope for the future of immunotherapy in this malignancy.
Citation Format: Sarah A. Penny, Jennifer G. Abelin, Abu Z. Saeed, Stacy A. Malaker, Paisley D. Trantham, Jeffrey Shabanowitz, Stephen T. Ward, Donald F. Hunt, Mark Cobbold. Phosphopeptides as novel T cell epitopes in colorectal cancer. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3627. doi:10.1158/1538-7445.AM2014-3627
Abstract
While tumor-reactive CD4+ T cells have been associated with effective immunotherapy responses, accurate prediction of MHC class II-restricted ligands remains a challenge, limiting our ...ability to harness CD4+ immunity for cancer therapy. To this end, we have developed a state-of-the-art MHC class II binding predictor, neonmhc2, trained on HLA-II ligands identified from mass spectrometry (MS) of cell lines engineered to express a single affinity-tagged HLA-II allele. Over 60 HLA-II alleles have been characterized to date. We demonstrate that neonmhc2 outperforms NetMHCIIpan, the current benchmark for class II prediction, in distinguishing 1) HLA-II ligands presented in cell lines and tissues, and critically, 2) immunogenic CD4+ epitopes identified with tetramer-guided epitope mapping. Furthermore, we show that numerous neoantigen peptides that were ranked highly by neonmhc2 but poorly by NetMHCIIpan were immunogenic in an ex vivo induction. We next studied HLA-II antigen processing in order to further boost our ability to predict CD4+ epitopes. We determined a gene-level bias by comparing transcript expression and gene length to the frequency of observations in MS, finding that secreted genes are over-represented in HLA-II ligandomes from tissues. We built numerous primary sequence-based processability predictors trained on MS data, but only achieved significant prediction improvement when using the simple feature of determining if a candidate peptide contained sequence that overlapped a previously observed HLA-II ligand. By combining neonmhc2 binding prediction, transcript expression, gene bias, and the overlap feature in an integrated presentation predictor, we were able to achieve up to a 61-fold increase in ability to predict HLA-II peptides presented from tissue MS data over NetMHCIIpan alone. We also sought to understand which cells are presenting HLA-II ligands in the tumor microenvironment to elucidate the presentation pathway most relevant to immunotherapy. By leveraging publicly available RNA-seq (bulk and single cell), we found that professional antigen-presenting cells rather than tumor cells are primarily responsible for HLA-II presentation. We developed a novel SILAC-based MS workflow to directly interrogate peptides derived from phagocytosed tumor cells that are presented by dendritic cells. The experiment revealed that mitochondrial genes are preferentially presented from phagocytosed cells. In conclusion, by integrating proteomics and genomics data at large scale, we have defined new rules for understanding HLA-II processing and presentation, particularly in the context of the tumor microenvironment. This work should enhance our ability to predict CD4+ epitopes for immunotherapy.
Citation Format: Dewi Harjanto, Jennifer G. Abelin, Matthew Malloy, Prerna Suri, Tyler Colson, Scott P. Goulding, Amanda L. Creech, Lia R. Serrano, Gibbs Nasir, Yusuf Nasrullah, Christopher D. McGann, Diana Velez, Ying S. Ting, Asaf Poran, Daniel A. Rothenberg, Sagar Chhangawala, Alex Rubinsteyn, Jeff Hammerbacher, Richard B. Gaynor, Edward F. Fritsch, Rob C. Oslund, Dominik Barthelme, Terri A. Addona, Christina M. Arieta, Michael S. Rooney. Enhanced HLA-II epitope prediction for immunotherapy with novel proteomics and genomics approaches abstract. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B23.
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