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Sensitive and quantitative detection of MHC-I displayed neoepitopes using a semi-automated workflow and TOMAHAQ mass spectrometry

ABSTRACT: Advances in several key technologies, including MHC peptidomics, has helped fuel our understanding of basic immune regulatory mechanisms and identify T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. This multi-step analytical process remains challenging, and throughput and reproducibility are paramount for rapidly characterizing multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines, can be enriched in a semi-automated fashion on reusable, dry-storage, customized antibody cartridges. TOMAHAQ, a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation.

ORGANISM(S): Mus musculus

PROVIDER: GSE163326 | GEO | 2021/07/20

REPOSITORIES: GEO

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Sensitive and quantitative detection of MHC-I displayed neoepitopes using a semi-automated workflow and TOMAHAQ mass spectrometry

Project description:Advances in several key technologies, including MHC peptidomics, has helped fuel our understanding of basic immune regulatory mechanisms and identify T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. This multi-step analytical process remains challenging, and throughput and reproducibility are paramount for rapidly characterizing multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines, can be enriched in a semi-automated fashion on reusable, dry-storage, customized antibody cartridges. TOMAHAQ, a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation.

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Sensitive and quantitative detection of MHC-I displayed neoepitopes using a semi-automated workflow and TOMAHAQ mass spectrometry

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Project description:Personalized cancer immunotherapies such as vaccines and T cell receptor (TCR)-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen (HLA) class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. For their detection by liquid chromatography mass spectrometry (LC-MS), we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this for to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft (PDX) from as little as 2.5×106 cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, we here present optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, which allowed for the identification of actionable neoepitopes from sample sizes usually available in the clinic.

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Project description:Strategies for maximizing the potency and specificity of cancer immunotherapies have sparked efforts to identify recurrent epitopes presented in the context of defined tumor-associated neoantigens. Discovering these neoepitopes can be difficult owing to the limited number of peptides that arise from a single point mutation, a low number of copies presented on the cell surface, and variable binding specificity of the human leukocyte antigen (HLA) class I complex. Due to these limitations, many discovery efforts focus on identifying neoepitopes from a small number of cancer neoantigens in the context of few HLA alleles. Here we describe a systematic workflow to characterize binding and presentation of neoepitopes derived from 47 shared cancer neoantigens in the context of 15 HLA alleles. Through the development of a high-throughput neoepitope-HLA binding assay, we surveyed 24,149 candidate neoepitope-HLA combinations resulting in 587 stable complexes. These data were supplemented by computational prediction that identified an additional 257 neoepitope-HLA pairs, resulting in a total of 844 unique combinations. We used these results to build sensitive targeted mass spectrometry assays to validate neoepitope presentation on a panel of HLA-I monoallelic cell lines engineered to express neoantigens of interest as a single polypeptide. Altogether, our analyses detected 84 unique neoepitope-HLA pairs derived from 37 shared cancer neoantigens and presented across 12 HLA alleles. We subsequently identified multiple TCRs which specifically recognized two of these neoantigen-HLA combinations. Finally, these novel TCRs were utilized to elicit a T cell response suggesting that these neoepitopes are likely to be immunogenic. Together these data represent a validated, extensive resource of therapeutically relevant neoepitopes and the HLA context in which they can be targeted.

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