Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification.

Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types for proteogenomic-based discovery of neoantigens. By using an optimized computational approach, we discover a large number of tumor-specific and tumor-associated antigens. To create a pipeline for the identification of neoantigens in our cohort, we combine DNA and RNA sequencing with MS-based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity and an in-depth validation process. We detect a broad variety of non-canonical HLA-binding peptides in the majority of patients demonstrating partially immunogenicity. Our validation process allows for the selection of 32 potential neoantigen candidates. The majority of neoantigen candidates originates from variants identified in the RNA data set, illustrating the relevance of RNA as a still understudied source of cancer antigens. This study underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates.

The CAR-HEMATOTOX score as a prognostic model of toxicity and response in patients receiving BCMA-directed CAR-T for relapsed/refractory multiple myeloma.

BACKGROUND: BCMA-directed CAR T-cell therapy (CAR-T) has altered the treatment landscape of relapsed/refractory (r/r) multiple myeloma, but is hampered by unique side effects that can lengthen hospital stays and increase morbidity. Hematological toxicity (e.g. profound and prolonged cytopenias) represents the most common grade ≥ 3 toxicity and can predispose for severe infectious complications. Here, we examined the utility of the CAR-HEMATOTOX (HT) score to predict toxicity and survival outcomes in patients receiving standard-of-care idecabtagene vicleucel and ciltacabtagene autoleucel. METHODS: Data were retrospectively collected from 113 r/r multiple myeloma patients treated between April 2021 and July 2022 across six international CAR-T centers. The HT score-composed of factors related to hematopoietic reserve and baseline inflammatory state-was determined prior to lymphodepleting chemotherapy. RESULTS: At lymphodepletion, 63 patients were HTlow (score 0-1) and 50 patients were HThigh (score ≥ 2). Compared to their HTlow counterparts, HThigh patients displayed prolonged severe neutropenia (median 9 vs. 3 days, p < 0.001), an increased severe infection rate (40% vs. 5%, p < 0.001), and more severe ICANS (grade ≥ 3: 16% vs. 0%, p < 0.001). One-year non-relapse mortality was higher in the HThigh group (13% vs. 2%, p = 0.019) and was predominantly attributable to fatal infections. Response rates according to IMWG criteria were higher in HTlow patients (≥ VGPR: 70% vs. 44%, p = 0.01). Conversely, HThigh patients exhibited inferior progression-free (median 5 vs. 15 months, p < 0.001) and overall survival (median 10.5 months vs. not reached, p < 0.001). CONCLUSIONS: These data highlight the prognostic utility of the CAR-HEMATOTOX score for both toxicity and treatment response in multiple myeloma patients receiving BCMA-directed CAR-T. The score may guide toxicity management (e.g. anti-infective prophylaxis, early G-CSF, stem cell boost) and help to identify suitable CAR-T candidates.

Functional analysis of peripheral and intratumoral neoantigen-specific TCRs identified in a patient with melanoma.

BACKGROUND: Neoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation. METHODS: Three neoantigens, which we identified either by immunopeptidomics or in silico prediction, were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient's immune repertoire recognizing these antigens. TCRs were compared in vitro by multiparametric analyses including functional avidity, multicytokine secretion, and cross-reactivity screenings. A xenograft mouse model served to study in vivo functionality of selected TCRs. We investigated the patient's TCR repertoire in blood and different tumor-related tissues over 3 years using TCR beta deep sequencing. RESULTS: Selected mutated peptide ligands with proven immunogenicity showed similar binding affinities to the human leukocyte antigen complex and comparable disparity to their wild-type counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs recognizing these antigens demonstrated distinct patterns in functionality and frequency. TCRs with lower functional avidity showed at least equal antitumor immune responses in vivo. Moreover, they occurred at high frequencies and particularly demonstrated long-term persistence within tumor tissues, lymph nodes and various blood samples associated with a reduced activation pattern on primary in vitro stimulation. CONCLUSIONS: We performed a so far unique fine characterization of neoantigen-specific T-cell responses revealing defined reactivity patterns of neoantigen-specific TCRs. Our data highlight qualitative differences of these TCRs associated with function and longevity of respective T cells. Such features need to be considered for further optimization of neoantigen targeting including adoptive T-cell therapies using TCR-transgenic T cells.

Deep learning boosts sensitivity of mass spectrometry-based immunopeptidomics.

Characterizing the human leukocyte antigen (HLA) bound ligandome by mass spectrometry (MS) holds great promise for developing vaccines and drugs for immune-oncology. Still, the identification of non-tryptic peptides presents substantial computational challenges. To address these, we synthesized and analyzed >300,000 peptides by multi-modal LC-MS/MS within the ProteomeTools project representing HLA class I & II ligands and products of the proteases AspN and LysN. The resulting data enabled training of a single model using the deep learning framework Prosit, allowing the accurate prediction of fragment ion spectra for tryptic and non-tryptic peptides. Applying Prosit demonstrates that the identification of HLA peptides can be improved up to 7-fold, that 87% of the proposed proteasomally spliced HLA peptides may be incorrect and that dozens of additional immunogenic neo-epitopes can be identified from patient tumors in published data. Together, the provided peptides, spectra and computational tools substantially expand the analytical depth of immunopeptidomics workflows.

Key Features Relevant to Select Antigens and TCR From the MHC-Mismatched Repertoire to Treat Cancer.

Adoptive transfer of T cells transgenic for tumor-reactive T-cell receptors (TCR) is an attractive immunotherapeutic approach. However, clinical translation is so far limited due to challenges in the identification of suitable target antigens as well as TCRs that are concurrent safe and efficient. Definition of key characteristics relevant for effective and specific tumor rejection is essential to improve current TCR-based adoptive T-cell immunotherapies. We here characterized in-depth two TCRs derived from the human leukocyte antigen (HLA)-mismatched allogeneic repertoire targeting two different myeloperoxidase (MPO)-derived peptides presented by the same HLA-restriction element side by side comprising state of the art biochemical and cellular in vitro, in vivo, and in silico experiments. In vitro experiments reveal comparable functional avidities, off-rates, and cytotoxic activities for both TCRs. However, we observed differences especially with respect to cytokine secretion and cross-reactivity as well as in vivo activity. Biochemical and in silico analyses demonstrate different binding qualities of MPO-peptides to the HLA-complex determining TCR qualities. We conclude from our biochemical and in silico analyses of peptide-HLA-binding that rigid and high-affinity binding of peptides is one of the most important factors for isolation of TCRs with high specificity and tumor rejection capacity from the MHC-mismatched repertoire. Based on our results, we developed a workflow for selection of such TCRs with high potency and safety profile suitable for clinical translation.

T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target.

Cancer immunotherapy has proven high efficacy in treating diverse cancer entities by immune checkpoint modulation and adoptive T-cell transfer. However, patterns of treatment response differ substantially from conventional therapies, and reliable surrogate markers are missing for early detection of responders versus non-responders. Current imaging techniques using 18F-fluorodeoxyglucose-positron-emmission-tomograpy (18F-FDG-PET) cannot discriminate, at early treatment times, between tumor progression and inflammation. Therefore, direct imaging of T cells at the tumor site represents a highly attractive tool to evaluate effective tumor rejection or evasion. Moreover, such markers may be suitable for theranostic imaging. Methods: We mainly investigated the potential of two novel pan T-cell markers, CD2 and CD7, for T-cell tracking by immuno-PET imaging. Respective antibody- and F(ab´)2 fragment-based tracers were produced and characterized, focusing on functional in vitro and in vivo T-cell analyses to exclude any impact of T-cell targeting on cell survival and antitumor efficacy. Results: T cells incubated with anti-CD2 and anti-CD7 F(ab´)2 showed no major modulation of functionality in vitro, and PET imaging provided a distinct and strong signal at the tumor site using the respective zirconium-89-labeled radiotracers. However, while T-cell tracking by anti-CD7 F(ab´)2 had no long-term impact on T-cell functionality in vivo, anti-CD2 F(ab´)2 caused severe T-cell depletion and failure of tumor rejection. Conclusion: This study stresses the importance of extended functional T-cell assays for T-cell tracer development in cancer immunotherapy imaging and proposes CD7 as a highly suitable target for T-cell immuno-PET imaging.

Identification and Characterization of Neoantigens As Well As Respective Immune Responses in Cancer Patients.

Cancer immunotherapy has recently emerged as a powerful tool for the treatment of diverse advanced malignancies. In particular, therapeutic application of immune checkpoint modulators, such as anti-CTLA4 or anti-PD-1/PD-L1 antibodies, have shown efficacy in a broad range of malignant diseases. Although pharmacodynamics of these immune modulators are complex, recent studies strongly support the notion that altered peptide ligands presented on tumor cells representing neoantigens may play an essential role in tumor rejection by T cells activated by anti-CTLA4 and anti-PD-1 antibodies. Neoantigens may have diverse sources as viral and mutated proteins. Moreover, posttranslational modifications and altered antigen processing may also contribute to the neoantigenic peptide ligand landscape. Different approaches of target identification are currently applied in combination with subsequent characterization of autologous and non-self T-cell responses against such neoantigens. Additional efforts are required to elucidate key characteristics and interdependences of neoantigens, immunodominance, respective T-cell responses, and the tumor microenvironment in order to define decisive determinants involved in effective T-cell-mediated tumor rejection. This review focuses on our current knowledge of identification and characterization of such neoantigens as well as respective T-cell responses. It closes with challenges to be addressed in future relevant for further improvement of immunotherapeutic strategies in malignant diseases.

Tools to define the melanoma-associated immunopeptidome.

Immunotherapies have been traditionally applied in malignant melanoma, which represent one of the most immunogenic tumours. Recently, immune checkpoint modulation has shown high therapeutic efficacy and may provide long-term survival in a significant proportion of affected patients. T cells are the major players in tumour rejection and recognize tumour cells predominantly in an MHC-dependent way. The immunopeptidome comprises the peptide repertoire presented by MHC class I and II molecules on the surface of the body's cells including tumour cells. To understand characteristics of suitable rejection antigens as well as respective effective T-cell responses, determination of the immunopeptidome is of utmost importance. Suitable rejection antigens need to be further characterized and validated not only to systematically improve current therapeutic approaches, but also to develop individualized treatment options. In this review, we report on current tools to explore the immunopeptidome in human melanoma and discuss current understanding and future developments to specifically detect and select those antigens that may be most relevant and promising for effective tumour rejection.

Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry.

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.