A comprehensive proteogenomic pipeline for neoantigen discovery to advance personalized cancer immunotherapy.

The accurate identification and prioritization of antigenic peptides is crucial for the development of personalized cancer immunotherapies. Publicly available pipelines to predict clinical neoantigens do not allow direct integration of mass spectrometry immunopeptidomics data, which can uncover antigenic peptides derived from various canonical and noncanonical sources. To address this, we present an end-to-end clinical proteogenomic pipeline, called NeoDisc, that combines state-of-the-art publicly available and in-house software for immunopeptidomics, genomics and transcriptomics with in silico tools for the identification, prediction and prioritization of tumor-specific and immunogenic antigens from multiple sources, including neoantigens, viral antigens, high-confidence tumor-specific antigens and tumor-specific noncanonical antigens. We demonstrate the superiority of NeoDisc in accurately prioritizing immunogenic neoantigens over recent prioritization pipelines. We showcase the various features offered by NeoDisc that enable both rule-based and machine-learning approaches for personalized antigen discovery and neoantigen cancer vaccine design. Additionally, we demonstrate how NeoDisc's multiomics integration identifies defects in the cellular antigen presentation machinery, which influence the heterogeneous tumor antigenic landscape.

Tumor-reactive T cell clonotype dynamics underlying clinical response to TIL therapy in melanoma.

Adoptive cell therapy (ACT) using in vitro expanded tumor-infiltrating lymphocytes (TILs) has inconsistent clinical responses. To better understand determinants of therapeutic success, we tracked TIL clonotypes from baseline tumors to ACT products and post-ACT blood and tumor samples in melanoma patients using single-cell RNA and T cell receptor (TCR) sequencing. Patients with clinical responses had baseline tumors enriched in tumor-reactive TILs, and these were more effectively mobilized upon in vitro expansion, yielding products enriched in tumor-specific CD8+ cells that preferentially infiltrated tumors post-ACT. Conversely, lack of clinical responses was associated with tumors devoid of tumor-reactive resident clonotypes and with cell products mostly composed of blood-borne clonotypes that persisted in blood but not in tumors post-ACT. Upon expansion, tumor-specific TILs lost tumor-associated transcriptional signatures, including exhaustion, and responders exhibited an intermediate exhausted effector state after TIL engraftment in the tumor, suggesting functional reinvigoration. Our findings provide insight into the nature and dynamics of tumor-specific clonotypes associated with clinical response to TIL-ACT, with implications for treatment optimization.

Machine learning methods and harmonized datasets improve immunogenic neoantigen prediction.

The accurate selection of neoantigens that bind to class I human leukocyte antigen (HLA) and are recognized by autologous T cells is a crucial step in many cancer immunotherapy pipelines. We reprocessed whole-exome sequencing and RNA sequencing (RNA-seq) data from 120 cancer patients from two external large-scale neoantigen immunogenicity screening assays combined with an in-house dataset of 11 patients and identified 46,017 somatic single-nucleotide variant mutations and 1,781,445 neo-peptides, of which 212 mutations and 178 neo-peptides were immunogenic. Beyond features commonly used for neoantigen prioritization, factors such as the location of neo-peptides within protein HLA presentation hotspots, binding promiscuity, and the role of the mutated gene in oncogenicity were predictive for immunogenicity. The classifiers accurately predicted neoantigen immunogenicity across datasets and improved their ranking by up to 30%. Besides insights into machine learning methods for neoantigen ranking, we have provided homogenized datasets valuable for developing and benchmarking companion algorithms for neoantigen-based immunotherapies.

TCR sequencing and cloning methods for repertoire analysis and isolation of tumor-reactive TCRs.

T cell receptor (TCR) technologies, including repertoire analyses and T cell engineering, are increasingly important in the clinical management of cellular immunity in cancer, transplantation, and other immune diseases. However, sensitive and reliable methods for repertoire analyses and TCR cloning are still lacking. Here, we report on SEQTR, a high-throughput approach to analyze human and mouse repertoires that is more sensitive, reproducible, and accurate as compared with commonly used assays, and thus more reliably captures the complexity of blood and tumor TCR repertoires. We also present a TCR cloning strategy to specifically amplify TCRs from T cell populations. Positioned downstream of single-cell or bulk TCR sequencing, it allows time- and cost-effective discovery, cloning, screening, and engineering of tumor-specific TCRs. Together, these methods will accelerate TCR repertoire analyses in discovery, translational, and clinical settings and permit fast TCR engineering for cellular therapies.

Development of an optimized closed and semi-automatic protocol for Good Manufacturing Practice manufacturing of tumor-infiltrating lymphocytes in a hospital environment.

BACKGROUND AIMS: Several studies report on Good Manufacturing Process (GMP)-compliant manufacturing protocols for the ex vivo expansion of tumor-infiltrating lymphocytes (TILs) for the treatment of patients with refractory melanoma and other solid malignancies. Further opportunities for improvements in terms of ergonomy and operating time have been identified. METHODS: To enable GMP-compliant TILs production for adoptive cell therapy needs, a simple automated and reproducible protocol for TILs manufacturing with the use of a closed system was developed and implemented at the authors' institution. RESULTS: This protocol enabled significant operating time reduction during TILs expansion while allowing the generation of high-quality TILs products. CONCLUSIONS: A simplified and efficient method of TILs expansion will enable the broadening of individualized tumor therapy and will increase patients' access to state-of-the-art TILs adoptive cell therapy treatment.

Guillain-Barré syndrome after adoptive cell therapy with tumor-infiltrating lymphocytes.

BACKGROUND: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) is a promising experimental immunotherapy that has shown high objective responses in patients with melanoma. Current protocols use a lymphodepletive chemotherapy before infusion of ex vivo expanded TILs, followed by high-dose interleukin-2 (IL-2). Treatment-related toxicities are mainly attributable to the chemotherapy regimen and to the high-dose IL-2 and are generally reversible. Neurological side effects have rarely been described. Nevertheless, due to improvements in cell production techniques and due to combinations with other immunomodulating molecules, side effects not previously described may be encountered. CASE PRESENTATION: We report the case of a 53-year-old heavily pretreated patient with melanoma who developed Guillain-Barré syndrome (GBS) 19 days after ACT using autologous TILs, given in the context of a phase I trial. He presented with dorsal back pain, unsteady gait and numbness in hands and feet. Lumbar puncture showed albuminocytological dissociation, and nerve conduction studies revealed prolonged distal motor latencies in median, ulnar, tibial and peroneal nerves, compatible with a GBS. The patient was treated with intravenous immunoglobulins and intensive neurological rehabilitation, with progressive and full recovery at 21 months post-TIL-ACT. Concomitant to the onset of GBS, a cytomegalovirus reactivation on immunosuppression was detected and considered as the most plausible cause of this neurological side effect. CONCLUSION: We describe for the first time a case of GBS occurring shortly after TIL-ACT for melanoma, even though we could not identify with certainty the triggering agent. The report of such rare cases is of extreme importance to build on the knowledge of immune cellular therapies and their specific spectrum of toxicities.

Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes.

Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides derived from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single-cell transcriptomics, ribosome profiling, and two MS/MS search tools in combination. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides, including an immunogenic peptide derived from an open reading frame downstream of the melanoma stem cell marker gene ABCB5. These findings hold great promise for the discovery of previously unknown tumor antigens for cancer immunotherapy.

Tumor-associated factors are enriched in lymphatic exudate compared to plasma in metastatic melanoma patients.

Liquid biopsies allow monitoring of cancer progression and detection of relapse, but reliable biomarkers in melanoma are lacking. Because secreted factors preferentially drain to lymphatic vessels before dilution in the blood, we hypothesized that lymph should be vastly enriched in cancer biomarkers. We characterized postoperative lymphatic exudate and plasma of metastatic melanoma patients after lymphadenectomy and found a dramatic enrichment in lymphatic exudate of tumor-derived factors and especially extracellular vesicles containing melanoma-associated proteins and miRNAs, with unique protein signatures reflecting early versus advanced metastatic spread. Furthermore, lymphatic exudate was enriched in memory T cells, including tumor-reactive CD137+ and stem cell-like types. In mice, lymph vessels were the major route of extracellular vesicle transport from tumors to the systemic circulation. We suggest that lymphatic exudate provides a rich source of tumor-derived factors for enabling the discovery of novel biomarkers that may reflect disease stage and therapeutic response.