Common phenotypic dynamics of tumor-infiltrating lymphocytes across different histologies upon checkpoint inhibition: impact on clinical outcome.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the cancer therapeutic landscape and our perception of interactions between the immune system and tumor cells. Despite remarkable progress, disease relapse and primary resistance are not uncommon. Understanding the biological processes that tumor-infiltrating lymphocytes (TILs) undergo during ICI, how this affects the tumor microenvironment (TME) and, ultimately, clinical outcome is, therefore, necessary to further improve treatment efficacy. AIM: In the current study, we sought to characterize TILs from patients with metastatic solid tumors undergoing ICI correlating flowcytometric findings with clinical outcome. METHODS: In total, 20 patients with 10 different metastatic solid tumors treated with ICIs targeting programmed-cell death-1 (PD-1)/PD-L1 axis were included in this study. The phenotype of T cells deriving from biopsies obtained prior to treatment initiation and on-treatment was investigaded. Analyses were focused on T cells' degree of differentiation and activity and how they correlate with transcriptomic changes in the TME. RESULTS: Data indicate that patients benefitting from ICIs accumulate CD8+central memory T cells. TILs developed an effector-like phenotype over time, which was also associated with a cytolytic gene signature. In terms of modulation of T-cell responses, we observed that high expression of checkpoint molecules pre-treatment (i.e., PD-1, lymphocyte activation gene-3 [LAG-3], B and T-lymphocyte attenuator [BTLA] and T-cell immunoglobulin and mucin domain containing-3 [TIM-3]) was associated with similar gene signature and correlated to treatment benefit. Increasing expression of LAG-3 and BTLA in the CD8 compartment and their co-expression with PD-1 during treatment were, however, a common feature for patients who failed to respond to ICIs. CONCLUSIONS: Besides identifying immune profiles suggestive of response to ICI, our results provide a more nuanced picture regarding expression of checkpoint molecules that goes beyond T-cell anergy.

Nivolumab reaches brain lesions in patients with recurrent glioblastoma and induces T-cell activity and upregulation of checkpoint pathways.

Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. Although immunotherapy is being explored as a potential treatment option for patients with GBM, it is unclear whether systemic immunotherapy can reach and modify the tumor microenvironment in the brain. We evaluated immune characteristics in patients receiving the anti-PD1 immune checkpoint inhibitor Nivolumab one week prior to surgery, compared to control patients receiving salvage resection without prior Nivolumab treatment. We observed saturating levels of Nivolumab bound to intratumorally- and tissue-resident T cells in the brain, implicating saturating levels of Nivolumab reaching brain tumors. Following Nivolumab treatment, significant changes in T-cell activation and proliferation were observed in the tumor resident T-cell population, and peripheral T cells upregulated chemokine receptors related to brain homing. A strong Nivolumab-driven upregulation in compensatory checkpoint inhibition molecules, TIGIT, LAG-3, TIM-3 and CTLA-4 was observed, potentially counteracting the treatment effect. Finally, tumor-reactive tumor-infiltrating lymphocytes (TILs) were found in a subset of Nivolumab-treated patients with prolonged survival, and neoantigen-reactive T cells were identified in both TILs and blood. This indicates a systemic response towards GBM in a subset of patients, which was further boosted by Nivolumab, with T-cell responses towards tumor-derived neoantigens. Our study demonstrates that Nivolumab does reach the GBM tumor lesion and enhances antitumor T-cell responses both intratumorally and systemically. However, various anti-inflammatory mechanisms mitigate the clinical efficacy of the anti-PD1 treatment.

Dose escalation study of a personalized peptide-based neoantigen vaccine (EVX-01) in patients with metastatic melanoma.

BACKGROUND: Neoantigens can serve as targets for T cell-mediated antitumor immunity via personalized neopeptide vaccines. Interim data from our clinical study NCT03715985 showed that the personalized peptide-based neoantigen vaccine EVX-01, formulated in the liposomal adjuvant, CAF09b, was safe and able to elicit EVX-01-specific T cell responses in patients with metastatic melanoma. Here, we present results from the dose-escalation part of the study, evaluating the feasibility, safety, efficacy, and immunogenicity of EVX-01 in addition to anti-PD-1 therapy. METHODS: Patients with metastatic melanoma on anti-PD-1 therapy were treated in three cohorts with increasing vaccine dosages (twofold and fourfold). Tumor-derived neoantigens were selected by the AI platform PIONEER and used in personalized therapeutic cancer peptide vaccines EVX-01. Vaccines were administered at 2-week intervals for a total of three intraperitoneal and three intramuscular injections. The study's primary endpoint was safety and tolerability. Additional endpoints were immunological responses, survival, and objective response rates. RESULTS: Compared with the base dose level previously reported, no new vaccine-related serious adverse events were observed during dose escalation of EVX-01 in combination with an anti-PD-1 agent given according to local guidelines. Two patients at the third dose level (fourfold dose) developed grade 3 toxicity, most likely related to pembrolizumab. Overall, 8 out of the 12 patients had objective clinical responses (6 partial response (PR) and 2 CR), with all 4 patients at the highest dose level having a CR (1 CR, 3 PR). EVX-01 induced peptide-specific CD4+ and/or CD8+T cell responses in all treated patients, with CD4+T cells as the dominating responses. The magnitude of immune responses measured by IFN-γ ELISpot assay correlated with individual peptide doses. A significant correlation between the PIONEER quality score and induced T cell immunogenicity was detected, while better CRs correlated with both the number of immunogenic EVX-01 peptides and the PIONEER quality score. CONCLUSION: Immunization with EVX-01-CAF09b in addition to anti-PD-1 therapy was shown to be safe and well tolerated and elicit vaccine neoantigen-specific CD4+and CD8+ T cell responses at all dose levels. In addition, objective tumor responses were observed in 67% of patients. The results encourage further assessment of the antitumor efficacy of EVX-01 in combination with anti-PD-1 therapy.

IMPROVE: a feature model to predict neoepitope immunogenicity through broad-scale validation of T-cell recognition.

Background: Mutation-derived neoantigens are critical targets for tumor rejection in cancer immunotherapy, and better tools for neoepitope identification and prediction are needed to improve neoepitope targeting strategies. Computational tools have enabled the identification of patient-specific neoantigen candidates from sequencing data, but limited data availability has hindered their capacity to predict which of the many neoepitopes will most likely give rise to T cell recognition. Method: To address this, we make use of experimentally validated T cell recognition towards 17,500 neoepitope candidates, with 467 being T cell recognized, across 70 cancer patients undergoing immunotherapy. Results: We evaluated 27 neoepitope characteristics, and created a random forest model, IMPROVE, to predict neoepitope immunogenicity. The presence of hydrophobic and aromatic residues in the peptide binding core were the most important features for predicting neoepitope immunogenicity. Conclusion: Overall, IMPROVE was found to significantly advance the identification of neoepitopes compared to other current methods.

Neoepitope load, T cell signatures and PD-L2 as combined biomarker strategy for response to checkpoint inhibition immunotherapy.

Immune checkpoint inhibition for the treatment of cancer has provided a breakthrough in oncology, and several new checkpoint inhibition pathways are currently being investigated regarding their potential to provide additional clinical benefit. However, only a fraction of patients respond to such treatment modalities, and there is an urgent need to identify biomarkers to rationally select patients that will benefit from treatment. In this study, we explore different tumor associated characteristics for their association with favorable clinical outcome in a diverse cohort of cancer patients treated with checkpoint inhibitors. We studied 29 patients in a basket trial comprising 12 different tumor types, treated with 10 different checkpoint inhibition regimens. Our analysis revealed that even across this diverse cohort, patients achieving clinical benefit had significantly higher neoepitope load, higher expression of T cell signatures, and higher PD-L2 expression, which also correlated with improved progression-free and overall survival. Importantly, the combination of biomarkers serves as a better predictor than each of the biomarkers alone. Basket trials are frequently used in modern immunotherapy trial design, and here we identify a set of biomarkers of potential relevance across multiple cancer types, allowing for the selection of patients that most likely will benefit from immune checkpoint inhibition.

Neoantigen-specific CD8 T cell responses in the peripheral blood following PD-L1 blockade might predict therapy outcome in metastatic urothelial carcinoma.

CD8+ T cell reactivity towards tumor mutation-derived neoantigens is widely believed to facilitate the antitumor immunity induced by immune checkpoint blockade (ICB). Here we show that broadening in the number of neoantigen-reactive CD8+ T cell (NART) populations between pre-treatment to 3-weeks post-treatment distinguishes patients with controlled disease compared to patients with progressive disease in metastatic urothelial carcinoma (mUC) treated with PD-L1-blockade. The longitudinal analysis of peripheral CD8+ T cell recognition of patient-specific neopeptide libraries consisting of DNA barcode-labelled pMHC multimers in a cohort of 24 patients from the clinical trial NCT02108652 also shows that peripheral NARTs derived from patients with disease control are characterised by a PD1+ Ki67+ effector phenotype and increased CD39 levels compared to bystander bulk- and virus-antigen reactive CD8+ T cells. The study provides insights into NART characteristics following ICB and suggests that early-stage NART expansion and activation are associated with response to ICB in patients with mUC.

Neoantigen-reactive CD8+ T cells affect clinical outcome of adoptive cell therapy with tumor-infiltrating lymphocytes in melanoma.

BACKGROUNDNeoantigen-driven recognition and T cell-mediated killing contribute to tumor clearance following adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs). Yet how diversity, frequency, and persistence of expanded neoepitope-specific CD8+ T cells derived from TIL infusion products affect patient outcome is not fully determined.METHODSUsing barcoded pMHC multimers, we provide a comprehensive mapping of CD8+ T cells recognizing neoepitopes in TIL infusion products and blood samples from 26 metastatic melanoma patients who received ACT.RESULTSWe identified 106 neoepitopes within TIL infusion products corresponding to 1.8% of all predicted neoepitopes. We observed neoepitope-specific recognition to be virtually devoid in TIL infusion products given to patients with progressive disease outcome. Moreover, we found that the frequency of neoepitope-specific CD8+ T cells in TIL infusion products correlated with increased survival and that neoepitope-specific CD8+ T cells shared with the infusion product in posttreatment blood samples were unique to responders of TIL-ACT. Finally, we found that a transcriptional signature for lymphocyte activity within the tumor microenvironment was associated with a higher frequency of neoepitope-specific CD8+ T cells in the infusion product.CONCLUSIONSThese data support previous case studies of neoepitope-specific CD8+ T cells in melanoma and indicate that successful TIL-ACT is associated with an expansion of neoepitope-specific CD8+ T cells.FUNDINGNEYE Foundation; European Research Council; Lundbeck Foundation Fellowship; Carlsberg Foundation.

T Cell Epitope Prediction and Its Application to Immunotherapy.

T cells play a crucial role in controlling and driving the immune response with their ability to discriminate peptides derived from healthy as well as pathogenic proteins. In this review, we focus on the currently available computational tools for epitope prediction, with a particular focus on tools aimed at identifying neoepitopes, i.e. cancer-specific peptides and their potential for use in immunotherapy for cancer treatment. This review will cover how these tools work, what kind of data they use, as well as pros and cons in their respective applications.

Tumor-Infiltrating T Cells From Clear Cell Renal Cell Carcinoma Patients Recognize Neoepitopes Derived From Point and Frameshift Mutations.

Mutation-derived neoantigens are important targets for T cell-mediated reactivity toward tumors and, due to their unique tumor expression, an attractive target for immunotherapy. Neoepitope-specific T cells have been detected across a number of solid cancers with high mutational burden tumors, but neoepitopes have been mostly selected from single nucleotide variations (SNVs), and little focus has been given to neoepitopes derived from in-frame and frameshift indels, which might be equally important and potentially highly immunogenic. Clear cell renal cell carcinomas (ccRCCs) are medium-range mutational burden tumors with a high pan-cancer proportion of frameshift mutations. In this study, the mutational landscape of tumors from six RCC patients was analyzed by whole-exome sequencing (WES) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs), and tumor-infiltrating lymphocytes (TILs, germline reference). Neopeptides were predicted using MuPeXI, and patient-specific peptide-MHC (pMHC) libraries were created for all neopeptides with a rank score < 2 for binding to the patient's HLAs. T cell recognition toward neoepitopes in TILs was evaluated using the high-throughput technology of DNA barcode-labeled pMHC multimers. The patient-specific libraries consisted of, on average, 258 putative neopeptides (range, 103-397, n = 6). In four patients, WES was performed on two different sources (TF and TCL), whereas in two patients, WES was performed only on TF. Most of the peptides were predicted from both sources. However, a fraction was predicted from one source only. Among the total predicted neopeptides, 16% were derived from frameshift indels. T cell recognition of 52 neoepitopes was detected across all patients (range, 4-18, n = 6) and spanning two to five HLA restrictions per patient. On average, 21% of the recognized neoepitopes were derived from frameshift indels (range, 0-43%, n = 6). Thus, frameshift indels are equally represented in the pool of immunogenic neoepitopes as SNV-derived neoepitopes. This suggests the importance of a broad neopeptide prediction strategy covering multiple sources of tumor material, and including different genetic alterations. This study, for the first time, describes the T cell recognition of frameshift-derived neoepitopes in RCC and determines their immunogenic profile.

Human endogenous retroviruses form a reservoir of T cell targets in hematological cancers.

Human endogenous retroviruses (HERV) form a substantial part of the human genome, but mostly remain transcriptionally silent under strict epigenetic regulation, yet can potentially be reactivated by malignant transformation or epigenetic therapies. Here, we evaluate the potential for T cell recognition of HERV elements in myeloid malignancies by mapping transcribed HERV genes and generating a library of 1169 potential antigenic HERV-derived peptides predicted for presentation by 4 HLA class I molecules. Using DNA barcode-labeled MHC-I multimers, we find CD8+ T cell populations recognizing 29 HERV-derived peptides representing 18 different HERV loci, of which HERVH-5, HERVW-1, and HERVE-3 have more profound responses; such HERV-specific T cells are present in 17 of the 34 patients, but less frequently in healthy donors. Transcriptomic analyses reveal enhanced transcription of the HERVs in patients; meanwhile DNA-demethylating therapy causes a small and heterogeneous enhancement in HERV transcription without altering T cell recognition. Our study thus uncovers T cell recognition of HERVs in myeloid malignancies, thereby implicating HERVs as potential targets for immunotherapeutic therapies.