Immunogenic Neoantigens Research Articles

DeepNeo: a webserver for predicting immunogenic neoantigens.

Non-self epitopes, whether originated from foreign substances or somatic mutations, trigger immune responses when presented by major histocompatibility complex (MHC) molecules and recognized by T cells. Identification of immunogenically active neoepitopes has significant implications in cancer and virus medicine. However, current methods are mostly limited to predicting physical binding of mutant peptides and MHCs. We previously developed a deep-learning based model, DeepNeo, to identify immunogenic neoepitopes by capturing the structural properties of peptide-MHC pairs with T cell reactivity. Here, we upgraded our DeepNeo model with up-to-date training data. The upgraded model (DeepNeo-v2) was improved in evaluation metrics and showed prediction score distribution that better fits known neoantigen behavior. The immunogenic neoantigen prediction can be conducted at https://deepneo.net.

Abstract LB199: A personalized neoantigen vaccine is well tolerated and induces specific T-cell immune response in patients with resected melanoma

Abstract Tumor mutations giving rise to neoantigens have recently emerged as promising targets for cancer immunotherapy. Vaccines delivering tumor-specific neoantigens have demonstrated favorable efficacy and safety results in numerous preclinical and early clinical studies. However, selecting therapeutically relevant neoantigens amongst the myriad of cancer mutations has proven challenging. To overcome this, we have developed a proprietary AI-based target discovery platform, PIONEER, with enhanced predictive performance and increased precision. The PIONEER top-ranked neoantigens were selected and included in the personalized DNA vaccine candidate EVX-02. To assess the ability of the PIONEER designed neoantigen vaccine candidate to induce neoantigen immunogenicity and anti-tumor effect in preclinical models, mice were immunized with a mouse surrogate EVX-02 molecule, mEVX-02. mEVX-02 vaccination completely prevented establishment of tumors and induced neoantigen-specific, polyfunctional CD4+ and CD8+ T cells in the blood and spleen of immunized mice. An enhanced antitumor effect was obtained when combining mEVX-02 with anti-PD-1 mAb treatment. Encouraged by the preclinical results, we conducted a first-in-human multicenter Phase I clinical study of EVX-02 in combination with nivolumab in patients with resected malignant melanoma. The objectives of the trial were to investigate safety and tolerability, operational and clinical feasibility, and immunogenicity of the applied personal neoantigens. Each patient received a fully personalized drug that was produced in a complex process, from biopsy, through genome sequencing, AI-profiling, vaccine design, manufacturing, quality testing, and drug product release. This was succeeded with every single step for each patient. In all patients, EVX-02 treatment was well tolerated, and only very mild adverse events (AEs) have been observed in relation to immunization with EVX-02. Interim data demonstrated neoantigen-specific T-cell immune responses upon EVX-02 treatment and that the responses were mediated by activated CD4+ and CD8+ T cells. The measured T-cell responses were robust and long lasting. Together, these findings validate the precision and predictive power of our proprietary AI platform, PIONEER, and provide proof of mechanism for the DNA-delivery technology in that the encoded neoantigens gave rise to significant immune reactions. Citation Format: Daniela Kleine-Kohlbrecher, Nadia Viborg Petersen, Michail Angelos Pavlidis, Thomas Trolle, Stine Friis, Jens Kringelum, Anders Bundgaard Soerensen, Thomas Strandet Jepsen, Camilla Højgaard, Anders Jespersen, Erik D. Heegaard, Britt Winding Lauenborg, Birgitte Rønø. A personalized neoantigen vaccine is well tolerated and induces specific T-cell immune response in patients with resected melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB199.

Abstract 1125: Neoantigen immunogenicity landscapes and evolution of tumor ecosystems during immunotherapy with nivolumab

Abstract Immune checkpoint blockade (ICB) therapy is a cornerstone of oncologic treatment for patients with advanced stage non-small cell lung cancer (NSCLC) and other malignancies. Neoantigen immunoediting drives ICB efficacy, yet how tumor evolution and neoantigen immunogenicity shape treatment response remains poorly understood. To address these questions, we conducted a prospective clinical trial of NSCLC patients treated with nivolumab. We assessed genomic alterations in tumors from 58 patients and performed large-scale immune epitope analyses, before and during treatment (CheckMate153, CA209-153). Tumors were analyzed by whole-exome and transcriptome sequencing. In responding patients, loss of mutation and neoantigen burden early during therapy was associated with clinical benefit. We evaluated the immunogenicity of 1,453 candidate neoantigens and identified 502 neopeptides that bound to MHC I and 196 neopeptides that were immunogenic and recognized by T cells in the setting of nivolumab treatment. These T cell reactive neoantigens were differentially present in clonal populations that underwent distinctive evolutionary trajectories across responders and nonresponders. Mapping these neoantigens to tumor clonal dynamics and clinical response revealed strong selection against immunogenic mutations compared to non-immunogenic mutations. We used this large collection of neoantigens to build ImmunoSEEK, a machine learning-based neoantigen prediction model and validated it using an orthogonal set of neoantigens. Changes in the genomic and neoantigen immunogenicity landscapes were accompanied by temporal changes in the tumor microenvironment. Nivolumab-induced microenvironmental evolution in NSCLC shared some similarities with that in melanoma, yet critical differences in immunologic programs were apparent from comparative network analysis between tumor types. This study provides unprecedented molecular portraits of the genomic and neoantigen landscapes underlying nivolumab’s mechanism of action. Citation Format: Tyler Joseph Alban, Nadeem Riaz, Prerana Parthasarathy, Vlad Makarov, Slav Kendall, Seong-Keun Yoo, Rachna Shah, Nils Weinhold, Raghvendra Srivastava, Xiaoxiao Ma, Chirag Krishna, Edward Garon, Wallace Akerley, Ben Creelan, Nivedita Aanuren, Diego Chowell, William Geese, Naiyer Rizvi, Timothy Chan. Neoantigen immunogenicity landscapes and evolution of tumor ecosystems during immunotherapy with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1125.

Abstract 5472: Utilizing response in immune checkpoint inhibitor treated cohorts improves clinical applicability of neoantigen immunogenicity predictions

Abstract Introduction: Neoantigen-based biomarkers have improved predictions of response to immune checkpoint blockade (ICB) therapy, highlighting the importance of accurate prediction of immunogenic neoantigen candidates. A challenge in developing robust immunogenicity prediction models is limited availability of sequencing-associated immunogenicity data for evaluating methods due to the complexity of generating such datasets. We propose a novel approach to optimize prediction models of immunogenic neoantigens using a meta-analysis framework based on multiple ICB cohorts. Methods: To build on the 110 mono-allelic immunopeptidomics-derived SHERPA® MHC binding prediction framework, we engineered T-cell recognition features on two datasets: peptide-centric data aggregated by Schmidt et al. and patient-specific exome and transcriptome sequencing data from the TESLA consortium. We developed two-tiered models based on the feature landscapes of both datasets to predict peptide-MHC (pMHC) immunogenicity, incorporating features with significant performance gains. We systematically re-processed publicly available DNA and RNA sequencing data from over 500 ICB treated patients spanning 12 different cohorts across five different cancer types with a harmonized bioinformatics pipeline. We then evaluated the performance of each model consisting of a unique combination of immunogenic features across the ICB training (N=7) and validation (N=5) cohorts using a meta-analysis framework. Results: We evaluated iterations of SHERPA-Immunogenicity (SI) models using the Schmidt et al. and TESLA datasets, resulting in a range of performance metrics (area under the precision recall curves of 0.74-0.84 and positive predictive values of 0.32-0.54). After aggregating pMHC predictions into patient-specific scores based on the most immunogenic peptide present (SHERPA-Immunogenicity Maximum - SIM) or the quantity of immunogenic peptides identified (SHERPA-Immunogenicity Burden - SIB), we observed that responders had higher SIM and SIB scores compared to non-responders across the melanoma training cohorts. We found SIM scores outperformed SIB scores, suggesting the degree of epitope immunogenicity may be a critical factor in predicting response. The model with the most significant meta p-value for ICB response in melanoma cohorts (OR=2.43, p=0.006) also predicted overall survival in 3/5 melanoma cohorts (p&lt0.05). Conclusions: We developed a novel framework to predict neoantigen immunogenicity utilizing meta-analysis of ICB cohorts to overcome dataset limitations and gain prediction performance confidence. We look forward to supporting personalized cancer vaccine development with our pMHC immunogenicity predictions and applying our predictive biomarker on additional ICB cohorts. Citation Format: Hima Anbunathan, Neeraja Ravi, Rachel Marty Pyke, Steven Dea, Richard O. Chen, Sean Michael Boyle. Utilizing response in immune checkpoint inhibitor treated cohorts improves clinical applicability of neoantigen immunogenicity predictions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5472.

Abstract 5114: Development of personalized RNA-based immunotherapeutics for glioblastom

Abstract Background: The development of successful immunotherapy targeting antigens in glioblastoma multiforme (GBM) remains a challenge owing to antigen heterogeneity and the low mutation burden in GBM. Cancer immunogenomics represents a complementary approach to the application of genomics in developing novel immunotherapies for GBM. Our goal is to develop a personalized RNA-based therapeutic vaccine that simultaneously targets a selected pool of tumor rejection antigens including all neoantigens and tumor-associated antigens (TAAs) as identified using cancer immunogenomics. We evaluated the treatment efficacy of these RNA therapeutics in combination with adoptive cellular therapy and checkpoint inhibitors. Methods: We identified immunogenic neoantigens and TAAs that are aberrantly overexpressed in KR158-luc and GL261 murine GBM tumors using our cancer immunogenomics pipeline called the Open Reading Frame Antigen Network (O.R.A.N.). A tumor antigen-specific RNA library was created for each tumor using a novel gene enrichment strategy and validated by RNAseq. Tumor-bearing animals were treated with adoptively transferred ex vivo expanded lymphocytes and dendritic cells loaded with the tumor antigen-specific RNA as vaccines. Additionally, we treated animals with the tumor antigen-specific RNA vaccines in combination with anti-PD-1 checkpoint-blockade therapy. Tumor progression and survival outcomes were analyzed to evaluate the treatment efficacies. We determined the peripheral and tumor-infiltrating lymphocyte responses using flow cytometry, TCR sequencing, and single cell RNAseq. Results: The tumor antigen-specific RNA vaccines were significantly effective in slowing the progression of murine GBM tumors in combination with both the adoptive cellular therapy and the checkpoint blockade therapy and improved the outcome of these therapies as compared to monotherapy alone. Additionally, we identified antigen-specific T cells targeting several of our predicted antigens and an increase in tumor-infiltrating lymphocytes and memory T cells in the treated animals. Conclusion: Here, we developed an RNA-based immunotherapy that simultaneously targets numerous tumor-specific antigens and demonstrated its therapeutic efficacy in preclinical models of GBM. Citation Format: Vrunda Trivedi, Changlin Yang, Oleg Yegorov, Kelena Klippel, Graeme Fenton, Christina von Roemeling, Lan Hoang-Minh, Elizabeth Ogando-Rivas, Paul Castillo, Kyle Dyson, Ginger Moore, Duane Mitchell. Development of personalized RNA-based immunotherapeutics for glioblastom. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5114.

Abstract 611: Sex disparities in cancer immunoediting

Abstract Cancer immunoediting is the process through which tumor specific T cell populations both prevent cancer development and apply selective pressure for the outgrowth of less immunogenic tumors with reduced neoantigen burden. Male and female immune systems demonstrate distinct differences that drive characterized disparities in response to viral infection and immunization. While several recent publications have shown that anti-tumor immune responses are inhibited by male sex hormone signaling, a critical gap in knowledge exists surrounding sex-based differences in the larger-scale process of immunoediting. Because the residual neoantigen burden expressed after tumor escape from immune surveillance is an important determinant of rejection following immune checkpoint blockade, disparities in immunoediting between males and females could have profound impacts on both cancer incidence and response to therapy. The purpose of our studies is to determine if the enhanced functionality of female T cells results in more effective immunoediting of tumors that evolve in females versus those that evolve in males. We have used the 3-methylcholanthrene carcinogen-induced tumor model and several transplant tumor models to determine if T cell function regulates differential cancer development between the sexes. We have also analyzed predicted neoantigen burden in male and female patients with melanoma and lung cancer to identify sex-based differences in tumor immunogenicity. We have found that female T cell responses are more effective at preventing tumor occurrence and delaying tumor outgrowth compared to male T cell responses in mice, which may help to explain established sex-based disparities in cancer incidence in the human population. In comparison, male patient tumors were more likely to express high neoantigen burden compared to their female counterparts. These results suggest that while stronger anti-tumor T cell responses in females are helpful in driving lower cancer incidence in females compared to males, this disparity may also result the development of tumors that express greater numbers of immunogenic neoantigens in males versus females. A better understanding of how sex impacts the entire process of immunoediting will help to explain cancer disparities between the sexes and identify potential new therapeutic strategies that can benefit all patients. Citation Format: Katey Hunt, Elise Alspach. Sex disparities in cancer immunoediting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 611.

Abstract 610: Modeling tumor immunoediting coupled with T-cell repertoire dynamic

Abstract As tumors evolve, cancer cells acquire somatic mutations and these mutations generate tumor specific neoantigens that are different from self and can be recognized by T-cells, inducing anti-tumor immune responses. Such immunogenic antigens can be effective targets for neoantigen-based cancer vaccines. However, the identification of immunogenic neoantigens remains limited with the current prediction models. Here we designed mouse experiments to model tumor evolution under immune pressure and its association with T-cell clones to identify these antigens agnostically and improve current prediction models. We injected three lung cancer cell lines (KPA, KPC, and HKP1) in wildtype immune-competent C57BL/6 mice and immune-deficient Rag1 defective (Rag1 KO) mice, respectively. The three tumor cell lines have common driver mutations, Kras G12D/+ and p53 flox/flox. Two weeks post tumor implantation, whole exome sequencing and RNA-seq was performed on tumor samples. We also performed T cell receptor (TCR) sequencing to profile tumor infiltrated TCR clones. Despite the shared driver mutations, tumor growth patterns in vivo over time are very distinct for each cell line. Interestingly, KPA tumors show differential evolutionary patterns in wild type mice versus Rag1 KO mice, regressing in the former but rapidly progressing in the latter, suggesting that the difference can be attributed to immunoediting of KPA specific neoantigens. HKP1 tumors do not show substantial differences in that aggressive tumor progression is observed in both types of mice. Using WES data, we estimated the cancer cell fraction of each mutation based on phylogeny re-construction. We found that evolved KPA tumors (immunogenic) had significantly lower mutation burden in wild type mice compared to Rag1 KO mice (3410 vs 4199 on average), and the fraction of cell line mutations edited in vivo is significantly higher in wild type than in Rag1 KO (26% vs 14% on average), suggesting again the active on-going immunosurveillance in immune competent mice. In contrast, HKP1 tumors (non-immunogenic) do not show significant differences in mutation burden nor fraction of mutations edited between wild type and Rag1 KO mice. Furthermore, TCR-seq data show that the clonal expansion of T-cells is strongly associated with tumor immunogenicity. KPA tumor samples show significantly higher TCR clustering but with less diversity, quantified by modified Simpson’s Index, that is strongly correlated with the suppression of mutational burden (r=0.74). In summary, we found that tumor evolution in immune competent mice is dependent on mutational context of the tumor. By analyzing longitudinal tumor and TCR sequencing data, we showed that the immunogenicity of tumors is associated with significant decrease in mutational burden and higher T-cell clustering that is indicative of tumor mutation and TCR clone expansion co-evolution in immune competent mice. Citation Format: Jayon Lihm, Hyejin Choi, Mariam M. George, Yuval Elhanati, David Hoyos, Marta Luksza, Taha Merghoub, Benjamin D. Greenbaum. Modeling tumor immunoediting coupled with T-cell repertoire dynamic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 610.

Dominant neoantigen verification in hepatocellular carcinoma by a single-plasmid system coexpressing patient HLA and antigen

BackgroundPrevious studies confirmed that most neoantigens predicted by algorithms do not work in clinical practice, and experimental validations remain indispensable for confirming immunogenic neoantigens. In this study, we identified the...

TLimmuno2: predicting MHC class II antigen immunogenicity through transfer learning.

Major histocompatibility complex (MHC) class II molecules play a pivotal role in antigen presentation and CD4+ T cell response. Accurate prediction of the immunogenicity of MHC class II-associated antigens is critical for vaccine design and cancer immunotherapies. However, current computational methods are limited by insufficient training data and algorithmic constraints, and the rules that govern which peptides are truly recognized by existing T cell receptors remain poorly understood. Here, we build a transfer learning-based, long short-term memory model named 'TLimmuno2' to predict whether epitope-MHC class II complex can elicit T cell response. Through leveraging binding affinity data, TLimmuno2 shows superior performance compared with existing models on independent validation datasets. TLimmuno2 can find real immunogenic neoantigen in real-world cancer immunotherapy data. The identification of significant MHC class II neoantigen-mediated immunoediting signal in the cancer genome atlas pan-cancer dataset further suggests the robustness of TLimmuno2 in identifying really immunogenic neoantigens that are undergoing negative selection during cancer evolution. Overall, TLimmuno2 is a powerful tool for the immunogenicity prediction of MHC class II presented epitopes and could promote the development of personalized immunotherapies.

Genome, Validity of Adjuvants & Misterming Adjuvants as Cancer Vaccines

The term adjuvant comes from the Latin word ’adjuvare’ meaning help. Thus, adjuvants help vaccines improve the antigen-specific immune response by inducing pathogen-associated molecular patterns (PAMPs) that can activate various pattwern recognition receptors (PRRs) of innate immune cells.The key objective of this study is to verify that what are called therapeutic vaccines of cancers are totally the types of adjuvants that increase the immunogenicity of neoantigens (autovaccines) found on surfaces of cancered cells such as those of metastatic tumors. The contents of Figures & Table were designed to serve as Results derived from the targetful and strategic methods implemented, under the title of Methodology & Results. The contents of Figures & Table under the title of Methodology & Results represent the result. Represented by 31 conclusive statements listed (enumerated) under the conclusion section of this paper.

Abstract OT2-10-03: HCRN BRE 19-433: A Multi-institutional Phase II Study to Evaluate Efficacy and Safety of TAlazoparib, Radiotherapy and Atezolizumab in gBRCA 1/2 negative Patients with PD-L1+ Metastatic Triple Negative Breast Cancer (TARA)

Abstract Background Although immunotherapy (IO) in combination with chemotherapy has improved progression free and overall survival in patients with PD-L1+ metastatic triple negative breast cancer (mTNBC), prognosis remains poor. A potential therapeutic strategy to restore sensitivity to IO in patients with progressive disease is to introduce agents that re-sensitize the immune system to IO leading to a more tumor-specific and less toxic treatment in the second-line setting or beyond. Both talazoparib, a PARP inhibitor, and radiation (XRT) independently increase PD-L1 expression on the tumor cell surface resulting in heightened sensitivity to IO agents like atezolizumab, a PD-L1 inhibitor. Although a local treatment, XRT has the ability to produce an abscopal effect resulting in systemic shrinkage of non-irradiated tumors outside/distant to the XRT field, a phenomenon observed in patients receiving concurrent IO. While talazoparib is standard treatment in patients with gBRCA1 and 2 mutations, it is also a potent radiosensitizing agent that suppresses homologous recombination and PARP-1-dependent nonhomologous end joining (NHEJ) repair while promoting error-prone alt-NHEJ. When combined with IO, talazoparib can amplify immune responses by generating immunogenic neo-antigens independent of gBRCA1/2 status. We, therefore, hypothesize that the combination of talazoparib, XRT, and atezolizumab will re-sensitize mTNBC tumors to IO and promote a durable tumor-specific response that spares patients from toxicities associated with traditional chemotherapy regimens. Methods This is a Phase II multi-institutional study designed to assess efficacy and safety of talazoparib, high dose XRT, and atezolizumab given in the second-, third-, or fourth-line settings to patients with mTNBC that is PD-L1 positive. A total of 23 patients with mTNBC who do not carry gBRCA pathogenic variants will be enrolled. All patients will be treated with induction talazoparib of 1mg PO daily starting Day 1 of a 28-day cycle. Patients will then receive 8 Gy x 3 fractions to 1-4 metastatic lesions QOD beginning Day 12, 13, or 14. Atezolizumab will be given intravenously (840 mg)) on Day 15 of the 1st cycle and then on Day 1 and Day 15 of the subsequent cycles. Talazoparib and atezolizumab treatment will continue until progression or severe toxicity. The primary endpoint is objective response rate (ORR) in non-irradiated lesions 8 weeks after the first dose of atezolizumab. Key inclusion criteria include biopsy proven mTNBC (ER< 10%, PR< 10%, Her2-) with at least 2 extracranial metastatic lesions. Patients must have at least 1 extracranial metastatic lesion amenable to high dose radiotherapy and at least one additional extracranial lesion of measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST v1.1) that will not receive radiotherapy. Tumors must be PD-L1 positive as defined as >1% on IHC using the SP142 Ventana Assay. Key exclusion criteria include patients with germline BRCA pathogenic variants, more than three previous lines of chemotherapy treatment in the advanced setting with or without IO, and breast cancer progression within the first 3 months of previous IO treatment for non-metastatic or metastatic breast cancer. Sample size was determined using Simon’s 2-stage Minimax design to detect a 20% increase in ORR. The null hypothesis that the true response rate among gBRCA1/2 negative patients of 10% will be tested against a one-sided alternative. Inflammatory cytokines, circulating B cells, and ctDNA will be collected for correlative analysis. Enrollment began in April 2021. The study is managed by the Hoosier Cancer Research Network and is open to accrual at Emory University and University of Alabama at Birmingham. Clinical trial information: NCT04690855 Citation Format: Mylin A. Torres, Kevin Kalinsky, Erica Stringer-Reasor, Ahmed Elkhanany, Jolinta Lin, David M. Schuster, Sarah Friend, Jeffrey Switchenko, Manali Bhave. HCRN BRE 19-433: A Multi-institutional Phase II Study to Evaluate Efficacy and Safety of TAlazoparib, Radiotherapy and Atezolizumab in gBRCA 1/2 negative Patients with PD-L1+ Metastatic Triple Negative Breast Cancer (TARA) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-10-03.

Nanovaccine that activates the NLRP3 inflammasome enhances tumor specific activation of anti-cancer immunity

Neoantigen cancer vaccines that target tumor specific mutations are emerging as a promising modality for cancer immunotherapy. To date, various approaches have been adopted to enhance efficacy of these therapies, but the low immunogenicity of neoantigens has hindered clinical application. To address this challenge, we developed a polymeric nanovaccine platform that activates the NLRP3 inflammasome, a key immunological signaling pathway in pathogen recognition and clearance. The nanovaccine is comprised of a poly (orthoester) scaffold engrafted with a small-molecule TLR7/8 agonist and an endosomal escape peptide that facilitates lysosomal rupture and NLRP3 inflammasome activation. Upon solvent transfer, the polymer self-assembles with neoantigens to form ∼50 nm nanoparticles that facilitate co-delivery to antigen-presenting cells. This polymeric activator of the inflammasome (PAI) was found to induce potent antigen-specific CD8+ T cell responses characterized by IFN-γ and GranzymeB secretion. Moreover, in combination with immune checkpoint blockade therapy, the nanovaccine stimulated robust anti-tumor immune responses against established tumors in EG.7-OVA, B16·F10, and CT-26 models. Results from our studies indicate that NLRP3 inflammasome activating nanovaccines demonstrate promise for development as a robust platform to enhance immunogenicity of neoantigen therapies.

Reprogramming the tumor microenvironment leverages CD8+ T cell responses to a shared tumor/self antigen in ovarian cancer

Tumor antigen-driven responses to weakly immunogenic self-antigens and neoantigens directly affect treatment efficacy following immunotherapy. Using orthotopically grown SV40T antigen+ ovarian carcinoma in antigen-naive wild-type or TgMISIIR-TAg-Low transgenic mice expressing SV40T antigen as a self-antigen, we investigated the impact of CXCR4-antagonist-armed oncolytic virotherapy on tumor progression and antitumor immunity. Immunostaining and single-cell RNA sequencing analyses of the peritoneal tumor microenvironment of untreated tumors in syngeneic wild-type mice revealed the presence of SV40T antigen-specific CD8+ Tcells, a balanced M1/M2 transcriptomic signature of tumor-associated macrophages, and immunostimulatory cancer-associated fibroblasts. This contrasted with polarized M2 tumor-associated macrophages, immunosuppressive cancer-associated fibroblasts, and poor immune activation in TgMISIIR-TAg-Low mice. Intraperitoneal delivery of CXCR4-antagonist-armed oncolytic vaccinia virus led to nearly complete depletion of cancer-associated fibroblasts, M1 polarization of macrophages, and generation of SV40T antigen-specific CD8+ Tcells in transgenic mice. Cell depletion studies revealed that the therapeutic effect of armed oncolytic virotherapy was dependent primarily on CD8+ cells. These results demonstrate that targeting the interaction between immunosuppressive cancer-associated fibroblasts and macrophages in the tolerogenic tumor microenvironment by CXCR4-A-armed oncolytic virotherapy induces tumor/self-specific CD8+ Tcell responses and consequently increases therapeutic efficacy in an immunocompetent ovarian cancer model.

Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in patients with lung cancer.

Although most characterized tumor antigens are encoded by canonical transcripts (such as differentiation or tumor-testis antigens) or mutations (both driver and passenger mutations), recent results have shown that noncanonical transcripts including long noncoding RNAs and transposable elements (TEs) can also encode tumor-specific neo-antigens. Here, we investigate the presentation and immunogenicity of tumor antigens derived from noncanonical mRNA splicing events between coding exons and TEs. Comparing human non-small cell lung cancer (NSCLC) and diverse healthy tissues, we identified a subset of splicing junctions that is both tumor specific and shared across patients. We used HLA-I peptidomics to identify peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides were immunogenic in vitro, and CD8+ T cells specific for junction-encoded epitopes were present in tumors and tumor-draining lymph nodes from patients with NSCLC. We conclude that noncanonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in patients with NSCLC.

Nonsense-Mediated mRNA Decay as a Mediator of Tumorigenesis.

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved and well-characterized biological mechanism that ensures the fidelity and regulation of gene expression. Initially, NMD was described as a cellular surveillance or quality control process to promote selective recognition and rapid degradation of erroneous transcripts harboring a premature translation-termination codon (PTC). As estimated, one-third of mutated and disease-causing mRNAs were reported to be targeted and degraded by NMD, suggesting the significance of this intricate mechanism in maintaining cellular integrity. It was later revealed that NMD also elicits down-regulation of many endogenous mRNAs without mutations (~10% of the human transcriptome). Therefore, NMD modulates gene expression to evade the generation of aberrant truncated proteins with detrimental functions, compromised activities, or dominant-negative effects, as well as by controlling the abundance of endogenous mRNAs. By regulating gene expression, NMD promotes diverse biological functions during development and differentiation, and facilitates cellular responses to adaptation, physiological changes, stresses, environmental insults, etc. Mutations or alterations (such as abnormal expression, degradation, post-translational modification, etc.) that impair the function or expression of proteins associated with the NMD pathway can be deleterious to cells and may cause pathological consequences, as implicated in developmental and intellectual disabilities, genetic defects, and cancer. Growing evidence in past decades has highlighted NMD as a critical driver of tumorigenesis. Advances in sequencing technologies provided the opportunity to identify many NMD substrate mRNAs in tumor samples compared to matched normal tissues. Interestingly, many of these changes are tumor-specific and are often fine-tuned in a tumor-specific manner, suggesting the complex regulation of NMD in cancer. Tumor cells differentially exploit NMD for survival benefits. Some tumors promote NMD to degrade a subset of mRNAs, such as those encoding tumor suppressors, stress response proteins, signaling proteins, RNA binding proteins, splicing factors, and immunogenic neoantigens. In contrast, some tumors suppress NMD to facilitate the expression of oncoproteins or other proteins beneficial for tumor growth and progression. In this review, we discuss how NMD is regulated as a critical mediator of oncogenesis to promote the development and progression of tumor cells. Understanding how NMD affects tumorigenesis differentially will pave the way for the development of more effective and less toxic, targeted therapeutic opportunities in the era of personalized medicine.

Melanoma antigens recognized by T cells and their use for immunotherapy.

Melanoma has been a prototype for cancer immunology research, and the mechanisms of anti-tumor T-cell responses have been extensively investigated in patients treated with various immunotherapies. Individual differences in cancer-immune status are defined mainly by cancer cell characteristics such as DNA mutations generating immunogenic neo-antigens, and oncogene activation causing immunosuppression, but also by patients' genetic backgrounds such as HLA types and genetic polymorphisms of immune related molecules, and environmental and lifestyle factors such as UV rays, smoking, gut microbiota and concomitant medications; these factors have an influence on the efficacy of immunotherapy. Recent comparative studies on responders and non-responders in immune-checkpoint inhibitor therapy using various new technologies including multi-omics analyses on genomic DNA, mRNA, metabolites and microbiota and single cell analyses of various immune cells have led to the advance of human tumor immunology and the development of new immunotherapy. Based on the new findings from these investigations, personalized cancer immunotherapies along with appropriate biomarkers and therapeutic targets are being developed for patients with melanoma. Here, we will discuss one of the essential subjects in tumor immunology: identification of immunogenic tumor antigens and their effective use in various immunotherapies including cancer vaccines and adoptive T-cell therapy.

Mutant and non-mutant neoantigen-based cancer vaccines: recent advances and future promises.

Major advances in cancer treatment have emerged with the introduction of immunotherapies using blocking antibodies that target T-cell inhibitory receptors, such as programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), known as immune checkpoints. However, most cancer patients do not respond to immune checkpoint blockade (ICB) therapies, suggesting the development of resistance mechanisms associated with either an insufficient number of preexisting tumor-specific T-cell precursors and/or inappropriate T-cell reactivation. To broaden clinical benefit, anti-PD-1/PD-1 ligand (PD-L1) neutralizing antibodies have been combined with therapeutic cancer vaccines based on non-mutant and/or mutant tumor antigens, to stimulate and expand tumor-specific T lymphocytes. Although these combination treatments achieve the expected goal in some patients, relapse linked to alterations in antigen presentation machinery (APM) of cancer cells often occurs leading to tumor escape from CD8 T-cell immunity. Remarkably, an alternative antigenic peptide repertoire, referred to as T-cell epitopes associated with impaired peptide processing (TEIPP), arises on these malignant cells with altered APM. TEIPP are derived from ubiquitous non-mutant self-proteins and represent a unique resource to target immune-edited tumors that have acquired resistance to cytotoxic T lymphocytes (CTLs) related to defects in transporter associated with antigen processing (TAP) and possibly also to ICB. The present review discusses tumor-associated antigens (TAAs) and mutant neoantigens and their use as targets in peptide- and RNA-based therapeutic cancer vaccines. Finally, this paper highlights TEIPP as a promising immunogenic non-mutant neoantigen candidates for active cancer immunotherapy and combination with TAA and mutant neoantigens. Combining these polyepitope cancer vaccines with ICB would broaden T-cell specificity and reinvigorate exhausted antitumor CTL, resulting in the eradication of all types of neoplastic cells, including immune-escaped subtypes.

Immunotherapies targeting neoantigens are effective in PD-1 blockade-resistant tumors.

Only a small fraction of tumor-infiltrating lymphocytes can specifically recognize and attack cancer cells in PD-1/PD-L1 blockade therapy. Here, we investigate approaches to expand the neoantigen-specific CD8+ T cells to overcome the difficulties in treating PD-1/PD-L1 blockade-resistant tumors. Mutation-associated neoepitopes of murine nonsmall cell lung cancer ASB-XIV were estimated by whole-exome and RNA sequencing and predicted by MHC-I binding affinity (FPKM >1) in silico. Using ASB-XIV-specific CD8+ T cells, we screened a panel of 257 neoepitope peptides derived from ASB-XIV missense and indel mutations. Mutated Phf3 peptide (mPhf3) was successfully identified as an immunogenic neoepitope. Prophylactic mPhf3-DC vaccination inhibited ASB-XIV tumor growth through CD8+ T cell-mediated antitumor immunity. Combining the mPhf3-DC vaccine and anti-PD-1 treatment elicited robust antitumor activity through the induction of mPhf3-specific CD8+ T cells in the tumor microenvironment. Furthermore, the adoptive transfer of mPhf3-specific CD8+ T cells eradicated ASB-XIV tumors. Likewise, the combination of mutated Cdt1 peptide (mCdt1)-DC vaccine and anti-PD-1 treatment or adoptive transfer of mCdt1-specific CD8+ T cells also led to significant regression of PD-1 blockade-resistant murine gastric YTN16 tumors. In conclusion, a novel immunogenic neoepitope of ASB-XIV was identified for immunotherapy targeting neoantigens. Identification of immunogenic neoantigens can extend the therapeutic strategies by increasing the frequency of neoantigen-specific T cells, even for PD-1/PD-L1 blockade-resistant tumors.

Combination TIGIT/PD-1 blockade enhances the efficacy of neoantigen vaccines in a model of pancreatic cancer.

Cancer neoantigens are important targets of cancer immunotherapy and neoantigen vaccines are currently in development in pancreatic ductal adenocarcinoma (PDAC) and other cancer types. Immune regulatory mechanisms in pancreatic cancer may limit the efficacy of neoantigen vaccines. Targeting immune checkpoint signaling pathways in PDAC may improve the efficacy of neoantigen vaccines. We used KPC4580P, an established model of PDAC, to test whether neoantigen vaccines can generate therapeutic efficacy against PDAC. We focused on two immunogenic neoantigens associated with genetic alterations in the CAR12 and CDK12 genes. We tested a neoantigen vaccine comprised of two 20-mer synthetic long peptides and poly IC, a Toll-like receptor (TLR) agonist. We investigated the ability of neoantigen vaccine alone, or in combination with PD-1 and TIGIT signaling blockade to impact tumor growth. We also assessed the impact of TIGIT signaling on T cell responses in human PDAC. Neoantigen vaccines induce neoantigen-specific T cell responses in tumor-bearing mice and slow KPC4580P tumor growth. However, KPC4580P tumors express high levels of PD-L1 and the TIGIT ligand, CD155. A subset of neoantigen-specific T cells in KPC4580P tumors are dysfunctional, and express high levels of TIGIT. PD-1 and TIGIT signaling blockade in vivo reverses T cell dysfunction and enhances neoantigen vaccine-induced T cell responses and tumor regression. In human translational studies, TIGIT signaling blockade in vitro enhances neoantigen-specific T cell function following vaccination. Taken together, preclinical and human translational studies support testing neoantigen vaccines in combination with therapies targeting the PD-1 and TIGIT signaling pathways in patients with PDAC.

Abstract IA018: Cancer immune-interception in Lynch Syndrome: Neoantigen-based vaccine development

Abstract Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer and also conveys significantly increased risks for several other malignancies, including endometrial, small bowel, gastric, ovarian, adrenocortical tumors, and others. LS results from a heterozygous germline mutation in one of four DNA mismatch repair (MMR) genes. The acquisition of a somatic “second hit” causes affected cells to lose the ability to repair DNA mismatch replication errors that frequently occur in microsatellite regions of the genome. Consequently, hundreds to thousands of small insertion/deletions (indels) accumulate in these regions, and when expressed, result in frameshift peptides that are recognized as neoantigens that are then presented on the cell surface via the major histocompatibility complexes (MHC-I/II) for immune cell recognition. Vaccine development efforts in this high-risk population have been challenged by the substantial inter-individual variability in both the set of expressed neoantigens and MHC I/II responses. Yet, recent advances in next-generation sequencing and associated bioinformatic approaches are now allowing for more accurate profiling of the most frequently recurring and shared mutated neoantigens in LS-associated tumors. This allows for identification of the most immunogenic neoantigens that can be incorporated into different vaccine platforms to test the development of a population-based vaccine. The Vilar Lab has collaborated with industry and academic partners such as Nouscom, s.r.l. and the National Cancer Institute to develop two different clinical trials to bring new vaccines to the LS population based on mutated neoantigens and tumor associated antigens. In this presentation, we will focus on mutated neoantigen-based strategies and present the Phase I clinical trial (NCT05078866) using a viral-based vaccine including 209 distinct neoantigens. The primary endpoint of this trial is safety and immunogenicity. A total of 45 participants will be enrolled to receive a prime and boost vaccination. Different secondary biomarkers will explore immunological aspects of this novel immune-interception strategy. Citation Format: Eduardo Vilar-Sanchez. Cancer immune-interception in Lynch Syndrome: Neoantigen-based vaccine development [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA018.