Neoantigen Peptides Research Articles

Abstract 6668: Immune infiltrate co-occurrence and neoantigen similarity are prognostic factors in early stage NSCLC

Abstract The prevalence of early-stage non-small cell lung cancer (NSCLC) with curative treatment options is expected to increase with recent implementation of annual screening programs. Predictors and molecular drivers of disease relapse, especially the role of intra-tumoral immune dysfunction, remains unclear but critical for the refinement of therapeutic decisions. By leveraging a comprehensive individual portrait of each patient's immune system potential novel mechanisms associated with tumor relapse in early-stage NSCLC may be identified. We profiled 11 non-relapsed (at least 2 year FU) lung adenocarcinoma patients and 11 covariate-matched (gender, age, stage) relapsed patients, who underwent curative treatment in stage IA-IIIB disease. We used NeXT SummitTM for variant and CNA calling, gene expression quantification, neoantigen prediction, HLA profiling (typing, mutation, and loss of heterozygosity), T-cell receptor and tumor microenvironment (TME) profiling. Neoantigen peptide sequences were subjected to further filtering and clustering based on between-patient similarity scores, with the goal of identifying shared clusters of relapse-associated neoantigens in each possible pair of patients. Differential network analyses were applied to the TME composition estimates to investigate relapse-associated patterns of cellular co-occurrence and interaction. When considering neoantigens selected on the basis of similarity, we found that those belonging to non-relapsed patients had significantly lower HLA binding rank (17.8 points) compared to that of relapsed patients (P=0.02), indicating weaker binding for relapsed cases. Clustering of both the most similar and frequently shared neoantigens correlated with relapse (P < 0.002). In the TME, we observed differential immune cell co-occurrence associated with relapse status, such as Tregs are positively correlated with B and CD4 T cells only in relapsed patients (Pearson’s R=0.7 and 0.74, both P<0.02 vs. R=0.18 and 0.35, both P>0.2 in non-relapsed patients), indicating suppressive anti-tumor immunity. Relapsed patients did not share significant enrichment of mutations in any biological pathway. Surprisingly, mutation purity (less mutations than expected by chance) was observed in relapsed patients, suggesting selective killing and escape. In this pilot cohort, we used an integrated platform to broadly characterize both the tumor and immune system, enabling identification of relapse-associated neoantigens that may share universal features which enhance HLA binding. Relapses in early-stage LUAD patients were associated with neoantigens with lower immunogenicity and an immunosuppressive TME. These findings demonstrate that deeper profiling of shared neoantigen features has the potential to become an early biomarker of relapse, informing patient therapy selection and surveillance. Citation Format: Martina M. Sykora, Jason Pugh, Bailiang Li, Finn O. Mildner, Hubert Hackl, Arno Amann, Fabienne I. Nocera, Rachel M. Pyke, Lee McDaniel, Charles W. Abbott, Sean M. Boyle, Richard O. Chen, Dominik Wolf, Sieghart Sopper, Gabriele Gamerith. Immune infiltrate co-occurrence and neoantigen similarity are prognostic factors in early stage NSCLC [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 6668.

Abstract 448: Computational discovery and experimental validation of splicing-derived neoantigens

Abstract RNA splicing is a central step in the regulation of gene expression, leading to the accurate assembly of viable mRNA isoforms. Errors in the splicing process are common in cancer and lead to the production of tumor-specific mRNAs that often encode for neoantigen peptides. Such neoantigens cannot be discovered using conventional DNA-based tools; they require specialized software for alternative splicing analysis using RNA-seq data. Envisagenics has developed SpliceIOTM, a proprietary software platform for splicing-derived neoantigen discovery. SpliceIO uses RNA-seq data to uncover extracellular neoantigens encoded by transmembrane proteins or presented to immune cells through the MHC complex. Using SpliceIO, we have analyzed roughly 8,000 RNA-seq samples from 4 different cancers and 14 normal tissue types. Each dataset first undergoes an unsupervised clustering analysis using SpliceSliceTM to stratify patients into subpopulations based on similar aberrations in splicing machinery. We then leverage SpliceIO to identify splicing-derived, tumor-specific neoantigens enriched in these cohorts. SpliceIO incorporates features, such as protein localization, MHC binding affinity, epitope accessibility and cancer relevance of the gene to help select tumor specific targets with the highest therapeutic potential. Here, we summarize the discovery and validation of novel splicing-derived neoantigens identified using SpliceIO. To validate neoantigen candidates, we performed isoform expression quantification at the RNA and protein levels using PCR, RNA-FISH, western blot and mass spectrometry. Our experiments confirmed several neoantigen candidates encoded by tumor-specific RNA isoforms, that are translated and localized to the cellular membrane. Interestingly, one of the candidates is an endoplasmic reticulum protein that exhibits cassette exon skipping in approximately 24% of TNBC tumors. Fluorescence microscopy experiments demonstrate that the TNBC-specific isoform of the protein is localized to plasma membrane but not the ER, suggesting that alternative splicing can alter protein localization. Together, our results demonstrate the potential of RNA splicing analysis for the discovery of novel immunotherapeutic targets. Citation Format: Hasan Ekrem Zumrut, Kendall Anderson, Miguel Manzanares, Shaleigh Smith, Sakshi Gera, Nayab Habib, Sanjana Shah, Devon Trahan, Martin Akerman, Gayatri Arun. Computational discovery and experimental validation of splicing-derived neoantigens [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 448.

PD25-09 TUMOR ASSOCIATED ANTIGEN VACCINE ENHANCES ANTI-TUMOR EFFECTS OF IMMUNE CHECKPOINT INHIBITORS AGAINST REFRACTORY CANCERS

You have accessJournal of UrologyCME1 Apr 2023PD25-09 TUMOR ASSOCIATED ANTIGEN VACCINE ENHANCES ANTI-TUMOR EFFECTS OF IMMUNE CHECKPOINT INHIBITORS AGAINST REFRACTORY CANCERS Shohei Ueda, Miho Ushijima, Atsushi Irie, Yasuharu Nishimura, and Masatoshi Eto Shohei UedaShohei Ueda More articles by this author , Miho UshijimaMiho Ushijima More articles by this author , Atsushi IrieAtsushi Irie More articles by this author , Yasuharu NishimuraYasuharu Nishimura More articles by this author , and Masatoshi EtoMasatoshi Eto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Progressive and chemoresistant bladder cancer is problematic because of the poor prognosis. Immune checkpoint blockade (ICB) has exhibited drastic anti-tumor efficacy in some patients, but the response rates were limited. Therefore, to improve efficacy of immunotherapy for refractory cancer, we have tried to develop a combination immunotherapy of tumor antigen vaccine and ICB in tumor-bearing mouse models. METHODS: We identified genes highly expressed in tumors but not in normal tissues by comparing RNA-seq data of mouse bladder cancer MB49, cisplatin-resistant MB49-CR, and lymphoma RMA cell lines, all of which are refractory to ICB, with datasets of 26 normal mouse tissues. Subsequently tumor-associated antigens-derived peptides predicted to have high binding affinities to MHC-I and -II molecules were identified by prediction algorithms. We also selected immunogenic and cancer-specific mutated Neo-antigenic peptides. MB49, MB49-CR, or RMA bearing mice were treated with these peptides and ICB. Finally, we investigated tumor infiltrating immune cells by flow cytometry, T cell receptor (TCR) repertoire analysis and RNA-seq of inoculated tumors. RESULTS: In 50-80% of mice pre-immunized with identified peptides, growths of MB49 and RMA inoculated s.c. were prevented and these immunized mice rejected re-transplanted tumors. Furthermore, combination therapy of these peptide vaccines and ICB synergistically inhibited tumor-growth of MB49-CR, MB49 and RMA indicating that the combination immunotherapy enhances therapeutic efficacy to treat refractory cancers. We also observed combination therapy with MHC-I antigen vaccination and ICB synergistically increased limited CD8+ TCR-alpha and beta genes in tumors, suggesting increased antigen-specific tumor-infiltrating CD8+ T cells. Furthermore, combination therapy with MHC-I/II antigen vaccine and ICB synergistically induced drastic changes of various types of tumor-infiltrating immune cells such as CD8+ T cells, CD4+ type 1 helper T cells, regulatory CD4+ T cells and natural killer cells. CONCLUSIONS: These results suggest that the combination immunotherapy described above shapes tumor-infiltrating immune cells and enhances therapeutic efficacy to treat refractory cancer. Source of Funding: This work was supported by KAKENHI. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e732 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shohei Ueda More articles by this author Miho Ushijima More articles by this author Atsushi Irie More articles by this author Yasuharu Nishimura More articles by this author Masatoshi Eto More articles by this author Expand All Advertisement PDF downloadLoading ...

KIT A502_Y503 duplication mutation serves as a potential and universal target for neoantigen peptide in Chinese GIST patients.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor with high prevalence of KIT and PDGFRA mutations. Few effective treatments can be exploited in imatinib or sunitinib resistant cases. While in immunotherapy, application of the highly individualized cancer neoantigen vaccines is hampered due to high economic and time cost. In this study we identified the most frequent mutation in Chinese GIST patients and predicted candidate neopeptide by next generation sequencing (NGS). Tumor tissues and matched blood samples of 116 Chinese GIST patients were collected. Genomic profile was detected through NGS, and 450 cancer genes were deeply sequenced. KIT mutations were identified, and long peptides containing the mutation were queried in NetMHCpan 4.0 tools to predict MHC class I binding of mutant peptides. The most frequent mutated genes in detected GIST patients were KIT (81.9%, 95/116), CDKN2A (18.97%, 22/116), and CDKN2B (15.52%, 18/116) in this cohort. The most common mutation of KIT was A502_Y503 duplication (15.93%, 18/113) in exon 9. Among the 116 cases, 103 were HLA I genotyped, and 101 were HLA II genotyped. In total, 16 samples with the mutation of KIT p.A502_Y503dup were identified to produce neoantigens with qualified HLA affinity. KIT hotspot mutation (p.A502_Y503dup) has the highest incidence, which may further eliminate the need for whole genome sequencing and patient-specific neoantigen prediction and synthesis. Therefore, for those carrying such mutation, accounting for around 16% of Chinese GIST patients and are usually less sensitive to imatinib, effective immunotherapies are in prospect.

Identification of T Cell Receptors Targeting a Neoantigen Derived from Recurrently Mutated FGFR3

Immunotherapies, including immune checkpoint blockades, play a critically important role in cancer treatments. For immunotherapies, neoantigens, which are generated by somatic mutations in cancer cells, are thought to be good targets due to their tumor specificity. Because neoantigens are unique in individual cancers, it is challenging to develop personalized immunotherapy targeting neoantigens. In this study, we screened "shared neoantigens", which are specific types of neoantigens derived from mutations observed commonly in a subset of cancer patients. Using exome sequencing data in the Cancer Genome Atlas (TCGA), we predicted shared neoantigen peptides and performed in vitro screening of shared neoantigen-reactive CD8+ T cells using peripheral blood from healthy donors. We examined the functional activity of neoantigen-specific T cell receptors (TCRs) by generating TCR-engineered T cells. Among the predicted shared neoantigens from TCGA data, we found that the mutated FGFR3Y373C peptide induced antigen-specific CD8+ T cells from the donor with HLA-A*02:06 via an ELISPOT assay. Subsequently, we obtained FGFR3Y373C-specific CD8+ T cell clones and identified two different sets of TCRs specifically reactive to FGFR3Y373C. We found that the TCR-engineered T cells expressing FGFR3Y373C-specific TCRs recognized the mutated FGFR3Y373C peptide but not the corresponding wild-type peptide. These two FGFR3Y373C-specific TCR-engineered T cells showed cytotoxic activity against mutated FGFR3Y373C-loaded cells. These results imply the possibility of strategies of immunotherapies targeting shared neoantigens, including cancer vaccines and TCR-engineered T cell therapies.

Driving the Chimeric Antigen Receptor: The Road Ahead for Cell Therapy

Driving the Chimeric Antigen Receptor: The Road Ahead for Cell Therapy

Mutated genes on ctDNA detecting postoperative recurrence presented reduced neoantigens in primary tumors in colorectal cancer cases

The detection and sequencing of the mutated ctDNA is one of the irreplaceable clinical measures in the postoperative management of colorectal cancer (CRC) cases. However, we are curious to comprehend the essential traits of mutated genes comprising metastatic sites out of whole mutated genes in primary sites. In the current retrospective study, we conducted target resequencing of ctDNA using 47 plasma samples and established a cancer panel carrying the commonly mutated genes between primary and recurrent tumors. We found that mutated genes in ctDNA indicated immune-resistance traits with respect to the impaired ability to present neoantigens by loss of expression or binding affinity to HLA in the primary tumor. Compared with the estimated neoantigens from all mutated genes in primary tumors, the neoantigen peptides from commonly mutated genes on the panel showed abundant expression but no binding affinity to HLA. Therefore, ctDNA mutations can be frequently and postoperatively detected to identify recurrence; however, these mutated genes were derived from immune-tolerated clones owing to the loss of neoantigen presentation in primary CRC tumors.

Lymph node-targeting adjuvant/neoantigen-codelivering vaccines for combination glioblastoma radioimmunotherapy.

Glioblastoma multiforme (GBM) is the most common and lethal type of adult brain cancer. Current GBM standard of care, including radiotherapy, often ends up with cancer recurrence, resulting in limited long-term survival benefits for GBM patients. Immunotherapy, such as immune checkpoint blockade (ICB), has thus far shown limited clinical benefit for GBM patients. Therapeutic vaccines hold great potential to elicit anti-cancer adaptive immunity, which can be synergistically combined with ICB and radiotherapy. Peptide vaccines are attractive for their ease of manufacturing and stability, but their therapeutic efficacy has been limited due to poor vaccine co-delivery and the limited ability of monovalent antigen vaccines to prevent tumor immune evasion. To address these challenges, here, we report GBM radioimmunotherapy that combines radiotherapy, ICB, and multivalent lymph-node-targeting adjuvant/antigen-codelivering albumin-binding vaccines (AAco-AlbiVax). Specifically, to codeliver peptide neoantigens and adjuvant CpG to lymph nodes (LNs), we developed AAco-AlbiVax based on a Y-shaped DNA scaffold that was site-specifically conjugated with CpG, peptide neoantigens, and albumin-binding maleimide-modified Evans blue derivative (MEB). As a result, these vaccines elicited antitumor immunity including neoantigen-specific CD8+ T cell responses in mice. In orthotopic GBM mice, the combination of AAco-AlbiVax, ICB, and fractionated radiation enhanced GBM therapeutic efficacy. However, radioimmunotherapy only trended more efficacious over radiotherapy alone. Taken together, these studies underscore the great potential of radioimmunotherapy for GBM, and future optimization of treatment dosing and scheduling would improve the therapeutic efficacy.

T cells of colorectal cancer patients’ stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells

BackgroundPatients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc).MethodsColorectal cancers (CRC) HROC113...

EXTH-27. TRANSLATABLE CCL3 THERAPY TO PROMOTE DC VACCINE MIGRATION AND EFFICACY AGAINST GLIOMA

Abstract INTRODUCTION Prior evidence from our studies have identified chemokine ligand 3 (CCL3) as a key modulator to increase DC vaccine migration to vaccine draining lymph nodes (VDLNs) which is associated with improved long-term survival clinically. METHODS The effect of CCL3 treatment on DC vaccine migration ex vivo and in vivo (n=5), antigen-specific T cell responses (n=5), and efficacy against orthotopic GL261-OVA and SMA560 tumors (n=10) was studied in C57Bl/6 and VMdK mice. CCL3 treatment of mouse and human DC vaccines ex vivo was also tested in transwell assays. DCs were electroporated with OVA-mRNA or pulsed with ODC1 neoantigen peptide. DC migration and T cell responses were quantified by flow cytometry. Median overall survival (mOS) was measure in days (d) post-intracranial implantation. RESULTS Ex-vivo treatment of DC vaccines with 20ng/mL CCL3 (18 and 1hr pre-injection) increased transwell (mouse and human) migration and to VDLN. In vivo systemic CCL3 treatment also resulted in a dose-dependent increase in migration of DC vaccine to VDLN (10µg, 20µg, 50µg). CCL3 added to DC vaccination generated more tumor antigen-specific CD8+IFNγ+ T cells at 7-days. CCL3 with tumor antigen-DC treatment also resulted in significantly greater survival compared to OVA-DC alone (GL261-OVA: mOS DCvac: 19.5d, CCL3+DCvac 37d, p=0.0174; SMA560: mOS: DCvac: 25d, CCL3+DCvac: 48d, p=0.002). CONCLUSIONS These data combined with previous success of our DC vaccine clinical trials reflect the potency of CCL3 to enhance DC vaccine-specific migration, immune responses, and survival. CCL3 is a novel and safe adjuvant to overcome prior limitations in DC vaccine therapy and may be translatable to increase heterogeneous tumor antigen presentation following vaccine-targeted tumor killing. CCL3 used as a drug could increase vaccine migration without potential systemic toxicity in patients with glioblastoma.

Immunoinformatics Analysis of Citrullinated Antigen as Potential Multi-peptide Lung Cancer Vaccine Candidates for Indonesian Population.

Non-small-cell lung cancer (NSCLC) is the most common lung cancer which has the highest mortality rate in Indonesia. One of the trends in treating cancer is by utilizing peptide vaccines, an immunotherapeutic approach that aims to stimulate the cell-mediated adaptive immune system to recognize cancer-associated peptides. Currently, no peptide vaccines are available in the market for NSCLC treatment. Therefore, this project aims to develop a multi-epitope peptide-based vaccine for NSCLC utilizing citrullinated peptides. Citrullination is a post-translational modification that occurs in cancer cells during autophagy that functions to induce immune responses towards modified self-epitopes such as tumor cells, through activation of PAD enzymes within the APC and target cells. It was found that introducing a common citrullinated neo-antigen peptide such as vimentin and enolase to the immune system could stimulate a higher specific CD4+ T cell response against NSCLC. Moreover, carcinoembryonic antigen (CEA), an antigen that is highly expressed in cancer cells, is also added to increase the vaccine's specificity and to mobilize both CD4+ and CD8+ T cells. These antigens bind strongly to the MHC Class II alleles such as HLA-DRB1*07:01 and HLA-DRB*11:01, which are predominant alleles in Indonesian populations. Through in silico approach, the peptides generated from CEA, citrullinated vimentin and enolase, were analyzed for their MHC binding strength, immunogenicity, ability to induce IFNγ response, and population coverage. It is expected that the immunodominant antigens presentation is able to induce a potent immune response in NSCLC patients in Indonesia, resulting in tumor eradication.

Comparison of Ridostin Pro and Poly(I:C) as adjuvant for a cancer neoantigen peptide vaccine

Background. The effectiveness of cancer neoantigen peptide vaccines depends on the presence of an adjuvant in their composition. Poly(I:C), a TRL-3 agonist, is used as an adjuvant in mouse models of cancer vaccines, but has limitations for use in humans. Therefore, the search for new effective adjuvants for inclusion in the composition of cancer neoantigen peptide vaccine is relevant. Ridostin Pro is a domestic drug that contains a natural complex of sodium salts of double-chiral and single-chiral ribonucleic acids, is an agonist of TLR-3, an inducer of interferon, its antiviral activity is shown. In this regard, the study of Ridostin Pro as an adjuvant in the composition of neoantigen peptide vaccines is of interest.Aim. To evaluate the ability of Ridostin Pro and Poly(I:C) adjuvants enhance the specific T-cell response to neoantigen synthetic peptides; to study the antitumor efficacy of a neoantigen peptide vaccine with Ridostin Pro or Poly(I:C) adjuvants.Materials and methods. Immunogenicity of peptides after vaccination with Ridostin Pro or Poly(I:C) adjuvants evaluated with ELISpot. Antitumor effect of Ridostin Pro or Poly(I:C) adjuvants were evaluated on a mouse model of the B16-F10 tumor by the effect on the tumor growth rate and survival of mice.Results. Vaccination of mice with Ridostin Pro or Poly(I:C) with neoantigen peptides contributed to the appearance of a specific immune response to peptides that were part of the vaccine. Ridostin Pro, both as part of a vaccine model and when administered without a peptide, inhibits tumor growth and increases the life expectancy of mice with melanoma B16-F10.Conclusion. Ridostin Pro promotes the formation of a specific immune response to the peptide vaccine, enhances the antitumor effect of the vaccine. These results confirm that Ridostin Pro may prove to be an effective adjuvant for personalized neoantigen peptide vaccines.

Heat-inactivated modified vaccinia virus Ankara boosts Th1 cellular and humoral immunity as a vaccine adjuvant

Protein or peptide-based subunit vaccines have generated excitement and renewed interest in combating human cancer or COVID-19 outbreak. One major concern for subunit vaccine application is the weak immune responses induced by protein or peptides. Developing novel and effective vaccine adjuvants are critical for the success of subunit vaccines. Here we explored the potential of heat-inactivated MVA (heat-iMVA) as a vaccine adjuvant. Heat-iMVA dramatically enhances T cell responses and antibodies responses, mainly toward Th1 immune responses when combined with protein or peptide-based immunogen. The adjuvant effect of Heat-iMVA is stronger than live MVA and is dependent on the cGAS/STING-mediated cytosolic DNA-sensing pathway. In a therapeutic vaccination model based on tumor neoantigen peptide vaccine, Heat-iMVA significantly extended the survival and delayed tumor growth. When combined with SARS-CoV-2 spike protein, Heat-iMVA induced more robust spike-specific antibody production and more potent neutralization antibodies. Our results support that Heat-iMVA can be developed as a safe and potent vaccine adjuvant for subunit vaccines against cancer or SARS-CoV-2.

Identification of neoantigens in oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) has a relatively poor long-term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumour mutations, presented to cytotoxic T-cells by human leucocyte antigen (HLA) molecules. Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumours from a cohort of seven patients with OAC. We directly identified one HLA-I presented neoantigen from one patient, and report functional T-cell responses from a predicted HLA-II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T-cell responses, emphasizing the need for improved strategies for neoantigen identification.

Redirecting Polyclonal T Cells against Cancer with Soluble T-Cell Receptors.

Cancer cells accumulate genetic mutations in coding proteins that may be presented by HLA as neoantigenic peptides (peptide HLA, pHLA). T cells scan for neoantigenic pHLA by the T-cell receptor (TCR):CD3 complex. This complex has the dual function of binding pHLA, by the TCR, and triggering T-cell activation by CD3. Checkpoint therapy activates exhausted T cells to kill cancer cells and generally work best against tumors with high neoantigen burden and in patients with neoantigenic-reactive T cells. TCR T-cell engagers (TCE) are a novel class of immunotherapy that bypasses these two requirements by redirecting polyclonal T cells, regardless of their native specificity, to kill a cancer cell independent of neoantigen burden. This is accomplished through deconstructing the membrane-bound TCR:CD3 complex into a soluble bispecific protein comprised of a targeting domain (TCR) and activating domain (usually anti-CD3 single-chain variable fragment). The pool of targets for TCR TCE is larger than for antibody therapeutics and includes >90% of human intra- or extracellular proteins. Most tumor-associated antigens for solid tumors are intracellular and accessible only by a TCR therapeutic. Tebentafusp, a TCR TCE directed to a peptide derived from the gp100 melanoma protein presented by HLA*A02:01, demonstrated a survival benefit in metastatic uveal melanoma (mUM). This survival benefit highlights the promise of TCR TCEs because mUM is a solid tumor with a very low neoantigen burden and has poor response to checkpoints and chemotherapy. Other TCR TCE programs are now in clinical studies for a broader range of tumors.

Autoantibody and T cell responses to oxidative post-translationally modified insulin neoantigenic peptides in type 1 diabetes

Aims/hypothesisAntibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides.MethodsoxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs.ResultsWe observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12–21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11–30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21–30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p<0.001) and CD8+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies.Conclusions/interpretationOur findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes.Graphical abstract

Abstract IA011: Identifying tumor-restricted target antigens for adoptive cellular immunotherapy to treat Ewing Sarcoma using multi-omic discovery platforms

Abstract Background: In the last four decades, no significant improvement in survival rates for children with Ewing Sarcoma (EWS) has been made. Less than 30% of patients with metastatic disease are long- term survivors and standard therapy incorporates dose-intensive cytotoxic chemotherapy risking lifelong late effects. Engineered T cells (TCRs/CARs) can mediate impressive clinical anti-tumor activity and would represent a promising treatment option for patients. Development of potent and safe adoptive cellular immunotherapies (ACT) relies on antigents that are absent from any normal, vital tissue in the body to avoid on-target/off-tumor recognition, which conventional methods often fail to identify. Therefore, novel pipelines for rational target antigen discovery are critical for advancing ACT for the treatment of the EWS. Methods: To identify tumor antigens that meet the requirement for ACT targets, we performed differential gene expression analysis of compiled transcriptomes from EWS tumors (n=120) and normal tissues (n=42). Bioinformatic cell-surface annotation identified top differentially expressed targets best suited for CAR T cells (Heitzeneder et. al, JNCI 2019). However, more than 70% of the human proteome represent intracellular molecules, a target-pool accessible to TCRs through presentation of peptides via MHC. To empirically determine the immunopeptidome, we profiled EWS cells by immunoprecipitation with a pan-MHC-I antibody, followed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). To identify peptide-MHC antigens best suited for TCRs, this data was incorporated with the differential tumor-to-normal transcriptome. Results: We identified 584 genes as overexpressed in EWS(LogFC&amp;gt;1, P&amp;lt;.01) compared to normal tissue. A total of 24 genes showed high abundance in EWS (AVElog2[TPM+1]&amp;gt;5) and low normal tissue expression (AVElog2[TPM+1]&amp;lt;2). Of those, 11 were predicted to be cell surface-associated and possess the potential to serve as CAR T-cell targets. This identified pregnancy-associated plasma protein-A (PAPP-A), a placental antigen expressed at the maternal-fetal interface (Heitzeneder et al, JNCI 2019). Immunopeptidome analysis of HLA-A*0201+EW8 cells showed that peptides of ~30% of the top 24 genes were presented by MHC-I. No neoantigenic peptides caused by common mutations in EWS or the EWS-Fli1 breakpoint region were found. Instead, immunopeptidome analysis identified oncofetal and cancer-testis antigens for TCR-based treatment approaches. Conclusions: Truly tumor-specific antigens are rare in pediatric solid tumors, yet, they often manifest stalled fetal developmental programs and continue to express placental, oncofetal and cancer testis antigens. Those represent an attractive pool of target antigens for the development of ACT to treat EWS, which is otherwise characterized by lofe immunogenicity. Comprehensive analysis of the surfaceome and immunopeptidome has proven an efficient method to identify such target antigens and facilitates the preclinical development of CAR-T cell and TCR-based immunotherapies. Citation Format: Sabine Heitzeneder. Identifying tumor-restricted target antigens for adoptive cellular immunotherapy to treat Ewing Sarcoma using multi-omic discovery platforms [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA011.

Poster session 2Peptide identification methodology introduces bias in the detected repertoire of MHC presented peptides

Immunopeptidomic profiling with mass spectrometry has the capacity to directly identify peptide neo-antigens and provide a better understanding of the relationship between oncogenic processes, antigen presentation and immunity. Key to this is the use of search engines for the interpretation of mass spectra acquired after HLA peptide purification and LC-MS. In this study we present a comparison of three commonly used search engines across several immunopeptidomic datasets. We found that choice of search engine influences not only sensitivity but remarkably the repertoire of peptide sequences identified. Using NetMHC we validate our results showing that additional peptides identified are highly likely to be bound to a HLA protein. Based on this we investigate how search engine bias affects the outcome of an immunopeptidomic analyses given a background of different HLA alleles. We look in detail at this effect profiling several tumour samples with diverse HLA types.

Transcriptional and functional analyses of neoantigen-specific CD4 T cells during a profound response to anti-PD-L1 in metastatic Merkel cell carcinoma

BackgroundMerkel cell carcinoma (MCC) often responds to PD-1 pathway blockade, regardless of tumor-viral status (~80% of cases driven by the Merkel cell polyomavirus (MCPyV)). Prior studies have characterized tumor-specific T...

Personalized neoantigen vaccine combined with PD-1 blockade increases CD8+ tissue-resident memory T-cell infiltration in preclinical hepatocellular carcinoma models

BackgroundPersonalized neoantigen vaccine could induce a robust antitumor immune response in multiple cancers, whose efficacy could be further enhanced by combining with programmed cell death 1 blockade (α-PD-1). However, the...