Neoepitope Peptide Research Articles

RTID-02. PERSONALIZED PEPTIDE VACCINATION AMONG 173 PATIENTS WITH GLIOBLASTOMA

Abstract Current treatment outcome of patients with glioblastoma (GBM) remains poor. Following standard therapy, recurrence is universal with limited survival. GBM tumors from 173 patients were analyzed for somatic mutations to generate a personalized peptide vaccine targeting tumor-specific neoepitopes. In agreement with their treating physician, patients added a personalized peptide vaccine to their treatment as an individual healing attempt. Patients were monitored from October 2015 until August 2023. We retrospectively evaluated their clinical courses and the results of their immune monitoring data. Among all patients, including 70 treated prior to progression (primary) and 103 treated after progression (recurrent), the median overall survival from first diagnosis was 31.9 months (95% CI: 25.0-36.5). Adverse events were infrequent and were predominantly grade 1 or 2. An immune response to at least one of the vaccinated peptides was detected in blood samples of 87 of 97 (90%) monitored patients. T-cell responses to vaccinated neoepitope peptides were durable in most patients. Significantly prolonged survival was observed for patients with multiple vaccine-induced immune responses (53 months) compared to those with no/low induced responses (27 months; P=0.03). Altogether, our results highlight that the application of personalized neoantigen-targeting peptide vaccine is feasible and represents a promising potential treatment option for GBM patients. This is the largest real-world observation involving GBM patients safely treated with a personalized peptide vaccine to date. This real-world observation will be translated into a clinical trial to evaluate the specific contribution of the neoantigen vaccine.

CTIM-12. A RETROLECTIVE ANALYSIS ON THE SAFETY, EFFICACY AND IMMUNOGENICITY OF 203 GLIOBLASTOMA PATIENTS TREATED WITH PERSONALIZED THERAPEUTIC CANCER VACCINES (PTCV)

Abstract BACKGROUND Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor. Despite of the current SoC, patients have a poor prognosis with a median OS of about 14-17 months.Method, RESULTS We report retrospectively analyzed data from 203 GBM patients (IDH wildtype) who added PTCV derived from patient individual tumor mutations to their treatment regimen in the setting of individual treatment attempts (ITA) in Germany. Data were analyzed re. vaccine induced T cell responses and safety acc. to CTCAE terminology version 5.0. Subsets of patients with and without hypermethylation of the MGMT promoter were further evaluated re. efficacy. Treated patients had a median age of 53 years at diagnosis, started vaccination after a median of 11 months after diagnosis, received a median of 8 immunizations with a median of 20 neoepitope peptides, and had a median follow-up time since first vaccination of 21 months. They usually received SoC (Stupp scheme), often accompanied by additional therapies. In general, the PTCV was well tolerated, and most patients experienced only Grade 1 and 2 AEs. Most frequently injection site reactions were observed (67%). Seven patients developed Grade 3 allergic or anaphylactic reactions after 7 or more vaccinations. For 119 patients T-cell responses against the vaccinated neoepitopes were investigated. Almost all patients developed vaccine induced T-cell responses (97%). 71% of patients showed both CD4+ and CD8+ T-cell responses towards vaccinated peptides. Immune responses usually increased over time peaking within 3-6 months. Median OS of the whole cohort was 26 mo (95% CI 24, 31). Primary GBM patients without recurrence events before the start of vaccination and with or without hypermethylated MGMT promoter status showed a mOS of 41 months (95% CI: 32, NA) or 23 months (95% CI: 20, NA), respectively. CONCLUSION The retrospective data analysis from a real-world cohort of GBM patients suggests that PTCV are safe, immunogenic, and may prolong the survival of patients.

TERT-associated DNA polymerases genes link CAF and CD8+ T Cells to improve immunotherapy response rate across multiple cancers.

e14669 Background: The cancer cells exhibit heightened basal levels of responses to DNA breaks, accumulating DNA polymerases family (DNA-pol) as repair protein, thereby increasing DNA mutation and neoantigens. Based on this, we investigated the potential of DNA-pol as an immunotherapy biomarker and its underlying mechanism. Methods: We conducted a pan-cancer analysis of DNA-pol gene dysregulation in 6366 samples. Normal fibroblast, cancer-associated fibroblast, and CD8+ T cells were cultured from cancer patients (3 esophagus, 5 lung, 5 renal, and 3 liver cancer), then treated them with PD-L1 inhibitor or overexpressing TERT for RNA-seq and proteomics. 996 multiple cancer cases with ICIs were collected to test biomarker efficiency. A phase II clinical trial is evaluating and exploring our biomarker in mechanism prospectively. Results: We defined a distinct signature score of DNA-pol genes upregulated in cancer (DNA-pol-up) and linked to worse prognosis (HR=1.2). DNA-pol-up program dysregulation led to immune processes alteration and was related to upstream activation of TERT signaling (z-score=3.6) in ingenuity pathway analysis and linked to immunologically active tumors by recruiting CD8+ T cell and downregulating CAF. DNA-pol-up score was elevated in PD-L1-treated CAF and TERT-overexpressed CD8+ T cell, in MSI-H cancer, also related to more neoepitope peptides, TMB (Rho=0.54), cancer stemness (Rho=0.78), and tumor purity (Rho=0.29). Cancers that activate this program carried distinct genomic profiles with PIK3CA, KRAS, and TP53 mutations. This signature was an independent predictor for ICIs response (OR=2.52), even outperforming cytolytic activity, MSI, TMB (OR=0.9). In 12 clinical trial datasets, this signature could stratify the responding patients (maxAUC=0.762). In our phase II clinical trial, 34 liver cancer were enrolled with 4-year follow-up, it also has a satisfactory performance in predicting the ORR (AUC=0.699) and classifies mortality rate (HR=2.5), meanwhile verified its relationship with detailed potential molecular mechanism. Conclusions: Our findings identify a distinct transcriptional pattern of DNA-pol genes across cancers, which highlighted the role of DNA-pol family genes in predicting the immunotherapy response for the first time, and TERT could be a novel vaccine candidate for improving immunotherapy response.

A phase 1b study of tbio-4101 (autologous selected and expanded tumor-infiltrating lymphocytes [TILs]) with pembrolizumab in patients with anti-PD-1–resistant, advanced head and neck squamous cell carcinoma (aHNSCC).

TPS6115 Background: Despite the excitement and approval of anti-PD1 therapy in first line setting of patients with aHNSCC, prognosis remains poor, with the majority experiencing disease progression after therapy. Consequently, novel strategies to improve treatment activity represent a critical and unmet need for this patient population. Cell therapies offer the advantage of a more personalized therapy for patients over standard immune checkpoint inhibitors. Historically, cell therapies have relied on bulk products that may not effectively select a tumor-reactive T cell population. TBio-4101 is produced by an industrialized manufacturing approach leveraging single-cell sorting of patient-specific neoepitope-reactive TIL with the potential to address immunologically ‘cold’ tumors. The TBio-4101 process specifically enriches polyclonal, polyfunctional, tumor-reactive T cells with an endogenous neoantigen-reactive T cell receptor repertoire that can target multiple tumor antigens. Non-tumor-reactive bystander T cells, which may negatively impact treatment efficacy via product dilution, and typically account for ≥90% of bulk TIL, are substantially reduced by this process. Inspired by the early academic clinically efficacious NCI selection and enrichment strategies (Tran et al 2014, 2016; Zacharakis et al, 2022), TBio-4101 is refined to better identify and potentially enrich to >70% tumor-reactive CD8+ and CD4+ T cells (median < 3% in bulk TIL). Methods: This is a Phase 1b, open-label, single-center study of TBio-4101 and pembrolizumab. Eligible patients are 18 to 75 years of age with aHNSCC. Prior to receiving pembrolizumab with or without chemotherapy as first line standard-of-care (SOC), these patients undergo tumor harvest for TILs followed by apheresis to collect monocytes. TILs are isolated from the tumor and expanded in vitro. Patient-specific neoepitopes are defined by whole exome and RNA sequencing of tumor versus germline to manufacture a peptide pool representing potential tumor-specific mutations. Monocytes isolated from apheresis are differentiated into dendritic cells, pulsed with the neoepitope peptides, and co-cultured with the TILs. Tumor-reactive T cells are specifically activated and selected based on upregulation of activation markers prior to expansion in culture. This process results in a patient-specific T cell product enriched for tumor-reactive T cells. Enrolled patients who progress on first line SOC therapy are preconditioned with 5 days of nonmyeloablative lymphodepletion followed by TBio-4101 infusion. Thereafter, patients receive ≤6 doses of IL-2 (600,000 IU/kg) every 8h, and pembrolizumab (200 mg) every 3 weeks until disease progression. The trial is currently open, with a target enrollment of 15 patients (NCT06236425). Clinical trial information: NCT06236425 .

Abstract 6212: A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden that leads to the generation of few neo-epitopes derived from mutated genes that can be recognized by cytotoxic T cells. The immunosuppressive microenvironment of PDAC comprises suppressive cell populations that include myeloid cells, cancer associated fibroblast subpopulations, and regulatory T cells that reduce T cell infiltration and cytotoxic function. We have developed a murine personalized cancer vaccine to enhance cytotoxic T cell function against PDAC, PancVAX (Kinkead et al, JCI Insight 2018). Survival benefit with PancVAX was dependent on the addition of immune checkpoint inhibitor (ICI) therapy to increase the number of cytotoxic CD8 T cells infiltrating tumors and maintain their cytotoxic function (Huff et al, JCI Insight 2023). Yet, the impact of cell-cell communication on effector T cell function arising from the immunosuppressive microenvironment of PDAC remains unknown. We previously developed a ligand-receptor inference method called Domino that infers inter- and intra-cellular signaling networks in single-cell RNA-seq data (Cherry et al, Nat Biomed Eng 2021). However, this technique is limited to data from single conditions. Understanding how this combination immunotherapy alters cellular signaling networks requires new computational tools that identify changes in regulatory networks between treatment conditions. We developed capabilities for assessment of differential signaling with Domino founded on a basis of differential signal receipt. Active receptors are identified by receptor expression in recipient cells that is correlated with downstream transcription factors in the intracellular signaling cascade. Identification of causative ligands for receptor activation is assessed by a probability of interaction for ligand-receptor interactions between cell types. This probability metric considers mean expression of ligand and receptor genes as well as the number of sender cells for comparison across treatment groups. Comparing communication networks in PancVAX alone to PancVAX with ICI, PancVAX with ICI enhances cell-cell adhesion and interferon gamma signaling in cytotoxic CD8 T cells towards exhausted CD8 T cells. Macrophages in PancVAX with ICI enhance expression of immune checkpoint ligands towards CD8 T cells, despite the presence of blocking ICI antibodies. Intracellular signaling downstream of PD-1 in exhausted T cells shows diminished numbers of transcription factors linked to PD-1 signaling. This software informs prioritization of inhibitory signals for modulation in future immunotherapy regimens and can be applied generally to other tumor subtypes and treatment contexts. Citation Format: Jacob T. Mitchell, Atul Deshpande, Amanda L. Huff, Chris Cherry, Sushma Nagaraj, Kavita Krishnan, Dmitrijs Lvovs, Jennifer H. Elisseeff, Elizabeth M. Jaffee, Neeha Zaidi, Elana J. Fertig. A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6212.

MediMer: a versatile do-it-yourself peptide-receptive MHC class I multimer platform for tumor neoantigen-specific T cell detection.

Peptide-loaded MHC class I (pMHC-I) multimers have revolutionized our capabilities to monitor disease-associated T cell responses with high sensitivity and specificity. To improve the discovery of T cell receptors (TCR) targeting neoantigens of individual tumor patients with recombinant MHC molecules, we developed a peptide-loadable MHC class I platform termed MediMer. MediMers are based on soluble disulfide-stabilized β2-microglobulin/heavy chain ectodomain single-chain dimers (dsSCD) that can be easily produced in large quantities in eukaryotic cells and tailored to individual patients' HLA allotypes with only little hands-on time. Upon transient expression in CHO-S cells together with ER-targeted BirA biotin ligase, biotinylated dsSCD are purified from the cell supernatant and are ready to use. We show that CHO-produced dsSCD are free of endogenous peptide ligands. Empty dsSCD from more than 30 different HLA-A,B,C allotypes, that were produced and validated so far, can be loaded with synthetic peptides matching the known binding criteria of the respective allotypes, and stored at low temperature without loss of binding activity. We demonstrate the usability of peptide-loaded dsSCD multimers for the detection of human antigen-specific T cells with comparable sensitivities as multimers generated with peptide-tethered β2m-HLA heavy chain single-chain trimers (SCT) and wild-type peptide-MHC-I complexes prior formed in small-scale refolding reactions. Using allotype-specific, fluorophore-labeled competitor peptides, we present a novel dsSCD-based peptide binding assay capable of interrogating large libraries of in silico predicted neoepitope peptides by flow cytometry in a high-throughput and rapid format. We discovered rare T cell populations with specificity for tumor neoepitopes and epitopes from shared tumor-associated antigens in peripheral blood of a melanoma patient including a so far unreported HLA-C*08:02-restricted NY-ESO-1-specific CD8+ T cell population. Two representative TCR of this T cell population, which could be of potential value for a broader spectrum of patients, were identified by dsSCD-guided single-cell sequencing and were validated by cognate pMHC-I multimer staining and functional responses to autologous peptide-pulsed antigen presenting cells. By deploying the technically accessible dsSCD MHC-I MediMer platform, we hope to significantly improve success rates for the discovery of personalized neoepitope-specific TCR in the future by being able to also cover rare HLA allotypes.

In silico designed mRNA vaccines targeting CA-125 neoantigen in breast and ovarian cancer.

Somatic mutation-derived neoantigens are associated with patient survival in breast and ovarian cancer. These neoantigens are targets for cancer, as shown by the implementation of neoepitope peptides as cancer vaccines. The success of cost-effective multi-epitope mRNA vaccines against SARS-Cov-2 in the pandemic established a model for reverse vaccinology. In this study, we aimed to develop an in silico pipeline designing an mRNA vaccine of the CA-125 neoantigen against breast and ovarian cancer, respectively. Using immuno-bioinformatics tools, we predicted cytotoxic CD8+T cell epitopes based on somatic mutation-driven neoantigens of CA-125 in breast or ovarian cancer, constructed a self-adjuvant mRNA vaccine with CD40L and MHC-I -targeting domain to enhance cross-presentation of neoepitopes by dendritic cells. With an in silico ImmSim algorithm, we estimated the immune responses post-immunization, showing IFN-γ and CD8+T cell response. The strategy described in this study may be scaled up and implemented to design precision multi-epitope mRNA vaccines by targeting multiple neoantigens.

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.

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

Neoepitope fragments as biomarkers for different phenotypes of intervertebral disc degeneration.

BackgroundDuring the intervertebral disc (IVD) degeneration process, initial degenerative events occur at the extracellular matrix level, with the appearance of neoepitope peptides formed by the cleavage of aggrecan and collagen. This study aims to elucidate the spatial and temporal alterations of aggrecan and collagen neoepitope level during IVD degeneration.MethodsBovine caudal IVDs were cultured under four different conditions to mimic different degenerative situations. Samples cultured after 1‐ or 8‐days were collected for analysis. Human IVD samples were obtained from patients diagnosed with lumbar disc herniation (LDH) or adolescent idiopathic scoliosis (AIS). After immunohistochemical (IHC) staining of Aggrecanase Cleaved C‐terminus Aggrecan Neoepitope (NB100), MMP Cleaved C‐terminus Aggrecan Neoepitope (MMPCC), Collagen Type 1α1 1/4 fragment (C1α1) and Collagenase Cleaved Type I and II Collagen Neoepitope (C1,2C), staining optical density (OD)/area in extracellular matrix (OECM) and pericellular zone (OPCZ) were analyzed. Conditioned media of the bovine IVD was collected to measure protein level of inflammatory cytokines and C1,2C.ResultsFor the bovine IVD sections, the aggrecan MMPCC neoepitope was accumulated in nucleus pulposus (NP) and cartilage endplate (EP) regions following mechanical overload in the one strike model after long‐term culture; as for the TNF‐α induced degeneration, the OECM and OPCZ of collagen C1,2C neoepitope was significantly increased in the outer AF region after long‐term culture; moreover, the C1,2C was only detected in conditioned medium from TNF‐α injection + Degenerative loading group after 8 days of culture. LDH patients showed higher MMPCC OECM in NP and higher C1,2C OECM in AF region compared with AIS patients.ConclusionsIn summary, aggrecan and collagen neoepitope profiles showed degeneration induction trigger‐ and region‐specific differences in the IVD organ culture models. Different IVD degeneration types are correlated with specific neoepitope expression profiles. These neoepitopes may be helpful as biomarkers of ECM degradation in early IVD degeneration and indicators of different degeneration phenotypes.

Identification of Neoantigens in Two Murine Gastric Cancer Cell Lines Leading to the Neoantigen-Based Immunotherapy.

Simple SummaryDespite the success of immune checkpoint inhibitors (ICI) for treating a variety of solid cancers, most gastric cancer patients are resistant to ICI monotherapies. Combinations of ICI with other therapies may be able to overcome this resistance. In order to develop combination immunotherapies, immunologically well-characterized preclinical gastric cancer models are required. To this end, in the present study, we characterized two murine gastric cancer cell lines, namely, YTN2 which spontaneously regresses, and YTN16 which grows progressively. Although anti-CTLA-4 monotherapy eradicated most YTN16 tumors, these were resistant to either anti-PD-1 or anti-PD-L1 treatment. Furthermore, we identified neoantigens in YTN2 and YTN16 tumors and conducted neoantigen-based immunotherapy for these tumors. In addition, the information on neoantigens facilitates the evaluation of tumor-specific immune responses induced by the combination therapies. These immunologically well-characterized gastric cancer models will contribute to the development of novel combination immunotherapies.To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8+ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.

A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells.Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation.Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG controlConclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. DisclosuresRoss: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Interrogation of neoantigen-specific CD8 T cells in peripheral blood following PD-L1 blockade in patients with metastatic urothelial carcinoma (mUC).

3075 Background: Proliferation of CD8 T cells can be detected in the blood of cancer patients (pts) following a single dose of immune checkpoint blockade (ICB) and tends to be more robust in responding pts. Furthermore, tumor mutational burden (TMB) is seen to predict outcome to ICB across cancers. Mutation-derived neoepitopes presented on the tumor cell surface is believed to be recognized by T cells and are thus critical for tumor clearance. However, the capacity to mount a neoantigen T cell response and the kinetics in relation to ICB remain poorly understood. Methods: 24 pts with mUC were treated with atezolizumab (anti-PD-L1) 1200mg q3w on IMVigor 210 at MSKCC and included in here. Pt-specific neoepitopes were predicted based on whole-exome and RNA sequencing of pre-treatment archival tumors using the MuPeXI platform. Using DNA-barcode labelled pMHC multimers, we investigated CD8 T cell recognition of mutation-derived neoepitopes by screening pt PBMC samples pre- and post-treatment with atezolizumab (n = 85 PBMC samples). The kinetics of neoepitope-specific CD8 T cells were assessed for association with durable clinical benefit (DCB; defined as progression free survival &gt; 6 mo). Results: Neoepitope peptide libraries of between 200-587 peptides were generated per pt (mean = 260 peptides per pt). 31 out of a combined 56 possible pt HLA types across the cohort were utilized for T cell analyses (mean four HLAs per pt). MHC multimer-based screening of pt PBMCs revealed detection of neoepitope-specific CD8 T cells in 22 of 24 pts pre-treatment (range one to 14 neoepitope responses) and 21 of 22 pts post-treatment (up to 273 weeks after trial start; one to 19 neoepitope responses). There were large inter- and intra-patient variations of neoepitope-specific CD8 T cell responses during treatment with the largest increases occurring at the 3-wk, post-treatment initiation timepoint. We observed that pts with DCB tend to raise a broader neoantigen T cell response than patients without DCB. 38% of pts without DCB and 67% of pts with DCB exhibited an increase in neoepitope-specific CD8 T cell responses within 3 wks of treatment initiation. Conclusions: Using high-throughput screening, pt-specific neoepitope reactive CD8 T cells could be detected pre- and post-treatment in pts with mUC treated with atezolizumab. Phenotypic characterization of neoepitope reactive CD8 T cells is ongoing. These data may help elucidate the dynamics and characteristics of the T cells of highest relevance to the ICB-induced, anti-tumor immune response.

Abstract PR04: Anti peptide-HLA (TCR-like) antibodies specific for the KRAS G12V neoantigen

Abstract The remarkably high prevalence of Ras driver mutations in tumors of diverse origins and stages indicates that Ras neoantigens are poor substrates for HLA-mediated detection by the native immune system. Nevertheless, immunotherapies based upon tumor-infiltrating lymphocytes targeting Ras neoantigens have been reported, indicating that such mutations are subject to HLA display and immune surveillance. Antibodies capable of recognizing specific peptide-HLA complexes at the cell surface have the potential to target intracellular tumor neoantigens for therapeutic intervention. However, such reagents have proved to be challenging to develop. Using a novel antibody discovery platform in combination with a proprietary human germline scFv library, we have developed a rapid method for the isolation of antibodies against peptide-HLA complexes. Applying this technology to KRAS G12V, we have isolated two scFvs that recognize the peptide VVVGAVGVGK in complex with HLA-A*11:01. This peptide-HLA combination forms functional complexes both in vitro and at the cell surface and has been reported to elicit CTL responses in vivo. In contrast, the native peptide is a poor substrate for binding to HLA-A*11:01. Binding affinities of the two scFv binders for the target HLA complex are in the sub-100nM range. Both show highly specific, peptide-dependent recognition of the KRAS-HLA-A*11:01 complex, as demonstrated by the failure to bind complexes containing wild-type KRAS peptides or other control peptides. Alanine scanning mutagenesis reveals multiple residues within the neoepitope peptide that are critical for antibody binding, including the mutated valine residue. The extended binding footprint encompassing the neoepitope and the poor presentation of the unmutated peptide are likely to contribute to the on-target specificity of these antibodies. Incorporation of the scFvs into a bispecific T-cell engager (BiTE) format has demonstrated the simultaneous engagement of both the KRAS HLA-A*11:01 complex and the CD3 surface marker of T cells. Ongoing work is aimed to demonstrate BiTE-dependent CTL-mediated lysis of target cells displaying the KRAS HLA-A*11:01 complex. We are currently developing CAR-T cells carrying peptide-HLA specific scFvs and are screening further mutant Ras peptides in the context of other HLA-A subtypes. This abstract is also being presented as Poster B48. Citation Format: Avisa Maserati, Dahna Makris, Ben Kiefel, Matt Beasley, Brad McColl, Ben Clifton. Anti peptide-HLA (TCR-like) antibodies specific for the KRAS G12V neoantigen [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR04.

Evidence for selective silencing of MHC-binding neoepitopes to avoid immune surveillance.

2591 Background: Overall response rates to immune checkpoint inhibition (ICI) are &lt; 50% even in TMB-high patients (e.g. Checkmate-227), suggesting other mechanisms of immune escape exist beyond expressing checkpoints. At least 18% of somatic-specific exonic DNA variants are not expressed into mRNA (Rabizadeh, 2018), yet the selection criteria for which variants to silence remains unclear. We sought to determine if immunogenicity of variants factors into their suppression. Methods: Somatic-specific single nucleotide variants (SNVs) were identified from paired tumor/normal whole-exome sequencing (WES), and annotated as expressed if observed in &gt; = 2 RNAseq reads. MHC1 binding affinity for 9-mer neoepitope peptides resulting from said SNVs were predicted using NetMHC within presented HLA-types. Cases with &gt; 200 non-synonymous exonic mutations were designated as TMB-high in accordance with Rizvi et al, 2015. Tumor immune activity was inferred by RNAseq expression of 6 checkpoint/TME markers, as well as by estimating immune infiltration using RNAseq deconvolution of immune genesets (Bindea et al 2013). Significant associations between TMB, neoantigen-load, expressed neoepitope binding affinities, and immune activity were analyzed. Results: Within a clinical database of 1,363 cases with T/N/R sequencing, a total of 147,015 potential neoepitopes were identified. A small but significant enrichment was observed for silencing neoepitopes that are predicted to bind MHC1 (OR = 1.22, p = 2.4e-78 one-sided Fishers test). The silencing rate was similar between the 17% of patients with high TMB vs others, but was increased in 35% of all patients with high inferred immune infiltration (N = 490, OR = 1.30, p = 1.8e-31). A further silencing enrichment was observed in 19% of all patients displaying high immune activity but low PDL1 expression (N = 263, OR = 1.44, p = 4.0e-45). Conclusions: We observe significant preferential silencing of MHC binding neoepitopes. Specifically, when tumor infiltrating immune cells are activated, silencing neoepitopes may be an alternative to checkpoint expression for avoiding an immune cascade. Patients with TILs and silenced neoepitopes may benefit from epigenetic priming therapy prior to ICI therapy.

Abstract B33: Neoepitope peptide vaccines and immune checkpoint blockade in a new preclinical ovarian cancer model

Abstract Objective: Therapeutic success currently seen with immune checkpoint blockade (ICB) occurs primarily in immunogenic tumors and can be partly explained by the high rates of non-synonymous genetic mutations (single nucleotide variants, SNV) in tumor cells, leading to neoepitope presentations and robust T-cell infiltration. Despite showing mutations in fewer genes (most frequently TP53), high-grade serous ovarian cancer (HGSOC) displays signs of T-cell inflammation and responds, albeit modestly, to ICB. We focus on immune therapy tailored to the genomic characteristics of the tumor, especially to structural aberrations (SAs), like gene fusions. Our hypothesis is that SAs that are expressed as proteins are antigenic and immunogenic, and represent a source of neoantigens for combination immunotherapies that can overcome locoregional immune suppression. DNA structural aberrations can result in intergenic fusions and intragenic rearrangements, such as exon duplications (dup) and deletions (del). Here we focus on identifying gene fusion and exon dup/del-derived neoepitopes based on RNA-seq data for a combination of retrospective and prospective sample collections. Our hypothesis is that these gene rearrangements resulting from SVs may contribute to the immunogenicity and antigenicity of HGSOC. Methods: FusionCatcher was used to evaluate RNA sequencing data for in-frame gene fusions events in banked human HGSOC. HLAminer was used to determine individual haplotypes. Upon detection of fusion events (validated via PCR) we identified the MHC-I and MHC-II restricted peptides with predicted high or intermediate (IC50&amp;lt;500nM) binding affinity. The peptides (with sequences across the fusion point) were synthesized and pulsed onto HLA-matched PBMCs, for in vitro T cells stimulation assay. Following antigen priming and in vitro restimulation, IFNγ production by T cells was measured by ELISA. The efficacy of SA-derived neoepitopes was tested in vivo with two new preclinical mouse models based on MKP-Lung and MKP-Liver cells ovarian cancer cell lines. RNA-seq data of their complete mutanome were used to generate neoepitopes. RNA from normal mouse OSE isolated from healthy mice was used as reference for mutation calling. For vaccine generation, peptides were pulsed onto dendritic cells matured into Th1-inducing phenotype (DC1). Mice were treated with Rat IgG, anti-PDL1, or vaccine + anti PD-L1. Objective response was measured by tumor volume. Results: Our results demonstrate neoepitopes-specific human T-cell responses in vitro. RNA-seq data were available for nine patients; eight MHC-I and five MHC-II neoantigens were synthesized from gene fusions from three different patients represented by six sets of haplotype specific PBMCs. In vitro, neoantigen-treated PBMCs showed increased cell clumping. An objective IFN gamma response on ELISA was seen for six peptides at two different concentrations and five peptides at one concentration. Analysis of RNA-seq of murine tumors shows that the metastatic MKP-Lung cells (which carry molecular signatures associated with recurrent ovarian cancer), have an increased number of mutations (SNVs, indels) including gene fusions, in line with findings from human tumors. Using in silico prediction tools, we identified 16 potential peptide binders (IC50&amp;lt; 500), two with strong and 14 with moderate binding. The transcript variants were validated via PCR. The vaccine comprised the top seven MHC-I restricted peptide candidates with IC50&amp;lt;500Nm and one large (MHC-II) candidate peptide for CD4 T-cell responses. No difference was seen between treatment groups (ANOVA p=0.35). Conclusion: Our results demonstrate that PBMC pulsed with a combination of short and large peptides release increased IFNγ, supporting the proposed vaccine design. Structural variants contribute to tumor immunogenic potential. Immunogenicity data from this project will be further used to refine the target selection for individualized, mutanome-based vaccines. Citation Format: Malcolm S. Ross, Ma Tianzhou, Lixin Zhang, Nolan Priedigkeit, George Tseng, Adrian V. Lee, Robert P. Edwards, Anda M. Vlad. Neoepitope peptide vaccines and immune checkpoint blockade in a new preclinical ovarian cancer model. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B33.

Abstract 5631: Personal neoantigen-targeting vaccination generates neoepitope-specific T cell responses in tumors of patients with glioblastoma

Abstract Personal vaccines directed at neoantigens arising from tumor mutations can induce neoepitope-specific T cell responses in patients with highly mutated tumors such as melanoma. It remains unknown if this approach can be successfully applied in tumors with low mutation frequency. We conducted a phase 1/1b study to determine the safety and feasibility of patient-specific neoantigen-targeting vaccination in patients with newly diagnosed, methylguanine methyltransferase (MGMT) unmethylated glioblastoma, administered following maximum surgical resection and conventional radiotherapy. Tumor-specific mutations were identified by whole-exome sequencing. Each vaccine, composed of up to 20 synthetic long peptides containing predicted tumor neoepitopes admixed with poly-ICLC, was administered subcutaneously on a prime-boost schedule. Among 8 treated patients, adverse events were limited to mild injection site reactions. Seven patients (88%) received a vaccine with ≥10 neoepitope peptides (median 12, range, 7-20), with median time to vaccine initiation of 18.6 weeks from surgery. All patients died from progressive disease. Median progression-free and overall survival was 7.5 months (95% CI: 6.2, 9.7) and 16.8 months (90% CI: 9.6, 21.3), respectively. 3 patients dropped out of the study due to disease progression. We analyzed vaccine responses on 5 patients that got at least one booster immunization. All 3 patients who required dexamethasone during vaccine priming failed to generate interferon-γ responses in peripheral blood mononuclear cells. In contrast, 2 patients who did not receive dexamethasone during vaccine priming, generated robust de novo immune responses against multiple personal neoantigens. Circulating vaccine-induced polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses were enriched for memory and activated phenotypes, and increased numbers of tumor-infiltrating CD4+ and CD8+ T cells were detected in these 2 patients. T cell receptor analysis identified identical clonotypes isolated from post-vaccination glioblastoma tissue and peripheral blood including a clonotype specific for ARHGAP35, a neoantigen targeted by vaccination. To our knowledge we provide the first evidence that tumor specific T cells can traffic from the periphery into glioblastoma tumors and that neoantigen-targeting vaccines can favorably alter the tumor immune milieu of glioblastoma. In conclusion, individualized, multi-neoepitope vaccination is feasible, safe, and generates neoantigen-specific T cell responses in the periphery and intracranial tumors of patients with glioblastoma. Citation Format: Derin B. Keskin, Itay Tirosh, Annabelle Anandappa, Jing Sun, Nathan D. Mathewson, Sachet A. Shukla, Evisa Gjini, Shuqiang Li, Letitia Li, Anita Giobbie-Hurder, Phuong M. Le, Zhuting Hu, Wandi Zhang, Oriol Olive, Christine McCluskey, Heather Daley, Patrick Y. Wen, Antonio E. Chiocca, Maegan Harden, Niall Lennon, Stacey Gabriel, Gad Getz, Donna Neuberg, Jerome Ritz, Eric S. Lander, Aviv Regev, Kai Wucherpfennig, Mario Suva, Edward F. Fritsch, Rodig Scott, Keith L. Ligon, Kenneth J. Livak, Hacohen Nir, Patrick A. Ott, Catherine J. Wu, David A. Reardon. Personal neoantigen-targeting vaccination generates neoepitope-specific T cell responses in tumors of patients with glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5631.

Constrained De Novo Sequencing of neo-Epitope Peptides using Tandem Mass Spectrometry.

Neoepitope peptides are newly formed antigens presented by major histocompatibility complex class I (MHC-I) on cell surfaces. The cells presenting neoepitope peptides are recognized and subsequently killed by cytotoxic T-cells. Immunopeptidomic approaches aim to characterize the peptide repertoire (including neoepitope) associated with the MHC-I molecules on the surface of tumor cells using proteomic technologies, providing critical information for designing effective immunotherapy strategies. We developed a novel constrained de novo sequencing algorithm to identify neo-epitope peptides from tandem mass spectra acquired in immunopeptidomic analyses. Our method incorporates prior probabilities to putative peptides according to position specific scoring matrices (PSSMs) representing the sequence preferences recognized by MHC-I molecules. We implemented a dynamic programming algorithm to determine the peptide sequences with an optimal posterior matching score for each given MS/MS spectrum. Similar to the de novo peptide sequencing, the dynamic programming algorithm allows an efficient searching in the entire peptide sequence space. On an LC-MS/MS dataset, we demonstrated the performance of our algorithm in detecting the neoepitope peptides bound by the HLA-C*0501 molecules that were superior to database search approaches and existing general purpose de novo peptide sequencing algorithms.

Integrated analysis of whole exome and RNA sequencing for Neo-epitope peptide prediction in buccal cancer

Integrated analysis of whole exome and RNA sequencing for Neo-epitope peptide prediction in buccal cancer

Abstract 1695: Exploiting the pancreatic cancer mutome for immune interception

Abstract This study aims to translate tumor-specific mutations identified by next-generation sequencing techniques into an effective anti-tumor vaccine for a classically non-immunogenic tumor, pancreatic ductal adenocarcinoma (PDA). Pancreatic ductal adenocarcinomas develop an average of 63 mutations during tumorigenesis, collectively referred to as the “mutome.” Of these, approximately 45 are amino acid point mutations. These mutations are not present in normal cells and provide a source of tumor-specific neoepitopes for targeted immunotherapy. Next-generation sequencing technologies allow for rapid identification of mutations present in patient tumors; however, methods for rapid identification of immunogenic neoepitopes which can be effectively targeted for tumor clearance, and optimized vaccination strategies for targeting them, still need to be developed. In this study, we are using a transplantable murine PDA model, Panc02, to develop vaccination strategies for inducing neoepitope-specific T cell antitumor responses, and to define parameters for selecting appropriate targets. Exome sequencing identified 878 nonsynonymous mutations in the Panc02 cell line. Three immunological epitope prediction servers identified 878 potentially immunogenic peptides in the Panc02 model, 29 of which were strong candidates (scores &amp;lt;50) and another 240 candidate epitopes within the limits of prediction (scores &amp;lt;1000). Panc02 neoepitope peptide vaccinations in mice confirmed 14% of the predicted epitopes to be immunogenic by IFNγ ELISPOT, which may include both CD8+ and CD4+ T cell responses. Tumor transplant experiments demonstrate that therapeutic vaccination against neoepitopes in combination with a human STING-activating cyclic dinucleotide adjuvant is capable of inducing a temporary regression of implanted tumors. After tumor escape and regrowth, flow cytometry reveals a large population of infiltrating lymphocytes, the majority of which are co-expressing the checkpoint molecules PD-1, Tim3, and Lag3, indicating that treatment of vaccinated animals with checkpoint blockade therapy may potentiate infiltrating lymphocytes and ultimately result in permanent tumor regression. Thus, a broad and unbiased approach for screening vaccine targets will allow for the evaluation of predictive algorithms and ultimately the development of more effective vaccination approaches for targeting the tumor mutome. Citation Format: Heather Kinkead, Eric Lutz, Thomas W. Dubensky, Elizabeth Jaffee. Exploiting the pancreatic cancer mutome for immune interception [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1695. doi:10.1158/1538-7445.AM2017-1695