Glioblastoma is a fatal disease with a median prognosis of 12-18 months. Recent studies have shown encouraging results using neoantigen-based vaccines to stimulate glioblastoma-directed immune responses, but overall immunogenicity has been low. Here, we report the results of an open-label, single-arm, phase 1 clinical trial (GT-20) to evaluate the safety and feasibility (primary endpoints) as well as immunogenicity and preliminary clinical activity (secondary endpoints) of GNOS-PV01 monotherapy, a DNA-based personalized therapeutic cancer vaccine administered following surgical resection and radiation for patients with MGMT unmethylated glioblastoma. The GT-20 study vaccinated nine patients, using up to 40 neoantigens per patient (range, 17-40) without causing any serious adverse events, unexpected toxicities or dose-limiting toxicities. The vaccine induced activation and expansion of circulating peripheral T cells in all evaluated patients, except one who was being treated with dexamethasone. The secondary endpoint was to evaluate 6 month progression-free survival and 12 month overall survival; each observed in 66.7% of patients. Median progression-free survival was 8.5 months, median overall survival was 16.3 months and survival at 24 months was 33%, including one long-term survivor still alive 4 years from the time of initial surgery. This study met the pre-specified endpoints and supports the use of GNOS-PV01 as a potentially impactful component of glioblastoma immunotherapy. ClinicalTrials.gov: NCT04015700 .

T cell lymphomas constitute approximately 10

Neoantigens—mutated peptides arising from somatic changes specific to tumor cells—represent a unique class of immunogenic targets. These non-self-antigens can stimulate potent anti-tumor responses due to their high affinity for T cells and absence of central tolerance, unlike tumor-associated antigens. In this review, we discuss potential of neoantigens as vaccine targets, the advantages of a vaccine approach targeting personalized neoantigens, and the challenges of neoantigen identification and selection. Updates on current neoantigen-based vaccine platforms and clinical trial outcomes are summarized. The emerging synergy between personalized neoantigen vaccines and immune checkpoint inhibitors is highlighted. Future directions and challenges in neoantigen vaccine development are also discussed.

Abstract Glioblastoma (GBM) is the most common malignant central nervous system (CNS) tumor in adults. Despite multimodality treatment including surgery, radiation, and chemotherapy, patients with GBM have a median survival of less than 2 years. Recent clinical trials have reported promising results using cancer vaccines to stimulate a tumor-directed immune response in several histologies. The majority of trials targeted tumor neoantigens derived from a single tumor sample, which limits the antigen pool in spatially heterogeneous cancers like GBM. Our group previously implemented multisector sequencing into the design of personalized peptide vaccines to increase the candidate pool of targetable neoantigens (NCT03422094). Although the peptide vaccine treatment was successful in stimulating an effector T cell response, it was limited by a long turnaround time from vaccine design to administration and a low peptide production rate. Therefore, we incorporated multisector sampling into the design of a personalized DNA-based GBM vaccine (NCT04015700). An average of 33% of the candidate targetable neoantigens were spatially restricted to a single sampled region, which would have been missed without multisector sequencing. DNA-based vaccines are potentially advantageous over peptide-based vaccines because they enable a higher neoantigen payload and faster manufacturing time with potent immunogenicity. Here, we report the results from 9 subjects enrolled onto the study. Spatially distinct tumor regions were subjected to whole exome sequencing (WES) and RNA-sequencing (RNA-seq), data from which were used to identify neoantigens through the pVACseq algorithm (http://pvactools.org). An average of 32 neoantigens were included in each DNA vaccine, compared to an average of only 9 for the peptide vaccine, and the turnaround time from date of surgery to administration of the DNA vaccine was about half the time. Three subjects had radiographic evidence of tumor progression prior to vaccination, highlighting the aggressive nature of GBM and the need for short manufacturing timeframes. PBMCs pre- and post-DNA vaccination were collected from 7/9 subjects. All subjects presented detectable responses and 6/7 tested revealed a sustained increment in T cell reactivity against tumor neoantigens post-treatment by IFN-γ ELISpot. Intracellular staining showed a neoantigen-specific antitumor CD8/CD4 T cell cytolytic (CD69+, CD107a+) and proliferative (Ki67+) profile. Post-vaccination tumor resections were performed for 2 subjects who also were treated with PD1 blockade upon progression, providing an opportunity to explore vaccine-induced changes in the tumor microenvironment. Herein, we demonstrate the advantages of incorporating multisector sequencing into the development of DNA-based GBM vaccines and provide insights into the resulting immune responses. Citation Format: Elizabeth A. Garfinkle, Katherine E. Miller, Alexandra J. Livingstone, Renzo Perales-Linares, Neil Cooch, Alfredo Perales-Puchalt, Sarah Rochestie, Joann Peters, Niranjan Y. Sardesai, William E. Gillanders, Elaine R. Mardis, Gavin P. Dunn, Tanner M. Johanns. Incorporation of multisector analysis into the design of personalized DNA vaccines for patients with newly diagnosed glioblastoma [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 1174.

Abstract Circulating tumor DNA (ctDNA) has enabled the non-invasive monitoring of molecular residual disease (MRD) which reflects therapeutic response/resistance prior to conventional imaging approaches. However, little is known about the applications of ctDNA in the neoantigen-targeted personalized cancer vaccine setting, potentially due to the limited sensitivity of current ctDNA assays. NeXT Personal®, an ultra-sensitive tumor-informed ctDNA assay was used to longitudinally track ctDNA in advanced hepatocellular carcinoma (HCC) patients treated with GNOS-PV02 (a personalized therapeutic DNA cancer vaccine) in combination with pembrolizumab. Advanced unresectable or metastatic HCC patients that progressed on, or were intolerant to, first-line TKI therapy were enrolled into the Phase 1b/2a GT-30 study [NCT04251117]. Whole exome/transcriptome tumor sequencing was used for the design of GNOS-PV02. Patients were treated with GNOS-PV02 (1mg; Q3W x 4, Q9W) and plasmid-encoded IL-12 (0.3mg; Q3W x 4, Q9W) in combination with pembrolizumab (200mg; Q3W). Clinical Response was evaluated by RECIST 1.1 at baseline and Q9W. The ctDNA analysis and tracking for progression was performed as an exploratory objective. Over 200 prospective baseline and on-treatment plasma samples from 31 patients were collected and analyzed using NeXT Personal. WGS was performed to identify up to ~1,800 selected tumor variants to create a personalized panel for MRD detection. NeXT Personal has been analytically validated to detection thresholds down to 1-3 parts per million (PPM) of ctDNA with >99.95% specificity. ctDNA was detected in 152 of 207 plasma samples with a dynamic range of 1.76 - 166,968 PPM. The limit of ctDNA detection ranged down to 1.05 PPM. 96% (24/25) of patients were baseline ctDNA+. The percentage change of ctDNA relative to baseline at week 9 (C4D1) significantly correlated with best overall response (CR/PR/SD vs. PD: p = 0.0076). The ctDNA change from baseline to week 9 was predictive of OS (P=0.008), with 31.2% 2-yr OS (95% CI: 13.2 - 73.7%) for molecular non-responders vs 100% 2-yr OS (95%CI: 100 - 100%) for molecular responders. In addition, the ctDNA levels at week 9 were predictive of best overall response (P = 0.017). ctDNA burden at week 9 was significantly correlated with overall survival (HR = 5.46, 95%CI: 1.79 - 16.65; P=0.003; C-index = 0.886). ctDNA clearance was observed in all 3 CR patients with a lead time of 483, 126 and 105 days compared with MRI. This ultra-sensitive ctDNA assay shows significant association between ctDNA change and clinical response and survival, and can be used to accurately predict clinical outcome. The convenience of non-invasive liquid biopsy and rapid availability of data could enable the use of ctDNA to allow real-time monitoring of personalized cancer immunotherapy. Citation Format: Jian Yan, Bailiang Li, Josette Northcott, Charles W. Abbott, Rachel M. Pyke, Renzo Perales-Linares, Neil Cooch, Sarah Rochestie, Joann Peters, Edward J. Gane, Mark Yarchoan, Thomas U. Marron, Sean M. Boyle, Ildiko Csiki, Richard Chen, Niranjan Y. Sardesai. Detection of circulating tumor DNA predicts survival in advanced HCC patients treated with personalized therapeutic DNA cancer vaccine in combination with immune checkpoint blockade [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 976.

Abstract Background: PD-1 inhibitors have modest efficacy as monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) tailored against neoantigens identified in an individual’s tumor may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. Here, we present results from a single-arm Phase Ib/2a trial evaluating a DNA plasmid (GNOS-PV02) encoding up to 40 neoantigens co-administered with plasmid-encoded IL-12 (pIL12) in combination with pembrolizumab (PEMBRO) in patients (pts) with advanced HCC.Methods: Pts were eligible for study therapy upon progression or intolerance with a 1L tyrosine kinase inhibitor (TKI). The PTCV [GNOS-PV02 (1mg) and pIL12 (0.34mg)] was administered intradermally via in vivo electroporation Q3w x 4 doses, and Q9w thereafter. PEMBRO was administered at 200mg IV Q3w. The primary endpoints were safety and immunogenicity. To evaluate the secondary endpoint of ORR per RECIST 1.1 by investigator review with a null hypothesis of an ORR of 16.9% (KN-240, Finn et al ASCO 2019), 36 pts were included to provide 80% power to reject the null hypothesis at the one-sided 0.10 level, assuming the true ORR rate of 33.1%. The data cut date was August 18, 2023.Results: Among the 36 enrolled pts who received at least one dose of treatment, there were no DLTs or treatment-related grade ≥3 events. The most common treatment-related adverse events were injection site reactions, observed in 41.6% of pts. ORR (mITT) per RECIST 1.1 was 30.6% (11/36; 9 confirmed and 2 unconfirmed) with 8.3% (3/36) of pts achieving a CR. This achieved statistical significance with a one-sided p-value = 0.031 (1-sided 90% CI 20.4%-100%) The mOS was 19.9 months. ctDNA changes correlated with radiographic responses and preceded them. A complete molecular response (100% ctDNA clearance) was detected in 7 pts including the 3 radiographic CRs, and 4 additional pts who continue to show durable tumor control (3PR, 1 SD). Immunological analyses confirmed the induction of neoantigen-specific T cell responses by IFNγ-ELISpot in 19/22 (86.4%) evaluable pts, and pts with a larger ELISpot response showed a trend towards longer OS. Multi-parametric cellular profiling and single-cell analysis revealed active, proliferative, and cytolytic vaccine-specific CD4+ and CD8+ effector T cells in the blood of immunized pts. In 14/14 (100%) of pts with paired pre- and on-treatment blood and tumor biopsies, we identified by TCRβ bulk sequencing expanded T cell clones in the peripheral blood that also trafficked into the tumor. Conclusions: Our results show that a PTCV plus PEMBRO is well tolerated and has clinical activity in pts with advanced HCC, and support the PTCV mechanism of action based on the induction of anti-tumor T cells in peripheral blood and tumor. A confirmatory phase 3 clinical study assessing OS is planned. Citation Format: Mark Yarchoan, Edward J. Gane, Thomas U. Marron, Renzo Perales-Linares, Jian Yan, Neil Cooch, Daniel Shu, Elana J. Fertig, Luciane T. Kagohara, Gabor Bartha, Josette Northcott, John Lyle, Sarah Rochestie, Joann Peters, Jason Connor, Elizabeth Jaffee, Alfredo Perales-Puchalt, David B. Weiner, Ildiko Csiki, Niranjan Y. Sardesai. Personalized neoantigen DNA vaccine GNOS-PV02 and pembrolizumab as second-line treatment for advanced hepatocellular carcinoma [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 1191.

Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4 + and CD8 + effector T cells. T cell receptor β-chain (TCRβ) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV’s mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117 .

2629 Background: Tumor-specific neoantigens can be identified from cancer biopsies and used to develop personalized therapeutic cancer vaccines (PTCV) to prime neoantigen-specific T cell responses. Here, we characterized the antitumor neoantigen-specific reactivity of tumor-infiltrating, high-frequency TCR clones in a patient treated with personalized therapeutic DNA cancer vaccine GNOS-PV02 in the ongoing GT-30 advanced hepatocellular carcinoma single-arm open-label multi-center phase Ib/IIa trial (NCT04251117). Methods: Paired blood and tumor biopsy samples from patient #8 enrolled in the GT-30 study were collected before and after treatment with GNOS-PV02 (1mg) + plasmid-encoded IL-12 (0.3mg) + Pembrolizumab (200mg). GNOS-PV02 and IL-12 were administered at Q3W for the first 4 doses, then, at Q9W. Pembrolizumab was administered Q3W. Neoantigen positivity was evaluated by IFNγ-ELISpot. TCRβ sequencing was performed on all 4 samples and single-cell TCR and transcriptome sequencing was performed from T cells isolated from the post-treatment blood sample. After vaccination, three newly identified TCRs in blood and tumor were inserted into a pMXs-IRES-GFP retroviral plasmid vector and used to generate engineered TCR T cells. Engineered T cells were tested against the neoantigens included in PTCV by flow cytometry. Results: The treatment resulted in a partial response with a decrease in tumor size of 44% by RECIST1.1. Five vaccine-encoded responding neoantigens were identified. Differential abundance frame network analysis revealed that 27 of 42 (64.28%) significantly expanded peripheral TCR clones were also found enriched in the tumors post-treatment. Importantly, we observed an increase in cumulative frequency (from 0.4 to 7.7%), and absolute numbers (from 3 to 14) of significantly expanded vaccine-specific TCR clones in the tumor. Increased TCR clonality confirmed a focused tumor repertoire response. Single-cell sequencing data analysis revealed that the 6 most expanded clones in blood were activated CD8+CD69+ T cells (81.82%). Three full TCR sequences from T cell clones newly present in the tumor post-vaccination were selected, synthesized and cloned. TCR-engineered patient-specific T cells showed a dose-dependent CD8+ and CD4+ T cell activation (CD69+) upon stimulation with a pool of epitopes covering all the neoantigens in the patient’s PTCV. Conclusions: PTCV treatment resulted in neoantigen-specific T cell responses, clonal expansion in the periphery and primary lesion, and tumor infiltration of T cells with an activated phenotype. TCR-engineered high-frequency T cells found in the tumor are reactive to PTCV-encoded neoantigens post-treatment. These results may account for the observed objective decrease in the primary tumor size.

e15062 Background: ctDNA analysis has enabled non-invasive detection and monitoring of potentially actionable mutations, and can identify therapeutic response/resistance prior to confirmation by radiographic imaging. Little is known about ctDNA in the neoantigen-targeted personalized cancer DNA vaccine setting, potentially due to the limited sensitivity of current ctDNA assays. We used an ultra-sensitive, tumor-informed ctDNA platform to longitudinally track tumor neoantigen targets and monitor molecular residual disease (MRD) in advanced HCC patients (pts) being treated with a DNA personalized therapeutic cancer vaccine (GNOS-PV02). Methods: Pts with unresectable or metastatic HCC with progression on, or intolerance to, first-line therapy with TKI were enrolled into the Phase 1b/2a GT-30 study [NCT04251117]. GNOS-PV02 was designed based on whole exome/transcriptome tumor sequencing. Pts were treated with GNOS-PV02 (1mg; Q3W x 4, Q9W) and plasmid-encoded IL-12 (0.3mg; Q3W x 4, Q9W) in combination with pembrolizumab (200mg; Q3W). Response was evaluated by RECIST 1.1 at baseline (bl) and Q9W. Over 100 prospective bl and on-treatment (ot) plasma samples were collected and analyzed using NeXT Personal, a tumor-informed ctDNA assay that leverages whole genome sequencing of tumor/normal samples to generate personalized liquid biopsy panels. Each panel included personalized neoantigen targets, up to 1,800 selected variants for ultra-sensitive detection of MRD, and a fixed set of 2,100 known clinically actionable and resistance loci for detection of variants emerging under therapeutic pressure. Results: ctDNA was detected across a broad dynamic range (3-100,000 PPM; minimum limit of detection = 2.5 PPM) with frequent positive detections below 100 PPM. Bl ctDNA was detected in 100% (12/12) of patients. Ot changes in ctDNA relative to bl correlated with disease status. ctDNA tracking of a patient with a target liver lesion reduction of 36% at W9 deepening to -59% at W54 by RECIST1.1 showed ctDNA clearance of all liver-specific neoantigen targets between W12 to w21. New adrenal lesions were observed on W18 by MRI and retrospective ctDNA analysis revealed an increase of adrenal-specific ctDNA measures by W9. These data indicate ctDNA can be used to track tumor neoantigens and provide important data supporting immune pressure-induced tumor escape. Conclusions: Highly sensitive tracking of MRD and neo-antigenic variants over the course of therapy was achieved using a single assay. We show that ctDNA can sensitively monitor disease status non-invasively, potentially leading to accurate clinical outcome prediction. Additionally, the ease of sample handling, analysis, and rapid availability of data could enable the use of ctDNA monitoring to allow real-time dynamic personalized cancer immunotherapy.

Background Recent advances in circulating tumor DNA (ctDNA) analysis have enabled the non-invasive detection of mutations that lead to resistance mechanisms and therapeutic and disease monitoring in cancer patients. Improvements in whole exome sequencing with high sensitivity and specificity has made it feasible to interrogate a large number of genes simultaneously. We sought to evaluate the clinical utility of ctDNA analysis for longitudinal tracking of cancer neoantigen targets and for monitoring of disease status in advanced cancer patients being treated with aDNA personalized cancer vaccine (PCV). Our results also inform the kinetics of somatic variants included in the PCV (neoantigen vaccine targets) as well as the larger set of all variants identified in the tumor (MRD targets). Methods Patients with unresectable or metastatic HCC with progression on, or intolerance to, first-line therapy with tyrosine kinase inhibitors were enrolled into the Phase Ib/IIa GT-30 study [NCT04251117]. Tumors were biopsied for exome and transcriptome sequencing and a patient-specific neoantigen DNA vaccine (GNOS-PV02) was designed. Patients were treated with GNOS-PV02 (1mg) and DNA plasmid encoded IL-12 (0.34mg) in combination with pembrolizumab (200mg). Treatment response was evaluated by RECIST 1.1. Prospectively collected pre- and post-treatment samples from 17 patients were batched and analyzed by personalized ctDNA assays. Somatic mutation calls were made using Personalis ACE® exome data from tumor tissue biopsies. Capture probe panels were designed for the two sets of targets. ctDNA was extracted from the plasma samples; up to 50ng was used as input for deep sequencing. Advanced noise suppression, mutation calling, aggregate tumor tracking and MRD calling was performed using NeXT ctDNA technology. Results Pre-treatment ctDNA magnitude varied widely across patients for both neoantigen targets and MRD targets. Although analyzed retrospectively, changes in ctDNA magnitude over time correlated well with disease status in most patients. ctDNA broadly tracked with MRI scans for monitoring objective responses (CR and PR). In patients with tumor recurrence and/or emergence of de novo metastatic lesions, an increase in target copy number or mean tumor molecules/ml was detected prior to confirmation by MRI. Patient level data from the ongoing GT-30 clinical trial will be discussed at the meeting. Conclusions Our analysis indicated that ctDNA can be a useful tool for monitoring disease in a patient specific manner. The ease of sample handling and analysis, and rapid availability of data, could enable the use of ctDNA monitoring to allow real time clinical treatment decision making for personalized cancer immunotherapy. Trial Registration NCT04251117 Ethics Approval For GT-30 trial, the protocols were approved by Johns Hopkins Medicine Review Boards (CR00039002/IRB00227771), Icahn School of Medicine-Program for the Protection of Human Subjects (20-00076 GCO#1), and Northern A Health and Disability Ethics committee (Ethics ref: 20/NTA), respectively. Written informed consent was obtained from each patient prior to the patient participating in the trial.

2638 Background: Tumor neoantigens are epitopes derived from tumor-specific mutations that can be incorporated in personalized vaccines to prime T cell responses. DNA vaccines delivered with electroporation have recently shown strong CD8 and CD4 T cell responses in clinical trials. In preclinical studies, DNA-encoded neoantigen vaccines have shown induction of CD8 T cells against 50% of predicted high affinity epitopes with the ability to impact tumor growth. Methods: Paired blood and tumor biopsy samples were collected from a patient with hepatocellular carcinoma before and after treatment with GNOS-PV02 (DNA neoantigen targeted vaccine) + plasmid IL-12 + pembrolizumab. Treatment resulted in a partial response with a decrease in tumor size of 44% by RECIST (168 mm to 94 mm). TCRbeta sequencing was performed on all 4 samples and single cell TCR and transcriptome sequencing was performed from T cells isolated from the post-treatment blood sample. Newly identified TCRs in blood and tumor after vaccination were inserted into an expression vector and used to generate engineered TCR T cells. Engineered TCR T cells were tested against the neoantigens included in the vaccine and their responses characterized by flow cytometry. Results: We identified 67,893 new clones in PBMC after vaccination, 3 of which comprised between 0.1 to 1% of the total T cell clones. Moreover, we identified 5126 new clones in the tumor post vaccination, out of these, 3878 (75.68%) were not found within the patient’s pre vaccination PBMCs and 556 (10.86%) were identified within the pre vaccination PBMC pool. Importantly, of the newly identified T cells infiltrating the tumor post vaccination, we observed high frequency TCR clones of which 44 and 7 clones were above 0.1% and 1%, respectively. The majority of the newly identified T cell clones were CD8 T cells (68.75%) with an activated phenotype. Importantly, the 6 most expanded clones in blood were identified to be activated CD8+CD69+ T cells (81.82%). Engineered TCR T cells generated encoding the TCRs of these newly identified CD8 T cells showed activation when exposed to the tumor neoantigens encoded in the neoantigen DNA vaccine GNOS-PV02. Conclusions: GNOS-PV02, a neoantigen DNA vaccine, in combination with plasmid IL-12 and pembrolizumab resulted in expansion of newly identified T cells, primarily activated CD8, which trafficked to the tumor. These new tumor infiltrating T cells showed TCR specificity against tumor neoantigens encoded in GNOS-PV02 and may account for the observed objective decrease in tumor size.

Background Hepatocellular carcinoma (HCC) is a low TMB tumor with largely immune-excluded phenotype. Anti-PD1 monotherapy for 2L HCC has response rates of 12-18%. Therapeutic cancer vaccines targeting neoantigens can generate tumor-specific T-cell immunity, potentially enhancing responses to anti-PD1 therapy. GNOS-PV02 is a personalized cancer DNA vaccine encoding up to 40 patient-specific neoantigens. GT-30 trial is an ongoing single-arm open-label multi-center phase Ib/IIa study to evaluate safety, immunogenicity, and efficacy of GNOS-PV02 administered in combination with plasmid-encoded IL-12 (pIL12) and pembrolizumab. Methods Patients with unresectable or metastatic HCC and progression or intolerance on first-line therapy with tyrosine kinase inhibitors (sorafenib or lenvatinib) are enrolled. Tumors are biopsied for exome and transcriptome sequencing, peripheral blood is collected for germline sequencing, and a patient-specific vaccine is designed, optimized and manufactured, all in 6-8 weeks. GNOS-PV02 (1mg) and pIL12 (0.34mg) are administered via intradermal injection and electroporation Q3w x 4 doses, Q9w thereafter. Pembrolizumab 200mg is administered IV Q3w. Treatment response is evaluated Q9w by RECIST 1.1. Blood samples are collected pre-treatment, Q3w until w12, then Q12w for immunological analyses. Tumor biopsy is obtained at w9 for TME assessment. Results As of cutoff date of June 30, 2022, 24 patients were enrolled with median age 66.5 years (range 40-78 years). There were no DLTs, GNOS-PV02+pIL12 related SAEs, or Grade 3 or 4 AEs reported. Two cases of hypothyroidism and immune nephritis, likely immune-mediated were noted, however no increase in irAEs or SAEs was seen with the combination therapy relative to previously known pembrolizumab monotherapy data. ORR (mITT) per RECIST 1.1 was 29.2% (7/24). Disease control rate was 54.2% (13/24) consisting of 2 CR, 5 PR, 6 SD, 10 PD. One patient early-terminated due to a non-treatment-related SAE six days after their sole PCV dose and was deemed unevaluable but included in the mITT analysis. One patient with a radiological PR after five PCV doses achieved secondary resectability, and discontinued therapy to pursue resection without disease recurrence. Novel and expanded T cell clones, predominantly CD8+ with activated phenotype, were identified in all evaluated patients via pre-/post-vaccination analysis of TCR repertoire in peripheral blood and tumor tissue. These clones trafficked to the TME by w9, potentially mediating the observed tumor regressions. Conclusions GNOS-PV02 + INO-9012 combined with pembrolizumab in the 2L setting was well tolerated and induced tumor-neoantigen-directed CD8+ T cells and TILs. Data to date suggest clinical benefit relative to PD1 monotherapy in patients with advanced HCC. Trial Registration NCT04251117 Ethics Approval For GT-30 trial, the protocols were approved by Johns Hopkins Medicine Review Boards (CR00039002/IRB00227771), Icahn School of Medicine-Program for the Protection of Human Subjects (20-00076 GCO#1), and Northern A Health and Disability Ethics committee (Ethics ref: 20/NTA), respectively. Written informed consent was obtained from each patient prior to the patient participating in the trial.

Background Immune checkpoint inhibition (ICI) has revolutionized cancer therapy and significantly improved survival of patients across several cancer types. However, ICI is only effective in some patients and most patients don't respond to ICI.1 2 Neoantigens are tumor specific antigens derived from either point mutations or gene/RNA fusions in cancer cells, and can be recognized by the host immune system as foreign antigens. Several studies have shown that the success of ICI is linked to the number of neoantigens in the patient's tumor[3][4]. Here, we demonstrate that DNA immunogens designed to target 40 neoantigens derived from MC38 mouse model of colon cancer synergizes with anti-PD1 antibody and improves the efficacy of anti-PD1 therapy. Methods We performed whole exome sequencing on MC38 tumors to identify neoantigens. Through the sequencing data, we identified 40 neoantigens based on predicted affinity to class I MHC binding. All 40 neoantigens were encoded into a single plasmid vector, we designed each neoantigen separated by a furin cleavage site. Immune responses were measured in C57/Bl6 mice via IFN-γ ELISPOT assay and flow cytometry. Finally, we tested immunization with MC38vax to impact tumors in vivo and whether co-treatment with anti-PD1 antibody treatment further impacted tumor control. Results In ELISPOT data, we observed that 11/40 neoantigens generated immune responses in mice. We also studied immune response to WT peptides and observed that the immune response was specifically induced against mutated peptides. Using flow cytometry, we observed that the vaccine induced predominantly CD8+ T cell responses, although CD4+ T cell responses were also observed. In a therapeutic tumor challenge, both anti-PD1 antibody and MC38vax as single treatment partially controlled the growth of MC38 tumors. However, co-treatment with both therapies was synergistic, demonstrating a 100% tumor control rate and improved animal survival. Conclusions Large collections of neoantigens in a DNA immunization platform drive CD8+ T cell immunity against a diverse set of tumor antigens resulting in significant impact on tumor growth and improving survival. In combination with anti-PD1 these vaccines allow for tumor clearance and 100% survival from challenge, significantly improving the outcome of anti-PD1 therapy alone. These studies establish the importance and feasibility of improving patient specific T cell immunity, providing new tools for improving immunotherapy of, in this case colon adenocarcinoma, that is worth considering in other cold tumors that respond poorly to ICI. Acknowledgements This work is supported in part by a grant from Geneos Therapeutics References Robert C, Ribas A, Wolchok JD, Hodi FS, Hamid O, et al. Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial. Lancet 2014;384(9948):1109–17 Robert C, Schachter J, Long GV, Arance A, Grob JJ, et al. Pembrolizumab versus ipilimumab in advanced melanoma. N Engl J Med. 2015;372(26):2521–32 Goodman AM, Kato S, Bazhenova L, Patel SP, Frampton GM, et al. Tumor mutational bur-den as an independent predictor of response to immunotherapy in diverse cancers. Mol. Cancer Ther 2017;16(11):2598–608 Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, et al. Pan-tumor genomic biomarkers forPD-1 checkpoint blockade-based immunotherapy. Science. 2018;362(6411):eaar3593

Background Tumor immune editing and escape are key mechanisms of cancer progression and metastatic dissemination. However, immune editing in response to therapeutic cancer vaccines has been challenging to demonstrate in patients. Neoantigens derived from tumor-specific mutations are promising targets for immunotherapy. They can be incorporated in personalized cancer vaccines (PCV) to prime T cell activation. Here, we report evidence of immune editing in a patient treated with a therapeutic neoantigen DNA PCV from the ongoing GT-30 advanced hepatocellular carcinoma single-arm open-label multi-center phase Ib/IIa trial. Methods A 74 yo white male, having progressed on multiple prior lines of therapy including ablation, TACE and lenvatinib, was enrolled in the GT-30 study. Following WES and transcriptome analysis of the primary liver tumor biopsy, a DNA PCV encoding 29 neoantigens (GNOS-PV02) was manufactured. GNOS-PV02 (1mg) and pIL12 (0.3mg) were administered intradermally Q3w x 4 doses; Q9w thereafter. Pembrolizumab 200mg IV was administered Q3w. Treatment response was evaluated Q9w by RECIST 1.1. Pre-treatment and on-treatment biopsy samples and periodic blood samples were evaluated retrospectively for neoantigen repertoire, immune responses and ctDNA. Results GNOS-PV02+pIL12+pembrolizumab treatment resulted in a partial response, with target lesion reduction of -36% at w9 deepening to -59% at w54 by RECIST1.1. TCR/TIL analysis of w9 biopsy versus screening biopsy samples revealed the expansion and infiltration of multiple new T-cell clones post-vaccination. PBMC analysis by IFNg ELISpot detected strong T-cell response to 4/29 vaccine epitopes. Flow cytometry analysis showed antigen-specific, activated (CD69+, Ki67+) CD8 and CD4 T-cells at high frequency. A new, distal adrenal lesion was detected at w18 that increased in size by w54. Sequencing of the adrenal lesion at w54 identified 25 neoantigens, including 16 shared with the primary liver lesion. However all 4 of the vaccine epitopes with strongest ELISpot responses were absent in the adrenal lesion, consistent with neoantigen loss resulting from immune editing and subsequent clonal escape. ctDNA analysis was consistent with complete response of the liver-specific tumor clone by w21 persisting through w57 but showed an increasing frequency of the adrenal specific tumor ctDNA over time. Conclusions We documented evidence of PCV immune pressure induced clonal escape via the emergence and growth of a new distal lesion despite the primary lesion showing continued and deepened response. Such ongoing analysis of immune response and ctDNA for monitoring tumors offers a means to dynamic cancer therapy, whereby therapeutic vaccines with evolved neoantigen panels may be designed against new, or newly unresponsive, lesions. Trial Registration NCT04251117 Ethics Approval For GT-30 trial, the protocols were approved by Johns Hopkins Medicine Review Boards (CR00039002/IRB00227771), Icahn School of Medicine-Program for the Protection of Human Subjects (20-00076 GCO#1), and Northern A Health and Disability Ethics committee (Ethics ref: 20/NTA), respectively. Written informed consent was obtained from each patient prior to the patient participating in the trial.

BackgroundHepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death. Immune checkpoint inhibitors targeting PD-1 have limited activity in HCC as monotherapy, with response rates ranging from 14–17%. Tumor neoantigens derived from tumor-specific mutations can be incorporated into personalized therapeutic cancer vaccines to generate tumor-specific T cell immunity, potentially priming the immune system for anti-PD1 therapy. DNA vaccines have been shown to elicit strong CD8 and CD4 T cell responses in preclinical and clinical trials. GNOS-PV02 is a personalized DNA vaccine, encoding up to 40 patient-specific neoantigens. In the GT-30 trial, it is used in combination with INO-9012 (plasmid-encoded IL-12) and pembrolizumab for the treatment of advanced HCC.MethodsGT-30 is a single-arm phase I/II clinical trial to assess the safety, immunogenicity, and preliminary efficacy of GNOS-PV02 in combination with INO-9012 and pembrolizumab in patients with advanced HCC. Twenty-four patients are anticipated to be enrolled. Patients are recruited upon diagnosis or during first-line treatment with tyrosine kinase inhibitors (TKI). Tumors are biopsied for exome and transcriptome sequencing, and peripheral blood collected for germline sequencing and histogenetics. The tumor specific vaccine is designed, optimized and manufactured during first-line therapy. Each vaccine encodes up to 40 neoantigens. After progression or intolerance with first-line therapy, patients commence concurrent personalized vaccine and pembrolizumab. GNOS-PV02 + INO-9012 are administered Q3w for the first 4 doses and Q9w thereafter. Pembrolizumab is delivered Q3w.ResultsWe performed a data cut-off on the first 12 patients. The median age was 66 years (range 55–75 years). GNOS-PV02 + INO-9012 with pembrolizumab has had no reported DLTs or drug related SAEs. The most common treatment-related AE were grade 1 fatigue (25%) and grade 1 injection site reactions (17%). By including up to 40 epitopes in the vaccine we were able to target all neoantigens present in 83% of the patients. The objective response rate was 25% (3/12 partial response, 5/12 stable disease, 4/12 progressive disease). Analysis of the TCR repertoire in peripheral blood and tumor tissue identified novel and significantly expanded T cell clones post-vaccination in all patients analyzed. Many of the novel peripheral T cell clones were also identified to have trafficked to the TME at week 9, potentially mediating the observed tumor regressions.ConclusionsThese data demonstrate the potential of GNOS-PV02 + INO-9012 with pembrolizumab to target multiple neoepitopes, and provide initial support for the safety and efficacy of this regimen in patients with advanced HCC.Trial RegistrationNCT04251117Ethics ApprovalThe study obtained IRB approval (IRB) and all patients signed informed consent prior to taking part in the clinical trial. NZCR EC: 20/NTA/6; JHU: IRB00227771; Mount Sinai: HS#: 20–00076

BackgroundTumor neoantigens are epitopes derived from tumor-specific mutations. Such mutations can be incorporated in personalized vaccines to prime T cell responses against tumor specific antigens. DNA vaccines delivered with electroporation have recently shown strong CD8 and CD4 T cell responses in clinical trials. In preclinical studies, DNA-encoded neoantigen vaccines have shown induction of CD8 T cells against 50% of predicted high affinity epitopes with the ability to impact tumor growth.MethodsTwo resection samples from a patient with IDH+ MGMT-methylated anaplastic astrocytoma were subject to whole exome and transcriptome sequencing. Epitopes derived from 27 neoantigens and 3 shared tumor-associated antigens were prioritized and included in a personalized vaccine. The patient was treated with surgery, radiotherapy and temozolomide starting June 2018 and received the first dose of the personalized vaccine in June 2019 under a compassionate use single patient IND application with the FDA.ResultsAs of July 23rd, 2021, the patient has received 11 doses of the DNA personalized vaccine. No serious adverse events have been reported. Related adverse events are limited to grade 1 injection site reactions. The patient remains progression-free 37 months after surgery and 25 months after starting vaccination. Three weeks following the 3rd dose, a hyperintense image on the tumor bed was identified, which disappeared on the following MRI, 2 weeks following dose 5, being catalogued as pseudo progression. Ex vivo ELISpot have identified T cell responses to 28/30 epitopes (93.3%), including 25/27 (92.6%) neoantigens and 3/3 (100%) shared antigens. Flow cytometry analysis has determined that T cell responses are 92.3% CD8 and 69.2% CD4 (30.8% CD8 only; 61.5% both CD8 and CD4; and 7.7% CD4 only).ConclusionsThis compassionate use treatment in an adjuvant setting demonstrates manufacturing feasibility, safety, tolerability, immunogenicity, and suggests potential for persistent clinical response of DNA encoded personalized vaccines. The data supports further investigation of DNA-encoded personalized vaccines into newly diagnosed high-grade gliomas.Ethics ApprovalThe study was approved by Washington University's IRB. The participant gave informed consent before taking part in the study.

Neoantigens are tumor-specific antigens that arise due to somatic mutations in the DNA of tumor cells. They represent ideal targets for cancer immunotherapy since there is minimal risk for on-target, off-tumor toxicities. Additionally, these are foreign antigens that should be immunogenic due to lack of central immune tolerance. Tumor neoantigens are predominantly passenger mutations, which do not contribute to tumorigenesis. In cases of multi-focal or metastatic tumors, different foci can have significantly different mutation profiles. This suggests that it is important to target as many neoantigens as possible to better control tumors and target multi-focal tumors within the same patient. Herein, we report a study targeting up to 40 neoantigens using a single DNA plasmid. We observed significant plasticity in the epitope strings arranged in the vaccine with regard to immune induction and tumor control. Different vaccines elicited T cell responses against multiple epitopes on the vaccine string and controlled growth of multi-focal, heterogeneous tumors in a therapeutic tumor challenge. Additionally, the multi-epitope antigens induced long-term immunity and rejected a tumor re-challenge several weeks after the final vaccination. These data provide evidence that DNA-encoded long antigen strings can be an important tool for immunotherapeutic vaccination against neoantigens with implications for other in vivo-delivered antigen strings.