Personalized Neoantigen Vaccines Research Articles

Breaking Ground in Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma: Novel Therapies Beyond PD-L1 Immunotherapy.

The treatment for recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) with immune checkpoint inhibitors (anti-PD1) with or without chemotherapy has led to an improvement in survival. Yet, despite this therapeutic advancement, only 15%-19% of patients remain alive at four years, highlighting the poor survival and unmet need for improved therapies for this patient population. Some of the key evolving novel therapeutics beyond anti-PD1 in R/M HNSCC have included therapeutic vaccine therapies, bispecific antibodies/fusion proteins and multitargeted kinase inhibitors, and antibody-drug conjugates (ADCs). Multiple concurrent investigations of novel therapeutics for patients with R/M HNSCC beyond anti-PD(L)1 inhibition are currently underway with some promising early results. Beyond immune checkpoint inhibition, novel immunotherapeutic strategies including therapeutic vaccines ranging from targeting human papillomavirus-specific epitopes to personalized neoantigen vaccines are ongoing with some early efficacy signals and large, randomized trials. Other novel weapons including bispecific antibodies, fusion proteins, and multitargeted kinase inhibitors leverage multiple concurrent targets and modulation of the tumor microenvironment to harness antitumor immunity and inhibition of protumorigenic signaling pathways with emerging promising results. Finally, as with other solid tumors, ADCs remain a promising therapeutic intervention either alone or in combination with immunotherapy for patients with R/M HNSCC. With early enthusiasm across novel therapies in R/M HNSCC, results of larger randomized trials in R/M HNSCC are eagerly awaited.

State-Of-The-Art Advancements on Cancer Vaccines and Biomarkers.

The origins of cancer vaccines date back to the 1800s. Since then, there have been significant efforts to generate vaccines against solid and hematologic malignancies using a variety of platforms. To date, these efforts have generally been met with minimal success. However, in the era of improved methods and technological advancements, supported by compelling preclinical and clinical data, a wave of renewed interest in the field offers the promise of discovering field-changing paradigms in the management of established and resected disease using cancer vaccines. These include novel approaches to personalized neoantigen vaccine development, as well as innovative immune-modulatory vaccines (IMVs) that facilitate activation of antiregulatory T cells to limit immunosuppression caused by regulatory immune cells. This article will introduce some of the limitations that have affected cancer vaccine development over the past several decades, followed by an introduction to the latest advancements in neoantigen vaccine and IMV therapy, and then conclude with a discussion of some of the newest technologies and progress that are occurring across the cancer vaccine space. Cancer vaccines are among the most promising frontiers for breakthrough innovations and strategies poised to make a measurable impact in the ongoing fight against cancer.

NeoAgDT: optimization of personal neoantigen vaccine composition by digital twin simulation of a cancer cell population.

Neoantigen vaccines make use of tumor-specific mutations to enable the patient's immune system to recognize and eliminate cancer. Selecting vaccine elements, however, is a complex task which needs to take into account not only the underlying antigen presentation pathway but also tumor heterogeneity. Here, we present NeoAgDT, a two-step approach consisting of: (i) simulating individual cancer cells to create a digital twin of the patient's tumor cell population and (ii) optimizing the vaccine composition by integer linear programming based on this digital twin. NeoAgDT shows improved selection of experimentally validated neoantigens over ranking-based approaches in a study of seven patients. The NeoAgDT code is published on Github: https://github.com/nec-research/neoagdt.

Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial

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.

Personalized Cancer Vaccines Directed against Tumor Mutations: Building Evidence from Mice to Humans.

Personalized vaccines directed to tumor mutations have recently gained significant momentum. On the basis of the concept of stimulating T-cell responses against neoantigens encoded by a tumor's host of personal mutations, these vaccines utilize genome or exome sequencing, mutation calling, and epitope prediction followed by manufacturing of a customized vaccine for each patient. In their 2012 Cancer Research publication, Castle and colleagues provided evidence that vaccinating with long peptide vaccines encompassing neoantigens can generate robust immune responses and induce antitumor activity in a mouse B16F10 melanoma. This approach, harnessing the exquisite specificity of mutations to the tumor and thus providing an effective target for cancer vaccines, was subsequently shown to be safe and immunogenic in a series of small first in man trials in patients with melanoma. The field has accelerated and expanded substantially over the last 5 years, propelled by increasing evidence for vaccine-mediated clinical efficacy, leading to ongoing registrational trials using personalized RNA neoantigen vaccines in patients with melanoma and several other malignancies. See related article by Castle and colleagues, Cancer Res 2012;72:1081-91.

Abstract 5121: Next-generation immunocompetent and humanized neuroblastoma murine models for the discovery and validation of novel immunotherapies

Abstract The success of CD19-directed chimeric antigen receptor (CAR)-T cell therapy for ALL has yet to be matched in solid pediatric malignancies. Moreover, immune checkpoint blockade has shown limited benefit in pediatric cancer. Despite this unmet need, there is a lack of reliable preclinical models to facilitate novel target discovery, validate therapeutic benefit, anticipate on- or off-target toxicity, and understand mechanisms of resistance. Syngeneic murine model systems are a critical intermediate for preclinical translation, preserving the complex and heterogenous immune landscape and host-immunotherapy interactions. However, model interpretation and validity are limited as expression and function of murine targets and immune effector cells are not always conserved. We have developed a syngeneic immunocompetent C57BL/6 neuroblastoma system to study CAR efficacy, toxicity, and mechanisms of resistance, as well a variety of vaccination strategies. Specifically, we have built two murine CAR T constructs containing a 100% conserved antigen-directed scFv joined to orthologous murine TCR signaling machinery. In parallel, we engineered the murine neuroblastoma cell line 9464D to stably express surface antigen Gpc2 (Bosse, Cancer Cell 2017), or a novel chimeric single chain trimer of a 9mer peptide from intracellular oncoprotein PHOX2B discovered on HLA-A*24:02 (Yarmarkovich, Nature 2023) as Phox2b/A24*02/H2-kb to generate tumor-bearing B6 or B6-A24-Tg mice. We are currently using this system to demonstrate how vaccination approaches enhance the persistence and potency of CART directed to both targets and are extending our syngeneic murine constructs to additional TH-MYCN allograft models. To further study the innate and adaptive immune response to various therapeutics, we have developed peripheral blood mononuclear cell (PBMC) humanized models. Our autologous humanized PDX mouse models of neuroblastoma use immunocompromised NSG mice engrafted with a PDX and humanized with patient-matched PBMCs to evaluate personalized vaccines in vivo. We have shown the ability to stably engraft both lymphoid and myeloid populations in various human cytokine transgenic NSG variants (i.e. SGM3, Flt3L), allowing for T/B cell and APC crosstalk, thus providing a robust means to study targeted interventions. We are currently using this autologously humanized PDX model to test the ability of multivalent, personalized neoantigen vaccines developed specific to the neoantigen landscape of individual PDXs, with a focus on endogenous T cell responses of patient PBMC against their own PDX in vivo. Citation Format: Timothy T. Spear, Elisabeth Posthill, David Groff, Anna M. Giudice, Kristopher R. Bosse, Leyuan Ma, Stephen P. Schoenberger, John M. Maris. Next-generation immunocompetent and humanized neuroblastoma murine models for the discovery and validation of novel immunotherapies [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 5121.

Abstract 4107: Evaluating the efficacy of personalized neoantigen cancer vaccine in preclinical model: A promising step towards precision immunotherapy

Abstract Background: In the past decade, immunotherapy has transformed cancer treatment, with the development of immune-checkpoint inhibitors and adoptive cell therapies. A novel frontier is personalized vaccines targeting neoantigens unique to tumor cells. Vaccines based on neoantigens have several advantages. First, neoantigens are exclusively expressed by tumor cells and can, therefore, elicit truly tumor-specific T cell responses, thereby preventing ‘off-target’ damage to nonmalignant tissues. Second, neoantigens are de novo epitopes derived from somatic mutations, which presents the possibility to circumvent T cell central tolerance of self-epitopes and thus induce immune responses to tumors. One of the major challenges is the optimal neoantigen discovery/selection. Our study combines in silico HLA binding models with the 'MHC-PepSeq' platform to enhance neoantigen prediction, creating a personalized neoantigen peptide vaccine. We aim to demonstrate its efficacy in a mouse CT26 colon cancer model. Methods: Our method involved whole exome sequencing of tumor and normal DNA to identify neoantigens expressed in CT26 tumors, generating a peptide library. The peptides were further filtered using an in-silico prediction algorithm and our proprietary peptide-MHC binding assay (PepSeq assay), to determine those exhibiting the highest binding affinity with mice HLAs. Vaccines were prepared by dissolving these peptides in sterile PBS. Mice (BALB/C) were subcutaneously implanted with CT26 tumors, then immunized with the vaccine via IV injection in combination with a TLR7/8 agonist. Mice received a second vaccine dose on Day 14, and some groups were treated with an anti-PD-1 agent. Tumors and spleens from the mice were collected, processed into single-cell suspensions, and analyzed by single-cell sequencing, TCR analysis, IHC, and flow cytometry assays. Peripheral blood mononuclear cells (PBMCs) were collected and utilized in MHC tetramer assays. Results: Preliminary in vivo studies demonstrated significant tumor growth restrictions (TGI around 70%) and prolonged tumor-free survival in some mice. Our data showed the generation of neoantigen-specific T cells in vaccinated mice. Remarkably, vaccinated, tumor-free mice, when re-challenged with the same tumor cells, maintained their tumor-free status, suggesting the vaccine induced post-treatment immunological memory. Combining the vaccine with checkpoint inhibition resulted in better tumor growth restriction in mice (TGI around 93%). Conclusion: Overall, our data suggests that our personalized neoantigen vaccine platform has the potential to evoke robust and durable immune responses and, when paired with suitable complementary therapies and immune adjuvants, may serve as an effective cancer treatment. Citation Format: Tithi Ghosh Halder, Kate Gutowsky, Serina Ng, Trason Thode, Alexis Weston, Erin Kelley, Jorge Soria-Bustos, Jorge A. Giron, Taylor Bargenquast, Mohan Kaadige, Sanjana Tripuraneni, Brian Durbin, Shelby Rheinschmidt, Sydney Adamson, Michael Gordon, Justin Moser, John Altin, Raffaella Soldi, Sunil Sharma. Evaluating the efficacy of personalized neoantigen cancer vaccine in preclinical model: A promising step towards precision immunotherapy [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 4107.

An Advanced Intrahepatic Cholangiocarcinoma Patient Benefits from Personalized Immunotherapy.

Advanced intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignancy characterized by limited response to standard therapeutic modalities, such as radiotherapy, chemotherapy, and targeted therapy. The prognosis for patients with advanced ICC is exceedingly bleak, with an overall survival of less than 1 year. In recent years, personalized neoantigen vaccines have emerged as a promising approach to augment the immune response against tumors. Clinical investigations are currently underway to evaluate the efficacy of neoantigen-based peptide, DNA, and dendritic cell vaccines. Herein, we present a noteworthy case of advanced ICC patients who experienced disease progression following relapse and subsequently received immunotherapy with a personalized neoantigen nanovaccine. This innovative treatment strategy involved the administration of a custom-designed neoantigen-based peptide nanovaccine tailored to the patient's specific gene mutation profile subsequent to failure of first-line therapy. The clinical efficacy and anti-tumor immune responses were evaluated using various methods, including imaging, interferon-γ ELISPOT assay, and intracellular cytokine staining. Notably, the neoantigen nanovaccine elicited a robust and specific tumor-killing effect mediated by T cells, resulting in a durable response lasting up to 25 months. These findings highlight the potential of neoantigen-based immunotherapy as a novel therapeutic avenue for the management of advanced ICC.

Abstract A083: Personalized targeted circulating tumor DNA (ctDNA) and T-cell responses after combined neoantigen vaccine and immune checkpoint blockade in ovarian cancer

Abstract A083: Personalized targeted circulating tumor DNA (ctDNA) and T-cell responses after combined neoantigen vaccine and immune checkpoint blockade in ovarian cancer

Personalized neoantigen vaccine enhances the therapeutic efficacy of bevacizumab and anti-PD-1 antibody in advanced non-small cell lung cancer

Clinically, a considerable number of non-small cell lung cancer (NSCLC) patients are unable to receive or resist chemotherapy, and the efficacy of non-chemotherapy treatment strategies based on anti-angiogenic agents combined with immune checkpoint blockade is still unsatisfactory. Neoantigen vaccine, based on personalized tumor DNA mutations, could elicit tumor specific T cell infiltration into the tumor site, exerting potent anti-tumor efficacy. Here, we evaluated the feasibility and safety of a new antitumor strategy by adding neoantigen vaccine to the regimen of bevacizumab and anti-PD-1 antibody. Firstly, 7 novel immunogenic neoantigen peptides were identified and developed for neoantigen vaccine (LLCvac), which can elicit strong antitumor immune response in vivo. Then, in orthotopic lung cancer model, LLCvac further combining with bevacizumab and anti-PD-1 antibody exerted a stronger antitumor effect, exhibiting significant decrease of tumor volume without obvious toxicity. Furthermore, tumor immune microenvironment assessment also showed that the proportion of neoantigen-specific T cells in blood could be induced dramatically by the combined therapy. And a large amount of neoantigen-specific Ki67-positive CD8+ T cells were found in tumor tissues, which infiltrated tumor tissues effectively to kill tumor cells expressing identified neoantigens. Overall, these results suggested that this combined therapy could safely induce robust antitumor efficacy, serving as an effective chemotherapy-free strategy for NSCLC treatment.

CD4 T cell-activating neoantigens enhance personalized cancer vaccine efficacy.

Personalized cancer vaccines aim to activate and expand cytotoxic antitumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a vaccine (PancVAX2) targeting both major histocompatibility complex class I- (MHCI-) and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T cells into the tumor with lower PD-1 expression after reactivation compared with the CD8+ vaccine alone. Vaccine-induced neoantigen-specific Th1 CD4+ T cells in the tumor were associated with decreased Tregs. Consistent with this, PancVAX2 was associated with more proimmune myeloid-derived suppressor cells and M1-like macrophages in the tumor, demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4+ T cell-specific neoantigens for personalized cancer vaccine modalities.

BIOM-56. THE PRIVATE AND RECURRING ANTIGENIC PROFILE OF MOST AGGRESIVE PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Efficient immunotherapies for pediatric brain tumors face a significant challenge in identifying tumor rejection antigens. To overcome this hurdle, we utilized a comprehensive computer algorithm called the Open Reading Frame Antigen Network (O.R.A.N.). METHODS Our focus was on expressed epitopes, including single nucleotide variations, small indels, gene fusions, novel junctions from alternative splicing, and Tumor Associated Antigens (TAAs), for antigenic epitope predictions. To ensure uniqueness, we compared immunogenic TAA epitopes against a personalized human proteomic library, ensuring they were not shared by other expressed isoforms or genes. The immunological landscape of medulloblastoma (n = 170) and DIPG (n = 30) was explored using immune deconvolution techniques combined with single-cell RNA sequencing integration. RESULTS Our hypothesis was that medulloblastomas and DIPG exhibit diverse immune profiles and express genes that are rarely found in normal tissues, making them potential targets for immunotherapy. In this study, we found that most neoantigens identified in medulloblastoma patients were unique to each individual, emphasizing the need for personalized neoantigen-based therapies. Interestingly, recurring neoantigens were predominantly present in the MB SHH subgroup, while they constituted only a small fraction of putative antigens in WNT, Group 3, and Group 4 patients. On the other hand, recurring Tumor Associated Antigens (TAAs) were more frequently expressed in the latter three molecular subgroups but not in the SHH subgroup. Thus, a combination of personalized neoantigen vaccines and targeting recurring TAAs could potentially maximize the anti-tumor effects. CONCLUSION Medulloblastoma is a disease characterized by its heterogeneity, and current standard treatments do not adequately address this aspect. In the case of DIPG, surgical resection is not a viable option due to its diffuse and infiltrative nature within the brainstem. By leveraging the power of the immune system, cellular immunotherapy holds promise in providing more targeted and personalized treatment options for pediatric brain tumor patients.

Personalized DNA Vaccine Immunogenic Against Melanoma.

A personalized neoantigen vaccine built around a DNA backbone, EVX-02, elicited robust and lasting T-cell responses in patients with melanoma after surgery. The first-in-human data, presented at the Society for Immunotherapy of Cancer Annual Meeting, have inspired a next-generation DNA vaccine candidate, EVX-03, that includes an additional payload and a more sophisticated antigen-selection process.

Abstract PR015: Tumor-specific immunity generated by a personalized neoantigen vaccination incorporating locally delivered ipilimumab in renal cell carcinoma

Abstract Personalized cancer vaccines can generate circulating immune responses against predicted neoantigen targets, but whether such responses lead to actual immune recognition of a patient’s tumor and consequent clinical activity is largely unknown. To investigate this, we conducted a phase I clinical trial of a personalized neoantigen vaccine as adjuvant therapy in high-risk, completely surgically resected, stage III/IV clear cell renal cell carcinoma (RCC; NCT02950766). This first-in-disease study included several modifications from prior neoantigen vaccine studies, including: (i) the use of an improved antigen prediction tool (HLAthena), a neural network model that was trained on a large HLA class I immunopeptidome dataset; (ii) the subcutaneous and intradermal administration of the vaccine to engage a broader repertoire of antigen-presenting cells in the skin; and (iii) the inclusion of ipilimumab administered subcutaneously next to the vaccination site (for pre-specified cohorts) intended to improve T cell priming and activation at the local draining lymph node. Across all nine patients, a median of 15 vaccinating peptides (range: 8 – 19) were successfully manufactured and administered per study subject, targeting a median of 13 unique mutations (range: 7 – 17). Six of nine patients were vaccinated against a neoantigen formed through a mutation in at least one known RCC driver gene (VHL, PBRM1, BAP1, KDM5C, or PIK3CA). Single-cell transcriptomic and T cell receptor (TCR) sequencing analysis of vaccine site immune populations in the skin revealed a substantial increase in the number of T cells (p = 0.003) and T cell clonotypes (p < 0.001) following vaccination. No patient had a pre-existing ex vivo peripheral T cell response against any of the neoantigen peptides in the vaccine. Following vaccination, all nine patients developed detectable ex vivo peripheral T cell response (by IFN-gamma ELISPOT) against neoantigen peptides in the vaccine. Nearly all detectable ex vivo responses were derived from CD4+ T cells as assessed by flow cytometry analysis, and a substantial proportion of responses (27.4% - 99.8% of total responses for an individual pool of vaccine peptides) were polyfunctional (with T cells producing at least 2 of the following cytokines: IFN-gamma, IL-2, or TNF-alpha). Among peripheral immune responses, there were no observable differences in the quantity or phenotype of T cells responding to vaccine alone versus the vaccine co-administered with ipilimumab. In seven of nine patients, neoantigen vaccine-specific T cells exhibited in vitro anti-tumor reactivity against autologous RCC cells. Clinically, there were no dose limiting toxicities, and no disease recurrences were observed in this high-risk population (median follow-up of 23.9 months at time of data cutoff). Overall, these data suggest that personalized neoantigen vaccines (with or without co-administered subcutaneous CTLA-4 blockade) can induce anti-tumor immune responses in patients with RCC. Citation Format: David A. Braun, Derin B. Keskin, Sachet A. Shukla, Bradley A. McGregor, Nicholas R. Schindler, Eryn Blass, Susan Klaeger, Lucas Pomerance, Siranush Sarkizova, Shuqiang Li, Jackson Southard, Giorgia Moranzoni, Christina B. Pedersen, Yiwen Liu, Steven L. Chang, Michelle S. Hirsch, Nicole R. LeBoeuf, Matthew Mossanen, Vipheaviny Chea, Isabel Carulli, Oriol Olive, Ambica Mehndiratta, Haley Greenslade, Giacomo Oliveira, J. Bryan Iorgulescu, Sabina Signoretti, Jon C. Aster, Liudmila Elagina, Ignaty Leshchiner, Gad Getz, Maegan Harden, Stacey Gabriel, Lars R. Olsen, Donna S. Neuberg, Edward F. Fritsch, Nir Hacohen, Kenneth J. Livak, Steven Carr, Patrick A. Ott, Catherine J. Wu, Toni K. Choueiri. Tumor-specific immunity generated by a personalized neoantigen vaccination incorporating locally delivered ipilimumab in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR015.

Spleen-targeted neoantigen DNA vaccine forpersonalized immunotherapy of hepatocellularcarcinoma.

Neoantigens are emerging as attractive targets to develop personalized cancer vaccines, but their immunization efficacy is severely hampered by their restricted accessibility to lymphoid tissues where immune responses are initiated. Leveraging the capability of red blood cells (RBCs) to capture and present pathogens in peripheral blood to the antigen-presenting cells (APCs) in spleen, we developed a RBC-driven spleen targeting strategy to deliver DNA vaccine encoding hepatocellular carcinoma (HCC) neoantigen. The DNA vaccine-encapsulating polymeric nanoparticles that were intentionally hitchhiked on the preisolated RBCs could preferentially accumulate in the spleen to promote the neoantigen expression by APCs, resulting in the burst of neoantigen-specific T-cell immunity to prevent tumorigenesis in a personalized manner, and slow down tumor growth in the established aggressively growing HCC. Remarkably, when combined with anti-PD-1, the vaccine achieved complete tumor regression and generated a robust systemic immune response with long-term tumor-specific immunological memory, which thoroughly prevented tumor recurrence and spontaneous lung metastasis. This study offers a prospective strategy to develop personalized neoantigen vaccines for augmenting cancer immunotherapy efficiency in immune "cold" HCC.

Final results: Dose escalation study of a personalized peptide-based neoantigen vaccine in patients with metastatic melanoma.

9551 Background: Despite the advances made with CPIs such as aPD-1/aPD-L1, aCTL4 and aLAG-3 there still is a significant unmet medical need for patients with metastatic cancers. As it has been shown that the effect of CPIs are mediated by tumor specific T cells, one strategy for increasing treatment success is to actively induce tumor specific T cells. Here, we report on a first-in-human clinical trial evaluating a personalized neoantigen vaccine (EVX-01) in patients with metastatic melanoma. Methods: Stage-IV metastatic melanoma patients were treated with standard aPD-1 in combination with a personalized tumor-specific neo-antigen vaccine. The vaccine consisted of multiple 15-27mer peptides comprising one or more patient specific neoantigens identified from tumor DNA and mRNA sequencing data by an integrated AI-platform; PIONEER and delivered together with the novel liposomal adjuvant CAF09b to potentiate immune responses. Results: Vaccines were designed and manufactured for each patient in less than 7 weeks. In total, 12 eligible patients received either 500ug (5 patients, reported in PMID: 35036074), 1000ug (3 patients) or 2000ug (4 patients) total peptide load keeping the peptide:CAF09b ratio constant. Four patients had stable disease on aPD-1 treatment for at least 4 months before enrolment in the study, whereas the remaining patients were treatment naïve. All patients received monotherapy with pembrolizumab (11) or nivolumab (1) during the vaccine manufacturing period. The personalized vaccine was shown to be safe and well tolerated with fatigue and injection site reactions being the most frequently reported events and only grade 1-2 events being related to the vaccine. Response was evaluated by RECIST 1.1, according to the investigator’s assessment with 8 patients (67%) having objective response (CR: n = 2, PR: n = 6), of which 1 patients had response initiation before addition of the vaccine. Importantly the vaccine induced neoantigen specific T cells in all patients analyzed. Both CD4+ and CD8+ T cells were detected, with the majority of responses being CD4+ T cell mediated. Interestingly, we report a significant correlation between the PIONEER prediction score and induced immunogenicity. Furthermore, the breadth of the neoantigen recognition seemed to be important for the clinical effect of the vaccine as responders demonstrated T cell recognition of more epitopes within the vaccine compared to non-responders. We found, in our cohort, that the neo-antigen scores effectively separated responders and non-responders, as opposed to the TMB (by FDA guidelines). Conclusions: aPD-1 treatment combined with a peptide-based personalized neo-antigen-based vaccine was shown to be feasible and safe with promising signs of efficacy, warranting further study. Further, the neo-antigen selection method appeared crucial for designing an efficacious vaccine. Clinical trial information: NCT03715985 .

RNA-based Personalized Cancer Vaccine for AML immunotherapy

Abstract Acute Myeloid Leukemia (AML) is a rapidly progressing malignancy relying primarily on chemotherapy as the frontline treatment option. Success of personalized neoantigen vaccines in melanoma patients have garnered great attention in the field of immunotherapy. We hypothesize that personalized cancer vaccine, in the form of mRNA, can be a viable treatment strategy for AML patients. Next generation sequencing was performed on both C57BL6Jinv mouse’s and C1498 cell line’s nucleic acid materials. Three different neoepitope prediction pipelines were utilized to identify potential immunogenic neoepitopes. Candidate neoepitopes within intron polyadenlyation sites (iPAS), retained intron (RI) sites and somatic mutation (SM) sites were found. A total of 285 SM-derived neoepitopes were identified. Top 38 highly expressed genes were selected for further analysis. Using Sanger sequencing, we confirmed 37 out of the 38 mutations. A total of 5 iPAS-derived neoepitopes were identified. A total of 26 RI-derived neoepitopes were identified in our first RNAseq analysis and a separate 476 RI-derived neoepitopes were identified in our second RNAseq analysis. With these neoepitope candidates, we narrowed down our selection and did a preliminary screen for immunogenic epitopes using a total of 71 synthetic peptides in an ELISpot assay. 10 neoepitope candidates were short-listed for further in-vivoevaluation. mRNA vaccine was synthesized and evaluated in a murine AML model. T cell responses against the 10 neoepitopes were analyzed to provide pre-clinical evidence for patient studies. Neoepitope screening platform with human PBMCs has also been established to validate the prediction pipelines in AML patients. Supported by grant from NMRC (MOH-CIRG21jun-0008)

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

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

Abstract CT270: Immunogenicity of PGV_001 neoantigen vaccine in a Phase-I clinical trial, across various types of cancers in adjuvant setting

Abstract Introduction: Immunotherapies such as checkpoint blockade, have demonstrated remarkable clinical efficacy yet a large percentage of patients do not respond, potentially due to a paucity of pre-existing immune priming against neoantigens. We developed a personalized genome vaccine (PGV_001) platform to generate neoantigen vaccines targeting each patient’s unique mutanome. Primary objectives of the study were to determine 1) the safety and tolerability; 2) the feasibility of PGV_001 production and administration; and 3) the immunogenicity of PGV_001. Secondary objectives included immunophenotyping vaccine driven cellular and soluble immune milieu in peripheral blood. We previously reported on the clinical efficacy, and here we report, analysis of vaccine-driven immune responses in all treated patients. Methods: The study (Trial Registration NCT02721043) enrolled patients with resected malignancies, including Head and neck squamous cell carcinomas, breast cancer and bladder cancer, or, in the case of multiple myeloma treated with autologous stem cell transplant; all patients determined to have a high risk of disease recurrence (>30% over 5 years). Tumor-derived and germline RNA and DNA was sequenced to predict neoantigens utilizing our custom computation pipeline, OpenVax. Approximately 10 peptides were synthesized per patient, and a mixture of these peptides was administered as 10 subcutaneous and intradermal vaccines over 27 weeks in combination with poly-ICLC and helper Tetanus peptide as adjuvants. Immune responses were analyzed utilizing assays including IFN-gamma ELISPOT, antigen specific T cell expansion followed by flow cytometry, etc. Results: In total 148 neoantigen peptides were manufactured for 15 patients. Overall, 136 PGV_001 doses were administered to 13 patients. Vaccine-specific T cell immunity was observed against multiple vaccine neoepitopes in all 13 subjects. Of the peptides administered, 45% of vaccine antigens (57/126) induced de novo immunity, starting as early as Week8 and often sustaining past last vaccination. Notably, while the vaccine driven T cell immunity was CD4 T cell dominant, most evaluated subjects also displayed vaccine induced polyfunctional CD8-T cell responses. Additional studies are ongoing to define qualities of reactive T cells, evaluate vaccine-induced humoral responses and probe the circulating inflammatory immune milieu. These will be presented. Conclusions: We have established a platform for generating personalized neoantigen vaccines. 100% of the vaccinated patients developed an immune response specific to the vaccine neoantigens predicted by OpenVax. Subjects who received treatment experienced mild Grade 1 or 2 adverse reactions as per the CTEP v 4.0 NCI CTCAE. This vaccine trial reached the primary endpoint of safety, tolerability, feasibility and immunogenicity. Based on the PGV_001 platform two clinical trials, one in patients with glioblastoma multiforme (NCT03223103) in combination with TT fields and second in patients with urothelial carcinoma (NCT03359239) in combination with Atezolizumab have been performed. Data from these trials is under evaluation. Citation Format: Mansi Saxena, Thomas Marron, Julia Kodysh, Alex Rubinsteyn, John Finnigan, Ana Blasquez, Marcia Meseck, Tim O'Donnell, Daniela Delbeau, Mathew Galsky, Deborah Doroshow, Brett Miles, Krzysztof Misiukiewicz, Hanna Irie, Amy Tiersten, Samir Parekh, Marshall Posner, Andrea Wolf, John Mandeli, Rachel Brody, Sacha Gnjatic, Eric Schadt, Philip Friedlander, Nina Bhardwaj. Immunogenicity of PGV_001 neoantigen vaccine in a Phase-I clinical trial, across various types of cancers in adjuvant setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT270.

Abstract 5076: Combination PancVAX neo-epitope vaccine with anti-CTLA-4 and anti-PD-1 antibodies enhances infiltration of cytotoxic T cells and mitigates T cell exhaustion in a murine model of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden and therefore few neoantigen targets that can be recognized by cytotoxic T cells. Most PDACs are thus insensitive to either single or dual immune checkpoint inhibitor (ICI) therapy. Personalized neoantigen vaccines can expand the number and repertoire of anti-tumor T cells that infiltrate the tumor and mediate cytotoxicity. To model a personalized neoantigen vaccine treatment strategy in PDAC, we previously developed PancVAX, a peptide-based vaccine targeting 12 neoantigens expressed in the murine pancreatic cell line Panc02 (Kinkead et al, JCI Insight 2018). Although we observed increased T cell infiltration present in the tumor post-vaccination, these cells expressed high levels of exhaustion markers. We therefore hypothesized that sequential administration of anti-CTLA-4 and anti-PD-1 would enhance the pool of T cells primed by the neoantigen vaccine and maintain activation of antigen-experienced T cells, respectively, to yield optimal and durable neoantigen-specific anti-tumor immunity in PDAC. To address this, mice bearing subcutaneous Panc02 tumors were vaccinated with two rounds of the PancVAX neoantigen vaccine followed by anti-CTLA-4 and anti-PD-1 3 days later. Anti-PD-1 maintenance was given twice weekly beginning at the first vaccine dose. Twelve days after the last peptide vaccine dose, tumors were harvested and dissociated into single-cell suspensions for paired single-cell RNA-sequencing and TCR-sequencing. Mice that were untreated or given ICIs without PancVAX had the highest proportions of CD8+ T cells expressing exhaustion markers. PancVAX-treated mice had more intratumoral cycling CD8 T cells and effector CD8+ T cells with high cytotoxic gene expression. Among mice treated with PancVAX, tumors from mice treated with PancVAX + anti-PD1 or PancVAX + anti-PD1 + anti-CTLA-4 had the highest proportions of effector CD8+ T cells. Ongoing analyses include differential gene expression and pathway analysis between treatment conditions in the T cell compartment in mice treated with combination ICI and PancVAX. Additionally, we will assess changes in T cell clonality and diversity within the tumors when mice are treated with single or combination therapy. These results will define a transcriptional signature associated with the generation of a productive anti-tumor immune response when neoantigen vaccines and ICI are used in combination. This work demonstrates how the addition of ICIs to personalized neo-epitope vaccines for PDAC can further enhance the quality of vaccine-induced T cell effector function in an otherwise immunologically cold tumor type and supports their inclusion in neoantigen vaccination strategies for patients with PDAC. Citation Format: Jacob T. Mitchell, Amanda Huff, Emily Davis-Marcisak, Fangluo Chen, Todd D. Armstrong, Luciane T. Kagohara, James Leatherman, Rulin Wang, Srinivasan Yegnasubramanian, Elizabeth M. Jaffee, Elana J. Fertig, Neeha Zaidi. Combination PancVAX neo-epitope vaccine with anti-CTLA-4 and anti-PD-1 antibodies enhances infiltration of cytotoxic T cells and mitigates T cell exhaustion in a murine model of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5076.