Neoantigen Vaccines Research Articles

Early Intervention and Favorable Biologic Effects of Personalized Neoantigen Vaccines on the Tumor Immune Microenvironment in Smoldering Waldenstrom Macroglobulinemia

Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade B-cell lymphoma, characterized by the presence of clonal tumor cells infiltrating the bone marrow. Despite a cumulative risk of progression, there is no approved therapy for patients in the asymptomatic phase. We conducted a first-in-human clinical trial of a novel individualized therapeutic DNA idiotype neoantigen vaccine as an early intervention in nine patients with untreated asymptomatic LPL. The vaccine platform encoded the autologous LPL-derived Ig single chain variable fragment fused to human chemokine CCL20 (macrophage inflammatory protein-3, MIP-3a), which was designed to trigger T-cell immunity by targeting delivery of the expressed fusion protein to antigen presenting cells. The vaccine was well tolerated with no dose limiting toxicities. One patient achieved a minor response, and all remaining patients experienced stable disease, with median time to disease progression of 61+ months, which compares favorably to median time to progression of 3.9 years in published series. Direct interrogation of the tumor microenvironment by single-cell transcriptome analysis revealed explanations for lack of more robust objective clinical responses. Comparing paired pre- and post- vaccine bone marrow samples, we observed a striking dichotomous pattern of significantly reduced numbers of clonal tumor cells, identified by their unique BCR sequences, and downregulation of genes involved in signaling pathways critical for B-cell survival among the mature B cells subpopulation post-vaccine in the majority of patients, but no change in clonally related plasma cell-like clusters of any patient (Figure 1). Downregulation of HLA molecule expression suggested intrinsic resistance by tumor plasma cell subpopulations and cell-cell interaction analyses predicted paradoxical upregulation of IGF signaling post vaccine by plasma cell, but not mature B-cell subpopulations, suggesting a potential mechanism of acquired resistance. Vaccine therapy induced dynamic changes in bone marrow T-cells, including upregulation of signaling pathways involved in T-cell activation, expansion of T-cell clonotypes (Figure 2), increased T-cell clonal diversity, and functional tumor idiotype-specific cytokine production, with little change in co-inhibitory pathways or Treg. Vaccine therapy also globally altered cell-cell communication networks across various bone marrow cell types. CellChat analysis inferred significant downregulation of signaling pathways post-vaccine that likely directly promote growth of LPL cells, such as APRIL and IL-6 which are known to promote B- or plasma cell survival. Other pathways were reduced post-vaccine, such as RESISTIN, which has a known role in supporting proliferation of solid cancers. A role for RESISTIN in supporting LPL has not been previously inferred, but it has been reported to induce multidrug resistance in multiple myeloma. Bioinformatic analysis also identified a predominant role for myeloid cells in the tumor microenvironment as a source of vaccine-induced, downregulated pro-tumoral signaling to LPL cells. The pathways affected were primarily associated with monocytic, rather than granulocytic or dendritic cell subpopulations, particularly non-classical CD14 -CD16 + monocytes, and to a lesser extent classical CD14 +CD16 - monocytes . Recent reports indicate that an increased pro-inflammatory myeloid signature is an early step in the development of LPL and in monoclonal gammopathy of undetermined significance. Taken together, these results suggest that this prototype neoantigen vaccine favorably perturbed the tumor immune microenvironment, resulting in reduction of clonal tumor mature B-cell, but not plasma cell subpopulations. Future functional studies of the pathways affected are needed to confirm mechanisms of resistance elucidated and to design combination strategies to circumvent them. Such strategies could include adding IFN gamma or epigenetic drugs, designed to increase HLA molecule expression on plasma cell-like LPL subpopulations and combining neoantigen vaccines with agents that specifically target plasma cells or pathways known to promote their growth, such as IGF-1 receptor inhibitors. Finally, our data suggest that combinations of these vaccines with myeloid cell checkpoint blockade may be worthwhile.

EXTH-70. UTILIZING ONCOLYTIC VIROTHERAPY AS AN APPROACH TO ENHANCE NEOANTIGEN SPECIFIC IMMUNE RESPONSES IN GLIOBLASTOMA

Abstract Despite the success in treating many solid cancers, immunotherapy has not shown significant efficacy in patients with glioblastoma (GBM). The lack of tumor-infiltrating T cells recognizing tumor antigens displayed with major histocompability complex I (MHC I) is one of the reasons for GBM’s evasion from immunotherapy. Our preclinical GBM models and an ongoing clinical trial with oncolytic herpes simplex virus-1 (oHSV) at our institution (NCT03152318) demonstrated that oHSV treatment leads to an increase influx of CD8+ and CD4+ T cells into tumor. However, it remained unclear whether these T cells are specific to the tumor antigens (neoantigens) or reactive to only viral antigens. To determine whether infiltrating CD8 T cells target tumor antigens, we first performed whole-exome and RNA sequencing of CT2A, a preclinical model of GBM impervious to immunotherapies for identification of neoantigens. We screened immunogenicity of identified neoantigens by ELISPOT assay. TILs from the oHSV-treated (but not from control group) reacted to two of the neoantigens that were identified with our pipeline and this activation was similar in magnitude to the reaction to the immunodominant HSV antigen. FACS analyses of TILs seven days after virotherapy showed that oHSV-treated tumor had higher level of TILs than vehicle control tumors, a finding that mirrored what we observed in our phase 1 clinical trial with oHSV. High dose of oHSV treatment not just induces the infiltration of viral specific T cells, but infiltration of T cells targeting neoantigens that were not presented without OV infection. Phosphoproteomic analysis of bulk tumor revealed activation of key inflammatory pathways, including that of T cells receptor signaling upon virus treatment. We evaluated the efficacy of therapeutic neoantigen vaccination in combination with oncolytic intratumoral virotherapy. Survival analysis showed that combination therapy not just delays the tumor progression but also provides survival advantage compared to monotherapies.

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.

Recent advances in neoantigen vaccines for treating non-small cell lung cancer.

The breakthrough of programmed cell death protein 1 (PD-1) blockade therapy has changed the clinical treatment of non-small cell lung cancer (NSCLC) in the past few years. The success of PD-1 blockade therapy has been attributed to high tumor mutation burden and high immunogenicity of lung cancer cells. To further improve the efficacy of NSCLC immunotherapy and overcome the resistance of lung cancer cells to immune checkpoint blockade, new approaches that enhance the active immune response, such as neoantigen vaccines and cellular-based therapies, are urgently required. Neoantigens are considered ideal targets for cancer immunotherapy because of their high immunogenicity and specificity. In this mini review, we first discuss the current advances in neoantigen vaccines for treating cancers and then review the results of preclinical studies and early-phase human clinical trials of neoantigen-based therapies for NSCLC. Finally, we focus on the identification of neoantigens in patients with NSCLC and review the candidate mutations reported by recent studies and our investigations. The review concludes that, in addition to immune checkpoint blockade, approaches targeting neoantigens are promising for improving the efficacy of NSCLC immunotherapy.

Neo-intline: integrated pipeline enables neoantigen design through the in-silico presentation of T-cell epitope

Neoantigen vaccines are one of the most effective immunotherapies for personalized tumour treatment. The current immunogen design of neoantigen vaccines is usually based on whole-genome sequencing (WGS) and bioinformatics prediction that focuses on the prediction of binding affinity between peptide and MHC molecules, ignoring other peptide-presenting related steps. This may result in a gap between high prediction accuracy and relatively low clinical effectiveness. In this study, we designed an integrated in-silico pipeline, Neo-intline, which started from the SNPs and indels of the tumour samples to simulate the presentation process of peptides in-vivo through an integrated calculation model. Validation on the benchmark dataset of TESLA and clinically validated neoantigens illustrated that neo-intline could outperform current state-of-the-art tools on both sample level and melanoma level. Furthermore, by taking the mouse melanoma model as an example, we verified the effectiveness of 20 neoantigens, including 10 MHC-I and 10 MHC-II peptides. The in-vitro and in-vivo experiments showed that both peptides predicted by Neo-intline could recruit corresponding CD4+ T cells and CD8+ T cells to induce a T-cell-mediated cellular immune response. Moreover, although the therapeutic effect of neoantigen vaccines alone is not sufficient, combinations with other specific therapies, such as broad-spectrum immune-enhanced adjuvants of granulocyte-macrophage colony-stimulating factor (GM-CSF) and polyinosinic-polycytidylic acid (poly(I:C)), or immune checkpoint inhibitors, such as PD-1/PD-L1 antibodies, can illustrate significant anticancer effects on melanoma. Neo-intline can be used as a benchmark process for the design and screening of immunogenic targets for neoantigen vaccines.

Use of Stromal Intervention and Exogenous Neoantigen Vaccination to Boost Pancreatic Cancer Chemo-Immunotherapy by Nanocarriers.

Despite the formidable treatment challenges of pancreatic ductal adenocarcinoma (PDAC), considerable progress has been made in improving drug delivery via pioneering nanocarriers. These innovations are geared towards overcoming the obstacles presented by dysplastic stroma and fostering anti-PDAC immune reactions. We are currently conducting research aimed at enhancing chemotherapy to stimulate anti-tumor immunity by inducing immunogenic cell death (ICD). This is accomplished using lipid bilayer-coated nanocarriers, which enable the attainment of synergistic results. Noteworthy examples include liposomes and lipid-coated mesoporous silica nanoparticles known as "silicasomes". These nanocarriers facilitate remote chemotherapy loading, as well as the seamless integration of immunomodulators into the lipid bilayer. In this communication, we elucidate innovative ways for further improving chemo-immunotherapy. The first is the development of a liposome platform engineered by the remote loading of irinotecan while incorporating a pro-resolving lipoxin in the lipid bilayer. This carrier interfered in stromal collagen deposition, as well as boosting the irinotecan-induced ICD response. The second approach was to synthesize polymer nanoparticles for the delivery of mutated KRAS peptides in conjunction with a TLR7/8 agonist. The dual delivery vaccine particle boosted the generation of antigen-specific cytotoxic T-cells that are recruited to lymphoid structures at the cancer site, with a view to strengthening the endogenous vaccination response achieved by chemo-immunotherapy.

Immunological analysis of hybrid neoantigen peptide encompassing class I/II neoepitope-pulsed dendritic cell vaccine.

Neoantigens/ are tumor-specific antigens that evade central immune tolerance mechanisms in the thymus. Long-term tumor-specific cytotoxic T lymphocyte activity maintenance requires class II antigen-reactive CD4+ T cells. We had previously shown that intranodal vaccination with class I neoantigen peptide-pulsed dendritic cells (DCs) induced a robust immune response in a subset of patients with metastatic cancer. The present study aimed to perform a detailed ex vivo analysis of immune responses in four patients receiving an intranodal hybrid human leukocyte antigen class II neoantigen peptide encompassing a class I neoantigen epitope (hybrid neoantigen)-pulsed DC vaccine. After vaccination, strong T-cell reactions against the hybrid class II peptide and the class I-binding neoantigen peptide were observed in all four patients. We found that hybrid class II neoantigen peptide-pulsed DCs stimulated CD4+ T cells via direct antigen presentation and CD8+ T cells via cross-presentation. Further, we demonstrated that hybrid class II peptides encompassing multiple class I neoantigen epitope-pulsed DCs could present multiple class I peptides to CD8+ T cells via cross-presentation. Our findings provide insight into the mechanisms underlying hybrid neoantigen-pulsed DC vaccine therapy and suggest future neoantigen vaccine design.

Challenges in developing personalized neoantigen cancer vaccines.

The recent success of cancer immunotherapies has highlighted the benefit of harnessing the immune system for cancer treatment. Vaccines have a long history of promoting immunity to pathogens and, consequently, vaccines targeting cancer neoantigens have been championed as a tool to direct and amplify immune responses against tumours while sparing healthy tissue. In recent years, extensive preclinical research and more than one hundred clinical trials have tested different strategies of neoantigen discovery and vaccine formulations. However, despite the enthusiasm for neoantigen vaccines, proof of unequivocal efficacy has remained beyond reach for the majority of clinical trials. In this Review, we focus on the key obstacles pertaining to vaccine design and tumour environment that remain to be overcome in order to unleash the true potential of neoantigen vaccines in cancer therapy.

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.

Abstract A013: Tumor-specific immunity generated by a personalized neoantigen vaccination incorporating locally delivered ipilimumab in renal cell carcinoma

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR015) of the Conference Program/Proceedings. 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 A013.

α-d-Glucose-1,6-Biphosphate Induces Dendritic Cell Homing to Enhance the Antitumor Effect of Neoantigen Vaccines.

Neoantigen vaccines have achieved good therapeutic effects in animal experiments and early clinical trials on certain malignant tumors. However, their overall objective effectiveness in clinical trials still needs to be improved. Low-efficiency dendritic cell (DC) migration (<5%) to lymph nodes is one of the factors that limits vaccine effectiveness. For neoantigen vaccines, improving the homing efficiency of DCs is expected to further improve the immunotherapeutic effect. In this study, we used α-d-glucose-1,6-biphosphate (α-d-Glu), a metabolite that successfully enhanced C57BL/6J mouse bone marrow-derived DC homing induced by neoantigen peptide, mRNA, and DC vaccines during the administration process and improved the antitumor effects in the mouse C57BL/6J model with a neoantigen vaccine. We clarified that α-d-Glu activated MAPK8IP1 by inhibiting the expression of microRNA-10a-5p, thereby activating the MAPK signaling pathway to promote DC homing. Excitingly, the efficiency of α-d-Glu in promoting DC migration is not weaker than that of PGE2, which is the gold standard used to promote DC migration in clinical trials of DC vaccines. Thus, this study lays the foundation for further enhancing the objective clinical response rate of neoantigen vaccines and overcoming the limitation of an insufficient clinical response rate for neoantigen vaccines caused by low DC homing efficiency.

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.

Novel neoantigen peptides and tumor regression in the MC38 colorectal cancer model.

28 Background: Neoantigen vaccine is an emerging modality to treat cancer by unleashing cytotoxic T cells against tumor-specific mutant antigens. Neoantigens are de-novo changes in tumor cells and are hence more immunogenic and less susceptible to off-target toxicities. In building our own pipeline for neoantigen discovery, we used the mouse colon adenocarcinoma cell line MC38 as a validation model. Methods: We sequenced MC38 cells by whole exome and RNA sequencing to shortlist variants. Candidates were confirmed by sanger sequencing, then analyzed for binding affinity to MHC class I H2-Kb and H2-Db. We selected top 10 epitopes for facile modification to maximize immunogenicity by taking into account peptide characteristics, such as hydrophobicity. In-vivo immunogenicity was tested by subcutaneous injections of pooled peptides in C57BL/6J mice. Amongst the 10 peptides tested, we picked five that elicited greatest IFNγ secretion in re-stimulated splenocytes. Predicted binding affinity did not correlate with in-vivo immunogenicity. We inoculated MC38 cells into the left flank of C57BL/6J mice and started treatment four days later. Mice were injected twice weekly with PBS (group 1), CpG only (group 2), anti-PDL1 only (group 3), Peptide pool + CpG (group 4) and Peptide pool + CpG + anti-PDL1 (group 5) (n=8 per group). CpG ODN 1826 is a TLR9 agonist and acts as an adjuvant, while anti-PDL1 is an immune checkpoint inhibitor. We monitored tumor size, animal weights and food intake every 2-3 days. Results: In control group 1, tumor size exponentially increased by 36 folds on the 18th day of treatment. Groups 2, 3, 4 and 5 saw 16, 13, 8 and 5-fold increases in tumor size respectively, 21 days post-treatment. No tumors were found in 3/7 mice in group 5 upon sacrifice, suggesting tumor regression. Tumor control in groups 4 and 5 corroborated with significant IFNγ secretion from neoantigen re-stimulated splenocytes. We observed 38-45 fold expansion in the number of CD8+ KLRG1+ short-lived effector cells and significant increase in PD-1+ TIM3+ exhausted T cells in group 4 and 5 relative to control. Reps1 and Adpgk neoantigen peptides are widely reported and used in MC38 syngenic model. However, they were not included because both were undetected by sequencing. Cell line authentication by STR profiling confirmed that our cells matched that of MC38 from Kerafast. Conclusions: Using an in-house pipeline, we derived a set of novel neoantigen peptides that robustly controlled tumor growth in the MC38 tumor challenge model. In-silico binding predictions did not correlate with in-vivo patterns of IFNγ secretion. In-vivo tests for IFNγ induction after peptide vaccination is a better measure for potential in tumor control. Upregulation of T-cell exhaustion markers after peptide vaccination explains the improved response with anti-PDL1. Reps1 and Adpgk mutations were absent in our cells, which calls caution to researchers conducting similar research.

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 .

H3.3-K27M neoantigen vaccine elicits anti-tumor T cell immunity against diffuse intrinsic pontine glioma: The phase I ENACTING trial.

2052 Background: Diffuse intrinsic pontine glioma (DIPG) harboring H3.3-K27M mutation is a malignant pediatric brain tumor with a &gt;90% mortality rate within two-year of diagnosis. Current therapeutic options for DIPG are limited. Aiming to improve therapeutic outcomes, we herein report the preliminary findings of a phase I trial studying a neoantigen peptide vaccine targeting H3.3-K27M. Methods: ENACTING is an open-label, single center, two-armed phase 1 trial to assess the safety and T cell immunity of a neoantigen peptide vaccine against H3.3-K27M. Patients aged ≥ 5 years old with newly diagnosed DIPG were consented and screened. HLA-A*02+/H3.3-K27M+ patients were enrolled to a two-arm study: Arm A consists of subjects receiving open debulking surgery, and Arm B consists of subjects without surgery eligibilities who received stereotactic biopsy. All patients subsequently received conformal radiotherapy and neoantigen vaccine treatment designed to elicit both CD4+ and CD8+ T cell immune response. Vaccine was administered intramuscularly in combination with polyinosinic-polycytidylic acid-poly-L-Iysine carboxymethylcellulose (Poly-ICLC). The primary objective is to evaluate the safety (AEs graded by CTCAE v4.03) and survival outcomes. Secondary objectives include maximum tolerated dose (MTD) and immunological responses. Results: As of Jan 2023, 11 patients have been treated, with 7 in Arm A and 4 in Arm B. No grade 3-4 treatment-related adverse events have been observed, with fever (81.9%) and injection site pain (54.5%) being the most common AEs. Among 10 efficacy-assessable patients, median progression-free survival (mPFS) and median overall survival (mOS) were 11.4 months (95% CI: 5.8~14.7) and 15.4 months (95% CI: 7.53~not reached), respectively. One-year OS rate was 66.7% (95% CI: 42~100%). One patient was assessed as complete response (CR). T cell responses against neoantigen were detected and H3.3-K27M mutation-specific CD4+ and CD8+ TCR clones were validated. Conclusions: The H3.3-K27M neoantigen vaccine was well tolerated. Initial results from this ongoing study suggest that, compared with other current therapies against DIPG, H3.3-K27M peptide vaccination may provide superior patient survival outcomes. Clinical trial information: NCT04749641 . [Table: see text]

A pilot trial of personalized neoantigen pulsed autologous dendritic cell vaccine as adjuvant treatment in hepatocellular carcinoma.

e14658 Background: The primary means of treating hepatocellular carcinoma (HCC) is radical surgery. However, HCC has a high propensity to recur post-operatively, and no standard adjuvant therapies have been approved. Tumor neoantigens, which are epitopes derived from tumor-specific mutations, may be utilized in personalized vaccines to activate T cell responses. This trial aims to assess the safety and efficacy of personalized neoantigen pulsed autologous dendritic cell vaccine (Neo-DCVac-02) in patients with HCC. Methods: Eligible patients had histologically confirmed high-risk HCC after radical surgery. Upon obtaining the baseline biopsy specimens and PBMCs of appropriate quality for DNA and RNA sequencing, neoantigen prediction, screening, and synthesis were performed. Subsequently, autologous dendritic cells (DCs) were generated and pulsed with neoantigen peptides to produce Neo-DCVac-02.On day 0, patients were treated with cyclophosphamide at a dose of 250 mg/m2. The prepared Neo-DCVac-02 vaccine was administered subcutaneously on day 1, followed by GM-CSF administered daily for 5 days. The primary objectives of this study were to evaluate the feasibility and safety/tolerability. Secondary objectives included assessment of immune responses (via vaccine response: IFNγ ELISpot), recurrence-free survival (RFS), and overall survival (OS). Results: A total of 32 patients provided informed consent to initiate the process of personalized neoantigen discovery, of which 26 (81%) met the requirements for successful product selection for clinical manufacturing. And feasibility was shown with 13 patients receiving Neo-DCVac-02. Thirteen patients did not receive neoantigen vaccines (7 for progressive disease,6 for withdrawal of informed consent because of COVID-19). Treatment was well-tolerated with no grade 2+ treatment-related adverse events (TRAEs). The common grade 1TRAEs were neutropenia (8.3%), injection site reactions (75%), rash (16.7%), fatigue (8.3%), and headache (8.3%). The median follow-up to date is 19.30 (14.4-31.9) months, with median RFS and OS not yet reached and a 1-year RFS of 83%. Five of 13 evaluable patients showed elevated antigen-specific T-cell activity by IFNγ ELISpot for more than 50% of neoantigen peptides in Neo-DCVac-02, and persistent cellular responses were noted out to 1 year. Increases in activation/co-stimulatory of T cells seen after treatment with Neo-DCVac-02 revealed by Flow-cytometric analyses, suggest immune priming. Conclusions: These data demonstrate that Neo-DCVac-02 is safe, well tolerated, and able to generate durable antigen-specific lymphocyte immune responses. Early signs of clinical efficacy were observed in HCC patients. Clinical trial information: NCT04147078 .

An engineered influenza virus to deliver antigens for lung cancer vaccination.

The development of cancer neoantigen vaccines that prime the anti-tumor immune responses has been hindered in part by challenges in delivery of neoantigens to the tumor. Here, using the model antigen ovalbumin (OVA) in a melanoma model, we demonstrate a chimeric antigenic peptide influenza virus (CAP-Flu) system for delivery of antigenic peptides bound to influenza A virus (IAV) to the lung. We conjugated attenuated IAVs with the innate immunostimulatory agent CpG and, after intranasal administration to the mouse lung, observed increased immune cell infiltration to the tumor. OVA was then covalently displayed on IAV-CPG using click chemistry. Vaccination with this construct yielded robust antigen uptake by dendritic cells, a specific immune cell response and a significant increase in tumor-infiltrating lymphocytes compared to peptides alone. Lastly, we engineered the IAV to express anti-PD1-L1 nanobodies that further enhanced regression of lung metastases and prolonged mouse survival after rechallenge. Engineered IAVs can be equipped with any tumor neoantigen of interest to generate lung cancer vaccines.

Induction of cancer neoantigens facilitates development of clinically relevant models for the study of pancreatic cancer immunobiology

Neoantigen burden and CD8 T cell infiltrate are associated with clinical outcome in pancreatic ductal adenocarcinoma (PDAC). A shortcoming of many genetic models of PDAC is the lack of neoantigen burden and limited T cell infiltrate. The goal of the present study was to develop clinically relevant models of PDAC by inducing cancer neoantigens in KP2, a cell line derived from the KPC model of PDAC. KP2 was treated with oxaliplatin and olaparib (OXPARPi), and a resistant cell line was subsequently cloned to generate multiple genetically distinct cell lines (KP2-OXPARPi clones). Clones A and E are sensitive to immune checkpoint inhibition (ICI), exhibit relatively high T cell infiltration, and have significant upregulation of genes involved in antigen presentation, T cell differentiation, and chemokine signaling pathways. Clone B is resistant to ICI and is similar to the parental KP2 cell line in terms of relatively low T cell infiltration and no upregulation of genes involved in the pathways noted above. Tumor/normal exome sequencing and in silico neoantigen prediction confirms successful generation of cancer neoantigens in the KP2-OXPARPi clones and the relative lack of cancer neoantigens in the parental KP2 cell line. Neoantigen vaccine experiments demonstrate that a subset of candidate neoantigens are immunogenic and neoantigen synthetic long peptide vaccines can restrain Clone E tumor growth. Compared to existing models, the KP2-OXPARPi clones better capture the diverse immunobiology of human PDAC and may serve as models for future investigations in cancer immunotherapies and strategies targeting cancer neoantigens in PDAC.

Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients1, yet harbours mutation-derived T cell neoantigens that are suitable for vaccines 2,3. Here in a phase I trial of adjuvant autogene cevumeran, an individualized neoantigen vaccine based on uridine mRNA–lipoplex nanoparticles, we synthesized mRNA neoantigen vaccines in real time from surgically resected PDAC tumours. After surgery, we sequentially administered atezolizumab (an anti-PD-L1 immunotherapy), autogene cevumeran (a maximum of 20 neoantigens per patient) and a modified version of a four-drug chemotherapy regimen (mFOLFIRINOX, comprising folinic acid, fluorouracil, irinotecan and oxaliplatin). The end points included vaccine-induced neoantigen-specific T cells by high-threshold assays, 18-month recurrence-free survival and oncologic feasibility. We treated 16 patients with atezolizumab and autogene cevumeran, then 15 patients with mFOLFIRINOX. Autogene cevumeran was administered within 3 days of benchmarked times, was tolerable and induced de novo high-magnitude neoantigen-specific T cells in 8 out of 16 patients, with half targeting more than one vaccine neoantigen. Using a new mathematical strategy to track T cell clones (CloneTrack) and functional assays, we found that vaccine-expanded T cells comprised up to 10% of all blood T cells, re-expanded with a vaccine booster and included long-lived polyfunctional neoantigen-specific effector CD8+ T cells. At 18-month median follow-up, patients with vaccine-expanded T cells (responders) had a longer median recurrence-free survival (not reached) compared with patients without vaccine-expanded T cells (non-responders; 13.4 months, P = 0.003). Differences in the immune fitness of the patients did not confound this correlation, as responders and non-responders mounted equivalent immunity to a concurrent unrelated mRNA vaccine against SARS-CoV-2. Thus, adjuvant atezolizumab, autogene cevumeran and mFOLFIRINOX induces substantial T cell activity that may correlate with delayed PDAC recurrence.