Neoantigen Vaccines Research Articles

GEN-009, a neoantigen vaccine containing ATLAS selected neoantigens, to generate broad sustained immunity against immunogenic tumor mutations and avoid inhibitory peptides.

3107 Background: Tumor-specific neoantigens provide personalized targets for immunotherapy. Vaccines against epitopes predicted by in silico approaches very rarely induce CD4+ and CD8+ ex vivo T cell responses regardless of formulation. ATLAS selects neoantigens for vaccine inclusion using ex vivo screening of all patient-specific mutations to identify pre-existing CD4+ or CD8+ T cell responses and to exclude Inhibigens, which are inhibitory peptides that suppress immunity and accelerate tumor progression. The Inhibigen burden correlates with patient outcomes in observational studies and rapid tumor progression in mouse models. Methods: GEN-009-101 is a phase 1/2a study testing safety, immunogenicity and clinical activity in immune responsive tumors. After next-generation tumor sequencing and ATLAS testing of autologous leukocytes, up to 20 stimulatory synthetic long peptides adjuvanted with poly-ICLC comprise each personalized vaccine. Eight vaccinated patients have been followed for sustained immunological responses and clinical outcomes. Results: The 40 doses given across patients have induced only mild local discomfort and no DLT. Vaccination has generated immune responses against 99% of administered peptides, with both CD8+ and CD4+ responses in ex vivo fluorospot assays. To date, no patients have developed recurrent disease. Broad immunity develops as early as Day 29 and is sustained for over 12 months. Immune response against individual peptides is correlated with peptide concentration (OR = 1.26, p≤0.0001) but not with other classifiers such as GRAVY index (Grand Average of Hydropathy), tumor type, injection site or sex. The Inhibigen burden prior to treatment again correlates with disease progression. Conclusions: GEN-009 identifies tumor specific immune targets from the individual patient’s tumor mutagens. Initial clinical data show that ATLAS antigen selection may be critical to the induction of broad, rapid and sustained immunity against tumor specific neoantigens. Clinical vaccination with PD-1 blockade is in process. Clinical trial information: NCT03633110 . [Table: see text]

Neoantigen-based vaccination as a practical measure for head and neck cancer treatment.

e18528 Background: The view that neoantigens serve as potential vaccine targets has arisen in the last decade. Clinical and computational efforts have been done to increase the practicality of its application in real world. With these advances, we conducted a retrospective study on a Chinese population to explore the clinical feasibility of neoantigen-based vaccines for head and neck cancer treatment. Methods: Tumor and normal samples were profiled using a 1021-gene panel. Sequencing data were pre-analyzed according to our in-house standard procedures. Class I HLA typing was completed using OptiType v1.0. Curated somatic mutations in coding regions (SNVs and non-frameshift Indels with an allele frequency ≥ 5%) were collected and altered peptides produced by these mutations were analyzed using NetMHCpan v4.0. Peptides with an IC50 < 500 nM were considered potential binders, and especially, those with an IC50 < 50 nM were considered strong binders. An altered peptide was considered a neoantigen if IC50 altered is < IC50 wildtype. Results: We analyzed a total of 243 patients and detected 114 unique HLA alleles. By carrier percentage, the top three alleles are C*01:02 (44%), B*46:01 (36%), and A*11:01 (33%). In total, 743 mutations were deemed eligible for neoantigen prediction and 223 unique neoantigens were found. Of these neoantigens, 67 (carried by 21% of patients) were strong binders, among which 26 (carried by 9% patients) exhibited a great fold change (≥ 5 folds) of binding affinity. Moreover, the neoantigens in these patients are unique, as only two neoantigens were shared. A search for shared neoantigens revealed a combination of mutation PIK3CA p.E542K and HLA A*11:01, which was detected in 0.54% of all patients. Additionally, 43.6% (106/243) of patients were diagnosed with nasopharyngeal carcinoma, among whom 42% (44/106) possessed predicted neoantigens, including 15 patients with strong-binder neoantigens. Conclusions: (1) Neoantigen-based vaccination is a practical measure to treat patients with head and neck cancer, as indicated by the percentage of patients harboring strong-binder neoantigens. (2) Off-the-shelf neoantigen vaccines may not be practical, given the result that the most common combination of a neoantigen-producing mutation and the corresponding HLA was only found in 0.54% of all patients.

An educated approach: AI-enabled neoantigen vaccine R&D

An educated approach: AI-enabled neoantigen vaccine R&D

Identification of predicted neoantigen vaccine candidates in follicular lymphoma patients.

8054 Background: Follicular lymphoma (FL) is incurable with conventional therapies and poorly responsive to immune checkpoint blockade. There is a need for new therapies without long-term complications of chemotherapy and with curative potential. We hypothesize that FL contains tumor-specific mutant antigens (TSMAs) that can be targeted by the immune system by vaccination. Recent reports have highlighted the potential for unique immunoglobulin peptides to elicit immune response in lymphomas. We utilized whole exome sequencing (WES) and RNA sequencing (RNA-Seq) of FL patient samples to infer HLA genotype, and predict TSMAs with the goal of designing a personalized cancer vaccine, supported by recent reports of this approach in solid cancers. Methods: DNA and RNA from 58 patients’ FL biopsies underwent WES and RNA-Seq. pVACtools and MiXCR predicted potential somatic and B-cell clonotype neoantigens, which were filtered to identify high quality TSMAs. B-cell oligoclonality was determined by comparison to B-cell receptor (BCR) repertoire profiling of healthy individual lymph nodes. RNA-seq data allowed us to identify expressed TSMAs. Complementary in silico analysis based on mRNA-based peptide reconstruction and custom HLA affinity binding predictions were performed. Results: An average of 52 somatic mutations per patient (range: 2-172) were identified. At least one high quality TSMA was predicted for 57 of 58 patients. Five or more TSMA candidates were identified for 52 (90%) patients with a mean of 17 predicted peptides per patient (range: 0-45). 81% (813/1,004) of the total predicted TSMA peptides arose from missense mutations, 9% (94/1,004) from indels, and 10% (97/1,004) from BCR. 78% (45/58) of patients have both somatic and BCR vaccine candidates, while 21% (12/58) of patients had only somatic vaccine candidates. Predicted TSMAs were identified in multiple genes recurrently mutated in lymphoma (e.g., BCL2). There was a high prediction concordance with the orthogonal BostonGene Vaccine Module V1 pipeline. These pre-clinical results led to a first-in-human pilot trial of personalized TSMA vaccine combined with anti-PD-1 mAb for rel/ref FL patients (NCT03121677), with one response observed within 4 patients evaluable for response to date. Conclusions: TSMA peptides suitable for cancer vaccines were identified for most FL patients via next-generation sequencing, MiXCR and pVACtools. This pre-clinical study suggests that FL patients will be candidates for TSMA vaccine clinical trials and pilot clinical results provide proof of concept for this approach.

Comparison of personal and shared frameshift neoantigen vaccines in a mouse mammary cancer model

BackgroundIt is widely hoped that personal cancer vaccines will extend the number of patients benefiting from checkpoint and other immunotherapies. However, it is clear creating such vaccines will be challenging. It requires obtaining and sequencing tumor DNA/RNA, predicting potentially immunogenic neoepitopes and manufacturing a one-use vaccine. This process takes time and considerable cost. Importantly, most mutations will not produce an immunogenic peptide and many patient’s tumors do not contain enough DNA mutations to make a vaccine. We have discovered that frameshift peptides (FSP) created from errors in the production of RNA rather than from DNA mutations are potentially a rich source of neoantigens for cancer vaccines. These errors are predictable, enabling the production of a FSP microarray. Previously we found that these microarrays can identify both personal and shared neoantigens. Here, we compared the performance of personal cancer vaccines (PCVs) with that of a shared antigen vaccine, termed Frameshift Antigen Shared Therapeutic (FAST) vaccine, using the 4 T1 breast cancer model. Sera from 4 T1-tumor bearing mice were assayed on the peptide microarray containing 200 Fs neoantigens, for the PCV, the top 10 candidates were select and personal vaccines constructed and administrated to the respective mice. For the FAST, we selected the top 10 candidates with higher prevalence among all the mice challenged. Seven to 12 days challenged mice were immunized, combined or not with immune checkpoint inhibitor (ICI) (αPD-L1 and αCTLA-4). Primary and secondary tumor clearance and growth were evaluated as well as cellular and humoral immune response against the vaccine targets by IFN-γ ELISPOT and ELISA. Lastly, we analyzed the immune response of the FAST-vaccinated mice by flow cytometry in comparison to the control group.ResultsWe found that PCVs and FAST vaccines both reduced primary tumor incidence and growth as well as lung metastases when delivered as monotherapies or in combination with ICI. Additionally, the FAST vaccine induces a robust and effective T-cell response.ConclusionsThese results suggest that FSPs produced from RNA-based errors are potent neoantigens that could enable production of off-the-shelf shared antigen vaccines for solid tumors with efficacy comparable to that of PCVs.

Immune Therapy Opportunities in Ovarian Cancer.

Immunotherapy has emerged as a highly promising approach in the treatment of epithelial ovarian cancer (EOC). Immune checkpoint blockade (ICB) therapy, PARP inhibitors (PARPis), neoantigen vaccines, and personalized T-cell therapy have been associated with encouraging clinical activity in a small subset of patients. To increase the proportion of patients who are likely to derive benefit, it will be important not only to generate sufficient numbers of antitumor T cells but also to overcome multiple inhibitory networks in the ovarian tumor microenvironment (TME). Therefore, a major direction is to develop biomarkers that would predict responsiveness to different types of immunotherapies and allow treatment selection based on the results. Moreover, such biomarkers would allow rational combination of immunotherapies while minimizing toxicities. In this review, we provide progress on immune therapies and future directions for maximally exploiting immune-based strategies for the treatment of ovarian cancer.

Immunogenicity comparison of neoantigen vaccines through different delivery platform

Abstract Neoantigens are promising cancer vaccine candidates because they’re cancer-specific and can be predicted from the patient’s genomic data. A major challenge for developing potent neoantigen vaccines is the low immunogenicity of neoepitopes. DNA vaccines can deliver multiple epitopes and cytokines as an adjuvant in a single plasmid, and its immunogenicity can be further enhanced through heterologous prime-boost strategy. Here, we hypothesized utilizing DNA prime, adenovirus boost (refer to the DDA approach) strategy can improve the immunogenicity of neoantigen vaccines. To investigate whether vaccination through the DDA approach or DDA combine GM-CSF as adjuvant can improve the immunogenicity of neoantigen vaccines. Using IEDB analysis resource to predict and rank the top 21 MHC class I epitopes (short peptide) for the B16F10 tumor while 27mer peptides (long peptide) were synthesized to cover both MHC class I and class II epitopes. The 27mer neoantigen sequences of the selected 21 MHC class I epitopes were cloned into one plasmid and adenoviral vector with or without GM-CSF sequence linked by p2a. Mice were immunized through the DDA approach or peptides with polyI:C. In the peptide vaccine, the majority of the response were mediated by CD4+ T cells, only one peptide can induce CD8+ T cells response. For the DDA approach, 14% epitopes can induce CD8+ T cell response in mice immunized with DNA vaccine. Moreover, in the group with genetic encoded GM-CSF. it has significantly enhanced CD8+T cell response compare to the former, which dramatically improved the immunogenicity of neoantigen vaccines. Our preliminary data also showed that immunization of DNA vaccines significantly delayed tumor growth.

Synthetic long peptide vaccines possessing a universal helper epitope can unmask the therapeutic effects of MHC I-restricted neoepitopes

Abstract Synthetic long peptide (SLP) vaccines targeting neoantigens arising from patient-specific missense mutations may offer a means to address the antigenic heterogeneity of glioblastoma (GBM) tumors by simultaneously targeting a multitude of tumor-specific antigens expressed throughout these heterogenous tumors. This approach is challenged by uncertainties pertaining to vaccine design, including peptide composition required for immunogenicity and efficacy. To this end, we investigated the mechanism of an efficacious SLP vaccine targeting the neoantigen Odc1, expressed within the mouse astrocytoma SMA560. This led to the identification of three generalizable principles governing the effectiveness of neoantigen-targeting SLPs: (1) SLPs containing an MHC I-restricted neoepitope may activate neoantigen-reactive CD8+ T cells, which drive direct antitumor effects; (2) to induce robust neoantigen-reactive CD8+ T-cell responses, CD40L-mediated T cell "help" is required; and, (3) CD40L interactions are conferred by an SLP only when a “helper” epitope is physically conjoined to an MHC I-restricted neoepitope. To leverage these findings for clinical translation, we developed a rationally-designed vaccine comprised of an MHC I-restricted neoepitope linked to a universal “helper” epitope. This design was capable of maintaining the effects of the Odc1 vaccine and, remarkably, unmasked the therapeutic effects of, otherwise, poorly immunogenic MHC I-restricted neoepitopes. Together, these findings are significant because they elucidate mechanisms required for efficacious SLP vaccines and demonstrate a clinically-tractable approach with the potential to expand the therapeutic breadth of neoantigen vaccines.

Amplification of neoantigen-specific antitumor responses using a long-lasting IL-2 fusion protein

Abstract Low frequency of tumor-specific T cells represents one major reason for failed immunotherapy. IL-2 can boost tumor-reactive T cells but its benefit is limited due to toxicity and potential off-target effects. We developed a novel IL-2/CD25 fusion protein which exhibits more favorable pharmacology when compared to IL-2. At low doses, IL-2/CD25 targets the high affinity IL-2R and selectively boosts Tregs. Here we show that a single administration of high dose mouse IL-2/CD25 in conjunction with cognate peptide and LPS amplified tumor-specific Pmel-1 CD8+ and TRP-1 CD4+ Teff cells and Pmel-1 memory cells while only transiently increasing Tregs. This approach led to effective anti-tumor responses against B16.F10 melanoma. This scheme was modified to amplify endogenous polyclonal melanoma-reactive T cells, which is translationally more relevant. Development of tumor-specific T cells required priming and boosting with a mixture of B16.F10 neoantigens and poly (I:C) followed by application of IL-2/CD25 after the boost. This resulted in largely a CD4+ anti-neoantigen specific T cell response. This immunization scheme delayed B16.F10 growth and extend survival time not only when immunized mice were challenged with B16.F10 but also for mice with pre-established B16.F10. The immune response within the tumor microenvironment was accompanied by increased frequencies of granzyme B-expressing CD4+ and CD8+ T cells. Our data show that limited application of high-dose IL-2/CD25 in the context of neoantigen vaccines offers potential advantages over IL-2 for melanoma treatment by avoiding toxicities seen with IL-2 therapy and minimizing Treg-associated tolerance.

A review of glioblastoma immunotherapy.

Glioblastoma is a very aggressive cancer with dismal prognosis despite standard of care including surgical resection, radiation therapy, and chemotherapy. There is interest in applying immunotherapy to glioblastoma as this modality has demonstrated remarkable improvements in the management of several solid tumors including melanoma, renal cell carcinoma, and non-small cell lung cancer. This review aims to provide an overview of the current state of glioblastoma immunotherapy. Literature search was performed on PubMed between 1961 and 2020. Initial clinical trials of checkpoint inhibitors and vaccine therapy for glioblastoma have largely been disappointing for both primary and recurrent glioblastoma. This failure has been attributed to glioblastoma's highly immunosuppressive environment and multiple mechanisms of therapy resistance including high tumor heterogeneity, low mutational burden, systemic immunosuppression, and local immune dysfunction. Current clinical trials are exploring combination therapy and novel treatment strategies beyond immune checkpoint therapies and vaccine therapy such as CAR T cells. There is also an effort to establish synergy between immunotherapy and current standard of care. Furthermore, recent advances in personalized neoantigen vaccines suggest a shift towards personalized, patient-specific GBM treatment.

Abstract B67: Synthetic DNA multi-neoantigen vaccine drives predominately MHC class I CD8+ T cell-mediated effector immunity impacting tumor challenge

Abstract T-cell recognition of cancer neoantigens is important for effective immune checkpoint blockade therapy, and there is increasing interest in developing personalized tumor neoantigen vaccines. Previous studies utilizing RNA and synthetic long peptide neoantigen vaccines in preclinical and early-phase clinical studies have shown immune responses predominantly driven by MHC class II CD4+ T cells. Although it has been established that CD4+ T cells are able to recognize tumor neoantigens, the majority of naturally occurring tumor antigen-specific killer T cells identified in patients have been of CD8+ origin, indicating that additional immunization strategies aimed to stimulate neoantigen-specific CD8+ T cells may be useful. Here, we report the first preclinical study utilizing a synthetic DNA vaccine platform to target tumor neoantigens in mice. For this study, we identified neoepitopes by sequencing syngeneic mouse tumors, and designed optimized DNA plasmids encoding long strings of neoepitopes separated by highly efficient cleavage sites preserving epitope integrity. We tested the immunogenicity of 84 independent neoepitopes encoded into optimized DNA plasmids delivered by adaptive electroporation in mice in vivo. We observed that this approach generated robust T-cell immunity against a similar proportion of epitopes compared to other vaccine platforms (20/84, 24%). Strikingly, however, the synthetic neoantigen DNA vaccine platform generated a much larger proportion of CD8+ T-cell responses compared to the prior studies. Synthetic neoantigen DNA vaccines generated 75% CD8+ only or CD4/CD8+ T-cell responses, and 25% CD4+ only T-cell responses, showing a dramatic and important CD8+ T-cell bias. Inclusion of only high-affinity MHC class I (<500nM) epitopes selected for a larger proportion of immunogenic epitopes, and for 100% CD8+ or CD8+/CD4+ T-cell epitopes. These neoantigen vaccines were able to control tumor growth therapeutically in vivo in both lung and ovarian cancer models, and T cells expanded from immunized mice were able to kill tumor cells ex vivo. Because of the potential for rapid synthesis of vaccine constructs, the capacity to deliver a large number of neoepitopes simultaneously, and the potent CD8+ stimulatory capacity, this advanced DNA vaccine platform represents a likely important new approach for cancer immunotherapy. Citation Format: Elizabeth K. Duperret, Alfredo Perales-Puchalt, Regina Stoltz, G.H. Hiranjith, Nitin Mandloi, James Barlow, Amitabha Chaudhuri, Niranjan Y. Sardesai, David B. Weiner. Synthetic DNA multi-neoantigen vaccine drives predominately MHC class I CD8+ T cell-mediated effector immunity impacting tumor challenge [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B67.

Abstract PR20: A novel combination immunotherapy with a vaccine targeting tumor neoepitopes that mediates immune cascade in murine tumor models

Abstract Clinical successes utilizing checkpoint inhibitors demonstrate the potential of immune-based therapies for the treatment of cancer. Research suggests that these successes are a result of T cells recognizing nonsynonymous mutations uniquely expressed by a patient’s tumor. The advent of comparative tumor-normal DNA sequencing and tumor RNA sequencing for accurate prediction of presented somatic mutations has made it possible to rapidly identify these neoepitopes, allowing for the development of personalized cancer vaccines. However, such vaccines are often ineffective due to the immunosuppressive nature of the tumor microenvironment, and their clinical efficacies would be improved when used in an orchestrated combination approach with additional immune-oncology agents. In the present study, we investigated the combination of three immune-oncology agents, each targeting a unique mechanism of immune therapy, with a vaccine targeting tumor neoepitopes. We combined a neoepitope vaccine, delivered as a peptide or via a novel E2b-deleted adenovirus vehicle, with N-IL15 (previously called ALT-803), an IL-15 superagonist, to enhance the development of antigen-specific immunity; NHS-IL12, a tumor-targeting IL-12 molecule, to promote the maintenance and expansion of T cells within the tumor microenvironment; and PD-L1 blockade to decrease immune cell exhaustion within the tumor microenvironment. This combination therapy resulted in the regression of established tumors, which associated with the generation of robust immunity against neoepitopes contained in the vaccine. It also showed evidence of inducing a neoepitope cascade by causing development of T-cell responses to neoepitopes expressed by the tumor but not contained within the vaccine. This report is the first demonstration of an E2b-deleted adenovirus neoantigen vaccine generating robust immunity. Together, this study demonstrates the importance of a multifaceted, orchestrated treatment regimen to promote the generation of effective antitumor immunity. This abstract is also being presented as Poster A18. Citation Format: Karin L. Lee, Claudia Palena, John Lee, Kayvan Niazi, Andrew Nguyen, Frank Jones, Shahrooz Rabizadeh, Patrick Soon-Shiong, Jeffrey Schlom, Duane H. Hamilton. A novel combination immunotherapy with a vaccine targeting tumor neoepitopes that mediates immune cascade in murine tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR20.

A bi-adjuvant nanovaccine that potentiates immunogenicity of neoantigen for combination immunotherapy of colorectal cancer.

Neoantigen vaccines have been enthusiastically pursued for personalized cancer immunotherapy while vast majority of neoantigens have no or low immunogenicity. Here, a bi-adjuvant neoantigen nanovaccine (banNV) that codelivered a peptide neoantigen (Adpgk) with two adjuvants [Toll-like receptor (TLR) 7/8 agonist R848 and TLR9 agonist CpG] was developed for potent cancer immunotherapy. Specifically, banNVs were prepared by a nanotemplated synthesis of concatemer CpG, nanocondensation with cationic polypeptides, and then physical loading with hydrophobic R848 and Adpgk. The immunogenicity of the neoantigen was profoundly potentiated by efficient codelivery of neoantigen and dual synergistic adjuvants, which is accompanied by reduced acute systemic toxicity. BanNVs sensitized immune checkpoint programmed death receptor 1 (PD-1) on T cells, therefore, a combination of banNVs with aPD-1 conspicuously induced the therapy response and led to complete regression of 70% neoantigen-specific tumors without recurrence. We conclude that banNVs are promising to optimize personalized therapeutic neoantigen vaccines for cancer immunotherapy.

Relationships Between Immune Landscapes, Genetic Subtypes and Responses to Immunotherapy in Colorectal Cancer.

Colorectal cancer (CRC) is highly heterogeneous at the genetic and molecular level, which has major repercussions on the efficacy of immunotherapy. A small subset of CRCs exhibit microsatellite instability (MSI), a molecular indicator of defective DNA mismatch repair (MMR), but the majority are microsatellite-stable (MSS). The high tumor mutational burden (TMB) and neoantigen load in MSI tumors favors the infiltration of immune effector cells, and antitumor immune responses within these tumors are strong relative to their MSS counterparts. MSI has emerged as a major predictive marker for the efficacy of immune checkpoint blockade over the last few years and nivolumab or pembrolizumab targeting PD-1 has been approved for patients with MSI refractory or metastatic CRC. However, some MSS tumors show DNA polymerase epsilon (POLE) mutations that also confer a very high TMB and may also be heavily infiltrated by immune cells making them amenable to respond to immune checkpoint inhibitors (ICI). In this review we discuss the role of the different immune landscapes in CRC and their relationships with defined CRC genetic subtypes. We discuss potential reasons why immune checkpoint blockade has met with limited success for the majority of CRC patients, despite the finding that immune cell infiltration of primary non-metastatic tumors is a strong predictive, and prognostic factor for relapse and survival. We then consider in which ways CRC cells develop mechanisms to resist ICI. Finally, we address the latest advances in CRC vaccination and how a personalized neoantigen vaccine strategy might overcome the resistance of MSI and MSS tumors in patients for whom immune checkpoint blockade is not a treatment option.

Treatment of an aggressive orthotopic murine glioblastoma model with combination checkpoint blockade and a multivalent neoantigen vaccine.

Although clinical trials testing immunotherapies in glioblastoma (GBM) have yielded mixed results, new strategies targeting tumor-specific somatic coding mutations, termed "neoantigens," represent promising therapeutic approaches. We characterized the microenvironment and neoantigen landscape of the aggressive CT2A GBM model in order to develop a platform to test combination checkpoint blockade and neoantigen vaccination. Flow cytometric analysis was performed on intracranial CT2A and GL261 tumor-infiltrating lymphocytes (TILs). Whole-exome DNA and RNA sequencing of the CT2A murine GBM was employed to identify expressed, somatic mutations. Predicted neoantigens were identified using the pVAC-seq software suite, and top-ranking candidates were screened for reactivity by interferon-gamma enzyme linked immunospot assays. Survival analysis was performed comparing neoantigen vaccination, anti-programmed cell death ligand 1 (αPD-L1), or combination therapy. Compared with the GL261 model, CT2A exhibited immunologic features consistent with human GBM including reduced αPD-L1 sensitivity and hypofunctional TILs. Of the 29 CT2A neoantigens screened, we identified neoantigen-specific CD8+ T-cell responses in the intracranial TIL and draining lymph nodes to two H2-Kb restricted (Epb4H471L and Pomgnt1R497L) and one H2-Db restricted neoantigen (Plin2G332R). Survival analysis showed that therapeutic neoantigen vaccination with Epb4H471L, Pomgnt1R497L, and Plin2G332R, in combination with αPD-L1 treatment was superior to αPD-L1 alone. We identified endogenous neoantigen specific CD8+ T cells within an αPD-L1 resistant murine GBM and show that neoantigen vaccination significantly augments survival benefit in combination with αPD-L1 treatment. These observations provide important preclinical correlates for GBM immunotherapy trials and support further investigation into the effects of multimodal immunotherapeutic interventions on antiglioma immunity. 1. Neoantigen vaccines combined with checkpoint blockade may be promising treatments.2. CT2A tumors exhibit features of human GBM microenvironments.3. Differential scanning fluorimetry assays may complement in silico neoantigen prediction tools.

Abstract PR14: Emergence of quality metrics for neoantigens: Dissimilarity to the self-proteome as a novel determinant of immunogenicity

Abstract Fewer than 2% of computationally predicted neoantigens are immunogenic, a proven barrier to the efficacy of neoantigen vaccines. Currently, strong MHC affinity is dominant among neoantigen selection criteria for targeted therapies. To enrich for neoantigens with the greatest likelihood of immunogenicity, we developed an analytic method to parse neoantigen quality through rational biologic criteria and applied this method across five clinical datasets for 318 cancer patients. We assessed absolute MHC affinity, pathogen homology, and a novel analysis of dissimilarity to the self-proteome. We hypothesized that sequences with high dissimilarity to wild-type sequences would be more likely to perceived as “non-self” by immunosurveilling T cells. In a validation dataset, dissimilarity enriched for immunogenic peptides with an odds ratio of 34.9 (95% CI: 30.9 to 39.5, p < 0.001). Pathogen homology enriched 3.9-fold (95% CI: 3.6 to 4.3, p < 0.001), whereas selection on the basis of strong MHC-binding affinity alone enriches only 1.4-fold (95% CI: 1.2 to 1.5, p < 0.001). In patient tumors, these newly classified high-dissimilarity neoantigens were rare (1.18% of predicted MHC binders), did not overlap with the other metrics of neoantigen quality, and were enriched for hydrophobic sequences. While conventionally predicted neoantigens did not correlate with survival in two trials of PD-1 checkpoint therapy in patients with non-small cell lung cancer, high-dissimilarity neoantigens, even independent of MHC affinity, were predictive (HR - 3.29, 95% CI: 1.50 – 7.22, p = 0.014 and 3.40, 95% CI: 1.37 – 8.47, p = 0.021). Furthermore, we found that dissimilar peptides were enriched among known immunogenic neoantigens reported in multiple independent settings, including vaccine clinical trials (p = 0.001 – 0.023). This enrichment was not explained by a correlation with reported MHC binding affinities. Dissimilarity also enriched for immunogenicity in a dataset of human autoantigens, tumor-associated antigens, and cancer-testis antigens (odds ratio: 10.44, p < 2.2e-16). Dissimilarity to the self-proteome is therefore an orthogonal approach to MHC affinity for predicting CD8 T cell immunogenicity in both neoantigens and self-antigens. Overall, we show that neoantigen quality, including our novel analysis of dissimilarity to the self-proteome, identifies immunogenic peptides from conventionally predicted neoantigens for which the immunogenic “hit rate” is currently low and facilitates the more rapid development of neoantigen vaccines for cancer immunotherapy. Our pipeline, antigen.garnish, is open source and available on GitHub. This abstract is also being presented as Poster B30. Citation Format: Lee P. Richman, Andrew J. Rech, Robert H. Vonderheide. Emergence of quality metrics for neoantigens: Dissimilarity to the self-proteome as a novel determinant of immunogenicity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR14.

Abstract A28: A comparison of a DNA-sourced neoantigen vaccine to an RNA-sourced frameshift vaccine in the mouse ovarian cancer model

Abstract Development of cancer vaccines is currently focused on using neoantigens arising from mutations in the DNA of tumors. We have proposed, and demonstrated, that errors in RNA processing that create frameshift (FS) neoantigens are also a good source of vaccine components, even in tumors that are DNA mutation poor. Here we directly compare the two vaccine approaches in the mouse ovarian model, ID8. Martin et al. (2016) reported that 7 DNA neoepitopes in the ovarian tumor line (ID8-G7) failed to show any protection when tested individually as peptide vaccines even though the peptides elicited an immune response. We created a pooled peptide vaccine consisting of these 7 neoantigens (NeoAg vax). We compared this vaccine to one composed of 13 RNA-sourced FS neoantigens (FS vax). These FSs had conferred protection when tested in the 4T1 mammary and/or B16 melanoma models. 10 of the FSs arise from mis-transcription thru microsatellites and 3 from mis-splicing of exons. In a prophylactic vaccination protocol, both vaccines induce ~20% extended survival compared to mock controls. The FS vax elicited higher numbers of ELISpots than the ID8 control while the NeoAg vax did not when splenocytes were peptide stimulated. The FS vax and NeoAg vax induced T-cells were only significantly cytotoxic to the ID8 cells if anti-PD-L1/CTLA-4 antibodies were included. In the therapeutic vaccination protocol, both the FS vax and the NeoAg vax conferred extended survival in ~20% of the mice compared to controls. Neither vaccine induced more ELISpots compared to controls using peptides. However, the FS vax did elicit more ELISpots in response to ID8 stimulation and more ID8 cytotoxicity than the NeoAg vax. From this work we first conclude that the DNA-sourced neoantigens that did not protect individually can when pooled. Secondly, and more importantly, the RNA-sourced, FS neoantigens can perform as well as the DNA-sourced neoantigens, at least in this model. This may be important as we have shown that all tumors surveyed to date, in contrast to DNA neoantigens, have abundant RNA-sourced FS neoantigens. Citation Format: Milene Tavares Batista, Sierra Murphy, John Lainson, Lui Shen, Stephen Johnston. A comparison of a DNA-sourced neoantigen vaccine to an RNA-sourced frameshift vaccine in the mouse ovarian cancer model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A28.

Comprehensive mutanome analysis of Lewis lung cancer reveals immunogenic neoantigens for therapeutic vaccines

Personalized neoantigen vaccines are capable of eliciting vigorous T-cell responses and have been demonstrated to achieve striking therapeutic effects against cancer. Here we performed comprehensive mutanome analysis of the mouse Lewis lung cancer cells to identify tumor neoantigens followed by prediction of their MHC affinity and immunogenicity. We adopted a strategy that enables us to select neoantigens that were predicted to have high affinity to both MHC I and MHC II. Ten neoantigens were selected to synthesize peptide vaccines and tested in vivo for immunogenicity. Four neoantigen peptide vaccines were found to elicit robust immune reactivity and were further examined for tumor inhibition in mice with xenografted LLC tumors. Two neoantigen peptide vaccines showed significant inhibition on tumor growth and prolonged the survival of tumor-bearing mice. Our studies explored the neoantigen peptide vaccines to treat lung cancer and provide rationale for the optimization of tumor neoantigen selection for therapeutic vaccines.

Neoantigens in Hematologic Malignancies.

T cell cancer neoantigens are created from peptides derived from cancer-specific aberrant proteins, such as mutated and fusion proteins, presented in complex with human leukocyte antigens on the cancer cell surface. Because expression of the aberrant target protein is exclusive to malignant cells, immunotherapy directed against neoantigens should avoid “on-target, off-tumor” toxicity. The efficacy of neoantigen vaccines in melanoma and glioblastoma and of adoptive transfer of neoantigen-specific T cells in epithelial tumors indicates that neoantigens are valid therapeutic targets. Improvements in sequencing technology and innovations in antigen discovery approaches have facilitated the identification of neoantigens. In comparison to many solid tumors, hematologic malignancies have few mutations and thus fewer potential neoantigens. Despite this, neoantigens have been identified in a wide variety of hematologic malignancies. These include mutated nucleophosmin1 and PML-RARA in acute myeloid leukemia, ETV6-RUNX1 fusions and other mutated proteins in acute lymphoblastic leukemia, BCR-ABL1 fusions in chronic myeloid leukemia, driver mutations in myeloproliferative neoplasms, immunoglobulins in lymphomas, and proteins derived from patient-specific mutations in chronic lymphoid leukemias. We will review advances in the field of neoantigen discovery, describe the spectrum of identified neoantigens in hematologic malignancies, and discuss the potential of these neoantigens for clinical translation.

Personal tumor antigens in blood malignancies: genomics-directed identification and targeting.

Hematological malignancies have long been at the forefront of the development of novel immune-based treatment strategies. The earliest successful efforts originated from the extensive body of work in the field of allogeneic hematopoietic stem cell transplantation. These efforts laid the foundation for the recent exciting era of cancer immunotherapy, which includes immune checkpoint blockade, personal neoantigen vaccines, and adoptive T cell transfer. At the heart of the specificity of these novel strategies is the recognition of target antigens presented by malignant cells to T cells. Here, we review the advances in systematic identification of minor histocompatibility antigens and neoantigens arising from personal somatic alterations or recurrent driver mutations. These exciting efforts pave the path for the implementation of personalized combinatorial cancer therapy.