Neoantigen Vaccines Research Articles

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.

Immunogenicity of an AI-designed personalized neoantigen vaccine, EVX-01, in combination with anti-PD-1 therapy in patients with metastatic melanoma.

9561 Background: Personalized vaccines, targeting mutation-derived neoantigens (neoAg), represent a promising frontier in cancer immunotherapy. Here, we characterize the vaccine-induced immune response in seven melanoma patients treated with the AI-generated personalized cancer vaccine, EVX-01, in the ongoing single arm multicenter trial (NCT05309421). Data from an additional five patients will be available at the time of presentation. Methods: Tumor-specific neoAgs were identified and selected using the proprietary vaccine target discovery AI-Immunology platform based on tumor DNA- and RNA-sequencing data. The top-ranked neoAgs for each patient were manufactured as synthetic long peptides and formulated with an adjuvant, creating the personalized cancer vaccine, EVX-01, tailored to the individual tumor and immune system characteristics. Patients initiated anti-PD1 therapy (Pembrolizumab) 12 weeks prior to EVX-01 administration. Each patient received six priming doses of EVX-01 administered bi-weekly and will be followed by four booster immunizations at later timepoints. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples collected at different timepoints to evaluate the effect of anti-PD1 therapy, EVX-01 priming immunizations and EVX-01 booster immunizations on T-cell immunogenicity. NeoAg-specific T-cell responses were evaluated by IFN-γ ELISpot assay and intracellular cytokine staining after T cell in vitro expansion with vaccine neoAgs. Results: Immunogenicity analysis of PBMC samples from seven patients demonstrated that EVX-01 induced neoAg-specific immune responses in all patients after the priming phase. The effect of booster immunizations on neoAg-specific T-cell responses was analyzed in the patients who had reached these late visits of the study, and it demonstrated a clear trend towards increased immune responses after the first booster dose. The observed responses were mediated by both CD4+ and CD8+ neoAg-specific T-cells. Investigations of the response induced by the individual vaccine neoAgs revealed that the majority of the EVX-01 neoAgs triggered a specific T-cell response, affirming the ability of the AI-Immunology™ platform to precisely select effective vaccine targets. Data from an additional five patients will be available at the time of presentation. Importantly EVX-01 was safe and well-tolerated, with only grade 1 and 2 ADRs related to EVX-01 even after boosting. The combination of EVX-01 and anti-PD1 also appeared safe and well tolerated. Conclusions: EVX-01 induced neoAg-specific immune responses in all analyzed patients and a broad response against the neoAgs. These results further validate the precision and predictive power of our proprietary vaccine target discovery AI-Immunology platform. The combination of EVX-01 and anti-PD1 was safe and well tolerated. Clinical trial information: NCT05309421 .

Breaking the Mold: Trailblazing Melanoma Therapy Beyond Checkpoint Through Innovative Approaches.

Melanoma has long been a difficult malignancy to treat with low response rates to standard chemotherapies. In recent years, the use of immune checkpoint inhibitors have demonstrated promising results, paving the way for the use of the rapidly developing novel immune targeting therapies. In this review, we look beyond immune checkpoint inhibitor treatments and summarize several emerging treatment strategies for melanoma, including neoantigen vaccines, conventional antibody drug-conjugates, and bispecific T-cell engager therapies.

29P Characterization of T cell responses induced by the individualized mRNA neoantigen vaccine autogene cevumeran in adjuvant stage II (high risk)/stage III colorectal cancer (CRC) patients (pts) from the biomarker cohort of the phase II BNT122-01 trial

29P Characterization of T cell responses induced by the individualized mRNA neoantigen vaccine autogene cevumeran in adjuvant stage II (high risk)/stage III colorectal cancer (CRC) patients (pts) from the biomarker cohort of the phase II BNT122-01 trial

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.

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

e16264 Background: The primary treatment for hepatocellular carcinoma (HCC) is radical surgery. However, HCC has a high propensity to recur postoperatively and no standard adjuvant therapies have been approved. This Phase I trial aims to evaluate the safety and efficacy of an adjuvant 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. After obtaining the necessary baseline biopsy specimens and PBMCs of appropriate quality for DNA and RNA sequencing, neoantigen prediction, screening and synthesis were performed. Subsequently, leukapheresis was performed and DCs were isolated and cultured. DCs were then 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 to the axillary and inguinal regions bilaterally on day 1, followed by daily administration of GM-CSF at a dose of 0.075 mg for 5 days (days 2-6). The primary objectives of this study were to assess feasibility and safety/tolerability. Secondary objectives included evaluation of immune responses (via vaccine response: IFNγ ELISpot), relapse-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. Feasibility was demonstrated with 13 patients receiving Neo-DCVac-02. 13 patients did not receive the neoantigen vaccine (7 due to progressive disease, 6 due to withdrawal of informed consent because of COVID-19). The treatment was well tolerated with no grade 2+ treatment-related adverse events (TRAEs). The most common Grade 1 TRAEs were injection site reactions (75%), rash (16.7%), fatigue (8.3%), neutropenia (8.3%), and headache (8.3%). Five of 13 evaluable patients showed increased antigen-specific T-cell activity and more than 50% of the neoantigen peptides in Neo-DCVac-02, and sustained cellular responses were observed up to one year. Increases in T-cell activation/co-stimulation seen after treatment with Neo-DCVac-02, as demonstrated by flow cytometric analysis, suggest immune priming. At a median follow-up of 29.7 months, median RFS was not reached, with 1- and 2-year RFS of 84.6% and 60%, respectively, and all patients are alive. Patients with a strong immune response had a longer median relapse-free survival (not reached) compared to patients with a weak immune response (17.6 months, P = 0.003). Conclusions: These data demonstrate that Neo-DCVac-02 is safe, well tolerated and capable of inducing durable antigen-specific lymphocyte immune responses that may correlate with delayed HCC recurrence. Clinical trial information: NCT04147078 .

Neoantigen landscape supports feasibility of personalized cancer vaccine for follicular lymphoma

AbstractPersonalized cancer vaccines designed to target neoantigens represent a promising new treatment paradigm in oncology. In contrast to classical idiotype vaccines, we hypothesized that “polyvalent” vaccines could be engineered for the personalized treatment of follicular lymphoma (FL) using neoantigen discovery by combined whole-exome sequencing (WES) and RNA sequencing (RNA-seq). Fifty-eight tumor samples from 57 patients with FL underwent WES and RNA-seq. Somatic and B-cell clonotype neoantigens were predicted and filtered to identify high-quality neoantigens. B-cell clonality was determined by the alignment of B-cell receptor (BCR) CDR3 regions from RNA-seq data, grouping at the protein level, and comparison with the BCR repertoire from healthy individuals using RNA-seq data. An average of 52 somatic mutations per patient (range, 2-172) were identified, and ≥2 (median, 15) high-quality neoantigens were predicted for 56 of 58 FL samples. The predicted neoantigen peptides were composed of missense mutations (77%), indels (9%), gene fusions (3%), and BCR sequences (11%). Building off of these preclinical analyses, we initiated a pilot clinical trial using personalized neoantigen vaccination combined with PD-1 blockade in patients with relapsed or refractory FL (#NCT03121677). Synthetic long peptide vaccines targeting predicted high-quality neoantigens were successfully synthesized for and administered to all 4 patients enrolled. Initial results demonstrate feasibility, safety, and potential immunologic and clinical responses. Our study suggests that a genomics-driven personalized cancer vaccine strategy is feasible for patients with FL, and this may overcome prior challenges in the field. This trial was registered at www.ClinicalTrials.gov as #NCT03121677.

Dose escalation study of a personalized peptide-based neoantigen vaccine (EVX-01) in patients with metastatic melanoma

BackgroundNeoantigens can serve as targets for T cell-mediated antitumor immunity via personalized neopeptide vaccines. Interim data from our clinical study NCT03715985 showed that the personalized peptide-based neoantigen vaccine EVX-01, formulated...

Expanding the Potential of Neoantigen Vaccines: Harnessing Bacille Calmette-Guérin Cell-Wall-Based Nanoscale Adjuvants for Enhanced Cancer Immunotherapy.

Personalized antitumor immunotherapy utilizing neoantigen vaccines holds great promise. However, the limited immunogenicity of existing recognized neoantigens and the inadequate stimulation of antitumor immune responses by conventional adjuvants pose significant challenges. To address these limitations, we developed a nanovaccine that combines a BCG bacterial cell wall skeleton (BCG-CWS) based nanoscale adjuvant (BCNA) with peptide neoantigens (M27 and M30). This integrated approach provides an efficient translational strategy for cancer immunotherapy. The BCNA nanovaccine, formulated with PLGA as an emulsifier, exhibits excellent biocompatibility and superior antigen presentation compared with conventional BCG-CWS adjuvants. Subcutaneous immunization with the BCNA-based nanovaccine effectively targets lymph nodes, eliciting robust innate and tumor-specific immune responses. Importantly, our findings demonstrate that BCNAs significantly enhance neoantigen immunogenicity while minimizing acute systemic toxicity. Furthermore, when combined with a mouse PD-L1 antibody, our strategy achieves complete tumor elimination in 60% of cases and prevents 25% of tumor growth in a melanoma mouse model. In conclusion, our BCNA-based nanovaccine represents a promising avenue for advancing personalized therapeutic neoantigen vaccines and holds significant implications for enhancing personalized immunotherapy and improving patient outcomes in the field of cancer treatment.

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.

Abstract LB113: Immunogenicity of Atezolizumab plus personalized neoantigen vaccination (PGV-001) in patients with urothelial cancer in adjuvant vs metastatic setting

Abstract Background: Urothelial cancer (UC) of the genitourinary tract, most commonly arising from the bladder, has historically been a challenging area for the development of new therapeutics. Immune checkpoint blockade inhibitors (CBIs) induce durable responses only in a subset of patients with UC, potentially due to lack of neoantigen primed T cells. To build on this hypothesis, we designed a study to evaluate safety and immunogenicity of a combination treatment with atezolizumab and PGV-001, a personalized genomic neoantigen vaccine. Having previously reported clinical outcomes, here we describe analysis of immunogenicity induced by the treatment. Methods: In this phase I trial (NCT03359239) UC patients were enrolled to receive PGV-001 with atezolizumab in the adjuvant (undergone radical cystectomy or nephroureterectomy with a high risk of recurrence) or metastatic setting. Each PGV001 vaccine included up to 10 neoantigen (predicted using OpenVax pipeline) synthetic long peptides (SLPs). Each subject received 10 PGV-001 vaccines and 1200 mg i.v. atezolizumab every 3 weeks for up to 12 months (adjuvant) or 24 months (metastatic). To investigate T cell immunity, ex vivo IFNγ ELISPOT and T cell expansion assays were performed using 15mer overlapping peptide (OLP) pools corresponding to each neoantigen SLP. Results: Samples from 9 subjects (3 adjuvant and 6 metastatic) were evaluable for ex vivo ELISPOT analysis. In 100% of the subjects, at least 1 neoantigen was found to elicit an immune response. Overall, out of 87 evaluated neoantigens, 47% were immunogenic, with the immunogenic peptides inducing an average of 16.7 fold increase in ex vivo ELISPOT response over baseline. While 39% (11/28) of the peptides in adjuvant cohort were immunogenic vs 51% (39/50) in metastatic cohort, it is not possible to determine statistically significant difference in response between metastatic and adjuvant cohort. The T cell expansion assay was performed for 6 subjects, 3 each from the adjuvant and metastatic cohorts. Among 57 evaluated neoantigens, 46% and 54% of the peptides elicited a CD8+ and CD4+ T cell targeted immune response, respectively. A greater proportion of neoantigens induced both CD8+ and CD4+ T cell responses in subjects treated in the metastatic setting vs adjuvant setting. Upon epitope mapping, for 5 evaluable subjects, 141 OLPs were analyzed and a similar number of OLPs were found to be immunogenic for CD8+ (31.2%, 44/141) and CD4+ response (30%, 43/141). Conclusions: These data indicate that vaccination with personalized neoantigens in combination with systemic anti-PD-L1 can induce neoantigen-focused T cell responses in both adjuvant and metastatic disease settings. However, the single arm nature of the trial, and sample size, preclude definitive assessment of the contribution of the components of the combination therapy. Future studies will investigate the efficacy of this approach on a larger scale with pre- and on-treatment analyses of treatment-driven immunomodulation. Citation Format: Mansi Saxena, Jonathan Anker, Julia Kodysh, Timothy O'Donnell, Marcia Meseck, Olivia Hapanowicz, Scot Niglio, Hardik Shah, Yayoi Kinoshita, Rachel Brody, Alex Rubinsteyn, Robert Sebra, Nina Bhardwaj, Mathew Galsky. Immunogenicity of Atezolizumab plus personalized neoantigen vaccination (PGV-001) in patients with urothelial cancer in adjuvant vs metastatic setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB113.

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.

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.

Melanoma neoantigen vaccines: Are we getting more personal now?

KEYNOTE-9421 is a randomized phase II adjuvant study in patients with resected stage III melanoma investigating a personalized neoantigen mRNA vaccine in combination with anti-PD-1. The study gave a clear signal of superiority for the vaccine plus anti-PD-1 in relapse-free and distant-metastasis-free survival but is not yet conclusive, and important questions remain.

Personalized Neoantigen DNA Cancer Vaccines: Current Status and Future Perspectives

Tumor mutation-derived neoantigens are considered promising targets for cancer immunotherapy. Personalized vaccines have emerged as an approach to deliver neoantigens and thereby trigger the induction of specific T-cell responses that can find and eliminate tumor cells based on the cell-surface presence of neoantigens. To this end, several neoantigen vaccine formats have provided encouraging results in clinical trials, resulting in neoantigen immunogenicity and clinical benefit. DNA offers a versatile and safe platform to deliver neoantigens and immune stimulants in a single entity through vaccination. Herein, we provide an overview of how DNA vaccines are being used as a means to deliver personalized neoantigens to cancer patients. We summarize the developments in DNA vaccine formulation and delivery technologies that contribute to elicit robust immune responses after vaccination. We outline the main results from central preclinical and clinical investigations, showing that neoantigen DNA vaccines induce a specific immune response directed against tumor neoantigens. Lastly, we discuss the opportunities and challenges for neoantigen DNA vaccines as an individualized approach to immunotherapy of cancer.

Integrating Reinforcement Learning and Monte Carlo Tree Search for enhanced neoantigen vaccine design.

Recent advances in cancer immunotherapy have highlighted the potential of neoantigen-based vaccines. However, the design of such vaccines is hindered by the possibility of weak binding affinity between the peptides and the patient's specific human leukocyte antigen (HLA) alleles, which may not elicit a robust adaptive immune response. Triggering cross-immunity by utilizing peptide mutations that have enhanced binding affinity to target HLA molecules, while preserving their homology with the original one, can be a promising avenue for neoantigen vaccine design. In this study, we introduced UltraMutate, a novel algorithm that combines Reinforcement Learning and Monte Carlo Tree Search, which identifies peptide mutations that not only exhibit enhanced binding affinities to target HLA molecules but also retains a high degree of homology with the original neoantigen. UltraMutate outperformed existing state-of-the-art methods in identifying affinity-enhancing mutations in an independent test set consisting of 3660 peptide-HLA pairs. UltraMutate further showed its applicability in the design of peptide vaccines for Human Papillomavirus and Human Cytomegalovirus, demonstrating its potential as a promising tool in the advancement of personalized immunotherapy.

Nanomaterial Delivery Vehicles for the Development of Neoantigen Tumor Vaccines for Personalized Treatment.

Tumor vaccines have been considered a promising therapeutic approach for treating cancer in recent years. With the development of sequencing technologies, tumor vaccines based on neoantigens or genomes specifically expressed in tumor cells, mainly in the form of peptides, nucleic acids, and dendritic cells, are beginning to receive widespread attention. Therefore, in this review, we have introduced different forms of neoantigen vaccines and discussed the development of these vaccines in treating cancer. Furthermore, neoantigen vaccines are influenced by factors such as antigen stability, weak immunogenicity, and biosafety in addition to sequencing technology. Hence, the biological nanomaterials, polymeric nanomaterials, inorganic nanomaterials, etc., used as vaccine carriers are principally summarized here, which may contribute to the design of neoantigen vaccines for improved stability and better efficacy.

Abstract 6755: NKT activation enhanced the therapeutic efficacy of neoantigen vaccine in hepatocellular carcinoma

Abstract One sentence introduction: This study indicated the potential of combinational therapy of neoantigen vaccine and iNKT activator α-GalCer in Hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is a leading cause of death around the world. Current immune checkpoint blockade therapies only show effectiveness in some cancer patients, so there is an urgent need for more effective immunotherapy strategies. One alternative is the use of neoantigen-based cancer vaccines, which target tumor-specific antigens (neoantigens) on cancer cells and induce strong T-cell responses. Advances in technology now allow for precise identification of tumor neoantigens using bioinformatic analysis or mass spectrometry. While cancer vaccines have demonstrated strong anti-tumor immune responses and therapeutic effectiveness in some cancer types, their success in HCC remains limited. A contributing factor is the suppressive tumor microenvironment (TME) found in advanced HCC. Some studies suggest that combining neoantigen vaccines with anti-PD1/PD-L1 therapies can significantly enhance HCC treatment outcomes. Overcoming the suppressive immune microenvironment is crucial in cancer treatment, and reactivating intratumoral immunity is essential. In our study, we developed an HCC cancer vaccine using transcriptome sequencing and bioinformatics analysis. Our ELISpot assay results showed that this vaccine induced strong tumor-specific immune responses and significantly inhibited tumor growth in early-stage orthotopic HCC mouse models when administered five days after tumor inoculation. However, the effectiveness decreased in advanced tumors when administered ten days after tumor inoculation. We also discovered a significant relationship between the therapeutic effectiveness of the neoantigen vaccine and the activation of intratumoral NKT cells in HCC. Our study suggested that the type-I NKT cell activator, α-Galactosylceramide (α-GalCer), significantly improved the therapeutic outcomes of the cancer neoantigen vaccine in advanced orthotopic HCC mouse models. In some cases, tumors were almost completely eliminated. This indicates the potential of combining iNKT activators with neoantigen vaccines for HCC treatment. To further understand the role of NKT cells in HCC's TME, we plan to investigate intratumoral NKT cell subtypes following neoantigen vaccine therapy. Additionally, we will develop a new delivery system to deliver type I NKT cell activators and type II NKT inhibitors to tumor sites, aiming to reactivate the suppressive TME in HCC after neoantigen vaccine therapy. Citation Format: Renhao Li, Jing-Chu Hu, Li Rong, Yangfan Wu, Yige He, Jiandong Huang. NKT activation enhanced the therapeutic efficacy of neoantigen vaccine in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6755.

Abstract 4955: An analytic pipeline of neoantigen selection for both MHC-I and MHC-II pathways

Abstract Background: Accurate neoantigen selection is pivotal for assessing mutation burden and optimizing cancer vaccine formulation thereby playing a crucial role in cancer immunotherapy. The concurrent activation of CD4+ and CD8+ T cells engenders a robust immune response, which is key to effective treatment. However, currently analytic tools predominantly focus on the MHC-I pathway, typically relying on metrics such as gene expression level and MHC-binding affinity of mutated peptides. To address this gap, our study presents a comprehensive pipeline that encompasses both MHC-I and MHC-II pathways and integrates three critical dimensions of immune response: neoantigen abundance, presentation, and immunogenicity. Methods: We estimated mutated peptide abundance by analyzing genomic allelic frequency, transcriptomic allelic frequency, and RNA expression level. Antigen presentation was assessed using the patient harmonic binding rank (PHBR, a composite binding score for all potential MHC-peptide pairs) and MHC diversity (the variety of MHC alleles involved in peptide binding), with additional consideration for the loss of heterozygosity of MHC-I alleles, potentially indicative of immune escape. For immunogenicity, we factored in agretopicity (comparison of MHC-binding affinity between mutated and wild-type peptides), foreignness (similarity to immunogenic foreign antigens), and dissimilarity (sequence disparity with self-peptides, reflecting T cell negative selection process). Results: Implementing our pipeline on a dataset from Parkhurst et al. (2019), we analyzed immune reactivity in relation to 11,537 somatic mutations from 75 gastrointestinal cancer patients. Key metrics—RNA expression, transcriptomic allelic frequencies, PHBR, and MHC diversity—demonstrated significant correlations with both CD4+ and CD8+ activity (p<0.05). Notably, genomic allelic frequency was a significant factor in CD8+ reactivity, whereas agretopicity was crucial for CD4+ reactivity. Conclusion: Our pipeline elucidates the necessity for implementing both MHC-I and MHC-II pathway analyses to effectively select neoantigen candidates for CD8+ and CD4+ T cells respectively. The study confirms that abundance and antigen presentation metrics are robust indicators of neoantigen immunogenicity, supporting their use as potential markers in crafting more effective cancer vaccines. The findings highlight the critical role of this dual-pathway selection approach in generating a comprehensive immune response through neoantigen vaccination. Citation Format: Ko-Han Lee, Timothy Sears, Andrea Castro, Maurizio Zanetti, Hannah Carter. An analytic pipeline of neoantigen selection for both MHC-I and MHC-II pathways [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 4955.

Abstract 6752: Targeting of neoantigen cancer vaccines to antigen presenting cells results in faster and broader T cell responses whether encoded in DNA or mRNA

Abstract Individualized cancer vaccines based on patient-specific neoantigens represent a groundbreaking approach in oncology, offering a highly individualized treatment option for cancer patients. Unlike traditional vaccines, which are designed to prevent diseases by stimulating an immune response against a specific pathogen, personalized cancer vaccines are therapeutic, aiming to treat cancer by training the immune system to recognize and attack the patient’s own cancer cells. The key is to induce a fast, strong, broad, and persistent CD8+ T cell response. Nykode Therapeutics has developed a platform that targets antigens directly to antigen presenting cells (APCs) using a modular dimeric protein format known as a VaccibodyTM. Individualized neoantigen vaccines delivered in a DNA-encoded Vaccibody format have been shown to induce strong and broad T cell responses in pre-clinical and clinical settings. Here, we wanted to determine if the APC-targeted approach leads to superior T cell responses if Vaccibody vaccines are encoded in mRNA and administered in a lipid nanoparticle (LNP) formulation. For this purpose, we designed and evaluated a model cancer vaccine composed of 20 unstructured 27 amino acid long neoantigens derived from the murine MC38 tumor model. A DNA-encoded Vaccibody vaccine elicited faster T cell responses compared to an untargeted mRNA-LNP-encoded vaccine. DNA-encoded Vaccibody responses were also broader, targeting a substantially greater number of neoantigens. Next, we compared the APC-targeting Vaccibody vaccine to an untargeted vaccine when both were formulated in an mRNA-LNP format. The APC-targeting Vaccibody vaccine elicited a greater breath and magnitude of T cell responses compared to the untargeted vaccine. Further studies to evaluate the functional differences associated with the various formats, in terms of T cell phenotype and response to tumor challenge, are ongoing. In summary, the data demonstrate that an APC-targeted Vaccibody vaccine containing MC38 neoantigens can be encoded in both DNA and mRNA-LNP to induce faster, broader, and stronger T cell responses compared to a non-targeted neoantigen vaccine. Citation Format: Brian T. Weinert, Lise Skullerud, Eirik Solbakken, Ana Textor, Mirjam Dürkoop, Inga Patrycja Winge, Gunnstein Norheim, Elisabeth Stubsrud, Stine Granum, Mikkel W. Pedersen, Håkan Norell. Targeting of neoantigen cancer vaccines to antigen presenting cells results in faster and broader T cell responses whether encoded in DNA or mRNA [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 6752.