Simultaneous analysis of pMHC binding and reactivity unveils virus-specific CD8 T cell immunity to a concise epitope set.

CD8 T cells provide immunity to virus infection through recognition of epitopes presented by peptide major histocompatibility complexes (pMHCs). To establish a concise panel of widely recognized T cell epitopes from common viruses, we combined analysis of TCR down-regulation upon stimulation with epitope-specific enumeration based on barcode-labeled pMHC multimers. We assess CD8 T cell binding and reactivity for 929 previously reported epitopes in the context of 1 of 25 HLA alleles representing 29 viruses. The prevalence and magnitude of CD8 T cell responses were evaluated in 48 donors and reported along with 137 frequently recognized virus epitopes, many of which were underrepresented in the public domain. Eighty-four percent of epitope-specific CD8 T cell populations demonstrated reactivity to peptide stimulation, which was associated with effector and long-term memory phenotypes. Conversely, nonreactive T cell populations were associated primarily with naive phenotypes. Our analysis provides a reference map of epitopes for characterizing CD8 T cell responses toward common human virus infections.

IMPROVE: a feature model to predict neoepitope immunogenicity through broad-scale validation of T-cell recognition.

Background: Mutation-derived neoantigens are critical targets for tumor rejection in cancer immunotherapy, and better tools for neoepitope identification and prediction are needed to improve neoepitope targeting strategies. Computational tools have enabled the identification of patient-specific neoantigen candidates from sequencing data, but limited data availability has hindered their capacity to predict which of the many neoepitopes will most likely give rise to T cell recognition. Method: To address this, we make use of experimentally validated T cell recognition towards 17,500 neoepitope candidates, with 467 being T cell recognized, across 70 cancer patients undergoing immunotherapy. Results: We evaluated 27 neoepitope characteristics, and created a random forest model, IMPROVE, to predict neoepitope immunogenicity. The presence of hydrophobic and aromatic residues in the peptide binding core were the most important features for predicting neoepitope immunogenicity. Conclusion: Overall, IMPROVE was found to significantly advance the identification of neoepitopes compared to other current methods.

T antigen-specific CD8+ T cells associate with PD-1 blockade response in virus-positive Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel cell polyomavirus, which is driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the nonresponding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class I haplotypes. We observed a broad T cell recognition of T-Ags, including identification of 20 T-Ag-derived epitopes we believe to be novel. Broadening of the T-Ag recognition profile and increased T cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor-rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.

Cross-reactive CD8+ T cell responses to tumor-associated antigens (TAAs) and homologous microbiota-derived antigens (MoAs).

BACKGROUND: We have recently shown extensive sequence and conformational homology between tumor-associated antigens (TAAs) and antigens derived from microorganisms (MoAs). The present study aimed to assess the breadth of T-cell recognition specific to MoAs and the corresponding TAAs in healthy subjects (HS) and patients with cancer (CP). METHOD: A library of > 100 peptide-MHC (pMHC) combinations was used to generate DNA-barcode labelled multimers. Homologous peptides were selected from the Cancer Antigenic Peptide Database, as well as Bacteroidetes/Firmicutes-derived peptides. They were incubated with CD8 + T cells from the peripheral blood of HLA-A*02:01 healthy individuals (n = 10) and cancer patients (n = 16). T cell recognition was identified using tetramer-staining analysis. Cytotoxicity assay was performed using as target cells TAP-deficient T2 cells loaded with MoA or the paired TuA. RESULTS: A total of 66 unique pMHC recognized by CD8+ T cells across all groups were identified. Of these, 21 epitopes from microbiota were identified as novel immunological targets. Reactivity against selected TAAs was observed for both HS and CP. pMHC tetramer staining confirmed CD8+ T cell populations cross-reacting with CTA SSX2 and paired microbiota epitopes. Moreover, PBMCs activated with the MoA where shown to release IFNγ as well as to exert cytotoxic activity against cells presenting the paired TuA. CONCLUSIONS: Several predicted microbiota-derived MoAs are recognized by T cells in HS and CP. Reactivity against TAAs was observed also in HS, primed by the homologous bacterial antigens. CD8+ T cells cross-reacting with MAGE-A1 and paired microbiota epitopes were identified in three subjects. Therefore, the microbiota can elicit an extensive repertoire of natural memory T cells to TAAs, possibly able to control tumor growth ("natural anti-cancer vaccination"). In addition, non-self MoAs can be included in preventive/therapeutic off-the-shelf cancer vaccines with more potent anti-tumor efficacy than those based on TAAs.

Herpes Virus Infections in Kidney Transplant Patients (HINT) - a prospective observational cohort study.

BACKGROUND: Kidney transplant recipients receive maintenance immunosuppressive therapy to avoid allograft rejection resulting in increased risk of infections and infection-related morbidity and mortality. Approximately 98% of adults are infected with varicella zoster virus, which upon reactivation causes herpes zoster. The incidence of herpes zoster is higher in kidney transplant recipients than in immunocompetent individuals, and kidney transplant recipients are at increased risk of severe herpes zoster-associated disease. Vaccination with adjuvanted recombinant glycoprotein E subunit herpes zoster vaccine (RZV) prevents herpes zoster in older adults with excellent efficacy (90%), and vaccination of kidney transplant candidates is recommended in Danish and international guidelines. However, the robustness and duration of immune responses after RZV vaccination, as well as the optimal timing of vaccination in relation to transplantation remain unanswered questions. Thus, the aim of this study is to characterize the immune response to RZV vaccination in kidney transplant candidates and recipients at different timepoints before and after transplantation. METHODS: The Herpes Virus Infections in Kidney Transplant Patients (HINT) study is a prospective observational cohort study. The study will include kidney transplant candidates on the waiting list for transplantation (n = 375) and kidney transplant recipients transplanted since January 1, 2019 (n = 500) from all Danish kidney transplant centers who are offered a RZV vaccine as routine care. Participants are followed with repeated blood sampling until 12 months after inclusion. In the case of transplantation or herpes zoster disease, additional blood samples will be collected until 12 months after transplantation. The immune response will be characterized by immunophenotyping and functional characterization of varicella zoster virus-specific T cells, by detection of anti-glycoprotein E antibodies, and by measuring cytokine profiles. DISCUSSION: The study will provide new knowledge on the immune response to RZV vaccination in kidney transplant candidates and recipients and the robustness and duration of the response, potentially enhancing preventive strategies against herpes zoster in a population at increased risk. TRIAL REGISTRATION: ClinicalTrials.gov (NCT05604911).

TCR-engaging scaffolds selectively expand antigen-specific T-cells with a favorable phenotype for adoptive cell therapy.

BACKGROUND: Adoptive cell therapy (ACT) has shown promising results for the treatment of cancer and viral infections. Successful ACT relies on ex vivo expansion of large numbers of desired T-cells with strong cytotoxic capacity and in vivo persistence, which constitutes the greatest challenge to current ACT strategies. Here, in this study, we present a novel technology for ex vivo expansion of antigen-specific T-cells; artificial antigen-presenting scaffolds (Ag-scaffolds) consisting of a dextran-polysaccharide backbone, decorated with combinations of peptide-Major Histocompatibility Complex (pMHC), cytokines and co-stimulatory molecules, enabling coordinated stimulation of antigen-specific T-cells. METHODS: The capacity of Ag-scaffolds to expand antigen-specific T-cells was explored in ex vivo cultures with peripheral blood mononuclear cells from healthy donors and patients with metastatic melanoma. The resulting T-cell products were assessed for phenotypic and functional characteristics. RESULTS: We identified an optimal Ag-scaffold for expansion of T-cells for ACT, carrying pMHC and interleukin-2 (IL-2) and IL-21, with which we efficiently expanded both virus-specific and tumor-specific CD8+ T cells from peripheral blood of healthy donors and patients, respectively. The resulting T-cell products were characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion, a multifunctional cytokine profile upon antigen-challenge and superior tumor killing capacity. This demonstrates that the coordinated stimuli provided by an optimized stoichiometry of TCR engaging (pMHC) and stimulatory (cytokine) moieties is essential to obtain desired T-cell characteristics. To generate an 'off-the-shelf' multitargeting Ag-scaffold product of relevance to patients with metastatic melanoma, we identified the 30 most frequently recognized shared HLA-A0201-restricted melanoma epitopes in a cohort of 87 patients. By combining these in an Ag-scaffold product, we were able to expand tumor-specific T-cells from 60-70% of patients with melanoma, yielding a multitargeted T-cell product with up to 25% specific and phenotypically and functionally improved T cells. CONCLUSIONS: Taken together, the Ag-scaffold represents a promising new technology for selective expansion of antigen-specific CD8+ T cells directly from blood, yielding a highly specific and functionally enhanced T-cell product for ACT.

Engineered E. coli Nissle 1917 for delivery of bioactive IL-2 for cancer immunotherapy.

In this study we performed a step-wise optimization of biologically active IL-2 for delivery using E. coli Nissle 1917. Engineering of the strain was coupled with an in vitro cell assay to measure the biological activity of microbially produced IL-2 (mi-IL2). Next, we assessed the immune modulatory potential of mi-IL2 using a 3D tumor spheroid model demonstrating a strong effect on immune cell activation. Finally, we evaluated the anticancer properties of the engineered strain in a murine CT26 tumor model. The engineered strain was injected intravenously and selectively colonized tumors. The treatment was well-tolerated, and tumors of treated mice showed a modest reduction in tumor growth rate, as well as significantly elevated levels of IL-2 in the tumor. This work demonstrates a workflow for researchers interested in engineering E. coli Nissle for a new class of microbial therapy against cancer.

Ex vivo modulation of intact tumor fragments with anti-PD-1 and anti-CTLA-4 influences the expansion and specificity of tumor-infiltrating lymphocytes.

Checkpoint inhibition (CPI) therapy and adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL-based ACT) are the two most effective immunotherapies for the treatment of metastatic melanoma. While CPI has been the dominating therapy in the past decade, TIL-based ACT is beneficial for individuals even after progression on previous immunotherapies. Given that notable differences in response have been made when used as a subsequent treatment, we investigated how the qualities of TILs changed when the ex vivo microenvironment of intact tumor fragments were modulated with checkpoint inhibitors targeting programmed death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Initially, we show that unmodified TILs from CPI-resistant individuals can be produced, are overwhelmingly terminally differentiated, and are capable of responding to tumor. We then investigate these properties in ex vivo checkpoint modulated TILs finding that that they retain these qualities. Lastly, we confirmed the specificity of the TILs to the highest responding tumor antigens, and identified this reactivity resides largely in CD39+CD69+ terminally differentiated populations. Overall, we found that anti-PD-1 will alter the proliferative capacity while anti-CTLA4 will influence breadth of antigen specificity.

DNA based neoepitope vaccination induces tumor control in syngeneic mouse models.

Recent findings have positioned tumor mutation-derived neoepitopes as attractive targets for cancer immunotherapy. Cancer vaccines that deliver neoepitopes via various vaccine formulations have demonstrated promising preliminary results in patients and animal models. In the presented work, we assessed the ability of plasmid DNA to confer neoepitope immunogenicity and anti-tumor effect in two murine syngeneic cancer models. We demonstrated that neoepitope DNA vaccination led to anti-tumor immunity in the CT26 and B16F10 tumor models, with the long-lasting presence of neoepitope-specific T-cell responses in blood, spleen, and tumors after immunization. We further observed that engagement of both the CD4+ and CD8+ T cell compartments was essential to hamper tumor growth. Additionally, combination therapy with immune checkpoint inhibition provided an additive effect, superior to either monotherapy. DNA vaccination offers a versatile platform that allows the encoding of multiple neoepitopes in a single formulation and is thus a feasible strategy for personalized immunotherapy via neoepitope vaccination.

Improved T cell receptor antigen pairing through data-driven filtering of sequencing information from single cells.

Novel single-cell-based technologies hold the promise of matching T cell receptor (TCR) sequences with their cognate peptide-MHC recognition motif in a high-throughput manner. Parallel capture of TCR transcripts and peptide-MHC is enabled through the use of reagents labeled with DNA barcodes. However, analysis and annotation of such single-cell sequencing (SCseq) data are challenged by dropout, random noise, and other technical artifacts that must be carefully handled in the downstream processing steps. We here propose a rational, data-driven method termed ITRAP (improved T cell Receptor Antigen Paring) to deal with these challenges, filtering away likely artifacts, and enable the generation of large sets of TCR-pMHC sequence data with a high degree of specificity and sensitivity, thus outputting the most likely pMHC target per T cell. We have validated this approach across 10 different virus-specific T cell responses in 16 healthy donors. Across these samples, we have identified up to 1494 high-confident TCR-pMHC pairs derived from 4135 single cells.

Neoepitope load, T cell signatures and PD-L2 as combined biomarker strategy for response to checkpoint inhibition immunotherapy.

Immune checkpoint inhibition for the treatment of cancer has provided a breakthrough in oncology, and several new checkpoint inhibition pathways are currently being investigated regarding their potential to provide additional clinical benefit. However, only a fraction of patients respond to such treatment modalities, and there is an urgent need to identify biomarkers to rationally select patients that will benefit from treatment. In this study, we explore different tumor associated characteristics for their association with favorable clinical outcome in a diverse cohort of cancer patients treated with checkpoint inhibitors. We studied 29 patients in a basket trial comprising 12 different tumor types, treated with 10 different checkpoint inhibition regimens. Our analysis revealed that even across this diverse cohort, patients achieving clinical benefit had significantly higher neoepitope load, higher expression of T cell signatures, and higher PD-L2 expression, which also correlated with improved progression-free and overall survival. Importantly, the combination of biomarkers serves as a better predictor than each of the biomarkers alone. Basket trials are frequently used in modern immunotherapy trial design, and here we identify a set of biomarkers of potential relevance across multiple cancer types, allowing for the selection of patients that most likely will benefit from immune checkpoint inhibition.

Herpesvirus immunology in solid organ transplant recipients - liver transplant study (HISTORY): a retrospective and prospective observational cohort study.

BACKGROUND: Life-long immunosuppressive treatment after liver transplantation (LT) prevents graft rejection but predisposes the LT recipient to infections. Herpesvirus infections are associated with morbidity and mortality among LT recipients. Among those, especially cytomegalovirus (CMV) and varicella-zoster virus (VZV) pose challenges after LT. The aim of this study is to provide an in-depth characterization of the cellular immune response against CMV and VZV infections in LT recipients and identify potential risk factors for infection. METHODS: The Herpesvirus Infections in Solid Organ Transplant Recipients - Liver Transplant Study (HISTORY) consists of an epidemiological and immunological substudy. The epidemiological substudy is a retrospective observational cohort study that includes all patients who underwent LT in Denmark between 2010 and 2023 (N ≈ 500). Using data from nationwide hospital records and national health registries, the incidence of and clinical risk factors for CMV and VZV infections will be determined. The immunological substudy is an explorative prospective observational cohort study including patients enlisted for LT in Denmark during a 1.5-year period (N > 80). Participants will be followed with scheduled blood samples until 12 months after LT. CMV- and VZV-derived peptides will be predicted for their likelihood to be presented in participants based on their HLA type. Peptide-MHC complexes (pMHC) will be produced to isolate CMV- and VZV-specific T cells from peripheral blood mononuclear cells before and after CMV and VZV infection. Their frequency, T cell receptor sequences, and phenotypic characteristics will be examined, and in a subset of participants, CMV- and VZV-specific T cells will be expanded ex vivo. DISCUSSION: This study will provide novel insight into T cell immunity required for viral control of CMV and VZV and has the potential to develop a prediction model to identify LT recipients at high risk for infection based on a combination of clinical and immunological data. Furthermore, this study has the potential to provide proof-of-concept for adoptive T cell therapy against CMV and VZV. Combined, this study has the potential to reduce the burden and consequence of CMV and VZV infections and improve health and survival in LT recipients. TRIAL REGISTRATION: ClinicalTrials.gov (NCT05532540), registered 8 September 2022.

First in man study: Bcl-Xl_42-CAF®09b vaccines in patients with locally advanced prostate cancer.

Background: The B-cell lymphoma-extra-large (Bcl-XL) protein plays an important role in cancer cells' resistance to apoptosis. Pre-clinical studies have shown that vaccination with Bcl-XL-derived peptides can induce tumor-specific T cell responses that may lead to the elimination of cancer cells. Furthermore, pre-clinical studies of the novel adjuvant CAF®09b have shown that intraperitoneal (IP) injections of this adjuvant can improve the activation of the immune system. In this study, patients with hormone-sensitive prostate cancer (PC) received a vaccine consisting of Bcl-XL-peptide with CAF®09b as an adjuvant. The primary aim was to evaluate the tolerability and safety of IP and intramuscular (IM) administration, determine the optimal route of administration, and characterize vaccine immunogenicity. Patients and methods: Twenty patients were included. A total of six vaccinations were scheduled: in Group A (IM to IP injections), ten patients received three vaccines IM biweekly; after a three-week pause, patients then received three vaccines IP biweekly. In Group B (IP to IM injections), ten patients received IP vaccines first, followed by IM under a similar vaccination schedule. Safety was assessed by logging and evaluating adverse events (AE) according to Common Terminology Criteria for Adverse Events (CTCAE v. 4.0). Vaccines-induced immune responses were analyzed by Enzyme-Linked Immunospot and flow cytometry. Results: No serious AEs were reported. Although an increase in T cell response against the Bcl-XL-peptide was found in all patients, a larger proportion of patients in group B demonstrated earlier and stronger immune responses to the vaccine compared to patients in group A. Further, we demonstrated vaccine-induced immunity towards patient-specific CD4, and CD8 T cell epitopes embedded in Bcl-XL-peptide and an increase in CD4 and CD8 T cell activation markers CD107a and CD137 following vaccination. At a median follow-up of 21 months, no patients had experienced clinically significant disease progression. Conclusion: The Bcl-XL-peptide-CAF®09b vaccination was feasible and safe in patients with l hormone-sensitive PC. In addition, the vaccine was immunogenic and able to elicit CD4 and CD8 T cell responses with initial IP administration eliciting early and high levels of vaccine-specific responses in a higher number og patients. Clinical trial registration: https://clinicaltrials.gov, identifier NCT03412786.

Safety, Outcomes, and T-Cell Characteristics in Patients with Relapsed or Refractory MDS or CMML Treated with Atezolizumab in Combination with Guadecitabine.

PURPOSE: We hypothesized that resistance to hypomethylating agents (HMA) among patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) would be overcome by combining a programmed death-ligand 1 antibody with an HMA. PATIENTS AND METHODS: We conducted a Phase I/II, multicenter clinical trial for patients with MDS not achieving an International Working Group response after at least 4 cycles of an HMA ("refractory") or progressing after a response ("relapsed") with 3+ or higher risk MDS by the revised International Prognostic Scoring System (IPSS-R) and CMML-1 or -2. Phase I consisted of a 3+3 dose-escalation design beginning with guadecitabine at 30 mg/m2 and escalating to 60 mg/m2 Days 1 to 5 with fixed-dose atezolizumab: 840 mg intravenously Days 8 and 22 of a 28-day cycle. Primary endpoints were safety and tolerability; secondary endpoints were overall response rate (ORR) and survival. RESULTS: Thirty-three patients, median age 73 (range 54-85), were treated. Thirty patients had MDS and 3 had CMML, with 30% relapsed and 70% refractory. No dose-limiting toxicities were observed in Phase I. There were 3 (9%) deaths in ≤ 30 days. Five patients (16%) came off study for drug-related toxicity. Immune-related adverse events (IRAE) occurred in 12 (36%) patients (4 grade 3, 3 grade 2, and 5 grade1). ORR was 33% [95% confidence interval (CI), 19%-52%] with 2 complete remission (CR), 3 hematologic improvement, 5 marrow CR, and 1 partial remission. Median overall survival was 15.1 (95% CI, 8.5-25.3) months. CONCLUSIONS: Guadecitabine with atezolizumab has modest efficacy with manageable IRAEs and typical cytopenia-related safety concerns for patients with relapsed or refractory MDS and CMML.

Dynamics of Melanoma-Associated Epitope-Specific CD8+ T Cells in the Blood Correlate With Clinical Outcome Under PD-1 Blockade.

Immune checkpoint blockade (ICB) is standard-of-care for patients with metastatic melanoma. It may re-invigorate T cells recognizing tumors, and several tumor antigens have been identified as potential targets. However, little is known about the dynamics of tumor antigen-specific T cells in the circulation, which might provide valuable information on ICB responses in a minimally invasive manner. Here, we investigated individual signatures composed of up to 167 different melanoma-associated epitope (MAE)-specific CD8+ T cells in the blood of stage IV melanoma patients before and during anti-PD-1 treatment, using a peptide-loaded multimer-based high-throughput approach. Additionally, checkpoint receptor expression patterns on T cell subsets and frequencies of myeloid-derived suppressor cells and regulatory T cells were quantified by flow cytometry. Regression analysis using the MAE-specific CD8+ T cell populations was applied to identify those that correlated with overall survival (OS). The abundance of MAE-specific CD8+ T cell populations, as well as their dynamics under therapy, varied between patients. Those with a dominant increase of these T cell populations during PD-1 ICB had a longer OS and progression-free survival than those with decreasing or balanced signatures. Patients with a dominantly increased MAE-specific CD8+ T cell signature also exhibited an increase in TIM-3+ and LAG-3+ T cells. From these results, we created a model predicting improved/reduced OS by combining data on dynamics of the three most informative MAE-specific CD8+ T cell populations. Our results provide insights into the dynamics of circulating MAE-specific CD8+ T cell populations during ICB, and should contribute to a better understanding of biomarkers of response and anti-cancer mechanisms.

Neoantigen-specific CD8 T cell responses in the peripheral blood following PD-L1 blockade might predict therapy outcome in metastatic urothelial carcinoma.

CD8+ T cell reactivity towards tumor mutation-derived neoantigens is widely believed to facilitate the antitumor immunity induced by immune checkpoint blockade (ICB). Here we show that broadening in the number of neoantigen-reactive CD8+ T cell (NART) populations between pre-treatment to 3-weeks post-treatment distinguishes patients with controlled disease compared to patients with progressive disease in metastatic urothelial carcinoma (mUC) treated with PD-L1-blockade. The longitudinal analysis of peripheral CD8+ T cell recognition of patient-specific neopeptide libraries consisting of DNA barcode-labelled pMHC multimers in a cohort of 24 patients from the clinical trial NCT02108652 also shows that peripheral NARTs derived from patients with disease control are characterised by a PD1+ Ki67+ effector phenotype and increased CD39 levels compared to bystander bulk- and virus-antigen reactive CD8+ T cells. The study provides insights into NART characteristics following ICB and suggests that early-stage NART expansion and activation are associated with response to ICB in patients with mUC.

Personalized therapy with peptide-based neoantigen vaccine (EVX-01) including a novel adjuvant, CAF®09b, in patients with metastatic melanoma.

The majority of neoantigens arise from unique mutations that are not shared between individual patients, making neoantigen-directed immunotherapy a fully personalized treatment approach. Novel technical advances in next-generation sequencing of tumor samples and artificial intelligence (AI) allow fast and systematic prediction of tumor neoantigens. This study investigates feasibility, safety, immunity, and anti-tumor potential of the personalized peptide-based neoantigen vaccine, EVX-01, including the novel CD8+ T-cell inducing adjuvant, CAF®09b, in patients with metastatic melanoma (NTC03715985). The AI platform PIONEERTM was used for identification of tumor-derived neoantigens to be included in a peptide-based personalized therapeutic cancer vaccine. EVX-01 immunotherapy consisted of 6 administrations with 5-10 PIONEERTM-predicted neoantigens as synthetic peptides combined with the novel liposome-based Cationic Adjuvant Formulation 09b (CAF®09b) to strengthen T-cell responses. EVX-01 was combined with immune checkpoint inhibitors to augment the activity of EVX-01-induced immune responses. The primary endpoint was safety, exploratory endpoints included feasibility, immunologic and objective responses. This interim analysis reports the results from the first dose-level cohort of five patients. We documented a short vaccine manufacturing time of 48-55 days which enabled the initiation of EVX-01 treatment within 60 days from baseline biopsy. No severe adverse events were observed. EVX-01 elicited long-lasting EVX-01-specific T-cell responses in all patients. Competitive manufacturing time was demonstrated. EVX-01 was shown to be safe and able to elicit immune responses targeting tumor neoantigens with encouraging early indications of a clinical and meaningful antitumor efficacy, warranting further study.

Epigenetic therapy in combination with a multi-epitope cancer vaccine targeting shared tumor antigens for high-risk myelodysplastic syndrome - a phase I clinical trial.

BACKGROUND: Standard care for patients with high-risk myelodysplastic syndrome (MDS) is hypomethylating agents such as azacitidine (AZA), which can induce expression of methylated tumor-associated antigens and therefore potentiate immunotherapeutic targeting. METHOD: In this phase 1 trial, we combined AZA with a therapeutic peptide vaccine targeting antigens encoded from NY-ESO-1, MAGE-A3, PRAME, and WT-1, which have previously been demonstrated to be upregulated by AZA treatment. RESULT: Five patients who had responded to AZA monotherapy were included in the study and treated with the vaccine. The combination therapy showed only few adverse events during the study period, whereof none classified as serious. However, no specific immune responses could be detected using intracellular cytokine staining or ELISpot assays. Minor changes in the phenotypic composition of immune cells and their expression of stimulatory and inhibitory markers were detected. All patients progressed to AML with a mean time to progression from inclusion (TTP) of 5.2 months (range 2.8 to 7.6). Mean survival was 18.1 months (range 10.9 to 30.6) from MDS diagnosis and 11.3 months (range 4.3 to 22.2) from inclusion. Sequencing of bone marrow showed clonal expansion of malignant cells, as well as appearance of novel mutations. CONCLUSION: The patients progressed to AML with an average time of only five months after initiating the combination therapy. This may be unrelated to the experimental treatment, but the trial was terminated early as there was no sign of clinical benefit or immunological response. Why the manuscript is especially interesting This study is the first to exploit the potential synergistic effects of combining a multi-peptide cancer vaccine with epigenetic therapy in MDS. Although our results are negative, they emphasize challenges to induce immune reactivity in patients with high-risk MDS.