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Over recent decades, efforts to explain the heritability of developing cancer have focused on identifying high-risk genes and common genetic variants. However, no studies considered genetic factors regulating tumor-specific immune responses. By developing a novel human leukocyte antigen (HLA) score, determined by autologous HLA allele-binding epitopes of tumor-specific antigens, we showed that individuals with HLA allele sets supporting broader tumor-specific T cell responses have lower risk of developing melanoma. The risk ratio between the highest risk and most protected groups was 5.69 comparing the top and bottom 20% of the HLA score distribution (p

BackgroundVaccines have little chance of destroying heterogeneous tumor cells since they rarely induce polyclonal T-cell responses against the tumor. The main challenge is the accurate identification of tumor targets recognizable by T cells. Presently, 6–8% of neoepitopes selected based on the patients‘ tumor biopsies are confirmed as real T cell targets.1 2. To overcome this limitation, we developed a computational platform called Personal Antigen Selection Calculator (PASCal) that identifies frequently presented immunogenic peptide sequences built on HLA-genetics and tumor profile of thousands of real individuals.3 Here we show the performance of PASCal for the identification of both shared and personalized tumor targets in metastatic colorectal cancer (mCRC) and breast cancer subjects.MethodsExpression frequency of the tumor-specific antigens (TSAs) ranked in PASCal’s database (based on 7,548 CRC specimen) was compared to the RNA-sequencing data of CRC tumors obtained from TCGA. Using PASCal, 12 shared PEPIs (epitopes restricted to at least 3 HLA class I alleles of a subject from an in silico cohort) derived from 7 TSAs were selected as frequent targets (calculated probability: average 2.5 [95%CI 2.4–2.8] TSAs/patient). Spontaneous immune responses against each of the twelve 9mer peptides were determined by ELISpot using PBMCs of 10 mCRC subjects who participated in the OBERTO-101 study.4 PEPIs selected for a breast cancer subject based on her HLA genotype were also tested.ResultsEach of the 106 tumors analyzed expressed at least 13, average 15 of the 20 top-ranked TSAs in PASCal’s database confirming their prevalence in CRC. 7/10 subjects had spontaneous CD8+ T-cell responses against at least one peptide selected with PASCal. Each peptide (12/12) was recognized by at least one patient. Patients‘ T-cells reacted with average 3.6/12 (30%) peptides confirming the expression of average 2.8 [95%CI 1.0–4.6] TSAs (n=10). After HLA-matching, among the subjects for whom we could select at least 4 PEPIs (average 5) from the list of 12 peptides (n=6), average 2.5 (50%) peptides were positive. Of the 12 PEPIs selected with PASCal for a breast cancer subject, we detected spontaneous T-cell responses against 9 PEPIs, indicating that at least 75% of the selected peptides were present in the subject’s tumor and were recognized by T-cells.ConclusionsPASCal platform accommodates both tumor- and patient heterogeneity and identifies non-mutated tumor targets that may trigger polyclonal cytotoxic T-cell responses. It is a rapid tool for the design of both off-the-shelf and personalized cancer vaccines negating the need for tumor biopsy.ReferencesWells DK, van Buuren MM, Dang KK, et al. Key parameters of tumor epitope immunogenicity revealed through a consortium approach improve neoantigen prediction. Cell 2020:183(3):818–34.e13.Bulik-Sullivan B, Busby J, Palmer CD, et al. Deep learning using tumor HLA peptide mass spectrometry datasets improves neoantigen identification. Nat Biotech 2018:37:55–63.Somogyi E, Csiszovszki Z, Lorincz O, et al. 1181PDPersonal antigen selection calculator (PASCal) for the design of personal cancer vaccines. Annal Oncol 2019:30(Supplement_5):v480-v81.Hubbard J, Cremolini C, Graham R, et al. P329 PolyPEPI1018 off-the shelf vaccine as add-on to maintenance therapy achieved durable treatment responses in patients with microsatellite-stable metastatic colorectal cancer patients (MSS mCRC). J ImmunoTher Cancer 2019:7(1):282.

Background The current challenge in developing effective cancer vaccines is the accurate prediction of epitopes that induce CD8+ cytotoxic T-cell responses. Recent technological advances have enabled development of patient-specific therapeutic vaccines. However, in these vaccines only about 10-20% of the predicted neoepitopes induced CD8+ T-cell responses in patients. To overcome this limitation, we developed PASCal for improved selection of peptides (epitopes) that induce T-cell responses. Methods PASCal operates by 3 moduls: (1) a validated epitope database containing 108 true HLA-epitope pairs derived from 1300 tumor antigens and HLA class I and II molecules covering the HLA genotype of 26000 subjects. (2) Expression frequency-based shared tumor antigen database established for 19 indications based on > 96000 tumor biopsies. (3) Algorithm for the dentification of immunogenic peptides by the selection of personal epitopes (PEPIs) binding to ≥ 3 autologous HLA alleles. Using PASCal, personal 20mer peptide vaccines were designed for 3 HLA-genotyped cancer patients (with ovarian-, breast- and colorectal cancer). Immunogenicity of the vaccines was tested by ELISPOT and Intracellular Citokine Staining (ICS). Results Personalized cancer vaccines contained PEPIs from 12 disease specific tumor-antigens most frequently expressed in the patients disease. T-cell responses were induced by 100% of peptides. An average of 11/12 PEPIs induced CD8+ T-cell responses and 12/12 induced CD4+ T-cell responses in each patient. Pre-existing antigen specific T-cell reactivities were detectable against 25% of vaccine antigens (demonstrating the expression of the target vaccine antigens by the patient’s tumor), the others were induced de novo. Both CD8+ and CD4+ T-cells were polyfunctional, as evident by secretion of multiple cytokines determined by ex vivo ICS. Conclusions We used the largest validated database of tumor epitopes reported to-date along with an algorithm successfully selecting immunogenic peptides to develop personalized cancer vaccines. PEPIs outperform reported immunogenicity of personalized neoantigen vaccines and induced unprecedented immune responses in cancer patients. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure E. Somogyi: Shareholder / Stockholder / Stock options: Treos Bio Ltd. Z. Csiszovszki: Shareholder / Stockholder / Stock options: Treos Bio Ltd. O. Lorincz: Shareholder / Stockholder / Stock options: Treos Bio Ltd. J. Toth: Shareholder / Stockholder / Stock options: Treos Bio Ltd. L. Molnar: Shareholder / Stockholder / Stock options: Treos Bio Ltd. W. Schonharting: Shareholder / Stockholder / Stock options: PMCR GmbH. S. Urban: Shareholder / Stockholder / Stock options: PMCR GmbH. K. Pantya: Shareholder / Stockholder / Stock options: Treos Bio Ltd. M. Megyesi: Shareholder / Stockholder / Stock options: Treos Bio Ltd. E.R. Tőke: Shareholder / Stockholder / Stock options: Treos Bio Ltd.; Leadership role: Treos Bio Zrt. All other authors have declared no conflicts of interest.

Background Human Leucocyte Antigen (HLA) molecules are encoded by the most polymorphic genes in the human genome. The genetic variation of these genes are considerable across different geographic subpopulations. We hypothesised that this genetic variation might contribute to the risk of melanoma both at population and subject level. Methods We developed a cancer risk predictor based on the complete HLA class I genotype of individuals. The HLA-score, used in the predictor describes the ability of the HLA class I alleles of an individual to bind epitopes derived from 48 selected tumor antigens as an indicator of the breadth of the tumor-specific T-cell responses. We collected HLA data for subjects from 20 different geographic regions (ethnic populations) (n = 3278) as well as the corresponding melanoma incidence rates. The average HLA-scores were compared to the incidence rates. We also classified a mixed US population consisting of melanoma and healthy subjects based on their HLA-score. Results On population level, we found significant correlation between the incidence rates of melanoma and average HLA-scores in different geographic regions (R2 = 0.5005; p Conclusions By developing a novel HLA-score determined by autologous HLA allele binding epitopes of tumor antigens, we showed that individuals with HLA allele sets supporting broader tumor-specific T-cell responses have lower risk of developing melanoma. These results imply that the HLA genotype and HLA-score could be used to determine the immunogenetic risk of melanoma. Legal entity responsible for the study Treos Bio Zrt. Funding Treos Bio Zrt. Disclosure L. Molnar: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. J. Toth: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. O. Lorincz: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. Z. Csiszovszki: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. P. Pales: Full / Part-time employment: Treos Bio Ltd. K. Pantya: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. M. Megyesi: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. E. Somogyi: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. E.R. Tőke: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: Treos Bio Ltd. All other authors have declared no conflicts of interest.

Background This study evaluated the safety, tolerability, immunogenicity and initial efficacy of PolyPEPI1018 as an add-on to maintenance therapy in subjects with mCRC. PolyPEPI1018 peptide vaccine contains 12 unique epitopes derived from 7 conserved cancer antigens frequently expressed in mCRC. The predictive value of the novel Personal EPItopes (PEPI) test was also explored. Methods mCRC patients in first line setting received up to 3 doses of PolyPEPI1018 vaccine (0.2 mg/peptide) 12 weeks apart, just after the transition to maintenance therapy with fluoropyrimidine and bevacizumab. Vaccine-specific T cell responses were first predicted by PEPI test (using the patient’s complete HLA genotype) then measured by ELISpot and Intracellular Cytokine Staining (ICS) after one cycle of vaccination. Tumour responses were evaluated by RECIST. Results Eleven patients were vaccinated with PolyPEPI1018. The vaccine was well tolerated; common adverse events were transient skin reactions and flu-like syndrome. No grade 3+ adverse events related to the vaccine occurred. Initial analysis of 8 patients after a single dose demonstrated that 100% of patients had CD4+ T cell response and 75% had CD8+ T cell responses against at least 3 antigens. Both CD8+ and CD4+ T cells were polyfunctional based on the secretion of multiple cytokines determined by ex vivo ICS. PEPI test correctly predicted ELISpot-measured CD8+ T cell responses (PPV = 65%, p = 0.049). Of the 5 patients who received at least 2 doses of the vaccine, 3 experienced Stable disease and 2 had unexpected tumour size reduction. Patients experiencing tumour shrinkage had higher number of predicted antigens than those without tumour response. Conclusions PolyPEPI1018 was safe, well-tolerated and induced unprecedented broad polyfunctional CRC-specific T cell responses, similar to personalized neoantigen vaccines. The candidate biomarker demonstrated high accuracy for the prediction of subject’s vaccine-specific CD8+ T cell responses and indicated patient’s clinical responses. Based on these encouraging results further development of the PolyPEPI1018 vaccine with companion diagnostic is planned. Clinical trial identification NCT03391232. Legal entity responsible for the study TreosBio. Funding Treos Bio. Disclosure J. Hubbard: Research grant / Funding (institution): Taiho Oncology; Research grant / Funding (institution): TreosBio; Advisory / Consultancy, Honoraria to Mayo Clinic: Bayer; Research grant / Funding (institution): Senhwa Pharmaceuticals; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Effector Therapeutics; Research grant / Funding (institution): Merck. E.R. Tőke: Full / Part-time employment: TreosBio. Z. Csiszovszki: Full / Part-time employment: TreosBio. O. Lorincz: Full / Part-time employment: TreosBio. L. Molnar: Full / Part-time employment: TreosBio. E. Somogyi: Full / Part-time employment: TreosBio. M. Megyesi: Full / Part-time employment: TreosBio. K. Pantya: Full / Part-time employment: TreosBio. J. Toth: Full / Part-time employment: TreosBio. P. Pales: Full / Part-time employment: TreosBio. I. Miklos: Full / Part-time employment: TreosBio. All other authors have declared no conflicts of interest.

Background Most therapeutic vaccine clinical trials (CTs) have failed to prove efficacy, even if immunogenicity was confirmed earlier. It was already shown that immune responses generated against multiple antigens are indicative of clinical responses. We aimed to find association between the heterogeneity of immune response and clinical efficacy and, based on this develop a tool that can predict the clinical outcome of cancer vaccines. Methods In an extensive literature search we collected the immune- and objective response rates (IRR, ORR) of 94 CTs in which 2,338 patients were treated with 64 different vaccines. Vaccine sequences were used to predict personal epitopes (PEPIs) that bind to at least 3 HLA alleles of the same subject, for all patients of a representative model population. Then we determined the percentage of subjects with at least 1 vaccine specific PEPIs (PEPI Score) and at least 2 vaccine specific PEPIs derived from different antigens (MultiAg PEPI-Score) and compared to the published IRR and ORR. Results PEPI Score was able to predict the immunogenicity of therapeutic vaccines; significant correlation was found with IRR (p = 0.002). As expected, no correlation was found between ORR and IRR (p = 0.294), neither between PEPI Score and ORR (p = 0.302), suggesting, that immune response against a single epitope is not enough for efficient tumor response. However, we found that MultiAg PEPI-Score significantly correlates with ORR (p Conclusions Our results demonstrate that both IRR and ORR can be predicted by PEPIs. For clinical efficacy it is crucial to target and generate immune response against multiple antigens. Based on our analysis our computational model is useful and accurate for the prediction of the clinical outcome of cancer vaccines and can even be suitable for rescuing CTs with insufficient or missing responder selection. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure O. Lorincz: Shareholder / Stockholder / Stock options: Treos Bio Ltd; Full / Part-time employment: Treos Bio Zrt. J. Toth: Shareholder / Stockholder / Stock options: Treos Bio Ltd; Full / Part-time employment: Treos Bio Zrt. M. Megyesi: Shareholder / Stockholder / Stock options: Treos Bio Ltd; Full / Part-time employment: Treos Bio Zrt. K. Pantya: Shareholder / Stockholder / Stock options: Treos Bio Ltd; Full / Part-time employment: Treos Bio Zrt. E. Somogyi: Shareholder / Stockholder / Stock options: Treos Bio Ltd; Full / Part-time employment: Treos Bio Zrt. Z. Csiszovszki: Shareholder / Stockholder / Stock options: Treos Bio Ltd; Full / Part-time employment: Treos Bio Zrt. P. Pales: Full / Part-time employment: Treos Bio Zrt. L. Molnar: Shareholder / Stockholder / Stock options: Treos Bio Ltd; Full / Part-time employment: Treos Bio Zrt. E.R. Tőke: Shareholder / Stockholder / Stock options, Officer / Board of Directors: Treos Bio Ltd; Full / Part-time employment: Treos Bio Zrt. All other authors have declared no conflicts of interest.

West Nile virus (WNV) is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI) covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime - boost regime) with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical). In parallel a heterologous boost with purified recombinant WNV envelope (E) protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8(+) specific IFN-gamma expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection.