Multi-peptide Vaccine Research Articles

Targeting LAG3/GAL-3 to overcome immunosuppression and enhance anti-tumor immune responses in multiple myeloma

Immune profiling in patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma (MM) provides the framework for developing novel immunotherapeutic strategies. Here, we demonstrate decreased CD4+ Th cells, increased Treg and G-type MDSC, and upregulation of immune checkpoints on effector/regulatory and CD138+ cells in MM patients, compared MGUS/SMM patients or healthy individuals. Among the checkpoints profiled, LAG3 was most highly expressed on proliferating CD4+ Th and CD8+ Tc cells in MM patients BMMC and PBMC. Treatment with antibody targeting LAG3 significantly enhanced T cells proliferation and activities against MM. XBP1/CD138/CS1-specific CTL generated in vitro displayed anti-MM activity, which was further enhanced following anti-LAG3 treatment, within the antigen-specific memory T cells. Treg and G-type MDSC weakly express LAG3 and were minimally impacted by anti-LAG3. CD138+ MM cells express GAL-3, a ligand for LAG3, and anti-GAL-3 treatment increased MM-specific responses, as observed for anti-LAG3. Finally, we demonstrate checkpoint inhibitor treatment evokes non-targeted checkpoints as a cause of resistance and propose combination therapeutic strategies to overcome this resistance. These studies identify and validate blockade of LAG3/GAL-3, alone or in combination with immune strategies including XBP1/CD138/CS1 multipeptide vaccination, to enhance anti-tumor responses and improve patient outcome in MM.

Immunopeptidomics-Guided Warehouse Design for Peptide-Based Immunotherapy in Chronic Lymphocytic Leukemia

Antigen-specific immunotherapies, in particular peptide vaccines, depend on the recognition of naturally presented antigens derived from mutated and unmutated gene products on human leukocyte antigens, and represent a promising low-side-effect concept for cancer treatment. So far, the broad application of peptide vaccines in cancer patients is hampered by challenges of time- and cost-intensive personalized vaccine design, and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. In this study, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics. Comparative mass spectrometry-based immunopeptidome analyses of primary chronic lymphocytic leukemia (CLL) samples, as representative example of low-mutational burden tumor entities, and a dataset of benign tissue samples enabled the identification of high-frequent non-mutated CLL-associated antigens. These antigens were further shown to be recognized by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, and were evaluated as pre-manufactured warehouse for the construction of personalized multi-peptide vaccines in a first clinical trial for CLL (NCT04688385). This workflow for the design of peptide warehouses is easily transferable to other tumor entities and can provide the foundation for the development of broad personalized T cell-based immunotherapy approaches.

Abstract SY17-02: Multiantigen vaccines for colon cancer prevention

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US when men and women are combined. While overall CRC incidence has declined over the years, there has been an increase in CRC diagnosis in young and middle-aged adults. The US Preventive Services Task Force has recently issued draft recommendations for CRC screening to begin at 45 years of age instead of the previously recommended age of 50 for asymptomatic healthy individuals, who are at average risk of CRC, i.e. without a family history of hereditary CRC syndromes such as Lynch syndrome and familial adenomatous polyposis, or with no prior diagnosis of CRC, adenomatous polyps, inflammatory bowel disease. While the evidence supports that CRC-screening and early detection confers a substantial benefit, additional CRC prevention strategies can greatly help reduce the cancer burden in the US and around the world. A number of previous studies have shown that the use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancers, including CRC. However, their extended use can be associated with serious side effects, including gastrointestinal bleeding. Safer and more efficacious preventive approaches are needed for durable long-term CRC prevention. The majority of newly diagnosed CRC are non-familial (sporadic) and non-hypermutated/microsatellite-stable (MSS). Compared to CRC with high microsatellite instability, MSS CRC and adenomas have significantly lower somatic mutation frequencies, thus lower neoantigen load. Furthermore, MSS CRC have generally lower levels of tumor-infiltrating lymphocytes (TILs), higher density of myeloid derived suppressor cells (MDSC), and downregulation of HLA molecules. These are characteristic of immunologically “cold” tumors, making it highly challenging to immunologically control tumor growth. If cancer vaccines can successfully prime the immune system, in particular before the development of invasive cancer, vaccine-induced adaptive antitumor immunity may render “cold” tumors “hot” and achieve a favorable tumor control. One of the key questions, however, is what tumor antigens can be targeted by vaccines if tumors have lower levels of neoantigens that are known to elicit robust antitumor immune responses. Another approach for cancer vaccine-mediated immune priming is to target tumor associated antigens (TAAs). Unlike tumor-specific neoantigens, TAAs are self-antigens that are overexpressed in tumors, but may also be expressed in normal cells at various levels. Therefore, it is highly critical to identify T cell epitopes that break tolerance and help elicit effective antitumor immunity. Clinically beneficial antitumor immunity is characterized by high-density infiltrates of effector CD8+ cytotoxic T lymphocytes and effector memory T cells and the low abundance of immunosuppressive elements (e.g. regulatory T cells, MDSC, and M2-polarized tumor-associated macrophages) in the tumor microenvironment. Disis and colleagues from University of Washington have previously established a systematic and stepwise approach to identify Th1-selective epitopes for efficacious cancer vaccines while eliminating Th2-epitopes. They have shown cancer vaccines targeting TAAs thus developed could elicit robust Th1 immune response associated with antitumor effects. The NCI PREVENT Cancer Preclinical Drug Development Program (PREVENT) is a peer-reviewed agent development program focused on preclinical development of innovative interventions, including cancer vaccines and drugs, for precision cancer prevention-interception towards clinical applications (https://prevention.cancer.gov/major-programs/prevent-cancer-preclinical). PREVENT and Disis team collaborated on the development of a multi-antigen multi-peptide colon cancer vaccine, named Colovac, for immunoprevention of CRC. It consists of highly immunogenic Th1-promoting epitopes from three TAAs that are selected through CRC genomic database and literature search. TAAs selected are overexpressed in CRC, known to play a role in CRC oncogenesis, and associated with poor disease outcomes. This presentation will cover the background of Colovac development, preclinical antitumor efficacy data on Colovac used alone or in combination with naproxen, characterization of immune correlates of protection, and current ongoing development efforts toward clinical trials. Citation Format: Shizuko Sei. Multiantigen vaccines for colon cancer prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY17-02.

Abstract 440: Potentiation of EGFR peptide cancer vaccine by an orally bioavailable glutamine antagonist prodrug JHU-083

Abstract Lung cancer is the leading cause of cancer mortality worldwide. More than 85% of lung cancers are non-small cell lung cancer (NSCLC). EGFR mutations occur in 47.9% of Asia-Pacific patients with NSCLC and 19.2% of Western patients. The most common EGFR mutations (>90%) are deletions in exon 19 and/or point mutations in exon 21 (L858R). Th1 helper cellular immunity is critical for immunotherapy-mediated tumor regression. We have previously characterized an MHC-II-restricted EGFR multi-peptide vaccine (two peptides: “SCVRACGADSYEMEEDGVRK” and “VWSYGVTVWELMTFGSKPY”) (EGFR-V) that targets the EGFR protein and decreases EGFR-driven lung tumorigenesis by ~80% in EGFRL858R transgenic mice that were vaccinated before doxycycline-induction of the EGFR protein [1]. However, diminished efficacy was observed when this MHC-II-restricted EGFR multi-peptide vaccine was given two weeks after doxycycline induction of the EGFR protein, suggesting that expression of the EGFR oncoprotein significantly increased the immunosuppressive microenvironment. JHU083, an orally bioavailable glutamine antagonist, has recently been shown to not only inhibit tumor growth but also boost anti-cancer immunity [2]. To determine if JHU083 could potentiate the efficacy of EGFR vaccine in a post-initiation setting, we evaluated in vivo antitumor efficacy of EGFR vaccine combined with JHU083 using an EGFRL858R transgenic mouse model. JHU083 inhibited tumor burden by 31% and EGFR vaccine suppressed tumor burden by 33%, whereas combining JHU083 with anti-EGFR peptide vaccine had an additive effect (54% tumor inhibition). Mechanistically, JHU083 by itself, markedly reduced the immunosuppressive monocytic myeloid-derived suppressor cells (MDSCs) in lung tissues. The combination of JHU083 and the vaccine significantly reduced Tregs in lung tissues. The anti-EGFR vaccine primarily induced expansion of antigen specific antitumor CD4+ effector T cells. Long term administration of JHU-083 did not decrease bodyweight in mice. Together with previous results, these data suggest that JHU083 could be used to reshape the tumor microenvironment toward one that enhances antitumor T cell responses and could further enhance the efficacy of the EGFR anticancer vaccine.

Computational formulation and immune dynamics of a multi-peptide vaccine candidate against Crimean-Congo hemorrhagic fever virus

The sole objective of this research is to devise an epitope-based vaccine candidate as prophylaxis for the Crimean-Congo hemorrhagic fever virus (CCHFV) using the knowledge of immunoinformatics and structural biology. Importantly, CCHFV outbreaks have increased in several countries resulting in increased mortality up to 40% due to the lack of prospective medication and an efficient vaccine. In this study, we have used several immunoinformatic tools and servers to anticipate potent B-cell and T-cell epitopes from the CCHFV glycoprotein with the highest antigenicity. After a comprehensive evaluation, a vaccine candidate was designed using 6 CD8+, 3 CD4+, and 7 B-cell epitopes with appropriate linkers. To enhance the vaccine's efficiency, we added Mycobacterium tuberculosis lipoprotein LprG (Rv1411c) to the vaccine as an adjuvant. The final construct was composed of a total of 468 amino acid residues. The epitope included in the construct showed 98% worldwide population coverage. Importantly, the construct appeared as antigenic, immunogenic, soluble, and non-allergenic in nature. To explore further, we modelled the three-dimensional (3D) structure of the constructed vaccine. Our chimeric vaccine showed stable and strong interactions for toll-like receptor 2 (TLR2) found on the cell surface. Moreover, the dynamics simulation of immune response showed elevated levels of cellular immune activity and faster clearance of antigen from the body upon repetitive exposure. Finally, the optimized codon (CAI≈1) ensured the marked translation efficiency of the vaccine protein in E. coli strain K12 bacterium followed by the insertion of construct DNA into the cloning vector pET28a (+). We believe that the designed vaccine chimera could be useful in vaccine development to fight CCHFV outbreaks.

CTIM-08. COMBINATION OF THE IMA950/POLY-ICLC MULTIPEPTIDE VACCINE WITH PEMBROLIZUMAB IN RELAPSING GLIOBLASTOMA PATIENTS

Abstract The IMA950 peptide vaccine is composed of 9 HLA-A2-restricted peptides eluted from the surface of GBM samples and of two HLA class II-binding peptides1. It was tested in combination with poly-ICLC in patients with newly diagnosed GBM, demonstrating safety. The vaccine was able to elicit CD4 and CD8 T cell responses in the peripheral blood in the majority of patients, with however an overall low magnitude of T cell responses and suboptimal migration of elicited T cells to the brain, probably limiting clinical efficacy2,3. With the aim to improve homing of vaccine-specific T cell to the tumor, we are conducting a phase II clinical trial in patients with recurrent GBM testing the IMA950/poly-ICLC multipeptide vaccine with or without the anti-PD1 antibody pembrolizumab (NCT03665545). 24 patients will be included (12 patients in each arm) and pre- and post-vaccination tumor sample will be available, allowing assessing effect of the vaccine at the tumor site. The primary objective of this trial is safety of IMA950/poly-ICLC given together with pembrolizumab. Secondary objectives include (i) estimation of 6, 9 and 12-month progression-free survival (PFS), (ii) overall survival, (iii) analysis of patient quality of life and (iv) of the synergy/immunogenicity of IMA950/poly-ICLC and pembrolizumab. Immunomonitoring will include measure of vaccine-induced immune responses, IHC for immune cell markers, RNA/TCR sequencing and methylome analysis, to assess vaccine-induced T cell responses, immune modulation and potential signatures predictive of response. Thus far, 6 patients have been included (3 in each arm). Preliminary results show CD4 and CD8 T cell responses to the vaccine are detected in both groups in the peripheral blood. Analysis at the tumor site and comparison between arms will be performed once all patients have been included. 1. Dutoit, V. et al. Brain (2012); 2. Migliorini, D. et al. Neuro Oncol (2019); 3. Rampling, R. et al. Clin Cancer Res(2016)

Therapeutic Cancer Vaccination Targeting Shared Tumor Associated Antigens in Combination with Azacitidine 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). AZA can induce expression of silenced genes, including methylated tumor associated antigens. Such tumor associated antigens may be recognized by T cells, and therefore exploited for immunotherapeutic targeting. To our knowledge, this is the first clinical study that combine hypomethylating agents with a multi-peptide therapeutic cancer vaccine in a hematological malignancy. Method: In this open label phase 1 trial (ClinicalTrials.gov NCT02750995), we combine AZA with a 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. Four long synthetic peptides containing previously described class I and class II epitopes for a variety HLA types was emulsified in Incomplete Freund's Adjuvant for subcutaneous injection. Patients were included following verified treatment response to six courses of AZA monotherapy. Result: Five patients 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. There was one instance of grade 4 toxicity; a case of neutrophil count decrease, requiring administration of prophylactic antibiotics, and two instances of grade 3 toxicity; platelet count decrease and neutrophil count decrease. No vaccine-specific immune response could be detected using intracellular cytokine staining or ELISpot assays, however changes in the phenotypic composition of immune cells and their expression of stimulatory and inhibitory markers were identified in individual patients. All patients progressed to AML with a mean time to progression from inclusion (TTP) of 4.9 months (range 2.8 to 7.6). Survival was 17 months (range 10.9 to 30.6) from MDS diagnosis. Sequencing of bone marrow showed clonal evolution 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. Figure 1. (a) Trial design. All participants received six courses of AZA prior to inclusion and were evaluated with bone marrow biopsy for treatment response. Vaccination was given together with the next three courses of AZA. (b) Vaccine composition. Synthetic long peptides from NY-ESO-1, PRAME, MAGE-A3 and WT-1 were emulsified in adjuvant Montanide ISA 51. Figure 1 Disclosures No relevant conflicts of interest to declare.

Single-cell derived tumor organoids display diversity in HLA class I peptide presentation

Tumor heterogeneity is a major cause of therapeutic resistance. Immunotherapy may exploit alternative vulnerabilities of drug-resistant cells, where tumor-specific human leukocyte antigen (HLA) peptide ligands are promising leads to invoke targeted anti-tumor responses. Here, we investigate the variability in HLA class I peptide presentation between different clonal cells of the same colorectal cancer patient, using an organoid system. While clone-specific differences in HLA peptide presentation were observed, broad inter-clone variability was even more prevalent (15–25%). By coupling organoid proteomics and HLA peptide ligandomics, we also found that tumor-specific ligands from DNA damage control and tumor suppressor source proteins were prominently presented by tumor cells, coinciding likely with the silencing of such cytoprotective functions. Collectively, these data illustrate the heterogeneous HLA peptide presentation landscape even within one individual, and hint that a multi-peptide vaccination approach against highly conserved tumor suppressors may be a viable option in patients with low tumor-mutational burden.

Multi-epitope based vaccine design against Sarcoptes scabiei paramyosin using immunoinformatics approach

Scabies is a parasitic skin infestation caused by mite Sarcoptes scabiei and is listed as one of the top 50 epidemic diseases known to affect >200 million people globally. The scabies mites have shown resistance to the many of currently available chemotherapeutics, a prerequisite to identify potential targets for vaccine development. Paramyosin has been identified as a potential vaccine candidate for parasite control and appears as an important immunogen in S. scabiei. In the present study, we used different immune-informatics tools to analyze S. scabiei paramyosin sequence and screen potential B and T cells epitopes. Epitope prioritization was done based on reference allele coverage, antigenicity, high antigenicity, adhesive ability, non-allergenicity and potential of binding to the DRB*0101 allele. Further, it was ensured that the selected candidates were feasible for experimental cloning and expression analysis. The predicted epitopes were also used in multi-peptide vaccine construction to maximize immunogenicity and efficient recognition and processing by the host immune system. Host immune system dynamics in response to the vaccine construct antigen were also elaborated in silico, revealing robust cellular and humoral immunity. To understand cellular interaction mechanisms, molecular docking followed by molecular dynamic simulation was performed to probe the predominant binding mode of the vaccine construct to the innate immune receptor TLR5. Estimation of binding free energy for the final construct was accomplished and revealed the system highly stable with enriched electrostatic and van der Waals energy. In summary, this study provides a gateway to understand the protein function analysis of a potential vaccine candidate for S. scabiei.

Abstract 4563: Immunoprevention of triple negative breast cancer by a multi-antigen, multi-peptide vaccine

Abstract Triple negative breast cancer (TNBC) is the most aggressive sub-type of breast cancer, comprising approximately 20% of all breast cancers, and has a poor prognosis due to its aggressive behavior and lack of effective targeted therapies. In the present study, we employed transcriptome and protein analyses to select genes that are uniquely overexpressed in human triple-negative breast cancer. Overexpression of selected genes, Cyclin A2, CDK1, Cyclin E2, PRC1, Kif15, TPX2, Ki67, Top2A, were confirmed on human breast cancer tissue microarrays and in TNBC derived cell lines from C3(1)/TAg mice. Antigen-specific MHC class II epitopes with promiscuous high-binding affinity were identified using the “combined scoring system”, based on their potential to elicit a selective Th1 immunity. Furthermore, all peptides were selected to have over 85% amino acid sequence homology between human and mice for potential clinical translation. Strong immunogenic peptides from five antigens were selected by immunogenicity testing in tumor naïve mice based on high IFN-γ and low IL-10 ELISpot response data. The selected peptides, Cyclin A2 (#335), CDK1 (#113), Cyclin E2 (#93, 298, 334), KIF15 (#129), and Top2A (#236, 410, 606), were combined to form a multi-antigen, multi-peptide vaccine (MAMPV). The tumor preventive efficacy of the MAMPV was evaluated in syngraft study using a TNBC model: C3(1)/TAg-REAR mice and a TNBC cell line derived from C3(1)/TAg (M6). MAMPV-vaccinated animals demonstrated a significant reduction in tumor volume, and significantly higher tumor infiltration of CD4+ T cells compared to the control animals. Pathological examination of major organs did not reveal any toxicity associated with the vaccine. Similar tumor preventive efficacy was demonstrated in genetically engineered mouse model (GEMM) using C3(1)/TAg mice. Taken together, our data indicate that formulation of the multi-antigen multi-peptide vaccine is highly immunogenic and has the potential to be safe and efficacious in the immunoprevention of TNBC. Citation Format: Yian Wang. Immunoprevention of triple negative breast cancer by a multi-antigen, multi-peptide vaccine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4563.

Abstract LB-094: Hepavac-101 first-in-man clinical trial of a multi-peptide-based vaccine for hepatocellular carcinoma

Abstract Background and Aims: Hepatocellular carcinoma (HCC) is the third leading cause of death from cancer globally with an extremely variable 5-year survival rate. Immunotherapy strategies for HCC may represent a key therapeutic tool to improve clinical outcome in HCC patients. HepaVac-101 (EudraCT Number: 2015-003389-10; NCT03203005), is a single-arm, first-in-man Phase I/II clinical trial evaluating a therapeutic cancer vaccine in patients affected by HCC. It is a highly innovative, novel approach based on a multi-peptide vaccine (IMA970A) combined with the TLR7/8/RIG I agonist CV8102 as an adjuvant. Method: The IMA970A off-the-shelf vaccine includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides identified and selected from native human HCC tumor tissue by applying the XPRESIDENT® discovery platform. CV8102 is a novel ribonucleic acid (RNA) based immunostimulatory agent inducing a balanced Th1/Th2 immune response.HLA-A*02 and/or A*24-positive patients with very early, early and intermediate stage HCCs have been enrolled to be treated with 9 intradermal vaccinations consisting of IMA970A plus CV8102 following a single pre-vaccination infusion of low-dose cyclophosphamide acting as an immunomodulator. Patients have completed standard of care treatments at time of enrolment and received study drugs without concomitant anti-tumor therapy with no evidence of disease reactivation. The primary endpoints of the HepaVac-101 clinical trial are safety, tolerability, and antigen specific T cell response. Secondary/exploratory endpoints are additional immunological parameters in blood, infiltrating T-lymphocytes in tumor tissue, biomarkers in blood and tissue, disease-free survival/progression-free survival and overall survival. Results: Patients were enrolled in 6 centers located in 5 European countries i.e. Italy (Naples and Negrar/Varese), Germany (Tübingen), UK (Birmingham), Spain (Pamplona) and Belgium (Antwerp). The end of trial (EOT) was reached on last December 20, 2019. 82 HCC patients have been screened for suitable HLA haplotypes, 22 patients were put on study treatment (i.e. received at least the pre-treatment with cyclophosphamide). The vaccination showed a good safety profile with no major SAE nor AESI reported during the protocol. Immunogenicity data are currently evaluated and will be available at time of the meeting. Conclusion: The HEPAVAC-101 clinical trial is the achievement of the HEPAVAC Consortium supported by the European Commission's 7th framework program with contract No. 602893 (www.hepavac.eu). Citation Format: Luigi Buonaguro, Andrea Mayer, Markus Loeffler, Sarah Missel, Roberto Accolla, Yuk Ting Ma, Tanguy Chaumette, Regina Heidenreich, Alfred Königsrainer, Joerg Ludwig, Diego Alcoba, Heiko Schuster, Cecile Gouttenfangeas, Greta Forlani, Maria Tagliamonte, Paolo A. Ascierto, Antonio Avallone, Marco Borrelli, Hans-Georg Rammensee, Bruno Sangro, Mercedes Iñarrairaegui, Sven Francque, Luisa Vonghia, Stefania Gori, Tony Weinschenk, Carsten Reinhardt, Ulrike Gnad-Vogt, Harpreet Singh. Hepavac-101 first-in-man clinical trial of a multi-peptide-based vaccine for hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-094.

Abstract 4584: A novel combination of a multipeptide KRAS vaccine and an ACAT1 inhibitor to prevent lung cancer

Abstract Kirsten rat sarcoma viral oncogene homolog (KRAS) gene encodes a small GTPase that cycles between GDP and GTP-bound states, which modulates important cell surface receptors essential for cell proliferation and survival. It is one of the most common drivers for many types of cancers, is present in up to 25% all human tumors, and 80% of mutation occurs in codon12. Efforts to target KRAS-driven cancers preventively or therapeutically have been unsuccessful. We recently formulated a multi-peptide KRAS vaccine (KVAX), which targets both wild-type and G12D mutant forms of KRAS and has 100% sequence homology between human and mouse. KVAX elicited strong immunologic response and exhibited striking cancer preventive efficacy when administered prior to KRAS mutation induction. However, clinical high-risk individuals may already express mutant KRAS, which has known to promote an immunosuppressive environment. Therefore, we combined KVAX with avasimibe (AVA), an ACAT1 inhibitor that modulates lipid metabolism in T cells and facilitates better TCR/peptide/MHC interactions, in syngeneic lung cancer mouse model and genetically engineered mouse model, in which mutant KRAS initiated lung tumorigenesis before vaccination. We found that the combination significantly decreased the presence of regulatory T cells in the tumor microenvironment, facilitated CD8+ T cell infiltration in tumor sites, and ultimately led to enhanced anti-cancer efficacy. Using 10X Genomics, we performed single cell RNA-seq on lung tumors from mice treated with KVAX and AVA and found significant increases in APC monocytes and CD4+ Th1 cells, especially increases in the effector/memory T cell population. Meanwhile, KVAX also decreased Tregs and M2 macrophages. These studies include the evaluation of tumor-infiltrating immune cells by single-cell RNA-seq (scRNA-seq) and provide a more detailed mechanisms of action of KVAX alone or in combination with AVA in modulating the tumor immune microenvironment. Citation Format: Jing Pan, Qi Zhang, Katie Palen, Bryon Johnson, Li Wang, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. A novel combination of a multipeptide KRAS vaccine and an ACAT1 inhibitor to prevent lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4584.

Evaluation of safety, immunogenicity, and preliminary efficacy of PolyPEPI1018 off-the-shelf vaccine with fluoropyrimidine/bevacizumab maintenance therapy in metastatic colorectal cancer (mCRC) patients.

4048 Background: PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine containing 12 immunogenic epitopes derived from 7 cancer testis antigens (CTAs) frequently expressed in patients with CRC. Here we report the final results of the phase I study of PolyPEPI1018 vaccine as an add-on to maintenance therapy in mCRC patients. Methods: 11 patients with MSS mCRC were vaccinated with PolyPEPI1018 just after the transition to maintenance therapy with fluoropyrimidine/bevacizumab after first-line combo chemotherapy and bevacizumab. Part A: n = 5, single dose; Part B: n = 6, 3 doses, Q12W. Primary endpoint was safety. Immunomonitoring was performed at both blood and tumor levels, as well as prospectively predicted. Results: The vaccine was well tolerated; most common side effects were transient skin reactions. No vaccine-related SAE occurred. Pre-existing immune responses were boosted by the vaccine for 7/10 patients. De novo responses were also induced, overall, 80% of patients had CD8+ T cell responses against at least 3 CTAs. The magnitude of immune responses as well as the density and the ratio of CD8+/CD3+ tumor infiltrating T cells increased with multiple vaccine doses. Three patients had objective tumor response according to RECIST v1.1: one of them in the single dose group and two of them in the 3 doses group. Both patients in the 3 doses group qualified for curative surgery. One of them had no viable tumor cells in his primary tumor at the time of surgery. Post-trial follow-up revealed PFS of at least 12 months for 3 patients. mPFS was longer for patients receiving multiple doses (9.9 months) compared to single dose (6.1 months). Both measured and predicted multiantigenic immune responses tend to correlate with PFS and tumor volume reduction. Conclusions: PolyPEPI1018 was effective in restoring immunological responses to CTAs in patients’ with spontaneous immunity against. Treatment with PolyPEPI1018 vaccine and maintenance therapy was safe and demonstrated evidence of early clinical activity in MSS mCRC tumors. Data support further randomized trials with PolyPEPI1018. Clinical trial information: NCT03391232 .

Multipeptide vaccine to prevent epithelial ovarian cancer relapse by microenvironment immunomodulation.

e15268 Background: Typically cancer vaccines for relapse prevention in epithelial ovarian cancer (EOC) are administrated in distant anatomical sites away from the primary tumor location. Ideally for EOC relapse prevention vaccines we should explore different anatomical sites related with the original tumor location and nearby lymph nodes, in order to observe if the administration would have better clinical impact due to a potential access to the tumor microenvironment. Moreover most trials for EOC relapse prevention use one or few targets and is mainly unknown if they are relevant to promote EOC relapse. We treated EOC patients with a multi-peptide vaccine administered in lymph nodes (LN) and in the peritoneum area and were able to suppress cancer relapse with not significant adverse effects after eight years. Methods: We enrolled n = 25 EOC patients in our study and identified by systematic review and validated by siRNA six proteins involved in EOC relapse and fourteen peptides were predicted. Twelve peptides were immunogenic by Granzyme B ELISPOT, DTH and ELISA. Patients were immunized with the vaccine in axillary and inguinal LN subcutaneously every week for four weeks, every two-weeks four times and finally every month for a total of thirty-six months. Clinical and laboratory evaluations were performed every 2-months and at the end of the study. Results: 100% of the patients developed both antibodies against all the peptides by ELISA and CD8 specific cells. The peptides of the six proteins were able to induce T cell expansion in both peptides and purified protein (p = 0.0001) and (p = 0.05), respectively for Ape-1. The selected targets were all able to induce DTH and ELISA responses with statistical significance. Peptides predicted from Bcl-2, survivin, Ape-1, Sox2, EGFR and RCAS1/EBAG9 were able to increase and sustain the levels of Granzyme B ELISPOT. Bcl-2 A peptide (p = 0.001), Bcl-2 B (p = 0.005), Bcl-2 C (p.0002), survivin A (p = 0.001), Survivin C (0.05), Ape-1 A (0.0001), Ape-1 D (p = 0.001), Sox2 A (p = 0.003), Sox2 B (p = 0.0001), EGFR B (p = 0.0001) and EGFR D (p = 0.001). Conclusions: Treating EOC patients in remission in zones hardly explored previously in order to potentially improve the activation of antigen-specific Th1 and CD8 cells with specific peptides seems promising as a feasible and safe approach to prevent EOC relapse.

Design of a peptide-based subunit vaccine against novel coronavirus SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that was first reported in Wuhan, China, and has subsequently spread worldwide. In the absence of any antiviral or immunomodulatory therapies, the disease is spreading at an alarming rate. A possibility of a resurgence of COVID-19 in places where lockdowns have already worked is also developing. Thus, for controlling COVID-19, vaccines may be a better option than drugs. An mRNA-based anti-COVID-19 candidate vaccine has entered a phase 1 clinical trial. However, its efficacy and potency have to be evaluated and validated. Since vaccines have high failure rates, as an alternative, we are presenting a new, designed multi-peptide subunit-based epitope vaccine against COVID-19. The recombinant vaccine construct comprises an adjuvant, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes joined by linkers. The computational data suggest that the vaccine is non-toxic, non-allergenic, thermostable, with the capability to elicit a humoral and cell-mediated immune response. The stabilization of the vaccine construct is validated with molecular dynamics simulation studies. This unique vaccine is made up of 33 highly antigenic epitopes from three proteins that have a prominent role in host-receptor recognition, viral entry, and pathogenicity. We advocate this vaccine must be synthesized and tested urgently as a public health priority.

Potentiation of Kras peptide cancer vaccine by avasimibe, a cholesterol modulator.

BackgroundNo effective approaches to target mutant Kras have yet been developed. Immunoprevention using KRAS-specific antigenic peptides to trigger T cells capable of targeting tumor cells relies heavily on lipid metabolism. To facilitate better TCR/peptide/MHC interactions that result in better cancer preventive efficacy, we combined KVax with avasimibe, a specific ACAT1 inhibitor, tested their anti-cancer efficacy in mouse lung cancer models, where Kras mutation was induced before vaccination.MethodsControl of tumor growth utilizing a multi-peptide Kras vaccine was tested in combination with avasimibe in a syngeneic lung cancer mouse model and a genetically engineered mouse model (GEMM). Activation of immune responses after administration of Kras vaccine and avasimibe was also assessed by flow cytometry, ELISpot and IHC.FindingsWe found that Kras vaccine combined with avasimibe significantly decreased the presence of regulatory T cells in the tumor microenvironment and facilitated CD8+ T cell infiltration in tumor sites. Avasimibe also enhanced the efficacy of Kras vaccines target mutant Kras. Whereas the Kras vaccine significantly increased antigen-specific intracellular IFN-γ and granzyme B levels in CD8+ T cells, avasimibe significantly increased the number of tumor-infiltrating CD8+ T cells. Additionally, modulation of cholesterol metabolism was found to specifically impact in T cells, and not in cancer cells.InterpretationAvasimibe complements the efficacy of a multi-peptide Kras vaccine in controlling lung cancer development and growth. This treatment regimen represents a novel immunoprevention approach to prevent lung cancer.

MHCquant: Automated and Reproducible Data Analysis for Immunopeptidomics

Personalized multipeptide vaccines are currently being discussed intensively for tumor immunotherapy. In order to identify epitopes-short, immunogenic peptides-suitable for eliciting a tumor-specific immune response, human leukocyte antigen-presented peptides are isolated by immunoaffinity purification from cancer tissue samples and analyzed by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Here, we present MHCquant, a fully automated, portable computational pipeline able to process LC-MS/MS data automatically and generate annotated, false discovery rate-controlled lists of (neo-)epitopes with associated relative quantification information. We could show that MHCquant achieves higher sensitivity than established methods. While obtaining the highest number of unique peptides, the rate of predicted MHC binders remains still comparable to other tools. Reprocessing of the data from a previously published study resulted in the identification of several neoepitopes not detected by previously applied methods. MHCquant integrates tailor-made pipeline components with existing open-source software into a coherent processing workflow. Container-based virtualization permits execution of this workflow without complex software installation, execution on cluster/cloud infrastructures, and full reproducibility of the results. Integration with the data analysis workbench KNIME enables easy mining of large-scale immunopeptidomics data sets. MHCquant is available as open-source software along with accompanying documentation on our website at https://www.openms.de/mhcquant/ .

Evaluation of immunogenicity of novel multi-epitope subunit vaccines in combination with poly I:C against Brucella melitensis and Brucella abortus infection

Brucellosis is a worldwide zoonotic disease affecting domestic animals and humans. Due to several safety problems associated with live attenuated vaccines (Rev1 and RB51), it is necessary to produce an efficient and safer vaccine against Brucella. In this study, we evaluated efficacy of two novel multi-peptide vaccine candidates of FliC, 7α-HSDH, BhuA antigens with and without poly I:C adjuvant. Hence, humoral and cellular immune responses and protective efficacy were determined in immunized mice. Our investigation indicated that multi-epitope antigens showed a significant induction of Th1 immunity with high levels of specific IgG (especially the IgG2a), as well as IFN-γ and IL-2 compared to the control group. The addition of poly I:C to multi-epitope antigens improved the humoral and cellular immune responses. The multi-epitope antigens with and without poly I:C also provided cross protection against B. melitensis16M and B. abortus544 infections. The present study suggests that the novel multi-epitope vaccine candidates based on B cell, CD4+ and CD8+T-cell epitopes of FliC, 7α-HSDH, BhuA proteins would be potential vaccine candidate against B. melitensis and B. abortus infections. Furthermore, poly I:C could be considered as a strong Th1-inducing adjuvant in designing vaccine formulation against brucellosis.

Abstract 647: Immunoprevention of triple negative breast cancer by a TOP2A multi-peptide vaccine

Abstract Triple negative breast cancer (TNBC) comprises approximately 20% of all breast cancers, and it has a poor prognosis due to aggressive growth characteristics and lack of effective targeted therapies. Emerging data suggest vaccination as a promising approach to prevent TNBC as well as other cancer types, but appropriate vaccine targets need to be identified. In the present study, we employed in silico analyses to select genes that are uniquely expressed in TNBC. Topoisomerase 2 alpha (Top2A) was found to be highly expressed in human TNBC. Top2A is a key enzyme in DNA replication and has been identified as a target of anti-cancer therapy. Overexpression of Top2A was confirmed in human breast cancer tissue microarrays and in mouse TNBCs derived from C3(1)/TAg mice. Top2A-specific MHC class II epitopes with optimal binding affinity were identified using the “combined scoring system”, based on their potential to elicit a Th1 immune response. Furthermore, all peptides were selected to have 100% amino acid sequence homology between humans and mice for potential clinical translation. Four Top2A peptides with strong immunogenic responses were selected by immunization of tumor naïve mice followed by IFN-γ-based ELISPOT assays. These peptides were combined to form a multi-peptide Top2A vaccine. The anti-tumor efficacy of the Top2A vaccine was tested in a preventive setting using a syngeneic TNBC model in C3(1)/TAg-REAR mice. Vaccinated animals demonstrated a significant reduction in tumor growth when compared to the adjuvant control with tumor size decreasing from 278.1 mm3 in adjuvant control to 150.4 mm3 in vaccinated group (p < 0.05). Taken together, our data clearly demonstrate that the Top2A multi-peptide vaccine is highly immunogenic and provides immunopreventive efficacy against TNBC in mice. Citation Format: Sang Beom Lee, Jing Pan, Yueqiang Jiang, Katie Palen, Bryon Johnson, Romaine Ingrid Fernando, Shizuko Sei, Ronald Lubet, Ming You, Yian Wang. Immunoprevention of triple negative breast cancer by a TOP2A multi-peptide vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 647.

Enhanced CD138 peptide-specific cytotoxic T lymphocyte activities against breast, colon and pancreatic cancers in combination with pembrolizumab (anti-PD1).

e14302 Background: We investigated the ability of CD138 antigen-specific CD8+ cytotoxic T lymphocytes (CD138-CTL), generated using a novel HLA-A2-specific CD138260-268 (GLVGLIFAV) peptide, in combination with various immune checkpoint inhibitors, to target breast, colon, and pancreatic cancer. Methods: This study was designed to evaluate anti-tumor activities of CD138-CTL, alone or in combination with Pembrolizumab (anti-PD1), against solid tumors. Results: Upregulation of CD138 protein expression was detected on breast, colon, and pancreatic cancer cells. CD138-CTL induced ex vivo by repeated stimulations with HLA-A2-specific CD138260-268 (GLVGLIFAV) peptide were enriched for CD45RO+ memory CTL and demonstrated antigen-specific and HLA-A2-restricted anti-tumor activities. Treatment of CD138-CTL with anti-PD1, anti-TIM3, or anti-LAG3 or tumor cells with anti-PDL1, anti-Gal9 or anti-LAG3 resulted in an increased proliferation of CD138-specific CD8+ CTL, which express costimulatory and activation markers (CD28, CD40L, 41BB, CD69, CD38). However, expression of immune checkpoints (PD1, LAG3, CTLA4) was also induced on these CD138-CTL upon stimulation. Importantly, anti-tumor activities of CD138 peptide-CTL was significantly enhanced in combination with Pembrolizumab, especially within the antigen-specific memory CD8+ T cell subsets (central memory > effector memory), directed against HLA-A2+ breast cancer, colon cancer and pancreatic cancer. Conclusions: These data provide the framework for an immunotherapeutic strategy encompassing a CD138260-268 (GLVGLIFAV) peptide-based cancer vaccine in combination with Pembrolizumab to enhance antigen-specific central memory cells with more potent and long-lasting CD8+ CTL immune responses in patients with solid tumors. Based on these result, clinical trials evaluating CD138260-268 (GLVGLIFAV) in multi-peptide cancer vaccine are on-going in patients with triple negative breast cancer in combination with anti-PD1 (Pembrolizumab) in metastatic setting (NCT 03362060) or anti-PD-L1 (Durvalumab) in stage II/III, adjuvant setting (NCT 02826434).