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Overlapping peptides covering the entire NY-ESO-1 protein

Descriptions of Supplementary Figures 1, 2, and 3, and supplementary table 1

Supplementary Fig 2

Supplementary Fig 1

Given its ability to induce both humoral and cellular immune responses, NY-ESO-1 has been considered a suitable antigen for a cancer vaccine. Despite promising results from early-phase clinical studies in patients with melanoma, NY-ESO-1 vaccine immunotherapy has not been widely investigated in larger trials; consequently, many questions remain as to the optimal vaccine formulation, predictive biomarkers, and sequencing and timing of vaccines in melanoma treatment. We conducted an adjuvant phase I/II clinical trial in high-risk resected melanoma to optimize the delivery of poly-ICLC, a TLR-3/MDA-5 agonist, as a component of vaccine formulation. A phase I dose-escalation part was undertaken to identify the MTD of poly-ICLC administered in combination with NY-ESO-1 and montanide. This was followed by a randomized phase II part investigating the MTD of poly-ICLC with NY-ESO-1 with or without montanide. The vaccine regimens were generally well tolerated, with no treatment-related grade 3/4 adverse events. Both regimens induced integrated NY-ESO-1–specific CD4+ T-cell and humoral responses. CD8+ T-cell responses were mainly detected in patients receiving montanide. T-cell avidity toward NY-ESO-1 peptides was higher in patients vaccinated with montanide. In conclusion, NY-ESO-1 protein in combination with poly-ICLC is safe, well tolerated, and capable of inducing integrated antibody and CD4+ T-cell responses in most patients. Combination with montanide enhances antigen-specific T-cell avidity and CD8+ T-cell cross-priming in a fraction of patients, indicating that montanide contributes to the induction of specific CD8+ T-cell responses to NY-ESO-1.

Supplementary Fig 3

One of the several impediments to effective oncolytic virus therapy of cancer remains a lack of tumor-specific targeting. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells induced by tumor factors in tumor-bearing hosts. The biodistribution kinetics of MDSC and other immune cell types in a murine hepatic colon cancer model was investigated through the use of tracking markers and MRI. MDSCs were superior to other immune cell types in preferential migration to tumors in comparison with other tissues. On the basis of this observation, we engineered a strain of vesicular stomatitis virus (VSV), an oncolytic rhabdovirus that bound MDSCs and used them as a delivery vehicle. Improving VSV-binding efficiency to MDSCs extended the long-term survival of mice bearing metastatic colon tumors compared with systemic administration of wild-type VSV alone. Survival was further extended by multiple injections of the engineered virus without significant toxicity. Notably, direct tumor killing was accentuated by promoting MDSC differentiation towards the classically activated M1-like phenotype. Our results offer a preclinical proof-of-concept for using MDSCs to facilitate and enhance the tumor-killing activity of tumor-targeted oncolytic therapeutics. Cancer Res; 73(16); 5003–15. ©2013 AACR.

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PDF file - 653K, MDSC purity (S1); Effective Feridex labeling of MDSCs (S2); VSV does not alter the ability of MDSCs to migrate to tumor sites (S3); Antibody conjugation improves viral delivery to tumor sites (S4); VSV-MDSCs exhibit more tumor specificity than free virus (S5); VSV-MDSC treatment does not result in any appreciable neuropathological toxicity (S6); The survival curves of LLC lung cancer-bearing mice after various treatments (S7).

Background aims Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34+ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP). Methods cGMP VPA-mediated expansion was initiated with CD34+ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts. Results The authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products. Conclusions The authors’ results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34+ cells for clinical utilization.

Pharmacological and genetic studies over the past decade have established the follicle-stimulating hormone (FSH) as an actionable target for diseases affecting millions, namely osteoporosis, obesity, and Alzheimer’s disease. Blocking FSH action prevents bone loss, fat gain, and neurodegeneration in mice. We recently developed a first-in-class, humanized, epitope-specific FSH-blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a Good Laboratory Practice (GLP)-compliant platform, we now report the efficacy of MS-Hu6 in preventing and treating osteoporosis in mice and parameters of acute safety in monkeys. Biodistribution studies using 89Zr-labeled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone and bone marrow. The MS-Hu6 displayed a β phase t½ of 7.5 days (180 hr) in humanized Tg32 mice. We tested 217 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze-thaw and at different temperatures, with minimal aggregation, and without self-, cross-, or hydrophobic interactions or appreciable binding to relevant human antigens. The MS-Hu6 showed the same level of “humanness” as human IgG1 in silico and was non-immunogenic in ELISpot assays for IL-2 and IFN-γ in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable, and manufacturable, and is therefore poised for future human testing.

Introduction: Immunotherapies such as checkpoint blockade, have demonstrated remarkable clinical efficacy yet a large percentage of patients do not respond, potentially due to a paucity of pre-existing immune priming against neoantigens. We developed a personalized genome vaccine (PGV_001) platform to generate neoantigen vaccines targeting each patient’s unique mutanome. Primary objectives of the study were to determine 1) the safety and tolerability; 2) the feasibility of PGV_001 production and administration; and 3) the immunogenicity of PGV_001. Secondary objectives included immunophenotyping vaccine driven cellular and soluble immune milieu in peripheral blood. We previously reported on the clinical efficacy, and here we report, analysis of vaccine-driven immune responses in all treated patients. Methods: The study (Trial Registration NCT02721043) enrolled patients with resected malignancies, including Head and neck squamous cell carcinomas, breast cancer and bladder cancer, or, in the case of multiple myeloma treated with autologous stem cell transplant; all patients determined to have a high risk of disease recurrence (>30% over 5 years). Tumor-derived and germline RNA and DNA was sequenced to predict neoantigens utilizing our custom computation pipeline, OpenVax. Approximately 10 peptides were synthesized per patient, and a mixture of these peptides was administered as 10 subcutaneous and intradermal vaccines over 27 weeks in combination with poly-ICLC and helper Tetanus peptide as adjuvants. Immune responses were analyzed utilizing assays including IFN-gamma ELISPOT, antigen specific T cell expansion followed by flow cytometry, etc. Results: In total 148 neoantigen peptides were manufactured for 15 patients. Overall, 136 PGV_001 doses were administered to 13 patients. Vaccine-specific T cell immunity was observed against multiple vaccine neoepitopes in all 13 subjects. Of the peptides administered, 45% of vaccine antigens (57/126) induced de novo immunity, starting as early as Week8 and often sustaining past last vaccination. Notably, while the vaccine driven T cell immunity was CD4 T cell dominant, most evaluated subjects also displayed vaccine induced polyfunctional CD8-T cell responses. Additional studies are ongoing to define qualities of reactive T cells, evaluate vaccine-induced humoral responses and probe the circulating inflammatory immune milieu. These will be presented. Conclusions: We have established a platform for generating personalized neoantigen vaccines. 100% of the vaccinated patients developed an immune response specific to the vaccine neoantigens predicted by OpenVax. Subjects who received treatment experienced mild Grade 1 or 2 adverse reactions as per the CTEP v 4.0 NCI CTCAE. This vaccine trial reached the primary endpoint of safety, tolerability, feasibility and immunogenicity. Based on the PGV_001 platform two clinical trials, one in patients with glioblastoma multiforme (NCT03223103) in combination with TT fields and second in patients with urothelial carcinoma (NCT03359239) in combination with Atezolizumab have been performed. Data from these trials is under evaluation. Citation Format: Mansi Saxena, Thomas Marron, Julia Kodysh, Alex Rubinsteyn, John Finnigan, Ana Blasquez, Marcia Meseck, Tim O'Donnell, Daniela Delbeau, Mathew Galsky, Deborah Doroshow, Brett Miles, Krzysztof Misiukiewicz, Hanna Irie, Amy Tiersten, Samir Parekh, Marshall Posner, Andrea Wolf, John Mandeli, Rachel Brody, Sacha Gnjatic, Eric Schadt, Philip Friedlander, Nina Bhardwaj. Immunogenicity of PGV_001 neoantigen vaccine in a Phase-I clinical trial, across various types of cancers in adjuvant setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT270.

Pharmacological and genetic studies over the past decade suggest that FSH is an actionable target for diseases affecting millions, notably osteoporosis, obesity and Alzheimer's disease (AD). Blocking FSH action prevents bone loss (1, 2), fat and energy metabolism (3) and AD–like features in mice (4). We recently developed a first–in–class, humanized, epitope–specific FSH blocking antibody that binds to a 13–amino–acid–long sequence of FSHβ—"MS-Hu6"—with a KD of 7.52 nM (5). We showed that MS-Hu6 bound specifically to FSHβ and its different glycosylated forms, namely FSHβ21/18 and FSHβ24, without binding to LH and TSH. Here, using a GLP–compliant platform, we report the efficacy of MS-Hu6 in preventing obesity, osteoporosis and AD in mice. Notably, MS-Hu6-treated mice showed lower body weight and fat mass, increased lean mass (qNMR) and evidence of beiging in ThermoMice (IVIS imaging) compared with IgG–treated mice. Consistent with this, the thermogenic genes Ucp1 and Cidea were upregulated, whereas Pparg expression was attenuated in fat depots. Treatment of ThermoMice for 8 weeks also increased bone mineral density (BMD), improved microstructure (micro-CT), elevated bone formation (dynamic histomorphometry), and upregulated the osteoblastic genes Alp and Col1a1. The increase in bone mass and improved microstructure were replicated in C.J.R's lab using female mice 24 weeks post–ovariectomy. Preliminary testing using AD mice, namely APP/PS1 mice, showed that MS-Hu6 prevented the impairment in recognition and contextual memory. Biodistribution studies using 89Zr–labelled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone, bone marrow and fat depots. MS-Hu6 displayed a β phase t½ of 13 days (316 hours) in humanized Tg32 mice, and bound endogenous FSH. In monkeys, an acute single injection of MS-Hu6 did not affect vitals, and biochemical parameters remained within the normative range. We tested 215 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze–thaw and at different temperatures, with minimal aggregation, and without self–, cross–, or hydrophobic interactions or appreciable binding to relevant human antigens. MS-Hu6 showed the same level of "humanness" as human IgG1 in silico, and was non–immunogenic in ELISPOT assays for IL-2 and IFNγ in human peripheral blood mononuclear cell cultures. In conclusion, MS-Hu6 is efficacious, durable and manufacturable, and is therefore poised for future human testing as a multipurpose therapeutic for obesity, osteoporosis, and perhaps for AD.References: 1Sun et al., Cell, 2006, PMID: 16630814; Ji et al, PNAS, 2018, PMID: 29440419; 3Liu et al., Nature, 2017, PMID: 28538730; 4Xiong et al., Nature (In press); 5Gera et al., PNAS, 2020, PMID: 33127753 Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Pharmacological and genetic studies over the past decade have established FSH as an actionable target for diseases affecting millions, notably osteoporosis, obesity and Alzheimer’s disease (AD). Blocking FSH action prevents bone loss, fat gain and AD–like features in mice. We recently developed a first–in–class, humanized, epitope–specific FSH blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a GLP–compliant platform, we now report the efficacy of MS-Hu6 in preventing obesity and osteoporosis in mice, and parameters of acute safety in monkeys. Biodistribution studies using 89Zr–labelled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone, bone marrow and fat depots. MS-Hu6 displayed a β phase t½ of 13 days (316 hours) in humanized Tg32 mice, and bound endogenous FSH. We tested 215 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze–thaw and at different temperatures, with minimal aggregation, and without self–, cross–, or hydrophobic interactions or appreciable binding to relevant human antigens. MS-Hu6 showed the same level of “humanness” as human IgG1 in silico, and was non–immunogenic in ELISPOT assays for IL-2 and IFNγ in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable and manufacturable, and is therefore poised for future human testing as a multipurpose therapeutic.

SummaryBackgroundProduction of affordable coronavirus disease 2019 (COVID-19) vaccines in low- and middle-income countries is needed. NDV-HXP-S is an inactivated egg-based Newcastle disease virus vaccine expressing the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It’s being developed in Thailand, Vietnam, and Brazil; herein are initial results from Thailand.MethodsThis phase 1 stage of a randomised, dose-escalation, observer-blind, placebo-controlled, phase 1/2 trial was conducted at the Vaccine Trial Centre, Mahidol University (Bangkok). Healthy adults aged 18-59 years, non-pregnant and negative for SARS-CoV-2 antibodies were eligible. Participants were block randomised to receive one of six treatments by intramuscular injection twice, 28 days apart: 1 µg±CpG1018 (a toll-like receptor 9 agonist), 3 µg±CpG1018, 10 µg, or placebo. Participants and personnel assessing outcomes were masked to treatment. The primary outcomes were solicited and spontaneously reported adverse events (AEs) during 7 and 28 days after each vaccination, respectively. Secondary outcomes were immunogenicity measures (anti-S IgG and pseudotyped virus neutralisation). An interim analysis assessed safety at day 57 in treatment-exposed individuals and immunogenicity through day 43 per protocol. ClinicalTrials.gov (NCT04764422).FindingsBetween March 20 and April 23, 2021, 377 individuals were screened and 210 were enrolled (35 per group); all received dose one; five missed dose two. The most common solicited AEs among vaccinees, all predominantly mild, were injection site pain (InterpretationNDV-HXP-S had an acceptable safety profile and potent immunogenicity. The 3 µg and 3 µg+CpG1018 formulations advanced to phase 2.FundingNational Vaccine Institute (Thailand), National Research Council (Thailand), Bill & Melinda Gates Foundation, National Institutes of Health (USA)

Background: Dendritic cells (DCs) play a critical role in anti-tumor immune response. However, tumor-induced immunosuppression promotes DC dysfunction and T cell exhaustion resulting in evasion of tumor immunity. In this study, we aimed to compare two approaches for engaging DCs and inducing an immune response to tumor antigens in the absence of tumor (melanoma) Methods: This is a Phase II open-label, randomized, two-arm study to compare Arm A: Poly-ICLC-matured DC+PolyICLC to Arm B: Montanide ISA-51-VG+Poly-ICLC, as adjuvants for NY-ESO-1 and Melan-A/MART-1 long peptides in patients with melanoma in complete clinical remission but at high risk of disease recurrence (NCT02334735). Each arm also received helper peptide, keyhole limpet hemocyanin (KLH) with the first vaccine and Poly-ICLC on day 2 of each vaccine. 36 patients were consented and randomized. Of these, 31 subjects received treatment, 16 in Arm A and 15 in Arm B. Immunohistochemistry (IHC) was used to determine expression of NY-ESO-1 and Melan-A/MART-1 and to determine the immune infiltrate landscape in the primary tumors. Humoral responses, TCR clonality, and inflammatory pathways were assessed by ELISA, bulk TCR sequencing, and Olink, respectively. Functional T-cell responses were investigated ex-vivo by interferon (IFN)-g enzyme-linked immunospot assay (ELISPOT) and after expansion by intracellular cytokine staining. Results: Arm B induced a stronger humoral response vs Arm A against both Melan-A/MART-1 and NY-ESO-1. A stronger ex vivo IFN-g response as well as expanded CD4+ T cell response against NY-ESO-1 was also induced in Arm B. However, the response to Melan-A/MART-1, as measured by ex vivo ELISPOT and expanded CD4+T cell assay, was comparable in both arms. Interestingly, while similar proportions of patients in each arm displayed a CD8+ T cell response to NYESO-1, more patients in Arm A vs Arm B responded with a CD8+ T cell response to Melan-A/MART-1 (9/16 responders in Arm A vs 4/14 in Arm B). Melan-A expression was observed in 81% of the patients but did not correlate with antigen specific immune response. TCR sequencing, Olink analysis and evaluation of NY-ESO-1 expression and immune infiltrates in the primary tumors is ongoing. Conclusion: This trial reached the primary endpoint of safety and tolerability. Arm B induced a stronger antibody and CD4+ T cell response, especially to NY-ESO-1, whereas the Arm A vaccine appears to be more efficient at eliciting CD8+ T cell responses against MelanA/MART1. The seemingly enhanced CD8 T cell response in Arm A versus Arm B, could be attributed to frequency of specific HLA-I alleles in either arm or pre-existing immune response in select patients. A deeper investigation into correlation between HLA type and cellular immune response to vaccine is ongoing. Citation Format: Mansi Saxena, Ashleih Burke, Anna Pavlick, Ana Blazquez, Gustavo Gimenez, Marcia Meseck, Michael Donovan, Denise Rodriguez, Mireia Castillo-Martin, Tin Htwe Thin, Rachel Sabado, John Mandeli, Sacha Gnjatic, Philip Friedlander, Nina Bhardwaj. Immunogenicity of Poly-ICLC matured dendritic cells as an adjuvant for NY-ESO-1 and Melan-A-MART-1 peptide vaccination compared to Montanide® ISA-51 VG, in study subjects with melanoma in complete clinical remission but at high risk of disease recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT108.

Background The efficacy of T cell directed immunotherapies relies on adequate priming of T cells to tumor-specific neoantigens, which some studies have augmented with synthetic neoantigen vaccines. This is the first report of a personalized genomic vaccine (PGV-001) in multiple histologies in the adjuvant setting. Methods Tumor and germline RNA and DNA were sequenced, and neoantigen peptides were selected using our OpenVax custom computation pipeline that identifies and ranks mutant sequences by a combination of predicted MHC-I binding affinity and neoantigen abundance within tumor. Up to 10 peptides were synthesized per patient and were administered over the course of 27 weeks in combination with the poly-ICLC. Primary objectives were to determine 1) the safety and tolerability; 2) the feasibility of PGV-001 production and administration; and 3) the immunogenicity of PGV-001. Secondary objectives included immunophenotyping neoantigen-specific T cells in peripheral blood, and characterization of peripheral blood lymphoid, myeloid and humoral responses. We report here for the first time on the primary endpoints. Results Vaccine was synthesized for 15 patients. A mean of 1619 somatic variants (range 521–5106) were detected. Our pipeline identified a mean of 67.1 neoantigens/patient (range 8–193) and 9.7 peptides/patient were synthesized (range 7–10). 13 patients received PGV-001 (11 patients received all 10 doses and 2 patients received at least 8 doses) while 2 had progressive disease before vaccine initiation. Transient grade 1 injection site reactions were seen in 31% of patients, and one patient experienced grade 1 fever. There were no other significant adverse events. Ex vivo ELISpot analysis of patient blood demonstrated significant induction of T cell responses following receipt of 10 vaccines that were not present after the 6th vaccine, supporting the need for a prolonged dosing schedule. Robust responses were seen in both CD4 and CD8 T cells by intracellular cytokine staining for TNF-a, IFN-a, and IL-2 following in vitro expansion in the presence of vaccine antigens. Additional studies are ongoing to define the most immunogenic antigens. Conclusions A personalized neoantigen vaccine of synthetic mutant peptides and adjuvant poly-ICLC was successfully synthesized for 15 patients and administered successfully to 87% patients over the course of 27 weeks. The vaccine was well tolerated, and T cell expansion and reactivity to synthetic neoantigens confirms immunogenicity of neoantigens identified with OpenVax. Trial Registration NCT02721043 Ethics Approval This study was approved by the IRB of The Mount Sinai Hospital in accordance with Federal law. HSM #15-00841.

Background: The majority of novel cancer immunotherapies rely on adequate priming of T cells to tumor-specific neoantigens, which is believed to be lacking in patients who do not respond to therapy. We developed a personalized genomic vaccine (PGV-001) in which patient-specific synthetic neoantigen peptides (25 mer),are formulated and administered to patients with multiple cancer types in the adjuvant setting (NCT02721043). Methods: This trial enrolled patients whom had undergone curative-intent surgery (solid tumor patients) or autologous stem cell transplant (multiple myeloma patients), and for whom there was >30% chance of recurrence. Sequencing of tumor and germline DNA and RNA was performed and the OpenVax custom computation pipeline was used to identify candidate neoantigens; this platform ranks transcribed mutations using predicted MHC-I binding affinity and neoantigen abundance. A maximum of 10 peptides were synthesized per patient. Peptides were administered over the course of 27 weeks with poly-ICLC and a tetanus helper peptide. Primary objectives were to determine the safety and tolerability of vaccination, feasibility of vaccine production and administration, and immunogenicity. Results: Within 15 patients enrolled, the OpenVax pipeline identified an average of 67.1 neoantigens/patient (range 8-193), only two patients did not have adequate number of neoantigens identified to synthesize 10 peptides. 13 of the 15 patients received PGV-001, including 10 patients with solid tumor diagnoses and 3 patients with multiple myeloma, 11 of whom received all 10 doses, while 1 experienced progression of disease while on treatment. The vaccine was well tolerated, with grade 1 injection site reactions in 31% of patients, and grade 1 fever in one patient; there were no other significant adverse events. While one patient was lost to follow-up, of the remaining 12 patients the median progression-free survival from the time of their surgery or transplant of 618 days. With a mean follow-up of 925 days, 4 patients remain without evidence of disease, 4 patients are receiving subsequent lines of therapy, and 4 patients have died, though notably only two with documented recurrence of their malignancy. Initial analysis of the patient samples analyzed confirms immunogenicity. T cell responses were measured using ex vivo ELISpot and intracellular cytokine staining following expansion with neoantigen peptide libraries, both demonstrating induction of IFN-gamma, TNF-alpha and IL-2. Notably, robust T cell reactivity was only seen at the completion of all 10 vaccines, supporting the need for a prolonged schedule. Conclusions: PGV-001 was successfully synthesized for 15 patients and administered successfully to 13 patients without significant adverse events. Immune monitoring of immunogenicity is ongoing, with initial analysis demonstrating induction of neoantigen-specific CD4 and CD8 T cell expansion. Citation Format: Thomas Urban Marron, Mansi Saxena, Nina Bhardwaj, Marcia Meseck, Alex Rubinsteyn, John Finnigan, Julia Kodysh, Ana Blazquez, Tim O'Donnel, Mathew Galsky, Deborah Doroshow, Brett Miles, Krysztof Misiukiewicz, Hanna Irie, Amy Tiersten, Samir Parekh, Marshall Posner, Andrea Wolf, John Mandeli, Rachel Brody, Sacha Gnjatic, Eric Schadt, Philip Friedlander, Jeffrey Hammerbacher. An adjuvant personalized neoantigen peptide vaccine for the treatment of malignancies (PGV-001) [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 LB048.

Given its ability to induce both humoral and cellular immune responses, NY-ESO-1 has been considered a suitable antigen for a cancer vaccine. Despite promising results from early-phase clinical studies in patients with melanoma, NY-ESO-1 vaccine immunotherapy has not been widely investigated in larger trials; consequently, many questions remain as to the optimal vaccine formulation, predictive biomarkers, and sequencing and timing of vaccines in melanoma treatment. We conducted an adjuvant phase I/II clinical trial in high-risk resected melanoma to optimize the delivery of poly-ICLC, a TLR-3/MDA-5 agonist, as a component of vaccine formulation. A phase I dose-escalation part was undertaken to identify the MTD of poly-ICLC administered in combination with NY-ESO-1 and montanide. This was followed by a randomized phase II part investigating the MTD of poly-ICLC with NY-ESO-1 with or without montanide. The vaccine regimens were generally well tolerated, with no treatment-related grade 3/4 adverse events. Both regimens induced integrated NY-ESO-1–specific CD4+ T-cell and humoral responses. CD8+ T-cell responses were mainly detected in patients receiving montanide. T-cell avidity toward NY-ESO-1 peptides was higher in patients vaccinated with montanide. In conclusion, NY-ESO-1 protein in combination with poly-ICLC is safe, well tolerated, and capable of inducing integrated antibody and CD4+ T-cell responses in most patients. Combination with montanide enhances antigen-specific T-cell avidity and CD8+ T-cell cross-priming in a fraction of patients, indicating that montanide contributes to the induction of specific CD8+ T-cell responses to NY-ESO-1.

Background: Mutation-derived tumor antigens (MTAs) arise as a result of somatic mutations, such as nucleotide substitutions and small insertions/deletions. MTAs can serve as specific targets for antitumor therapy, including neoantigen vaccines. The goal of neoantigen vaccination is to help prime T cells to recognize such tumor-specific mutations. Here we describe a phase I trial testing a personalized genomic vaccine (PGV-001) in multiple histologies in the adjuvant setting (NCT02721043). Methods: This trial included patients with histologic diagnosis of solid malignancies or multiple myeloma with a >30% risk of recurrence but no measurable disease at the time of first vaccination. For each patient, HLA typing was performed, and the patient's tumor and germline DNA and tumor RNA were sequenced. Mutated peptides containing predicted neoantigens were selected using the OpenVax computational pipeline, which prioritizes somatic mutations by expression of the mutant allele in the tumor RNA and predicted MHC class I epitope binding for the patient's HLA type. A maximum of 10 peptides was included in each patient's personalized vaccine. The vaccine was administered over the course of 6 months, given in combination with poly-ICLC as the adjuvant. The primary objectives were to determine safety and tolerability, feasibility, and immunogenicity of the PGV-001 neoantigen vaccine. Results: The neoantigen vaccine was successfully administered to 13 patients, spanning 5 different tumor types. For each patient, an average of 1730 somatic mutations were identified (range 521-5106), of which 349 were coding variants (range 68-1493), 88 were coding and expressed in the tumor RNA (range 9-233), and 71 were coding, expressed and resulted in predicted MHC class I ligands (range 8-193). The urothelial, head & neck, and lung tumors resulted in larger numbers of predicted neoantigens than in the cases of multiple myeloma or breast tumors. Despite this difference, each of the 13 patient tumor samples showed enough of a neoantigen load to enable vaccination. We have also initiated analysis of immunogenicity, and early reports demonstrate neoantigen-specific CD4 and CD8 T-cell responses. Conclusions: The PGV-001 personalized neoantigen vaccine was successfully administered to 13 patients, where each patient's neoantigen load was adequate for vaccine synthesis. While we have demonstrated the computational feasibility of identification of neoantigens for inclusion in the PGV-001 genomic vaccine, clinical outcomes will be reported separately. Citation Format: Julia Kodysh, Thomas Marron, Alex Rubinsteyn, Tim O'Donnell, John Finnigan, Ana Blazquez, Mansi Saxena, Marcia Meseck, Philip Friedlander, Nina Bhardwaj. PGV-001: A phase I trial of a multipeptide personalized neoantigen vaccine in the adjuvant setting [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 CT173.

e14307 Background: Mutation-derived tumor antigens (MTAs) arise as a direct result of somatic variations that occur during carcinogenesis and can be characterized via genetic sequencing and used to identify MTAs. We developed a platform for a fully-personalized MTA-based vaccine in the adjuvant treatment of solid and hematological malignancies. Methods: This is a single-arm, open label, proof-of-concept phase I study designed to test the safety and immunogenicity of Personalized Genomic Vaccine 001 (PGV001) that targets up to 10 predicted personal tumor neoantigens based on patient’s HLA profile (ClinicalTrials.gov: NCT02721043). Results: Patients who completed vaccination with PGV001_002 (head and neck squamous cell cancer) received 10 doses of vaccine comprising 10 long peptides (LP) combined with poly-ICLC (toll-like receptor-3 agonist) intradermally. Vaccine-induced T-cell responses were determined at weeks 0 and 27 (before and after treatment, respectively), ex vivo by interferon (IFN)-g enzyme-linked immunospot assay and after expansion by intracellular cytokine staining. Overlapping 15-mer peptides (OLPs) spanning the entirety of each LP and 9-10-mer peptides corresponding to each predicted class I epitope (Min) were pooled. Ex vivo responses to these peptide pools were undetectable at week 0 but were evident at week 27 against 2 OLPs out of 10 (20%) and in 5 Min out of 10 (50%). After in vitro expansion, neoantigen-specific CD4+ and CD8+ T-cell responses were found in 5 out of 10 pooled peptides (50%). 7 out of 10 (70%) epitopes elicited polyfunctional T-cell responses (secretion of INF-g, TNF-a, and/or IL-2) from either CD4+ or CD8+ T cells. Conclusions: The PGV001 vaccine in our first patient showed both safety and immunogenicity, eliciting CD4+ and CD8+ responses to the vaccine peptides. As we enroll additional patients in this clinical trial, and perform deeper phenotyping of their tumor-reactive T cells, we will learn the determinants necessary for the successful generation of MTA-based vaccines, while informing future immunotherapeutic approaches and rational combinations. Clinical trial information: NCT02721043.

9538 Background: Dendritic cells (DC) play a critical role in tumor immune-surveillance. Combination therapies by utilizing check point inhibitors may revert tumor-induced-T cell exhaustion; however, DCs are necessary to prime/activate T cells to target tumor cells. Montanide is a mineral oil-based adjuvant that enhances the immune response to vaccination. In this study, we compared the immunogenicity of Montanide and poly-ICLC-matured DCs. Methods: This is a Phase II open label, randomized two arm study to compare Poly-ICLC matured DC with systemic administration of Poly-ICLC on days 1 and 2 (ARM A) to Montanide ISA-51 and Poly-ICLC as adjuvants for NY-ESO-1 and Melan-A/MART-1 peptide vaccination with systemic administration of Poly-ICLC on day 2 (ARM B) in study subjects with melanoma in complete clinical remission but at high risk of disease recurrence (NCT02334735). Evaluation of primary tumor expression of NY-ESO-1 and Melan-A tumor was determined by immunohistochemistry (IHC). Humoral responses were assessed by Seromics (ELISA) and T-cell responses were performed ex-vivo by interferon (IFN)-g enzyme-linked immunospot assay (ELISPOT) and after expansion by intracellular cytokine staining (ICS). Results: Twenty-nine patients have been enrolled in this study. IHC studies demonstrated tumor expression of NY-ESO-1 and Melan-A in 78% and 81% of the patients, respectively. 100% of patients within arm B became seropositive for NY-ESO-1 peptide by cycle 2 day 8 (C2D8). 80% of patients within arm A also seroconverted to this antigen but titers were significantly lower. Melan-A-specific antibody responses were also found in arm B patients, but to a lesser degree. However, arm A patients failed to develop seroreactivity to Melan-A. Cellular responses are under analysis. Preliminary data show that subjects in both arms develop T cell responses to both antigens. Conclusions: This vaccine trial reached the primary endpoint of safety and tolerability. Patients vaccinated with either DC or Montanide had demonstrable antibody titers to immunizing antigens, although the latter reproducibly induced higher titers. Evaluation of cellular responses is ongoing. Clinical trial information: NCT02334735.

Introduction: Mutation-derived tumor antigens (MTAs) arise as a direct result of somatic variations, including nucleotide substitutions, insertions, and deletions that occur during carcinogenesis. These somatic variations can be characterized via genetic sequencing and used to identify MTAs. We developed a platform for a fully-personalized MTA-based vaccine in the adjuvant treatment of solid and hematologic malignanicies. Methods: This is a single-arm, open label, proof-of-concept phase I study designed to test the safety and immunogenicity of Personalized Genomic Vaccine 001 (PGV001) that targets up to 10 predicted personal tumor neoantigens. The single-center study will enroll 20 eligible subjects with histologic diagnosis of solid and hematologic malignancies. Subjects must have no measurable disease at time of first vaccine administration, and 5-year disease recurrence risk of > 30%. Toxicity will be defined by CTCAE v5.0. Blood samples will be collected at various time points for immune response monitoring. Each patient’s vaccine peptides are selected by identifying somatic mutations from comparison of tumor and normal exome sequencing data, phasing somatic variants with co-occurring germline variants using tumor RNA sequencing data, and ranking mutated peptide sequences ”Openvax pipeline.” The process for determining somatic variants hews closely to the Broad Institute’s “Best Practices” for cancer SNVs and indels. The phasing of somatic and germline variants is implemented in a custom bioinformatics tool called Isovar. Mutated protein sequences containing phased variants are ranked according to two criteria: expression of the mutant allele in tumor RNA and aggregated predicted affinity to the patient’s Class I MHCs. Both quantities are normalized and multiplied together to create single ranked ordering of the candidate mutant sequences. Results: PGV001_002 (head and neck squamous cell cancer), who has completed vaccination, received 10 doses of vaccine comprising 10 long peptides (25 amino acid length) combined with poly-ICLC (toll-like receptor-3 agonist) intradermally. Vaccine-induced blood T-cell responses were determined, at weeks 0 (before-treatment) and 27 (after-treatment), ex vivo by interferon (IFN)-g enzyme-linked immunospot (ELISPOT) assay and after in vitro expansion by intracellular cytokine staining (ICS). Overlapping 15-16-mer assays peptides (OLPs) spanning the entirety of each ILP and 9-10-mer peptides corresponding to each predicted class I epitope (Min) were pooled and used to monitor immunogenicity. Ex vivo responses to these peptide pools were undetectable at week 0 but were evident at week 27 against 2 OLPs out of 10 (20%) and in 5 Min out of 10 (50%). After in vitro expansion, neoantigen-specific CD4+ and CD8+ T-cell responses were found in 5 out of 10 pooled peptides (50%). 7 out of 10 (70%) epitopes elicited polyfunctional T-cell responses (secretion of INF-α, TNF-α, and/or IL-2) from either CD4+ or CD8+ T-cells. Conclusion: To identify which predicted epitopes within the peptides pools stimulated the T-cell responses, we deconvoluted all the pools by either ex vivo and in vitro expansion. Ex vivo IFN-α production was detected in 1 (15-mer) peptide out of 15 (6.7%) and in 4 (9-10-mer) peptides out of 22 (18.2%). After expansion with single peptides, of 22 (9-10-mer) peptides tested, CD8+ T-cells were reactive against 13 peptides (59%), while CD4+ responses were seen in response to 11 of 15 (15-16-mer) peptides tested. Both CD4+ and CD8+ T-cell responses were polyfunctional. The PGV001 vaccine in our first patient showed both safety and immunogenicity, eliciting both CD4+ and CD8+ responses to the vaccine peptides. As we are enrolling additional patients, the information learned from this clinical trial will instruct the next generation of MTA-based vaccines, future development of immunotherapeutic approaches and rational combinations. Citation Format: Ana B. Blazquez, Alex Rubinsteyn, Julia Kodysh, John P. Finnigan, Thomas Marron, Rachel L. Sabado, Marcia Meseck, Timothy J. O'Donnell, Jeffrey Hammerbacher, Michael Donovan, John Holt, Milind Mahajan, John Mandeli, Krysztof Misiukiewicz, Eric M. Genden, Brett A. Milles, Hooman Khorasani, Peter R. Dottino, Hanna Irie, Amy B. Tiersten, Elisa R. Port, Andrea S. Wolf, Hern J. Cho, Ashutosh Tewari, Samir S. Parekh, Sujit Nair, Matthew D. Galsky, William K. Oh, Sacha Gnjatic, Eric E. Schadt, Phillip A. Friedlander, Nina Bhardwaj. A phase I study of the safety and immunogenicity of a multipeptide personalized genomic vaccine in the adjuvant treatment of solid cancers [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A005.

Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the second leading cause of cancer-related mortality in the United States. The liver and lung are the most common sites of distant metastasis of CRC. The approval of newer chemotherapeutic agents such as oxaliplatin, irinotecan, bevacizumab, cetuximab and panitumumab has significantly improved survival, yet the majority of patients still succumb to the disease in less than 2 years. Novel therapeutic agents that can provide significant clinical benefit for metastatic CRC patients are needed. Oncolytic vesicular stomatitis virus (VSV) is a promising tool as a cancer therapeutic agent. In this study, we examined the feasibility of repeated intravenous infusions of rVSV in multiple CRC lung metastases, compared with repeated hepatic arterial administration in multifocal CRC liver metastasis in immune competent rats. We established a multifocal liver metastases model or the multiple lung metastases model using a CRC cell line, RCN-H4, implanted into syngeneic F344/DuCrj rats. 4.0x10(6) plaque-forming units (pfu) of recombinant VSV vectors expressing mutant (L289A) Newcastle disease virus fusion protein [rVSV-NDV/F(L289A)] were administered 3 times for 3 consecutive days locally via the hepatic artery for liver metastases or systemically via the penial vein for lung metastases. In the liver metastasis model, significantly enhanced survival was observed with rVSV-NDV/F(L289A)-treated rats (P=0.0196). Median survival was 110 and 25 days, respectively. In addition, 4 out of 7 of the rVSV-NDV/F(L289A)-treated rats demonstrated long-term survival exceeding 100 days. The long-term surviving rats were sacrificed to evaluate for residual malignancy. Liver tumors were not detected. In the lung metastasis model, median survival was 10 [VSV-NDV/F(L289A)-treated rats] and 7 days (control). Although survival was significantly prolonged (P

Exploitation of the patient's own immune system to induce antitumor immune responses using dendritic cell (DC) immunotherapy has been established in early clinical trials as a safe and promising therapeutic approach for cancer. However, their limited success in larger clinical trials highlights the need to optimize DC vaccine preparations. This chapter describes the methodologies utilized for the preparation of the DC vaccine most commonly used in clinical trials. Optional variations at different stages in DC vaccine preparation, based on the nature of antigen, delivery of antigen, maturation stimuli, and mode of administration for DC vaccines, are also presented for consideration as these are often dependent on the disease setting, desired immune response, and/or resources available.

Exploitation of the patient's own immune system to induce antitumor immune responses using dendritic cell (DC) immunotherapy has been established in early clinical trials as a safe and promising therapeutic approach for cancer. However, their limited success in larger clinical trials highlights the need to optimize DC vaccine preparations. This chapter describes the methodologies utilized for the preparation of the DC vaccine most commonly used in clinical trials. Optional variations at different stages in DC vaccine preparation, based on the nature of antigen, delivery of antigen, maturation stimuli, and mode of administration for DC vaccines, are also presented for consideration as these are often dependent on the disease setting, desired immune response, and/or resources available.

In tumor-bearing hosts, myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) play important roles in immune suppression, the reversal of which is vitally important for the success of immune therapy. We have shown that ckit ligand is required for MDSC accumulation and Treg development. We hypothesized that sunitinib malate, a receptor tyrosine kinase inhibitor, could reverse MDSC-mediated immune suppression and modulate the tumor microenvironment, thereby improving the efficacy of immune-based therapies. Treatment with sunitinib decreased the number of MDSC and Treg in advanced tumor-bearing animals. Furthermore, it not only reduced the suppressive function of MDSCs but also prevented tumor-specific T-cell anergy and Treg development. Interestingly, sunitinib treatment resulted in reduced expression of interleukin (IL)-10, transforming growth factor-β, and Foxp3 but enhanced expression of Th1 cytokine IFN-γ and increased CTL responses in isolated tumor-infiltrating leukocytes. A significantly higher percentage and infiltration of CD8 and CD4 cells was detected in tumors of sunitinib-treated mice when compared with control-treated mice. More importantly, the expression of negative costimulatory molecules CTLA4 and PD-1 in both CD4 and CD8 T cells, and PDL-1 expression on MDSC and plasmacytoid dendritic cells, was also significantly decreased by sunitinib treatment. Finally, sunitinib in combination with our immune therapy protocol (IL-12 and 4-1BB activation) significantly improves the long-term survival rate of large tumor-bearing mice. These data suggest that sunitinib can be used to reverse immune suppression and as a potentially useful adjunct for enhancing the efficacy of immune-based cancer therapy for advanced malignancies. [Cancer Res 2009;69(6):2514–22]

Oncolytic vesicular stomatitis virus (VSV) is being developed as a novel therapeutic agent for cancer treatment, although it is toxic in animals when administered systemically at high doses. Its safety can be substantively improved by an M Delta 51 deletion in the viral genome, and yet VSV(M Delta 51) induces a much greater, robust cellular inflammatory response in the host than wild-type VSV, which severely attenuates its oncolytic potency. We have reported that the oncolytic potency of wild-type VSV can be enhanced by vector-mediated expression of a heterologous viral gene that suppresses cellular inflammatory responses in the lesions. To develop an effective and safe VSV vector for cancer treatment, we tested the hypothesis that the oncolytic potency of VSV(M Delta 51) can be substantively elevated by vector-mediated expression of M3, a broad-spectrum and high-affinity chemokine-binding protein from murine gammaherpesvirus-68. The recombinant vector rVSV(M Delta 51)-M3 was used to treat rats bearing multifocal lesions (1-10 mm in diameter) of hepatocellular carcinoma (HCC) in their liver by hepatic artery infusion. Treatment led to a significant reduction of neutrophil and natural killer cell accumulation in the lesions, a 2-log elevation of intratumoral viral titer, substantively enhanced tumor necrosis, and prolonged animal survival with a 50% cure rate. Importantly, there were no apparent systemic and organ toxicities in the treated animals. These results indicate that the robust cellular inflammatory responses induced by VSV(M Delta 51) in HCC lesions can be overcome by vector-mediated intratumoral M3 expression, and that rVSV(M Delta 51)-M3 can be developed as an effective and safe oncolytic agent to treat advanced HCC patients in the future.

Oncolytic virotherapy is a promising strategy for treatment of malignancy, although its effectiveness is hampered by host antiviral inflammatory responses. The efficacy of treatment of oncolytic vesicular stomatitis virus (VSV) in rats bearing multifocal hepatocellular carcinoma (HCC) can be substantially elevated by antibody-mediated depletion of natural killer (NK) cells. In order to test the hypothesis that the oncotyic potency of VSV can be exponentially elevated by evasion of inflammatory responses in vivo, we constructed a recombinant VSV vector expressing equine herpes virus-1 glycoprotein G, which is a broad-spectrum viral chemokine binding protein (rVSV-gG). Infusion of rVSV-gG via the hepatic artery into immune-competent rats bearing syngeneic and multifocal HCC in their livers, resulted in a reduction of NK and NKT cells in the tumors and a 1-log enhancement in intratumoral virus titer in comparison with a reference rVSV vector. The treatment led to increased tumor necrosis and substantially prolonged animal survival without toxicities. These results indicate that rVSV-gG has the potential to be developed as an effective and safe oncolytic agent to treat patients with advanced HCC. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host antiviral inflammatory responses could have general applicability in the field of oncolytic virotherapy for cancer.

4T1 breast carcinoma is a highly malignant and poorly immunogenic murine tumor model that resembles advanced breast cancer in humans, and is refractory to most immune stimulation-based treatments. We hypothesize that the ineffectiveness of immune stimulatory treatment is mediated by the suppressive effects of CD4(+)CD25(+) regulatory T (Treg) cells, which can be attenuated by engaging the glucocorticoid-induced tumor necrosis factor receptor family-related protein with its natural ligand (GITRL); further, combination treatment with existing immune stimulation regimens will augment anti-tumor immunity and eradicate metastatic 4T1 tumors in mice.A soluble homodimeric form of mouse GITRL (mIg-mGITRLs) was molecularly constructed and used to treat orthotopic 4T1 tumors established in immune-competent, syngeneic Balb/c mice. When applied in combination with adenovirus-mediated intratumoral murine granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-12 (IL-12) gene delivery plus systemic 4-1BB activation, mIg-mGITRLs attenuated the immune-suppressive function of splenic Treg cells, which led to elevated interferon-gamma (IFN-gamma) production, tumor-specific cytolytic T-cell activities, tumor rejection and long-term survival in 65% of the animals without apparent toxicities. The results demonstrate that addition of mIg-mGITRLs to an immune-stimulatory treatment regimen significantly improved long-term survival without apparent toxicity, and could potentially be clinically translated into an effective and safe treatment modality for metastatic breast cancer in patients.

We have previously shown that the local-membrane bound 4-1BB ligand and IL-12 gene transfer induced a significant antitumor response in a mouse colon carcinoma model. However, a high viral dose was required in order to achieve the best efficacy. In this study, we hypothesize that the systemic administration of soluble Ig-4-1BB ligand can give rise to better T-cell immune activation than local gene delivery. With potential clinical applications in mind, we further compare whether the natural 4-1BB ligand fused to mouse IgG2a (Ig-4-1BBL) would be as effective as the agonistic anti-4-1BB antibody. The dimeric form of Ig-4-1BBL was purified from HeLa cells transduced with a recombinant adenovirus (ADV/Ig-4-1BBL) expressing Ig-4-1BBL. Functional activity was confirmed by the ligand's ability to bind to activated splenic T cells or bone marrow (BM)-derived dendritic cells (DCs) that express 4-1BB receptor. The soluble Ig-4-1BBL efficiently costimulated CD3-activated T-cell proliferation in vitro. More importantly, it induced tumor-specific CTLs as effectively as the agonistic anti-4-1BB antibody. When combined with IL-12 gene transfer, systemic administration of the Ig-4-1BBL proved to be more potent than local gene delivery. In addition, the Ig-4-1BBL is as potent as the agonistic anti-4-1BB antibody for the treatment of hepatic MCA26 colon carcinoma, resulting in 50% complete tumor regression and long-term survival. In long-term surviving mice, both treatment modalities induced persistent tumor-specific CTL activity. In summary, these results suggest that the systemic delivery of Ig-4-1BBL can generate a better antitumor response than local gene delivery. Ig-4-1BBL had equivalent biological functions when compared to the agonistic anti-4-1BB antibody. Thus, soluble 4-1BBL dimmer can be developed as a promising agent for cancer therapy in humans.

Previously, we showed that the eradication of murine hepatic metastatic colon carcinomas was achieved by using a combination therapy with adenovirus-mediated gene delivery of interleukin 12 (IL-12) and anti-4-1BB agonistic antibody. However, the therapeutic efficacy was compromised severely when mice with tumors larger than 8 x 8 mm(2) were treated. In this report, we studied the effect of OX40 costimulation through agonistic anti-OX40 in combination with the IL-12 + anti-4-1BB immunotherapy on primary and memory anti-tumor cytotoxic T lymphocyte responses in a large-tumor setting (8 x 8 to 12 x 12 mm(2)). Tumor-infiltrating leukocytes (TILs) isolated from mice treated with the combination therapy of IL-12, anti-4-1BB, and anti-OX40 showed a significantly higher ex vivo direct cytotoxic T lymphocyte (CTL) activity against parental tumors, as compared with those from mice treated with IL-12 and anti-4-1BB. In vivo depletion of CD4(+)T cells during combination therapy significantly decreased the number of tumor-infiltrating CD8(+)T cells and their cytotoxic activity in mice treated with IL-12 + anti-4-1BB + anti-OX40 combination therapy but not in the group treated with IL-12 + anti-4-1BB, which indicated that in vivo OX40 engagement on CD4(+) T cells led to the higher CTL responses observed in animals treated with IL-12 + anti-4-BB + anti-OX40 combination therapy. Furthermore, the combination therapy of IL-12, anti-4-1BB, and anti-OX40 resulted in a significantly higher survival rate in treated mice, as compared with treatment with IL-12 and anti-4-1BB. More importantly, long-term surviving mice from the combination therapy with IL-12, anti-4-1BB, and anti-OX40 exhibited higher memory CTL responses against parental tumor cells. The results demonstrated that OX40 ligation of CD4(+) T cells facilitated the development of primary and memory CTL responses against tumorcells and that coordinated immune activation by IL-12, anti-4-1BB, and anti-OX40 resulted in a significantly higher survival rate in mice with large tumor burdens. Thus, the combination therapy with the adenovirus encoding IL-12 (Adv.mIL-12) + anti-4-1BB + anti-OX40 antibodies may provide a better treatment modality for patients with advanced cancers, often associated with a state of immune suppression or tolerance.

Gene expression arrays show that human epithelial cells and human arthritis-affected cartilage lack detectable amounts of mRNA for IL-1 antagonizing molecules: IL-1Ra and IL-1RII, but constitutively express IL-1. Functional genomic analysis was performed by reconstituting human IL-1RII expression in various IL-1RII-deficient cell types to examine its antagonist role using gene therapy approaches. Adenovirus-expressing IL-1RII when transduced into human and bovine chondrocytes, human and rabbit synovial cells, human epithelial cells, and rodent fibroblasts expressed membrane IL-1RII and spontaneously released functional soluble IL-1RII. The IL-1RII+ (but not IL-1RII−) cells were resistant to IL-1β-induced, NO, PGE2, IL-6, and IL-8 production or decreased proteoglycan synthesis. IL-1RII inhibited the function of IL-1 in chondrocytes and IL-1- and TNF-α-induced inflammatory mediators in human synovial and epithelial cells. IL-1RII+ chondrocytes were more resistant to induction of NO and PGE2 by IL-1β compared with IL-1RII− cells incubated with a 10-fold (weight) excess of soluble type II IL-1R (sIL-1RII) protein. In cocultures, IL-1RII+ synovial cells released sIL-1RII, which in a paracrine fashion protected chondrocytes from the effects of IL-1β. Furthermore, IL-1RII+ (but not IL-1RII−) chondrocytes when transplanted onto human osteoarthritis-affected cartilage in vitro, which showed spontaneous release of sIL-1RII for 20 days, inhibited the spontaneous production of NO and PGE2 in cartilage in ex vivo. In summary, reconstitution of IL-1RII in IL-1RII− cells using gene therapy approaches significantly protects cells against the autocrine and paracrine effects of IL-1 at the signaling and transcriptional levels.

Low levels of hepatic insulin production have been shown to prevent lethal ketoacidosis associated with type 1 diabetes. To assess the beneficial effects of sustained hepatic production of insulin on glycemic control in type 1 diabetes, we have employed the adenovirus-mediated gene delivery system to transfer an engineered rat preproinsulin gene to the livers of streptozotocin-induced diabetic nude rats. Hepatic insulin production resulted in the reduction of blood glucose in treated diabetic rats, the degree of blood glucose reduction correlated with both the vector dose and the level of hepatic insulin expression. At moderate vector doses, 0.3–0.7 ng/ml of plasma insulin was produced in treated diabetic animals, resulting in significant reduction of nonfasting hyperglycemia and improvement in glucose tolerance. Furthermore, these animals maintained euglycemia after 12-h fast. At higher vector doses, greater than 1 ng/ml of plasma insulin was produced, completely reversing nonfasting hyperglycemia in treated rats. However, all of the treated animals developed severe hypoglycemia upon fasting. This study has defined the maximal tolerable level of hepatic insulin production that is sufficient to reduce the degree and ameliorate the adverse effects of nonfasting hyperglycemia without risk of fasting hypoglycemia in type 1 diabetic rats.

Paradigms of insulin gene therapy for type 1 diabetes should incorporate vigorous control for insulin gene expression to be effective in correcting postprandial hyperglycemia and to be safe in preventing fasting hypoglycemia. We hypothesize that hepatic insulin gene expression auto-regulated positively by glucose and negatively by insulin might be both effective and safe in the treatment of type 1 diabetes. Expression of the glucose 6-phosphatase (G6Pase) gene in the liver is both stimulated by glucose and suppressed by insulin. The G6Pase promoter incorporated with intronic enhancers of the aldolase B gene was used to direct insulin gene expression in the liver of streptozotocin-induced diabetic nude rats. In the treated animals, blood insulin levels were elevated after feeding, and nonfasting hyperglycemia was significantly reduced. Glucose tolerance testing also illustrated that the treated animals exhibited accelerated glucose utilization rates. Upon fasting, blood glucose was reduced to normoglycemic range within 4 h and maintained at that level during the prolonged fasting of 16 h. No hypoglycemia was observed in any treated animals at any time throughout the fasting period, as blood insulin gradually declined to the normal range. These results suggest that auto-regulated hepatic insulin expression can potentially be developed as an effective and safe treatment modality for type 1 diabetes.

The liver is an attractive target organ for insulin gene expression in type 1 diabetes as it contains appropriate cellular mechanisms of regulated gene expression in response to blood glucose and insulin. We hypothesize that insulin production regulated by both glucose and insulin may be achieved using the promoter of the glucose 6-phosphatase gene (G6Pase), the expression of which in the liver is induced by glucose and suppressed by insulin. Recombinant adenoviral vectors expressing the reporter gene CAT or insulin under transcriptional direction of the G6Pase promoter were constructed. Glucose-stimulated as well as self-limiting insulin production was achieved in vector-transduced hepatoma cells in which expression of the insulin gene was controlled by the G6Pase promoter. While insulin strongly inhibited the G6Pase promoter activity under low glucose conditions, its inhibitory capacity was attenuated when glucose levels were elevated. At the physiologic glucose level of 5.5 mM glucose, vector-transduced hepatoma cells produced a self-limited level of insulin at approximately 0.2-0.3 ng/ml, which is within the range of fasting levels of insulin in normal animals. These results indicate that the G6Pase promoter possesses desirable features and may be developed for regulated hepatic insulin gene expression in type 1 diabetes.

One of the several impediments to effective oncolytic virus therapy of cancer remains a lack of tumor-specific targeting. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells induced by tumor factors in tumor-bearing hosts. The biodistribution kinetics of MDSC and other immune cell types in a murine hepatic colon cancer model was investigated through the use of tracking markers and MRI. MDSCs were superior to other immune cell types in preferential migration to tumors in comparison with other tissues. On the basis of this observation, we engineered a strain of vesicular stomatitis virus (VSV), an oncolytic rhabdovirus that bound MDSCs and used them as a delivery vehicle. Improving VSV-binding efficiency to MDSCs extended the long-term survival of mice bearing metastatic colon tumors compared with systemic administration of wild-type VSV alone. Survival was further extended by multiple injections of the engineered virus without significant toxicity. Notably, direct tumor killing was accentuated by promoting MDSC differentiation towards the classically activated M1-like phenotype. Our results offer a preclinical proof-of-concept for using MDSCs to facilitate and enhance the tumor-killing activity of tumor-targeted oncolytic therapeutics. Cancer Res; 73(16); 5003–15. ©2013 AACR.

Costimulatory factors hold great promise for development into novel anticancer biotherapeutics. An agonist to 4-1BB is ranked number 8 by National Cancer Institute on the list of 20 agents with high potential for use in treating cancer. We earlier reported on a recombinant murine 4-1BB ligand fusion protein that binds 4-1BB receptor on murine T cells and stimulates their proliferation in tumor-bearing mice. To facilitate clinical translation,we constructed a corresponding recombinant human 4-1BB ligand fusion protein (hIg-h4-1BBLs) and showed its ability to activate human T cells in vitro. Using Chinese hamster ovary cells transformed with a plasmid coexpressing hIg-h4-1BBLs and rat glutamine synthetase, we generated a high-producing clone by sequential selection with methionine sulfoximine. The hIg-h4-1BBLs was partially purified by protein A column chromatography and characterized biochemically and functionally, using human 4-1BB binding and human T-cell proliferation assays, in vitro.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western Blot confirmed that the hIg-h4-1BBLs is expressed predominantly as a functionally active multimeric protein with the ability to specifically bind to cells expressing human 4-1BB receptor and induce significant T-cell proliferation in vitro using both human and monkey peripheral blood mononuclear cells. The hIg-h4-1BBLs can be produced in large quantities from the high producer clone and developed as a novel immune costimulatory biotherapeutic to treat, alone and in combination with other modalities, various malignant diseases in patients through T-cell activation. Process development of this clinical agent has been discussed with the Food and Drug Administration in a pre-Investigational New Drug meeting and presented to the Office of Biotechnology Activities in a public hearing.

Recombinant oncolytic viruses represent a promising alternative option for the treatment of malignant cancers. We have reported earlier the safety and efficacy of recombinant vesicular stomatitis virus (VSV) vectors in a rat model of hepatocellular carcinoma (HCC). However, the full potential of VSV therapy is limited by a sudden decline in intratumoral virus replication observed early after viral administration, a phenomenon that coincides with an accumulation of inflammatory cells within infected lesions. To overcome the antiviral function of these cells, we present a recombinant virus, rVSV-UL141, which expresses a protein from human cytomegalovirus known to downregulate the natural killer (NK) cell-activating ligand CD155. The modified vector resulted in an inhibition of NK cell recruitment in vitro, as well as decreased intratumoral accumulations of NK and NKT cells in vivo. Administration of rVSV-UL141 through hepatic artery infusion in immune-competent Buffalo rats harboring orthotopic, multi-focal HCC lesions resulted in a one-log elevation of intratumoral virus replication over a control rVSV vector, which translated to enhance tumor necrosis and substantial prolongation of survival. Moreover, these results were achieved in the absence of apparent toxicities. The present study suggests the applicability of this strategy for the development of effective and safe oncolytic agents to treat multi-focal HCC, and potentially a multitude of other cancers, in the future.

Excessive production of triglyceride-rich VLDL is attributable to hypertriglyceridemia. VLDL production is facilitated by microsomal triglyceride transfer protein (MTP) in a rate-limiting step that is regulated by insulin. To characterize the underlying mechanism, we studied hepatic MTP regulation by forkhead box O1 (FoxO1), a transcription factor that plays a key role in hepatic insulin signaling. In HepG2 cells, MTP expression was induced by FoxO1 and inhibited by exposure to insulin. This effect correlated with the ability of FoxO1 to bind and stimulate MTP promoter activity. Deletion or mutation of the FoxO1 target site within the MTP promoter disabled FoxO1 binding and resulted in abolition of insulin-dependent regulation of MTP expression. We generated mice that expressed a constitutively active FoxO1 transgene and found that increased FoxO1 activity was associated with enhanced MTP expression, augmented VLDL production, and elevated plasma triglyceride levels. In contrast, RNAi-mediated silencing of hepatic FoxO1 was associated with reduced MTP and VLDL production in adult mice. Furthermore, we found that hepatic FoxO1 abundance and MTP production were increased in mice with abnormal triglyceride metabolism. These data suggest that FoxO1 mediates insulin regulation of MTP production and that augmented MTP levels may be a causative factor for VLDL overproduction and hypertriglyceridemia in diabetes., This study was supported in part by the American Diabetes Association and NIH grant DK066301.

FoxO1 plays an important role in mediating the effect of insulin on hepatic metabolism. Increased FoxO1 activity is associated with reduced ability of insulin to regulate hepatic glucose production. However, the underlying mechanism and physiology remain unknown. We studied the effect of FoxO1 on the ability of insulin to regulate hepatic metabolism in normal vs. insulin-resistant liver under fed and fasting conditions. FoxO1 gain of function, as a result of adenovirus-mediated or transgenic expression, augmented hepatic gluconeogenesis, accompanied by decreased glycogen content and increased fat deposition in liver. Mice with excessive FoxO1 activity exhibited impaired glucose tolerance. Conversely, FoxO1 loss of function, caused by hepatic production of its dominant-negative variant, suppressed hepatic gluconeogenesis, resulting in enhanced glucose disposal and improved insulin sensitivity in db/db mice. FoxO1 expression becomes deregulated, culminating in increased nuclear localization and accounting for its increased transcription activity in livers of both high fat-induced obese mice and diabetic db/db mice. Increased FoxO1 activity resulted in up-regulation of hepatic peroxisome proliferator-activated receptor-γ coactivator-1β, fatty acid synthase, and acetyl CoA carboxylase expression, accounting for increased hepatic fat infiltration. These data indicate that hepatic FoxO1 deregulation impairs the ability of insulin to regulate hepatic metabolism, contributing to the development of hepatic steatosis and abnormal metabolism in diabetes., This work was supported by National Institutes of Health Grant DK066301.

The apolipoprotein apoC-III plays an important role in plasma triglyceride metabolism. It is predominantly produced in liver, and its hepatic expression is inhibited by insulin. To elucidate the inhibitory mechanism of insulin in apoC-III expression, we delivered forkhead box O1 (Foxo1) cDNA to hepatocytes by adenovirus-mediated gene transfer. Foxo1 stimulated hepatic apoC-III expression and correlated with the ability of Foxo1 to bind to its consensus site in the apoC-III promoter. Deletion or mutation of the Foxo1 binding site abolished insulin response and Foxo1-mediated stimulation. Likewise, Foxo1 also mediated insulin action on intestinal apoC-III expression in enterocytes. Furthermore, elevated Foxo1 production in liver augmented hepatic apoC-III expression, resulting in increased plasma triglyceride levels and impaired fat tolerance in mice. Transgenic mice expressing a constitutively active Foxo1 allele exhibited hypertriglyceridemia. Moreover, we show that hepatic Foxo1 expression becomes deregulated as a result of insulin deficiency or insulin resistance, culminating in significantly elevated Foxo1 production, along with its skewed nuclear distribution, in livers of diabetic NOD or db/db mice. While loss of insulin response is associated with unrestrained apoC-III production and impaired triglyceride metabolism, these data suggest that Foxo1 provides a molecular link between insulin deficiency or resistance and aberrant apoC-III production in the pathogenesis of diabetic hypertriglyceridemia.