Antitumor Efficacy Of Combination Therapy Research Articles

Abstract 2534: Single-cell dissection of heterogeneity and dynamics of tumor ecosystem upon immune-checkpoint blockade in hepatocellular carcinoma

Abstract Background: Therapy-induced alteration of tumor ecosystem poses a major barrier for cancer regression. As the majority of patients with hepatocellular carcinoma (HCC) develops resistance to immune checkpoint blockade (ICB) therapy, we aimed to characterize heterogeneity and dynamics of multicellular tumor ecosystem following anti-PD-1 treatment using scRNA-seq. Methods: In a phase II clinical trial of pembrolizumab on advanced hepatitis B virus-related HCC patients (NCT03419481), we performed a comprehensive single-cell transcriptomic profiling of biopsy samples from 26 patients before and during treatment. The composition and distribution of immune aggregates in responsive tumors was characterized by scRNA-seq and spatial transcriptome analyses. The anti-tumor efficacy of combined therapy with anti-PD-1 and inhibition of immunosuppressive macrophage were further determined in RIL-175-derived PD-1-resistant mouse model. Results: After initial data processing and quality control, we obtained 353,329 high-quality cells that included tumor cells, myeloid cells, lymphoid cells, endothelial cells and fibroblasts. Our analysis revealed that baseline tumor-reactive CD8+T cells exhibited a strong association with improved prognosis. Notably, treatment-induced expansion and differentiation of tumor-reactive CD8+T cells with memory-like phenotype highly correlated with durable ICB response. Moreover, we observed a co-occurrence pattern of key anti-tumor immune subsets in the tumor microenvironment (TME), which associated with ligand-receptor pair interactions. Spatial transcriptome analysis further validated the presence of immune aggregates in responsive tumors. Importantly, we derived an responsive signature from immune aggregates, which showed a strong association with improved prognosis in pan-cancer ICB datasets. Paired comparisons of pre- and on-treatment macrophage percentages in non-responders revealed a specific expansion of an immunosuppressive macrophage subset. Inhibition of this macrophage subset could overcome ICB resistance by enhancing T cell function in our PD-1 resistant model. We further observed that a tumorous epithelial-mesenchymal transition (EMT) program might contribute to therapeutic resistance via TME remodeling. Conclusions: Our study suggests that tumor-reactive CD8+T cells and immune aggregates may be associated with durable ICB response, while ICB-induced upregulation of immunosuppressive macrophages and EMThi tumor cells contributed to therapeutic evasion. These findings provide valuable insights for identification of predictive biomarkers and therapeutic strategies for ICB therapy of HCC. This project is supported by the General Research Fund (14119023, 14120621 and 14115820), the Li Ka Shing Foundation, and the CUHK Strategic Seed Funding for Collaborative Research Scheme. Citation Format: Alfred S. L. Cheng, Zhewen Xiong, Haoran Wu, Jianquan Cao, Zhixian Liang, Jingying Zhou, Joseph Jao-Yiu Sung, Kevin Yip, Stephen Chan. Single-cell dissection of heterogeneity and dynamics of tumor ecosystem upon immune-checkpoint blockade in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2534.

Abstract LB201: A novel combination of Listeria-based immunotherapy and repurposed drug combats colorectal cancer

Abstract Background: Colorectal cancer (CRC) has been a difficult-to-treat cancer with a significant proportion of microsatellite stable (MSS-CRC) patients who do not respond to immunotherapy. Currently, various strategies are being investigated to sensitize MSS-CRC tumors by converting them to immunologically “hot” tumors and improve responsiveness to immunotherapy. In this study, we demonstrate a novel strategy of combining a repurposed FDA-approved compound, Pimavanserin tartrate (PVT), with a Listeria monocytogenes (Lm)-based vaccine targeting Interferon-Stimulated Gene 15(ISG15), Lm-LLO-ISG15, against MSS-CRC tumors. Our findings demonstrate that both agents synergize to produce CD8+ T cell-dependent therapeutic efficacy in immunotherapy-resistant MSS-CRC tumors. Methods: The direct killing effect of PVT in vitro was confirmed by cytotoxic SRB assay. BALB/c mice bearing CT26, a murine model of MSS-CRC, were randomly assigned to receive either PBS, PVT, Lm-LLO-ISG15, or PVT+Lm-LLO-ISG15. Mice were monitored for tumor growth kinetics to study the anti-cancer effect of each monotherapy and the combination therapy. Changes in immune-related gene and protein expression were analyzed by qPCR and Western blot, respectively. Various immune cell subsets in the tumors and spleens were evaluated by multi-color flow cytometry and immunohistochemistry. In addition, organs such as the liver, kidney, heart, lungs, spleen, brain, and pancreas were collected for safety analysis. Results: PVT efficiently induced apoptosis and directly killed both human and mouse MSS-CRC cell lines in vitro. Further, PVT treatment enhanced T-cell activation and induced ISG15 expression, thus, sensitizing the tumor cells to targeting by the CTL-mediated immunotherapy, Lm-LLO-ISG15. While each monotherapy failed to demonstrate anti-tumor efficacy, treatment with the combination therapy (PVT + Lm-LLO-ISG15) significantly controlled tumor burden and extended the median survival. The therapeutic efficacy of the combination approach was associated with a higher infiltration of CD8+ T cells in the TME and an increased population of effector memory T cells (CD4+CD44hiCD62Llo) in the spleen. The anti-tumor efficacy of combination therapy on subcutaneous CT26 tumor growth was completely abrogated after the depletion of CD8+ T cells. Moreover, the safety profile of dual therapy was comparable to that of each monotherapy and PBS. Conclusions: MSS-CRCs are considered “immunologically cold” tumors that represent great challenges for any standalone therapies. In this study, we evaluated, for the first time, the therapeutic efficacy of a novel combination of PVT and Lm-LLO-ISG15 in an aggressive MSS-CRC mouse model. Combination therapy demonstrated a synergistic effect that led to a significant reduction of tumor burden and an extension of median survival. While the underlying mechanism for synergism requires further examination, our findings strongly suggest that combining PVT and Lm-LLO-ISG15 could be a promising approach for MSS-CRC patients. Citation Format: Shreyas Ramchandra Gaikwad, Hong My Nguyen, Sanjay K. Srivastava, Laurence Wood. A novel combination of Listeria-based immunotherapy and repurposed drug combats colorectal cancer [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 LB201.

Abstract 6217: Inhibiting ataxia-telangiectasia mutated and RAD3-related (ATR) by BAY 1895344 overcomes chemoresistance to oxaliplatin and promotes synergistic anti-tumor effect in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive disease with a poor prognosis and a limited response to most of the treatments. Despite a platinum-based drug such as oxaliplatin or cisplatin is one of the most effective chemotherapy drugs for PDAC, resistance to it is a major limiting factor in PDAC treatment, indicating an urgent need for new approaches. Recently, targeting major DNA damage response (DDR) regulators such as ATM (Ataxia-telangiectasia mutated) or ATR (Ataxia telangiectasia mutated and Rad3-related) kinase has shown therapeutic potential in cancer treatment. This shows that it may be possible to enhance the responsiveness of platinum medicines via a DDR inhibition strategy. The most recently developed ATR inhibitor with the greatest potency, BAY 1895344, showed an anti-proliferative effect in clinical trials. Here, we aimed to evaluate the effect of ATR inhibition using BAY 1895344 on responsiveness to oxaliplatin in pancreatic cancer, for the first time. CFPAC-1 and Capan-2 are selected among six kinds of pancreatic cancer cell lines as oxaliplatin-sensitive and -resistant cells, respectively. According to BRAID analysis, combining the BAY 1895344 and oxaliplatin resulted in strong synergistic effects in both cell lines, particularly in Capan-2. The synergism is also confirmed in all four organoids derived from PDAC patients. We found that p-Chk1, coordinating DDR and cell cycle checkpoint, was considerably suppressed by the combined treatments, which was associated with elevated γ-H2AX intensity, cell cycle arrest and apoptosis. Moreover, we investigated the in vivo synergistic anti-tumor efficacy of combination therapy using a tumor-bearing nude mice model with CFPAC-1 and Capan-2 cells, demonstrating a substantial reduction of tumor growth in combination therapy when compared to single treatment. In conclusion, ATR inhibition enhanced the anticancer effect of oxaliplatin, and this combined therapeutic strategy may be effective in overcoming chemo-resistance in PDAC. (This study is supported by National Cancer Center, Republic of Korea (No. 2212470, 2010330)) Citation Format: Hye Won Shon, Jung Won Chun, Jeong Eun Gong, Mi Rim Lee, Yu-Sun Lee, Sumin Kang, Sunshin Kim, Sang Myung Woo, In Rae Cho, Woo Hyun Paik, Woo Jin Lee, Ji Kon Ryu, Yong-Tae Kim, Sang Hyub Lee, Yun-Hee Kim. Inhibiting ataxia-telangiectasia mutated and RAD3-related (ATR) by BAY 1895344 overcomes chemoresistance to oxaliplatin and promotes synergistic anti-tumor effect in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6217.

Abstract CT529: A Phase 1b Study of CG0070 combined with nivolumab in cisplatin ineligible patients with muscle invasive bladder cancer

Abstract Background: Up to 50% of patients with muscle invasive bladder cancer (MIBC) are unable to receive neoadjuvant chemotherapy, creating an unmet clinical need for alternative systemic treatment. Results from recent neoadjuvant immune checkpoint blockade (ICB) studies signal pCR rates between 31-42%. However, there was a clear dichotomy in response between patients with high vs. low pre-treatment lymphocytic infiltration. In tumors exhibiting immune excluded or immune desert phenotypes, therapeutic strategies to attract CD8+ T cells infiltration prior to ICB may be beneficial. CG0070 is an oncolytic adenovirus designed to preferentially replicate through a transcriptionally regulated promoter that is up-regulated in RB-pathway-defective cells (>70% of MIBC). In addition, CG0070 expresses GM-CSF to attract antigen presenting cells to the tumor microenvironment. We hypothesize that combination therapy using intravesical CG0070 and systemic Nivolumab is 1) safe and 2) can enhance local and systemic treatment response in the neoadjuvant setting against MIBC. Methods: This is a phase Ib trial evaluating 2 doses of nivolumab (4 weeks apart) with 6 weekly intravesical CG0070 followed by cystectomy. Dose-limiting toxicities (DLTs) will be assessed in 6 DLT-evaluable patients in a safety lead-in phase. The combination would be declared tolerable if the incidence of DLTs is <33%, and the study expanded to 30 patients. Key eligibility: cT2-T4aN0-1M0 stage, no autoimmume disease within 2 years, and no concurrent invasive upper tract carcinoma or other active cancer. The primary objective is to evaluate the safety of combination therapy according to CTCAE v5.0. The secondary objective is to assess the changes in the lymphocytic infiltration following combination therapy and to assess the antitumor efficacy of combination therapy. Exploratory endpoints include assessing the predictive value of E2F expression, tumor mutational burden, immune infiltration, and various peripheral blood for treatment response. Study Progress: Study is actively accruing. Clinical trial information: NCT04610671. An update on the study will be provided at the time of presentation. Citation Format: Roger Li, Wade Sexton, Phillipe Spiess, Scott Gilbert, Julio Pow-Sang, Jingsong Zhang, Gustavo Borjas, Paola Grandi, James Burke, Rohit Jain, Jose Conejo-Garcia. A Phase 1b Study of CG0070 combined with nivolumab in cisplatin ineligible patients with muscle invasive bladder cancer [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 CT529.

Efficacy of birinapant in combination with carboplatin in targeting platinum‑resistant epithelial ovarian cancers.

Patients diagnosed with epithelial ovarian cancers (EOCs) often suffer from disease relapse associated with the emergence of resistance to standard platinum-based chemotherapy. Treatment of patients with chemo-resistant disease remains a clinical challenge. One mechanism of chemoresistance includes overexpression of pro-survival proteins called inhibitors of apoptosis (IAP) which enable cancer cells to evade apoptosis. Due to their anti-apoptotic activity, association with poor prognosis, and correlation with therapy resistance in multiple malignancies, IAP proteins have become an attractive target for development of anticancer therapeutics. Second mitochondrial activator of caspase (SMAC) mimetics are the most widely used IAP antagonists currently being tested in clinical trials as a monotherapy and in combination with different chemotherapeutic drugs to target different types of cancer. In the present study, the antitumor efficacy of combination therapy with birinapant, a bivalent SMAC mimetic compound, and carboplatin to target platinum-resistant EOC cells was investigated. A 3D organoid bioassay was utilized to test the efficacy of the combination therapy in a panel of 7 EOC cell lines and 10 platinum-resistant primary patient tumor samples. Findings from the in vitro studies demonstrated that the birinapant and carboplatin combination was effective in targeting a subset of ovarian cancer cell lines and platinum-resistant primary patient tumor samples. This combination therapy was also effective in vitro and in vivo in targeting a platinum-resistant patient-derived xenograft (PDX) model established from one of the patient tumors tested. Overall, our study demonstrated that birinapant and carboplatin combination could target a subset of platinum-resistant ovarian cancers and also highlights the potential of the 3D organoid bioassay as a preclinical tool to assess the response to chemotherapy or targeted therapies in ovarian cancer.

Sequential Release of Paclitaxel and Imatinib from Core-Shell Microparticles Prepared by Coaxial Electrospray for Vaginal Therapy of Cervical Cancer.

To optimize the anti-tumor efficacy of combination therapy with paclitaxel (PTX) and imatinib (IMN), we used coaxial electrospray to prepare sequential-release core–shell microparticles composed of a PTX-loaded sodium hyaluronate outer layer and an IMN-loaded PLGA core. The morphology, size distribution, drug loading, differential scanning calorimetry (DSC), Fourier transform infrared spectra (FTIR), in vitro release, PLGA degradation, cellular growth inhibition, in vivo vaginal retention, anti-tumor efficacy, and local irritation in a murine orthotopic cervicovaginal tumor model after vaginal administration were characterized. The results show that such core–shell microparticles were of spherical appearance, with an average size of 14.65 μm and a significant drug-loading ratio (2.36% for PTX, 19.5% for IMN, w/w), which might benefit cytotoxicity against cervical-cancer-related TC-1 cells. The DSC curves indicate changes in the phase state of PTX and IMN after encapsulation in microparticles. The FTIR spectra show that drug and excipients are compatible with each other. The release profiles show sequential characteristics in that PTX was almost completely released in 1 h and IMN was continuously released for 7 days. These core–shell microparticles showed synergistic inhibition in the growth of TC-1 cells. Such microparticles exhibited prolonged intravaginal residence, a >90% tumor inhibitory rate, and minimal mucosal irritation after intravaginal administration. All results suggest that such microparticles potentially provide a non-invasive local chemotherapeutic delivery system for the treatment of cervical cancer by the sequential release of PTX and IMN.

Vitamin C sensitizes BRAFV600E thyroid cancer to PLX4032 via inhibiting the feedback activation of MAPK/ERK signal by PLX4032

BackgroundBRAFV600E mutation is the most common mutation in thyroid cancer. It strongly activates MAPK/ERK pathway and indicates an invasive subtype of thyroid cancer. PLX4032 is a selective oral inhibitor of the BRAFV600 kinase although with limited effect in treating this panel of thyroid cancer, due to the feedback activation of MAPK/ERK as well as PI3K/AKT pathways. It was investigated that Vitamin C plays a positive role in inhibiting these pathways in thyroid cancer. However, whether Vitamin C could enhance the antitumor effect of PLX4032 remains largely unclear.MethodsThe antitumor efficacy of combination therapy with PLX4032 and Vitamin C on BRAFMT thyroid cancer cell was assessed by the MTT assay, EdU assay and colony formation, Chou-Talalay way was employed to analyze the synergistic effect. Flow cytometry were employed to assess cells’ apoptosis and cell cycle arrest in response to combination therapy. Xenograft models were used to test its in vivo antitumor activity. Western blot and IHC were applied to investigate the mechanism underlying synergistic effect.ResultsPLX4032 or Vitamin C monotherapy was mildly effective in treating BRAFMT thyroid cancer cell and xenografts model. The combination therapy significantly inhibited cancer cell proliferation and tumor growth in nude mice, and induced cell apoptosis and cell cycle arrest compared to either monotherapy. PLX4032 monotherapy induced feedback activation of MAPK/ERK as well as PI3K/AKT pathway; while combination therapy significantly relieved this feedback.ConclusionVitamin C promotes the antitumor effect of PLX4032 in BRAFMT thyroid cancer cell and xenografts model via relieving the feedback activation of MAPK/ERK as well as PI3K/AKT pathway. PLX4032/Vitamin C combination may be a potential therapeutic approach to treat BRAFMT thyroid cancer.

Abstract OT2-03-04: A trial of induction Talazoparib followed by a combination of Talazoparib and Avelumab in advanced breast cancer: The TALAVE study

Abstract Background: Patients with advanced breast cancer (ABC) have recently gained access to promising new therapies, including PARP inhibitors (PARPi) and immunotherapy. However, not all patients benefit from these approaches, with response rates highest in patients characterized by a particular biomarker (ie BRCA1/2 mutation or PD-L1 expression). Combination strategies may be more efficacious than single agents and may induce responses in otherwise non-responders. PARPi activate the STING pathway leading to T cell recruitment and stimulate antigen presentation via increased T cell cytotoxic activity, creating a tumor microenvironment that may be more susceptible to immunotherapy. A number of trials have assessed the antitumor efficacy of this combination, though the optimal drug scheduling and the impact of BRCA1/2 status on the effect of PARP inhibition on immunomodulation are unknown. In the TALAVE study, the PARPi talazoparib is combined with the PD-L1 inhibitor avelumab. Talazoparib is an oral small molecule selective inhibitor of PARP-1/2 with potent in vitro PARP trapping capacity. Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody that prevents the interaction between PD-L1 and PD-1 and allows for an engagement of Fc-γ receptors on NK cells to induce tumor-directed ADCC in vitro. Trial design: This is an open-label, multi-institutional trial (NCT03964532) for patients with ABC. During the phase I portion, 6 patients are enrolled regardless of BRCA1/2 mutation status to assess safety of the combination. A maximum of 24 patients will be enrolled. Eligibility criteria include willingness to undergo serial biopsies and no previous exposure to PARPi or prior disease progression on anti-PD1 or anti-PDL1 therapy or within 6 months of use. Patients will be enrolled to two pre-defined cohorts: cohort 1 - BRCA1/2 mutant and HER2 negative ABC (pre-identified presence of somatic or germline BRCA1/2 deleterious mutation) and cohort 2 - BRCA1/2 wild type and TNBC (patients with previous somatic or germline testing for BRCA1/2 that did not reveal a deleterious mutation). Enrolled patients will receive a 4-week induction of talazoparib (1mg orally daily, D1-D28), followed by a combination of talazoparib and avelumab (800mg IV D1 and D15). To assess the efficacy and immunomodulatory effects of PARP inhibition induction followed by anti-PD-L1 therapy, patients will undergo serial tumor biopsies (baseline, post 4 weeks of talazoparib, post 4 weeks of talazoparib and avelumab and at progression for patients who clearly benefitted from therapy) to assess tumor infiltrating lymphocytes (TILs) and PD-L1 expression. Patients will also undergo serial blood sample collection to gauge the peripheral immunoscore by flow cytometry, including an assessment of the number of immune cells and their function. Specific aims: The primary objective is to evaluate the safety and tolerability of this combination. Secondary objectives includeassessment of anti-tumor efficacy of combined therapy as determined by measurement of ORR, OS, PFS, DOR and DCR and evaluation of the effect of BRCA1/2 mutation, talazoparib alone, and talazoparib plus avelumab on immunomodulation. Statistical methods: The projected sample size is 24 patients. Sample size is based on a feasibility analysis. Statistics will be primarily descriptive, and toxicities will be tabulated according to grade. PFS and OS will be estimated using the Kaplan Meier method. DCR and DOR will be calculated according to RECIST v1.1 and compared descriptively to historical outcomes. Cox regression models will assess whether measures of immune activation are associated with ORR, PFS or OS. Accrual: Patient accrual started in April 2019. To date, 3 of the maximum target accrual of 24 patients have been enrolled in the phase I portion of the study (2 on cohort 1 and 1 on cohort 2). Citation Format: Arielle L Heeke, Michael Pishvaian, Hongkun Wang, Adam Cohen, Jeffrey Schlom, Renee Donahue, Caroline Jochems, Margaret Gatti-Mays, Paula Pohlmann, Antoinette Tan, Claudine Isaacs, Filipa Lynce. A trial of induction Talazoparib followed by a combination of Talazoparib and Avelumab in advanced breast cancer: The TALAVE study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-03-04.

Liposomal simvastatin sensitizes C26 murine colon carcinoma to the antitumor effects of liposomal 5-fluorouracil in vivo.

5‐Fluorouracil‐based therapy remains the main approach in colorectal cancer, even though there are still some drawbacks, such as chemoresistance. In this study we combined 5‐fluorouracil encapsulated in long‐circulating liposomes with simvastatin, also encapsulated in long‐circulating liposomes, that was previously proved to exert antitumor actions on the same tumor model. The production of angiogenic/inflammatory proteins was assessed by protein array and the production of markers for tumor aggressiveness (Bcl‐2, Bax, and nuclear factor [NF]‐κB) were determined by western blot analysis. Intratumor oxidative stress was evaluated through measurement of malondialdehyde level by HPLC, and through spectrophotometric analysis of catalytic activity of catalase and of total antioxidant capacity. Immunohistochemical analysis of tumors for CD31 expression was assessed. Intratumor activity of MMP‐2 by gelatin zymography was also carried out. Our results revealed that combined therapies based on liposomal formulations exerted enhanced antitumor activities compared with combined treatment with free drugs. Sequential treatment with liposomal simvastatin and liposomal 5‐fluorouracil showed the strongest antitumor activity in C26 colon carcinoma in vivo, mainly through inhibition of tumor angiogenesis. Important markers for cancer progression (Bcl‐2, Bax, NF‐κB, and intratumor antioxidants) showed that liposomal simvastatin might sensitize C26 cells to liposomal 5‐fluorouracil treatment in both regimens tested. The outcome of simultaneous treatment with liposomal formulations was superior to sequential treatment with both liposomal types as the invasive capacity of C26 tumors was strongly increased after the latest treatment. The antitumor efficacy of combined therapy in C26 colon carcinoma might be linked to the restorative effects on proteins balance involved in tumor angiogenesis.

Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response.

Patients with metastatic HER2 breast cancer (MBC) often become resistant to HER 2 targeted therapy and have recurrence of disease. The Panacea trial suggested that HER2 MBC patients were more likely to respond to checkpoint therapy if TIL were present or if tumor expressed PD-L1. We assessed whether type I polarized dendritic cells (DC1) could improve checkpoint therapy in a preclinical model of HER2+ breast cancer. TUBO bearing mice were vaccinated with either MHC class I or class II HER2 peptide pulsed DC1 (class I or class II HER2-DC1) concurrently or sequentially with administration of anti-PD-1 or anti-PDL1. Infiltration of tumors by immune cells, induction of anti-HER2 immunity and response to therapy was evaluated. Class I or class II HER2-DC1 vaccinated mice generated anti-HER2 CD8 or CD4+ T cell immune responses and demonstrated delayed tumor growth. Combining both MHC class I and II HER2-pulsed DC1 did not further result in inhibition of tumor growth or enhanced survival compared to individual administration. Interestingly class II HER2-DC1 led to both increased CD4 and CD8 T cells in the tumor microenvironment while class I peptides typically resulted in only increased CD8 T cells. Anti-PD-1 but not anti-PD-L1 administered sequentially with class I or class II HER2-DC1 vaccine could improve the efficacy of HER2-DC1 vaccine as measured by tumor growth, survival, infiltration of tumors by T cells and increase in systemic anti-HER2 immune responses. Depletion of CD4+ T cells abrogated the anti-tumor efficacy of combination therapy with class II HER2-DC1 and anti-PD-1, suggesting that tumor regression was CD4 dependent. Since class II HER2-DC1 was as effective as class I, we combined class II HER2-DC1 vaccine with anti-rat neu antibodies and anti-PD-1 therapy. Combination therapy demonstrated further delay in tumor growth, and enhanced survival compared to control mice. In summary, Class II HER2-DC1 drives both a CD4 and CD8 T cell tumor infiltration that leads to increased survival, and in combination with anti-HER2 therapy and checkpoint blockade can improve survival in preclinical models of HER2 positive breast cancer and warrants exploration in patients with HER2 MBC.

Abstract 3231: Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma

Abstract Background: Osteosarcoma (OS) is the most frequent primary malignant tumor of bone in children and adolescents. Although immune checkpoint inhibitor, anti-programmed death protein 1 (PD-1) antibody, has dramatically improved the clinical outcome in some cancer patients, OS patients are less sensitive to PD-1 blockade due to poor immune responses. Recently, oncolytic virotherapy has been shown to stimulate the immune system through induction of immunogenic cell death (ICD). We recently developed a RGD fiber-modified telomerase-specific oncolytic adenovirus OBP-502, which can enter into tumor cells by binding to cell surface integrin and induce oncolytic cell death in a telomerase-dependent manner. In this study, we assessed the in vitro and in vivo antitumor efficacy of combination therapy with PD-1 blockade and OBP-502 in OS cells. Methods: We used 2 murine OS cell lines, K7M2 and NHOS. The expression of PD-L1, coxsackie and adenovirus receptor (CAR), and integrin on the cell surface was analyzed by flow cytometric analysis. We analyzed the in vitro antitumor effect of OBP-502 using XTT assay and western blot analysis. Virus-induced ICD was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). To evaluate the therapeutic potential of oncolytic immunotherapy, we investigated the in vivo antitumor effect of combination therapy with anti-PD-1 antibody and OBP-502 using a subcutaneous K7M2 xenograft tumor model. Moreover, the number of tumor-infiltrating CD8+, CD4+ and Foxp3+ T cells was analyzed by immunohistochemistry. Results: Flow cytometric analysis demonstrated that K7M2 and NHOS cells had the expression of PD-L1 and integrin, but not CAR. XTT assay showed that OBP-502 efficiently suppressed the viability of K7M2 and NHOS cells in a dose-dependent manner. Western blot analysis revealed that OBP-502 induced the apoptosis-related cell death in K7M2 and NHOS cells. ELISA assay demonstrated that OBP-502 significantly increased the level of extracellular ATP and HMGB1 in K7M2 and NHOS cells. In vivo experiment using a subcutaneous K7M2 xenograft tumor model showed that combination of anti-PD-1 antibody and OBP-502 significantly reduced tumor growth compared to mock treatment or monotherapy. Immunohistochemical analysis revealed that OBP-502 significantly increased the number of tumor-infiltrating CD8+ T cells compared to mock treatment or monotherapy. By contrast, OBP-502 did not affect the number of CD4+ and Foxp3+ T cells in tumor tissues. Conclusion: These results suggest that oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus is a promising antitumor strategy to promote the therapeutic potential of PD-1 blockade in OS through stimulation of antitumor immune response. Citation Format: Koji Demiya, Hiroshi Tazawa, Yusuke Mochizuki, Miho Kure, Joe Hasei, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma [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 3231.

ERK Inhibitor Enhances Everolimus Efficacy through the Attenuation of dNTP Pools in Renal Cell Carcinoma

The clinical efficiency of everolimus, an mammalian target of rapamycin (mTOR) inhibitor, is palliative as sequential or second-line therapy for renal cell carcinoma (RCC). However, the limited response of everolimus in RCC remains uncertain. In the present study, everolimus-resistant RCC models were established to understand the mechanisms and to seek combination approaches. Consequently, the activation of ERK was found to contribute toward everolimus-acquired resistance and poor prognosis in patients with RCC. In addition, the efficacy and mechanism of combination treatment underlying RCC using everolimus and ERK inhibitors was investigated. The ERK inhibitor in combination with everolimus synergistically inhibited the proliferation of RCC cells by arresting the cell cycle in the G1 phase. The combination treatment markedly attenuated the deoxyribonucleoside triphosphate (dNTP) pools by downregulating the mRNA expression of RRM1 and RRM2 through E2F1. The overexpression of E2F1 or supplementation of dNTP rescued the anti-proliferation activity of the everolimus-SCH772984 combination. The antitumor efficacy of combination therapy was reiterated in RCC xenograft models. Thus, the current findings provided evidence that the everolimus-ERK inhibitor combination is a preclinical therapeutic strategy for RCC.

Abstract 4570: Telomerase-specific oncolytic virotherapy promotes therapeuticefficacy of PD-1 blockade in murine osteosarcoma

Abstract Background: Treatment with immune checkpoint inhibitor anti-programmed death protein 1 (PD-1) antibody has dramatically improved the clinical outcome in cancer patients. Then immunotherapy is the most exciting field for cancer treatment in recent years. However, PD-1 blockade therapy is limited to certain cancer types. Therefore, the enhancement of therapeutic efficacy in PD-1 blockade therapy is required. Recently, it has been suggested that the effect of PD-1 blockade therapy can be enhanced when combined with immunogenic antitumor therapy including chemotherapy and virotherapy. We recently developed a RGD fiber-modified telomerase-specific oncolytic adenovirus OBP-502, which can enter into tumor cells by binding to cell surface integrin and induce oncolytic cell death in a telomerase-dependent manner. In this study, we assessed the in vitro and in vivo antitumor efficacy of combination therapy with anti-PD-1 antibody and OBP-502 in murine osteosarcoma. Methods: We used 2 murine osteosarcoma cell lines, K7M2 and NHOS. The expression of PD-L1, coxsackie and adenovirus receptor (CAR), and integrin on the cell surface was analyzed by flow cytometric analysis. The in vitro antitumor effect of OBP-502 was evaluated using a XTT assay. Virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). To evaluate the therapeutic potential of anti-PD-1 antibody and OBP-502, we assessed the antitumor effect of combination therapy with anti-PD-1 antibody and OBP-502 using a subcutaneous K7M2 xenograft tumor model. Moreover, the number of tumor-infiltrating CD8+ and Foxp3+ T cells was analyzed by immunohistochemistry. Results: K7M2 and NHOS cells showed the expression of PD-L1 and integrin, but not CAR, in flow cytometric analysis. OBP-502 efficiently suppressed the viability of K7M2 and NHOS cells in a dose-dependent manner. OBP-502 significantly increased the release of HMGB1 in K7M2 cells and the secretion of ATP in NHOS cells. Intratumoral injection of OBP-502 significantly induced the number of tumor-infiltrating CD8+ T cells and enhanced the suppression of tumor growth by PD-1 blockade in a subcutaneous K7M2 xenograft tumor model. Moreover, tumor free was observed in 6 out of 7 mice treated with anti-PD-1 antibody and OBP-502 even at 4 weeks after treatment. Conclusion: These results suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to promote the therapeutic efficacy of PD-1 blockade therapy in osteosarcoma. Citation Format: Yusuke Mochizuki, Hiroshi Tazawa, Koji Demiya, Tadashi Komatsubara, Kazuhisa Sugiu, Joe Hasei, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Telomerase-specific oncolytic virotherapy promotes therapeuticefficacy of PD-1 blockade in murine osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4570.

Antitumor Efficacy of Combination Therapy Consisting of S-1, Leucovorin, and Oxaliplatin against Human Gastric Cancer Xenografts

Background/Aim: A phase 3 trial of S-1, leucovorin (LV), and oxaliplatin for treating gastric cancer is now underway. However, the antitumor efficacy of the combination has not yet been examined in an in vivo preclinical study. This study examined the antitumor efficacy of combination therapy consisting of S-1, LV, and oxaliplatin against 4 human gastric cancer xenografts: NUGC-4, St-40, SC-2, and SC-4. Methods: The antitumor efficacy was evaluated using human gastric cancer xenograft-bearing nude mice. S-1 and LV were administered orally once daily on days 1-7 at doses of 6.9 and 10 mg/kg, respectively. Oxaliplatin was administered intravenously at a dose of 8.3 mg/kg on day 1. The tumor volume was measured on day 15, and the relative tumor volume (RTV) was calculated. Results: In all 4 xenograft models, S-1 alone and oxaliplatin alone, but not LV alone, had significant antitumor activities (p < 0.001). Combination therapy consisting of S-1 and LV resulted in a significantly smaller RTV than S-1 alone (p < 0.001). Combination therapy consisting of S-1 and oxaliplatin also resulted in a significantly smaller RTV than either S-1 alone (p < 0.001) or oxaliplatin alone (p < 0.001). Furthermore, combination therapy consisting of S-1, LV, and oxaliplatin resulted in the highest antitumor activity in these models (p < 0.001 vs. S-1 + LV; p < 0.001 or p = 0.003 vs. S-1 + oxaliplatin). Conclusion: Combination therapy consisting of S-1, LV, and oxaliplatin administered according to a 1-week-on/1-week-off schedule may be useful for the treatment of patients with gastric cancer.

Abstract 4564: Synergistic anti-tumor efficacy of combination therapy with APS001F, a cytosine deaminase (CD) expressing Bifidobacterium, 5-fluorocytosine (5-FC) and anti-mPD-1 antibody in syngeneic mice model

Abstract Immune checkpoint blockers like anti-PD-1 antibody are leading the new generation in anti-cancer drug development. A lot of scientific evidences have demonstrated that immune checkpoint antibodies have promising clinical response in patients with multiple cancer types. Nevertheless, limited fraction of patients responds to the antibody immunotherapy, and moreover, immune related serious adverse events caused by nonspecific reaction with immune checkpoint blockers result in treatment discontinuation. Many approaches have been attempted to improve therapeutic outcomes of antibody immunotherapy, particularly a combination of antibody treatment and other therapies is one of worth-trying ideas. APS001F is a live recombinant Bifidobacterium longum expressing cytosine deaminase (CD), which is an enzyme to catalyze the hydrolytic deamination of cytosine to uracil. Bifidobacterium is a non-pathogenic anaerobic bacterium derived from normal human intestinal flora. It can only survive and grow at low oxygen environment such as solid tumor. Intravenous administration of APS001F to patients leads to colonization of the B.longum and production of CD enzyme only inside the solid tumor. Locally expressed CD enzyme converts orally taken prodrug, 5-FC, into anti-tumor drug 5-FU, which increases 5-FU concentration specifically inside tumor and results in alleviation of side effects caused by attacking normal organs. The phase 1 clinical trial for APS001F is conducted in the US sponsored by us. 5-FU has been shown to alleviate Myeloid Derived Suppressor Cells (MDSC) in the tumor site and release immune suppression in tumor [1]. Furthermore, Van Der Kraak et. al. have reported that 5-FU upregulated PD-L1 expression on the surface of tumor cells [2]. These findings drove us to combine anti-PD-1 antibody with the APS001F therapy. At this present study, systemic administration of APS001F and 5-FC significantly suppressed tumor growth in CT26 bearing syngeneic mice model, which is consistent with our previous finding. Additionally, anti-murine PD-1 antibody combined with APS001F and 5-FC further suppressed tumor progression and some of individual samples achieved complete regression of CT26 tumors. The combination treatment effect of anti-murine PD-1 antibody and APS001F with 5-FC showed statistical significance in comparison with either treatment group, which encourages us to expect much effective results and less side effects in clinic. Reference: 1. Vincent J. et. al., Cancer Res. 2010 Apr 15;70(8):3052-61, 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity. 2. Van Der Kraak et. al., J Immunother Cancer., 2016 Oct 18;4:65. 5-Fluorouracil upregulates cell surface B7-H1 (PD-L1) expression in gastrointestinal cancers. Citation Format: Koichiro Shioya, Yuji Seki, Tomio Matsumura, Yuko Shimatani, Shun'ichiro Taniguchi, Minoru Fujimori. Synergistic anti-tumor efficacy of combination therapy with APS001F, a cytosine deaminase (CD) expressing Bifidobacterium, 5-fluorocytosine (5-FC) and anti-mPD-1 antibody in syngeneic mice model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4564. doi:10.1158/1538-7445.AM2017-4564

Tumor-Homing and Penetrating Peptide-Functionalized Photosensitizer-Conjugated PEG-PLA Nanoparticles for Chemo-Photodynamic Combination Therapy of Drug-Resistant Cancer.

The combination of photodynamic therapy (PDT) and chemotherapy holds great potential in combating drug-resistant cancers. However, the major challenge that lies ahead is how to achieve high coloading capacity for both photosensitizer and chemo-drugs and how to gain efficient delivery of drugs to the drug-resistant tumors. In this study, we prepared a nanovehicle for codelivery of photosensitizer (pyropheophorbide-a, PPa) and chemo-drugs (paclitaxel, PTX) based on the synthesis of PPa-conjugated amphiphilic copolymer PPa-PLA-PEG-PLA-PPa. The obtained nanoparticles (PP NP) exhibited a satisfactory high drug-loading capacity for both drugs. To achieve effective tumor-targeting therapy, the surface of PP NP was decorated with a tumor-homing and penetrating peptide F3. In vitro cellular experiments showed that F3-functionalized PP NP (F3-PP NP) exhibited higher cellular association than PP NP and resulted in the strongest antiproliferation effect. In addition, compared with the unmodified nanoparticles, F3-PP NP exhibited a more preferential enrichment at the tumor site. Pharmacodynamics evaluation in vivo demonstrated that a longer survival time was achieved by the tumor-bearing mice treated with PP NP (+laser) than those treated with chemotherapy only or PDT only. Such antitumor efficacy of combination therapy was further improved following the F3 peptide functionalization. Collectively, these results suggested that targeted combination therapy may pave a promising way for the therapy of drug-resistant tumor.

IS1-5 - Exploring the Anti-Tumor Efficacy of Combination Therapy with Inhibitors Targeting the Insulin-Like Growth Factor (IGF) and PI3K/AKT/MTOR Signaling Pathways for Hepatocellular Carcinoma (HCC)

ABSTRACT Background The IGFR signaling pathway plays important roles in carcinogenesis and drug resistance of many cancers, including HCC, but inhibitors targeting the IGF signaling pathway showed limited therapeutic efficacy. This study explored the efficacy of combining inhibitors targeting the IGF and PI3K/AKT/mTOR signaling pathways in HCC. Methods HCC cell lines tested included Hep3B, Huh7, and PLC5. The MTT agents tested included NVP-AEW541 (IGF receptor kinase inhibitor, Novartis), MK2206 (Akt inhibitor, MSD), BEZ235 (PI3K/mTOR inhibitor, Novartis), and RAD001 (mTOR inhibitor, Novartis). The potential synergistic antitumor effects were tested by MTT assay and median dose effect analysis in vitro and by xenograft models in vivo. Apoptosis was analyzed by flow cytometry and western blotting. The activity of pertinent signaling pathways and expression of apoptosis-related proteins were measured by western blotting and by an apoptosis protein array (Proteome ProfilerTM, R&D Systems). Results Synergistic growth-inhibitory and apoptosis-inducing effects in HCC cells were found most prominently in NVP-AEW541/ MK2206 combination, followed by NVP-AEW541/BEZ235 combination. These synergistic anti-tumor effects correlated with a more sustained inhibition of Akt and 4EBP-1 phosphorylation. Potential downstream mediators identified by the apoptosis array included survivin and clapsin. The synergistic anti-tumor effects of NVP-AEW541/MK2206 and NVP-AEW541/BEZ235 combinations were confirmed in vivo by xenograft models, with acceptable toxicity profiles. Conclusions Combination therapy of IGF receptor inhibitors with inhibitors targeting the PI3K/AKT/mTOR pathway may have therapeutic efficacy in HCC (supported by grants NHRI-EX100-9911BC, DOH100-TD-B-111-001, and NSC 100-2314-B-002-058-MY3).