Immunotherapy Of Ovarian Cancer Research Articles

Abstract A15: Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy

Abstract The natural occurrence of immune cells in the tumor microenvironment of ovarian cancer (OC) facilitates the ex vivo generation and expansion of tumor-infiltrating lymphocytes (TIL) for infusion in the context of adoptive T-cell transfer. Yet, the lack of tumor-reactive TILs and the suppressive microenvironment have hindered the translation of TIL therapy into meaningful clinical responses in OC patients. Therefore, generating a proinflammatory tumor microenvironment with oncolytic adenoviruses, in order to induce a steady and potent antitumor activity, may prove an effective add-on to TIL therapy for OC. Besides their well-established efficacy in OC, oncolytic adenoviruses genetically modified to carry the interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFa) transgenes may further support and promote T-cell mediated antitumor immunity. In this study, we assessed the capability of an oncolytic virus coding for IL-2 and TNFa (Ad5/3-E2F-D24-hIL-2-IRES-TNFa; TILT-123) to enhance the reactivity of TILs towards OC in a suppressive microenvironmental context. Fresh tumor tissue from regional metastatic or primary sites was obtained from stage III-IV OC patients. Part of the samples were diced in small fragments for the generation and rapid expansion of TILs under clinically relevant in vitro conditions. Phenotypic characterization of TILs was performed by flow cytometry after rapid expansion. Following TIL production and characterization, TILs were co-cultured with T cell-depleted autologous OC single-cell suspensions in the presence or absence of oncolytic virus. TIL reactivity was determined by quantifying Interferon (IFN)-gamma in culture supernatants through ELISA. The remainder of the tumor sample was dissociated overnight and cultured in the presence or absence of oncolytic virus. Supernatants were harvested at different days post-infection for detection of relevant cytokines. Following a 14-day rapid expansion period, the final TIL product was composed mostly of CD8+ T cells (56%), CD4+ T cells (27%) and a small fraction of CD3+CD56+ NK-like T cells (2%). When TILs were cocultured with T-cell depleted OC single-cell suspensions, treatment with oncolytic virus coding for IL-2 and TNFa caused a significant increase in the release of IFNg by TILs as compared to conditions where no virus or the backbone oncolytic virus (without cytokine transgenes) was added (p<0.05). Microenvironmental changes by the cytokine-encoding oncolytic adenovirus were evidenced by increased release of proinflammatory cytokines such as, TNFa, IL-2 and IFNg in 7-day cell cultures of ex vivo single-cell suspensions derived from OC patients. In summary, tailoring the tumor microenvironment with IL-2 and TNFa using oncolytic viruses sharpens the TIL reactivity in an immune-suppressive OC microenvironmental context. Hence, this approach may enable effective responses in OC patients treated with TIL therapy. This evidence supports further clinical validation. Citation Format: João Manuel Santos, Camilla Hëinio, Victor Cervera-Carrascon, Mikko Siurala, Dafne Quixabeira, Tanja De Gruijl, Heini Lassus, Anna Kanerva, Akseli Hemminki. Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A15.

Abstract A29: The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies

Abstract Immunotherapy in ovarian cancer has been disappointing, with only ~10% of patients responding to checkpoint blockade. The determinants of this low response rate remain poorly understood, and there is a pressing need for immune-competent preclinical models to elucidate the biology of immune evasion in ovarian cancer. One critical area of interest is the role of homologous recombination (HR) repair in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in homologous recombination-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations, which are TP53, BRCA1, PTEN, Myc, Cyclin E1 (CCNE1), Akt2, and Kras, to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune/stromal analysis using flow cytometry, single-cell RNA sequencing, and transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and regulatory compartments between HR-proficient and -deficient tumors within the primary and metastatic tumors themselves and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease. Citation Format: Sonia Iyer, Shuang Zhang, Anniina Farkkila, Sean G. Smith, David Pepin, Raghav Mohan, Esmee Hoefsmit, Tian Xia, Ferenc Reinhardt, Tony E. Chavarria, Shailja Pathania, Yunlan Zhou, Kevin M. Elias, Benjamin G. Neel, Robert A. Weinberg. The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A29.

Abstract B52: The systematic identification of novel immune checkpoint averting the metastasis of ovarian cancer: The role of IL-20/IL24-IL20RA axis

Abstract Ovarian cancer has high mortality among all gynecologic malignancies due to the lack of clear symptoms at early stage and the metastasis of cancer cells when diagnosed with advanced stages. The disseminated ovarian cancer cells have to overcome the immune surveillance in abdominal environment, which is rich in many types of immune cells, to survive and proliferate. However, the key genes and mechanisms preventing the metastasis of ovarian cancer cells into abdominal sites remain largely unexplored. Here, through an in vivo, genome-scale CRISPR/Cas9 knockout screening, we identified IL-20/IL24-IL20RA as a key checkpoint preventing the abdominal metastasis of ovarian cancer cells. In ovarian cancer patients, the level of IL20RA is dramatically decreased in metastatic cancer cells when compared with that in primary sites. Overexpression of IL20RA in highly metastatic ovarian cancer cells greatly suppressed the abdominal metastasis of ovarian cancer and the formation of ascites. In contrast, silencing IL20RA could increase the metastasis of ovarian cancer cells. We further examined IL02RA-mediated crosstalk between the disseminated cancer cells and the immuno-microenvironment in mouse in vivo ovarian cancer models and in vitro coculture experiments. Silencing IL20RA in ovarian cancer cells resulted in a dramatic decrease of M1 subtype of macrophages and increase of the M2 subtype of macrophages without affecting the population of T and B lymphocytes in ascites. Further mechanistic studies revealed that the ovarian cancer cells, when invaded into the abdominal sites, greatly induced the mesothelial cells in peritoneum to produce IL20RA ligands, i.e., IL-20 and IL-24, which in turn activated the IL20RA downstream signaling in ovarian cancer cells to trigger the production and maturation of IL-18. IL-18, when secreted into the microenvironment, was able to promote the polarization of macrophages into M1-subtype to clear the cancer cells. Thus, we identified a novel immune checkpoint IL20/IL24-IL20RA that was always downregulated in metastatic ovarian cancer cells to prevent the formation of an antitumor immune microenvironment, which could be utilized to develop new strategies for the immunotherapy of ovarian cancers. Citation Format: Jia Li, Xuan Qin, Mengyao Xu, Jie Yan, Longlong Wang, Yi Shi, Rong Xiang. The systematic identification of novel immune checkpoint averting the metastasis of ovarian cancer: The role of IL-20/IL24-IL20RA axis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B52.

Targeting the immunosuppressive tumor microenvironment: Synergistic effect of low-dose IL12 in combination with dual immune checkpoint inhibition in a preclinical model of ovarian cancer.

7 Background: Epithelial ovarian cancer (OC) is the most lethal gynecologic cancer with ~22,000 women diagnosed annually in the US. The impact of immune checkpoint inhibition (ICI) in the treatment of solid tumors has been significant. However, the response rates for OC are low ranging from 11-15%. It is critical to explore strategies to enhance the efficacy of ICI immunotherapy in OC. Targeting immunosuppressive factors and cells within the tumor microenvironment (TME) represents a feasible approach. The use of IL12 is attractive because induces potent antitumor activity by targeting myeloid cells and lymphocytes. However its clinical application has been hindered by its potential systemic toxicity. Here we explore the use of low dose intraperitoneal IL12 to enhance the antitumor activity of dual ICI in OC. Methods: Mice bearing ID8-VEGF tumors implanted intraperitoneally received either anti-PD1 alone or dual ICI treatment of anti-PD1 plus anti-CTLA4 with or without low dose IL12. Ascites accumulation was used as surrogate for tumor progression and determined by assessing weight increase. Blood and ascites were analyzed by flow cytometry for frequency of PMN-MDSC, M-MDSC, and activated T cells. Results: Low dose IL12 alone induced a significant delay in ascites accumulation when compared to untreated controls or mice treated with PD1 monotherapy or dual ICI. Addition of IL12 to dual ICI resulted in significant tumor regression and extended survival benefit compared to dual ICI alone. A synergistic effect of IL12 was not observed when combined with PD1 monotherapy. Antitumor responses associated with a marked decrease in the frequency of M-MDSC in blood and a decrease in both PMN- and M-MDSC in ascites. Decrease in MDSC associated with elevated levels of activated T cells. Conclusions: Low dose IL12 can induce regression of ID8-VEGF tumors. However, durable responses were only observed when IL12 was added to dual ICI. This suggests that IL12 can induce changes in the TME, particularly on MDSC, that can potentiate the antitumor activity of dual ICI. Our findings also suggest a crucial role of CTLA4 blockade perhaps via Treg targeting.

Artificial Mini Dendritic Cells Boost T Cell-Based Immunotherapy for Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy with high recurrence rates and low survival rates, remaining a disease of high unmet need. Cancer immunotherapy, which harnesses the potential of the immune system to attack tumors, has emerged as one of the most promising treatment options in recent years. As an important form of immunotherapy, dendritic cell (DC)–based vaccines have demonstrated the ability to induce an immune response, while clinical efficacy of DC vaccines remains unsubstantiated as long‐term benefit is only reported in a restricted proportion of patients. Here, a biomimetic nanovaccine derived from DCs is developed through cell membrane coating nanotechnology. This nanovaccine, denoted “mini DC,” inherits the ability of antigen presentation and T cells' stimulation from DCs and is shown to elicit enhanced activation of T cells both in vitro and in vivo. In a mouse model of ovarian cancer, mini DCs exhibit superior therapeutic and prophylactic efficacy against cancer including delayed tumor growth and reduced tumor metastasis compared with DC vaccine. These findings suggest that mini DCs may serve as a facile and potent vaccine to boost anticancer immunotherapy.

Heterogeneity of immune microenvironment in ovarian cancer and its clinical significance: a retrospective study

ABSTRACT Treatment of ovarian cancer (OC) remains the biggest challenge among gynecological malignancies. Immune checkpoint blockade therapy is promising in many cancers but shows low response rates in OC because of its heterogeneity. Although the biological and molecular heterogeneity of OC has been extensively investigated, heterogeneity of immune microenvironment remains elusive. We have collected the expression profiles of 3071 OC patients from 22 publicly available datasets. CIBERSORT was applied to infer the infiltration fraction of 22 immune cells among 2086 patients with CIBERSORT P < .05. We then explored the heterogeneity landscape of immune microenvironment in OC at three levels (immune infiltration, prognostic relevance of immune infiltration, immune checkpoint expression patterns). Multivariable Cox regression model was used to investigate the associations between survival risk and immune infiltration. Constructed immune risk score stratified patients with significantly different survival risk (HR: 1.47, 95% CI: 1.31–1.66, P < .0001). The immune infiltration landscape, prognostic relevance of immune cells, and expression patterns of 79 immune checkpoints exhibited remarkable clinicopathological heterogeneity. For instance, M1 macrophages were significantly associated with better outcomes among patients with high-grade, late-stage, type-II OC (HR: 0.77–0.83), and worse outcomes among patients with type-I OC (HR: 1.78); M2 macrophages were significantly associated with worse outcomes among patients with high-grade, type-II OC (HR: 1.14–1.17); Neutrophils were significantly associated with worse outcomes among patients with high-grade, late-stage, type-I OC (HR: 1.14–1.73). The heterogeneous landscape of immune microenvironment presented in this study provided new insights into prognostic prediction and tailored immunotherapy of OC.

An autologous humanized patient-derived-xenograft platform to evaluate immunotherapy in ovarian cancer

ObjectiveThe aim of this study was to “humanize” ovarian cancer patient-derived xenograft (PDX) models by autologous transfer of patient-matched tumor infiltrating lymphocytes (TILs) to evaluate immunotherapies. MethodsOrthotopic high-grade serous ovarian cancer (HGSOC) PDX models were established from three patient donors. Models were molecularly and histologically validated by immunohistochemistry. TILs were expanded from donor tumors using a rapid expansion protocol. Ex vivo TIL and tumor co-cultures were performed to validate TIL reactivity against patient-matched autologous tumor cells. Expression of TIL activation markers and cytokine secretion was quantitated by flow cytometry and ELISA. As proof of concept, the efficacy of anti-PD-1 monotherapy was tested in autologous TIL/tumor HGSOC PDX models. ResultsEvaluation of T-cell activation in autologous TIL/tumor co-cultures resulted in an increase in HLA-dependent IFNγ production and T-cell activation. In response to increased IFNγ production, tumor cell expression of PD-L1 was increased. Addition of anti-PD-1 antibody to TIL/tumor co-cultures increased autologous tumor lysis in a CCNE1 amplified model. Orthotopic HGSOC PDX models from parallel patient-matched tumors maintained their original morphology and molecular marker profile. Autologous tumor-reactive TIL administration in patient-matched PDX models resulted in reduced tumor burden and increased survival, in groups that also received anti-PD-1 therapy. ConclusionsThis study validates a novel, clinically relevant model system for in vivo testing of immunomodulating therapeutic strategies for ovarian cancer, and provides a unique platform for assessing patient-specific T-cell response to immunotherapy.

Abstract A044: A precision medicine platform to predict the clinical response to chemo- and immunotherapy for epithelial ovarian cancer

Abstract Introduction: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. First-line therapy in advanced EOC is surgery in combination with platinum-based chemotherapy and paclitaxel. 15-20% of patients do not respond and in 80% of advanced cases, the disease recurs within three years. PARP inhibitors synergize with platinum therapy and have been approved for platinum sensitive EOC. Clinical trials with immunotherapies, such as PD-1/PD-L1 blockade, have so far not been successful. Currently, the only approved companion diagnostic is BRCA gene mutations for PARP inhibitors. More diagnostic assays to predict the clinical response to chemo- or immunotherapies are needed. We have developed a biomarker discovery platform using ascites of ovarian cancer patients. Experimental procedures Ascites was gathered from patients by punction or during debulking surgery. Cells were collected by centrifugation and characterized by flow cytometry using specific antibodies. Genomic DNA was sequenced using Illumina cancer gene panels. Gene expression was analyzed by quantitative PCR (qPCR). Cellular activity of the tryptophan metabolizing enzymes IDO1 and TDO was measured with NFK Green [1]. Levels of L-tryptophan and its metabolite L-kynurenine in ascites fluid and blood were determined with LC-MS/MS. In vitro data were related to tumor histopathology and clinical response data. Results: Low passage cell samples from twenty patients were profiled for sensitivity to various cytotoxic agents and targeted anti-cancer therapies in cell proliferation assays. In parallel the mutation status of fifty cancer genes including BRCA1 and 2 was assessed by DNA sequencing. The expression of genes implicated in resistance to chemotherapy (CCNE1, ABCB1) or immunotherapy (PD-L1, IDO1, TDO) was determined with qPCR. The immune status of ascites was analyzed by measuring the relative proportion of different immune cell populations, i.e., cytotoxic and regulatory T cells, monocytes, dendritic and natural killer cells. The expression of the immune suppressive markers PD-L1, IDO1 and TDO was related to the immune cell composition of the ascites, kynurenine-tryptophan ratio, and clinical response data. A tumor cell sample derived from a patient with low grade serous ovarian cancer (LGSOC) was heterozygous for an oncogenic NRAS mutation and was much more sensitive to MEK inhibitors than other samples not harboring the mutation. The cells also expressed IDO1 and high levels of PD-L1 at the cell surface. Two other samples derived from high grade serous ovarian cancer (HGSOC) expressed high PD-L1 and one also IDO1. Several HGSOC samples expressed TDO. One HGSOC sample showed high expression of the ABCB1 gene, encoding the multidrug transporter P-glycoprotein. The sample was relatively resistant to paclitaxel, a known substrate of P-glycoprotein Conclusion: Our study shows that in vitro drug sensitivity assays with primary patient samples can be used to confirm or identify predictive drug response biomarkers. In an ongoing study, in which hundred patients with HGSOC will be included, the in vitro drug response of tumor cells from ascites to first-line cytotoxic anti-cancer agents will be determined and compared to the clinical response of patients bearing specific genomic biomarkers. [1] Seegers et al. (2014) J Biomol Screen 19, 1266-1272 Citation Format: Guido J.R. Zaman, Judith E. den Ouden, Jelle Dylus, Antoon M. van Doornmalen, Winfried R. Mulder, Diep Vu-Pham, Suzanne J.C. van Gerwen, Joost C.M. Uitdehaag, Rogier C. Buijsman, Leon F. Massuger, Anne M. van Altena. A precision medicine platform to predict the clinical response to chemo- and immunotherapy for epithelial ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A044. doi:10.1158/1535-7163.TARG-19-A044

Abstract PR04: The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies

Abstract High-grade serous ovarian cancer (HGSOC) is the most frequent and most aggressive histologic subtype of ovarian cancer. The cornerstone of the existing treatment of HGSOC is DNA-damaging chemotherapy; however, practically all patients eventually develop a progressive disease and the 5-year survival is only 40%. Immunotherapy would seem to be an attractive alternative treatment to chemotherapy, yet existing immunotherapies perform poorly in ovarian cancer, with only ~10% of patients responding to checkpoint blockade. Why this is the case remains poorly understood and there is a pressing need to understand the underlying biology of immune evasion in ovarian cancer. One critical area of interest is the role of homology-dependent DNA repair (HR) in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. Hence, we have modeled the biology of ovarian cancer using patient-relevant mutational landscapes in an immune-proficient, syngeneic mouse model in order to help us identify the contribution of common driver mutations to the immune repertoire in the tumor microenvironment, and thus to responses of HGSOC tumors to immunotherapy. We hypothesize that the immune composition and gene expression signatures of the resulting tumors will vary based on the combination of genetic alterations and the DNA repair proficiency of the transformed cells. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in HR-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations which are TP53, BRCA1, PTEN, MYC, Cyclin E1 (CCNE1), AKT2, and Kras to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune /stromal analysis using flow cytometry, scRNAseq transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and T-cell regulatory compartments between HR-proficient and -deficient primary and metastatic tumors and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease. Citation Format: Sonia Iyer, Shuang Zhang, Anniina Farkkila, Sean Smith, David Pepin, Raghav Mohan, Ferenc Reinhardt, Tian Xia, Tony E. Chavarria, Esmee Hoefsmit, Shailja Pathania, Yunlan Zhou, Kevin M. Elias, Benjamin G. Neel, Robert A. Weinberg. The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR04. doi:10.1158/1535-7163.TARG-19-PR04

Abstract KN01: Dissecting antitumor immune response in ovarian cancer

Abstract In epithelial ovarian cancer (EOC), intratumoral or intraepithelial tumor infiltrating lymphocytes (TILs), i.e. T cells specifically infiltrating tumor islets, occur in approximately half of the patients and correlate with longer survival. As with many other solid tumors, despite promising results in mouse models, response of EOC to PD-1 blockade has been quite limited in the clinic, but the mechanisms underlying therapeutic failures in immunoreactive human tumors harboring TILs are not well understood. Our work has sought to answer the following key questions: a) whether ovarian TILs are tumor-specific; b) if so, what are the relevant antigens recognized by TILs; c) whether the PD-1/PD-L1 pathway is relevant; d) if so, what are the mechanisms underlying response to PD-1 blockade; and finally e) what mechanisms govern TIL recruitment and engraftment. The data presented will show that there is indeed a spontaneous and tumor-specific T-cell response in a proportion of EOC, accurately heralded by intraepithelial CD8+ TILs. Tumor-specific antigens will be discussed. Our work also shows that the PD-1/PD-L1 is a central immune checkpoint pathway in these tumors. However, only a fraction of such tumors with ieTILs respond to PD-1. We dissected the mechanisms underlying response to PD-1 blockade and therapeutic approaches to enhance response to PD-1 in human and mouse preclinical models. Therapeutic approaches to achieve effective immunotherapy in ovarian cancer will be discussed, including combination checkpoint blockade, adoptive T cell therapy and new rational combinations. Finally, successful T-cell engraftment in ovarian cancer requires tumor cell-derived CCL5 and is amplified by myeloid cell-secreted IFNg-inducible CXCL9, dependent on activation of tumor-infiltrating lymphocytes (TILs). Spontaneous, epigenetically-mediated loss or stable knockdown of ccl5 leads to CD8+ T-cell loss and immune desertification in the mouse, while forced ccl5 expression prevents loss of cxcl9 and TILs, and leads to attenuated tumor growth. Accordingly, CCL5-CXCL9 co-expression reveals immunoreactive tumors with prolonged survival and response to PD-1 blockade. The impact of homologous recombination and BRCA loss on the establishment of immunoreactive tumors will be also discussed. Citation Format: George Coukos. Dissecting antitumor immune response in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr KN01. doi:10.1158/1535-7163.TARG-19-KN01

Abstract TMIM-077: MYXOMA VIRUS ENHANCES TREATMENT BENEFIT OF CHEMOTHERAPY AND DENDRITIC CELL IMMUNOTHERAPY IN OVARIAN CANCER PRECLINICAL MODEL

Abstract Myxoma virus (MYXV) is a poxvirus with oncolytic and immunotherapeutic potential in many cancer models, including ovarian cancer (OC). Moreover, MYXV synergizes with chemotherapy including cisplatin for OC treatment. In this study, we used a p53-/- ID8 mouse model that closely portrays human high-grade serous OC to test MYXV virotherapy. We combined MYXV regime with cisplatin treatment and an innovative Th17-inducing dendritic cell (Th17-DC) vaccine currently being tested in clinical trials with OC patients. For DC immunotherapy, bone marrow-derived DC were pulsed with peptides from Sp17, a cancer testis antigen highly expressed in human OC and the ID8 mouse model. For MYXV virotherapy, we examined both wild-type and a replication-defective MYXV that is engineered without an essential viral immunomodulator called M062 (M062R-null MYXV). We found that inoculation with replication-competent MYXV improves survival when administered before cisplatin therapy. Interestingly, when the replication-defective M062R-null MYXV was used, improved survival was observed with virotherapy after the cisplatin treatment. We further found that M062R-null MYXV treatment provided long-lasting survival in combination with cisplatin and Th17-DC immunotherapy. Related studies showed that M062R-null MYXV infection in murine and primary human OC cells stimulated robust type I IFN expression and pro-inflammatory cytokines. Moreover, M062R-null MYXV activates type I IFN in macrophages and expression of inflammatory cytokines (e.g., CXCL-10 and IL-6). In conclusion, we propose that MYXV virotherapy provokes a type I interferon response and inflammatory cytokines that modulate the tumor microenvironment and enhance protective immune responses to Th17-DC vaccination for OC treatment. Citation Format: Steven J Conrad, Bernice Nounamo, Martin Cannon, Jia Liu. MYXOMA VIRUS ENHANCES TREATMENT BENEFIT OF CHEMOTHERAPY AND DENDRITIC CELL IMMUNOTHERAPY IN OVARIAN CANCER PRECLINICAL MODEL [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-077.

Abstract TMIM-073: THE COMBINATION OF INTERFERONS ALPHA AND GAMMA AND MONOCYTES INDUCES OVARIAN CANCER CELL DEATH AND PROVIDE A RATIONALE FOR A NOVEL, ONGOING, IMMUNOTHERAPY PHASE 1 CLINICAL TRIAL

Abstract Standard of care for the treatment of ovarian cancer is surgical tumor debulking, followed by administration of a platinum based compound in combination with a taxane compound. While there is an initial good response to the therapy, especially in optimally debulked tumors, the disease is characterized by a high rate of relapse. There is no definitive second line treatment for patients. Ovarian cancer is largely retained in the peritoneal cavity, with metastases outside of the peritoneum occurring late in the course of the disease. The restriction of the bulk of the tumor burden to the peritoneal cavity makes intraperitoneal (IP) treatment a reasonable approach for ovarian cancer. This strategy was first employed for ovarian cancer using immunotherapy with IP administration of Interferon Alpha. However, one of the hallmarks of ovarian cancer a highly immunosuppressive environment. This environment includes the metastases themselves and the fluid of the peritoneum which contains a mix of pro and anti-inflammatory cells, cytokines and lipids. Tipping the balance towards a pro-inflammatory environment is necessary for the effective treatment of disease. Herein, we define the mechanisms by which IFNs and monocytes are potent killers of ovarian cancer cells. While patients with ovarian cancer have normal whole blood counts, the tumoricidal activity of their monocytes has never been measured. We demonstrate that ovarian cancer patient monocytes are more tumoricidal when cultured with IFNs than monocytes from sex and age matched controls. In this work, we expand on our previous observations of synergistic killing of ovarian cancer cell lines by monocytes and IFNs by showing that an important mechanism of cell death is mediated by TRAIL expressed on monocytes, and the target cells die in a Caspase-8 dependent mechanism. We also found that the tumoricidal effect of IFNs and monocytes was independent of IRF9 and STAT2 signaling, and was instead dependent on IRF-1 and STAT1 signaling. Together, these data support a new, innate immune based, approach to immunotherapy of ovarian cancer. We are currently determining the safety of using autologous monocytes treated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced ovarian cancer in a phase 1 clinical trial (NCT02948426). While the data presented here and previously published works show that innate mediators of the immune system can kill ovarian cancer cells and decrease disease burden, a durable clinical response is dependent on a strong adaptive immune response. In the clinical trial we will identify whether the highly pro-inflammatory properties of the combination of the innate immune effectors monocytes and IFNs can stimulate an existing, but tumor suppressed, adaptive anti-tumor immune response. Here we present a mechanistic understanding of how IFNs and monocytes can kill ovarian cancer cells and provide mechanistic insights into innate immune based immune therapy for the treatment of ovarian cancer. Citation Format: Daniel S. Green, Chase L. Johnson, Chen-Feng Qi, Kevin W. Tosh, Olena Kamenyeva, Kathryn C. Zoon, Christina M. Annunziata. THE COMBINATION OF INTERFERONS ALPHA AND GAMMA AND MONOCYTES INDUCES OVARIAN CANCER CELL DEATH AND PROVIDE A RATIONALE FOR A NOVEL, ONGOING, IMMUNOTHERAPY PHASE 1 CLINICAL TRIAL [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-073.

Tumour Microenvironment-Based Molecular Profiling Reveals Ideal Candidates for High-Grade Serous Ovarian Cancer Immunotherapy

Background: Due to limited immunological profiles of high-grade serous ovarian cancer (HGS-OvCa), we aimed to characterise its molecular features to determine whether a specific subset that can respond to immunotherapy exists. Methods: A training cohort of 418 HGS-OvCa samples from TCGA was analysed by consensus non-negative matrix factorization. We correlated the expression patterns with the presence of immune cell infiltrates, immune regulatory molecules and other genomic or epigenetic features. Two independent cohorts containing 482 HGS-OvCa samples and in vitro experiments were used for validation. Findings: We identified immune and normal groups where the former was enriched in signatures that reflect immune cells, infiltration, and PD-1 signalling (all, P<0·001), and presented with a lower burden of chromosomal aberrations but significantly increased neoantigens, tumour mutation burden, and microsatellite instability (all, P<0·05); this group was further refined into two microenvironment-based subtypes characterized by either immunoactivation or carcinoma-associated fibroblasts (CAFs) and distinct prognosis. The CAFs-immune subtype was enriched for factors that mediate immunosuppression and promote tumour progression, including highly expressed stromal signature, TGF-β signalling, epithelial-mesenchymal transition, and tumour-associated M2-polarized macrophages (all, P<0·001). The robustness of these immune-specific subtypes were verified in validation cohorts, and in vitro experiments indicated the activated-immune subtype can benefit from anti-PD1 antibody therapy (P<0·05). Interpretation: Our findings revealed two distinct immune subtypes with different responses to immunotherapy and indicate some HGS-OvCas may be susceptible to immunotherapies or combination therapies. Funding Statement: This work was supported by the Fundamental Research Funds for the Central Universities grants of China (Grant No. 2632018FY04); the “Double First-Class” University project (CPU2018GY09); the National Natural Science Foundation of China (81973145). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Not required.

Oligopeptides for Immunotherapy Approaches in Ovarian Cancer Treatment.

Anti-ovarian cancer vaccines based on minimal immune determinants uniquely expressed in ovarian cancer biomarkers appear to promise a high level of sensitivity and specificity for ovarian cancer immunodiagnostics, immunoprevention, and immunotherapy. Using the Pir Peptide Match program, three ovarian cancer biomarkers - namely, sperm surface protein Sp17, WAP four-disulfide core domain protein 2, and müllerian-inhibiting substance - were searched for unique peptide segments not shared with other human proteins. Then, the unique peptide segments were assembled to define oligopeptides potentially usable as synthetic ovarian cancer antigens. This study describes a methodology for constructing ovarian cancer biomarkerderived oligopeptide constructs that might induce powerful, specific, and non-crossreactive immune responses against ovarian cancer.

1026P - Preselecting tumour-infiltrating lymphocyte subsets to implement adoptive immunotherapy in ovarian cancer

BackgroundAdoptive cell therapy (ACT) of expanded in-vitro tumour specific T-cells isolated from fresh tumour infiltrates has shown promising results in melanoma patients, and in ovarian cancer (OC). The use of pre-enriched tumor-specific T cells may simplify the TIL production method and enhances the tumour-reactivity of the final cellular product. We have investigated the role of PD-1 as a biomarker for the isolation and expansion of tumour-specific CD8 TILs in OC. MethodsSamples from ten OC patients were analyzed. We used flow cytometry FACs Aria sorter for phenotyping and separate T-cells. For reactivity assay we performed co-cultures of TILs with autologous tumour and non-related tumor cell line, and measured INFγ released by ELISPOT. For immunofluorescence multiplex we used a Vectra® microscope and inForm® software to analyzed data. Tumour DNA sequencing was done using a TrueShight™ 170 gene panel. ResultsBoth CD8+PD-1+and CD8+PD-1- TIL subsets expanded efficiently and no difference in fold expansion was found. Tumor-reactive TILs were detected in 5/10 patients and this tumor-reactivity was exclusively present in the cells derived from the PD-1+-enriched fraction. There were a higher frequency of CD8+PD-1+CD137+ by FC (p=0,0079) in reactive group in fresh biopsy. Total CD8+and CD8+PD-1+were more frequent in reactive patients (p=0,0079, p=0,0317). CD8+PD-1+CD137+were more frequent (p=0,0437) by IF in the tumoral epithelium of reactive patients. There were more total CD4+ and CD4+PD-1+by FC in reactive group (p=0,0079, p=0,0159). By IF we observed more CD4+PD-1+in both epithelium and stroma of reactive group (p=0,0437). Patients with reactive TILs exhibited significantly high number of missense mutactions (p=0,0198). ConclusionsCD8+PD-1+ T-cell subset include tumour-specific T-cell in OC. CD8+PD-1+CD137+ cells in epithelium may work as a biomarker for reactivity. Higher mutation load is related with tumour-reactive TILs in OC. Legal entity responsible for the studyThe authors. FundingISCIII Spanish grant (PI15/02027). DisclosureAll authors have declared no conflicts of interest.

1875PD - Targeting molecular mediators of T cell exclusion for effective immunotherapy in ovarian cancer

BackgroundClose proximity between cytotoxic T lymphocytes and tumour cells is required for effective immunotherapy. However, what determines the spatial distribution of T cells in the tumour microenvironment is not well understood. Coupling digital pathology and transcriptome analysis on large ovarian tumour cohorts, here we report classification and functionally dissection of tumour-immune contexture in human ovarian cancer. MethodsCD8 IHC and RNAseq analysis were performed on 370 ovarian tumours from the ICON7 phase III clinical trial. Coupling digital pathology with transcriptome analysis, a random forest machine learning algorithm was developed and independently validated for classifying tumour-immune phenotypes in ovarian cancer. Anti-tumour activity of TGFβ blockade in combination with anti-PD-L1 was evaluated in an ovarian cancer mouse model. ResultsWe show the identified tumour-immune phenotypes are of biological and clinical importance with interconnection to molecular subtypes and association with clinical outcome in ovarian cancer. Two important hallmarks of T cell exclusion were identified: 1) loss of antigen presentation on tumour cells and 2) upregulation of TGFβ and activated stroma. We identified TGFβ as a key mediator of T cell exclusion. TGFβ reduced MHC class I expression in ovarian cancer cells and induced extracellular matrix (ECM) production and immunosuppressive molecules in human primary fibroblasts. Finally, we demonstrated that combination of anti-TGFβ and anti-PD-L1 in a mouse ovarian cancer model significantly improved the anti-tumour efficacy and survival. ConclusionsThis study provided the first systematic and in-depth characterization of the molecular features and mechanisms underlying the tumour-immune phenotypes in ovarian cancer. We illuminated a multi-faceted role of TGFβ in mediating consequential crosstalk between tumour cells and cancer associated fibroblasts to shape the tumour-immune contexture. Our findings support that targeting the TGFβ pathway represents a promising therapeutic strategy to overcome T cell exclusion and optimize response to cancer immunotherapy. Legal entity responsible for the studyThe authors. FundingGenentech/Roche. DisclosureAll authors have declared no conflicts of interest.

Application Of Adoptive Immunotherapy In Ovarian Cancer.

Ovarian cancer (OC) has been the most fatal gynecological disease that threatens women’s health. Surgery and platinum-based chemotherapy are the basic ovarian cancer treatments that can improve survival, but the five-year survival rate has not improved because of delayed diagnosis, drug resistance, and recurrence. Novel treatments are needed to improve the prognosis and survival rate of ovarian cancer patients. In recent years, adoptive cell therapy (ACT) has received increasing attention as an emerging therapeutic strategy in the treatment of solid tumors including OC. ACT has shown promising results in many preclinical and clinical trials of OC. The application of ACT depends on different effector cells, such as lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TILs), and genetically modified T cells. In this review, we focus on adoptive immunotherapies in ovarian cancer and summarize completed and ongoing preclinical/clinical trials. The future development directions and obstacles for ACT in OC treatment are discussed.

Conventional and Chemically Programmed Asymmetric Bispecific Antibodies Targeting Folate Receptor 1.

T-cell engaging bispecific antibodies (biAbs) can mediate potent and specific tumor cell eradication in liquid cancers. Substantial effort has been invested in expanding this concept to solid cancers. To explore their utility in the treatment of ovarian cancer, we built a set of asymmetric biAbs in IgG1-like format that bind CD3 on T cells with a conventional scFv arm and folate receptor 1 (FOLR1) on ovarian cancer cells with a conventional or a chemically programmed Fab arm. For avidity engineering, we also built an asymmetric biAb format with a tandem Fab arm. We show that both conventional and chemically programmed CD3 × FOLR1 biAbs exert specific in vitro and in vivo cytotoxicity toward FOLR1-expressing ovarian cancer cells by recruiting and activating T cells. While the conventional T-cell engaging biAb was curative in an aggressive mouse model of human ovarian cancer, the potency of the chemically programmed biAb was significantly boosted by avidity engineering. Both conventional and chemically programmed CD3 × FOLR1 biAbs warrant further investigation for ovarian cancer immunotherapy.

Computed Tomography-Derived Radiomic Metrics Can Identify Responders to Immunotherapy in Ovarian Cancer.

To determine if radiomic measures of tumor heterogeneity derived from baseline contrast-enhanced computed tomography (CE-CT) are associated with durable clinical benefit and time to off-treatment in patients with recurrent ovarian cancer (OC) enrolled in prospective immunotherapeutic trials. This retrospective study included 75 patients with recurrent OC who were enrolled in prospective immunotherapeutic trials (n = 74) or treated off-label (n = 1) and had baseline CE-CT scans. Disease burden (total tumor volume, number of disease sites), radiomic measures of intertumor heterogeneity (cluster-site entropy, cluster-site dissimilarity), and intratumor heterogeneity of the largest lesion (Haralick texture features) were computed. Associations of clinical, conventional imaging, and radiomic measures with durable clinical benefit and time to off-treatment were examined. In univariable analysis, fewer disease sites, lower intertumor heterogeneity (lower cluster-site entropy, lower cluster-site dissimilarity), and lower intratumor heterogeneity of the largest lesion (higher energy) were significantly associated with durable clinical benefit (P ≤ .031). More disease sites, presence of pleural disease and/or distant metastases, higher intertumor heterogeneity (higher cluster-site entropy, higher cluster-site dissimilarity), and higher intratumor heterogeneity of the largest lesion (higher Contrastlargest-lesion) were significantly associated with shorter time to off-treatment (P ≤ .034). In multivariable analysis, higher Energylargest-lesion (indicator of lower intratumor heterogeneity; P = .006; odds ratio, 1.41) and fewer disease sites (P = .003; odds ratio, 1.64) remained significant indicators of durable clinical benefit (multivariable model C-index, 0.821). Higher cluster-site dissimilarity (indicator of higher intertumor heterogeneity) was a modest but single independent indicator of shorter time to off-treatment (P = .004; hazard ratio, 1.19; C-index, 0.6). Fewer disease sites and lower intra- and intertumor heterogeneity modeled from the baseline CE-CT may indicate better response of OC to immunotherapy.

Subsequent therapies and survival after immunotherapy in recurrent ovarian cancer

ObjectivesImmune checkpoint inhibitors (ICIs) have modest activity in ovarian cancer (OC), yet little is known about their effects on subsequent treatment. Preclinical studies suggest immunotherapy may enhance response to chemotherapy. We sought to evaluate the impact of ICIs on subsequent therapies and survival in recurrent OC. MethodsA retrospective review was conducted to identify women with recurrent OC who received ICI from 01/2013 to 5/2017 and ≥1 subsequent treatment. Treatment duration after ICI was calculated using time-to-event analysis. Kaplan-Meier survival analysis and Cox proportional hazards models were used to calculate overall survival (OS) from first treatment after ICI and to assess survival differences by clinical benefit from ICI, defined by long (≥24 weeks) versus short (<24 weeks) ICI treatment duration. ResultsOf 79 evaluable women identified, 66 (84%) had platinum-resistant OC. Median age at diagnosis was 57 years. Median time from diagnosis to ICI was 39.7 months, with median of 4 prior treatments (range, 1–12). Median number of treatments after ICI was 2 (range, 1–8). Median duration of first-line treatment after ICI was 3.7 months (95% CI, 2.9–6.0) and declined with each subsequent line. The most common therapies after ICI were taxanes, platinum-based regimens, and pegylated liposomal doxorubicin. Bevacizumab was used in 47 women (59%). Median OS after ICI was 18.3 months (95% CI, 11.8–22.7) and did not differ between long versus short ICI. ConclusionsIn this heavily pretreated population of patients with recurrent OC, therapies after ICI resulted in promising survival, suggesting that ICI may improve efficacy of subsequent chemotherapy.