Preclinical Models Of Ovarian Cancer Research Articles

Blockade of TGF-β and PD-L1 by bintrafusp alfa promotes survival in preclinical ovarian cancer models by promoting T effector and NK cell responses.

Failure of immunotherapy in high-grade serous ovarian cancer (HGSC) may be due to high levels of transforming growth factor-β (TGF-β) in ascites or tumour immune microenvironment (TIME). Here, we test whether coordinated blockade of TGF-β and PD-L1 with bintrafusp alfa (BA) can provoke anti-tumour immune responses in preclinical HGSC models. BA is a first-in-class bifunctional inhibitor of TGF-β and PD-L1, and was tested for effects on overall survival and altered TIME in syngeneic HGSC models. Using a mouse ID8-derived HGSC syngeneic model with IFNγ-inducible PD-L1 expression, BA treatments significantly reduced ascites development and tumour burden. BA treatments depleted TGF-β and VEGF in ascites, and skewed the TIME towards cytotoxicity compared to control. In the BR5 HGSC syngeneic model, BA treatments increased tumour-infiltrating CD8 T cells with effector memory and cytotoxic markers, as well as cytolytic NK cells. Extended BA treatments in the BR5 model produced ∼50% BA-cured mice that were protected from re-challenge. These BA-cured mice had increased peritoneal T-effector memory and NK cells compared to controls. Our preclinical studies of BA in advanced ovarian cancer models support further testing of BA as an improved immunotherapy option for patients with advanced ovarian cancer.

Abstract LB406: Synergistic antitumor effect of FRα TOP1i antibody drug conjugate, AZD5335, in combination with the PARP1 inhibitor, saruparib (AZD5305), in preclinical models of ovarian cancer

Abstract Folate receptor alpha (FRα) is a cell surface GPI-anchored protein overexpressed in several solid tumors with highest prevalence in ovarian cancer and lung adenocarcinoma but restricted expression in normal tissues. AZD5335, an FRα-targeting antibody conjugated to AZ’s proprietary topoisomerase 1 inhibitor (TOP1i) payload, AZ14170132, is currently being investigated in the FONTANA Phase 1/2 clinical trial (NCT05797168). Since homologous recombination repair deficiency (HRD) and mutations in genes involved with homologous recombination repair (HRRm+) are associated with sensitivity to DNA damaging agents, we evaluated the efficacy of AZD5335 in ovarian cancer patient-derived xenograft (PDX) models assessed for HRRm, as described1. Sixteen ovarian cancer PDX models were each treated with a single dose of AZD5335 (5 mg/kg IV). Of the HRRm- cohort, 8/11 (73%) cohort responded to treatment with a tumor regression >30% from baseline. In the HRRm+ cohort 4/5 (80%) responded to AZD5335. PARP1 can act as a positive regulator of genomic stability by counteracting TOP1-induced DNA damage (Malanga and Althaus, 2004) that led to the hypothesis that the efficacy of TOP1i drugs can be enhanced by combination with PARP inhibitors. To evaluate for potential synergy of AZD5335 with the PARP1 inhibitor, saruparib (AZD5305): Three FRα-expressing cancer cell lines, KB, IGROV-1 and OVCAR-3, were treated with AZD5335 +/- saruparib, revealing evidence of synergistic cytotoxicity. These results encouraged us to investigate this combination in pre-clinical HRRm+ in vivo xenograft models (OVCAR-3 and OV2022F). In OVCAR-3 xenograft models, single agent activity with saruparib (1 mg/kg PO QD x28) was 37% tumor growth inhibition (TGI) and with AZD5335 (0.625 mg/kg IV x1) was 50% TGI while combined administration at the same doses improved TGI to 86%. Similar combinatorial activity was seen in OV2022F with TGI 61%, 54%, and 97% for saruparib, AZD5335, and saruparib +AZD5335, respectively. In conclusion, AZD5335 exhibits a similar degree of single agent antitumor activity in both HRRm+ and HRRm- preclinical models, and the efficacy can be potentiated by combining with the PARP inhibitor, saruparib. 1. Clarke et al. 2022, NEJM Evid 2022;1(9) DOI: 10.1056/EVIDoa22000432. Malanga and Althaus 2004, JBC 2004; 279(7) DOI: 10.1074/jbc.C3004372003. Citation Format: Marco Gymnopoulos, Ravinder Tammali, Ana De Almeida, Jixin Wang, Claire Myers, Mark Albertella, Paula G. Fraenkel, Puja Sapra. Synergistic antitumor effect of FRα TOP1i antibody drug conjugate, AZD5335, in combination with the PARP1 inhibitor, saruparib (AZD5305), in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB406.

Abstract 3909: PARG inhibitor sensitivity is correlated with accumulation of single strand DNA gaps in preclinical models of ovarian cancer

Abstract Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair in conjunction with PARP1 by removal of poly (ADP-ribose) (PAR). Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA damaging agents, suggesting it is a potential therapeutic target for cancer therapy. Indeed, PARG inhibitors are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as ovarian and breast cancer patients with BRCA1/2-mutation. In this study, we investigated PARG inhibitor (PARGi)-PDD00017273 activity in preclinical models of ovarian cancer cell lines and primary patient-derived organoids. PARGi exhibited sensitivity in a subset of ovarian cancer cell lines that was distinct from PARP inhibitor sensitivity. Initially, we evaluated a PARGi-sensitive cell line, RMUGS. By gradually increasing the concentration of PARGi, we generated drug resistant cells. PARGi treatment resulted in increased S phase specific PAR, DNA-PARylation, DNA damage, and replication stress in the sensitive cells but not in cells with de novo or acquired resistance. In the sensitive cells, PARGi exposure also increased accumulation of PAR at the replication fork resulting from unligated Okazaki fragment processing. More importantly, PARGi treatment or PARG loss caused accumulation of single strand DNA (ssDNA) gaps in sensitive cells, as detected by the DNA fiber assays combined with S1 nuclease treatment. Regardless of the BRCA-status, the induction of ssDNA gaps in ovarian cancer cells correlated with PARGi sensitivity. Consistent with an increase in ssDNA gaps, PARGi exposure increased p-RPA, a ssDNA surrogate, at the replication fork in sensitive cells. p-RPA sites also colocalized with PAR suggesting that accumulation of PAR occurs at the ssDNA sites, perhaps, leading to the ssDNA gaps. Collectively, these results suggested that toxic accumulation of PAR at the replication fork leads to accumulation of ssDNA gaps due to Okazaki fragment processing defects and ovarian cancer cell killing after PARG inhibition. We further assessed PARG inhibition in patient-derived organoids (PDOs) and patient-derived xenograft (PDX)-derived organoids (PDXOs) as model systems of ovarian cancer. PARGi exhibited monotherapy activity in specific HR-deficient PDOs and PDXOs with BRCA1 or RAD51C mutation and in a HR-proficient PDXO model. Importantly, PARGi exposure resulted in accumulation of ssDNA gaps in organoids of ovarian cancer and this phenotype directly correlated with drug sensitivity. PDOs can therefore be a useful model system for testing PARGi sensitivity. The detection of ssDNA gaps in PDOs may provide a functional biomarker to predict the response to PARG inhibitors in clinical trials. Citation Format: Ramya Ravindranathan, Ozge Somuncu, Yuqing Jiao, Alexandre André B A da Costa, Benjamin P Lamarre, David B Martignetti, Gabriella A Zambrano, Sirisha Mukkavalli, Joyce Liu, Bose Kochupurakkal, Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I Shapiro, Alan D. D'Andrea. PARG inhibitor sensitivity is correlated with accumulation of single strand DNA gaps in preclinical models of ovarian cancer [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 3909.

Abstract 5909: Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer

Abstract Objectives: Given the intricate relationship between inflammation and ovarian cancer (OC) development and progression, anti-inflammatory drugs may have potential in the prevention and treatment of OC. Sulindac exhibits potent anti-inflammatory effects through inhibition of COX1/COX2 pathways and promising anti-tumor activity in pre-clinical models of colorectal and endometrial cancer. Thus, we investigated the efficacy of sulindac as an anti-tumorigenic agent in OC pre-clinical cell and mouse models. Methods: The human OC cell lines, MES and OVCAR5, and the K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mouse model of high-grade serous OC were used. Cellular proliferation was assessed by MTT and colony count assays. Apoptosis was assessed using cleaved caspase 3, 8, and 9 assays. To measure cellular stress, production of reactive oxygen species was measured by DCFH-DA assay, and the JC1 assay was used to assess changes in mitochondrial membrane potential. Cellular adhesion was evaluated by laminin assay, and cellular migration was determined by wound healing assay. Western immunoblotting was used to assess the effects of sulindac on downstream targets related to cellular stress, apoptosis, cellular invasion potential, cell cycle control, and inflammation. The KpB mice were treated with sulindac (7.5 mg/kg, oral gavage, daily) or placebo for four weeks. Results: Sulindac inhibited cellular proliferation in a dose-dependent manner in the MES and OVCAR5 cells, with IC50s of 75.3 and 75.69 μM, respectively. Sulindac (100 μM) reduced colony formation by 81% in the MES cells and by 49% in the OVCAR5 cells (p<0.01). Treatment of MES and OVCAR5 cells with sulindac increased ROS production and decreased mitochondrial membrane potential, in a dose-dependent manner (p<0.01). Sulindac induced activity of cleaved caspase 3, 8, and 9, in MES and OVCAR5 cells (p<0.01). In addition, sulindac (100 μM) effectively decreased cellular adhesion by 31% in MES cells and by 23% in OVCAR5 cells (p<0.01). These results were confirmed by Western immunoblotting demonstrating that sulindac downregulated the anti-apoptotic proteins Bcl-xl and Mcl-1 and upregulated the pro-apoptotic proteins Bax and cellular stress proteins Bip, ATF-4, and PDI, in both cell lines. Sulindac downregulated expression of several epithelial-mesenchymal transition proteins in both cell lines, including beta-catenin, snail, and slug. Sulindac also downregulated expression of cell cycle proteins CDK4, CDK5, and Cyclin D1 and inflammatory protein COX-2 in both cell lines. After 4 wks of treatment, sulindac significantly inhibited tumor growth by 71% (1.89 g vs 0.54 g, p<0.01) in KpB mice. Conclusions: Our results find that sulindac exhibited anti-tumorigenic effects in OC cell lines and the KpB OC mouse model. Given these promising pre-clinical results, further studies on the repurposing of sulindac for the prevention and treatment of OC is warranted. Citation Format: Nikita Sinha, Shuning Chen, Jennifer Haag, Xiaochang Shen, Boer Deng, Ziyi Zhao, Chunxiao Zhou, Victoria Bae-Jump. Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer [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 5909.

Abstract 3997: PARP inhibitors plus anlotinib as bridging therapy for TGFβ-insensitive CAR-T cell therapy targeting MSLN and CD19 in advanced ovarian cancer

Abstract Background: The efficacy of chimeric antigen receptor (CAR) T cell therapy remains modest for the treatment of solid tumors due to restricted T cell infiltration and immunosuppressive tumor microenvironment (TME). Leveraging the window of opportunity between leukapheresis and infusion using bridging therapy might improve TME and potentiate CAR-T cell therapy. Here, we assessed the potential of poly (ADP-ribose) polymerase (PARP) inhibitors plus anlotinib as bridging therapy for CAR-T cells with resistance to TGFβ and capability of rapid expansion by bait-and-switch strategy of dual-targeting mesothelin (MSLN) and CD19 in preclinical models of advanced ovarian cancer. Methods: Immunocompetent mice bearing ID8 tumors were treated with niraparib (21 days) plus anlotinib (14 days) and euthanized at different timepoints (0, 7, or 14 days since discontinuation) to quantify tumor-infiltrating T cells and profile T cell functions. CAR-T cells transfected with dominant-negative TGFβRII dual-targeting MSLN and CD19 were produced by IASO Biotherapeutics. NSG mice bearing MSLN-positive cell- or patient-derived xenografts (CDX or PDX) of ovarian cancer received CAR-T cells alone or after bridging therapy (niraparib plus anlotinib followed by a wash-out period of 7 days). Tumor volume was balanced before CAR-T cell infusion. Replication-deficient NALM6 cells pre-treated with mitomycin C were injected to provide CD19 antigen. A PET scan that visualized granzyme B release was used to track CAR-T cells and assess immune-mediated tumor killing. Results: Niraparib combined with anlotinib increased tumor-infiltrating T cells without impairing T cell functionality. The effects of promoting T cell infiltration remained significant at 14 days after discontinuation, which might associate with lasting activation of cGAS-STING pathway and secretion of downstream chemokines (CXCL10 and CCL5), and normalized tumor vasculature featured by better perfusion and reduced leakage. The engineered CAR-T cells could resist TGFβ and displayed improved proliferation, cytotoxicity, cytokine release, and in vivo antitumor activities upon CD19 stimulation. Importantly, bridging therapy significantly increased CAR-T cell infiltration, curbed tumor growth, and prolonged survival in mice bearing SKOV3 CDX, HRD-negative PDX, and another multidrug-resistant PDX. PET imaging showed that bridging therapy increased tumor-infiltrating CAR-T cells and boosted tumor-killing capability. Conclusions: PARP inhibitors plus anlotinib as bridging therapy facilitated CAR-T cell infiltration and enhanced antitumor activities in multiple preclinical models of advanced ovarian cancer, and an early phase I trial (NCT05141253) is ongoing to evaluate PARP inhibitors plus anlotinib as bridging therapy for CAR-T cell therapy in patients with refractory MSLN-positive ovarian cancer. Citation Format: Huayi Li, Minghua Xiang, Jiahao Liu, Wei Mu, Li Zhu, Xuejiao Zhao, Gordon B. Mills, Qinglei Gao, Yong Fang. PARP inhibitors plus anlotinib as bridging therapy for TGFβ-insensitive CAR-T cell therapy targeting MSLN and CD19 in advanced ovarian cancer [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 3997.

Abstract 4187: Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice

Abstract Background: Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human cancer and immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs, including immune checkpoint inhibitors (ICIs), which have demonstrated limited effectiveness in OC patients. Methods: We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). We assessed the efficacy of PD-1 blockade with pembrolizumab in the SKOV-3 Luc Hu-mouse model. The immune profiles of the tumors were characterized using colorimetric immunostaining and flow cytometry. Additionally, we analyzed RNA-seq data to investigate the gene expression signature of pembrolizumab refractory tumors. Results: We confirmed tumor development in both OC cell lines within CD34+ Hu-mice. In these models, human lymphocyte and myeloid cell subsets were present in the tumors, draining lymph nodes, blood, and spleens. The SKOV-3 Luc tumor-bearing Hu-mice did not respond to pembrolizumab monotherapy. These tumors exhibited a high presence of tumor-infiltrating macrophages. Tumors in Hu-mice unresponsive to pembrolizumab showed a lower abundance of CD8+ T-cells, memory B cells, plasma cells, and a higher proportion of naïve M0 macrophages and mast cells compared to the PBS control. Furthermore, we identified 43 significantly enriched gene sets in these tumors. The differentially expressed genes (DEGs) were predominantly enriched in HDAC class I, RB1, KLF1/3, TCF21, MYD88, SMARCE1 target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts. Conclusion: Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer potential avenues to explore mechanisms of resistance to PD-1/PD-L1 blockade in OC. Citation Format: Sung Wan Kang, Ji-young Lee, Ok-Ju Kang, Yong-Man Kim, Eun Kyung Choi, Shin-Wha Lee. Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice [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 4187.

Abstract 1858: Discovery of AT86474, a highly efficacious anti-ROR1 ADC utilizing a proprietary payload

Abstract ROR1 is a pseudo kinase that is involved in the non-canonical Wnt signaling pathway and is highly expressed during early embryonic development. In adult tissues, ROR1 is expressed at very low levels but its increased expression is observed in hematological cancers and a broad range of solid tumors. AT86474 is a highly homogeneous antibody drug conjugate incorporating AxcynCYSTM technology that allows reproducible site-specific conjugation with DAR4 composition greater than 97%. AT86474 utilizes a proprietary payload derived from an FDA approved drug that was optimized in-house for increased potency and with altered PK properties to limit systemic exposure. In vitro, the payload displays broad sub-nanomolar activity across multiple cancer cell lines, including those resistant to paclitaxel, DM1, MMAE and DXd. The payload is conjugated via an enzyme-cleavable linker optimized for solubility, stability, and high drug to antibody ratios. AT86474 uses a humanized and optimized monoclonal antibody that demonstrates selective binding to ROR1-positive cells and subsequent internalization, sub-nanomolar potency in vitro and strong bystander effects. AT86474 was evaluated in a preclinical CDX mouse model of ovarian cancer, where substantial tumor growth inhibition was observed with two weekly doses at as low as 1 mg/kg. As ROR1 over-expression is also observed in many solid tumors, such as pancreatic, gastric, breast and lung cancers, the use of AT86474 can be expanded to benefit a large patient population. Citation Format: Ban Xiong Tan, Xi Yun Zhang, You Xue Wu, Yin Wen Cheng, Shao Jun Liu, Ping Du, Chen Zhong, Bin Zou. Discovery of AT86474, a highly efficacious anti-ROR1 ADC utilizing a proprietary payload [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 1858.

The novel drug candidate S2/IAPinh improves survival in models of pancreatic and ovarian cancer

Cancer selective apoptosis remains a therapeutic challenge and off-target toxicity has limited enthusiasm for this target clinically. Sigma-2 ligands (S2) have been shown to enhance the cancer selectivity of small molecule drug candidates by improving internalization. Here, we report the synthesis of a novel drug conjugate, which was created by linking a clinically underperforming SMAC mimetic (second mitochondria-derived activator of caspases; LCL161), an inhibitor (antagonist) of inhibitor of apoptosis proteins (IAPinh) with the sigma-2 ligand SW43, resulting in the new chemical entity S2/IAPinh. Drug potency was assessed via cell viability assays across several pancreatic and ovarian cancer cell lines in comparison with the individual components (S2 and IAPinh) as well as their equimolar mixtures (S2 + IAPinh) both in vitro and in preclinical models of pancreatic and ovarian cancer. Mechanistic studies of S2/IAPinh-mediated cell death were investigated in vitro and in vivo using syngeneic and xenograft mouse models of murine pancreatic and human ovarian cancer, respectively. S2/IAPinh demonstrated markedly improved pharmacological activity in cancer cell lines and primary organoid cultures when compared to the controls. In vivo testing demonstrated a marked reduction in tumor growth rates and increased survival rates when compared to the respective control groups. The predicted mechanism of action of S2/IAPinh was confirmed through assessment of apoptosis pathways and demonstrated strong target degradation (cellular inhibitor of apoptosis proteins-1 [cIAP-1]) and activation of caspases 3 and 8. Taken together, S2/IAPinh demonstrated efficacy in models of pancreatic and ovarian cancer, two challenging malignancies in need of novel treatment concepts. Our data support an in-depth investigation into utilizing S2/IAPinh for the treatment of cancer.

Abstract A082: Targeting sterol regulatory element binding protein 1 (SREBP1) improves response to standard-of-care chemotherapy and alters the tumor microenvironment in pre-clinical models of obesity and ovarian cancer

Abstract Obesity is a recognized non-infectious pandemic that increases ovarian cancer (OvCa) incidence, enhances metastatic success and reduces survival. We have previously demonstrated a link between obesity and OvCa metastatic success in a diet-induced obesity (DIO) mouse model (Liu et al. Can.Res. 2015; 75:5046). DIO also elevated expression and nuclear localization of sterol regulatory element binding protein 1 (SREBP1), a master regulator of de novo lipogenesis and lipid homeostasis. A follow-up study using DIO models showed a significantly diminished response to standard-of-care (SOC) paclitaxel/carboplatin chemotherapy in high fat diet (HFD) mice relative to low fat diet (LFD) controls (Liu et al., J.Exp.Clin.Can.Res. 2023; 42:165). Both studies showed an effect of DIO on tumor fibrosis and immune landscape and these changes were also observed in the ovarian cancer tumors of women with high body mass index. In the current study, we have evaluated the effect of inhibitors of SREBP1 processing, in combination with SOC chemotherapy, in the context of DIO in pre-clinical models of OvCa. Our results show significantly diminished post-treatment tumor recurrence in HFD mice receiving SOC chemotherapy plus an SREBP1 processing inhibitor, with concomitant alterations in the peritoneal immune landscape. Together these data suggest that the reported negative impact of obesity on OvCa patient survival may be due obesity-induced changes in lipid regulatory factors (SREBP1) and the tumor microenvironment (immune landscape, fibrosis) that alter chemosensitivity. These data demonstrate a contribution of host obesity to ovarian tumor progression and therapeutic response and support future combination strategies targeting SREBP1, immune cells and/or fibrosis in the obese host. Citation Format: M. Sharon Stack, Yueying Liu, Jing Yang, Tyvette S. Hilliard, Zhikun Wang, Jeff Johnson, Wanrui Wang, Gena Dominique, Elizabeth I. Harper, Nicholas M. Stavrou, Anna Juncker-Jensen. Targeting sterol regulatory element binding protein 1 (SREBP1) improves response to standard-of-care chemotherapy and alters the tumor microenvironment in pre-clinical models of obesity and ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A082.

Functional enhancement of mesothelin-targeted TRuC-T cells by a PD1-CD28 chimeric switch receptor

T cells expressing a mesothelin (MSLN)-specific T cell receptor fusion construct (TRuC®), called TC-210, have demonstrated robust antitumor activity in preclinical models of mesothelioma, ovarian cancer, and lung cancer. However, they are susceptible to suppression by the programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) axis and lack intrinsic costimulatory signaling elements. To enhance the function of anti-MSLN TRuC-T cells, chimeric switch receptors (CSRs) have been designed to co-opt the immunosuppressive PD-1/PD-L1 axis and to deliver a CD28-mediated costimulatory signal. Here, we report that coexpression of the PD1-CD28 CSR in TRuC-T cells enhanced T cell receptor signaling, increased proinflammatory effector cytokines, decreased anti-inflammatory cytokines, and sustained effector function in the presence of PD-L1 when compared with TC-210. Anti-MSLN TRuC-T cells engineered to coexpress PD1-CD28 CSRs comprising the ectodomain of PD-1 and the intracellular domain of CD28 linked by the transmembrane domain of PD-1 were selected for integration into an anti-MSLN TRuC-T cell therapy product called TC-510. In vitro, TC-510 showed significant improvements in persistence and resistance to exhaustion upon chronic stimulation by tumor cells expressing MSLN and PD-L1 when compared with TC-210. In vivo, TC-510 showed a superior ability to provide durable protection following tumor rechallenge, versus TC-210. These data demonstrate that integration of a PD1-CD28 CSR into TRuC-T cells improves effector function, resistance to exhaustion, and prolongs persistence. Based on these findings, TC-510 is currently being evaluated in patients with MSLN-expressing solid tumors.

Turning up the heat: Oncolytic virus MEM-288 induces anti-tumoral immune response in pre-clinical models of high grade serous ovarian cancer (190)

Turning up the heat: Oncolytic virus MEM-288 induces anti-tumoral immune response in pre-clinical models of high grade serous ovarian cancer (190)

Targeting CD47-SIRPa axis shows potent preclinical anti-tumor activity as monotherapy and synergizes with PARP inhibition

The objective was to correlate CD47 gene expression with resistance to immune checkpoint inhibitors (ICI) in tumor tissue of gynecological cancer (GC). Further, we sought to assess the efficacy of targeting CD47 pathway alone and in combination in pre-clinical ovarian cancer (OC) models. We performed transcriptomic analyses in GC treated with ICI. Signaling pathway enrichment analysis was performed using Ingenuity Pathway Analysis. Immune cell abundance was estimated. CD47 expression was correlated with other pathways, objective response, and progression-free survival (PFS). Anti-tumor efficacy of anti-CD47 therapy alone and in combination was investigated both in-vitro and in-vivo using cell-line derived xenograft (CDX) and patient-derived xenograft (PDX) models. High CD47 expression associated with lower response to ICI and trended toward lower PFS in GC patients. Higher CD47 associated negatively with PDL1 and CTLA4 expression, as well as cytotoxic T-cells and dendritic cells but positively with TGF-β, BRD4 and CXCR4/CXCL12 expression. Anti-CD47 significantly enhanced macrophage-mediated phagocytosis of OC cells in-vitro and exhibited potent anti-tumor activity in-vivo in OC CDX and PDX models. In-vitro treatment with PARPi increased CD47 expression. Anti-CD47 led to significantly enhanced in-vitro phagocytosis, enhanced STING pathway and synergized in-vivo when combined with PARP inhibitors in BRCA-deficient OC models. This study provides insight on the potential role of CD47 in mediating immunotherapy resistance and its association with higher TGF-β, BRD4 and CXCR4/CXCL12 expression. Anti-CD47 showed potent anti-tumor activity and synergized with PARPi in OC models. These data support clinical development of anti-CD47 therapy with PARPi in OC.

A phase 1 study of the SYK inhibitor fostamatinib and weekly paclitaxel for recurrent platinum-resistant ovarian cancer.

5574 Background: Response to chemotherapy in patients (pts) with recurrent platinum-resistant ovarian cancer (PROC) is generally < 15%. Spleen tyrosine kinase (SYK) is overexpressed in PROC and is a key mediator of paclitaxel resistance in ovarian cancer cells. Fostamatinib (Fos), an orally available inhibitor of SYK, is currently FDA approved for the treatment of idiopathic thrombocytopenic purpura. The active form of Fos (R406) was shown to synergistically enhance taxane-mediated cytotoxicity in preclinical ovarian cancer models. This phase 1 study aimed to identify the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of Fos when combined with standard weekly paclitaxel (wPac) for pts with PROC. Methods: Pts with PROC, defined as progression ≤6 months of last platinum, were eligible. A modified toxicity probability interval (mTPI) dose escalation design was used to determine the MTD/RP2D and response was assessed using RECISTv1.1 criteria. All pts received paclitaxel (wPac) 80mg/m2 intravenously on Days 1, 8, and 15, of a 28-day cycle. Fos was administered orally twice a day (BID) on a continuous schedule. Three dose levels (DL) were planned: DL1 - 100mg, DL2 - 150mg, and DL3 - 200mg. MTD was determined based on cycle 1 dose-limiting toxicities (DLTs) and responses were assessed every 2 cycles. A dose expansion cohort to further assess adverse events (AEs) and tolerability was performed. Results: 27 eligible pts, median age 62 years (range 35-79 years) with high-grade serous (n = 18), clear cell (n = 4), carcinosarcoma (n = 3), or other (n = 2) histology were treated at 3 centers. Twelve pts were treated in the dose escalation cohort (DL1 = 6, DL2 = 3, DL3 = 3). Common AEs at least possibly attributed to Fos, wPac, or both included diarrhea (70%), fatigue (52%), anemia (44%), neutropenia (33%), nausea (33%), hypertension (30%), and dysgeusia (30%). Treatment-emergent grade 3-4 AEs were neutropenia (37%), anemia (26%), and thromboembolic event (22%). During DL1, updated toxicity management guidelines not consistent with the original protocol were released and 2 of the 3 pts treated prior to the amendment were not considered evaluable for DLT. Thus 3 more pts were enrolled to DL1 after the amendment. Of the 10 pts evaluable for DLT in dose escalation, no DLTs were reported on any of the 3 DLs (DL1 = 4, DL2 = 3, DL3 = 3). DL3 was selected for dose expansion. One of 15 pts in the dose expansion cohort had a DLT (neutropenia) and one patient had a grade 5 infection. Of 18 pts treated at DL3, 7 (39%) had partial or complete response (95% CI: 17.3, 64.3%). Overall, 27 patients received a median of 3 cycles (range 0-10). Analysis of pharmacokinetic and correlative molecular studies of target expression are ongoing. Conclusions: The RP2D of Fos will be 200 mg orally BID when combined with wPac. AE profile of the combination was as expected and the combination demonstrated promising efficacy in pts with recurrent PROC. Clinical trial information: NCT03246074 .

Abstract 2853: TP-6379, an investigational TGFBR1 inhibitor, shows improvement in survival and enhances activity of standard of care in preclinical ovarian cancer models

Abstract Ovarian cancer is still associated with poor prognosis and remains amongst the leading causes of cancer related deaths in females. High recurrence rates, resistance to chemotherapy, and meager outcome highlight the need for improved therapies that stem from understanding the complex and multifactorial etiology of ovarian malignancies. Transforming growth factor-β (TGF-β) signaling can regulate both oncogenesis and metastasis in ovarian cancer (Kumri et al. 2021). This, in part, is done by affecting apoptosis and survival mechanisms, epithelial to mesenchymal transition, immune cell recruitment and response. Inhibition of the TGF-β signaling pathway is a potential pharmaceutical approach for treating ovarian malignancies.TP-6379 is a potent and orally available investigational TGF-β receptor 1 (TGFBR1) inhibitor and is currently in preclinical development at Sumitomo Pharma Oncology, Inc. (SMP Oncology). We hypothesized that TP-6379 may inhibit the TGF-β signaling pathway and show activity in preclinical ovarian cancer models. TP-6379 was observed to inhibit TGFBR1 potently in a biochemical kinase assay. Dose dependent increase in compound concentration was observed in ES-2 tumor burdened animals treated with TP-6379. Preclinical data showed TP-6379 (150 mg/kg, BID) treatment significantly improved overall survival compared to vehicle treatment in an ES-2 mouse xenograft model of clear cell carcinoma. We hypothesize this result was, in part, due to alleviation of ovarian cancer induced cachexia, as animals treated with TP-6379 showed retention of muscle mass in their hind limbs compared to the vehicle treated animals. To investigate a combination effect with the standard of care in ovarian cancer, a SK-OV-3 ovarian cancer adenocarcinoma cell line was treated in vitro with TP-6379 and paclitaxel. Class III β-tubulin (TUBB3), a potential resistance mechanism to paclitaxel, showed induced expression in response to TGF-β, paclitaxel or both, after 24 hr treatment, and this effect was observed to be inhibited by TP-6379 treatment. In a SK-OV-3 xenograft model, TP-6379 (150 mg/kg, BID) treatment showed the trend of tumor growth inhibition (TGI) as a monotherapy and showed enhanced TGI in combination with paclitaxel (7.5 mg/kg, QW) when compared to vehicle treatment. Combination treatment in this xenograft model showed significant reduction of TUBB3 mRNA compared to paclitaxel treatment alone. In conclusion, our preliminary preclinical studies have shown promising activity for TP-6379 in ovarian malignancies as monotherapy and/or in combination with standard of care. TP-6379 may be a viable therapeutic option by targeting the TGF-β pathway in ovarian cancer. Citation Format: Tetyana V. Forostyan, David A. Kircher, Richard E. Heinz, Curtis Allred, Sal Sommakia, Yuta Matsumura, Adam Siddiqui, Jason M. Foulks, Steven L. Warner. TP-6379, an investigational TGFBR1 inhibitor, shows improvement in survival and enhances activity of standard of care in preclinical ovarian cancer models [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 2853.

Abstract 1133: A B7-H4 targeting antibody-drug conjugate shows anti-tumor activity in PARPi and platinum resistant cancers with B7-H4 expression

Abstract Purpose: Platinum and PARP inhibitors (PARPi) demonstrate activity in breast and ovarian cancers, but drug resistance ultimately emerges. B7-H4 is over expressed in breast and ovarian cancer compared to normal tissues making it a promising target for therapies, however, to date it has not been evaluated if the expression is maintained in drug resistant cancer. Here we examine B7-H4 expression in high grade serous ovarian carcinoma (HGSOC) tumors at diagnosis and post-platinum or PARPi-resistance. We also evaluate the activity of a novel B7-H4-directed antibody-drug conjugate (ADC) bearing the pyrrolobenzodiazepine-dimer payload tesirine, in preclinical models of breast and ovarian cancer, including those resistant to standard of care therapies. Experimental Design: B7-H4 expression was measured by quantitative flow cytometry and immunohistochemistry. ADC efficacy was tested against multiple cell lines in vitro and patient-derived xenografts (PDX) in vivo. The effect of ADC treatment on cell cycle, DNA damage, and apoptosis was measured using flow cytometry. Results: B7-H4 was over-expressed in 92% of HGSOC tumors at diagnosis (n = 12), persisted in recurrent matched samples after platinum treatment, and was expressed at similar levels at multiple metastatic sites after acquired multi-drug resistance (n = 4 donors). Treatment with the ADC resulted in target-specific growth inhibition in a panel of ten B7-H4 expressing ovarian and breast cancer cell lines (IC50 6.18-273.9 pM, median 48.3 pM). Co-cultures of B7-H4 +/- lines support bystander killing effects leading to further tumor cell death in B7-H4- clones. In platinum- or PARPi-resistant ovarian cancer cells, ADC treatment significantly decreased viability and colony formation (P < 0.001) while increasing S or G2/M phase cell cycle arrest (P < 0.001) and DNA damage (P < 0.0001), ultimately leading to apoptosis (P < 0.01). A single 0.3 mg/kg dose of the ADC resulted in tumor regression in 61% of breast and ovarian PDX models tested (n = 23), where lower activity was identified in B7-H4 low or negative expressing models (P = 0.048). In PARPi- and platinum-resistant HGSOC models (n = 3), continuous B7-H4 ADC treatment (dosed once every 28 days) resulted in sustained anti-tumor activity, leading to complete (7 of 16) or partial responses (3 of 16) and increased survival (P < 0.004). Conclusions: These data support B7-H4 as an attractive ADC target for treatment of drug-resistant HGSOC. Citation Format: Sarah B. Gitto, Margaret Whicker, Gareth Davies, Sushil Kumar, Krista Kinneer, Arthur Lewis, Srinivas Mamidi, Sergey Medvedev, Judith Anderton, Jessica Tang, Benjamin Ferman, Steve Coats, Robert W. Wilkinson, Eric J. Brown, Daniel J. Powell, Fiona Simpkins. A B7-H4 targeting antibody-drug conjugate shows anti-tumor activity in PARPi and platinum resistant cancers with B7-H4 expression [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 1133.

Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models

BackgroundLimited persistence of functional CAR T cells in the immunosuppressive solid tumor microenvironment remains a major hurdle in the successful translation of CAR T cell therapy to treat solid tumors....

Using genetically encoded fluorescent biosensors to interrogate ovarian cancer metabolism

BackgroundEpithelial ovarian cancer (OC) is the most lethal gynecological malignancy and patients present with significant metastatic burden, particularly to the adipose-rich microenvironment of the omentum. Recent evidence has highlighted the importance of metabolic adaptations in enabling this metastasis, leading to significant interest in evolving the arsenal of tools used to study OC metabolism. In this study, we demonstrate the capability of genetically encoded fluorescent biosensors to study OC, with a focus on 3D organoid models that better recapitulate in vivo tumor microenvironments.Materials and methodsPlasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval were transfected into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 μM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n = 8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 μM carboplatin.ResultsExposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients.ConclusionsOur study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC.

Dominant-negative transforming growth factor-β receptor-armoured mesothelin-targeted chimeric antigen receptor T cells slow tumour growth in a mouse model of ovarian cancer

Ovarian cancer is a major cause of death among all gynaecological cancers. Although surgery, chemotherapy and targeted therapy have yielded successful outcomes, the 5-year survival rate remains < 30%. Adoptive immunotherapy, particularly chimeric antigen receptor (CAR) T-cell therapy, has demonstrated improved survival in acute lymphoblastic leukaemia with manageable toxicity. We explored CAR T-cell therapy in a preclinical mouse model of ovarian cancer. Second-generation CAR T cells were developed targeting mesothelin (MSLN), which is abundantly expressed in ovarian cancer. Cytotoxicity experiments were performed to verify the lethality of CAR T cells on target cells via flow cytometry. The in vivo antitumour activity of MSLN CAR T cells was also verified using a patient-derived xenograft (PDX) mouse model with human tumour-derived cells. We also evaluated the potency of CAR T cells directed to MSLN following co-expression of a dominant-negative transforming growth factor-β receptor type II (dnTGFβRII). Our data demonstrate that anti-MSLN CAR T cells specifically eliminate MSLN-expressing target cells in an MSLN density-dependent manner. This preclinical research promises an effective treatment strategy to improve outcomes for ovarian cancer, with the potential for prolonging survival while minimizing risk of on-target off-tumour toxicity.

Adaptations of key metabolic pathways in primary and metastatic epithelial ovarian cancer (253)

Objectives: Patients with ovarian cancer (OC) often present with advanced-stage disease at diagnosis; however, the molecular mechanisms which drive the aggressive nature of OC metastases have not been well characterized. Metastasizing cancer cells undergo metabolic reprogramming to utilize available lipids in the metastatic niche of the omentum. This study aimed to understand the compensatory metabolic mechanisms that enable this OC metastasis. Our work focused on understanding the contributions of the pentose phosphate pathway (PPP)—a key mechanism responsible for redox homeostasis—in enabling this evolution. Methods: Using gene expression data of matched human primary and omental tumor samples (n=30), we performed targeted analysis of metabolic gene expression changes, which revealed upregulation of the PPP in omental metastases. We then performed in vitro and in vivo experiments using pre-clinical ovarian cancer models (SKOV3, IGROV, HEYA8). Given the canonical role of the PPP in maintaining redox homeostasis, we assessed oxidative stress in organoid cultures from omental metastases harvested at mouse necropsy and in cultures grown in omental conditioned media. Inhibition of the rate-limiting enzyme of the PPP, glucose-6-phosphate dehydrogenase (G6PD), was performed via shRNA knockdowns and treated with a pharmacological inhibitor. HeyA8 tumor-bearing mice were treated with the G6PD inhibitor, and the metastatic burden was evaluated using IVIS during treatment and in peritoneal organs following the sacrifice. Statistical significance was established using unpaired two-tailed Student’s t-tests. Results: Differential analysis of metabolic gene expression and pathway analysis revealed upregulation of the PPP in omental metastases compared to primary ovarian tumors (p<0.05, EnrichR Score = 800). Primary tumors and omental metastases were harvested from SKOV3, HeyA8, and IGROV1 tumor-bearing mice at necropsy and used to establish organoids. DCFH-DA, a fluorogenic probe for ROS, measured higher levels of oxidative stress in metastatic organoids than primary tumors (normalized F525= 11.3623, p<0.0005). SKOV3 organoids expressing HyPer, a ratiometric biosensor of intracellular H2O2, exhibited higher oxidative stress when grown in omental conditioned media versus basal organoid media (normalized F420/F500=1.27, p<0.05). Pharmacological and shRNA inhibition of G6PD sensitized cells to oxidative stress, reducing cell viability in vitro (p<0.05). Treatment with a G6PD inhibitor in vivo significantly decreased tumor growth and metastases compared to vehicle treatment (p<0.005). Objectives: Patients with ovarian cancer (OC) often present with advanced-stage disease at diagnosis; however, the molecular mechanisms which drive the aggressive nature of OC metastases have not been well characterized. Metastasizing cancer cells undergo metabolic reprogramming to utilize available lipids in the metastatic niche of the omentum. This study aimed to understand the compensatory metabolic mechanisms that enable this OC metastasis. Our work focused on understanding the contributions of the pentose phosphate pathway (PPP)—a key mechanism responsible for redox homeostasis—in enabling this evolution. Methods: Using gene expression data of matched human primary and omental tumor samples (n=30), we performed targeted analysis of metabolic gene expression changes, which revealed upregulation of the PPP in omental metastases. We then performed in vitro and in vivo experiments using pre-clinical ovarian cancer models (SKOV3, IGROV, HEYA8). Given the canonical role of the PPP in maintaining redox homeostasis, we assessed oxidative stress in organoid cultures from omental metastases harvested at mouse necropsy and in cultures grown in omental conditioned media. Inhibition of the rate-limiting enzyme of the PPP, glucose-6-phosphate dehydrogenase (G6PD), was performed via shRNA knockdowns and treated with a pharmacological inhibitor. HeyA8 tumor-bearing mice were treated with the G6PD inhibitor, and the metastatic burden was evaluated using IVIS during treatment and in peritoneal organs following the sacrifice. Statistical significance was established using unpaired two-tailed Student’s t-tests. Results: Differential analysis of metabolic gene expression and pathway analysis revealed upregulation of the PPP in omental metastases compared to primary ovarian tumors (p<0.05, EnrichR Score = 800). Primary tumors and omental metastases were harvested from SKOV3, HeyA8, and IGROV1 tumor-bearing mice at necropsy and used to establish organoids. DCFH-DA, a fluorogenic probe for ROS, measured higher levels of oxidative stress in metastatic organoids than primary tumors (normalized F525= 11.3623, p<0.0005). SKOV3 organoids expressing HyPer, a ratiometric biosensor of intracellular H2O2, exhibited higher oxidative stress when grown in omental conditioned media versus basal organoid media (normalized F420/F500=1.27, p<0.05). Pharmacological and shRNA inhibition of G6PD sensitized cells to oxidative stress, reducing cell viability in vitro (p<0.05). Treatment with a G6PD inhibitor in vivo significantly decreased tumor growth and metastases compared to vehicle treatment (p<0.005).

Clinical significance of cadherin-6 expression in primary and recurrent epithelial ovarian cancer and its association with outcomes: A potential therapeutic target for epithelial ovarian cancer (206)

<b>Objectives:</b> Cadherin-6 (CDH6) is a membrane glycoprotein, and aberrant expression of CDH6 has been reported in several human carcinomas, suggesting a possible role in metastasis and invasion. Recently, antibody-drug conjugates (ADCs) targeting CDH6 have drawn attention to new cancer therapies and demonstrated their antitumor activity in preclinical models of epithelial ovarian cancer (EOC). However, limited data are available regarding the frequency of CDH6 expression and its relationship to clinical factors in EOC. Our goal was to understand the clinical significance of CDH6 expression in EOC. <b>Methods:</b> We performed immunohistochemical (IHC) staining on CDH6 expression in a total of 232 EOC surgical samples (including 181 primary tumors and 51 recurrent tumors). CDH6 IHC was performed using the Leica BOND III staining platform with an anti-CDH6 antibody (HPA007456, ATLAS ANTIBODIES). The CDH6 IHC scoring standard refers to the HER2 detection guidelines for gastric cancer (0, 1+, 2+, 3+). We define a score greater than or equal to 1 as positive for the expression of CDH6. <b>Results:</b> We investigated 181 patients with primary EOC for CDH6 expression through IHC. Total 117 (64.6%) patients tested positive. In which, 18 (15.4%), 84 (71.8%) and 15 (12.8%) cases were scored as 1+, 2+ and 3+, respectively. We then investigated the relationship between CDH6 expression and clinical-pathologic features in EOC. CDH6 expression was observed more frequently in high-grade serous carcinomas (89.0%, p < 0.0001), stage III/IV tumors (84.8%, p < 0.0001) and gross residual tumors in primary surgery (84.7%, p < 0.0001). CDH6-positive patients showed shorter progression-free survival (PFS) and overall survival (OS) (p < 0.0001 and p=0.0006) than that of CDH6-negative patients in all EOC patients. In addition, patients with CDH6 3+ had shorter PFS and OS than that of patients who had 0, 1+ and 2+ (p =0.028 and p=0.015) in the high-grade serous subgroup. We also investigated the expression of CDH6 in 51 recurrent EOC; 38/51 (74.5%) recurrent tumors tested positive for CDH6 expression, and nine (23.7%), 17 (44.7%) and 12 (31.6%) received scores of 1+, 2+ and 3+, respectively. CDH6 expression from recurrent tumors was observed in 18/24 (75.0%) high-grade serous, 11/16 (68.8%) clear cell, 4/5 (80.0%) endometrioid and 5/6 (83.3%) other histotypes. <b>Conclusions:</b> CDH6 expression is frequently observed in primary and recurrent EOC of all histotypes, especially in high-grade serous carcinoma. The frequency of CDH6 expression is consistent between primary and recurrent tumors. CDH6 is a promising therapeutic target for the treatment of EOC.