Ovarian Cancer Xenograft Model Research Articles

Antitumor Activity of Dehydroxymethylepoxyquinomicin (DHMEQ) in Monotherapy and Combination with Cisplatin in the SKOV-3 Ovarian Cancer Model

The objective of this study was to evaluate the antitumor activity of DHMEQ as monotherapy and in combination with cisplatin in a human ovarian cancer xenograft model. Cisplatin was used as a comparator. To create the xenograft model, human ovarian cancer cells (SKOV-3 line) were subcutaneously implanted into immunodeficient mice. The study was conducted on female SCID Beige C.B-17 Cg-Prkdcscid Lystbg/Crl mice. Antitumor activity was determined by comparing tumor growth inhibition (TGI) in the treatment groups to that in the control group. Results showed that daily intraperitoneal administration of DHMEQ at a dose of 14 mg/kg following a single intraperitoneal dose of cisplatin at 4 mg/kg reduced tumor growth in the SKOV-3 cell line xenograft model.

Dual Inhibition of SYK and EGFR Overcomes Chemoresistance by Inhibiting CDC6 and Blocking DNA Replication.

Targeting multiple signaling pathways has been proposed as a strategy to overcome resistance to single-pathway inhibition in cancer therapy. A previous study in epithelial ovarian cancers identified hyperactivity of spleen tyrosine kinase (SYK) and epidermal growth factor receptor (EGFR), which mutually phosphorylate and activate each other. Given the potential for pharmacologic inhibition of both kinases with clinically available agents, this study aimed to assess the antitumor efficacy of both pharmacologic and genetic SYK and EGFR co-inhibition using a multifaceted approach to analyze the global phosphoproteome and chemoresistant ovarian cancer cell lines, patient-derived organoids, and xenograft models. Dual inhibition of SYK and EGFR in chemoresistant ovarian cancer cells elicited a highly synergistic antitumor effect. Notably, the combined inhibition strategy activated the DNA damage response, induced G1 cell cycle arrest, and promoted apoptosis. The phosphoproteomic analysis revealed that perturbation of SYK and EGFR signaling induced a significant reduction in both phosphorylated and total protein levels of cell division cycle 6 (CDC6), a crucial initiator of DNA replication. Together, this study offers preclinical evidence supporting dual inhibition of SYK and EGFR as a promising treatment for chemoresistant ovarian cancer that disrupts DNA synthesis by impairing formation of the prereplication complex. These findings warrant further clinical investigation to explore the potential of this combination therapy in overcoming drug resistance and improving patient outcomes.

The anthraquinone derivative KA-4s reduces energy metabolism and enhances the sensitivity of ovarian cancer cells to cisplatin.

Ovarian cancer is the leading cause of death from female gynecological cancers. Cisplatin (DDP)is a first-line drug for ovarian cancer treatment. Due to DDP resistance, there is an urgent need for novel therapeutic drugs with improved antitumor activity. AMPK-mediated metabolic regulatory pathways are related to tumor drug resistance. Our study aimed to determine the relationship between reversing DDP resistance with the anthraquinone derivative KA-4s and regulating AMPK energy metabolism in ovarian cancer. The results showed that KA-4s inhibited the proliferation of ovarian cancer cells. The combination of KA-4s with DDP effectively promoted drug-resistant ovarian cancer cell apoptosis and inhibited cell migration and invasion. Moreover, KA-4s decreased the intracellular ATP level and increased the calcium ion level, leading to AMPK phosphorylation. Further studies suggested that the AMPK signaling pathway may be involved in the mechanism through which KA-4s reduce drug resistance. KA-4s inhibited mitochondrial respiration and glycolysis; downregulated the glucose metabolism-related proteins GLUT1 and GLUT4; the lipid metabolism-related proteins SREBP1 and SCD1; and the drug resistance-related proteins P-gp, MRP1, and LRP. The inhibitory effect of KA-4s on GLUT1 was confirmed by the application of the GLUT1 inhibitor BAY-876. KA-4s combined with DDP significantly increased the expression of p-AMPKand reduced the expression of P-gp. In a xenograft model of ovarian cancer, treatment with KA-4s combined with DDP reduced energy metabolism and drug resistance, inducing tumor apoptosis. Consequently, KA-4s might be evaluated as a new agent for enhancing the chemotherapeutic efficacy of treatment for ovarian cancer.

HER2-CD3-Fc Bispecific Antibody-Encoding mRNA Delivered by Lipid Nanoparticles Suppresses HER2-Positive Tumor Growth.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor and tumor-associated antigen abnormally expressed in various types of cancer, including breast, ovarian, and gastric cancer. HER2 overexpression is highly correlated with increased tumor aggressiveness, poorer prognosis, and shorter overall survival. Consequently, multiple HER2-targeted therapies have been developed and approved; however, only a subset of patients benefit from these treatments, and relapses are common. More potent and durable HER2-targeted therapies are desperately needed for patients with HER2-positive cancers. In this study, we developed a lipid nanoparticle (LNP)-based therapy formulated with mRNA encoding a novel HER2-CD3-Fc bispecific antibody (bsAb) for HER2-positive cancers. The LNPs efficiently transfected various types of cells, such as HEK293S, SKOV-3, and A1847, leading to robust and sustained secretion of the HER2-CD3-Fc bsAb with high binding affinity to both HER2 and CD3. The bsAb induced potent T-cell-directed cytotoxicity, along with secretion of IFN-λ, TNF-α, and granzyme B, against various types of HER2-positive tumor cells in vitro, including A549, NCI-H460, SKOV-3, A1847, SKBR3, and MDA-MB-231. The bsAb-mediated antitumor effect is highly specific and strictly dependent on its binding to HER2, as evidenced by the gained resistance of A549 and A1847 her2 knockout cells and the acquired sensitivity of mouse 4T1 cells overexpressing the human HER2 extracellular domain (ECD) or epitope-containing subdomain IV to the bsAb-induced T cell cytotoxicity. The bsAb also relies on its binding to CD3 for T-cell recruitment, as ablation of CD3 binding abolished the bsAb's ability to elicit antitumor activity. Importantly, intratumoral injection of the HER2-CD3-Fc mRNA-LNPs triggers a strong antitumor response and completely blocks HER2-positive tumor growth in a mouse xenograft model of human ovarian cancer. These results indicate that the novel HER2-CD3-Fc mRNA-LNP-based therapy has the potential to effectively treat HER2-positive cancer.

Overcoming effector T cell exhaustion in ovarian cancer ascites with a novel adenovirus encoding for a MUC1 bispecific antibody engager and IL-2 cytokine

T cell-focused cancer immunotherapy including checkpoint inhibitors and cell therapies has been rapidly evolving over the past decade. Nevertheless, there remains a major unmet medical need in oncology generally and immuno-oncology specifically. We have constructed an oncolytic adenovirus, Ad5/3-E2F-d24-aMUC1aCD3-IL-2 (TILT-322), which is armed with a human aMUC1aCD3 T cell engager and IL-2. TILT-322 treatment stimulated T cell cytotoxicity through the increased presence of granzyme-B, perforin, and interferon-gamma. Additional immune profiling indicated TILT-322 increased gamma delta T cell activation and impacted other cell types such as natural killer cells, and natural killer-like T cells that are traditionally involved in cancer immunotherapy. TILT-322 treatment also decreased the proportion of exhausted CD8+ T cells as demarked by immune checkpoint expression in ovarian ascites samples. Overall, our data showed that TILT-322 treatment led to an enhanced T cell activation and reversed T cell exhaustion translating into high antitumor efficacy when given locally or intravenously. The analysis of blood and tumors isolated from an in vivo patient-derived ovarian cancer xenograft model suggested TILT-322 mediated tumor control through improved T cell functions. Therefore, TILT-322 is a promising novel anti-tumor agent for clinical translation.

HER2-targeted, enzyme-activated liposomes show superior in vivo efficacy in an ovarian cancer model

Liposomes carrying chemotherapeutic drugs can accumulate passively in solid tumors at high levels. However, additional targeting of the liposomes towards e.g. receptors expressed on cancer cells may improve their interaction and therapeutic properties. In this study, we designed a liposomal delivery system, which utilizes the intrinsic characteristics of HER2-positive tumors to ensure efficient delivery of oxaliplatin to the cancer cells. On the liposome surface, trastuzumab, an antibody specific to the HER2 receptor, was shown to facilitate internalization by the cancer cells. A polyethylene glycol (PEG) layer on the liposome surface provides protection from mononuclear phagocyte system uptake. To optimize the interaction between liposomes and cancer cells, a protease-sensitive cleavable peptide linker was inserted at the base of each PEG. The PEG layer is then cleaved off by intra- and extracellular matrix metalloproteinases (MMPs) upon accumulation in the tumor. Our data demonstrate that the removal of PEG significantly destabilizes the liposomes and leads to substantial oxaliplatin release. The proposed beneficial effect of combining antibody-mediated internalization with MMP sensitivity was confirmed in a series of in vivo studies using ovarian cancer xenograft models. The results demonstrated that HER2-targeted MMP-sensitive liposomes have superior anticancer activity compared to non-targeted and non-cleavable liposomes.

Crizotinib Enhances PARP Inhibitor Efficacy in Ovarian Cancer Cells and Xenograft Models by Inducing Autophagy.

Poly (ADP-ribose) polymerase inhibitors (PARPi) can encounter resistance through various mechanisms, limiting their effectiveness. Our recent research showed that PARPi alone can induce drug resistance by promoting autophagy. Moreover, our studies have revealed that anaplastic lymphoma kinase (ALK) plays a role in regulating the survival of ovarian cancer cells undergoing autophagy. Here, we explored whether the ALK-inhibitor crizotinib could enhance the efficacy of PARPi by targeting drug-induced autophagic ovarian cancer cell and xenograft models. Our investigation demonstrates that crizotinib enhances the anti-tumor activity of PARPi across multiple ovarian cancer cells. Combination therapy with crizotinib and olaparib reduced cell viability and clonogenic growth in two-olaparib resistant cell lines. More importantly, this effect was consistently observed in patient-derived organoids. Furthermore, combined treatment with crizotinib and olaparib led to tumor regression in human ovarian xenograft models. Mechanistically, the combination resulted in increased levels of reactive oxygen species (ROS), induced DNA damage, and decreased the phosphorylation of AKT, mTOR, and ULK-1, contributing to increased olaparib-induced autophagy and apoptosis. Notably, pharmacologic, or genetic inhibition or autophagy reduced the sensitivity of ovarian cancer cell lines to olaparib and crizotinib treatment, underscoring the role of autophagy in cell death. Blocking ROS mitigated olaparib/crizotinib-induced autophagy and cell death while restoring levels of phosphorylated AKT, mTOR and ULK-1. These findings suggest that crizotinib can improve the therapeutic efficacy of olaparib by enhancing autophagy. Implications: The combination of crizotinib and PARPi presents a promising strategy, that could provide a novel approach to enhance outcomes for patients with ovarian cancer.

Enhanced anti-tumor efficacy with multi-transgene armed mesenchymal stem cells for treating peritoneal carcinomatosis

BackgroundMesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime.MethodsIn this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking.ResultsFirstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively.ConclusionsTaken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC.

Overexpression of TRIM44 mediates the NF-κB pathway to promote the progression of ovarian cancer.

Ovarian cancer (OC) is the second most commonly seen cancer in the US, and patients with OC are commonly diagnosed in the advanced stage. Research into the molecular mechanisms and potential therapeutic targets of OC is becoming increasingly urgent. In our study, we worked to discover the role of TRIM44 in OC development. This study explored whether the overexpression of TRIM44 mediates the NF-kB pathway to promote the progression of OC. A TRIM44 overexpression model was constructed in SKOV3 cells, and the proliferation ability of the cells was detected using the CCK-8 assay. The migration healing ability of cells was detected using cell scratch assay. Cell migration and invasion were detected using Transwell nesting. TUNEL was applied to detect apoptosis, and ELISA and western blot were used to detect the expression of NF-κB signaling pathway proteins. The pathological changes of the tumor tissues were observed using HE staining in a mouse ovarian cancer xenograft model. Immunofluorescence double staining, RT-PCR, and western blot were used to determine the expression of relevant factors in tumour tissues. TRIM44 overexpression promoted the proliferation, migration, and invasion of SKOV3 cells in vitro and inhibited apoptosis while enhancing the growth of tumours in vivo. TRIM44 regulated the NF-κB signaling pathway. TRIM44 overexpression can regulate the NF-κB signaling pathway to promote the progression of OC, and TRIM44 may be a potential therapeutic target for OC.

Abstract LB048: Unleashing the power of p53 mRNA therapeutics to suppress proliferation of p53 mutant tumors

Abstract The p53 protein, recognized as the guardian of the genome, plays a pivotal role in suppressing tumor formation. The TP53 gene, which encodes the p53 protein, is commonly subject to mutations and deletions in many cancer types resulting in loss of tumor suppressor activity. A significant proportion of patients diagnosed with lung, pancreatic, ovarian, and head and neck cancers exhibit the expression of mutant p53 in their tumors, correlating with unfavorable prognosis and survival outcomes.This study endeavors to assess the efficacy of therapeutic tumor suppressive mRNA which induces expression of wild-type p53 protein across diverse cancer types. Optimization of the 5’ and 3’ untranslated regions (UTRs) and the 5’ cap has been undertaken to enhance the efficient expression of p53 mRNA. The growth inhibitory potential of p53 mRNA therapy has been evaluated across p53 mutant cancer cell lines representing various cancer origins, along with normal or p53 wild-type cancer cell lines. We have substantiated that the p53 mRNA therapeutics can selectively inhibit the proliferation of p53 mutant cancer cells. Furthermore, we have confirmed that p53 mRNA treatment upregulated modulator of apoptosis (PUMA) and B-cell translocation gene 2 (BTG2) and cell cycle arrest via p21 upregulation. In vivo efficacy assessments of the lipid nanoparticle (LNP)-encapsulated p53 mRNA have been conducted in lung, pancreatic, and ovarian cancer xenograft models. Both regional intratumoral (i.t) and systemic intravenous (i.v) administrations of the p53 mRNA therapeutics have shown substantial suppression of tumor growth, leading to prolonged mouse survival. These results underscore the potential of the p53 mRNA therapeutics as a promising avenue for targeted cancer treatment, offering insights into the selective inhibition of p53 mutant cancer cells. Citation Format: Yong Ho Heo, Seung-Hyun Shin, Youngjin Han, Chang Gyu Lim, Innah Kim, Ji Hee Lee, Gunwoo Lee, Seongju Jeong, Yunjae Kim, Jooyun Byun, Daejin Kim, In Young Choi. Unleashing the power of p53 mRNA therapeutics to suppress proliferation of p53 mutant tumors [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 LB048.

Abstract LB045: Exploiting biparatopic binding to deliver novel, differentiated ALPP/ALPPL2 targeting protein drug conjugates

Abstract The placental alkaline phosphatases ALPP and ALPPL2 are attractive targets for antibody and protein-drug conjugate (ADC and PDC) approaches. These GPI-anchored cell-surface proteins, which share very high sequence identity, are over-expressed in a number of different solid tumor indications including testicular, ovarian, endometrial, gastric, pancreatic and non-small cell lung cancers but have very little expression on normal adult tissues. Consequently, there is significant interest in exploiting ALPP/ALPPL2 for the development of new targeted oncology therapeutics, and an ALPP/ALPPL2 ADC, utilizing a classical full-length IgG antibody, is currently in Phase I clinical trials. The exploitation of biparatopic binding is an attractive approach for improving the therapeutic index of ADCs. This mode of target engagement increases the effective number of binding sites on the tumor cells and can promote internalisation and trafficking, thereby delivering increased efficacy for the same payload bolus and opening up patient populations with lower target expression. This can lead to a widening of the therapeutic window, by improving the efficacy achievable for a given amount of payload administered (often defined as the “platform” toxicity). For the development of biparatopic conjugates, the use of small protein domain binders offers advantages over the use of full-length antibodies, as they can readily be reformatted into a variety of biparatopic architectures to deliver homogeneous drug conjugates in high yield. Additionally, smaller protein formats have the capacity to penetrate deeper into solid tumors than large antibodies. We have identified a series of highly selective, high affinity ALPP/ALPPL2 single domain VHH binders, which target distinct epitopes on the protein and which, importantly, show no binding to the closely related ALPI or ALPL isoforms (which have high normal tissue expression). Using protein engineering approaches, a series of homogenous site-specifically labelled biparatopic PDCs have been developed employing the clinically validated vcMMAE (vedotin) payload. In pre-clinical studies, these PDC agents demonstrate excellent anti-tumor efficacy in vivo in ALPP/ALPPL2 positive cell-line derived xenograft models of gastric, pancreatic and ovarian cancer displaying a range of target expression. In benchmarking studies, lead biparatopic PDCs show increased internalisation and target-mediated in vitro cell kill potency compared to an ALPP/ALPPL2 targeting ADC. Additionally, in multiple CDX models, lead biparatopic PDCs demonstrate improved efficacy over an ALPP/ALPPL2 ADC both on a mg/kg and on a payload equivalents basis, in both single dose and multi-dose studies, providing complete regressions. Based on this excellent efficacy profile and favourable developability characteristics, preclinical development activities have been initiated. Citation Format: Graham Cotton, Estelle G. McLean, Paul Trumper, Mark Wappett, Stephanie Gatdula, Stacey Bell, Greg Papadakos, Kieron Lucas, Stephanie Burton, Chiara Saladino, Georgiana Parau, Aidan McCann, Jennifer Thom, Aaron N. Cranston, Tim Harrison. Exploiting biparatopic binding to deliver novel, differentiated ALPP/ALPPL2 targeting protein drug conjugates [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 LB045.

Abstract 2825: Experimental hyperthermic intraperitoneal chemotherapy in mice: Establishment of a novel ovarian cancer xenograft model

Abstract Introduction: The peritoneum is a common metastatic site for several abdominal cancers, including ovarian and colorectal cancer. Peritoneal metastasis is associated with poor prognosis and poor response to therapy. A potentially curative treatment is cytoreductive surgery (CRS) to remove all visible tumor tissue, followed by perfusion with hyperthermic intraperitoneal chemotherapy (HIPEC) to eliminate small residual tumors and free-floating tumor cells. Hyperthermia can enhance the cytotoxic effect of the drug, inhibit DNA repair and activate the immune system. At the current time, there is no standardized protocol for performing HIPEC, and there is a large variation regarding key parameters, such as choice of drug, drug concentration, treatment duration, carrier solution, volume and temperature. The impact of these parameters is unknown, and in vivo models can be a helpful tool to better understand some of these parameters to improve this treatment strategy. Methods: HIPEC was established in a murine xenograft model of ovarian cancer to evaluate the response to intraperitoneal perfusion of cisplatin and mitomycin C for 30 min at 41.5°C (HIPEC) and 37°C (NIPEC) compared to no treatment or intraperitoneal perfusion with saline at 41.5°C. The luciferase transfected B76 ovarian cancer cell line was injected intraperitoneally and treatment was performed on day 4 when the tumors were < 2 mm. Treatment efficacy was assessed by weekly luminescence measurement, tumor weight at an early time point (day 17), and overall survival. Results: Intraperitoneal perfusion with cisplatin or mitomycin C significantly inhibited tumor growth as assessed by luminescence, tumor weight on day 17, as well as increased overall survival of the mice compared to control treatment from 25-27 days to 36 days for cisplatin and 37 days for mitomycin C. No significant differences in tumor growth or survival were observed by the addition of hyperthermia. However, a slight reduction of tumor weight at day 17 was observed in the HIPEC groups. Conclusion: We have established a functional closed HIPEC model in mice bearing peritoneal metastases from the B76 ovarian cancer cell line which accurately mimics many features of the procedure in patients. Intraperitoneal perfusion of both cisplatin and mitomycin C efficaciously inhibited tumor growth and improved overall survival of the mice. Addition of hyperthermia caused an additional modest, temporary growth inhibition which did not translate into improved long-term outcome. Citation Format: Karianne Giller Fleten, Yvonne Andersson, Emil Løvstakken, Theodor M. Herud, Stein Waagene, Kjersti Flatmark. Experimental hyperthermic intraperitoneal chemotherapy in mice: Establishment of a novel ovarian cancer xenograft model [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 2825.

Abstract 3138: Discovery of AT65474, a highly selective anti-CLDN6 ADC with a proprietary payload

Abstract CLDN6 is a membrane-bound oncofetal protein and a promising target for cancers such as ovarian and testicular cancer. CLDN6 shares high sequence homology with other CLDN family members which are expressed in normal tissues, making a high degree of selectivity required for CLDN6-targeting therapies. In particular, CLDN6 is closest to CLDN9 in sequence with a difference of only three residues in the extracellular loops, making it challenging to generate a highly selective antibody. We report the development of a humanized anti-CLDN6 antibody with high affinity and high selectivity for targeted cells which displays > 800-fold higher binding affinity for CLDN6 over CLDN9. The antibody was refined for use as an ADC and incorporates AxcynCYSTM technology for site-specific conjugation which can reproducibly achieve high DAR4 ratios of greater than 97% by HIC analysis. AT65474 is an ADC candidate that incorporates a proprietary payload derived from an FDA approved drug that was optimized in-house for increased potency and improved PK properties to limit systemic exposure. In vitro, the payload displays broad sub-nanomolar activity across a wide range of 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. We provide evidence characterizing AT65474 as a highly potent ADC with high DAR4 homogeneity and strong in vivo activity at low doses. Compared with a benchmark ADC in clinical development, AT65474 shows stronger binding, higher internalization, superior in vitro cytotoxicity, and stronger bystander killing effect. When evaluated in PA-1 and OV90 xenograft models of ovarian cancer, AT65474 displays significantly higher efficacy over the benchmark ADC. Furthermore, we show that AT65474 displays an ADCC effect that contributes to tumor suppression in the OV90 model. AT65474 was shown to be well tolerated in all xenograft mouse models and a pre-tox study in non-human primates. The combined data support further development of AT65474.​ Citation Format: Chen Zhong, You Xue Wu, Xi Yun Zhang, Yin Wen Cheng, Qian Wang, Ping Du, Shao Jun Liu, Bin Zou. Discovery of AT65474, a highly selective anti-CLDN6 ADC with 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 3138.

Abstract 7451: Translational models of ovarian cancer-associated cognitive impairment and chemotherapy-induced peripheral neuropathy

Abstract Cisplatin is widely used to treat ovarian malignancies, and over 70% of ovarian cancer patients report experiencing cancer-related cognitive impairment (CRCI) and chemotherapy-induced peripheral neuropathy (CIPN) during and after chemotherapy treatment. Most pre-clinical CRCI/CIPN studies have been conducted on non-tumor bearing rodents. We developed two novel clinically relevant models of CRCI/CIPN: (1) the ID8 syngeneic mouse model in B6(Cg)-Tyrc-2J/J female mice, and (2) the SKOV3.ip1 ovarian cancer xenograft model in female Cr:NIH-RNU rats. We examined cognitive function and serum cytokine levels with respect to cancer progression and/or in response to cisplatin. B6 female mice were injected with 107 ID8 cells or 0.9% saline, i.p. Mice received cisplatin (2.3 mg/kg/day, i.p.) or 0.9% saline (OvT+CIS, OvT+VEH, respectively) for 5d, followed by 5d of rest for two cycles. RNU rats were injected with 107 SKOV3.ip1 cells or 0.9% saline, i.p. Rats received cisplatin (5 mg/kg, i.p) or 0.9% saline (OvT+CIS, OvT+VEH, respectively) every other week for four cycles. OvT+CIS and OvT+VEH mice and rats showed impairments in novel object recognition (NOR), with discrimination ratios ≤0.5. Cisplatin transiently increased hyperalgesia in OvT+CIS mice, which persisted longitudinally in OvT+VEH mice. Serum cytokine levels were elevated in OvT+VEH mice (IL-2, IL-10, INFγ) and OvT+VEH rats (IL-4, IL-10) compared to OvT+CIS (P<0.05). Ovarian cancer may evoke progressive sensory and neurocognitive deficits and increase systemic pro-inflammatory cytokine levels in the absence of chemotherapy. Future studies will address hyperalgesia and cognitive differences between healthy control and ovarian cancer models +/- cisplatin, and biological mechanisms underlying CRCI and CIPN. Citation Format: Naomi Lomeli, Donovan Argueta, Diana C. Pearre, Javier Lepe, Kalpna Gupta, Daniela A. Bota. Translational models of ovarian cancer-associated cognitive impairment and chemotherapy-induced peripheral neuropathy [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 7451.

Abstract 2622: TUB-040, a novel Napi2b-targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans

Abstract TUB-040 is a novel Antibody-Drug Conjugate (ADC) targeting Napi2b, a highly overexpressed target in ovarian cancer and lung adenocarcinoma. Napi2b is a member of the solute-carrier transporter family with eight transmembrane domains and an antibody-accessible extracellular loop, that regulates sodium-dependent phosphate homeostasis. Ethynylphosphonamidate conjugation chemistry was used to build TUB-040 with a homogenous DAR (drug-antibody-ratio) of 8 and the Topoisomerase I inhibitor Exatecan as payload, which is connected to a humanized, Fc-silenced IgG1 antibody targeting Napi2b using a cleavable linker system. In vitro, TUB-040 is characterized by sub-nanomolar affinity and specific binding to Napi2b, efficient target-mediated internalization and high cytotoxicity towards Napi2b-expressing ovarian and lung cancer cell lines as well as bystander activity against co-cultured target-negative cells. TUB-040 induces DNA damage and apoptosis in a dose-dependent fashion as well as markers of immunogenic cell death. In addition, no unspecific uptake and cytotoxicity in healthy, target-negative human cells could be detected, reducing the risk of target-independent toxicities. Pharmacokinetic analysis showed that - in contrast to maleimide-conjugated ADCs - TUB-040 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation, thus enabling most efficient and continued delivery of Exatecan to the tumor. Across a variety of cell line derived xenograft (CDX) and patient derived xenograft (PDX) models of ovarian and non-small cell lung cancer, including models with low target expression, single dose administration of TUB-040 results in long-lasting tumor growth inhibition with high complete remission rates in vivo. The minimally effective dose (MED) leading to complete remission is 1 mg/kg. Preliminary repeat-dose toxicological assessment of TUB-040 in the pharmacologically relevant species cynomolgus monkey demonstrated that TUB-040 is well tolerated with no signs of lung toxicity and thrombocytopenia. Allometric scaling and PK/PD modelling predicts a human serum half-life greater 10 days and a therapeutic index in humans up to 55. Based on these results, TUB-040, designed with differentiating technology for optimal efficacy and tolerability, is ready for testing in clinical trials. Citation Format: Saskia Schmitt, Isabelle Mai, Paul Machui, Sarah Herterich, Danila Hauswald, Philipp Ochtrop, Philipp Cyprys, Izabela Kozlowska, Annabel Kitowski, Marcus Gerlach, Olivier Marcq, Pamela A. Trail, Dominik Schumacher, Marc-André Kasper, Günter Fingerle-Rowson, Björn Hock, Jonas Helma-Smets, Annette M. Vogl. TUB-040, a novel Napi2b-targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans [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 2622.

Abstract 538: Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models

Abstract Introduction: Poly (ADP-ribose) polymerases (PARPs) can recruit DNA repair effectors and cope with DNA single-strand breaks. For DNA double-strand breaks (DSB), there are two main repair mechanisms: non-homologous end joining repair (NHEJ) which is prone to error due to infidelity events, and BRCA1/2 mediated homologous recombination repair (HR) restoring the original sequence. Therefore, inhibition of PARPs may cause both single stranded break repair deficiency and HR deficiency in BRCA1/2 deficient patients, leading to cell death. The concept of “synthetic lethality” has led to the development of PARP inhibitors, including Olaparib, Niraparib and Rucaparib in clinical practice. Despite encouraging efficacy of these PARP inhibitors, acquired resistance, such as HR repair restoration, drug efflux pump upregulation and replication fork protection, has limited their clinical benefits. Therefore, to investigate novel therapeutic strategies which can overcome the acquired resistance, we established patient-derived xenograft (PDX) ovarian cancer models with PARP inhibitor-induced resistance. Methods: OV9402, an ovarian cancer PDX model with BRCA 1 c.3155del, was implanted subcutaneously and treated with Niraparib at 35 mg/kg, daily, for 129 days. Relapse tumors (tumor #1 and tumor #2) were isolated and inoculated into naïve mice. Then the 2nd round of treatment (Niraparib, 35 mg/kg, daily) was given when the mean tumor volume reached 140 mm3. The tumors showing no significant response to Niraparib were collected on either day 32 (#1 derived tumor model) and day 22 (#2 derived tumor model) after randomization and their gene changes were analyzed by RNA sequencing (RNA-seq). Results: Niraparib significantly inhibited OV9402 tumor growth, with complete regression observed in nine out of ten mice on day 54 after the 1st round of treatment. However, tumor regrowth was observed in two out of the nine mice from day 68 onwards. The relapsed tumors (tumor #1 and tumor #2) from these two mice were collected and re-inoculated into 5 naïve mice to establish tumor models for the 2nd round of Niraparib treatment, separately. For #1 derived tumor model, all the tumors presented complete resistance to the Niraparib treatment. RNA-seq data showed that the gene expression of ABCB1, which encodes for efflux pumps, was upregulated. In parallel, complete resistance to Niraparib was observed in all the tumors derived from 2# tumor bearing mice. The RNA-seq data demonstrated that an acquired secondary genetic deletion on BRCA1 genes, which restored the functional protein expression loss in the parental line and restored HR repair, was found in the tumors. Conclusion: Ovarian cancer PDX models that have effectively developed resistance to PARP inhibitors have the potential to serve as valuable preclinical tools to evaluate potential next-generation PARP inhibitors and innovative combination strategies. Citation Format: Li Hua, Shuang Li, Chenpan Nie, Xiaobo Chen, Guo Sheng, Ludovic Bourre, Jingjing Wang. Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models [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 538.

Abstract 6441: Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in 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 6441.

Abstract 2913: Co-expression of extracellular matrix enzymes heparanase or PH-20 augments the anti-tumor efficacy of folate receptor 1-targeting CAR T cells in an ovarian cancer model

Abstract Introduction: The tumor extracellular matrix (ECM) is a physical barrier which obstructs trafficking of CAR T cells to tumors. We hypothesize that co-expressing ECM remodeling/degrading enzymes heparanase (HPSE) or PH-20 with a CAR directed against folate receptor 1 (FolR1) will overcome the physical tumor microenvironment barrier and improve anti-tumor efficacy of anti-FolR1 CAR in a human ovarian cancer model. Methods: ECM CARs were constructed by combining anti-FolR1 ScFv with a CD8 hinge and transmembrane domain, 4-1BB, and CD3ζ endodomains, followed by a P2A element and full-length HPSE or GPI-anchored PH-20 sequence. CAR T cells were prepared from CD4+ and CD8+ T cells of healthy donors by lentiviral transduction. CAR expression was evaluated by flow cytometry; HPSE and PH-20 expression were measured via ELISA and Western blotting, respectively. CAR-mediated killing and cytokine release were observed in overnight co-culture assays with FolR1-expressing tumor cells. ECM CARs’ migration through CultrexTM and sodium hyaluronate matrices were assessed for HPSE and PH-20 function, respectively. ECM CARs’ efficacy was evaluated in vivo using a FolR1-expressing subcutaneous OV90 ovarian cancer xenograft model in NOD SCID Gamma (NSG) mice, and representative OV90 tumors were harvested at day 7 post-CAR T treatment for immunohistochemical staining. Results: FolR1 expression was confirmed in ovarian cancer cell lines and substrates targeted by HPSE (perlecan, glypican 3, CD138) and PH-20 (hyaluronan) were detected by immunofluorescence in human ovarian tumors’ sections. Lentiviral transduced primary T cells yielded robust expression of FolR1 CAR and ECM enzymes. Anti-FolR1 CAR and ECM CARs secreted pro-inflammatory cytokines IL-2, TNFα, IFNƳ and lysed OVCAR3 tumor cells with similar efficacy. ECM CARs with HPSE or PH-20 exhibited enhanced migration through ECM-rich matrices as compared to anti-FolR1 CAR alone. In vivo, all anti-FolR1 CAR T cells rejected OV90 tumors and extended survival compared to tumor alone or untransduced T cells (UTD). However, ECM CARs co-expressing HPSE or PH-20 accelerated tumor regression as compared to anti-FolR1 CAR alone. Tumor histology revealed CAR T cell infiltration (CD3+, CD8+) in mice treated with anti-FolR1 CARs but not UTD, indicating that anti-FolR1 CARs can effectively infiltrate OV90 tumors. Imaging studies are underway to evaluate the function of ECM CARs in the tumor microenvironment. Conclusions: The anti-FolR1 CARs boosted with ECM enzymes HPSE or PH-20 demonstrated specific lysis of FolR1-expressing cell lines, improved penetration through ECM-rich matrices in vitro, and accelerated tumor regression in an ovarian cancer model in vivo. Therefore, ECM CARs may help improve clinical outcomes in patients with ovarian tumors. Citation Format: Brittany L. Steimle, Peirong Hu, Ngoc Tran, Jia-Jye Lee, Kathleen Baysac, Kun Luo, Xu Wang, Genqing Liang, Saule Nurmukhambetova, Tri Tran, Pradyot Dash, Dina Schneider. Co-expression of extracellular matrix enzymes heparanase or PH-20 augments the anti-tumor efficacy of folate receptor 1-targeting CAR T cells in an ovarian cancer model [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 2913.

Abstract 721: Novel and distinctive anti-HLA-G antibody, IMB-201, inhibits tumor progression by breaking immune escape mechanism and enhancing cytotoxicity

Abstract Human leukocyte antigen-G (HLA-G), a type of MHC class I, is known to be expressed only in the placenta and acts as an immune suppressor to protect the fetus from maternal immunity. Recent studies have revealed that HLA-G suppresses various tumor-infiltrated immune cells, such as T cells, NK cells, and macrophages, through interaction with inhibitory receptors Ig-like transcript 2 (ILT2). HLA-G has recently emerged as an attractive anticancer target because HLA-G is a highly specific tumor antigen and has a role in immune evasion of cancer cells. Additionally, the expression pattern of ILT2 differs from that of PD-1 in tumor-infiltrated CD8+ T cells. Therefore, an HLA-G targeting strategy is expected to be an alternative to address unmet medical needs for those not responding to anti-PD(L)1 therapies. In this study, we screened the novel anti-HLA-G antibody, IMB-201, using phage display technique and confirmed that IMB-201 selectively bound to the membrane HLA-G of cancer cells. It exhibited superior activities in ILT2 blockade, NK cell activation, and antibody-dependent cellular cytotoxicity (ADCC) compared to the reference antibody. Furthermore, IMB-201 demonstrated more efficient suppression of tumor growth compared to the reference antibody in subcutaneous gastric cancer xenograft model. Notably, the group administered IMB-201 with NK92MI cells (CD16-negative NK cells) in the ovarian cancer xenograft model showed stronger tumor growth inhibition compared to the group treated only IMB-201, indicating that NK92MI cells were activated by IMB-201. This result indicates that one of the mechanisms of action (MoA) of IMB-201 worked effectively in vivo. In addition, to maximize the efficacy of IMB-201 and differentiate it from competitors, various efficacy enhancing modality could be applicable. We aim to develop biopharmaceuticals by selecting the optimized modality. Based on these results, IMB-201 could overcome the limitations of existing cancer immunotherapy, potentially becoming highly influential in cancer treatment as a novel immune checkpoint inhibitor. It is also expected to exhibit higher anticancer efficacy in combination therapy with immune checkpoint inhibitors. Citation Format: Hyeonju Kang, Seungil Baek, Inyoung Lee, Manseok Ju, Seonggu Han, Eunjung Han, Sungmuk Kang, Jihye Koo, Yoojin Kim, Suho Park, Eunyoung Cho, Chungmin Lee, Gyongsik Ha. Novel and distinctive anti-HLA-G antibody, IMB-201, inhibits tumor progression by breaking immune escape mechanism and enhancing cytotoxicity [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 721.

A novel SIK2 inhibitor SIC-19 exhibits synthetic lethality with PARP inhibitors in ovarian cancer

PurposeOvarian cancer patients with HR proficiency (HRP) have had limited benefits from PARP inhibitor treatment, highlighting the need for improved therapeutic strategies. In this study, we developed a novel SIK2 inhibitor, SIC-19, and investigated its potential to enhance the sensitivity and expand the clinical utility of PARP inhibitors in ovarian cancer. MethodsThe SIK2 protein was modeled using a Molecular Operating Environment (MOE), and the most favorable model was selected based on a GBVI/WSA dG scoring function. The Chembridge Compound Library was screened, and the top 20 candidate compounds were tested for their interaction with SIK2 and downstream substrates, AKT-pS473 and MYLK-pS343. SIC-19 emerged as the most promising drug candidate and was further evaluated using multiple assays. ResultsSIC-19 exhibited selective and potent inhibition of SIK2, leading to its degradation through the ubiquitination pathway. The IC50 of SIC-19 correlated inversely with endogenous SIK2 expression in ovarian cancer cell lines. Treatment with SIC-19 significantly inhibited cancer cell growth and sensitized cells to PARP inhibitors in vitro, as well as in ovarian cancer organoids and xenograft models. Mechanistically, SIK2 knockdown and SIC-19 treatment reduced RAD50 phosphorylation at Ser635, prevented nuclear translocation of RAD50, disrupted nuclear filament assembly, and impaired DNA homologous recombination repair, ultimately inducing apoptosis. These findings highlight the crucial role of SIK2 in the DNA HR repair pathway and demonstrate the significant PARP inhibitor sensitization achieved by SIC-19 in ovarian cancer. ConclusionsSIC-19, a novel SIK2 inhibitor, effectively inhibits tumor cell growth in ovarian cancer by interfering with RAD50-mediated DNA HR repair. Furthermore, SIC-19 enhances the efficacy of PARP inhibitors, providing a promising therapeutic strategy to improve outcomes for ovarian cancer patients.