A Mucin 16 bispecific T cell–engaging antibody for the treatment of ovarian cancer

Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti–PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell–rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.

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Targeting galectin-3 with a high-affinity antibody for inhibition of high-grade serous ovarian cancer and other MUC16/CA-125-expressing malignancies

Scientific Reports ◽

10.1038/s41598-021-82686-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marina Stasenko ◽

Evan Smith ◽

Oladapo Yeku ◽

Kay J. Park ◽

Ian Laster ◽

...

Keyword(s):

Ovarian Cancer ◽

Tumor Growth ◽

Cancer Cells ◽

Cancer Therapeutics ◽

Ca 125 ◽

Ovarian Cancer Cells ◽

Carbohydrate Binding ◽

Matrigel Invasion ◽

Galectin 3

AbstractThe lectin, galectin-3 (Gal3), has been implicated in a variety of inflammatory and oncogenic processes, including tumor growth, invasion, and metastasis. The interactions of Gal3 and MUC16 represent a potential targetable pathway for the treatment of MUC16-expressing malignancies. We found that the silencing of Gal3 in MUC16-expressing breast and ovarian cancer cells in vitro inhibited tumor cell invasion and led to attenuated tumor growth in murine models. We therefore developed an inhibitory murine monoclonal anti–Gal3 carbohydrate-binding domain antibody, 14D11, which bound human and mouse Gal3 but did not bind human Galectins-1, -7, -8 or -9. Competition studies and a docking model suggest that the 14D11 antibody competes with lactose for the carbohydrate binding pocket of Gal3. In MUC16-expressing cancer cells, 14D11 treatment blocked AKT and ERK1/2 phosphorylation, and led to inhibition of cancer cell Matrigel invasion. Finally, in experimental animal tumor models, 14D11 treatment led to prolongation of overall survival in animals bearing flank tumors, and retarded lung specific metastatic growth by MUC16 expressing breast cancer cells. Our results provide evidence that antibody based Gal3 blockade may be a viable therapeutic strategy in patients with MUC16-expressing tumors, supporting further development of human blocking antibodies against Gal3 as potential cancer therapeutics.

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Ovarian Cancer Cells Induce Peripheral Mature Dendritic Cells to Differentiate Into Macrophagelike Cells In Vitro

International Journal of Gynecological Cancer ◽

10.1111/igc.0b013e3181bb70c6 ◽

2009 ◽

Vol 19 (9) ◽

pp. 1487-1493 ◽

Cited By ~ 12

Author(s):

Fangxue Chen ◽

Meng Hou ◽

Feng Ye ◽

Weiguo Lv ◽

Xing Xie

Keyword(s):

Ovarian Cancer ◽

Dendritic Cells ◽

T Cell ◽

Cancer Cells ◽

Fluorescein Isothiocyanate ◽

Interleukin 10 ◽

Interleukin 4 ◽

Ovarian Cancer Cells ◽

Macrophage Colony

Aims:Precursors of dendritic cells (DCs) are able to differentiate into macrophages induced by some tumor-associated molecules; however, whether peripheral mature DCs could differentiate into macrophages remains unknown. This study was designed to find out whether ovarian cancer cells could induce peripheral mature DCs to differentiate into macrophages.Main Methods:Mature DCs were cultured from monocytes with granulocyte-macrophage colony-stimulating factor and interleukin 4 (IL-4) for 6 days and lipopolysaccharide for another 24 hours and then were cocultured for 48 hours with ovarian cancer ascites or cell-free supernatants of SKOV3 and CAOV3 cell lines. In some experiments, mature DCs were cultured in the absence or presence of IL-10 or leukemia inhibitory factor (LIF) for the same time. In neutralization experiments, neutralizing monoclonal antibodies to IL-10 or LIF were added to the cultures. Cell phenotypes and phagocytosis were analyzed using flow cytometry; allogeneic T-cell proliferation assay was used to examine stimulatory activity of cells.Results and Conclusions:Mature DCs cocultured with ovarian cancer ascites or supernatants of SKOV3 and CAOV3 differentiated into a group of macrophagelike cells that exhibited increased expression of surface marker CD14+CD1a−, decreased expression of CD83, poorer T-cell costimulatory properties, and greater endocytosis of fluorescein isothiocyanate-dextran in vitro. Interleukin 10 but not LIF mediated this differentiation pathway.

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559 Fostriecin potentiates genome instability and anti-tumor immunity in ovarian cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0559 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A593-A593

Author(s):

Remya Raja ◽

Christopher Wu ◽

Kristina Butler ◽

Marion Curtis

Keyword(s):

Ovarian Cancer ◽

Dna Damage ◽

T Cell ◽

Cancer Cells ◽

Tumor Immunity ◽

T Cell Activation ◽

Cell Activation ◽

Genome Instability ◽

Ovarian Cancer Cells ◽

Syngeneic Mouse

BackgroundIncreased immune infiltration in ovarian tumors has been linked to improved patient outcome. Nonetheless, responses to checkpoint blockade therapies have been disappointing in ovarian cancer patients. This has been attributed to the low mutational burden present in ovarian tumors. However, many tumor antigens have been identified in ovarian cancer, which underscores the critical need to identify new treatment strategies that will trigger anti-tumor immunity in ovarian cancer. Recent studies have revealed that defects in DNA damage repair (DDR) pathways can contribute to improved responses to immune-directed therapies.1 2 We previously discovered that CT45 expression sensitizes ovarian cancer cells to chemotherapy via its interaction with the protein phosphatase 4 (PP4) complex.3 PP4 is known to play a key role in DDR pathways; however, its potential effects on anti-tumor immunity remain unknown.MethodsUsing fostriecin, a commercially available inhibitor of PP4, we studied the effect of fostriecin on chemosensitivity using cell cycle analysis and cell viability assays. To study the effect of fostriecin on DNA damage, we performed comet assays and measured micronuclei along with FANCD2 foci formation. Furthermore, using western blot, qPCR, and T cell activation assays, we assessed the role of fostriecin in promoting an inflammatory response. We tested the efficacy of combining fostriecin with carboplatin and PD-1 inhibition in a syngeneic mouse model of ovarian cancer.ResultsOur results show that fostriecin treatment combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using a panel of ovarian cancer cells, we show that fostriecin treatment triggers an anti-tumor immune response via STAT1 activation resulting in increased expression of pro-inflammatory cytokines. Furthermore, in a syngeneic mouse ID8 ovarian cancer cell line, we demonstrate that combination treatment of fostriecin and carboplatin significantly increased CD8 T cell activation over carboplatin treatment alone.ConclusionsOur work has identified a role for PP4 inhibition in promoting anti-tumor immunity. These findings form the groundwork for the rationale design of a clinical trial combining PP4 inhibitors with chemo-immunotherapy as a new approach in ovarian cancer treatment.ReferencesRodier F, Coppé J-P, Patil CK, Hoeijmakers WAM, Muñoz DP, Raza SR, et al. Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion. Nature Cell Biology 2009; 11: 973–979.Zhang H, Christensen CL, Dries R, Oser MG, Deng J, Diskin B, et al. CDK7 Inhibition potentiates genome instability triggering anti-tumor immunity in small cell lung cancer. Cancer cell 2020;37:37–54.e39.Coscia F, Lengyel E, Duraiswamy J, Ashcroft B, Bassani-Sternberg M, Wierer M, et al. Multi-level proteomics identifies CT45 as a chemosensitivity mediator and immunotherapy target in ovarian cancer. Cell 2018;175:159–170.e116.

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Effective antitumor activity of 5T4‐specific CAR‐T cells against ovarian cancer cells in vitro and xenotransplanted tumors in vivo

MedComm ◽

10.1002/mco2.34 ◽

2020 ◽

Vol 1 (3) ◽

pp. 338-350

Author(s):

Cuiyu Guo ◽

E Dong ◽

Qinhuai Lai ◽

Shijie Zhou ◽

Guangbing Zhang ◽

...

Keyword(s):

Ovarian Cancer ◽

T Cells ◽

Antitumor Activity ◽

Cancer Cells ◽

Ovarian Cancer Cells ◽

Car T Cells ◽

Car T

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Long Intergenic Noncoding RNA OIN1 Promotes Ovarian Cancer Growth by Modulating Apoptosis-Related Gene Expression

International Journal of Molecular Sciences ◽

10.3390/ijms222011242 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11242

Author(s):

Toshihiko Takeiwa ◽

Yuichi Mitobe ◽

Kazuhiro Ikeda ◽

Kosei Hasegawa ◽

Kuniko Horie ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Rna Sequencing ◽

Noncoding Rna ◽

Noncoding Rnas ◽

Advanced Ovarian Cancer ◽

Ovarian Cancer Cells ◽

Immunodeficient Mice ◽

Cancer Management ◽

Long Intergenic Noncoding Rna

Patients with advanced ovarian cancer usually exhibit high mortality rates, thus more efficient therapeutic strategies are expected to be developed. Recent transcriptomic studies revealed that long intergenic noncoding RNAs (lincRNAs) can be a new class of molecular targets for cancer management, because lincRNAs likely exert tissue-specific activities compared with protein-coding genes or other noncoding RNAs. We here show that an unannotated lincRNA originated from chromosome 10q21 and designated as ovarian cancer long intergenic noncoding RNA 1 (OIN1), is often overexpressed in ovarian cancer tissues compared with normal ovaries as analyzed by RNA sequencing. OIN1 silencing by specific siRNAs significantly exerted proliferation inhibition and enhanced apoptosis in ovarian cancer cells. Notably, RNA sequencing showed that OIN1 expression was negatively correlated with the expression of apoptosis-related genes ras association domain family member 5 (RASSF5) and adenosine A1 receptor (ADORA1), which were upregulated by OIN1 knockdown in ovarian cancer cells. OIN1-specifc siRNA injection was effective to suppress in vivo tumor growth of ovarian cancer cells inoculated in immunodeficient mice. Taken together, OIN1 could function as a tumor-promoting lincRNA in ovarian cancer through modulating apoptosis and will be a potential molecular target for ovarian cancer management.

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Transcriptional analysis of multiple ovarian cancer cohorts reveals prognostic and immunomodulatory consequences of ERV expression

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001519 ◽

2021 ◽

Vol 9 (1) ◽

pp. e001519

Author(s):

Marina Natoli ◽

John Gallon ◽

Haonan Lu ◽

Ala Amgheib ◽

David J Pinato ◽

...

Keyword(s):

Ovarian Cancer ◽

T Cells ◽

Cancer Cells ◽

Cell Lines ◽

Transcriptional Analysis ◽

Ovarian Cancer Cells ◽

Wild Type ◽

Patient Prognosis ◽

Independent Cohort

BackgroundEndogenous retroviruses (ERVs) play a role in a variety of biological processes, including embryogenesis and cancer. DNA methyltransferase inhibitors (DNMTi)-induced ERV expression triggers interferon responses in ovarian cancer cells via the viral sensing machinery. Baseline expression of ERVs also occurs in cancer cells, though this process is poorly understood and previously unexplored in epithelial ovarian cancer (EOC). Here, the prognostic and immunomodulatory consequences of baseline ERV expression was assessed in EOC.MethodsERV expression was assessed using EOC transcriptional data from The Cancer Genome Atlas (TCGA) and from an independent cohort (Hammersmith Hospital, HH), as well as from untreated or DNMTi-treated EOC cell lines. Least absolute shrinkage and selection operator (LASSO) logistic regression defined an ERV expression score to predict patient prognosis. Immunohistochemistry (IHC) was conducted on the HH cohort. Combination of DNMTi treatment with γδ T cells was tested in vitro, using EOC cell lines and patient-derived tumor cells.ResultsERV expression was found to define clinically relevant subsets of EOC patients. An ERV prognostic score was successfully generated in TCGA and validated in the independent cohort. In EOC patients from this cohort, a high ERV score was associated with better survival (log-rank p=0.0009) and correlated with infiltration of CD8+PD1+T cells (r=0.46, p=0.0001). In the TCGA dataset, a higher ERV score was found in BRCA1/2 mutant tumors, compared to wild type (p=0.015), while a lower ERV score was found in CCNE1 amplified tumors, compared to wild type (p=0.019). In vitro, baseline ERV expression dictates the level of ERV induction in response to DNMTi. Manipulation of an ERV expression threshold by DNMTi resulted in improved EOC cell killing by cytotoxic immune cells.ConclusionsThese findings uncover the potential for baseline ERV expression to robustly inform EOC patient prognosis, influence tumor immune infiltration and affect antitumor immunity.

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Cytotoxic effects of disulfiram and synergism with cisplatin on ovarian cancer cells in vitro

10.1055/s-0038-1671352 ◽

2018 ◽

Author(s):

F Guo ◽

Z Yang ◽

J Xu ◽

J Sehouli ◽

AE Albers ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Ovarian Cancer Cells ◽

Cytotoxic Effects

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Electrochemotherapy with Calcium Chloride and 17β-Estradiol Modulated Viability and Apoptosis Pathway in Human Ovarian Cancer

Pharmaceutics ◽

10.3390/pharmaceutics13010019 ◽

2020 ◽

Vol 13 (1) ◽

pp. 19

Author(s):

Zofia Łapińska ◽

Michał Dębiński ◽

Anna Szewczyk ◽

Anna Choromańska ◽

Julita Kulbacka ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Calcium Chloride ◽

Morphological Changes ◽

Cell Membrane Permeability ◽

Immunocytochemical Staining ◽

Ovarian Cancer Cells ◽

Apoptosis Pathway ◽

17Β Estradiol

Estrogens (Es) play a significant role in the carcinogenesis and progression of ovarian malignancies. Depending on the concentration, Es may have a protective or toxic effect on cells. Moreover, they can directly or indirectly affect the activity of membrane ion channels. In the presented study, we investigated in vitro the effectiveness of the ovarian cancer cells (MDAH-2774) pre-incubation with 17β-estradiol (E2; 10 µM) in the conventional chemotherapy (CT) and electrochemotherapy (ECT) with cisplatin or calcium chloride. We used three different protocols of electroporation including microseconds (µsEP) and nanoseconds (nsEP) range. The cytotoxic effect of the applied treatment was examined by the MTT assay. We used fluorescent staining and holotomographic imaging to observe morphological changes. The immunocytochemical staining evaluated the expression of the caspase-12. The electroporation process’s effectiveness was analyzed by a flow cytometer using the Yo-Pro™-1 dye absorption assay. We found that pre-incubation of ovarian cancer cells with 17β-estradiol may effectively enhance the chemo- and electrochemotherapy with cisplatin and calcium chloride. At the same time, estradiol reduced the effectiveness of electroporation, which may indicate that the mechanism of increasing the effectiveness of ECT by E2 is not related to the change of cell membrane permeability.

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627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0627 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A663-A663

Author(s):

Keegan Cooke ◽

Juan Estrada ◽

Jinghui Zhan ◽

Jonathan Werner ◽

Fei Lee ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Mouse Models ◽

T Cell Activation ◽

Phase 1 ◽

Phase 1 Study ◽

Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

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Genetic Perturbation of Pyruvate Dehydrogenase Kinase 1 Modulates Growth, Angiogenesis and Metabolic Pathways in Ovarian Cancer Xenografts

Cells ◽

10.3390/cells10020325 ◽

2021 ◽

Vol 10 (2) ◽

pp. 325

Author(s):

Carolina Venturoli ◽

Ilaria Piga ◽

Matteo Curtarello ◽

Martina Verza ◽

Giovanni Esposito ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Pyruvate Dehydrogenase ◽

Metabolic Pathways ◽

Negative Impact ◽

Digital Pathology ◽

Pyruvate Dehydrogenase Kinase ◽

Ovarian Cancer Cells ◽

Pyruvate Dehydrogenase Kinase 1

Pyruvate dehydrogenase kinase 1 (PDK1) blockade triggers are well characterized in vitro metabolic alterations in cancer cells, including reduced glycolysis and increased glucose oxidation. Here, by gene expression profiling and digital pathology-mediated quantification of in situ markers in tumors, we investigated effects of PDK1 silencing on growth, angiogenesis and metabolic features of tumor xenografts formed by highly glycolytic OC316 and OVCAR3 ovarian cancer cells. Notably, at variance with the moderate antiproliferative effects observed in vitro, we found a dramatic negative impact of PDK1 silencing on tumor growth. These findings were associated with reduced angiogenesis and increased necrosis in the OC316 and OVCAR3 tumor models, respectively. Analysis of viable tumor areas uncovered increased proliferation as well as increased apoptosis in PDK1-silenced OVCAR3 tumors. Moreover, RNA profiling disclosed increased glucose catabolic pathways—comprising both oxidative phosphorylation and glycolysis—in PDK1-silenced OVCAR3 tumors, in line with the high mitotic activity detected in the viable rim of these tumors. Altogether, our findings add new evidence in support of a link between tumor metabolism and angiogenesis and remark on the importance of investigating net effects of modulations of metabolic pathways in the context of the tumor microenvironment.

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