Nathalie Scholler

ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells

With the notable exception of B-cell malignancies, the efficacy of chimeric antigen receptor (CAR) T cells has been limited, and CAR T cells have not been shown to expand and persist in patients with nonlymphoid tumors. Here we demonstrate that redirection of primary human T cells with a CAR containing the inducible costimulator (ICOS) intracellular domain generates tumor-specific IL-17-producing effector cells that show enhanced persistence. Compared with CARs containing the CD3ζ chain alone, or in tandem with the CD28 or the 4-1BB intracellular domains, ICOS signaling increased IL-17A, IL-17F, and IL-22 following antigen recognition. In addition, T cells redirected with an ICOS-based CAR maintained a core molecular signature characteristic of TH17 cells and expressed higher levels of RORC, CD161, IL1R-1, and NCS1. Of note, ICOS signaling also induced the expression of IFN-γ and T-bet, consistent with a TH17/TH1 bipolarization. When transferred into mice with established tumors, TH17 cells that were redirected with ICOS-based CARs mediated efficient antitumor responses and showed enhanced persistence compared with CD28- or 4-1BB-based CAR T cells. Thus, redirection of TH17 cells with a CAR encoding the ICOS intracellular domain is a promising approach to augment the function and persistence of CAR T cells in hematologic malignancies.

Novel Recombinant Human B7-H4 Antibodies Overcome Tumoral Immune Escape to Potentiate T-Cell Antitumor Responses

B7-H4 (VTCN1, B7x, B7s) is a ligand for inhibitory coreceptors on T cells implicated in antigenic tolerization. B7-H4 is expressed by tumor cells and tumor-associated macrophages (TAM), but its potential contributions to tumoral immune escape and therapeutic targeting have been less studied. To interrogate B7-H4 expression on tumor cells, we analyzed fresh primary ovarian cancer cells collected from patient ascites and solid tumors, and established cell lines before and after in vivo passaging. B7-H4 expression was detected on the surface of all fresh primary human tumors and tumor xenotransplants, but not on most established cell lines, and B7-H4 was lost rapidly by tumor xenograft cells after short-term in vitro culture. These results indicated an in vivo requirement for B7-H4 induction and defined conditions for targeting studies. To generate anti-B7-H4-targeting reagents, we isolated antibodies by differential cell screening of a yeast-display single-chain fragments variable (scFv) library derived from patients with ovarian cancer. We identified anti-B7-H4 scFv that reversed in vitro inhibition of CD3-stimulated T cells by B7-H4 protein. Notably, these reagents rescued tumor antigen-specific T-cell activation, which was otherwise inhibited by coculture with antigen-loaded B7-H4+ APCs, B7-H4+ tumor cells, or B7-H4- tumor cells mixed with B7-H4+ TAMs; peritoneal administration of anti-B7-H4 scFv delayed the growth of established tumors. Together, our findings showed that cell surface expression of B7-H4 occurs only in tumors in vivo and that antibody binding of B7-H4 could restore antitumor T-cell responses. We suggest that blocking of B7-H4/B7-H4 ligand interactions may represent a feasible therapeutic strategy for ovarian cancer.

Blocking the B7-H4 pathway with novel recombinant antibodies enhances T cell-mediated antitumor responses

B7-H4 inhibits T-cell activation and is widely expressed by solid neoplasms. We have recently demonstrated that the expression of B7-H4 on the surface of malignant cells in vivo is inducible, and that novel anti-B7-H4 recombinant antibodies can reverse the inhibition of tumor-specific T cells. Thus, antibodies targeting the B7-H4 pathways may extend the survival of cancer patients by restoring T cell-mediated antitumor responses.

Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.

B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment.

Abstract TMEM-035: GLYCOMARKERS FOR PREDICTING PLATINUM–DRUG RESPONSE IN OVARIAN CANCER

Platinum-based drugs (pt-drugs) continue to be the mainstay of first-line therapy for a wide range of cancers including ovarian carcinoma. Despite common applications, resistance to pt-drugs is an ongoing dilemma in cancer treatments, because tumor cells have different molecular characteristics that affect their responses to drugs. A molecular test that can accurately identify pt-drug resistance would provide invaluable guidance for chemotherapy strategy and reduce the random use of ineffective drugs. This would be a significant advancement in treatment management. Our studies address this unmet need in cancer care by introducing an innovative method to predict pt-drug resistance, before administrating the drug. Our method is based on a new concept linking glycan cell surface expression with drug reactivity. We have identified a glycan structure, namely Glycomarker 1, whose expression level on the cancer cell surface is associated with a response to first-line chemotherapy. A patent for this discovery has been issued by the US patent office in 2009 (1) . Flow cytometry with a series of fluorescent lectins was initially used to profile the cell surface glycans on three isogenic pairs of ovarian carcinoma cell lines, each pair consisting of parental chemosensitive cell lines, 2008, A2780, IGROV-1, and their corresponding resistant cell lines 2008/C13*5.25, A2780/CP and IGROV-1/CP, respectively. The cell surface glycan comparison revealed that the expression levels of Glycomarker 1 on resistant phenotypes were at least ten times lower than that of sensitive parental cells, on all three pairs of cell lines. Further experiments by various methods of fluorescent confocal microscopy (with fluorescently labeled cisplatin), colony forming assay, glycan modifications, and mass spectrometry confirmed the association of Glycomarker 1 with drug uptake by ovarian carcinoma cell lines (1) . Lectin histochemistry (LHC) was adopted as a clinical method to test the Glycomarker 1 on human ovarian tissue samples. The LHC was optimized and validated for reproducibility, specificity, and sensitivity by colorimetric and fluorescent staining systems. The feasibility of LHC as a clinical test was evaluated on a training panel of 64 ovarian tissue sections. In retrospective studies, LHC correctly predicted drug-response in 22 out of 27 (81.4%) cancer specimens from patients with a known history of response to first-line chemotherapy. In the course of testing the clinical samples for Glycomarker 1, we identified another glycan structure, namely Glycomarker 2, whose expression pattern was strongly similar to that of the Glycomarker 1, suggesting the association of two glycan motifs with drug response. A patent for this discovery is pending in the US, Canada and European patent office (2) . The ultimate objective of this work is to translate the assays to clinical tests used to predict platinum response in ovarian cancer. This is a novel direct predictive method, truly different from existing procedures, which will provide molecular information for treatment strategy. Thus far, our experiments have focused on ovarian cancer. However, evidence indicates that this method may also apply to other cancers that are treated with pt-drugs. 1) US patent #7585503 2) PCT patent # 20150024409, 01/22/2015 pending in the US, Canada & Europe. Citation Format: Nahid Razi, Afshin Bahador, Nathalie Scholler and Nissi Varki. GLYCOMARKERS FOR PREDICTING PLATINUM–DRUG RESPONSE IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-035.

Abstract A041: Preclinical development of a preventive vaccine against ovarian cancer

Survival of cancer patients with high-grade serous carcinoma of the ovary (HGSCs) remains less than 30% five years after diagnosis, despite initial chemosensitivity of most HGSCs. We hypothesized that a tumor antigen-targeted vaccine capable of eliciting a robust cell-mediated immune response may help prevent ovarian cancer. Mesothelin (meso) is overexpressed by ovarian, lung and pancreatic cancers and is 57% conserved at the protein level between mouse and human. To elicit cell-mediated immune responses directed against mesothelin, we combined a human mesothelin recombinant protein with various adjuvants including: aluminum hydroxide gel (alum, for Th2 immune response); TLR4 ligand (synthetic lipid MPL, for Th1 response); squalene-oil-in-water (AddaVaxTM, for both Th1 and Th2 responses); and STING (stimulator of interferon genes) ligand (CDN) that activates innate immunity and triggers type 1 IFN response and NF-κB-dependent cytokine secretion. We detected robust anti-meso antibody (Ab) responses after 1 prime and 2 boosts with meso/alum/MPL or meso/CDN/AddaVax. The highest and most stable Ab titers were obtained with CDN/AddaVax-based adjuvants. Four groups of female C57BL/6 mice were immunized with alum/MPL or CDN/AddaVax with or without meso (n = 12 per group). Six weeks after the prime immunization, cells from a luciferase-transduced syngeneic mouse ovarian cancer cell line, ID8 (Luc-ID8) were injected orthotopically in the left ovary of the mice. In vivo bioluminescent imaging (BLI) suggested a 92% take rate of tumors 3 weeks after ID8-Luc injections in mice immunized with meso/alum/MPL or adjuvants only. However, in the group immunized with meso/CDN/Addavax only 30% of the mice showed a detectable signal 3 weeks after ID8-Luc implantation. After 10 weeks the average BLI signal was still significantly lower than in all the other groups. Furthermore, the majority of mice immunized with alum/MPL +/- meso developed ascites by 12 weeks; no significant difference of peritoneal leukocyte numbers or phenotype was observed between the groups immunized with alum/MPL vs. meso/alum/MPL. In contrast, 7 out 11 mice immunized with meso/CDN/Addavax did not develop ascites through 14 weeks post tumor challenge; most of the tumors were unilateral, smaller, with more T and B cells present in the peritoneal lavages compared to the other groups. Immunizations with CDN/AddaVax or meso/CDN/AddaVax strongly increased the frequency of CD62L-CD44+ effector memory T cells, stimulated innate immunity with M1 polarization, profoundly decreased MDSC levels in peritoneal lavages, and correlated with a less aggressive tumor phenotype (EpCAM+ PD-L1-). HE 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A041.

Abstract A56: Characterization of the tumor microenvironment of a humanized mouse model of ovarian cancer after treatment with anti-B7-H4 recombinant antibodies

Ovarian cancer is the deadliest gynecological malignancy. Disease recurrence occurs in 70% of patients diagnosed with stage III or IV epithelial ovarian cancer, in part due to immune evasion and suppression in the tumor microenvironment. Tumor infiltration with tumor-associated macrophages (TAMs) has been associated with poor patient survival and TAMs express B7-H4, an orphan ligand that modulates T cell responses. Common features observed in tumor microenvironments include hypoxic stress and other environmental cues that prompt the expression of immunomodulators such as CTLA4 and PD1, leading to increased tumor cell ability to escape immune recognition. We showed that B7-H4 is also expressed on ovarian cancer cells in vivo , suggesting that tumor cells may also hamper cell-mediated anti-tumor immune responses via inhibition of T cell co-receptors. We thus sought to determine whether targeting functional cell subsets expressing B7-H4 could restore an anti-tumor immune response and/or promote tumor cell death. We isolated novel recombinant anti-B7-H4 antibodies by differential cell screening of a yeast-display single-chain fragments variable (scFv) library that was derived from B cells of ovarian cancer patients. We demonstrated that B7-H4 scFv-mediated inhibition could rescue activation of tumor antigen-TCR-specific T cells in vitro , and we also showed that anti-B7-H4 scFv administration could delay tumor growth in a novel humanized NSG mouse model of ovarian cancer (Dangaj, D. et al., Novel recombinant human B7-H4 antibodies overcome tumoral immune escape to potentiate T cell anti-tumor responses, Cancer Research , 2013). We hypothesize that use of anti-B7-H4 scFv in vivo targeted functionally diverse tumor mass components (TAMs and tumor cells) leading to the reprogramming of the tumor microenvironment towards tumor rejection. Here we present a preliminary ex vivo analysis of the human tumors infiltrated with human leukocytes harvested from the B7-H4-treated humanized NSG mice. Tumors from mice treated with two anti-B7-H4 scFv or with a control scFv were sectioned and labeled by immunofluorescence to characterize tumor cells and immune cells. Tumor cells were co-stained with EpCAM and Ki-67, and leukocytes with CD45 and Ki-67. Preliminary studies suggested that in vivo delivery of anti-B7-H4 scFv decreased tumor cell proliferation as evidenced by a reduction of cells co-stained by EpCAM and Ki-67 compared with the staining of tumor cells from mice treated with the control antibody. Concurrently, tumors from mice receiving anti-B7-H4 scFv presented an increase in cells that stained positive for both CD45 and Ki-67 compared with tumors from mice receiving a control scFv. These results suggest that anti-B7-H4 scFv enhances leukocyte proliferation. Additional work will be performed to statistically analyze these results over all experimental groups, to functionally phenotype the tumor-infiltrating leukocytes, and to assess the impact of B7-H4 scFv administration on tumor hypoxia, apoptosis and angiogenesis. We tentatively conclude that B7-H4 is another immune checkpoint and its inhibition by antibody targeting may open novel immunotherapeutic avenues for ovarian cancer. Citation Format: Bhairavi (Vivi) Tolani, Denarda Dangaj, Daniel J. Powell, Jr., Nathalie Scholler. Characterization of the tumor microenvironment of a humanized mouse model of ovarian cancer after treatment with anti-B7-H4 recombinant antibodies. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A56. doi:10.1158/1538-7445.CHTME14-A56