Search

Ovarian cancer (OC) represents the deadliest gynecologic malignancy. Despite important advances in the field of OC therapy, recurrent OC still has very poor prognosis with a median survival of 1 year. Due to the close interaction between the ovarian cancer cells and tumor microenvironment, development of treatment strategies which not only target the tumor cells but also the components of the tumor microenvironment hold significance. Notably, a prime obstacle in the development of therapies is to identify targets with specific expression limited to the tumor surface and not the healthy tissues. The follicle-stimulating hormone receptor (FSHR) is one such target with selective expression in ovarian granulosa cells and thus, a potentially important therapeutic target in OC. Therefore, we developed monoclonal antibodies against FSHR and focused on studies of the most potent of these reagents. Anti-FSHR antibody bound to diverse FSHR expressing ovarian serous and clear cell adenocarcinoma cells suggesting these antibodies could be used to specifically target OC. We then designed a novel DNA encoded bispecific T cell engager targeting FSHR (FSHRxCD3) and evaluated this bispecific in therapeutic models for the treatment of OC. FSHRxCD3 bispecific in the presence of human PBMCs was highly specific in killing FSHR positive ovarian tumor lines. However, T cell approaches alone may not be fully effective in treating OC, referred as Immunologically “Cold” Tumors. Hence, we hypothesized that engaging the other components of immune system, would provide better tumor control. Increasing lines of evidence suggest that OC is receptive to Natural killer (NK) cell attack. We designed antibodies against human Siglec-7, an inhibitory receptor present on human NK cells and showed they could bind to NK cells. We then used these to create a novel class of bispecific NK engager (NKE) that simultaneously targets both Siglec-7 and FSHR (Siglec-7xFSHR). This NKE was potent at killing FSHR positive OC targets in both in vitro and in vivo assays. Multiple Ovarian tumors including BRCA mutated and PARPi resistant ovarian cancer cells could be targeted by this immune therapy. Our data demonstrate the therapeutic potential of these two novel bispecific molecules and initial studies suggest that their combination may be valuable in patients with OC. Citation Format: Devivasha Bordoloi, Pratik Bhojnagarwala, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, Alfredo Perales-Puchalt, Ryan P. O’Connell, Xizhou Zhu, Elizabeth M. Parzych, Rugang Zhang, Mohamed Abdel-Mohsen, David B. Weiner. Immunotherapy of ovarian cancer targeting FSHR by innate and adaptive immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4262.

Epstein Barr Virus (EBV) latent infection is associated with multiple types of cancer. Several clinical studies have targeted EBV antigens as immune therapeutic targets with limited efficacy of EBV+ malignancies, suggesting additional targets might be important. BamHI-A rightward frame 1 (BARF1) is an EBV antigen that is highly expressed in EBV-associated gastric carcinoma (EBVaGC) and EBV+ nasopharyngeal carcinoma (NPC) and has not been evaluated as an antigen in the context of therapeutic immunization. Here, we developed a synthetic DNA-based expression cassette as immunotherapy targeting BARF1 (pBARF1). Immunization with pBARF1 induced potent antigen-specific humoral and polyfunctional effector T cell responses in vivo. We observed significant antigen-specific increases of IFN-γ+, IFN-γ+ TNF-α+, and IFN-γ+ TNF-α+ IL-2+ populations in CD8+ T cells from the pBARF1 immunized animals in both C57BL/6 and BALB/c mice. Novel BARF1 expressing carcinoma lines that grow in immune-competent animals were developed as surrogates for human EBV+ tumors. We confirmed that BARF1 expression in these new cancer cell lines, MC38-BARF1 and CT26-BARF1, was within the range of human EBV+ cancer cell lines, SNU719 and C666-1. Immunization with pBARF1 plasmid demonstrated impact of tumor progression through induction of CD8+ T cell in these BARF1+ carcinoma models. We also observed long-term antitumor immunity, at least up to 446 days, against BARF1 in a CT26-BARF1 rechallenge study. Using IVIS in vivo imaging, we observed that pBARF1 immunized animals rapidly controlled and cleared cancer cells. These studies demonstrate the potential for pBARF1 immunity to induce antigen-specific immune responses impacting tumor progression. Further study of this immune target and these EBV+ tumor models is likely important as part of therapeutic approaches for EBV+ malignancies. Citation Format: Xizhou Zhu, Alfredo Perales-Puchalt, Krzysztof Wojtak, Ziyang Xu, Kun Yun, Pratik S. Bhojnagarwala, Devivasha Bordoloi, Daniel H. Park, Kevin Liaw, Mamadou A. Bah, Paul M. Lieberman, Ebony N. Gary, Ami Patel, David B. Weiner. DNA immunotherapy targeting BARF1 demonstrates therapeutic impact in novel murine carcinoma models expressing EBV latent antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3573.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with poor clinical prognosis and there is significant need for new, improved therapies. Immunotherapy is a growing therapeutic research area for patients with advanced brain cancers. DNA encoded Tumor Attacking Bridges (dTABs) are bispecific antibodies that redirect T cell activity against Tumor Associated Antigens (TAAs) of interest. Here, we describe the design and development of in vivo generated dTABs targeting IL13Rα2, which is expressed in tumors of approximately 75% of GBM patients. Four dTABs targeting 2 different epitopes of IL13Rα2 were designed to compare alternate orientations of heavy and light chains in the dTABs. All dTABs bound to U87 cells expressing target as well as CD3 on T cells. The dTABs induced T cell activation, cytokine production, and tumor killing in the presence of target expressing tumor cells in a dose dependent manner. An important issue in the field of antibody guided therapies is on target - off tumor T cell activity which can cause serious side effects in patients. In order to minimize such toxicities, we identified one arrangement of dTAB which induced high T cell activation with potent tumor killing in the presence of target expressing tumor cells, but not when co-cultured with tumors that do not express IL13Rα2. We were able to detect expression of this lead dTAB for up to 19 days in the serum of NSG mice after a single DNA injection followed by electroporation. Serum from mice injected with this dTAB activated T cells which potently killed target expressing U87 GBM cells. In a subcutaneous xenograft mouse model of human GBM, this in vivo produced dTAB demonstrated significant tumor control and improved survival of mice. This study illustrates the potential of dTABs targeting IL13Rα2 as a novel approach for immunotherapy of this difficult to treat brain cancer. Citation Format: Pratik Sanjivkumar Bhojnagarwala, Ryan O'Connell, Daniel Park, Ebony Gary, Xizhou Zhu, David Weiner. Novel DNA encoded tumor attacking bridges (dTABs) targeting IL13Rα2 display potent tumor control in an animal model of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1822.

Epstein-Barr Virus (EBV) is an oncogenic pathogen, with which more than 90% of world population has been infected. It is associated with several types of cancer, and no EBV-specific therapies are available for these diseases. Recent studies targeting EBV latent proteins have shown limited efficacy, for their moderate immunogenicity. Therefore, the development of effective anti-EBV immunotherapies is in need. In this study, we screened a group of EBV proteins and selected BARF1 as a vaccine candidate, for its strong immunogenicity and high expression in EBV positive nasopharyngeal and gastric carcinomas. The BARF1 vaccine was developed through a synthetic DNA approach and delivered in vivo by electroporation. The vaccine induced potent BARF1-specific antibody and polyfunctional effector T cell responses in both C57BL/6 and BALB/c mice. We next established two carcinoma models by generating BARF1-expressing CT26 and MC38 cell lines. In the therapeutic model where mice were challenged with cancer cells and then treated, BARF1 vaccine was able to suppress cancer progression alone and showed synergy when combined with anti-PD1 antibody. In the minimal residual disease model where mice were immunized and then challenged with cancer cells, one dose of BARF1 vaccine was able to generate 100% complete response in treated animals. CD8 T cell was shown to be necessary for this protection. Finally, using the in vivo imaging system, we found that the BARF1 vaccine-induced immune responses cleared tumor in 60% of subjects as soon as 48 hours post tumor challenge. Tumors were cleared in all animals by day 10. In summary, BARF1 DNA vaccine provides a novel tool of immunotherapy that is highly specific to EBV positive malignancies and combining it with checkpoint inhibitors might open up new avenues for precision and combination immunotherapy against EBV-associated cancer. In the future, we will also target other viral-specific cancer antigens with our DNA vaccine platform, exploiting its capability to induce potent cytotoxic T lymphocyte response. Citation Format: Xizhou Zhu. DNA vaccine targeting BARF1 induces potent antitumor immunity against Epstein-Barr virus-associated carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4569.