(T0930-04-21) Optimizing Immunotherapy of Ovarian Cancer — Nanomedicine Strategies for Heating ‘Cold’ Tumor with Concurrent PD-L1 Attenuation

Purpose: Ovarian Cancer (OC) is the leading cause of death from gynecological malignant tumors. The high lethality is attributed to the asymptomatic nature of early disease, with nearly 75% of patients presenting with stage III and IV. A major barrier to the successful deployment of cancer immunotherapy for OC patients is the immunosuppressive tumor microenvironment (TME) at the advanced stage, including extrinsic suppression of CD8+ effector cells by regulatory T cells (Tregs) [1,2], engagement of the inhibitory receptor PD-1 by the ligand PD-L1 and redundant myeloid-derived suppressor cells [3]. Overcoming this hurdle is crucial for the development of effective immunotherapeutic approaches. We have developed a new construct of antibody-drug conjugates (pADC) that consists of an antibody and several semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates (ST-P-drug) in a high drug-to-antibody ratio (DAR) [4]. In this study, we used PD-L1 as biomarker and designed anti-PD-L1 antibody-polymer-epirubicin conjugate. Polymer-epirubicin conjugate (P-EPI) has shown not only effective as an anticancer chemotherapeutics but also induced long-lasting immunogenic cell death [5]. We anticipate this new pADC may achieve dual chemo and immunotherapeutic benefits against the ovarian cancer.

Methods: The syngeneic murine ovarian cancer model was developed by injecting 5 to 10 million ID8-Luc cells (Ubigene Biosciences) in 8-week-old C57BL/6 female mice intraperitoneally. The tumor growth pattern was monitored weekly by bioluminescence imaging (BLI) by an in vivo imaging system (IVIS). For BLI activity measurement, tumor-bearing mice were administered 200 µl of luciferin substrate (D-Luciferin sodium salt) in a concentration of 15 mg/mL intraperitoneally (i.p.) to the mice 10 min before imaging. Mice were sacrificed at the early (before 4 weeks) and late (after 7 weeks ) stages of the tumor, respectively. Immune cells were isolated from the ascites fluid and stage-dependent tumor microenvironment was evaluated by flow cytometry. The single cell suspension was incubated with anti-mouse CD16/CD32 antibody to block non-specific binding of the immunoglobulin Fc receptors and then labeled with fluorochrome-conjugated antibodies: PE anti-mouse CD45, FITC anti-mouse CD3, PerCP anti-mouse CD4, APC anti-mouse CD8, FITC anti-mouse CD11b, APC anti-mouse F4/80, PE/Cy7 anti-mouse inos, PE anti-mouse CD206, PE anti-mouse CD11b, FITC anti-mouse Ly-6G/ Ly-6C (Gr-1), and APC anti-mouse CD274 (PD-L1). The therapeutic effect of our pADC was evaluated in advanced syngeneic model: when tumor growth reached a late stage (5~6 weeks after inoculation of tumor cells), tumor-bearing mice were treated at 3-day intervals four times in each of two cycles. Saline and mouse anti-PD-L1 Ab (BioXcell) were administered as controls. Bodyweight and survival were recorded.

Results: Tumor-bearing mice showed abdominal distention, increased volume of ascites fluid accumulation, and resulted in weight gain at the advanced stage of the tumor. Disease progression was confirmed by BLI activity, and an overall survival time was 52 ± 6 days. At necropsy, solid nodules of the tumor were found within the peritoneal cavity, and lesions were also detected in the liver, kidney, spleen, and abdominal wall. Diffuse tumor growth in the anterior abdominal was further confirmed by H & E stain on formalin-fixed tissue samples. Immune profiling from ascites demonstrated a higher PD-L1 expression and increased myeloid-derived suppressor cells (MDSC) in the late stage of the tumor compared to the early stage. Moreover, reduced CD8+ T cells, tumor-associated macrophages, and increased CD4+ were observed at the late stage of tumor-bearing mice. These results proved the success of OC model developed, which mimics the feature of clinical disease progress. Improved cellular uptake and significant cell-killing activity of pADC in the ID8-Luc cells were confirmed. To demonstrate the treatment efficacy, mice bearing ID8-Luc ovarian cancer xenograft were treated at advanced stage, and inhibition of tumor progress was monitored by bioluminescence imaging. Strikingly, pADC treatment significantly reduced tumor burden and extended survival time. On the contrary, due to immunosuppressive tumor microenvironment, anti-PD-L1 immunotherapy that simply blocks the PD-1/PD-L1 interaction was not efficient.

Conclusion: The in vivo study suggested our pADC is an effective immune-oncology agent in preclinical ovarian cancer model. Investigation of the mechanism of action is underway.

References: 1. Sato E et al., Proc Natl Acad Sci USA 2005; 102(51): 18538–18543.
2. Nishikawa H et al., Blood 2005; 106(3): 1008–1011.
3. Matsuzaki J et al., Proc Natl Acad Sci USA 2010; 107(17): 7875–7880.
4. Zhang L et al., Eur. J. Pharm. Sci. 103 (2017)36-46
5. Li L, et al., Adv Funct Mater 2020, 30, 1908961.

Acknowledgements: Huntsman Cancer Institute PCD Program Award to J. Yang


Figure 1. Development of immune suppressive of tumor microenvironment. (A) Murine ID8 cell-derived ovarian cancer development after i.p. injection of 5 x 106 cells into C57BL/6 mice. Early and advanced tumor was classified based on the presence of immune cells in the ascites-derived fluid. (B) Tumor stage-dependent PD-L1 expression, (C) CD3+ T cells, (D) CD4+, CD8+ cells, (E) macrophages, (F) M1 type macrophages (G) M2 type macrophages and H) Myeloid-derived suppressor cells (MDSCs) in the ascites fluid-derived cells from tumor-bearing C57BL/6 mice.


Figure 2. Therapeutic effect of pADC on ID8 cells derived ovarian cancer model. (A) Structure of pADC, (B) Uptake of pADC by ID8 cells. (C) Cytotoxicity of pADC on ID8 Luc cells, bioluminescence activity (BLI) after 72 h. (D) tumor development and treatment schedules (E) Survival of C57BL/6 mice bearing ovarian cancer under different treatments. (F) Body weight changes after treatment. (G) Bioluminescence imaging following different treatments.