Single Radiotherapy Fraction with Local Anti-CD40 Therapy Generates Effective Abscopal Responses in Mouse Models of Cervical Cancer

Abstract

Current treatment options for advanced cervical cancer are limited, especially for patients in poor-resource settings, with a 17% 5-year overall survival rate. Here, we report results in animal models of advanced cervical cancer, showing that anti-CD40 therapy can effectively boost the abscopal effect, whereby radiotherapy of a tumor at one site can engender therapeutically significant responses in tumors at distant untreated sites. In this study, two subcutaneous cervical cancer tumors representing one primary and one metastatic tumor were generated in each animal. Only the primary tumor was treated and the responses of both tumors were monitored. The study was repeated as a function of different treatment parameters, including radiotherapy dose and dosing schedule of immunoadjuvant anti-CD40. The results consistently suggest that one fraction dose of radiotherapy with a single dose of agonistic anti-CD40 can generate highly effective abscopal responses, with a significant increase in animal survival (p = 0.0004). Overall, 60% of the mice treated with this combination showed long term survival with complete tumor regression, where tumors of mice in other cohorts continued to grow. Moreover, re-challenged responders to the treatment developed vitiligo, suggesting developed immune memory for this cancer. The findings offer a potential new therapy approach, which could be further investigated and developed for the treatment of advanced cervical cancer, with major potential impact, especially in resource-poor settings.

Keywords: abscopal effect; anti-CD40; cervical cancer; immunotherapy; in situ vaccination; radiotherapy.

Figure 1

Study Design. (a) General timeline of the studies, including tumor inoculation 2 weeks prior to a treatment varying according to the study designs. In all studies, the treatment response was monitored over time by tumor volume measurements. (b) Schematic depiction of the treated mouse. Orange (right) tumor represents the treated side and green (left) tumor, the abscopal side. (c) Small Animal Radiation Research Platform set-up was used for RT and CT imaging. Image guided RT was only given to the right tumor. (d) Dose volume histogram showing RT dose distribution into the treated tumors and surrounding normal tissue, including contralateral non-treated tumors.

Figure 2

Investigating the abscopal effect in cervical cancer mouse model represented by tumor volume (mean ± SEM) in (a) treated and (b) untreated groups (n = 5). The abscopal effect was observed in combination treatment group with a significant tumor growth control in untreated and treated tumors. * p < 0.05.

Figure 3

Radiation dose titration study in cervical cancer. C57BL/6 mice were subcutaneously inoculated with TC-1 cells on both flanks. Once tumors reached a 3–4 mm size, the right tumors were treated with a single dose of 6 Gy, 10 Gy or 15 Gy, with a combination of agonistic anti-CD40 (20 µg/tumor) (a) Volumes of treated tumors over time. Higher doses of RT had a better treatment impact, but did not induce the abscopal effect. (b) Volumes of untreated (abscopal) tumors over time. Single dose of 6 Gy combined with anti-CD40 induced abscopal effect on contralateral tumor, by inhibiting tumor progression. Error bars are SEM. n = 5 mice/group. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 4

Immunoadjuvant dosing titration study on TC-1 tumors. Single dose of 6 Gy RT combined with anti-CD40 in single direct injection (concentration 20 µg/tumor) induced an abscopal effect, autoimmune memory, and autoimmune vitiligo. Other dosing regimens in combination with RT only effected the treated tumors. Once tumors reached treatable size, the right tumors were treated with single dose of 6 Gy or with a combination of agonistic anti-CD40, either in one dose or in three fractionated doses (day 0, 7, 14 post treatment) with a total application of 20 µg mAb. (a) Volumes of treated tumors over time. * p < 0.05; (b) Volumes of untreated (abscopal) tumors over time. Error bars are SEM. * p < 0.05; (c) Overall survival. n = 5 mice/group. *** p < 0.001 (d) Body weight averages of all groups. Increasing body weight corresponds to better mouse condition and smaller/no tumors. (e) Representative CT images of mice, two weeks after treatment. Orange color correlates to treated tumor and green to non-treated tumor. (f) Representative picture of vitiligo that occurred in mice in combination group of RT and single direct injection of anti-CD40 (20 µg) that had been re-challenged with TC-1 tumors.

Figure 5

Scheme of Abscopal Effect. Irradiation of the cancer cells leads to apoptosis and immunogenic cell death, which contributes to higher release of tumor neoantigens. Radiation causes decrease of myeloid-derived suppressor cells (MDSCs), increased expression of major histocompatibility complex I (MHC I) on tumor cells and production of cytokines and chemokines attracting T-cells. Released neoantigens are up-taken by APCs, migrate through lymphatic vessels to lymphatic nodules, where they are presented to naïve T-cells by interaction of MHC I with T cell receptor (TCR). Naïve T-cells become activated upon further co-stimulatory signals, such as CD80, CD40 and CD28. Activated T-cells, mainly effector T-cells, proliferate and differentiate into neoantigen-specific cytotoxic T cell. These cells then migrate from lymph nodes and accumulate in the places with the neoantigen “label”, meaning all primary and metastatic tumor lesions. The abscopal effect could be hampered by presence of cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) or programmed-death ligand 1 (PD-L1), which have immunosuppressive effect.