Exploiting the immunogenic potential of standard of care radiation or cisplatin therapy in preclinical models of HPV-associated malignancies

Abstract

Background: While radiation and chemotherapy are primarily purposed for their cytotoxic effects, a growing body of preclinical and clinical evidence demonstrates an immunogenic potential for these standard therapies. Accordingly, we sought to characterize the immunogenic potential of radiation and cisplatin in human tumor models of HPV-associated malignancies. These studies may inform rational combination immuno-oncology (IO) strategies to be employed in the clinic on the backbone of standard of care, and in so doing exploit the immunogenic potential of standard of care to improve durable responses in HPV-associated malignancies.

Methods: Retroviral transduction with HPV16 E7 established a novel HPV-associated sinonasal squamous cell carcinoma (SNSCC) cell line. Three established HPV16-positive cell lines were also studied (cervical carcinoma and head and neck squamous cell carcinoma). Following determination of sensitivities to standard therapies using MTT assays, flow cytometry was used to characterize induction of immunogenic cell stress following sublethal exposure to radiation or cisplatin, and the functional consequence of this induction was determined using impedance-based real time cell analysis cytotoxicity assays employing HPV16 E7-specific cytotoxic lymphocytes (CTLs) with or without N803 (IL-15/IL-15-Rα superagonist) or exogenous death receptor ligands. In vitro observations were translated using an in vivo xenograft NSG mouse model of human cervical carcinoma evaluating cisplatin in combination with CTL adoptive cell transfer.

Results: We showed that subpopulations surviving clinically relevant doses of radiation or cisplatin therapy were more susceptible to CTL-mediated lysis in four of four tumor models of HPV-associated malignancies, serving as a model for HPV therapeutic vaccine or T-cell receptor adoptive cell transfer. This increased killing was further amplified by IL-15 agonism employing N803. We further characterized that radiation or cisplatin induced immunogenic cell stress in three of three cell lines, and consequently demonstrated that upregulated surface expression of Fas and TRAIL-R2 death receptors at least in part mediated enhanced CTL-mediated lysis. In vivo, cisplatin-induced immunogenic cell stress synergistically potentiated CTL-mediated tumor control in a human model of HPV-associated malignancy.

Conclusion: Standard of care radiation or cisplatin therapy induced immunogenic cell stress in preclinical models of HPV-associated malignancies, presenting an opportunity poised for exploitation by employing IO strategies in combination with standard of care.

Keywords: Combined Modality Therapy; Immunotherapy, Adoptive; Radiotherapy.

Figure 1

Viability of HPV-associated tumor models after exposure to standard of care radiation or cisplatin. HPV-associated tumor models were treated with increasing concentrations of (A) radiation or (B) cisplatin, and viability was determined by MTT assays at 72 hours as a percentage relative to untreated control. Dotted lines represent sublethal clinically relevant concentrations of radiation (10 Gy) or cisplatin (1 µg/mL) used for subsequent studies. HPV, human papillomavirus.

Figure 2

HPV16 E7-specific cytotoxic lymphocyte effector function was significantly enhanced by IL-15 agonism. (A) SCCNC5 (parent, E7 negative) and E7-SCCNC5 (daughter, E7 transduced) were co-cultured with HLA-A2 restricted HPV16 E7-specific cytotoxic lymphocyte (CTL) at increasing effector to target (E:T) ratios with and without effector pretreatment with IL-15 superagonist (N803, 50 ng/mL) for 24 hours. Lysis was determined at 4 hours as a percentage relative to control without effector addition. (B) Three endogenous HPV16-positive tumor models were co-cultured with HPV16 E7-specific CTL, and lysis was determined at 4 hours, as above. Arrows represent E:T ratios used for subsequent studies (CaSki, 10:1; UPCI-SCC-90, 5:1; UPCI-SCC-152, 2:1; E7-SCCNC5, 2:1, not shown). ns p>0.05, ***p<0.001, ****p<0.0001. HPV, human papillomavirus; IL, interleukin.

Figure 3

Radiation or cisplatin exposure significantly enhanced CTL-mediated HPV-associated target cell lysis, which was further enhanced by IL-15 agonism. HPV-associated tumor models were treated with (A) radiation (10 Gy, 72 hours) or (B) cisplatin (1 µg/mL, 48 hours) followed by co-culture with HPV16 E7-specific CTL at established effector to target ratios (see figure 2) with and without effector pretreatment with IL-15 superagonist (N803, 50 ng/mL) for 72 hours after effector stimulation with α-CD2/CD3/CD28, as described in Materials and methods. Lysis was determined at 4 hours (CaSki, UPCI-SCC-90) or 18 hours (UPCI-SCC-152, E7-SCCNC5) as a percentage relative to control without effector addition in each respective group. Experiments were replicated at least twice with similar results. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CTL, cytotoxic lymphocyte; HPV, human papillomavirus; IL, interleukin.

Figure 4

Radiation or cisplatin exposure significantly upregulated death receptors Fas and TRAIL-R2, which functionally mediated enhanced HPV-associated target cell lysis. Representative HPV-associated tumor models for cervical carcinoma (CaSki), head and neck squamous cell carcinoma (UPCI-SCC-90), and sinonasal squamous cell carcinoma (E7-SCCNC5) were treated with (A, D) radiation (10 Gy, 72 hours) or (B, E) cisplatin (1 µg/mL, 48 hours) followed by flow cytometric interrogation of death receptors (A, B) Fas and (D, E) TRAIL-R2. Light shades and black, top boxes (−) depict untreated control groups. Dark shades and colored, bottom boxes (+) depict treatment groups. Gates represent per cent positive cells of the total viable population in the treatment group. Percentages indicate per cent positive cells of the total viable population. Numbers in parentheses indicate net mean fluorescent intensity (geometric mean of isotype subtracted from geometric mean of marker) of total viable population. Statistical significance determined by Kolmogorov-Smirnov test. (Bottom panel) Following radiation or cisplatin exposure, tumor models were co-incubated with (C) exogenous Fas ligand (α-Fas antibody, CH11 clone; 1 µg/mL, 10 µg/mL, 1 µg/mL, respectively) or (F) exogenous TRAIL-R2 ligand (KillerTRAIL; 20 ng/mL, 500 ng/mL, 500 ng/mL, respectively), and lysis was determined at 4 hours (KillerTRAIL, E7-SCCNC5) or 18 hours (others) as a percentage relative to control without effector addition in each respective group. Experiments were replicated twice with similar results. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. HPV, human papillomavirus.

Figure 5

Cisplatin significantly potentiated cytotoxic lymphocyte–mediated tumor control following in vivo adoptive cell transfer. (A) Pictogram representing experimental design. (B) NSG mice bearing CaSki tumors were treated with one cycle of cisplatin (5 mg/kg, ip) on day 12 and/or E7-specific CTL ACT (1×10⁶, ip) on day 14 post-tumor inoculation (sc). Inset demonstrating in vitro CTL lytic activity against CaSki at time of ACT at E:T ratios of 5:1, 10:1, and 20:1. Tumor volume (mm³) was monitored for 2 weeks following last treatment. Two-way analysis of variance with Tukey’s post hoc multiple comparisons test was used to compare tumor growth curves. ACT, adoptive cell transfer; CTL, cytotoxic lymphocyte; E:T, effector to target; ip, intraperitoneal; PBS, phosphate buffered saline; sc, subcutaneous. *p<0.05, ***p<0.001, ****p<0.0001.