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Generating the Abscopal Effect by Combining Proapoptotic Peptides With IL-12-Based Immunotherapy1

*Centre hospitalier universitaire vaudois (CHUV), Service et laboratoire central d'hématologie, département d'oncologie, rue du Bugnon 46, CH-1011 Lausanne, Switzerland; Centre hospitalier universitaire vaudois (CHUV), Centre d'immunothérapie et de la vaccinologie, rue du Bugnon 17, CH-1011 Lausanne, Switzerland; Université Pierre et Marie Curie, Faculté de Médecine Pitié-Salpêtrière, Centre Hospitalo-Universitaire Pitié-Salpêtrière, 91 Boulevard de l'Hôpital, F-75013 Paris, France

Improving the treatment of patients with advanced metastatic cancer is a daily challenge in clinical practice. The "renaissance" of immuno-oncology is mainly due to the success of the immune check-points inhibitors in prolonging the survival of metastatic cancer patients in different tumor types [1]. Very interestingly, some long lasting responses or even remissions have been reported [2].
Consequently, cancer immunotherapy became in the last decade, one of the major breakthrough in cancer treatment. Although active in monotherapy, anti-CTLA4 and anti-PD-1 monoclonal antibodies are not able to overcome every aspect of tumor immunosuppressive mechanisms. Several ongoing clinical trials attest that their combination may improve their clinical efficacy. Nevertheless, their associated autoimmune toxicities constitute a main reason of treatment discontinuation or abandon (NCT02477826, NCT03001882, NCT02982954 and NCT02231749) [3].
Thus, one of the main current challenge is how to improve cancer immunotherapy efficiency, without increasing autoimmune toxicities. An appropriate strategy may reside in a combinatorial approach able to overcome the numerous immunosuppressive networks of the tumor microenvironment (TME) [4] resulting in an "abscopal effect", a rare phenomenon first described in patients treated with radiotherapy, characterized by regression of tumor outside the radiation field [5].
The reproduction of such systemic reaction requires at least two major steps, as described in our published model [6]. First, inducing apoptosis of tumor cells and second, reprogramming the TME into a TH1 polarized response through an IL-12-based immunotherapy. One adequate translational approach would be to replace radiotherapy by proapoptotic peptides (PAPs) that also offer the possibility of targeting specifically the tumor through a systemic administration [7]. PAPs are sequences of amino acids that have the ability to induce apoptosis after binding cell-surface receptors. They are composed of two functional domains: a targeting domain designed to recognize tumor cell surface and a pro-apoptotic domain designed to be non-toxic outside of cells but toxic when internalized.
Here, we describe the combination of PAPs, composed of a protein transduction domain (PTD) peptide coupled to a killer peptide (KLAK) which disrupts eukaryotic mitochondrial membranes, along with an intratumor IL-12 based immunotherapy. This combination induces, in our murine model, an abscopal effect able to reduce massively the contralateral non-treated tumors ( Figure 1A) and consequently to prolong dramatically the mice-bearing tumor survival ( Figure 1B). In this report, we show that depletion of CD8+ T cells suppresses completely the abscopal effect on distant contralateral tumors, which in turn experience a dramatic progression (Figure 2A).
Our report shows for the first time the feasibility of inducing an abscopal effect through a synergistic combinatorial approach including locally administered PAPs and IL-12 based immunotherapy. This combination generates a potent antitumor immunity that acts as "in situ tumor vaccination" able to antagonize efficiently at multiple levels the tumor immunosuppressive environment ( Figure 3A). The mechanisms implicate a rebalance of cytokine profile leading to a TH1-dominant immune response, through an IL-12 induced differentiation of naïve T cells [8,9]. Consequently, immunosuppressive cytokines produced by TH2 pre-existing cells are reduced and cross presentation of dendritic cells (DCs) is enhanced by the up-regulation of interferon gamma (IFNγ) secretion from TH1 cells. This process also decreases the prevalence of immunosuppressive actors like myeloid-derived suppressor cells (MDSCs) and -old female BALB/c mice, as described previously [6,9]. The different treatments were initiated when tumor reach a volume~0.3cm 3 . Only tumor on one side was co-injected with mouse recombinant IL-12 (R&D systems) (delivered at 10 μg/day for a period of 7 days) in the presence or absence of 300 μg (30 μl final) of PAPs (PTD/KLAK) [6] for a period of 7 days only. After that, the contralateral tumor (non-treated side) volume were evaluated twice weekly using a caliper. (A), contralateral tumors (non-treated side) in mice treated concomitantly with IL-12 and PAPs were significantly smaller (Student's t-test; P b .001) than untreated tumors (treated with PBS, control) or those treated with either IL-12 or PAPs alone. (B), mice treated concomitantly with IL-12 and PAPs survived longer than the untreated mice (control) or those treated with IL-12 or PAPs alone, as shown by a Kaplan-Meier survival plot (n = 14 animals/group; P b .05, log-rank test). All animals were bred and maintained according the guidelines of both the Federation of European Laboratory Animal Science Associations (Tamworth, U.K.) and the Animal Experimental Ethics Committee (Paris, France). Animals were used at between 6 and 20 wk. of age. Animals bearing tumors in excess of 20-25% of the body mass were killed. T lymphocytes regulators (Tregs). IL-12 also promotes the proliferation and the cytolytic activity of NK cells and effector CD8+ T-cells. However, without the microenvironment reprogramming conferred by IL-12 co-administration, the apoptotic cell death is not by itself sufficient to allow an effective tumor rejection. Together, all these synergistic modifications on tumor microenvironment elicit a potent antitumor immunity leading to the rejection of distant metastatic tumors ( Figure 3B). As a translational extension from this observation, we postulate that the replacement of locally administered PAPs by an intravenous infusion (as described in [7]) may result in the same abscopal effect. Such scenario will undeniably open the door to the "democratization" of the abscopal effect in the clinical practice, which may bring new hope for cancer patients refractory to immune check-point blockade therapies.
Author Contributions MO conceived the study, performed the experiments and prepare the manuscript; FM prepared Figure 3; all authors wrote, commented and corrected the manuscript.