Abstract B134: Use of p19Arf/interferon-β immunotherapy in association with chemotherapy permits reduced drug dosage and avoids cardiotoxicity associated with doxorubicin
Authors: Strauss Bryan E. , Medrano Ruan F.V. , Tamura Rodrigo , Mendonça Samir A. , Feitosa Valker A. , Dariolli Rafael , Salles Thiago A. , Hunger Aline , Catani João P.P. , Rodrigues Elaine G. 
Published: February 2019

Abstract
Background: A variety of methods have been shown to induce immunogenic cell death (ICD) and overcome the immunosuppressive tumor microenvironment, thus generating an effective immunotherapy. In our previous work, we have shown that non-replicating adenovirus mediated gene transfer of p19Arf and interferon-β (IFNβ) forms a strategic alliance between gene function and the vector, resulting in a multi-modal mechanism of cell death consistent with necroptosis and with the liberation of ICD molecules HMGB1, ATP and calreticulin in B16F10 mouse melanoma cells. We have performed vaccine and immunotherapy assays as well as in situ vaccination in order to show that our gene transfer approach contributes to an antitumor immune response that involves neutrophils, NK, CD4+ and CD8+ T-cells and cytokine production that is consistent with a Th1 profile. While promising so far, we strive to boost tumor cell killing while preserving the well-being of the study subject. Objective: To use our gene transfer approach in association with doxorubicin (Dox), a well-known inducer of ICD, in order to promote cell killing while permitting the use of reduced drug dosages, thus ameliorating collateral effects, including cardiopathy, and possibly benefitting the antitumor immune response. Materials and Methods: MCA205 and B16F10 cells were used in this study. The AdRGDPG vectors are non-replicating recombinant Ad5 viruses containing the RGD tripeptide modification of the virus fiber protein and which utilize a p53-responsive promoter (termed PG) to control transgene expression. The AdRGDPG-GFP, AdRGDPG-p19, and AdRGDPG-IFNβ vectors encode enhanced green fluorescent protein (GFP), the mouse cDNAs for p19Arf (a functional partner of p53) and IFNβ, respectively. Using flow cytometry (Attune, Thermo Fisher), accumulation of hypodiploid cells was revealed upon fixation and propidium iodide staining while caspase 3/7 activity was visualized using Cell Event Caspase 3/7 (Thermo Fisher). Alternatively, BLI (IVIS Spectrum, Perkin Elmer) was used to detect a DEVD-dependent luciferase reporter construct in vivo. Cardiac function was monitored using echocardiography (VisualSonics Vevo 2100 Imaging System) and data analyzed using VevoCQ LV analysis software (VisualSonics). All animal assays were performed according to and with the approval of the Committee for the Ethical Use of Animals, FM-USP. Results: Association of p19Arf/IFNβ gene transfer with Dox treatment in vitro improved cell killing and central composite rotational design analysis revealed that indeed, the association permits the application of less drug and/or virus while preserving high levels of death in either cell line. In vitro, caspase 3/7 activity was additive upon exposure of MCA205 cells to the associated treatments, yet Dox was sufficient in B16F10. In vivo, Dox (20 mg/kg) alone or in combination with in situ gene therapy was correlated with inhibition of MCA205 tumor progression, increased caspase 3/7 activity and prolonged survival. However, 20 mg/kg Dox provoked reduced cardiac function (left ventricular ejection and systolic fractions, stroke and systolic volumes). Strikingly, the sub-therapeutic dose of 10 mg/kg Dox was not correlated with cardiopathy, yet only when used in combination with in situ gene transfer did it effectively block tumor progression. Finally, immunotherapy of previously established MCA205 tumors using a therapeutic vaccine consisting of dying cells treated ex vivo with both gene transfer and Dox resulted in effective challenge tumor inhibition. Conclusions: Here we have clearly shown that the association of gene transfer with Dox enables the use of lower drug doses in order to both preserve cardiac function and inhibit tumor progression. Early evidence also suggests that this combination increases the immunogenicity of treated cells, inviting the development of potential immunotherapeutic strategies. Funding: Sao Paulo Research Foundation (FAPESP).
Citation Format: Bryan E. Strauss, Ruan F.V. Medrano, Rodrigo Tamura, Samir A. Mendonça, Valker A. Feitosa, Rafael Dariolli, Thiago A. Salles, Aline Hunger, João P.P. Catani, Elaine G. Rodrigues. Use of p19Arf/interferon-β immunotherapy in association with chemotherapy permits reduced drug dosage and avoids cardiotoxicity associated with doxorubicin [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B134.