Abstract 3952: Localized nanodelivery combined with immunotherapy promotes curative anti-tumor responses in a murine breast cancer model
Authors: Kheirolomoom Azadeh , Silvestrini Matthew T. , Ingham Elizabeth S. , Mahakian Lisa M. , Tam Sarah M. , Tumbale Spencer K. , Foiret Josquin , Hubbard Neil E. , Borowsky Alexander D. , Murphy William J. , Ferrara Katherine W. 
Published: August 2019

Abstract
Optimal pairing of chemotherapy with immunotherapy can harness potential synergies and augment tumor immunity in solid tumors with low mutation burden by i) reducing tumor bulk, ii) releasing tumor antigen for cross-presentation and cross-priming, iii) releasing cancer-suppressive cytokines/chemokines, and iv) retaining circulating immune cell populations. Temperature-sensitive liposomes (TSL) have the potential to accomplish these goals as ultrasound (US) insonation or other treatment modalities can release the chemotherapy within bulky solid tumors in response to hyperthermia to maximize the local response and minimize off-target effects. Here we used Doxorubicin (Dox), stabilized within the core of TSL by forming a drug complex with copper (CuDox), to mediate immunogenic cell death (ICD) and release of type I interferons (IFNs). This activatable chemotherapy (CuDox-TSL+US) was combined with immunotherapy using CpG-ODN, a toll-like receptor 9 agonist, and a PD-1 antibody (αPD-1) for checkpoint blockade. We assessed the schedule and sequence of the chemo-immunotherapy protocol to maximize therapeutic outcome.
CuDox-TSL were made of DPPC:MPPC:DSPE-PEG2k, 86:10:4 in the presence of copper (II) gluconate and triethanolamine at 0.2 mg-Dox/mg-lipid. Mice with bilateral invasive neu deletion (NDL) tumors (4 mm) were treated with an i.v. administration of CuDox-TSL at 6 mg Dox/kg body weight. One tumor was insonified with a peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz at 42°C for 5 min prior to and 20 min post drug injection with a variable duty cycle. Upon completion of US-hyperthermia, 100 µg of CpG-ODN 1826 was injected intratumorally to the insonified tumor. Three days later αPD-1 (200 µg, i.p.) was administrated.
We found that Dox in combination with hyperthermia induced release of ICD markers and type I IFNs in cell culture of various murine cancer cell lines. Following intravenous administration
of CuDox-TSL, ultrasonic activation of liposomes in the treated tumors promoted release and cross-presentation of a model tumor antigen by antigen-presenting cells in distant tumors. While a variety of chemo-immunotherapy protocols achieved a complete response in more than 50% of treated mice, the complete response rate was greatest (90%) when one week of immunotherapy priming preceded activatable chemotherapeutic administration. Repeated chemotherapeutic delivery, while effectively reducing viable tumor, also reduced efficacy and the fraction of viable CD8+ T-cells as indicated by flow cytometric analysis. Taken together, the results suggest that a single-dose administration of activatable chemotherapy after immune priming enhances survival in a murine model of breast cancer. In clinical practice, however, assessment of macrophage and T-cell populations over the course of treatment could help inform dose timing and number of treatment repetitions.
Citation Format: Azadeh Kheirolomoom, Matthew T. Silvestrini, Elizabeth S. Ingham, Lisa M. Mahakian, Sarah M. Tam, Spencer K. Tumbale, Josquin Foiret, Neil E. Hubbard, Alexander D. Borowsky, William J. Murphy, Katherine W. Ferrara. Localized nanodelivery combined with immunotherapy promotes curative anti-tumor responses in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3952.