(M1530-05-31) Anti-cancer Sonosensitive DOX-Loaded Liposomes to Avoid Systemic Immunosuppression

Purpose: Recent studies have shown that doxorubicin (DOX), a conventionally used anticancer drug, can stimulate antitumor immunity through immunogenic cell death (ICD) and is also effective in eliminating myeloid-derived suppressor cells, a major contributor to the immunosuppressive environment. However, despite these excellent effects, non-specific delivery of DOX can also affect immune organs and immune cells, representing a limitation as an immunotherapeutic agent to build systemic immunity. In this study, we developed an IMP301 formulation capable of cancer-specific delivery of DOX using ultrasound (US)-sensitive liposomes to evaluate its antitumor efficacy in a 4T1 cancer animal model and its efficacy in combination therapy with immune checkpoint inhibitors.

Methods: After completing the physicochemical characterization, we confirmed the drug release behavior as a function of US irradiation time under the US conditions (power, 80 W; duty cycle, 2%; pulse repetition frequency, 250 Hz) that can collapse IMP301 without generating heat. In addition, in accordance with ethical animal experimentation procedures, 4T1 tumor tissues were formed in the left thigh of BALB/c female mice and evaluated for in vivo anticancer efficacy under the same US conditions. After the end of the evaluation, the recruitment of immune cells was observed by staining with CD80 and CD8 antibodies in the tumor tissue, and the efficacy of combination treatment with immune checkpoint inhibitors was also evaluated in the same animal model.

Results: In vivo immunogenicity and systemic toxicity of IMP301+US: IMP301 shows promising properties with a Z-average size of 94.23 ± 24.51 nm and DOX loading of 12.22 ± 0.33%. In the absence of US, IMP301 released 26.5 ± 4.28% DOX content at 48 h, which significantly increased to 55.3 ± 3.54% with US. We evaluated IMP301+US immunogenicity in 4T1 tumor-bearing mice, observing CD80 fluorescent signals in treated tumors, indicating successful APC maturation and DOX-mediated ICD. Although DOX-induced hemosiderosis can impair CTL generation, IMP301+US showed reduced hemosiderosis in the spleen due to less off-target DOX delivery. In addition, higher CD8 fluorescent signals in tumors treated with IMP301+US suggested increased CTL tumor infiltration. These results suggest that IMP301+US can induce antitumor immunity by promoting ICD and enhancing CTL infiltration while minimizing spleen cell damage by preventing off-target DOX release. IMP301+US has the potential to improve cancer treatment outcomes. In vivo anti-tumor efficacy of IMP301+US combination therapy in 4T1 tumor-bearing mice: The therapeutic efficacy of IMP301+US was evaluated in 4T1 tumor-bearing mice to compare its therapeutic efficacy with DOX alone. Thirty days after cancer cell injection, DOX inhibited tumor growth by 50.75% and delayed tumor doubling time by 15 days. In comparison, IMP301+US significantly improved therapeutic efficacy, inhibiting tumor growth by 88.89% and delaying the time to tumor doubling by more than 20 days. This improvement could be attributed to reduced hemosiderosis in the spleen and enhanced CTL infiltration into tumor tissue without significant side effects on major organs. Evaluation of IMP301+US in combination with immune checkpoint inhibitors: In addition, the therapeutic potential of IMP301+US in combination with PD-1 checkpoint blockade (aPD-1) was evaluated in a bilateral 4T1 tumor model. After 30 days, the DOX+aPD-1 arm inhibited 57.2% of tumor growth, whereas the IMP301+US+aPD-1 group inhibited 97.7% of tumor growth and prolonged the doubling time by more than 20 days, significantly enhancing the efficacy of aPD1. Notably, IMP301+US completely eliminated tumors in 75% of mice, demonstrating improved immunogenicity compared to DOX monotherapy.

Conclusion: In this study, we evaluated the therapeutic potential of IMP301+US to promote ICD-mediated antitumor immune responses. Compared with DOX, IMP301+US effectively stimulated APC maturation, DOX-mediated ICD and CTL infiltration, reducing splenic injury. These results highlight the ability of IMP301+US to overcome doxorubicin-induced systemic immunosuppression through targeted DOX delivery to tumors. Notably, IMP301+US demonstrated superior tumor growth inhibition compared to DOX alone, with or without PD1 immune checkpoint therapy, by reducing hemosiderosis in the spleen and enhancing CTL tumor infiltration into tumor tissue. Sonoimmunotherapy, which utilizes US for cancer immunotherapy, is showing promise in the clinic due to its precision, safety, and accessibility; therefore, IMP301+US has potential as a treatment option that can be combined with other immunotherapies such as ICD-mediated cancer therapy or immune checkpoint blockade therapy.

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2. The Unfolded Protein Response in Immunogenic Cell Death and Cancer Immunotherapy. Trends Cancer 2017, 3, 643–658.
3. Ultrasound-Responsive Liposomes for Targeted Drug Delivery Combined with Focused Ultrasound. Pharmaceutics 2022, 14, 1314.

Acknowledgements:

Funding: This work was supported by the Biohealth Technology Development Program (20018412, Development of ultrasound-sensitive drug delivery system for treatment of cancer) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) and the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (RS-2023-00236265). Conflicts of Interest: The authors declare no financial interest.