Purdue University West Lafayette, Indiana, United States
Purpose: Despite recent advances in cancer immunotherapy, immunotherapy benefits only a small fraction of cancer patients with identified tumor antigens and/or well-accessible tumors. New therapeutic strategies are needed to improve the efficacy of immunotherapeutics in broader patient populations with hard-to-reach, unidentified tumors. An approach gaining interest in the immuno-oncology community is to treat locatable and accessible tumors locally and stimulate antitumor immunity in situ to exert systemic effects against distant tumors. To develop an effective local immunotherapy, we have produced a polyethyleneimine derivative (2E’), which activates immune cells and co-delivers hydrophobic immunogenic cell death inducers and immunomodulatory nucleic acids or nucleotides for systemic therapy of solid tumors. Methods: The immunoactive polymeric assembly was formed by sequential complexation of 2E’, PTX, and siPD-L1. The assembly was characterized by tranmission electron microscopy. 2E’/PTX/siPD-L1 was tested for the cytotoxicity, cellular uptake, PD-L1 silencing, and immunostimulatory effects on two murine cancer cells (CT26 colorectal carcinoma and B16F10 melonoma cells) and immune cells [bone marrow-derived dendritic cells (BMDC) and bone marrow-derived macrophages (BMDM)]. The antitumor effects of locally administered 2E’/PTX/siPD-L1 and 2E’/PTX/CDN were evaluated in B16F10@C57BL/6, 4T1-Luc@Balb/c and CT26@Balb/c models. Surviving tumor-free mice were re-challenged with live cells on the contralateral side to test if systemic antitumor immunity was established. Results: In vitro characterization of 2E’/PTX/siPD-L1: 2E’/PTX/siPD-L1 showed a spherical shape with an average diameter of 50 nm. 2E’/PTX/siPD-L1 was more toxic to CT26 cancer cells than 2E’ or PTX alone, indicating a synergistic effect of 2E’ and PTX. However, 2E’/PTX/siPD-L1 did not show significant cytotoxicity to immune cells such as BMDC and splenocytes. 2E’/PTX/siPD-L1 entered cancer cells and inhibited the PD-L1 expression in IFN-γ-stimulated B16F10 cells. Furthermore, 2E’/PTX/siPD-L1 induced the secretion of immunostimulatory cytokine TNF-α from BMDC and BMDM.
Single injection of 2E’/PTX/siPD-L1 primes systemic antitumor immunity in poorly immunogenic B16F10 tumors: A single treatment of 2E’/PTX/siPD-L1 inhibited tumor growth in both treated and untreated distant tumors, yielding the smallest tumor size in both sides compared with other groups (Fig. 1a). 2E’/PTX/siPD-L1 also induced significant increase of melanoma antigen-specific T cells in spleen compared to control groups (Fig. 1b), supporting that 2E’/PTX/siPD-L1 activated systemic antitumor immunity in poorly immunogenic B16F10 tumor model.
2E’/PTX/siPD-L1 inhibits growth and metastasis of 4T1 tumors: To evaluate broad utility of 2E’/PTX/siPD-L1, we tested it in orthotopic 4T1-Luc tumors, another poorly immunogenic tumor model, along with three control groups (D5W, 2E’/PTX/siNeg, and complete surgical resection to mimic a clinical scenario). In the D5W group, tumors grew exponentially, and all animals reached physiological endpoints in 19 days with widespread metastasis. In the surgery group, 80% showed tumor relapse in the surgical site in 13 days from the surgery, and all showed lung metastasis in 17 days (Fig. 2a). In contrast, 2E’/PTX/siNeg and 2E’/PTX/siPD-L1-treated groups showed immediate loss of luminescence signal (in 1 day) and shrinkage of tumors (in 2 days) following treatment. The treated tumors regrew in all of the 2E’/PTX/siNeg group, but 2E’/PTX/siPD-L1 showed complete tumor regression in 25% (Fig. 2b). The occurrence of lung metastasis was also reduced by these treatments, with 55% in the 2E’/PTX/siNeg group and 88% in the 2E’/PTX/siPD-L1 group showing no luminescence signals in the lungs by 17 days after treatment. The tumor-free mice in the 2E’/PTX/siPD-L1 group resisted the rechallenged 4T1-Luc tumor growth, whereas all of the age-matched naïve mice grew challenged tumor in 11 days (Fig. 2c).
2E’/PTX combined with CDN eliminates established tumors and develops antitumor immunity in CT26@Balb/c model: To test the applicability of 2E’ to other drugs, we replaced siPD-L1 with CDN, an agonist of the stimulator of interferon genes pathway. 86% of the 2E’/PTX/CDN-treated mice survived tumor-free after a single treatment (Fig. 3). When rechallenged with live CT26 cells, none of the tumor-free mice grew the rechallenged tumor for 66 days (duration of observation), while all the age-matched naïve mice did in 11 days and died in 34 days (Fig. 3). These results indicate that the single intratumoral treatment of 2E’/PTX/CDN induced strong antitumor immune memory. The surviving animals did not reject unrelated syngeneic 4T1 tumors (Fig. 3), indicating that the antitumor immunity was tumor-specific. Conclusion: 2E’ showed selective toxicity against cancer cells relative to immune cells. 2E’/PTX/siPD-L1 and 2E’/PTX/CDN induced potent antitumor immunity by a single administration, causing immediate regression of large established tumors, tumor-free survival, abscopal effect on distant tumors, reduction of metastasis, and the resistance to rechallenge. 2E’/PTX/siPD-L1 and 2E’/PTX/CDN complexes are promising delivery systems for local cancer immunotherapy. References: Meng, F., Wang, J., He, Y., Creswell, G.M., Lanman, N.A., Lyle, L.T., Ratliff, T.L., Yeo, Y., 2022. Proceedings of the National Academy of Sciences, In press.
Acknowledgements:This work was supported by NIH R01 CA232419 and NIH R01 CA258737. This work was also supported by the Indiana CTSI, funded in part by NIH UL1 TR002529, and the Purdue University Center for Cancer Research.
Fig. 1 Effects of a single injection of 2E’/PTX/siPD-L1 on systemic anti-tumor effect in B16F10@CT57BL/6 tumor model. a. Schedule of bilateral B16F10 tumor inoculation in C57BL/6 mice and treatment injection; tumor growth curves and tumor sizes on day 11 post-treatment of treated and untreated tumors after a single treatment of D5W (n=6), 2E’/siPD-L1 + PTX NC (n=8), 2E’/PTX/siNeg (n=9) or 2E’/PTX/siPD-L1 (n=9); meanSD. b. The frequency of Trp2 (SVYDFFVWL)-specific CD8+ T cells in the splenic CD3+ cells. Spleens were collected on day 7 after treatment.
Fig. 2. Effects of a single administration of 2E’/PTX/siPD-L1 on the growth and metastasis of 4T1 tumors. Bioluminescence imaging of the 4T1 tumor-bearing Balb/c mice following the treatment with D5W (n=5), surgical resection (n=5), 2E’/PTX/siNeg (n=9), or 2E’/PTX/siPD-L1 (n=8).repeated tumor challenge in CT26@Balb/c. b. Individual growth curves of 4T1 tumors in response to different treatments; the size of tumors on day 19 post-treatment, and percentage of tumor-free mice following (re)challenge.
Fig. 3. Effects of a single injection of 2E’/PTX/CDN on growth of CT26 tumors and development of antitumor immunity.
