(M1230-03-16) Systemic Delivery of Paclitaxel by Find-Me Nanoparticles Activates Anti-tumor Immunity and Eliminates Tumors

Purpose: The premise of delivering immune-activating agents is to overcome the immunosuppressive environment of tumor and help the immune system to effectively attack cancer cells. The objective of the study is to enable systemic delivery of immune-activating agents to tumors. To this end, poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were loaded with paclitaxel (PTX), a chemotherapeutic agent inducing immunogenic cell death (ICD), and decorated with adenosine triphosphate (ATP), a damage-associated molecular pattern. The underlying hypothesis is that the ATP-modified NPs can be delivered to solid tumors by systemic administration, where PTX generates tumor antigens in situ and ATP recruits antigen-presenting cells (APCs) to facilitate cancer immunotherapy.

Methods: PTX-loaded PLGA NPs were prepared with the single emulsion method. The NPs were then coated with polydopamine by incubation in dopamine solution at pH 8.5 for 2h. Polydopamine-coated PLGA NPs (NP_pD) was further incubated with ATP to produce ATP-coated NP (NP_pD-ATP). NP_pD-ATP was tested with respect to the functionality of ATP to attract dendritic cells by the Boyden chamber assay. Cytotoxicity of PTX loaded in NP_pD-ATP was tested with murine CT26 colorectal carcinoma cells and human THP1 monocytes. The effects of PTX-loaded NP_pD-ATP (PTX@NP_pD-ATP) on tumor growth and tumor immune microenvironment were assessed in CT26 tumor-bearing immune-competent female BALB/c mice. Finally, the PTX@NP_pD-ATP was administered to CT26 tumor bearing mice by intravenous injection (q3dx4) in combination with anti-PD-l antibody (intraperitoneal injection, q3dx4).

Results: The z-average of PTX@NP_pD-ATP was 206 ± 7.2 nm. PTX and ATP loading contents were 3.2 wt% and 10.5 wt%, respectively. NP_pD-ATP was as effective as free ATP in attracting monocytes and dendritic cells across a Transwell insert with a pore size of 8 µm. PTX@NP_pD-ATP was selectively toxic to CT26 cells as compared to THP1 cells. The antitumor activity of PTX@NP_pD-ATP was first examined by intratumoral injection to CT26-tumor bearing BALB/c mice at a dose equivalent to 5 mg/kg PTX. Among the tested treatments, PTX@NP_pD-ATP demonstrated the best anti-tumor efficacy, resulting in complete tumor regression in 2 out of 5 mice after a single intratumoral injection (Fig. 1a). Other PTX formulations, such as PTX@NP_pD, a nanocrystal (NC) formulation of PTX¹ shown to be superior to the commercial benchmark Abraxane² in anti-tumor efficacy, and their mixtures with free ATP, showed no difference from each other and were not as effective as PTX@NP_pD-ATP. They led to a slight extension in survival but no complete regression. The same test was repeated with another set of CT26-bearing BALB/c mice and monitored for the immune cell population in tumor at 7 days after the intratumoral injection (Fig. 1b). PTX@NP_pD-ATP-treated mice showed a significantly higher number of dendritic cells than PTX@NP_pD + ATP (mixture) group or PBS group. The numbers of M1-like macrophages increased in both PTX@NP_pD-ATP and mixture groups compared to PBS. M2-like macrophages also increased in the mixture group, but not as much in the PTX@NP_pD-ATP group, resulting in a favorable M1/M2 ratio in the latter. PTX@NP_pD-ATP significantly increased the number of CD8⁺ T cells within the tumor compared to PBS but also increased regulatory T (Treg) cells. Since PTX@NP_pD-ATP administration increased the immunosuppressive Treg population, it was combined with anti-PD-1 antibody (Fig. 2a) to downregulate the PD-1/PD-L1 signaling implicated in Treg development and function.³ The PTX@NP_pD-ATP and anti-PD-1 antibody combination resulted in complete tumor regression in 75% (6/8) of the mice and delayed tumor growth in the other two mice (Fig. 2b). PTX@NP_pD-ATP alone delayed the tumor growth and extended the median survival time but did not achieve a complete regression. Anti-PD-1 antibody alone showed only a marginal effect with no complete regression of tumors. The surviving mice with complete tumor regression in the group treated with PTX@NP_pD-ATP and anti-PD-1 antibody combination were rechallenged with live CT26 cells to test whether the treatment helped them to develop immunological memory of CT26 tumor. All surviving tumor-free mice did not show tumor growth, whereas all age-matched naïve mice grew tumors in 9 days after the inoculation (Fig. 2c), indicating that systemically administered PTX@NP_pD-ATP and anti-PD-1 antibody induced anti-tumor immune memory to protect the mice from tumor rechallenge.

Conclusion: Systemically administered PTX@NP_pD-ATP induced the regression of tumors and extended the survival of animals with the assistance of the host immune system. Combined with the anti-PD-1 antibody, PTX@NP_pD-ATP led to the complete regression of CT26 tumors and the acquisition of anti-tumor immune memory.

References: (1) Park, J.; Sun, B.; Yeo, Y. Albumin-coated nanocrystals for carrier-free delivery of paclitaxel. Journal of Controlled Release 2017,263, 90-101. DOI: 10.1016/j.jconrel.2016.12.040.
(2) Park, J.; Park, J. E.; Hedrick, V. E.; Wood, K. V.; Bonham, C.; Lee, W.; Yeo, Y. A Comparative In Vivo Study of Albumin-Coated Paclitaxel Nanocrystals and Abraxane.Small 2018,14(16), e1703670. DOI: 10.1002/smll.201703670.
(3) Cai, J.; Wang, D.; Zhang, G.; Guo, X. The Role Of PD-1/PD-L1 Axis In Treg Development And Function: Implications For cancer Immunotherapy. Onco Targets Ther 2019,12, 8437-445.
DOI: 10.2147/OTT.S221340


Fig. 1. (a) Schedule of CT26 tumor inoculation and treatments in immune-competent BALB/c mice; changes in CT26 tumor size after an intratumoral injection of each treatment (PTX: 5 mg/kg); survival of the mice over time after the treatment, n=5 per group. (b) Immune cell population in CT26 tumors at day 7 after a single intratumoral treatment (PTX: 5 mg/kg), n=5 per group. Mix: PTX@NPpD + ATP; ATP-NP: PTX@NPpD-ATP. P-values were calculated by one-way ANOVA with Tukey’s multiple comparisons test.

Fig. 2. (a) Schedule of CT26 tumor inoculation and treatments in immune-competent BALB/c mice. Anti-PD-1 antibodies were given from 7 days after the initial treatment. IP: Intraperitoneal injection. PTX@NPpD-ATP: PTX 20 mg/kg/IV injection; anti-PD-1 antibodies: 150 μg/mouse/IP injection. (b) Individual growth curves of tumors and the survival of the mice over time after the treatment, n=8 per group. CR: Complete regression. Brackets at the bottom indicate the time of each treatment. (c) Individual growth curves of tumors and the percentage of tumor-free mice after a rechallenge with CT26 tumor cells. Surviving with CR: mice that were treated with PTX@NPpD-ATP and anti-PD-1 antibodies and reached complete regression (n=6); Age-matched control: age-matched tumor naïve mice (n=5).