(M1530-08-49) In Vivo Investigation of Double TLR4-TLR9 Activating Nanocomplexes for Cancer Immunotherapy

Purpose: In recent years, the immunomodulatory potential of many nanoparticles (NPs) has gained attention, not merely as an adverse effect, but as a promising avenue for therapeutic applications [1]. Previous work in our laboratory has demonstrated that ammonio methacrylate copolymer nanoparticles (AMCNPs) can stimulate the immune response by activating Toll-like receptor 4 (TLR4). The extent of the immune modulation depends on the physicochemical properties of the NPs, particularly their surface charge. The immunostimulatory properties of the NPs were successfully exploited to significantly retard tumor growth in a subcutaneous colorectal tumor model in mice [2]. Building on this, the present study aims to develop a nanoparticulate system capable of simultaneously activating two distinct TLRs, with the potential to enhance immunotherapeutic outcomes. To this end, nanocomplexes comprising two types of AMCNP with different degrees of cationic surface charge (acting as the TLR4 activator) and CpG oligonucleotides (CpG-ODN; acting as the TLR9 agonist) were formulated and evaluated for physicochemical properties, CpG-ODN complexation efficiency and release in vitro, and anticancer immunotherapeutic efficacy in vivo.

Methods: AMCNPs were prepared using types A and B AMCs with 10% and 5% quaternary ammonium content (coded RL-NP and RS-NP, respectively) via a modified instantaneous emulsification-solvent diffusion technique. After preparation, CpG-ODN2395 was complexed with the AMCNPs to form RL-CpG-NP and RS-CpG-NP. In brief, 100 µl of the CpG-ODN solution was added to 1 ml of the AMCNP formulation under sonication to achieve final concentrations of 10 mg/ml for AMCNPs and 300 µg/ml for CpG-ODN.The NPs were characterized in terms of size and zeta potential using a Nanoparticle Analyser SZ-100 (HORIBA). The CpG-ODN complexation efficiency was indirectly determined through the quantification of free CpG-ODN in the supernatant following the centrifugal sedimentation of the NPs, using a NanoDrop Microvolume UV Spectrometer (Thermo Scientific). The release of CpG from the RL-CpG-NP and RS-CpG-NP was investigated at both physiological pH (7.4) and the approximate pH of the tumor microenvironment (5.5) using a sample-and-separate method. Quantification of the free CpG-ODN was carried out as described above. The immunotherapeutic potential of double TLR-activating NPs was compared to that of the single components in vivo. Briefly, 6-week-old male BALB/c mice were injected with 3x10⁵ C26 colorectal cancer cells (National Cancer Institute) into their right flank. Treatment began once the tumor approached 40–50 mm³, with biweekly peritumoral injections of PBS, CpG-ODN, RL-NP, RS-NP, RL-CpG-NP or RS-CpG-NP. Tumor volume was calculated biweekly. Treatment was terminated once the tumor volume surpassed 1000 mm³, upon complete remission, or upon a body condition score of 1 combined with poor hair coat and severe lethargy, or upon more than 15% weight loss.

Results: Table 1 shows the size and zeta potential of the prepared NPs, as well as the complexation efficiency of the CpG-ODN. CpG-ODN release from both particles was in the form of a burst in the first 60 minutes, followed by a very slow release: almost a plateau for RL-CpG-NP and with a slight increase up to the last investigated time point for RS-CpG-NP. No significant difference was observed in the release at the tested pH values (7.4 and 5.5). Compared to RL-CpG-NPs, RS-CpG-NPs exhibited higher CpG-ODN release rates and extents (Figure 1A). When injected peritumorally, all NPs significantly delayed tumor growth compared to the PBS-treated control group (Figure 1B). While the tumor volume in most PBS-treated control animals surpassed 1000 mm³ within 38 days of tumor inoculation, significant retardation of tumor growth was observed in all other treatment groups (Figure 2B). Generally, animals treated with double TLR-activating NPs (RL-CpG-NP and RS-CpG-NP) survived longer than those treated with TLR4-activating RL-NP and RS-NP. However, the increase in estimated mean survival was only statistically significant for RS-CpG-NP (Figures 2C and 2D). One animal in the RS-CpG-NP group underwent complete remission.

Conclusion: This work involved the preparation of double TLR-activating nanocomplexes comprising the TLR4-activating RL-NP and RS-NP, as well as the TLR9-activating CpG-ODN. The particles were around 100 nm in size, had positive zeta potential values and a CpG-ODN complexation efficiency of over 80%. When injected peritumorally, both single- and double-TLR-activating formulations significantly delayed the growth of the subcutaneous colorectal tumor model compared to the PBS-treated control. Generally, double TLR-activating NPs led to a greater retardation of tumor growth than their single TLR-activating counterparts. However, the increase in estimated mean survival was only significant for the RS-CpG-NP formulation (p < 0.01 for RS-CpG-NP vs. RS-NP, and p ≈ 0.6 for RL-CpG-NP vs. RL-NP). This may be due to the greater release of CpG from RS-CpG-NP, which can promote systemic immune activation in addition to the localized immunotherapeutic effect. However, the veracity of this hypothesis is yet to be investigated. Future research should also focus on immunological readouts in the tumor microenvironment, such as immune cell infiltration, macrophage polarization, and the expression of markers associated with the activation of TLR4 and TLR9 pathways.

References: [1] B.S. Zolnik, Á. González-Fernández, N. Sadrieh, M.A. Dobrovolskaia, Minireview: Nanoparticles and the Immune System, Endocrinology. 151 (2010) 458–465. https://doi.org/10.1210/en.2009-1082.
[2] M.A. Shetab Boushehri, V. Stein, A. Lamprecht, Cargo-free particles of ammonio methacrylate copolymers: From pharmaceutical inactive ingredients to effective anticancer immunotherapeutics, Biomaterials. 166 (2018) 1–12. https://doi.org/10.1016/j.biomaterials.2018.02.053.

Table 1- Physicochemical properties of the single and double TLR activating NPs

Figure 1- (A) In vitro CpG-ODN release from RL-CpG-NP and RS-CpG-NP in physiological pH as well as the pH of the tumor microenvironment (5.5). RS-CpG-NP was associated with significantly higher release of CpG-ODN at both pHs. (B) Tumor volume (n=5) and (C) survival analysis of C26 tumor-bearing mice having received biweekly peritumoral injections of PBS, RL-NP, RS-NP, CpG-ODN, RL-CpG-NP, and RS-CpG-NP. (D) Log-rank analysis revealed a significant increase in the mean survival of the mice following treatment with all formulations (* p<0.05), while the mean estimated survival of RS-CpG-NP was highest compared to all other groups ($ p<0,05)