Poster X17 - Hybrid LNP Prime Dendritic Cells for Nucleotide Delivery

Introduction: Most cancers sustain an immunosuppressive microenvironment through elaborated mechanisms that are slowly being unraveled. Among the different immunotherapy approaches, cancer vaccination is of particular interest as it has the potential to elicit systemic and potentially durable anti-tumor effects. Cancer therapeutic vaccines take many forms, such as peptide admixed with adjuvant, adoptive cell therapy of antigen loaded dendritic cells (DCs), or mRNA delivery of encoded neo-antigens. Irrespective of the choice of tumor-associated antigens (TAA), immuno-stimulatory adjuvants are needed to boost vaccine effectiveness.

We hypothesized that it should be feasible to enhance double-stranded RNA (dsRNA) mediated toll-like receptor 3 (TLR3) effects through synergistic and complementary pathway activation in DC. Our prior work used a 37 nm cyclodextrin nanoparticle to deliver multiple small molecule immune modulators to tumor-associated macrophages and a lesser degree to DCs. Here we reasoned that an analogous strategy could be developed for dendritic cell-targeted vaccines but would have to be based on lipid nanoparticles (LNP) to improve dsRNA delivery. LNP has become the nanomaterial of choice for the delivery of nucleic acids given their considerable payload capacity, favorable pharmacokinetics, and ability to deliver cargo inside target cells.

Here we describe the synthesis and testing of a new hybrid LNP that contains a dsRNA and an LNP internal, cyclodextrin nanoparticle as a “sponge” for a small molecule cIAP antagonist to enhance DC stimulation. We show the synthesis of the ≈120 nm hybrid LNP featuring nanoparticles-in-nanoparticle concept and that this approach effectively eradicate tumors and generate long-lasting, durable anti-tumor immunity.

Materials and

Methods: The LNPs were prepared by rapidly mixing an ethanol solution of lipids and an aqueous solution of therapeutic payloads with a micro fluidic device. The lipid solution comprised of C12-200, cholesterol, DSPC and mPEG2000-DMG at a molar ratio of 35:16:46.5:2.5 in ethanol (by volume). For fluorescent LNPs, 0.5 mol.% of ATTO647N-DOPE was added to the ethanol solution. For all LNPs, the aqueous solution was mixed in a 3:1 water:ethanol volume ratio. To prepare LNPs, the lipid solution and aqueous solution were injected simultaneously into the micro fluidic device using a two syringe pumps at a volumetric flow rate of 1:3. Ethanol and non encapsulated payloads were removed by dialyzing LNPs with PBS using molecular cut-off filter, then concentrating the LNPs using centrifuge filters. LNPs generated were ≈120 nm in size.

Cyclodextrin nanoparticles (CDNPs) were synthesized using succinyl-𝛽-cyclodextrin per the previously reported method. LCL-161 (0.5 mg, payload) was dissolved in DMSO (10 μL) and subsequently added to the CDNPs (5 mg) to achieve a final concentration of 25 mg/mL (5% DMSO). The size of L-CANDI was 38.2 ± 0.1 nm with zeta potential of −5 mV.

To prepare hybrid LNP, The lipid solution was similar to the LNP1 formulation mentioned above and the prepared CDNPs were incorporated in the aqueous solution. C57/BL6 mouse models were used to study different aspects of LNP delivery and therapeutic efficacy.

Results, Conclusions, and Discussions: Our study shows that high-efficiency hybrid LNP (“nanoparticle-in-nanoparticle”) can be synthesized to dually stimulate DCs via different cellular pathways and that this stimulation can be sufficient by itself to provide tumor control. We used a commonly employed LNP formulation to encapsulate i) low molecular weight poly I:C for stimulation of the TLR3 pathway and ii) lipophilic cyclodextrin nanoparticles as a reservoir for small molecule payloads to affect complementary, immune stimulatory pathways inside DC. In particular, we started with cyclodextrin nanoparticle to entrap a cIAP inhibitor (LCL-161). To efficiently internalize the cyclodextrin nanoparticle into LNP, we modified its surface with different lipids and found palmitic acid to be the most efficient. We estimated that a mean LNP (120 nm) thus had ≈1 cyclodextrin nanoparticle each with an estimated maximum capacity of ≈1500 molecules of LCL-161 and ≈100 molecules of poly I:C calculated by using a molecular volume model according to previously published literature. Fluorescent versions were used to determine the PK and cellular distribution. These data show that LNP17 had a T1/2 of 30 min and accumulated in the tumor, lymph nodes, and liver These favorable PK likely contributed to the overall efficacy of the approach.

our LNP-based delivery approach achieves complete tumor eradication using doses of 5 μg with single administration, demonstrating ~40× increase in potency compared to Prior direct immunotherapy studies. In this study, we show the efficacy and lack of toxicity of the hybrid LNP immunostimulant at pharmacological concentrations. Several future experiments would be logical extensions to further improve effectiveness. For example, it is possible to further load the cyclodextrin nanoparticle with additional small molecule payloads to induce DC3, and type 1 cell-mediated immunity, effector pathways. Second, it is possible to target the hybrid LNP to DC through the use of antibodies. Such antibodies could be anchored on the surface of hybrid LNPs. Third, it is possible to arm LNP with tumor-associated antigens, including peptides or mRNA expressing tumor antigens. Irrespective of these additional modifications, the current first generation of hybrid LNP is already a highly efficient dendritic cell stimulant.

Acknowledgements (Optional): We thank Megan Mayer at the Harvard cryo-EM Facility for collecting the cryo-EM images, Yoshiko Iwamoto for help with tissue sectioning and Drs.
Lee and Im for helpful suggestions. This work was partly supported by the CSB Development Fund and R33 CA277820. R.D. and H.M.P. were
supported by T32CA079443. E.A.H. was supported by the Swiss National Science Foundation (P2EZP2_191831). I.R.F. was supported through the
Boehringer Ingelheim Fond supported IRF.