Poster X1 - Antibody/nanobody-nanoparticle conjugates for specific cell-type targeting of nucleic acid therapeutics

Introduction: Polymeric nanoparticles (NPs) are versatile carriers for nucleic acid delivery due to their extended cargo release properties, ease of formulation, and ability to encapsulate and protect a wide range of cargo molecules [1]. However, targeting of polymeric NPs to specific tissues and cell types in vivo remains challenging [2]. To improve the specificity of NP delivery, we have conjugated antibodies to the surface of polymeric NPs primarily composed of poly (lactic acid)-polyethylene glycol with conjugated maleimide functional groups (PLA-PEG-Mal). The goal is to increase the targeting efficiency of these delivery vehicles, while still taking advantage of polymer chemistry and NP characteristics with respect to avoiding phagocytic clearance and enhancing tissue tropism. Antibodies (Abs), for example, have a high affinity for their target ligands, leading to higher levels of Ab-NP conjugate uptake compared to non-specific Ab-NP conjugate or unconjugated NP uptake in the literature [3]. However, Abs can impact the surface chemistry of NPs, and therefore their biodistribution. Smaller targeting moieties such as nanobodies (Nbs) are alternative options to functionalize NPs. Lee et al. have shown the increased success of antibody fragment NP conjugates over full length antibody-nanoparticle conjugates [4]. Nb-NP conjugates may be advantageous for balancing targeting capabilities with the benefits of polymer and NP characteristics. Epithelial cell adhesion molecule (EpCAM) is of interest for targeting epithelial cell in the lung; here we investigate EpCAM Abs and Nbs for NP targeting airway epithelia.

Materials and

Methods: PLA-PEG-Mal NPs encapsulating a fluorescent DiI dye were formulated by nanoprecipitation using a blend of PLA-PEG and PLA-PEG-Mal polymers at an appropriate ratio to obtain the desired percent maleimide (5%-50%). PLA-PEG-Mal NP size and zeta potential were evaluated via dynamic light scattering using a Malvern Zetasizer. Subsequently, conjugation of PLA-PEG NPs to Abs/Nbs was performed via maleimide-thiol chemistry [5]. Briefly, thiol groups on cysteine residues of the Abs were reduced with tris(2-carboxyethyl(phosphine)) (TCEP). (L)-Dehydroascorbic acid (dHAA) was used to re-oxidize the reduced thiol groups to restore native disulfide bridges within the Ab structure. Reduced Abs were incubated with PLA-PEG-Mal NPs create Ab-NP conjugates (Figure 1A). We produced both EpCAM and IgG NP-Ab conjugates. Targeting capabilities of Ab/Nb-NPs were evaluated in human bronchial epithelial (HBE) cells. DiI-loaded NP uptake was evaluated by flow cytometry and fluorescence microscopy.

Results, Conclusions, and Discussions: To assess the targeting potential of our EpCAM Ab-conjugated NPs, we quantified uptake of EpCAM Ab-conjugated, IgG Ab-conjugated, and unconjugated PLA-PEG-Mal NPs in HBE cells. HBE cells were treated with different NP conjugates for 2 hours. By flow cytometry, 27.13% of HBE cells were EpCAM Ab-NP positive, whereas only 3.01% and 5.45% of cells were IgG Ab-NP positive or unconjugated NPs positive, respectively (Figure 1B). These data suggest that conjugation of a targeting antibody to the surface of NPs significantly increases the uptake in target cell types compared to isotype or unconjugated controls. We are currently optimizing the % maleimide composition of NPs enabling sufficient Ab/Nb conjugation and targeting while minimizing the extent of NP surface chemistry alteration.
While Ab-NP conjugates are effective in cell culture, they may not be as effective in vivo. For example, the Fc region of antibodies may be immunogenic, which can limit the circulation time of Ab-NP conjugates if administered intravenously. Additionally, surface-conjugated Abs covering much of the NP surface may hinder the natural tissue tropism of polymer chemistry and nanoparticle characteristics. We are now exploring engineered Nb-NP conjugates to evaluate whether this might be a viable strategy for targeting in vivo. Nanobodies are approximately 15 kDa in molecular weight making them around 10 times smaller than full length antibodies, while maintaining targeting capabilities. We have designed Nbs with a terminal cysteine residue to mediate conjugation to NPs via maleimide chemistry. Nb-NP conjugates are currently being compared to Ab-NP conjugates in cell culture and in vivo to evaluate cell uptake and blood circulation half-life.

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References:
1. AS. Piotrowski-Daspit et al. Adv Drug Deliv Rev. 2020;156:119-132
2. S. Sharma et al. Current Pathobiology Reports 2021; 9, 133–144
3. V.L. Luks et al. PLoS One. 2022. 17(4):e0266218.
4. NK Lee et al. Nano Converg. 2021; 16;8(1):24.
5. I. N. Gober et al. Journal of Peptide Science. 2021; 27(7):e3323