Poster X17 - Plug and play targeting strategy for polymeric and lipid nanoparticles via covalent attachment of IgG

Introduction: Nanoparticles (NPs) have become a leading drug delivery strategy for cancer therapies due to the enhanced permeability and retention (EPR) effect. The EPR effect describes how NPs passively accumulate in tumor tissue due to “leaky” tumor vasculature and are retained within the tumor interstitium due to weakened lymphatic drainage. Despite the EPR effect, less than 1% of an injected NP dose typically accumulates within tumor tissue. The use of targeting moieties, such as antibodies, can increase NP uptake at the intended target and provide a mechanism for tumor cell-specific uptake. However, improved methods for antibody-NP conjugation are needed. Covalent attachment methods, such as click chemistry, can result in random antibody orientation on the NP surface. They also typically require antibody modifications, which may lower the antibody’s affinity for its target. Conversely, protein-affinity based attachment methods require multiple preparation steps, and offer unstable, non-covalent binding.

We have developed an approach to covalently link nearly any IgG antibody to an NP surface, with no modifications to the antibody needed. This method uses a photoreactive antibody binding domain (pAbBD) that site-specifically conjugates to IgG after activation under UV light. pAbBD is expressed and purified with an azide or DBCO group and then linked to polymers or lipids via click chemistry. The resulting polymer/lipid-pAbBD conjugates are synthesized and purified without affecting pAbBD’s ability to photocrosslink with IgG. They are then incorporated into polymeric micelles or lipid nanoparticles (LNPs), providing an easy, cost-effective method to link IgG to NPs for targeted drug delivery.

Materials and

Methods: pAbBD was expressed and purified from bacterial culture with either an azide (N3) or DBCO group attached to the C-terminal end using sortase-tag expressed protein ligation (STEPL), following established methods. pAbBD-DBCO was attached to Poly(ε-caprolactone)[3kDa]-poly(ethylene glycol)[5kDa]-N3 (PCL-PEG-N3), and pAbBD-N3 was attached to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl(polyethylene glycol)-2000] (DSPE-PEG-DBCO) using click chemistry (Figure 1A-B). Reactions were done in dimethyl sulfoxide (DMSO) at a 1:8 molar ratio of protein:polymer/lipid and mixed overnight at 37C. The resulting solutions were added to an excess of toluene to precipitate the polymer/lipid-pAbBD product. The dried and purified product was resuspended in either DMSO or water for future characterization.

Polyacrylamide gel electrophoresis (PAGE) was used to verify click reaction completion and successful purification. Gels were stained for both protein (Coomassie) and PEG (barium chloride/iodine). To confirm pAbBD functionality after purification, pAbBD-polymer/lipid in water was mixed with the IgG trastuzumab and subjected to UV light at 4C for 2 hours for site-specific photocrosslinking. Success of the photocrosslinking reaction was confirmed via PAGE. To synthesize pAbBD-NPs, PCL-PEG-pAbBD was mixed with PCL-PEG to form micelles using the single emulsion solvent evaporation method. Similarly, DSPE-PEG-pAbBD was added to a lipid mixture of ionizable lipid (SM-102), cholesterol, DSPC, and DSPE-PEG(2k) in ethanol and added to PBS (pH 7) while sonicating and mixing to produce pAbBD-LNPs. pAbBD-NPs and pAbBD-LNPs were mixed with an excess of IgG and subjected to UV light for 2 hours at 4C for antibody-NP conjugation. Excess IgG was removed via size exclusion chromatography.

Results, Conclusions, and Discussions: The click chemistry reaction between pAbBD-DBCO/N3 and PCL-PEG-N3/DSPE-PEG-DBCO goes to completion at a 1:8 molar ratio of protein:polymer/lipid (Figure 1D-E). No free pAbBD is seen via PAGE following the reaction. The PCL-PEG-pAbBD product is a thick band at ~20kDa. This is as expected, given the combined molecular weight of pABbD (8kDa) and PCL-PEG-N3 (10kDa) should be 18kDa. Dual Coomassie (blue) and iodine (dark orange) staining verifies the presence of both protein and PEG in the product. Conversely, pAbBD alone stains only blue, and PCL-PEG-N3 alone stains only orange. The excess polymer appears as an orange smear below the PCL-PEG-pAbBD product, which is absent after purification. Similarly, the DSPE-PEG-pAbBD product is a clear band just above 10kDa, with an expected molecular weight of 11kDa. Again, no free pAbBD is seen in the product, and all excess lipid is removed after purification.

To confirm the functionality of pAbBD, the pAbBD-polymer/lipid conjugates were resuspended in water following purification. Each was mixed with increasing amounts of IgG and subjected to UV light. PAGE of the crosslinked products confirms pAbBD can still crosslink with IgG (Figure 1F-G). In excess amounts of IgG, both PCL-PEG-pAbBD and DSPE-PEG-pAbBD were fully saturated. Successful photocrosslinking was also confirmed by an observed ~20kDa or ~10kDa shift in the IgG heavy chain after attachment of PCL-PEG-pAbBD or DSPE-PEG-pAbBD, respectively (Figure 1H-I).

Following purification, the pAbBD-polymer/lipid conjugates were resuspended in DMSO. PCL-PEG-pAbBD can be incorporated into PCL-PEG micelles and DSPE-PEG-pAbBD can be incorporated into ionizable lipid nanoparticles upon addition to aqueous solvents. Following NP synthesis, any IgG can be conjugated to the nanoparticle surface (Figure 1C).

We have developed a simple, cost-effective method to site specifically attach antibodies to NPs. This method requires no antibody modifications, which helps conserve the antibody’s affinity for its target. We have also proven the robustness of pAbBD to undergo transition between multiple organic solvents and retain functionality. This covalent antibody-nanoparticle conjugation method can potentially achieve improved NP specificity and tumor accumulation in vivo. Additionally, it provides a “plug and play” approach that other scientists can adopt to develop and screen new targeted drug delivery systems.

Acknowledgements (Optional):