Poster N11 - Advancing targeted drug delivery with humanized antibodies and long-term storage strategy for maintaining in vivo efficacy

Introduction: Nanoparticles loaded with therapeutic reagents have emerged as a promising strategy in cancer research because of their enhanced biodistribution and ability to modify for targeted drug delivery. Despite the potential, translating the laboratory success to clinical application has remained a major challenge. This challenge includes reproducibility, lack of humanized targeting moiety and long-term storage strategies. Our lab has developed decreased non-specific adhesivity, receptor-targeted (DART) NPs with monoclonal antibodies as targeting ligands on the surface (Fig. 1). This innovative DART loaded with paclitaxel (PTX) is engineered to specifically target Fn14 receptor, an overexpressed marker in various cancers, has shown remarkable efficiency in targeting neoplastic cells in a preclinical model of triple-negative breast cancer brain metastasis and outperformed FDA-approved PTX formulation Abraxane. To advance clinical translation, we have formulated DART with humanized targeting moiety that can target Fn14 building upon our previous findings with mouse-derived targeting moiety. Simultaneously, we have studied a suitable storage condition for the DART with the aim of a stable, ‘ready-to-inject’ dosage at desired concentrations.

Materials and

Methods: The screening for humanized antibodies was carried out compared to mouse ITEM4 based on our previous studies. Fn14-specific binding and binding affinity of the screened humanized antibodies were conducted with Surface plasmon resonance (SPR). DART nanoparticles were formulated by using PLGA, mPEG-PLGA and PLGA-PEG-Maleimide via single emulsion method. The antibodies were modified with 2-Iminothiolane to add thiol groups to the antibodies and the thiolated antibody conjugation with the nanoparticles was carried out utilizing the thiol-maleimide reaction. The nanoparticles were stored in different conditions to assess long-term storage strategy. Finally, we tested our long-term stored DART with humanized antibodies on clinically relevant in vivo models to evaluate in vivo efficacy.

Results, Conclusions, and Discussions: In order to select the appropriate targeting antibody, we screened humanized glycosylated and aglycosylated antibodies and compared their specific binding affinity with Fn14 compared to mouse ITEM-4 which was used in our previous studies. The binding affinity of the thiolated antibodies was also evaluated along with thiol per antibody assessment. Based on the data, we selected aglycosylated antibody (IgG4 subtype) for further study. DART nanoparticles with humanized antibodies (~100 nm size) were stored in different temperature conditions with various sucrose concentrations for at least one month. The stored DART were evaluated for physicochemical properties such as size, PDI, zeta potential, drug loading and most importantly, Fn14-specific binding. The physicochemical properties of the stored samples clearly demonstrated that sucrose helps preserve DART nanoparticles' properties compared to freshly prepared samples. Further, we tested DART stored at our best storage condition for in vitro cytotoxicity, revealing that therapeutic efficacy is also preserved at the storage condition. Finally, the in vivo efficacy of the long-term stored DART nanoparticles was evaluated in a clinically relevant mouse model which showed that DART nanoparticles can be stored in a ‘ready-to-inject’ dose without deteriorating its in vivo efficacy.

In conclusion, our study successfully formulates nanoparticles with humanized targeting moiety which maintain its size, PDI, zeta potential and most importantly the Fn14-specific binding ability. The comprehensive storage study clearly explains the optimum storage conditions for antibody-based targeted nanoparticles. Moreover, this study shows that DART can be stored at a ‘ready-to-inject’ dose for several months. By bridging the gap between laboratory success and clinical translation, this study paves the way for the full therapeutic potential of DART nanoparticles in treating cancer.

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