(55b) ICAM-1 Nanobody Density on Liposomes Affects Selectivity for Triple Negative Breast Cancer and Inflamed Endothelium

Introduction: Triple negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of ER, PR, and HER2 receptors and a high rate of metastasis. There are currently no commercially available targeted therapies for the treatment of TNBC. To address this deficiency, we synthesized liposomes conjugated with nanobodies specific to ICAM1, a receptor that is overexpressed on both tumor-associated vasculature and TNBC. We hypothesized that liposome DDVs with higher anti-ICAM1 nanobody densities will bind a greater number of ICAM1 receptors to achieve stronger and more specific binding to target cells and increase vascular permeability through downstream ICAM1 signaling. We have employed a covalent conjugation chemistry that enables the DDV to retain binding specificity and biological activity in the presence of mouse plasma. Our data will show that ICAM1 nanobody density may be optimized to greatly improve TNBC targeting and penetration of tumor vasculature, resulting in improved tumor accumulation and growth inhibition.

Materials and Methods: Initial studies were performed with liposomes that were synthesized using lipids displaying nitriloacetic acid (NTA) and DiO, enabling conjugation of anti-ICAM1 nanobodies containing a his-tag and fluorescent detection of the liposomes respectively. Covalently-conjugated liposomes were synthesized using a published chemistry¹. The liposomes displayed nanobody densities ranging from 3,000 SdAb/μm² to 44,000 SdAb/μm². Binding experiments were performed at 4°C for 30 minutes to prevent internalization. Cell binding was analyzed by measuring DiO fluorescence of cells by flow cytometry. Endothelial cell (EC) monolayer penetration was assessed by performing transwell

experiments with TNF-α-stimulated and unstimulated confluent EC monolayers. To evaluate the downstream effect of ligand density on VE-cadherin dissociation from tight junctions, TNF-α stimulated HUVEC monolayers were treated with liposomes for 30 minutes at 37°C, followed by immunofluorescent (IF) staining for VE-cadherin and subsequent imaging on a confocal microscope.

Results and Discussion: Liposomes were synthesized with ligand surface densities ranging from 3,000 SdAb/μm² to 44,000 SdAb/μm². Cell binding studies with ICAM1-targeting NTA-liposomes indicated that the maximum relative binding to TNF-α-stimulated ECs and TNBC cells over corresponding control cell lines occurred at 33,000 SdAb/μm², where liposomes displayed approximately 4 and 3 fold higher binding respectively. Interestingly, the 33,000 SdAb/μm² formulation achieved the greatest penetration of both stimulated and unstimulated EC monolayers. IF staining of VE-cadherin on liposome-treated monolayers revealed that ligand densities of 22,000 SdAb/μm² and 33,000 SdAb/μm² resulted in 73% and 53% dissociation of VE-cadherin from tight junctions in TNF-α-stimulated EC monolayers, respectively.

Conclusions: We show that binding specificity for both TNBC and cytokine-stimulated ECs over non-diseased counterparts, EC permeability, and ICAM1 downstream signaling can be enhanced through optimization of anti-ICAM1 nanobody density on the surface of a liposome DDV, where the optimal surface density for all phenomena occurs at 22,000 – 33,000 SdAb/μm². This data can be used in the design of DDVs that permeabilize tumor-associated endothelium and bind cancer cells. Future research will be dedicated to elucidating the molecular mechanisms responsible for these observations and validating their influence on tumor targeting and therapeutic efficacy in vivo.

References:

1 Cong, Y. et al. Site-specific PEGylation at histidine tags. 23, 248-263, (2012).