Human epidermal growth factor receptor 2 (HER2) gene amplification and/or protein overexpression in tumors is a prerequisite for initiation of trastuzumab therapy. Although HER2 is a cell membrane receptor, differential rates of endocytosis and recycling engender a dynamic surface pool of HER2. Since trastuzumab must bind to the extracellular domain of HER2, a depressed HER2 surface pool hinders binding. Using in vivo biological models and cultures of fresh human tumors, we find that the caveolin-1 (CAV1) protein is involved in HER2 cell membrane dynamics within the context of receptor endocytosis. The translational significance of this finding is highlighted by our observation that temporal CAV1 depletion with lovastatin increases HER2 half-life and availability at the cell membrane resulting in improved trastuzumab binding and therapy against HER2-positive tumors. These data show the important role that CAV1 plays in the effectiveness of trastuzumab to target HER2-positive tumors.

Purpose: Interest in targeted alpha-therapy has surged due to a-particles' high cytotoxicity. However, the widespread clinical use of this approach could be limited by on-/off-target toxicities. Here, we investigated the inverse electron-demand Diels-Alder ligation between an 225 Ac-labeled tetrazine radioligand and a trans-cyclooctene-bearing anti-CA19.9 antibody (5B1) for pretargeted a-radioimmunotherapy (PRIT) of pancreatic ductal adenocarcinoma (PDAC). This alternative strategy is expected to reduce nonspecific toxicities as compared with conventional radioimmunotherapy (RIT).Experimental Design: A side-by-side comparison of 225 Ac-PRIT and conventional RIT using a directly 225 Ac-radiolabeled immunoconjugate evaluates the therapeutic efficacy and toxicity of both methodologies in PDAC murine models.Results: A comparative biodistribution study of the PRIT versus RIT methodology underscored the improved pharmacokinetic properties (e.g., prolonged tumor uptake and increased tumor-to-tissue ratios) of the PRIT approach. Cerenkov imaging coupled to PRIT confirmed the in vivo biodistribution of 225 Ac-radioimmunoconjugate but-importantly-further allowed for the ex vivo monitoring of 225 Ac's radioactive daughters' redistribution. Human dosimetry was extrapolated from the mouse biodistribution and confirms the clinical translatability of 225 Ac-PRIT. Furthermore, longitudinal therapy studies performed in subcutaneous and orthotopic PDAC models confirm the therapeutic efficacy of 225 Ac-PRIT with the observation of prolonged median survival compared with control cohorts. Finally, a comparison with conventional RIT highlighted the potential of 225 Ac-PRIT to reduce hematotoxicity while maintaining therapeutic effectiveness.Conclusions: The ability of 225 Ac-PRIT to deliver a radiotherapeutic payload while simultaneously reducing the off-target toxicity normally associated with RIT suggests that the clinical translation of this approach will have a profound impact on PDAC therapy.

Functional enzyme-nanoparticle bioconjugates are increasingly important in biomedical and biotechnology applications such as drug delivery and biosensing. Optimization of the function of such bioconjugates requires careful control and characterization of their structures and activity, but current methods are inadequate for this purpose. A key shortcoming of existing approaches is the lack of an accurate method for quantitating protein content of bioconjugates for low (monolayer) surface coverages. In this study, an integrated characterization methodology for protein-gold nanoparticle (AuNP) bioconjugates is developed, with a focus on site-specific attachment and surface coverage of protein on AuNPs. Single-cysteine-containing mutants of dihydrofolate reductase are covalently attached to AuNPs with diameters of 5, 15, and 30 nm, providing a range of surface curvature. Site-specific attachment to different regions of the protein surface is investigated, including attachment to a flexible loop versus a rigid α helix. Characterization methods include SDS-PAGE, UV-vis spectrophotometry, dynamic light scattering, and a novel fluorescence-based method for accurate determination of low protein concentration on AuNPs. An accurate determination of both protein and AuNP concentration in conjugate samples allows for the calculation of the surface coverage. We find that surface coverage is related to the surface curvature of the AuNP, with a higher surface coverage observed for higher surface curvature. The combination of these characterization methods is important for understanding the functionality of protein-AuNP bioconjugates, particularly enzyme activity.

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The inverse electron-demand Diels-Alder reaction between tetrazine (Tz) and trans-cyclooctene (TCO) facilitates the efficient radiosynthesis of Ac-labelled radioimmunoconjugates in a two-step method, outperforming conventional approaches based on isothiocyanate couplings.