Theoretical analyses of targeting agent pharmacokinetics provides specific guidance with respect to desirable design objectives such as agent size, affinity, and target antigen. These analyses suggest that IgG-sized macromolecular constructs exhibit the most favorable balance between systemic clearance and vascular extravasation, resulting in maximal tumor uptake. Quantitative predictions of the effects of dose and binding affinity on tumor uptake and penetration are also provided. The single bolus dose required for saturation of xenografted tumors in mice can be predicted from knowledge of antigen expression level and metabolic half-life. The role of high binding affinity in tumor uptake can be summarized as: essential for small peptides, less important for antibodies, and negligible for nanoparticles.

During directed cell migration (chemotaxis), cytoskeletal dynamics are stimulated and spatially biased by phosphoinositide 3-kinase (PI3K) and other signal transduction pathways. Live-cell imaging using total internal reflection fluorescence (TIRF) microscopy revealed that, in the absence of soluble cues, 3Ј-phosphoinositides are enriched in a localized and dynamic fashion during active spreading and random migration of mouse fibroblasts on adhesive surfaces. Surprisingly, we found that PI3K activation is uncoupled from classical integrin-mediated pathways and feedback from the actin cytoskeleton. Inhibiting PI3K significantly impairs cell motility, both in the context of normal spreading and when microtubules are dissociated, which induces a dynamic protrusion phenotype as seen by TIRF in our cells. Accordingly, during random migration, 3Ј-phosphoinositides are frequently localized to regions of membrane protrusion and correlate quantitatively with the direction and persistence of cell movement. These results underscore the importance of localized PI3K signaling not only in chemotaxis but also in basal motility/migration of fibroblasts. Journal of Cell Science 314 is largely PI3K dependent. This is seen most dramatically when microtubules are depolymerized using nocodazole, which induces rapid protrusion-retraction events as seen by TIRF. In the context of random fibroblast migration, formation of branched lamellipodia and turning events were found to coincide with localized enrichment of 3Ј-phosphoinositides. Results PI3K lipid products accumulate in a dynamic fashion during fibroblast spreadingTo assess the activation of PI3K signaling and its possible relation to adhesion-based motility, we established stable expression of the 3Ј-phosphoinositide-specific Akt pleckstrin-homology domain, fused with enhanced green fluorescent protein (EGFP-AktPH) in NIH3T3 fibroblasts and monitored these cells by TIRF microscopy as they attached and spread on glass coated with fibronectin or poly-D-lysine (Fig. 1). Glass coated with bovine serum albumin, which was also present in our imaging buffer, does not promote adhesion of these cells (results not shown). After allowing the cells to spread for 30-50 minutes, a saturating dose of PDGF was added to evaluate the maximal activation of PI3K in each cell, followed by a large dose of PI3K inhibitor to evaluate the fluorescence intensity associated with EGFP-AktPH in the cytosol; the latter is used to normalize the PI3K-dependent response (Schneider and Haugh, 2004).Cells spreading on fibronectin consistently showed dynamic enrichment of 3Ј-phosphoinositides, with transient bursts of signaling that were often localized in actively protruding regions at the cell periphery and other times showed a more global pattern ( Fig. 1A; supplementary material Movie 1). These patterns of PI3K signaling are distinct from the more stable, ring-like patterns seen in response to PDGF stimulation, which we have previously explained in quantitative detail (Schneider and Haugh, 2004). Analysis of a...

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts.

Small molecule ligands specific for tumor-associated surface receptors have wide applications in cancer diagnosis and therapy. Achieving high-affinity binding to the desired target is important for improving detection limits and for increasing therapeutic efficacy. However, the affinity required for maximal binding and retention remains unknown. Here, we present a systematic study of the effect of small molecule affinity on tumor uptake in vivo with affinities spanning a range of three orders of magnitude. A pretargeted bispecific antibody with different binding affinities to different DOTA-based small molecules is used as a receptor proxy. In this particular system targeting carcinoembryonic antigen, a small-molecule binding affinity of 400 pM was sufficient to achieve maximal tumor targeting, and an improvement in affinity to 10 pM showed no significant improvement in tumor uptake at 24 h post-injection. We derive a simple mathematical model of tumor targeting using measurable parameters that correlates well with experimental observations. We use relations derived from the model to develop design criteria for the future development of small molecule agents for targeted cancer therapeutics.