Comprehensive biophysical characterization of AAV-AAVR interaction uncovers serotype- and pH-dependent interaction.

After more than two decades of research and development, adeno-associated virus (AAV) has become one of the dominant delivery vectors in gene therapy. Despite the focused research, the cell entry pathway for AAV is still not fully understood. Universal AAV receptor (AAVR) has been identified to be involved in cellular entry of different AAV serotypes. With the unveiling of the high-resolution AAV-AAVR complex structure by cryogenic electron microscopy, the atomic level interaction between AAV and AAVR has become the focus of study in recent years. However, the serotype dependence of this binding interaction and the effect of pH have not been studied. Here, orthogonal approaches including bio-layer interferometry (BLI), size-exclusion chromatography coupled to multi-angle laser scattering (SEC-MALS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) were utilized to study the interaction between selected AAV serotypes and AAVR under different pH conditions. A robust BLI method was developed and the equilibrium dissociation binding constants (KD) between different AAV serotypes (AAV1, AAV5 and AAV8) and AAVR was measured. The binding constants measured by BLI together with orthogonal methods (SEC-MALS and SV-AUC) all confirmed that AAV5 has the strongest binding affinity followed by AAV1 while AAV8 binds the weakest. It was also observed that lower pH promotes the binding between AAV and AAVR and neutral or slightly basic conditions lead to very weak binding. These data indicate that for certain serotypes, AAVR may play a prominent role in trafficking AAV to the Golgi rather than acting as a host cell receptor. Information obtained from these combinatorial biophysical methods can be used to engineer future generations of AAVs to have better transduction efficiency.

A Biparatopic Antibody That Modulates MET Trafficking Exhibits Enhanced Efficacy Compared with Parental Antibodies in MET-Driven Tumor Models.

PURPOSE: Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy. EXPERIMENTAL DESIGN: Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors. RESULTS: In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling. CONCLUSIONS: Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.

Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes.

Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.

VEGF Trap complex formation measures production rates of VEGF, providing a biomarker for predicting efficacious angiogenic blockade.

VEGF is the best characterized mediator of tumor angiogenesis. Anti-VEGF agents have recently demonstrated impressive efficacy in human cancer trials, but the optimal dosing of such agents must still be determined empirically, because biomarkers to guide dosing have yet to be established. The widely accepted (but unverified) assumption that VEGF production is quite low in normal adults led to the notion that increased systemic VEGF levels might quantitatively reflect tumor mass and angiogenic activity. We describe an approach to determine host and tumor production of VEGF, using a high-affinity and long-lived VEGF antagonist now in clinical trials, the VEGF Trap. Unlike antibody complexes that are usually rapidly cleared, the VEGF Trap forms inert complexes with tissue- and tumor-derived VEGF that remain stably in the systemic circulation, where they are readily assayable, providing unprecedented capability to accurately measure VEGF production. We report that VEGF production is surprisingly high in non-tumor-bearing rodents and humans, challenging the notion that systemic VEGF levels can serve as a sensitive surrogate for tumor load; tumor VEGF contribution becomes significant only with very large tumor loads. These findings have the important corollary that anti-VEGF therapies must be sufficiently dosed to avoid diversion by host-derived VEGF. We further show that our assay can indicate when VEGF is optimally blocked; such biomarkers to guide dosing do not exist for other anti-VEGF agents. Based on this assay, VEGF Trap doses currently being assessed in clinical trials are in the efficacious range.