In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

Abstract

Human adenoviruses (Ads) have been classified into six species (A to F) containing 51 defined serotypes. Most Ad serotypes utilize the coxsackievirus Ad receptor (CAR) as a primary attachment receptor. Recently, we suggested a new grouping of species B Ads based on their receptor usage (32). Group 1 (Ad serotype 16 [Ad16], Ad21, Ad35, and Ad50) nearly exclusively utilize CD46 as a cellular receptor; group 2 (Ad3, Ad7, and Ad14) share the same nonidentified receptor, which we refer to as receptor X; group 3 (Ad11p) preferentially interacts with CD46 but also utilizes receptor X if CD46 is blocked (32).Recent studies performed in U.S. military training facilities indicate an emergence of diverse species B serotypes at the majority of sites. This included the group 1 serotype 21 and the group 2 serotypes 3, 7, and 14 (17, 39). Species B-derived, replication-deficient vectors have recently shown promise as vehicles for gene transfer into multiple human cell types including cancer cells and tissue stem cells (30,35).Because of the importance of species B Ads as pathogens and application of species B-derived vectors for gene transfer, we studied the interaction between group 2 Ad35 and CD46 in more detail. Ad35 engages CD46 via residues in the C-terminal trimeric fiber knob domain (7). Within CD46 the Ad35-interacting areas are located in the two distal extracellular domains of the receptor (6,8). More recently, we used an expression library of the Ad35 fiber knob with random mutations to identify the amino acid residues within the Ad35 knob that mediate binding to CD46. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization (36).In the present study we used the same Ad35 expression library to screen for Ad35 knob mutants with increased binding to CD46 compared to the wild-type Ad35 knob. Our goal was to construct Ad vectors with substantially increased affinity for CD46. The rationale for such vectors comes from studies with phage antibody expression libraries (33) and more recently from studies with aptamers, protein-binding oligonucleotides (23). The goal of phage and aptamer library screening is to identify variants with the highest affinity, because in in vitro and in vivo studies with single-chain variable fragment fragments and aptamers, higher affinity usually directly translates into more-efficient binding to receptor-positive cells. Along this line, attempts were undertaken to incorporate high-affinity ligands into measles virus (9) and Ad vectors (2,3,40) in order to increase efficacy and specificity of target cell infection in vivo or to establish new receptor-ligand systems for the propagation of vectors.

Development of a Targeted Gene Vector Platform Based on Simian Adenovirus Serotype 24

“…The goal of vector targeting is, therefore, to achieve a level of target-specific gene transfer that is substantially higher than that of off-target transduction. In a recent study, the efficacy of on-target gene transfer by a systemically injected Ad vector was significantly improved by increasing the affinity of the virus's fiber protein for its receptor (57). This success suggests that the in vivo target selectivity of an sAd24-derived vector can also be augmented by using ligands with higher affinity for the target receptor.…”

In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors

“…5 We identified CD46 as the high-affinity receptor for a number of adenoviruses (Ads), including serotype 11,16,21,35, and 50. 6 The receptor interacting moiety in the capsid of Ads is the C-terminal globular trimeric fiber domain, called the fiber knob.…”

Avidity Binding of Human Adenovirus Serotypes 3 and 7 to the Membrane Cofactor CD46 Triggers Infection

“…S3A and S3B in the supplemental material). Initial Biacore experiments using sensor chips had previously been described for immobilized FKs and monovalent CD46-SCR I-II as soluble analyte, thereby avoiding avidity effects of CD46 (10,47,48,71,72). The SCR I-II domain comprised the binding sites for the FKs (14,49,53,71).…”