A role for macromolecular crowding in off-target binding of therapeutic antibodies

The nonspecific binding of certain therapeutic antibodies to tissues or to soluble biomolecules can accelerate their clearance from the circulation and undermine their benefit to patients. This article proposes that tandem amino acid repeat sequences in antibody hypervariable segments, particularly the complementarity determining regions (CDRs), can enhance this off-target binding. This hypothesis is based on two sets of observations. First, in a limited number of cases, antibodies with clusters of amino acid repeats in their CDRs have significantly higher clearance rates in experimental animals than otherwise identical antibodies without the repeats. Second, tandem amino acid repeats are abundant in intracellular hub proteins where they appear to promote the promiscuous binding of these proteins to a wide variety of other molecules. These nonspecific hub protein interactions are highly favored by the intense macromolecular crowding that permeates the cytoplasm. A survey of the variable region sequences of 137 antibodies in various stages of development revealed that 26 have at least one CDR containing a cluster of three closely spaced amino acid repeats. If the overall hypothesis is valid, then it suggests strategies for site-directed mutagenesis to improve pharmacokinetic behavior and for the design of more reliable in vitro binding assays to predict off-target binding in vivo.