ABSTRACT
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.
Subject(s)
Antibodies/chemistry , Complementarity Determining Regions/genetics , Protein Binding/genetics , Repetitive Sequences, Amino Acid/genetics , Amino Acid Sequence , Animals , Antibodies/genetics , Antibodies/therapeutic use , Antibody Affinity/genetics , HumansABSTRACT
Immunoglobulin junctional diversity is concentrated in the third complementarity-determining region of the heavy chain (CDR-H3), which often plays a dominant role in antigen binding. The range of CDR-H3 lengths in mouse is shorter than in human, and thus the murine repertoire could be presumed to be a subset of the human one. To test this presumption, we analyzed 4751 human and 2170 murine unique, functional, published CDR-H3 intervals. Although tyrosine, glycine, and serine were found to predominate in both species, the human sequences contained fewer tyrosine residues, more proline residues, and more hydrophobic residues (p<0.001, respectively). While changes in amino acid utilization as a function of CDR-H3 length followed similar trends in both species, murine and human CDR-H3 intervals of identical length were found to differ from each other. These differences reflect both divergence of germline diversity and joining gene sequence and somatic selection. Together, these factors promote the production of a rather uniform repertoire in mice of tyrosine-enriched CDR-H3 loops with stabilized hydrogen bond-ladders versus a much more diverse repertoire in human that contains CDR-H3 loops sculpted by the presence of intra-chain disulfide bonds due to germline-encoded cysteine residues as well as the enhanced presence of somatically generated proline residues that preclude hydrogen bond ladder formation. Thus, despite the presumed need to recognize a similar range of antigen epitopes, the murine CDR-H3 repertoire is clearly distinct from its human counterpart in its amino acid composition and its predicted range of structures. These findings represent a benchmark to which CDR-H3 repertoires can be compared to better characterize and understand the shaping of the CDR-H3 repertoire over evolution and during immune responses. This information may also be useful for the design of species-specific CDR-H3 sequences in synthetic antibody libraries.