Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries

Abstract

Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on the surface of yeast, identifying both individual amino acids and motifs within the complementarity-determining regions which contribute to the phenotype. We find enrichment of glycine, valine, and arginine as both individual amino acids and as a part of motifs, and additionally enrichment of motifs containing tryptophan. Insertion of any of these motifs into the complementarity-determining region H3 of a "clean" antibody increased its nonspecificity, with greatest increases in antibodies containing Trp or Val motifs. We next applied these rules to the creation of a synthetic diversity library based on natural frameworks with significantly decreased incorporation of such motifs and demonstrated its ability to isolate binders to a wide panel of antigens. This work both provides a greater understanding of the drivers of nonspecificity and provides design rules to increase efficiency in the isolation of antibodies with drug-like properties.

Nature-inspired design and evolution of anti-amyloid antibodies

“…The need for glycine and phenylalanine in HCDR3 is consistent with the fact that both residues are commonly observed in the CDRs of human antibodies (37) and are expected to be important for affinity (phenylalanine) and CDR structure and flexibility (glycine). However, the lack of enrichment of these residues in AF1 is also consistent with previous observations that enrichment of these residues in antibody CDRs is linked to reduced specificity (32,34,44).…”

Sequence features of variable region determining physicochemical properties and polyreactivity of therapeutic antibodies

“…Moreover, these residues are abounded in Abs' paratopes and are unusually exposed at the molecular surface (Mian et al, 1991;Robin et al, 2014;Sundberg and Mariuzza, 2002;. In a recent study, it was demonstrated that the presence of motifs rich of Trp in CDR H3 is associated with a greater degree of antigen binding promiscuity as assessed by PSR assay (Kelly et al, 2018). This work was based on interrogation of single-chain variable fragment libraries expressed on the surface of yeast cells.…”

Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

“…Several types of polyspecificity reagents have been developed for eliminating non-specific variants and enabling the isolation of antibodies with specificities that rival those of natural antibodies [37, 38]. For example, one particularly effective polyspecificity reagent is composed of soluble membrane proteins generated from mammalian cell lysates [34, 37, 39, 40]. Negative selections performed with this polyspecificity reagent and positive selections performed with the target antigen led to the isolation of antibodies with greatly reduced levels of non-specific binding relative to antibodies obtained without negative selections [37].…”

“…The fact that measurements of antibody specificity (non-specific binding and self-association) are the best biophysical descriptors of the likelihood of antibody success in the clinic [67] highlights the importance of developing computational and bioinformatics methods for predicting antibody specificity. Some of the key factors that determine antibody specificity are becoming clearer, including the numbers of charged, hydrophobic and hydrophilic residues in CDRs [26, 27, 39, 41, 42, 44, 68–70] as well as the net charge of the variable regions [46, 47, 49]. However, methods are needed to collectively describe these disparate findings and provide guidelines for identifying antibody variants with high specificity based only on their amino acid sequences or their combined sequences and structures.…”