Improving Antibody Binding Affinity and Specificity for Therapeutic Development

Abstract

Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected. An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

“…Bound phage was eluted in 100 mM HCl for 20 min, then neutralized with 1/10 volume of 1 M Tris pH 11.0 and used to infect E. coli for amplification, followed by one round or two rounds of plate-based selection. VEGF-binding clones were selected by incubating the phage display libraries with 5, 0.5, and 0.1 nM biotinylated VEGF in solution in the successive rounds of selection with increasing stringency, as described previously (53). Bound clones were captured on ELISA wells coated with neutravidin, washed, eluted, and amplified as above.…”

Proprotein Convertase Substilisin/Kexin Type 9 Antagonism Reduces Low-Density Lipoprotein Cholesterol in Statin-Treated Hypercholesterolemic Nonhuman Primates

“…Mouse antibody J10 was humanized and affinity-matured to antibody J16 by using standard humanization and affinity maturation strategies (Bostrom et al, 2009).…”

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration

“…This is followed by affinitybased selection and screening a limited number of clones by affinity assay and sequencing (11). The positions to include for combinatorial mutation and the extent of randomization in the libraries are quite limited as the library size that can be generated and screened is finite.…”