Centaur antibodies: Engineered chimeric equine-human recombinant antibodies

Abstract

Hyper-immune antisera from large mammals, in particular horses, are routinely used for life-saving anti-intoxication intervention. While highly efficient, the use of these immunotherapeutics is complicated by possible recipient reactogenicity and limited availability. Accordingly, there is an urgent need for alternative improved next-generation immunotherapies to respond to this issue of high public health priority. Here, we document the development of previously unavailable tools for equine antibody engineering. A novel primer set, EquPD v2020, based on equine V-gene data, was designed for efficient and accurate amplification of rearranged horse antibody V-segments. The primer set served for generation of immune phage display libraries, representing highly diverse V-gene repertoires of horses immunized against botulinum A or B neurotoxins. Highly specific scFv clones were selected and expressed as full-length antibodies, carrying equine V-genes and human Gamma1/Lambda constant genes, to be referred as “Centaur antibodies”. Preliminary assessment in a murine model of botulism established their therapeutic potential. The experimental approach detailed in the current report, represents a valuable tool for isolation and engineering of therapeutic equine antibodies.

Exploiting V-Gene Bias for Rapid, High-Throughput Monoclonal Antibody Isolation from Horses

“…Based on the most recent IMGT ® classification, we confirm here that almost all expressed antibodies use just a handful of IgHV4-related genes (independent of antigen) and show that the majority of these derive from IgHV4-21 ORF . While our study was limited in sample size ( n = 2), reanalysis of 920 NCBI GenBank sequences (summarised in [ 29 ]) revealed the same gene bias predominated by IgHV4-21 ORF ( Supplementary Figure S1C ). Importantly, none of our antibodies which were assigned to IgHV4-22 had the rare framework region 2 (FWR2) cysteine residue described in the IMGT ® data.…”

“…The resulting products were used to transfect Expi293 cells in high-throughput 24-deepwell plates, and cell culture supernatants were harvested after five days for screening by ELISA. When compared with other equine mAb discovery platforms [ 12 , 29 ], this protocol is unique in the exploitation of V -gene bias to accelerate lead candidate identification (14 days) and allows for the isolation of full-length natively paired antibodies, with the ability to easily switch between human or equine IgG isotypes.…”