Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Abstract

In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility

“…Mouse RAG2 −/− pre-B cell lines 63-12 45 ectopically and stably expressing human ROR1 or human ROR2 were generated by transposition as described. 46 Parental 63-12, 63-12/hROR1, and 63-12/hROR2 cells were cultured in IMDM supplemented with 10% (v/v) heat inactivated FBS, 0.1% (v/v) β-mercaptoethanol, and 1 × penicillin-streptomycin (all from Thermo Fisher). HEK293F cells (Thermo Fisher) were maintained in FreeStyle Medium supplemented with 1% (v/v) heat inactivated FBS to support adherent culture or without FBS for suspension culture, and 1 × penicillin-streptomycin (all from Thermo Fisher).…”

From rabbit antibody repertoires to rabbit monoclonal antibodies

“…In general, humanization strategies that have worked for mouse mAbs also work for rabbit mAbs, whether it is by rational design, 17, 33, 187 directed evolution 188 or a combination of both. 155, 189 As is the case for mouse mAbs, grafting of all six CDRs followed by iterative fine-tuning of framework residues is the most frequently used method for humanizing rabbit mAbs. Borras et al used a general acceptor framework for the humanization of rabbit scFv.…”

Generation of an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins

“…Several reports have demonstrated the generation of tailor-made human lymphocytic cells lines using transposon-based gene transfer [39, 44, 47, 68–70]. We used Transpo-mAb TM technology to establish a human cell line displaying a TTC-reactive BCR on the cell surface.…”