Fig. 2: BiTE library generation and characterization of integrated library diversity.
From: A high throughput bispecific antibody discovery pipeline
a Design variables interrogated for the CD19 scFvs CD3 scFvs, and peptide linker connecting the scFvs. b Schematic representation of the integration of single copy BiTE variant in landing pad harboring HEK293 cell line (HEK293_LP) with promoterless BiTE gene encoding donor plasmid library. c Representation of diversity of BiTE design features obtained from long-read amplicon sequencing (PacBio SMRT) of PCR amplified BiTE genes from genomic DNA of BiTE integrated HEK293_LP cells, distribution of relative orientation of scFvs (i), Distribution of relative CD3 scFvs (ii), Distribution of CD19 scFvs (iii) and Distribution of scFv interconnecting linkers (iv). ~22,300 unique BiTE sequences obtained from long-read amplicon sequencing were used for analyzing the distribution of design parameters (relative scFv orientation, CD3 scFv type, CD19 scFv type, and scFv interconnecting linker type). The genomic DNA template used for amplifying integrated BiTE genes was obtained from 3 × 106 HEK_LPCD19/BiTE cells obtained from n = 1 integration experiment.
