SPLICELECT™: an adaptable cell surface display technology based on alternative splicing allowing the qualitative and quantitative prediction of secreted product at a single-cell level

Abstract

We describe a mammalian expression construct (SPLICELECT™) that allows the redirection of a proportion of a secreted protein onto the cell surface using alternative splicing: whereas the majority of the RNA is spliced into a transcript encoding a secreted protein, a weak splice donor site yields a secondary transcript encoding, in addition, a C-terminal transmembrane domain. The different sequence elements can be modified in order to modulate the level of cell surface display and of secretion in an independent manner. In this work, we demonstrated that the cell surface display of stable cell lines is correlated with the level of the secreted protein of interest, but also with the level of heterodimerization in the case of a bispecific antibody. It was also shown that this construct may be useful for rapid screening of multiple antibody candidates in binding assays following transient transfection. Thus, the correlation of product quantity and quality of the secreted and of membrane-displayed product in combination with the flexibility of the construct with regards to cell surface display/secretion levels make SPLICELECT™ a valuable tool with many potential applications, not limited to industrial cell line development or antibody engineering.

Alternative splicing for tuneable expression of protein subunits at desired ratios

“…This mechanism is used in nature to change the expression level of proteins or in order to modify protein activity during development. 10 Alternative splicing is usually controlled by complex interactions of many factors, including intron and exon size, 11 binding of regulatory proteins 12 , 13 and consensus sequences (i.e., branch point, splice donor and splice acceptor regions), 14–18 In the field of recombinant expression, alternative splicing has been described to generate variants of the same protein 19 or to allow expression of two different proteins from the same pre-mRNA, 20 including the expression of antibody heavy and light chain resulting in secretion of fully functional antibody, but with very low titers (in the µg/L range). 21 , 22 Although splicing is well known in the literature and consensus sequences of the splice sites have been published, as described above, the precise outcome of alternative splicing events depends on integration of multiple factors and this inherent complexity may be the reason for the low expression levels described so far for standard monoclonal antibodies (mAbs) using alternative splicing.…”

Immune Literacy: Reading, Writing, and Editing Adaptive Immunity

“…Another approach used the RIRR sequence that can be recognized and partially cleaved by the furin enzyme, leading to simultaneous secretion and display of antibodies ( Zhou et al., 2013 ). Yet another approach is based on alternative splicing, thus allowing titration of the secretion to display ratio ( Aebischer-Gumy et al., 2020 ; Horlick et al., 2013 ). More recently, several novel mammalian display platforms have been developed by taking advantage of CRISPR systems ( Figure 3 ).…”