Genetic rearrangement during site specific integration event facilitates cell line development of a bispecific molecule

Abstract

Site specific integration (SSI) expression systems offer robust means of generating highly productive and stable cell lines for traditional monoclonal antibodies. As complex modalities such as antibody-like molecules comprised of greater than two peptides become more prevalent, greater emphasis needs to be placed on the ability to produce appreciable quantities of the correct product of interest (POI). The ability to screen several transcript stoichiometries could play a large role in ensuring high amounts of the correct POI. Here we illustrate implementation of an SSI expression system with a single site of integration for development and production of a multichain, bi-specific molecule. A SSI vector with a single copy of all of the genes of interest was initially selected for stable Chinese hamster ovary transfection. While the resulting transfection pools generated low levels of the desired heterodimer, utilizing an intensive clone screen strategy, we were able to identify clones having significantly higher levels of POI. In-depth genotypic characterization of clones having the desirable phenotype revealed that a duplication of the light chain within the landing pad was responsible for producing the intended molecule. Retrospective transfection pool analysis using a vector configuration mimicking the transgene configuration found in the clones, as well as other vector configurations, yielded more favorable results with respect to % POI. Overall, the study demonstrated that despite the theoretical static nature of the SSI expression system, enough heterogeneity existed to yield clones having significantly different transgene phenotypes/genotypes and support production of a complex multi-chain molecule.

Generation of a host cell line containing a MAR‐rich landing pad for site‐specific integration and expression of transgenes

“…ampicillin resistance cassette) and also further optimize culture condition in fed-batch mode using DOE approach. 26,33,40,49 In summary, in this work, we developed a multi-copy MAR-rich landing pad system for the orthogonal, simultaneous integration of several copies of up to three GOIs. Generated LP-bearing host cell lines showed high stability and could be considered suitable for GOIs integration and expression.…”

“…Despite of that, cell lines generated in this study are not optimized yet and several points can be addressed to improve the system such as transfection conditions in order to find the best DNA amount and ratios for landing pad total occupancy, convert TI to recombinase‐mediated cassette exchange (RMCE) to avoid integration of additional DNA sequences (e.g. ampicillin resistance cassette) and also further optimize culture condition in fed‐batch mode using DOE approach 26,33,40,49 …”

“…Most of the developed systems have been examined by integrating msAb sequences into the landing pad to generate a production platform 34,36,37 . However, these TI‐generated cell lines resulted in cell‐specific productivities ranging between 1 and 20 pg/cell/day, hence not meeting the criteria for large industrial scale productions for which stable cell lines, generated by random integration, reaching commonly productivities well above 20 pg/cell/day 25,38–41 …”

“…34,36,37 However, these TI-generated cell lines resulted in cellspecific productivities ranging between 1 and 20 pg/cell/day, hence not meeting the criteria for large industrial scale productions for which stable cell lines, generated by random integration, reaching commonly productivities well above 20 pg/cell/day. 25,[38][39][40][41] To increase productivity of TI-based cell lines, the commonly used strategies are the inclusion of the landing pad itself into genomic regions, which promote a high and stable transcription rate (referred to as "genomic hot-spot") and the use of multi-copy expression vectors for transgene integration (donor vector) to increase GOIs copies that can be integrated into the host cell. 7,12,36,38,41,42 However, the process of hot-spot screening remains laborious and time-consuming, and the use of large donor vectors carrying multiple copies of the GOI reduces the efficiency of integration into the LP.…”

A genetic off‐target event in a site‐specific integration cell line expressing monoclonal antibody has no impact on commercial suitability

“…Most SSI cell lines are known for efficient genomic integration of exogenous DNA and prolonged genetic stability. 15,[17][18][19][20][21] However, the SSI system is still susceptible to off-target events (i.e. random gene mutations/ insertions/deletions) or potential vector rearrangements that may occur during or after transfection.…”