Abstract 1837: A human artificial chromosome (HAC)-based system as a powerful tool for stable co-expression of multiple proteins and therapeutic IgGs in mammalian cells
Authors: Kononenko Artem , Laster Ian , Yeku Oladapo , Spriggs David 
Published: July 2021

Abstract
Background: Currently, there is increasing demand for recombinant therapeutic proteins produced in mammalian cell lines, especially therapeutic antibodies. MUC16-directed IgG and bispecific diabodies are one such example that represents a promising strategy for treatment of ovarian cancer. HEK293 and CHO cells are mostly used for large-scale stable expression of therapeutic proteins, however low protein yield, transgene silencing, cell line instability, and the time-consuming process of cell line development represent serious challenges. Here, we are presenting an alternative system for stable, high yield expression of multiple genes including complex therapeutic antibodies (HC and LC genes) and other proteins within single HAC. HAC vector propagate independently in cells without integration in the host chromosomes and has unlimited cloning capacity. Furthermore, this particular HAC can be targeted by transcriptional transactivators like tetR-VP16, eliminating transgene silencing, thereby providing stable long-term high yield protein production in mammalian cells.
Methods: The targeting vector containing single or multiple genes is loaded at the unique LoxP site of tetO-HAC propagated in HPRT-deficient CHO cells by Cre-LoxP mediated recombination. As a result of recombination, functional HPRT gene will be reconstituted and HPRT-positive colonies are selected after 3 weeks growth in HAT medium. In the next step, HAC with therapeutic transgene(s) is transferred to the different high productive cell lines via MMCT technique following by high yield protein(s) expression in spinner flask. The alphoid tetO-HAC carries Bsr gene, so Blasticidin is used to select for the recipient cells that have taken up the HAC after Microcell-mediated chromosome transfer (MMCT).
Results: Targeting vector with His-tagged MUC16 BiTEs gene (or iBAC caring HC and LC of anti-MUC16 IgG) was inserted into the HAC propagated in CHO cells. HPRT gene reconstitution was confirmed by PCR using a set of specific primers. Expression of His-tagged MUC16 BiTEs or anti-MUC16 IgG in CHO cells was confirmed by western blot and SDS-PAGE analysis. The yield of Ni- purified MUC16 BiTEs was up to 6 mg/l in original CHO cells. Our data from cellular models confirmed high efficacy of Ni- purified MUC16 BiTEs against OVCAR3 tumor cells, - up to 80% of the tumor cells were killed by T-cells in the presence of MUC16 BiTEs protein. After that, HAC/MUC16 BiTEs was successfully transferred to high productive ExpiHEK293F cells by MMCT. Therapeutic HAC transferring was confirmed by expression DsRed transgene linked with MUC16BiTEs through IRES and FISH analysis. FISH reveals the HAC propagated independently in ExpiHEK293F cells.
Conclusions: Our data shows that the HAC is the new promising system for stable, high yield production of multiple therapeutic proteins.
Citation Format: Artem Kononenko, Ian Laster, Oladapo Yeku, David Spriggs. A human artificial chromosome (HAC)-based system as a powerful tool for stable co-expression of multiple proteins and therapeutic IgGs in mammalian cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1837.