Browsing by Author "Olayioye, Monilola A. (Prof. Dr.)"

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Open Access
Dual- and triple-targeting of the HER-family members using combinations of mono- and bispecific antibodies
(2023) Rau, Alexander; Olayioye, Monilola A. (Prof. Dr.)
Epidermal growth factor receptor (EGFR)-targeted cancer treatments with antibodies like Cetuximab are successfully used in the clinic for about 20 years. However, intrinsic, as well as newly developed resistance mechanisms to EGFR-targeted therapies, are the main reason for their failure. Activation of human epidermal growth factor receptor 3 (HER3)-signaling upon EGFR-targeted therapies is frequently observed and has motivated the development of combination therapies that simultaneously block EGFR and HER3. In this study, bispecific and bivalent, or tetravalent, respectively, single-chain diabody (scDb) and scDb-Fc molecules were developed comprising the antigen-binding sites of a humanized version of Cetuximab (hu225) as well as a recently developed anti-HER3 antibody (3-43). In total, eight molecules (two scDb and six scDb-Fc) with varying linkers were engineered. The scDb hu225x3 43 Fc showed the most favorable properties regarding production yield, purity, homogeneity and linker setup. Binding of the scDb-Fc to recombinant receptors, as well as to HER-family receptor expressing cell lines revealed retained binding properties, compared to parental antibodies. Furthermore, the scDb hu225x3 43 Fc showed strong and long-lasting inhibition of downstream signaling by EGF, HRG or combination of both ligands. Proliferation studies on head and neck squamous cell carcinoma (HNSCC), triple negative breast cancer (TNBC), and colorectal cancer (CRC) cell lines revealed either similar, or stronger inhibition, compared to parental antibodies as single or combination treatment, which translated into to long-lasting growth suppression in a s.c. xenograft tumor model. Treatment with the bispecific antibody inhibited in vitro HRG-stimulated oncosphere formation of two TNBC cell lines. In an orthotopic MDA-MB-468 tumor model, superior antitumor effects were observed compared to those obtained by the parental antibodies alone or in combination. Furthermore, this was associated with a reduced number of cells with stem-like properties demonstrating that the bispecific antibody not only efficiently blocks TNBC proliferation but also the survival and expansion of the cancer stem cell population. The high degree of plasticity and compensatory signaling within the HER-family not only leads to compensatory crosstalk by HER3 but also HER2 giving the rational to combine the EGFR- and HER3-targeting scDb-Fc with a HER2-targeting antibody like Trastuzumab. The triple-targeting approach with the scDb-Fc and Trastuzumab was superior in inhibition of HRG-stimulated proliferation of the CRC cell line LIM1215 compared to the combination of IgG hu225, Trastuzumab and IgG 3 43. This was also observed in primary and secondary CRC oncosphere formation assays. Finally, in CRC patient derived organoids (PDOs) grown in HRG-supplemented medium the triple-targeting of EGFR, HER2 and HER3 provided broader efficacy than dual- or mono-targeting of receptors of the HER family. In contrast to Afatinib (anti-EGFR, -HER2, -HER4), the triple-targeted antibody approach showed efficient inhibition in all tested PDOs. Thus, the bispecific scDb-Fc alone or in combination with Trastuzumab represents a superior strategy to deal with primary and acquired resistances compared to targeting a single receptor with different antibodies or any combination of antibodies targeting two receptors of the HER-family.
Open Access
MicroRNAs to boost the productivity of Chinese hamster ovary producer cells
(2013) Strotbek, Michaela; Olayioye, Monilola A. (Prof. Dr.)
MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate the expression of different target genes and, thus, potentially offer the opportunity to engineer networks of genes in order to achieve complex phenotypic changes in mammalian cells. The goal of this thesis was to explore whether this feature of miRNAs could be exploited as a strategy to improve therapeutic protein production processes by increasing the viable cell densities and/or productivity of mammalian producer cells. To identify miRNAs that increase the productivity of producer cells, a genome-wide functional miRNA screen was established and performed in Chinese hamster ovary (CHO) cells stably expressing an IgG1 that were grown in suspension in chemically defined medium. Using this approach nineteen human miRNAs were identified that significantly improved IgG titers in the primary screen. Almost half of these miRNAs could be validated to significantly increase the IgG concentrations and/or specific productivity when transiently introduced into CHO producer cells in a secondary screen. The increased titers of recombinant human serum albumin analyzed as a second secreted model protein pointed to product independent effects of most of the validated miRNAs. In addition, two of the validated miRNAs, hsa-miR-557 and hsa-miR-1978, also increased the secretion of an endogenously as well as a transiently expressed model protein in human cells, indicating that these miRNAs manipulate the cellular machinery by a universal mechanism. The strongest impact on the specific productivity of CHO cells was observed by a dead entry and thus artificial miRNA (hsa-miR-1978), which may represent a promising molecular tool for future synthetic biology approaches aimed at optimizing producer cells. For further studies, the two miRNAs hsa-miR-557 and hsa-miR-1287, positively impacting the viable cell density and specific productivity, respectively, were selected. Transient experiments supported the idea of combining these two miRNAs to further boost cellular productivity. Using a vector-based expression system, CHO pools stably overexpressing these human miRNAs were successfully generated allowing the study of miRNA-engineered producer cells under industrially relevant culture conditions such as fed-batch cultivation. Importantly, three independent cell pools stably coexpressing miR-557 and miR-1287 gave rise to significantly increased IgG titers by 30% in fed-batch cultures whilst product quality was conserved, proving the transferability of the transient results to a stable setting. Taken together, these results demonstrate that miRNA-based cell line engineering is an attractive approach toward the genetic optimization of CHO host cells for industrial applications.