Barbara Enenkel

A highly selective telomerase inhibitor limiting human cancer cell proliferation

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug‐treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non‐nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

Human telomerase contains two cooperating telomerase RNA molecules

Telomerase uses a short stretch of its intrinsic RNA molecule as template for telomere repeat synthesis. Reverse transcription of the RNA template is catalyzed by the telomerase reverse transcriptase (TERT) protein subunit. We demonstrate that human telomerase reconstituted from recombinant TERT and telomerase RNA runs as a dimer on a gel filtration column and that it contains two telomerase RNA molecules. Significantly, a telomerase heterodimer reconstituted from wild‐type and mutant telomerase RNA is barely active when compared with the wild‐type homodimer. We conclude that the telomerase RNA templates in the active enzyme are interdependent and functionally cooperate with each other. We discuss models that may explain the biological and enzymatic roles of telomerase dimerization.

Boosting antibody developability through rational sequence optimization

The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.

A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation

Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality.

Reformatting Rituximab into Human IgG2 and IgG4 Isotypes Dramatically Improves Apoptosis Induction In Vitro

The direct induction of cell death, or apoptosis, in target cells is one of the effector mechanisms for the anti CD20 antibody Rituximab. Here we provide evidence that Rituximab’s apoptotic ability is linked to the antibody IgG isotype. Reformatting Rituximab from the standard human IgG1 heavy chain into IgG2 or IgG4 boosted in vitro apoptosis induction in the Burkitt’s lymphoma B cell line Ramos five and four-fold respectively. The determinants for this behavior are located in the hinge region and CH1 domain of the heavy chain. By transplanting individual IgG2 or IgG4 specific amino acid residues onto otherwise IgG1 like backbones, thereby creating hybrid antibodies, the same enhancement of apoptosis induction could be achieved. The cysteines at position 131 of the CH1 domain and 219 in the hinge region, involved in IgG2 and IgG4 disulfide formation, were found to be of particular structural importance. Our data indicates that the hybrid antibodies possess a different CD20 binding mode than standard Rituximab, which appears to be key in enhancing apoptotic ability. The presented work opens up an interesting engineering route for enhancing the direct cytotoxic ability of therapeutic antibodies.

BI-HEX ® -GlymaxX ® cells enable efficient production of next generation biomolecules with enhanced ADCC activity

Background Despite the succes story of therapeutic monoclonal antibodies (mAbs), a medical need remains to improve their efficacy. One possibility to achieve this is to modulate important effector functions such as the antibody dependent cellular cytotoxicity (ADCC). The advantage of highly active biotherapeutic molecules is apart from the enhanced efficacy the reduction of side effects due to lower administered doses. Furthermore, these therapeutic antibodies may enable treatment of current nonresponders, e.g. patients with low antigen bearing tumors. Enhancement of the effector functions of antibodies can be achieved either by directly mutating the antibody’s amino acid sequence or by modifying its glycosylation pattern, e.g. by using a novel host cell line able to attach a desired glycostructure to the product. The latter approach has the advantage of not impacting the antibody structure itself, thereby avoiding negative effects on the PK/PD of the molecule. During the last decade it has been shown that antibodies with a reduced level of glycan fucosylation are much more potent in mediating ADCC, a mode of action particularly relevant for cancer therapeutics. Therefore, defucosylated antibodies are of major interest for biotherapeutics developers. To produce such antibodies, Boehringer Ingelheim has inlicensed the GlymaxX system from ProBioGen, Germany. This technology utilises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) which, when stably integrated into host cell lines, inhibits fucose de-novo biosynthesis. The enzyme deflects the fucosylation pathway by turning an intermediate (GDP-4-Keto-6-Deoxymannose) into GDP-Rhamnose, a sugar that cannot be metabolised by CHO cells. As a consequence, recombinant antibodies generated by such host cells exhibit reduced glycan fucosylation and 20100 fold higher ADCC activity. Here, we show the establishment of a new host cell line, termed BI-HEXGlymaxX which is capable of producing highly active therapeutic antibodies. We furthermore present data on the cell line properties concerning cell culture performance (e.g. titer, growth, transfection efficiency), process robustness and product quality reproducibility.

BI HEX™ – Platform for Fast Track Generation of High Producer Cell Lines Leading to High-Titer Processes for Production of Therapeutic Proteins from Mammalian Cells

The majority of biopharmaceuticals is currently being expressed in mammalian cells, mostly Chinese hamster ovary (CHO) and mouse myeloma (NS0) cells. The two most prominent challenges for mammalian cell-based systems relate to (i) product titer in the cell culture fluid at the end of cultivation and (ii) development times from final drug candidate selection to an established process to produce clinical grade material. The newest generations of mammalian cell culture production processes evolve rapidly to overcome these limitations. Boehringer Ingelheim’s current high expression (BI HEX™) cell line generation concept combines many improvements including novel or modified genetic elements to improve transcription rate, high throughput screening concepts to obtain highly productive clones reliably, and host cell lines that grow to high densities in serum-free chemically defined media. Specific productivities above 50 pg per cell and day for monoclonal antibodies have been achieved in CHO cells and were successfully translated into product titers of up to 4 g/L in an 11-day process. Furthermore, a strategy is presented for supply of material for toxicological studies produced from CHO cells in as little as 15 months starting with cloning of product-encoding genetic sequences into BI HEX vectors. At the same time it ensures that the production cell generated during this program will have the high expression potential needed to avoid a change in production cell line at later stages in development.

AMPLIFIED DICISTRONIC EXPRESSION UNITS MEDIATE APOPTOSIS PROTECTION IN CHO-DG44 CELLS ADAPTED FOR GROWTH IN SERUM-FREE MEDIA, IMPACT ON MITOCHONDRIA COPY NUMBER

We have constructed dicistronic expression vectors for one-step antiapoptosis engineering and product expression. Expression units encoding the soluble intercellular adhesion molecule 1 (sICAM), a potential therapeutic for treatment of the common cold, and bcl-2/bcl-xL were integrated and amplified in Chinese hamster ovary- (CHO) DG44 cells adapted for anchorage-independent growth in serum-free media. sICAM expression was translated in a cap-dependent and survival gene expression in a cap-independent manner based on the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). Batch cultivations of engineered CHO-DG44 cells containing amplified transgene expression units exhibited improved viability and delayed onset of apoptosis compared to cell lines harboring monocistronic control constructs. bcl-xL-mediated apoptosis protection was significantly higher compared to bcl-2-based survival engineering. High-level expression of bcl-2 and bcl-xL seem to be required to compensate for increased mitochondria numbers found to be associated with production cell lines grown in serum-free medium.

HTS-Based Development of High-Producing CHO Cell Lines

A major problem when establishing high-producing cell lines is the variability of integration sites. Integration of plasmid DNA into the genome of host cells occurs at random. As a consequence, selection of high producers is very time-consuming, costly and critical. Therefore, strategies which simplify and fasten the selection process are advantageous. Here, we present a system, that identifies and selects those of the transfected cells, which show the highest GFP-expression correlating with the expression of a product gene. This high expression system comprises expression vectors and serum-free transfection, selection and cultivation of CHO-DG44 cells.

Transient Bcl-x L Overexpression Has Different Effects on Protein Production in Various CHO-Derived Cell Lines

Most Chinese hamster ovary (CHO)-derived cell lines grown in serum- and protein-free suspension cultures for biopharmaceutical manufacturing are particularly sensitive to apoptosis. A wide variety of reports have demonstrated in the past years that overexpression of Bcl-2 significantly delays the onset of apoptosis and may have positive apoptosis-unrelated effects on production cell lines (viability, cell number, productivity). In contrast to Bcl-2, the impact of other anfi-apoptosis determinants of the Bcl-2 family such as Bcl-x L , on key cell characteristics has not yet been evaluated in great detail. However, pioneering work on Bcl-x L has suggested that its expression in a multicistronic expression configuration significantly enhances production of the model product protein SEAP (human placental secreted alkaline phosphatase). Here we show the influence of transient Bcl-x L overexpression on the production of the soluble intracellular adhesion molecule (sICAM) in various CHO-derived production cell lines.