Loss of a newly discovered microRNA in Chinese hamster ovary cells leads to upregulation of N-glycolylneuraminic acid sialylation on monoclonal antibodies.

Chinese hamster ovary (CHO) cells are known not to express appreciable levels of the sialic acid residue N-glycolylneuraminic acid (NGNA) on monoclonal antibodies. However, we actually have identified a recombinant CHO cell line expressing an IgG with unusually high levels of NGNA sialylation (>30%). Comprehensive multi-OMICs based experimental analyses unraveled the root cause of this atypical sialylation: (1) expression of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene was spontaneously switched on, (2) CMAH mRNA showed an anti-correlated expression to the newly discovered Cricetulus griseus (cgr) specific microRNA cgr-miR-111 and exhibits two putative miR-111 binding sites, (3) miR-111 expression depends on the transcription of its host gene SDK1, and (4) a single point mutation within the promoter region of the sidekick cell adhesion molecule 1 (SDK1) gene generated a binding site for the transcriptional repressor histone H4 transcription factor HINF-P. The resulting transcriptional repression of SDK1 led to a downregulation of its co-expressed miR-111 and hence to a spontaneous upregulation of CMAH expression finally increasing NGNA protein sialylation.

miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc.

Transcriptome analysis in primary B lymphoid precursors following induction of the pre-B cell receptor.

Pre-BCR signals are part of a checkpoint where early precursor (pre-) B cells with a pairing Ig muH chain (muHC) are clonally expanded before they differentiate into IgL-rearranging, resting pre-B cells. A pre-BCR consists of two muHCs, two surrogate L chains and the signal transducer Igalpha/Igbeta. The molecular circuits by which the pre-BCR controls proliferation and differentiation of pre-B cells are poorly characterized. Therefore, we identified the differential transcriptome by genome-wide expression profiling in progenitor (pro-) B cells from a Rag2-deficient mouse, in which the expression of a transgenic muHC and thus a pre-BCR as well as pre-BCR-mediated clonal expansion can be controlled by tetracycline (muHC-inducible mouse). This analysis revealed that pre-BCR signals upregulate components of the BCR signalosome, open the IgL chain (LC) locus and induce the krüppel-like transcription factor KLF2, a key regulator of quiescence and lymphocyte migration. Hence, pre-BCR signals establish the molecular network for BCR signaling even before the production of an IgLC and induce the expression of KLF2, a candidate for controlling clonal expansion and migration of functional pre-B cells.

Visualisation of intracellular production bottlenecks in suspension-adapted CHO cells producing complex biopharmaceuticals using fluorescence microscopy.

With the advance of complex biological formats such as bispecific antibodies or fusion proteins, mammalian expression systems often show low performance. Described determining factors may be accumulation or haltering of heterologous proteins within the different cellular compartments disturbing transport or secretion. In case of the investigated bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cell line neither impaired transcription nor decreased translation processes were identified and thus satisfactorily explained its low production capacity. Hence, we established a streamlined confocal microscopy-based methodology for CHO production cells investigating the distribution of the recombinant protein within the respective organelles of the secretory pathway and visualised the structure of the endoplasmic reticulum (ER) to be affected pinpointing towards an intra-ER bottleneck putatively hampering or limiting efficient secretion. The ER displayed not only a heavily altered morphology in comparison to a high immunoglobulin G (IgG)-producing cell line with a possibly inflated or overloaded structure, but the recombinant protein was also completely absent in the Golgi apparatus. Notably, the results obtained using an automated microscopy approach suggest the possible application of this methodology in cell line development and engineering.

A single-step FACS sorting strategy in conjunction with fluorescent vital dye imaging efficiently assures clonality of biopharmaceutical production cell lines.

The development of biopharmaceutical production cell lines typically starts with generation of heterogeneous populations of cells, from which then single cell clones are established. Several regulatory guidelines require that production cell lines are clonal, and the actual demonstration of clonality has been increasingly demanded by regulatory authorities over the last years. Here, the authors describe the relative contribution of flow cytometry mediated deposition of single cells in multiwell plates and subsequent imaging to assurance of clonality in a state of the art approach to single cell generation. Within the flow cytometry step, two unit operations are evaluated separately, doublet discrimination during event selection for deposition and droplet deposition accuracy. The imaging procedure is evaluated for the accuracy of detection of non-clonal populations. By employing mixing experiments of cell populations, the authors demonstrate that doublet discrimination is highly efficient, and that an appropriately set up flow cytometry system already can generate >99.5% true single cell clones. The efficiency of the described imaging process depends on several factors, reaching an optimal detection rate of non-clonal wells of about 99.8%. Our results demonstrate that one well characterized cloning step generate biopharmaceutical production cell lines with a probability of clonality of >99.99%.

Three-dimensional modeling of a pre-B-cell receptor.

Signals delivered by the immunoglobulin (Ig)-like pre-B-cell receptor (pre-BCR) are critical for efficient maturation of early precursor B (pre-B) cells. A pre-BCR contains two immunoglobulin mu-heavy chains (muHC), two surrogate light chains (SLC) consisting of the non-covalently associated polypeptides, VpreB and lambda5, and the heterodimeric signaling transducer Igalpha/beta. Although, it is generally accepted that signals initiated from the pre-BCR are required for efficient expansion and differentiation of pre-B cells, the three-dimensional structure of this receptor has not yet been determined by either NMR or X-ray spectroscopy. Therefore, we used indirect computer-assisted molecular modeling techniques to predict for the first time three-dimensional coordinates of the pre-BCR, the conformation of the SLC components, VpreB and lambda5, and the position and flexibility of the so-called non-Ig-like unique tails at the C-terminus of VpreB and the N-terminus of lambda5. Structure prediction revealed that these unique tails of VpreB and lambda5 protrude from the SLC at the position where the CDR3 of a conventional IgLchain would be located. Thus, the unique tails are accessible for ligand binding, which supports the recent finding that the lambda5 unique tail is required for pre-BCR/stroma cell interaction. Further, the non-covalent interaction of the extra beta-strand of lambda5 (beta8) with VpreB is predicted to result in a stabilization of the tertiary structure of VpreB. In summary, three-dimensional computer modeling suggests that the structure of a pre-BCR resembles that of a conventional B-cell receptor (BCR) and that the lambda5 unique tail could be a major binding site for pre-BCR ligands.

CD44 is dispensable for B lymphopoiesis.

Several studies have implicated a role for the cell surface glycoprotein CD44 in lymphocyte differentiation, adhesion to the matrix, homing, activation and apoptosis. However, the requirement of CD44 in B cell maturation remains elusive. To address this point, we analyzed the generation and activation of B lymphocytes in CD44-deficient mice. Unexpectedly, both maturation and autoreconstitution of early bone marrow B cell progenitors after sublethal irradiation as well as frequencies of splenic B cell subsets are indistinguishable in wild-type and CD44-deficient mice. Furthermore, splenic CD44-deficient B lymphocytes show a normal activation pattern after stimulation with LPS, anti-IgM/IL-4 or anti-CD40/IL-4. These results show for the first time that CD44 is clearly dispensable for development, reconstitution and activation of B lymphocytes.

Interaction of murine precursor B cell receptor with stroma cells is controlled by the unique tail of lambda 5 and stroma cell-associated heparan sulfate.

Efficient clonal expansion of early precursor B (pre-B) cells requires signals delivered by an Ig-like integral membrane complex, the so-called pre-B cell receptor (pre-BCR). A pre-BCR consists of two membrane micro H chains, two covalently associated surrogate L chains, and the heterodimeric signaling transducer Igalphabeta. In contrast to a conventional Ig L chain, the surrogate L chain is a heterodimer composed of the invariant polypeptides VpreB and lambda5. Although it is still unclear how pre-BCR signals are initiated, two recent findings support a ligand-dependent initiation of pre-BCR signals: 1) a pre-BCR/galectin-1 interaction is required to induce phosphorylation of Igalphabeta in a human precursor B line, and 2) soluble murine as well as human pre-BCR molecules bind to stroma and other adherent cells. In this study, we show that efficient binding of a soluble murine pre-BCR to stroma cells requires the non-Ig-like unique tail of lambda5. Surprisingly however, a murine pre-BCR, in contrast to its human counterpart, does not interact with galectin-1, as revealed by lactose blocking, RNA interference, and immunoprecipitation assays. Finally, the binding of a murine pre-BCR to stroma cells can be blocked either with heparin or by pretreatment of stroma cells with heparitinase or a sulfation inhibitor. Hence, efficient binding of a murine pre-BCR to stroma cells requires the unique tail of lambda5 and stroma cell-associated heparan sulfate. These findings not only identified heparan sulfate as potential pre-BCR ligands, but will also facilitate the development of appropriate animal models to determine whether a pre-BCR/heparan sulfate interaction is involved in early B cell maturation.

Selection of Ig mu heavy chains by complementarity-determining region 3 length and amino acid composition.

Although it is generally accepted that Ig heavy chains (HC) are selected at the pre-B cell receptor (pre-BCR) checkpoint, the characteristics of a functional HC and the role of pre-BCR assembly in their selection have remained elusive. We determined the characteristics of HCs that successfully passed the pre-BCR checkpoint by examining transcripts harboring V(H)81X and J(H)4 gene segments from J(H)(+/-) and lambda5(-/-)mice. V(H)81X-J(H)4-HC transcripts isolated from cells before or in the absence of pre-BCR assembly had no distinguishing complementarity-determining region 3 traits. In contrast, transcripts isolated subsequent to passage through the pre-BCR checkpoint had distinctive complementarity-determining regions 3 of nine amino acids in length (49%) and a histidine at position 1 (73%). Hence, our data define specific structural requirements for a functional HC, which is instrumental in shaping the diverse B cell repertoire.