Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a "caninized" version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer.

Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant production is still not common. In our study, stable monoclonal IgM producing CHO DG44 and HEK 293 cell lines, expressing two model IgM molecules (IgM-617 and IgM-012) were established. Recombinant cell lines were compared in regard of specific productivity, specific growth rate, maximal achieved antibody titer, gene copy numbers and transcription levels of transgene. IgM-617 cell lines were identified as high while IgM-012 clones were low producers. Although differences in gene copy numbers as well as in transcription levels were observed, they did not seem to be a limitation. Levels of relevant endoplasmic reticulumstress related proteins were analyzed and no indications of unfolded protein response were detected. This could indicate that the difference in the intrinsic protein stability of our model proteins (as was previously observed on purified samples) might cause lower yields of IgM-012. Transcriptomics and/or proteomics follow up studies might be necessary for identification of potential bottlenecks in IgM producing cell lines.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Recombinant CHO cell lines have integrated the expression vectors in various parts of the genome leading to different levels of gene amplification, productivity and stability of protein expression. Identification of insertion sites where gene amplification is possible and the transcription rate is high may lead to systems of sitedirected integration and will significantly reduce the process for the generation of stably and highly expressing recombinant cell lines. We have investigated a broad range of recombinant cell lines by FISH analysis and Giemsa-Trypsin banding and analysed their integration loci with regard to the extent of methotrexate pressure, transfection methods, promoters and protein productivities. To summarise, we found that the majority of our high producing recombinant CHO cell lines had integrated the expression construct on a larger chromosome of the genome. Furthermore, except from two cell lines, the exogene was integrated at a single site. The dhfr selection marker was colocalised to the target gene.

Recombinant human antibody production represents a major growing class of biopharmaceuticals based on the technological progress within the last decades especially in CHO cells. The HIV neutralizing human monoclonal antibody 2F5 was developed as hybridoma from human lymphocyte preparations. In order to estimate the potential of recombinant 2F5-expressing CHO cells, we generated different recombinant CHO cell lines by varying regulatory sequences, the codon usage, the signal peptides, and the transfection technique. These 2F5-expressing cell lines were developed by selection of the best producer, clone homogeneity, and clone stability. The gene copy number of the clones differed significantly due to methotrexate amplification. In one cell line, we identified only one copy of heavy chain and two copies of light chain. Neither the gene copy number nor the promoter was found to influence the amount of transcript exclusively emphasizing the positioning effect of the transgene. Messenger RNA levels were highest in 2F5/CO and may have resulted from a combination of the promoter and codon-optimized sequences, but unexpectedly, the amount of secreted product was not elevated in this configuration. In our example, translational and post-translational limitations are responsible for decreased antibody secretion.

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.

MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-014-5911-4) contains supplementary material, which is available to authorized users.