Characterisation of recombinant CHO cell lines by investigation of protein productivities and genetic parameters

We have generated a recombinant CHO cell line expressing the fusion protein EpoFc. After selection and screening, protein expression, gene and mRNA copy numbers were analysed in order to gain more information on the influence of genetic parameters on the productivity and stability of production cells. Results from semi-quantitative blot methods were compared to quantitative PCR (qPCR) analyses, whose advantage mainly lies in their higher sensitivity, and the cheaper and faster methodology. We developed stable and high producing clones with low gene copy numbers, in contrast to other cell lines where multiple steps of methotrexate amplification have lead to hundreds of copies of inserts with the risk of karyotypic instabilities and decreased growth rates that overcome the benefits of increased productivities. When comparing genetic parameters to productivity, a good correlation of mRNA levels with specific productivity was observed, whereas high gene copy numbers were not always accompanied by high protein expressions. Based on our data derived from a typical example of a cell line development process, genetic parameters are useful tools for the selection of scalable production clones. Nevertheless, a wider range of cell lines has to be investigated in order to implement genetic analyses into a screening process.     

Increased antigen binding strengths of hybrid antibodies produced by human hybrid hybridomas

Two cell lines of human hybridomas were fused to generate hybrid antibodies. One human hybridoma cell line was HT2 producing IgM monoclonal antibody (MAb) reactive to carboxy peptidase A (Cpase) and double stranded DNA (ds DNA) and another was SU-1-D2 secreting IgM MAb reactive to ds DNA but not to Cpase. Most hybrid hybridomas obtained by fusion of the two hybridomas secreted hybrid antibodies exhibiting increased antigen binding strengths. All of the hybrid antibodies with increased binding strengths against Cpase and ds DNA contained only the light chains derived from SU-1-D2. These results suggested that increase in the binding strength of the hybrid antibodies resulted from heterogeneous association of H and L chains derived from HT2 and SU-1-D2 cells.     

Overexpression of mutant cell division cycle 25 homolog B (CDC25B) enhances the efficiency of selection in Chinese hamster ovary cells

The effects of mutant cell division cycle 25 homolog B (CDC25B) overexpression on the generation of cells producing a monoclonal antibody were investigated in Chinese hamster ovary (CHO) cells. Mutant CDC25B (m-CDC25B) expression plasmids were transfected into CHO DG44-derived cells producing a monoclonal antibody, and the frequency of highly producing cells was assessed following gene amplification in the presence of 250 nM methotrexate. Most of the clones obtained from the m-CDC25B-overexpressing cells had higher antibody titers than did mock-transfected control cells. This arose from either higher transgene copy numbers or higher mRNA expression levels for the antibody. However, the high mRNA expression levels were not always accompanied by increases in transgene copy numbers. Our results suggest that cells producing high levels of a monoclonal antibody can be selected efficiently using m-CDC25B overexpression.     

HSV-IgM人-鼠嵌合抗体制备及稳定细胞株构建工艺开发

为实现体外大规模制备单纯疱疹病毒HSV-IgM(HSV1,HSV2)人鼠嵌合抗体,本研究通过RNA连接酶介导的cDNA末端快速扩增(RNA ligase-mediated rapid amplification of cDNA ends,RLM-RACE)技术获取其对应杂交瘤细胞基因序列,构建嵌合抗体至真核表达载体,在CHO-S细胞中稳定表达所需目的蛋白。同时优化稳定细胞株筛选工艺,对细胞池构建阶段和单克隆筛选阶段的加压条件进行摸索与探究,最后目的抗体采用蛋白L亲和纯化法进行纯化并进行生物活性检测;最终成功制备899 kDa和909 kDa的稳定高表达重组IgM抗体(HSV1,HSV2)细胞株。结果表明,最适筛选压力为20P200M(一轮加压)和50P1000M(二轮加压);使用加压培养基进行单克隆筛选抗体表达量较高,HSV1-IgM和HSV2-IgM单克隆最终表达量分别为1620 mg/L和623 mg/L。本研究为HSV1和HSV2的IgM系列重组抗体质控品开发以及体外高表达分泌IgM亚型抗体提供理论与实践基础。     

Selective gene therapy for prostate cancer cells using liposomes conjugated with IgM type monoclonal antibody against prostate-specific membrane antigen

Prostate cancer cells express prostate-specific membrane antigen (PSMA). We developed an IgM type monoclonal antibody against PSMA. The antibody was coupled to poly-L-lysine and thereafter this conjugate was mixed with cationic liposomes containing plasmid DNA. The antibody-liposome complex was tested whether it could deliver the gene of interest selectively to the PSMA positive cells. As assessed by beta-galactosidase reporter gene, the transfection efficiency was 13.2% with anti-PSMA-liposome complex as compared to 4% with control IgM liposome complex. In contrast, no such differences were observed in PSMA negative PC-3, DU145 and T24 cells. Furthermore, in the suicide gene therapy in vitro with thymidine kinase gene plus ganciclovir system, anti-PSMA liposome complex demonstrated a selective growth inhibitory effect on PSMA positive LNCaP cells but not on PSMA negative cell lines.     

Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering

The process of establishing high-producing cell lines for the manufacture of therapeutic proteins is usually both time-consuming and laborious due to the low probability of obtaining high-producing clones from a pool of transfected cells and slow cell growth under the strong selective pressure of screening to identify high-producing clones. We present a novel method to rapidly generate more high-producing cells by accelerating transgene amplification. A small interfering RNA (siRNA) expression vector against ataxia telangiectasia and Rad3 related (ATR), a cell cycle checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells. The influences of ATR downregulation on gene amplification and the productivity were investigated in CHO cells producing green fluorescent protein (GFP) and secreting monoclonal antibody (mAb). The ATR-downregulated cells showed up to a 6-fold higher ratio of GFP-positive cells than that of the control cell pool. Moreover, the downregulated mAb-producing cells had about a 4-fold higher specific production rate and a 3-fold higher volumetric productivity as compared with the mock cells. ATR-downregulated cells showed a much faster increase in transgene copy numbers during the gene amplification process via methotrexate (MTX) treatment in both GFP- and mAb-producing cells. Our results suggest that a pool of high-producing cells can be more rapidly generated by ATR downregulation as compared with conventional gene amplification by MTX treatment. This novel method may be a promising approach to reduce time and labor in the process of cell line development.     

IgM and IgG direct plaque-forming cells develop during the immune response of miniature swine to sheep erythrocytes.

A procedure for the direct determination of the class of antibody produced during the immune response of Minnesota miniature swine was developed by analyzing the distribution of Ig classes of direct hemolytic plaque-forming cells (PFC). The procedure involved cytoplasmic staining of fixed, direct PFC with class-specific fluorescent antibody reagents, utilizing the plaque assay methodology developed by Kennedy and Axelrad. Mean percentages of cytoplasmic IgM- and IgG-positive direct PFC determined during the immune response of conventional swine were found to be approximately 70% IgM positive and 30% IgG positive. Sheep red blood cell ghosts contained within the plaques stained positive for the same class of immunoglobulin as was expressed intracytoplasmically by the central PFC, indicating a specific correlation between cytoplasmic Ig class and the secreted class of antibody. Pronase treatment of spleen cells prior to assay, a procedure which removes surface Ig, produced no alteration in the frequencies of IgM- or IgG-positive direct PFC, indicating that our staining procedure was detecting genuine cytoplasmic Ig. The lack of significant overlap in the combined percentages of IgM- and IgG-positive PFC suggests that there should be few PFC producing antibody of both IgM and IgG classes simultaneously.     

Detection of a monoclonal human myeloma protein of IgM and IgG class by carp anti-idiotypic sera

In the Patient St. with a Morbus Waldenstr?m macroglobulinemia a double paraproteinemia could be detected. Besides the IgM myeloma protein an IgG myeloma protein was identified during the clinical course. A strong cross reactivity between the IgM and the IgG myeloma proteins was shown using anti-idiotypic antisera. This is an indirect indication for a common precursor cell clone of the IgM- and IgG-myeloma protein producing cells. The anti-idiotypic antisera were made in carp. The high specificity of these antisera could be confirmed by inhibition assays. The double paraproteinemia has been proved to be convenient model for testing the idiotypic specificity of anti-Id antisera of carp.     

Comparison of the production of a human monoclonal antibody against HIV-1 by heterohybridoma cells and recombinant CHO cells: A flow cytometric study

The production of human monoclonal antibodies for therapeutic use is of increasing importance for treatment of viral infections such as AIDS. As human x mouse heterohybridomas rarely reach the growth rates and cell specific production rates of mouse hybridomas the transfection of standard cell lines, such as CHO or BHK, is a promising alternative. This has the additional advantage that the IgG subtype can be changed to suit the desired application. However, the use of a cell line that has not originally developed to produce antibodies, as lymphocytes and myeloma cells have, might have unrecognised drawbacks. This will be especially significant in the case of antibodies as each molecule consists of 4 chains linked by disulphide bonds which require specific intracellular factors to be properly folded and processed (Heavy chain binding protein, Protein Disulfide Isomerase a.o.). In this study we have therefore compared two cell lines: a human x mouse heterohybridoma producing IAM-2F5, a human IgG₃ antibody specific for HIV-1 with neutralising properties and a Chinese Hamster Ovary cell transfected with dihydrofolate reductase and with the heavy and light chain genes of IAM-2F5 modified to IgG₁. From each cell line three subclones were selected with low, medium and high specific production rates. Batch cultures were performed and the following cellular parameters analysed by flow cytometry; 1) total RNA content (translational activity); 2) total protein content; 3) cell cycle phase distribution; 4) concentration of light and heavy chains; 5) concentration of helper proteins such as BiP and PDI. The production rate of heterohybridoma cells was best reflected in the intracellular concentration of kappa chain, while the gamma chain concentration was comparable for all three subclones. In the CHO cells the gamma chain expression and thus gene copy number appeared to be the limiting factor. The GRP78/BiP concentration in CHO remained unchanged in spite of a 5-fold higher concentration of gamma chain in the high producing subclone. The PDI concentration in CHO cells was much lower compared to the heterohybridoma cells, irrespective of production rates.Abbreviations PDI protein disulfide isomerase - GRP78/BiP Glucose regulated protein; Heavy chain binding protein