Humanization of chimeric anti-CD20 antibody by logical and bioinformatics approach with retention of biological activity.

AIM: To develop a fully bioactive humanized antibody from the chimeric rituximab for potential clinical applications using a relatively simpler and faster logical and bioinformatics approach. METHODS: From bioinformatics data, mismatched mouse amino acids in variable light and heavy chain amphipathic regions were identified and substituted with those common to human antibody framework. Appropriate synthetic DNA sequences inserted into vectors were transfected into HEK293 cells to produce the humanized antibody. RESULTS: Humanized antibodies showed specific binding to CD20 and greater cytotoxicity to cancer WIL2-NS cell proliferation than rituximab in vitro. CONCLUSION: A humanized version of rituximab with potential to be developed into a biobetter for treatment of B-cell disorders has been successfully generated using a logical and bioinformatics approach.

Stable expression of H1C2 monoclonal antibody in NS0 and CHO cells using pFUSE and UCOE expression system.

From our recent publications, it was found that the deimmunization method (Dharshanan et al. (2012) Sci Res Essays 7:2288-2299) should be applied for the development of humanized anti-C2 monoclonal antibody (H1C2 mAb). However, the overlapping-PCR mutagenesis procedure used to insert the variable regions into cloning vectors was laborious and time-consuming. Additionally, the expression of H1C2 mAb in NS0 cells was low in static culture vessels. Therefore H1C2 mAb was redeveloped by deimmunization method with the following modifications in order to optimize the production of H1C2 mAb. First, instead of the overlapping-PCR mutagenesis procedure, synthetic DNA coding the variable regions were used to express the mAb. Second, two expression vectors, pFUSE and UCOE, were used to express H1C2 mAb in NS0 cells and CHO cells in order to investigate the combination that gave the highest number of high producing stable clones. This will provide the highest chance of finding clones with the requisite high productivity and stability required for manufacturing. We found that transfection of UCOE in CHO cells generated the highest number of high producing stable clones. To our knowledge, this is the first time that H1C2 mAb has been expressed in CHO cells.

Rapid automated selection of mammalian cell line secreting high level of humanized monoclonal antibody using Clone Pix FL system and the correlation between exterior median intensity and antibody productivity

The selection of high-producing mammalian cell lines is a crucial step in process development for the production of biopharmaceuticals. Previously, cloning by limiting dilution method was used to isolate monoclonal NS0 cells secreting high levels of humanized-C2 monoclonal antibodies. However limiting dilution method is time consuming, has low probability of monoclonality and is significantly limited by the number of clones that can be feasibly screened. In order to minimize the duration and to increase the probability of obtaining high-producing clones with high monoclonality, an automated colony picker, Clone Pix FL system was used to replace limiting dilution method. We were able to screen 1 x 10(5) clones secreting humanized monoclonal antibodies and high producer clones were selected in just 7 days. Briefly, semi-solid media was used to immobilize single cells separately and allow them to proliferate into discrete clones. The high viscosity nature of the semi-solid media retains the secreted products in the vicinity of the associated clones. Using Clone Pix FL system, all clones were screened and the producer clones with different exterior fluorescent intensities were automatically isolated. We were able to isolate rare high-producers (> 3000 FU) with frequency of as low as 0.003 percent of the population. A quantitative ELISA was also performed to evaluate the correlation between the fluorescence intensity of clones with its corresponding antibody productivity. Clones with fluorescence intensity of < 1000 FU showed relatively low antibody productivity compared with those greater than 1000 FU; however above this there was no correlation of production with the increase in fluorescence intensity. Hence, although the high-throughput, rapid and automated nature of Clone Pix FL system allows the screening of large number of cells in a short period of time with also an increased in the probability of obtaining rare and precious high-producing clones...

Development of an indirect enzyme immunoassay using monoclonal antibodies for the measurement of 17alpha-hydroxyprogesterone in human serum.

An indirect enzyme immunoassay for the measurement of total 17alpha-hydroxyprogesterone (17OHP) in serum using monoclonal antibodies generated in our laboratory was developed. Here, (a) instead of extraction with solvents, serum was heated to free protein-bound 17OHP and assay was performed at pH 9.6, (b) to ensure uniform assay conditions for both standards and samples, buffer for standards contained charcoal-stripped pre-heated pooled cord serum. Assays were done in 96-well EIA microplates pre-coated with 17alpha-hydroxyprogesterone-3-(o-carboxymethyl)oxime: bovine serum albumin. Secondary antibody was horseradish peroxidase-linked sheep anti-mouse IgG polyclonal antibody. The method was accurate and suitable for screening for congenital adrenal hyperplasia.

Production and characterization of monoclonal antibodies against 17alpha-hydroxyprogesterone.

Hybridomas secreting monoclonal antibodies (MAbs) against 17alpha-hydroxyprogesterone (17OHP) have been generated. These MAbs are highly specific and have an affinity of 7-12 x 10(7) M(1). The hybridomas were obtained by fusion of spleen cells from immunized mice with mouse myeloma P3X63 Ag8.653 cells. The antigen used for immunization was 17OHP conjugated to bovine serum albumin (17OHP:BSA). Fused cells were plated and cloned in 96-well microtiter plates. Wells containing hybridomas were screened simultaneously for specific gamma globulin (IgG) and anti-17OHP activity using an enzyme-linked immunosorbent assay (ELISA)-based method, which is faster than the conventional radioimmunoassay (RIA) screening procedure. Limiting dilution methods were used to obtain single hybridoma clones producing MAb. The stable hybridomas secreting anti-17OHP MAbs were expanded into bioreactors or ascites fluid for large-scale production of the required antibodies. These MAbs will be used in the formulation of a 17OHP assay kit to screen for congenital adrenal hyperplasia (CAH) in local newborn human population.