Ofatumumab monoclonal antibody affinity maturation through in silico modeling

Background: Ofatumumab, an anti-CD20 mAb, was approved in 2009 for the treatment of chronic lymphocytic leukemia. This mAb acts through immune-mediated mechanisms, in particular complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity by natural killer cells as well as antibody-dependent phagocytosis by macrophages. Apoptosis induction is another mechanism of this antibody. Computational docking is the method of predicting the conformation of an antibody-antigen from its separated elements. Validation of the designed antibodies is carried out by docking tools. Increased affinity enhances the biological action of the antibody, which in turn improves the therapeutic effects. Furthermore, the increased antibody affinity can reduce the therapeutic dose of the antibody, resulting in lower toxicity and handling cost

Methods: Considering the importance of this issue, using in silico analysis such as docking and molecular dynamics, we aimed to find the important amino acids of the Ofatumumab antibody and then replaced these amino acids with others to improve antibody-binding affinity. Finally, we examined the binding affinity of antibody variants to antigen

Results: Our findings showed that variant 3 mutations have improved the characteristics of antibody binding compared to normal Ofatumumab antibodies. Conclusion: The designed anti- CD20 antibodies showed potentiality for improved affinity in comparison to commercial Ofatumumab

Development of Genetically Modified Chinese Hamster Ovary Host Cells for the Enhancement of Recombinant Tissue Plasminogen Activator Expression

Background: Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. However, the development of stable, high-yielding CHO cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. Therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. In order to examine the possibility of generating improved CHO host cells, cell line engineering approaches were developed based on ceramide transfer protein (CERT), and X-box binding protein 1s (XBP1s). Methods: CHO cells were transfected with CERT S132A, a mutant variant of CERT which is resistant to phosphorylation, or XBP1s expression plasmids, and then stable cell pools were generated. Transient expression of t-PA was examined in engineered cell pools in comparison to un-modified CHO host cells. Results: Overexpression of CERT S132A led to the enhancement of recombinant tissue plasminogen activator (t-PA) expression in transient expression by 50%. On the other hand, it was observed that the ectopic expression of the XBP1s, did not improve the t-PA expression level. Conclusion: The results obtained in this study indicate successful development of the improved CHO host cells through CERT S132A overexpression.

Retroviral transduction of fluonanobody and the variable domain of camelid heavy-chain antibodies to chicken embryonic cells

Single domain antibodies from camel heavy chain antibodies [VHH or nanobody], are advantages due to higher solubility, stability, high homology with human antibody, lower immunogenicity and low molecular weight. These criteria make them candidates for production of engineered antibody fragments particularly in transgenic animals. To study the development of transgenic chicken using a recombinant retrovirus containing fluonanobody. The retrovirus constructs containing nanobody genes along with secretory signals and GFP gene were established and packed. The virus particle containing the obtained fusion gene was injected into the eggs in stage X. Molecular detection and protein analysis was done in the G0 chickens. The rate of hatched chicken after gene manipulation was estimated to be about 33%. Real-Time PCR assay showed that the nanobody along with GFP gene were integrated in cells of 1.2% of chickens. We conclude that although the rate of gene transfer by recombinant viruses in chickens is low, it would be possible to transfect the target camel immunoglobulin gene into chicken genome.

[Enhancement of recombinant human tissue plasminogen activator expression in CHO cells using matrix attachment region containing vectors in combination with promoter activation strategy]

Development of high producing mammalian cell lines is a major bottleneck in manufacturing of recombinant therapeutic proteins. This study examines the effect of using the matrix attachment region from the human interferon beta gene in combination with promoter activation strategy with E1A 13S protein on human tissue plasminogen activator [t-PA] expression in Chinese hamster ovary [CHO] cells. The matrix attachment region was cloned in 3' and 5' flanking sides of the t-PA expression cassette in pTPA vector to generate pMTPA. After transfection of the cells with pTPA and pMTPA vectors, stable cell pools were developed and the t-PA expression level determined for each stable cell line. In the next step, E1A 13S expression plasmid was transfected to stable cell pools and t-PA titers were measured after 72 hours. Integration of pTPA and pMTPA vectors in the CHO genome was confirmed by PCR analysis on genomic DNA of stable cell pools. Analysis of the t-PA expression level showed a three-fold enhancement in pMTPA transfected cells compared to pTPAcontaining cells. t-PA expression was further enhanced up to 1771 U/ml by transient expression of E1A 13S in pMTPA stable cell pools. These results have shown that incorporation of matrix attachment region in an expression vector in combination with promoter activation can effectively enhance recombinant protein expression levels in CHO cells.

Periplasmic expression of a novel human bone morphogenetic protein-7 mutant in escherichia coli

Bone Morphogenetic Proteins [BMPs] belong to the transforming growth factor-beta [TGF-beta] superfamily, and play an important role in bone metabolism. Recombinant forms of BMP-2 and BMP-7 are the only BMPs used clinically. In this study the mature part of human bone morphogenetic protein-7 [BMP-7] was engineered through substitution of the BMP-7 Nterminal sequence by heparin-binding site of BMP-2. This targeted substitution was made to enhance the binding affinity of the novel protein to the extracellular matrix components such as heparin and heparan sulfate proteoglycans [HSPGs]. The engineered protein was expressed in Escherichia coli [E.coli]. The PelB signal sequence was used to translocate soluble proteins into the periplasmic space of E.coli. The protein was purified from periplasmic extract using Ni-NTA chromatography and the SDS-PAGE and western blot analysis confirmed the successful expression of the novel protein. The novel hBMP-7 mutant was produced as approximately 16 kDa monomer. It was found that the heparin binding of this protein was approximately 50% more than that of the wild-type at a protein concentration of 500 ng/ml. The findings showed that the periplasmic expression may be suitable to produce complex proteins like BMPs