Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic <it>P. pastoris </it>yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs.</p> <p>Results</p> <p>Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and <it>P. pastoris </it>cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from <it>Pichia </it>supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in <it>Pichia </it>more efficient than most other previously described trispecific or trivalent molecules produced in <it>E. coli</it>.</p> <p>Conclusion</p> <p>Bi- and tribody molecules are efficiently produced in <it>P. pastoris</it>. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of <it>P. pastoris </it>as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.</p

THE EXPRESSION OF THE RECOMBINANT LOW-AFFINED Fc-RECEPTOR OF THE MAN'S IMMUNOGLOBULIN E IN ESCHERICHIA COLI IN THE CELLS OF MAMMALS

The human protein CD23 was investigated. Obtained and characterized have been the different systems for the expression kDNA CD23 and its biologically active fragments (sCD23). For creating the expressing constructions, plasmides rKK223-3 and BMGNeo have been used. For the first time, the recombinant preparation sCD23(25kDa) in the form of the hybride protein with fragment of human IL3 in the cells E. coli with the output of about 10% of the total cellular protein has been received and the method of its isolation and cleaning has been developed. This preparation can be used for receiving the monoclonal antibodies and for the development of diagnostica. The polymorphism of chromosome gene CD23 has been revealed in the number of the man's cellular lines (RPM18866, U937, Raji, Namalva, Daudi). First, the absence of gene CD23 in line Daudi has been showh. For the first time the expression of the membrane form CD23 and recombinant sCD23 (29kDa) in the form of the secreting protein has been obtained in the cells of the mouse myeloma X63.Ag8.653Available from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides