title: Expression of an anti-CD33 single-chain antibody by Pichia pastoris.
abstract: CD33 is a cell surface glycoprotein expressed on cells of myelomonocytic lineage, leukaemic cells, but not haematopoietic stem cells. By virtue of its expression pattern, CD33 has become a popular target for new immunotherapeutic approaches to treat acute myeloid leukaemia. The methylotrophic yeast Pichia pastoris strain KM71H was used to produce an anti-CD33 single chain variable fragment (scFv), with the intention of conjugation to a radioisotope, for therapeutic use. To direct secreted expression of the anti-CD33-scFv the alpha-mating factor secretory signal sequence (alpha-MF) was used, with constructs containing a complete (CS) and incomplete (INCS) cleavage site to accommodate the potential outcomes of dibasic endopeptidase, Kex2, and dipeptidyl amino peptidase, Ste13, processing. The anti-CD33-scFv was expressed in BMMY cultures using both constructs, with a final yield of 48 mg/l (CS) and 11 mg/l (INCS). N-terminal sequencing showed that the CS-scFv had not been cleaved by Ste13, leaving amino acids EAEA at the N-terminus. The INCS-scFv construct produced a mixture of 50% authentic scFv and 50% with 11 amino acids from the alpha-MF remaining at the N-terminus. Despite the aberrations in alpha-MF processing, the anti-CD33-scFv's produced from both constructs were found to be functional. Flow cytometry and Biacore analysis demonstrated binding to target antigen CD33 on the surface of human leukaemic cell line HL-60, and to recombinant soluble CD33 respectively.
authors.affiliation: Department of Biosciences, University of Kent, Giles Lane, Canterbury, Kent, CT2 7NJ, UK.
authors.forename: Louise M
authors.initials: LM
authors.lastname: Emberson
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Trivett
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Blower
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Nicholls
mesh_terms: D015703:Antigens, CD / Q000276:immunology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology; D003001:Cloning, Molecular; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007167:Immunotherapy / Q000379:methods; D019169:Jurkat Cells; D015470:Leukemia, Myeloid, Acute / Q000276:immunology / Q000628:therapy; D010843:Pichia / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020539:Sequence Analysis, Protein; D063268:Sialic Acid Binding Ig-like Lectin 3; D020349:Surface Plasmon Resonance; D014170:Transformation, Genetic
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D063268:Sialic Acid Binding Ig-like Lectin 3