title: Potent anti-CD5 ricin A chain immunoconjugates from bacterially produced Fab' and F(ab')2.
abstract: We have used genetic engineering to obtain secretion of anti-human CD5 antibody fragments from Escherichia coli for conjugation to the 30-kDa form of ricin A chain (RTA30). This was accomplished by introducing stop codons at two positions in the hinge region of the human IgG1 gene so that coexpression of the truncated heavy-chain genes (Fd') with a light chain would result in Fab' and/or F(ab')2 proteins containing either one or two interheavy-chain cysteines. An Fd' gene encoding both interheavy-chain cysteines yielded a mixture of F(ab')2 and Fab', which could be separated by size-exclusion chromatography. An Fd' gene encoding only one interheavy-chain cysteine yielded primarily Fab'. Purified F(ab')2 protein was equivalent to unlabeled chimeric IgG in competing for binding of IgG with CD5 antigen, while the molar concentration of the monovalent Fab' required for 50% binding inhibition was 4- to 5-fold higher than IgG. An immunoconjugate was prepared with Fab' by direct coupling to the unique free cysteine on RTA30. The bivalent F(ab')2 was conjugated to RTA30 after derivatization with the crosslinking agent 5-methyl-2-iminothiolane. These immunoconjugates efficiently killed a CD5+ T-cell line and human peripheral blood T cells.
authors.affiliation: XOMA Corporation, Santa Monica, CA 90404.
authors.forename: M
authors.initials: M
authors.lastname: Better
authors.forename: S L
authors.initials: SL
authors.lastname: Bernhard
authors.forename: S P
authors.initials: SP
authors.lastname: Lei
authors.forename: D M
authors.initials: DM
authors.lastname: Fishwild
authors.forename: J A
authors.initials: JA
authors.lastname: Lane
authors.forename: S F
authors.initials: SF
authors.lastname: Carroll
authors.forename: A H
authors.initials: AH
authors.lastname: Horwitz
mesh_terms: D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D015703:Antigens, CD / Q000276:immunology*; D001483:Base Sequence; D001667:Binding, Competitive; D018956:CD5 Antigens; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D000922:Immunotoxins / Q000378:metabolism* / Q000633:toxicity; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012276:Ricin / Q000378:metabolism*; D013601:T-Lymphocytes / Q000187:drug effects
chemical_list: D015703:Antigens, CD; D018956:CD5 Antigens; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D012276:Ricin