title: Generation and characterization of an anti-CD19 single-chain Fv immunotoxin composed of C-terminal disulfide-linked dgRTA.
abstract: Our laboratory utilized two methods to produce the anti-CD19 immunotoxin containing a single-chain Fv (scFv) FVS191 and a ricin A chain (RTA). The first method produced the recombinant protein FVS191CDRTA from a fusing gene containing sequences encoding FVS191, catheptsin D proteinase digestion site (CD), and RTA. FVS191CDRTA did not show CD19 antigen binding and cytotoxic activity. The second method generated a disulfide-linked FVS191cys-dgRTA from a FVS191cys, the FVS191 with an additional C-terminal cysteine, and a deglycosylated RTA (dgRTA). The formation of FVS191cys-dgRTA is efficient; up to 70% of the proteins participating in the reaction had formed FVS191cys-dgRTA when the molar ratio of FVS191cys to dgRTA was 1:1. A competitive ELISA assay indicated that FVS191cys-dgRTA and the parental monoclonal antibody B43 possessed comparable CD19 binding abilities. The protein synthesis inhibition assay revealed that FVS191cys-dgRTA was toxic to CD19 positive cell lines, but it was less potent than the intact antibody-conjugated B43-dgRTA, which had an IC50 = 2 x 10(-11) M. 125I-Labeled FVS191 and 125I-labeled B43 were internalized by Nalm-6 cells at 37 degrees C as demonstrated by internalization studies; this result indicates that cross-linking of CD19 antigen is not required for the endocytosis of CD19 and raises the possibility that the lower cytotoxity of FVS191cys-dgRTA is not due to the monovalent binding of CD19 by FVS191cys-dgRTA. Our study with anti-CD19 scFv immunotoxin indicates that the formation of a disulfide-linked scFv immunotoxin is an alternative to the recombinant method of producing scFv immunotoxin.
authors.affiliation: University of Minnesota Cancer Center, Biotherapy Institute, Minneapolis 55455, USA.
authors.forename: D
authors.initials: D
authors.lastname: Wang
authors.forename: Q
authors.initials: Q
authors.lastname: Li
authors.forename: W
authors.initials: W
authors.lastname: Hudson
authors.forename: E
authors.initials: E
authors.lastname: Berven
authors.forename: F
authors.initials: F
authors.lastname: Uckun
authors.forename: J H
authors.initials: JH
authors.lastname: Kersey
mesh_terms: D018941:Antigens, CD19 / Q000737:chemistry* / Q000378:metabolism; D002402:Cathepsin D / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry; D000922:Immunotoxins / Q000378:metabolism* / Q000633:toxicity*; D012276:Ricin / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*
chemical_list: D018941:Antigens, CD19; D003432:Cross-Linking Reagents; D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D000922:Immunotoxins; D012276:Ricin; D002402:Cathepsin D