Generation and Characterization of an Anti-CD19 Single-Chain Fv Immunotoxin Composed of C-Terminal Disulfide-Linked dgRTA

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• 作者：Duo Wang ; Quanzhi Li ; Wendy Hudson ; Erica Berven ; Fatih Uckun ; and John H. Kersey
• 刊名：Bioconjugate Chemistry
• 出版年：1997
• 出版时间：November 1997
• 年：1997
• 卷：8
• 期：6
• 页码：878 - 884
• 全文大小：332K
• 年卷期：v.8,no.6(November 1997)
• ISSN：1520-4812

文摘

Our laboratory utilized two methods to produce the anti-CD19 immunotoxincontaining a single-chain Fv (scFv) FVS191 and a ricin A chain (RTA). The first methodproduced the recombinant proteinFVS191CDRTA from a fusing gene containing sequences encoding FVS191,catheptsin D proteinasedigestion site (CD), and RTA. FVS191CDRTA did not show CD19antigen binding and cytotoxicactivity. The second method generated a disulfide-linkedFVS191cys-dgRTA from a FVS191cys, theFVS191 with an additional C-terminal cysteine, and a deglycosylated RTA(dgRTA). The formationof FVS191cys-dgRTA is efficient; up to 70% of the proteinsparticipating in the reaction had formedFVS191cys-dgRTA when the molar ratio of FVS191cys to dgRTA was 1:1.A competitive ELISAassay indicated that FVS191cys-dgRTA and the parental monoclonalantibody B43 possessedcomparable CD19 binding abilities. The protein synthesisinhibition assay revealed that FVS191cys-dgRTA was toxic to CD19 positive cell lines, but it was less potent thanthe intact antibody-conjugatedB43-dgRTA, which had an IC₅₀ = 2 ×10^-11 M. ¹²⁵I-Labeled FVS191and ¹²⁵I-labeled B43 wereinternalized by Nalm-6 cells at 37 mages/entities/deg.gif">C as demonstrated byinternalization studies; this result indicatesthat cross-linking of CD19 antigen is not required for the endocytosisof CD19 and raises the possibilitythat the lower cytotoxity of FVS191cys-dgRTA is not due to themonovalent binding of CD19 byFVS191cys-dgRTA. Our study with anti-CD19 scFv immunotoxinindicates that the formation of adisulfide-linked scFv immunotoxin is an alternative to the recombinantmethod of producing scFvimmunotoxin.