Genetic Construction and Characterization of an Anti-Monkey CD3 Single-Chain Immunotoxin with a Truncated Diphtheria Toxin

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• 作者：Shenglin Ma ; Huaizhong Hu ; Jerry Thompson ; Scott Stavrou ; Joshua Scharff ; and David M. Neville ; Jr.
• 刊名：Bioconjugate Chemistry
• 出版年：1997
• 出版时间：September 1997
• 年：1997
• 卷：8
• 期：5
• 页码：695 - 701
• 全文大小：216K
• 年卷期：v.8,no.5(September 1997)
• ISSN：1520-4812

文摘

We have previously developed a chemically conjugated anti-rhesus monkeyCD3 immunotoxin FN18-CRM9 that can deplete in vivo T cells and induce long termtolerance of mismatched renal allograftin rhesus monkeys. This immunotoxin is a monkey analogue ofanti-human CD3 immunotoxinUCHT1-CRM9. In this study, we cloned the light and heavy chainvariable regions of anti-monkeyCD3 monoclonal antibody FN18 and constructed a single-chain Fv (sFv) bylinking variable light andvariable heavy regions with a (Gly₄Ser)₃linker. The single-chain immunotoxin DT390-FN18sFvwasconstructed by ligating the sFv to the carboxyl terminus of DT390, atruncated form of diphtheriatoxin. The DT390-FN18sFv fusion protein was expressed inEscherichia coli and purified with Ni-RTA affinity and anion exchange columns. Similar to the chemicallyconjugated immunotoxin FN18-CRM9, DT390-FN18sFv can also specifically inhibit protein synthesis inprimary monkey T cells ina dose-dependent manner. DT390-FN18sFv at 10^-7mol/L or FN18-CRM9 at 10^-8 mol/L issufficientto reduce protein synthesis of monkey primary T cells to less than 5%of the control. The 50% inhibitiondosage (IC₅₀) of FN18-CRM9 is 1 × 10^-10mol/L, while the IC₅₀ of DT390-FN18sFv is 1 ×10^-8 mol/L, reflecting the lowered affinity of monovalent Fab' FN18 to itsparental divalent antibody. Theavailability of functional FN18sFv will provide the basis for theconstruction of divalent anti-CD3immunotoxins for preclinical studies on the induction of tolerance inorgan transplantation andexperimental autoimmune diseases.