Construction, Expression, and Activities of L49-sFv--Lactamase, a Single-Chain Antibody Fusion Protein for Anticancer

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• 作者：Nathan O. Siemers ; David E. Kerr ; Susan Yarnold ; Mark R. Stebbins ; Vivekananda M. Vrudhula ; Ingegerd Hellstr&ouml ; m ; Karl Erik Hellstr&ouml ; m ; and Peter D. Senter
• 刊名：Bioconjugate Chemistry
• 出版年：1997
• 出版时间：July 1997
• 年：1997
• 卷：8
• 期：4
• 页码：510 - 519
• 全文大小：297K
• 年卷期：v.8,no.4(July 1997)
• ISSN：1520-4812

文摘

The L49 (IgG₁) monoclonal antibody binds to p97(melanotransferrin), a tumor-selective antigen thatis expressed on human melanomas and carcinomas. A recombinantfusion protein, L49-sFv-bL, thatcontains the antibody binding regions of L49 fused to theEnterobacter cloacae r2-1 mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-lactamase(bL)was constructed, expressed, and purified to homogeneity in anEscherichia coli soluble expressionsystem. The variable regions of L49 were cloned by reversetranscription-polymerase chain reactionfrom L49 hybridoma mRNA using signal sequence and constant regionprimers. Construction of thegene encoding L49-sFv-bL was accomplished by hybridization insertion ofV_H, V_L, and sFv linkersequences onto a pET phagemid template containing the bL gene fused tothe pelB leader sequence.Optimal soluble expression of L49-sFv-bL in E. coli wasfound to take place at 23 mages/entities/deg.gif">C with 50 mages/entities/mgr.gif">Misopropyl mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-D-thiogalactopyranoside induction and the useof the nonionic detergent Nonidet P-40 forisolation from the bacteria. Construction and expression of asoluble form of the p97 antigen in Chinesehamster ovary cells allowed affinity-based methods for analysis andpurification of the fusion protein.Surface plasmon resonance, fluorescent activated cell sorting, andMichaelis-Menten kinetic analysesshowed that L49-sFv-bL retained the antigen binding capability ofmonovalent L49 as well as theenzymatic activity of bL. In vitro experimentsdemonstrated that L49-sFv-bL bound to 3677 melanomacells expressing the p97 antigen and effected the activation of7-(4-carboxybutanamido)cephalosporinmustard (CCM), a cephalosporin nitrogen mustard prodrug. On thebasis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, andlocalization was determined bymeasuring bL activity. Tumor to blood conjugate ratios of 13 and150 were obtained 4 and 48 h postconjugate administration, respectively, and the tumor to liver, spleen,and kidney ratios were evenhigher. A chemically produced L49-Fab'-bL conjugate yielded a muchlower tumor to blood ratio (5.6at 72 h post administration) than L49-sFv-bL. Therapy experimentsestablished that well-tolerateddoses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumorsin nude mice. The favorablepharmacokinetic properties of L49-sFv-bL allowed prodrug treatment tobe initiated 12 h after theconjugate was administered. Thus, L49-sFv-bL appears to havepromising characteristics for site-selective anticancer prodrug activation.