Siemers N O, Kerr D E, Yarnold S, Stebbins M R, Vrudhula V M, Hellström I, Hellström K E, Senter P D
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
Bioconjug Chem. 1997 Jul-Aug;8(4):510-9. doi: 10.1021/bc9700751.
The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.
L49（IgG1）单克隆抗体与p97（黑素转铁蛋白）结合，p97是一种肿瘤选择性抗原，在人类黑色素瘤和癌组织上表达。构建了一种重组融合蛋白L49-sFv-bL，它包含L49的抗体结合区域，并与阴沟肠杆菌r2-1β-内酰胺酶（bL）融合，在大肠杆菌可溶性表达系统中表达并纯化至同质。利用信号序列和恒定区引物，通过逆转录-聚合酶链反应从L49杂交瘤mRNA中克隆L49的可变区。通过将VH、VL和sFv接头序列杂交插入到含有与pelB前导序列融合的bL基因的pET噬菌粒模板上，完成了编码L49-sFv-bL基因的构建。发现L49-sFv-bL在大肠杆菌中的最佳可溶性表达发生在23℃，用50μM异丙基β-D-硫代半乳糖苷诱导，并使用非离子去污剂Nonidet P-40从细菌中分离。在中国仓鼠卵巢细胞中构建并表达p97抗原的可溶性形式，使得能够采用基于亲和力的方法分析和纯化融合蛋白。表面等离子体共振、荧光激活细胞分选和米氏动力学分析表明，L49-sFv-bL保留了单价L49的抗原结合能力以及bL的酶活性。体外实验表明，L49-sFv-bL与表达p97抗原的3677黑色素瘤细胞结合，并激活了7-（4-羧基丁酰胺基）头孢菌素氮芥（CCM），一种头孢菌素氮芥前药。基于这些结果，将L49-sFv-bL注射到皮下接种3677肿瘤的裸鼠体内，并通过测量bL活性确定其定位。在给予缀合物后4小时和48小时，肿瘤与血液的缀合物比率分别为13和150，肿瘤与肝脏、脾脏和肾脏的比率甚至更高。化学合成的L49-Fab'-bL缀合物产生的肿瘤与血液比率（给药后72小时为5.6）比L49-sFv-bL低得多。治疗实验表明，耐受性良好剂量的L49-sFv-bL/CCM组合可治愈裸鼠体内的3677肿瘤。L49-sFv-bL良好的药代动力学特性使得在前药缀合物给药12小时后即可开始前药治疗。因此，L49-sFv-bL似乎具有用于位点选择性抗癌前药激活的良好特性。
