Stein R, Basu A, Chen S, Shih L B, Goldenberg D M
Garden State Cancer Center, Center for Molecular Medicine and Immunology, Newark, NJ 07103.
Int J Cancer. 1993 Dec 2;55(6):938-46. doi: 10.1002/ijc.2910550611.
The murine monoclonal antibody (MAb) RS7-3G11 is an IgG1 with pancarcinoma reactivity, which has been raised against human squamous-cell carcinoma of the lung. Immunoperoxidase staining of frozen tissue sections demonstrated that the antigen defined by RS7-3G11 is present in tumors of the lung, stomach, bladder, breast, ovary, uterus and prostate. The rate and extent of internalization of RS7-3G11 into Calu-3, an adenocarcinoma of the lung cell line, was investigated using unconjugated MAb, followed by fluorescence labelling, and by binding 125I-RS7-3G11 followed by acid removal of surface-bound antibody. Rapid internalization of MAb RS7-3G11 into target cells was observed. Antibody internalization was noted at 30 min, and by 2 hr virtually all MAb RS7-3G11 was internal. Although MAb RS7-3G11 was raised against non-small-cell carcinoma of the lung, ME-180, a cervical-carcinoma cell line, expresses higher quantities of the antigen than the lung-carcinoma cell lines. Due to the higher antigen density in ME-180 cells, this line was used for immunoprecipitation studies and antigen purification. Immunoprecipitation studies using the ME-180 cervical-carcinoma cell line metabolically labeled with [3H]leucine or [3H]glucosamine demonstrated that the antigen defined by RS7-3G11 is a glycoprotein of M(r) 46 kDa. Deglycosylation by treatment with endoglycosidase-F resulted in a protein with a M(r) of 35 kDa. RS7-3G11-antigen was purified from ME-180 tissue-culture cells using affinity-column chromatography. By SDS-PAGE it was seen that the antigen was highly purified. The major band appeared at M(r) of 45 to 48 kDa. This result is in agreement with the immunoprecipitation studies. The broad band observed in the SDS-PAGE is typical of many glycoproteins, and suggests heterogeneity of glycosylation. Chemical and enzymatic treatments of the antigen, followed by Western blot analyses, suggest that the RS7-3G11 antigenic determinant is composed of a conformation-dependent peptide.
鼠单克隆抗体（MAb）RS7-3G11是一种具有泛癌反应性的IgG1，它是针对人肺鳞状细胞癌产生的。对冷冻组织切片进行免疫过氧化物酶染色显示，RS7-3G11所定义的抗原存在于肺、胃、膀胱、乳腺、卵巢、子宫和前列腺的肿瘤中。使用未偶联的单克隆抗体，随后进行荧光标记，并通过结合125I-RS7-3G11，然后用酸去除表面结合的抗体，研究了RS7-3G11内化到肺腺癌细胞系Calu-3中的速率和程度。观察到单克隆抗体RS7-3G11迅速内化到靶细胞中。在30分钟时注意到抗体内化，到2小时时，几乎所有的单克隆抗体RS7-3G11都已内化。尽管单克隆抗体RS7-3G11是针对肺非小细胞癌产生的，但宫颈癌细胞系ME-180表达的该抗原量比肺癌细胞系更高。由于ME-180细胞中抗原密度更高，该细胞系被用于免疫沉淀研究和抗原纯化。使用用[3H]亮氨酸或[3H]葡糖胺代谢标记的ME-180宫颈癌细胞系进行免疫沉淀研究表明，RS7-3G11所定义的抗原是一种分子量为46 kDa的糖蛋白。用内切糖苷酶-F处理进行去糖基化后，得到一种分子量为35 kDa的蛋白质。使用亲和柱色谱从ME-180组织培养细胞中纯化RS7-3G11抗原。通过SDS-PAGE可以看出抗原得到了高度纯化。主要条带出现在分子量为45至48 kDa处。这一结果与免疫沉淀研究一致。在SDS-PAGE中观察到的宽带是许多糖蛋白的典型特征，表明糖基化存在异质性。对抗原进行化学和酶处理，随后进行蛋白质印迹分析，表明RS7-3G11抗原决定簇由一个构象依赖性肽组成。
