Couto J R, Blank E W, Peterson J A, Ceriani R L
Cancer Research Fund of Contra Costa, Walnut Creek, CA 94596.
Hybridoma. 1993 Aug;12(4):485-9. doi: 10.1089/hyb.1993.12.485.
We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of monoclonal antibody KC4G3. VL belongs to group II and resulted from a V kappa-J kappa 2 recombination. VH belongs to group IIId and arose from a V-D9-JH3 recombination. The VL and VH frameworks are respectively 84% and 83% identical to the corresponding VL and VH human consensus frameworks. The deduced amino acid sequence of VL contains an asparagine-linked glycosylation site in framework 3 (N74 I75 S76). We have determined that a large fraction of the light chains are indeed glycolysated. We constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine KC4G3 Fv-encoding cDNAs into plasmids encoding a human IgG1, kappa Fc domain) and expressed it in SP2/0-Ag14 mouse myeloma cells. This chimeric monoclonal antibody is designated ChiKC4. We have determined that the murine monoclonal antibody KC4G3 binds the human breast mucin with an affinity constant of 1.1 x 10(9) M-1. ChiKC4 binds the same antigen with an affinity constant of 1.2 x 10(9) M-1. ChiKC4 binds the carcinoma tissue sections by the ABC immunoperoxidase method in an identical manner as does KC4G3.
我们已克隆并测序了编码单克隆抗体KC4G3轻链（VL）和重链（VH）可变区的cDNA。VL属于第二组，由Vκ-Jκ2重排产生。VH属于第三组d，由V-D9-JH3重排产生。VL和VH框架分别与相应的人源共有框架有84%和83%的同源性。VL推导的氨基酸序列在框架3（N74 I75 S76）中含有一个天冬酰胺连接的糖基化位点。我们已确定很大一部分轻链确实发生了糖基化。我们构建了一种IgG1、κ人/鼠嵌合抗体（通过将编码鼠KC4G3 Fv的cDNA插入编码人IgG1、κ Fc结构域的质粒中），并在SP2/0-Ag14小鼠骨髓瘤细胞中进行表达。这种嵌合单克隆抗体被命名为ChiKC4。我们已确定鼠单克隆抗体KC4G3以1.1×10⁹ M⁻¹的亲和常数与人乳腺粘蛋白结合。ChiKC4以1.2×10⁹ M⁻¹的亲和常数结合相同抗原。ChiKC4通过ABC免疫过氧化物酶法与KC4G3以相同方式结合癌组织切片。
