Construction and characterization of a chimeric fusion protein consisting of an anti-idiotype antibody mimicking a breast cancer-associated antigen and the cytokine GM-CSF.
Construction and characterization of a chimeric fusion protein consisting of an anti-idiotype antibody mimicking a breast cancer-associated antigen and the cytokine GM-CSF.
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DOI：
10.1089/hyb.1999.18.193
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发表时间：
1999-04
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影响因子：
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通讯作者：
Pulak K. Tripathi;Hongxing Qin;M. Bhattacharya‐Chatterjee;Roberto L. Ceriani;K. Foon;S. Chatterjee
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文献类型：
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作者：
Pulak K. Tripathi;Hongxing Qin;M. Bhattacharya‐Chatterjee;Roberto L. Ceriani;K. Foon;S. Chatterjee
抗独特型抗体11 D10在生物学和抗原性上模拟高分子量人乳脂球（HMFG）的独特特异性表位，HMFG是一种存在于90%以上乳腺肿瘤样本中的癌症相关抗原。为了增强11 D10的免疫原性而无需载体蛋白或佐剂的帮助，我们制备了嵌合11 D10-GM-CSF融合蛋白用作疫苗。通过连接人κ恒定区上游的11 D10轻链可变区的DNA序列制备11 D10的表达质粒。携带GM-CSF的重链质粒是通过将人γ 1恒定区CH 1上游的重链可变区序列与编码成熟GM-CSF肽的DNA片段3'连接到CH 3外显子来制备的。通过电穿孔用轻链和重链载体转染NS 1浆细胞瘤细胞。纯化分泌在培养基中的融合蛋白，并通过凝胶电泳以及通过测定融合的GM-CSF的生物活性来表征。在非还原SDS-聚丙烯酰胺凝胶中，约200 Kd的单一条带与抗人κ、抗人IgG 1和抗GM-CSF抗体反应。在还原性聚丙烯酰胺凝胶中，约74 kd的蛋白质与抗人IgG 1和抗GM-CSF抗体反应。融合蛋白可诱导GM-CSF依赖性NFS-60细胞增殖。这些结果表明，该蛋白质是一种嵌合抗独特型抗体，由11 D10可变结构域，人κ和α 1恒定结构域组成，并与α 1恒定区融合的GM-CSF部分具有生物活性。
Anti-idiotype antibody, 11D10 mimics biologically and antigenically a distinct and specific epitope of the high molecular weight human milk fat globule (HMFG), a cancer-associated antigen present in over 90% of breast tumor samples. To augment the immunogenicity of 11D10 without the aid of a carrier protein or adjuvant, we made a chimeric 11D10-GM-CSF fusion protein for use as a vaccine. An expression plasmid for 11D10 was made by ligation of the DNA sequences of the 11D10 light-chain variable region upstream of the human kappa constant region. The heavy-chain plasmid carrying GM-CSF was made by ligation of the heavy-chain variable region sequences upstream of the human gamma1 constant region CH1 fused to the DNA fragment encoding the mature GM-CSF peptide 3' to the CH3 exon. NS1 plasmacytoma cells were transfected with the light and heavy-chain vectors by electroporation. Fusion protein secreted in the culture medium was purified and was characterized by gel electrophoresis as well as by determination of the biological activity of the fused GM-CSF. In nonreducing SDS-polyacrylamide gels, a single band approximately 200 Kd reacted with anti-human kappa, anti-human lambda1 and anti-GM-CSF antibodies. In reducing polyacrylamide gels, a approximately 74 kd protein reacted with anti-human lambda1 and anti-GM-CSF antibodies. The fusion protein induced proliferation of GM-CSF dependent NFS-60 cells. These results suggest that the protein is a chimeric anti-idiotype antibody consisting of 11D10 variable domains, human kappa and lambda1 constant domains and that the GM-CSF moiety fused to the constant region lambda1 is biologically active.