Purification, characterization and molecular cloning of novel cancer-reactive heavy-chain antibodies against tandem repeat region of MUC1
aDepartment of Clinical Biochemistry, School of Medical Sciences, bDepartment of Medical Biotechnology, School of Medical Sciences; Tarbiat Modarres University, Tehran, I.R. Iran, cInstitute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada
AIM: The membrane epithelial mucin MUC1 in which the extracellular domanin is formed by a repeating 20 amino acid sequence, is expressed at the luminal surface of most simple epithelial cells. However its expression is greatly increased in breast, ovary, pancreas, lung and prostate carcinomas. The increase in level of expression of MUC1 in cancers is accompanied by changes in the profile of glycosyl transferases involved in the synthesis of the O-glycans attached to the MUC1 core protein. Furthurmore this tumor-associated epitopes is situated on a high-molecular-weight (>400 KDa) glycoprotein molcule and best found in chemically deglycosylated form of human milk fat globule membrane (HMFG) and metastatic ascitic fluid.In order to prepare a tumor-targeting tool, we had produced heavy-chain antibodies in old world Camels against the MUC1 peptide and cloned them. METHODS: In this study we purified HMFG from human milk. This product then was chimically deglycosylated (D-HMFG). D-HMFG, synthetic MUC1 related peptide and homogenized cancerous tissues of several pations were used as a immunogen in order to produce heavy chain antibodies in camels. The produced heavy chain antibodies were characterized by reaction with synthetic peptide, homogenized cancerous tissues , HMFG and D-HMFG. The anti-MUC1 related peptide was purified by passing antiserum over a peptide affinity column with acidic elution. The purified specific anti - MUC1 antibodies were coupled to CN Br-activated Sepharose. The cancerous MUC1 was purified from ascitic fluid of a patient with aggressive small cell lung carcinoma and metastasis to peritoneum by a antibody-Sepharose affinity column. RESULTS: We have compared and characterized various form of MUC1 by ELISAs and Immunobloting. Total RNA from blood lymphocytes of these immune camels was isolated and mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR) with gene -specific primers. CONCLUSIONS: We report here the first example of a successfully raised heavy-chain antibodies in Camelus dromedarius and Camelus bactrianus against the MUC1 peptide and cloning of them. It may open prospective for their future and practical application as tumor-targeting tools, due to their small size , soluble behaviour and high stability.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 1091 (Vaccine trials).