| Colorectal cancer is one of most common malignancy worldwide. Whereas the 5-year survival for colorectal carcinoma is about 50%, it falls to less than 10% for stage IV disease. In China, with the change of lifestyle and diet, the incidence of colorectal carcinoma is gradually increasing and becomes the fourth common malignancy. Nevertheless, effective tools for the early detection of colorectal carcinoma and its high risk factor remain a major challenge. Approximately 25% of patients present with metastatic disease; the remaining 75% are treated surgically with cure as the objective, but even with complete resection the disease will eventually recur in 50% of these. Adjuvant therapy affords a risk reduction of these, but it remains problematic because of intrinsic or acquired drugs resistance. So there remains a compelling need for new tumor biomarker or therapeutic targets.Advances in our understanding of the molecular mechanism underlying the development and progression of cancer have resulted in the discovery of new therapeutic interventions that target specific molecular abnormalities, such as cancer vaccine, monoclonal antibody and small molecular antagonist. Among the numerous drug classes in development are the monoclonal antibodies (MAbs). Their specificity, and therefore their potential to bind preferentially and modify tumor-specific targets, sparing normal tissues and causing fewer side-effects than conventional cytotoxic agents, makes them an attractive therapeutic approach. However, the success of rituximab in the treatment of lymphomas has not been totally replicated in the case of solid tumors. Indeed, relatively few antibodies capable of effectively targeting solid tumors have been identified. Among the factors adduced to explain these limitations are the molecular heterogeneity that characterizes solid tumors as opposed to the clonality of lymphomas. Antibodies targeting colorectal carcinoma, such as edrecolomab (MAb 17-1A), bevacizumab (Abastin) and cetuximab (IMC-C225), reveal the same problem. They can lengthen median progression-free survival 5-11 months but still remain a great limit in effect and application. At this respect, the development of noval monoclonal antibodies specific and sensitive to colorectal carcinoma is of great interest in cancer research. Because antibody directed against cancer cell lines is not specific enough, we determined to improve immunogen and screening method to develop noval monoclonal antibodies. We collected fresh colorectal carcinoma tissue as immunogen and devided them into three groups: well-differentiated, moderately-differentiated and poorly differentiated, immunized mice with the cancer cell suspension from tissue. We also changed the screening method of conventional ELISA into immunohistochemiscal staining, using cryostat-prepared tissue sections of the colorectal carcinoma tissue and normal tissue, for better selection of specific MAbs targeting cancer in vivo.Results1. Fusion and specific MAbs resultsMice were immunized with cells suspension of colorectal carcinoma tissues. After a significant increase in potency test of antibodies serum, 96 hybridomas obtained by the three fusions of spleenocytes, 32 showed binding to colorectal carcinoma tissue in immunohistochemical reaction. Of those, only two, designated 2-3 and 15-8, were stable on expansion and secreted high titres of antibodies specific against colorectal carcinoma. They were further cloned by limited dilution. 2-3 antibody was more specific than 15-8 and was selected for further identification.2. Tissue specific distribution of monoclonal antibody 2-3Using immunohistochemical staining, we studied the 2-3 related antigen expression in the 30 colorectal carcinoma specimens, the 20 other cancer specimens, one lung cancer cell line, one colon cancer cell line, and the normal tissue specimens which taken from non-carcinoma regions adjacent to cancer tissue as control. Same experiments of CEA antibody(DAKO company) were carried out at the same time.Using cryostat-prepared section, 30 of 30 colorectal carcinoma showed positive staining with a dilution of 1:150000 of 2-3 antibody. Its reactive intensity and rate was much higher than that of non-carcinoma region adjacent to colorectal tissue(P<0.05). The incidence of 2-3 expression in non-carcinoma region far away from colorectal tissue is 14.3%. Of the 30 colorectal carcinoma cases, 2-3 antibody reacted strongly with moderate-differentiated and poorly–differentiated colorectal carcinoma. The immunoreactive substance was observed on luminal contents of colorectal carcinoma tubules and on apical surface of normal epithelium, indicating that the antigen recognized by 2-3 antibody may be a kind of protein secreted by colorectal carcinoma and normal epithelium. Paraffin section of colorectal carcinoma gave the same results, but its staining was a little weaker than cryostat-prepared section with a dilution of 1:100000 of 2-3.The tissue distribution of 2-3 antibody-reactive antigen was examined by immunoperoxidase staining. Weak positive staining of tissues adjacent to tumor tissues was observed in the mucosa of esophagus, stomach, colon, rectum and bile canaliculus, but the positive rate was low and the reactive region restricted to the superficial epithelium. The antigen was not detected in small bowel, cholecyst, spleen, lung, brain, breast, muscle and connective tissue, RBC, granulocyte or lymphocytes.Various tumor tissues tested showed different incidence rate: 1 of 2 esophagus tumors; 3 of 3 gastric cancer; 4 of 4 hepatocellular carcinoma; 6 of 7 lung cancers; 0 of 2 breast tumors; 1 of 1 brain metastasis from colorectal carcinoma; 1 of 1 pulmonary metastasis from colorectal carcinoma. The immunoreactive substance was observed in the part of grandular epithelium of adenocarcinoma, cancer nest of squamous cell carcinoma and cancer cell of signet-ring cell carcinoma. In all tumor tissue samples, stromal elements were always negative.By immunoperoxidase testing, 2-3 antibody reacted with lung cancer cell line A549 and colon cancer cell line SW480.Comparing with CEA, 2-3 antibody was similar in distribution of tissue and cell lines. But its reactive intensity and titre was higher than that of CEA with a dilution of 1:8000.3. Karyotype analyses of hybridoma, Western blot analyses and enzyme treatment of 2-3 related antigenKaryotype analyses of 2-3, 15-8 hydridoma indicated that they were all derived from the fusion of two cells. These two hydridomas, which had been frozen and recovered, were still stable and secreting specific antibodies over 9 months of culture. To determine the molecular weight of 2-3 related antigen, we performed western blot of colorectal carcinoma tissue lysates. We also carried out enzyme treatment experiments for analyses of 2-3 related antigenWestern blot analyses recognized a 180kDa protein from colorectal carcinoma tissue lysates using CEA but didn't recognized any band using 2-3 antibody. In order to address the effect of protein denaturation on epitope recognized by 2-3, we treated colorectal carcinoma tissue lysates with SDS and mercaptoethanol, the 2-3 antibody wasn't able to recognize the denatured target protein. Similar experiment using CEA antibody gave different result. These results suggest that the target region of antigen recognized by 2-3 antibody which is different from that of CEA.Treatment of colorectal carcinoma cryostat-prepared tissue with sodium periodate didn't decrease binding of 2-3, CEA antibody. Treatment of colorectal carcinoma lysates with trypsin completely diminished binding of 2-3, CEA antibody in dot blot. These results suggest that the antigen on immunizing tissue may be a protein. The exact antigen of 2-3 antibody was still unknown, further research of it enabled a better understanding of molecular events associated to the cancer development.
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