The Preparation And Characterization Of Monoclonal Antibodies Against Colon Tumor-associated Antigens

Colon cancer is a malignancy in the colon that seriously threatens mankind's health. It's the most common malignant carcinoma in digestive tract in most of western countries. Colorectal cancer is the third most common cancer in the United States and the second cause of cancer death. Both incidence and outcome in Europe are similar. In recent years, with the changes of the dietetic structure and life style, the incidence of colon cancer increases in China. Advances in our understanding of the molecular mechanisms underlying the development and progression of cancer have resulted in the discovery of new therapeutic interventions that target specific molecular abnormalities. Among the numerous drug classes in development are the monoclonal antibodies (mAbs). Besides the effects of CDC and ADCC, 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.TA (tumor antigen) is the basis of immune diagnosis and therapy, which includes two conceptions. One is TSA (tumor specific antigen) that emergs in the progress of carcinogenesis and the other is TAA (tumor associated antigen) that over-expresses in tumor cells while also exists in the normal counterpart.Most of TA belongs to proteoglycan, glycoprotein or glycolipid. The existence of TA is critical for the immune system to distinguish tumor tissues from the normal. For many years, research has been focused on the identification of cell-surface antigens with expression restricted to tumor cells. 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, the accessibility and the metabolism of tumor antigens, and the antibody affinity. The ideal antigen was long considered one that is preferentially expressed in high copies on the membrane of tumor cells, is genetically stable, is not shed or secreted, and plays a causal role in tumor development and/or progression. Concerned with the colon cancer, a number of tumor antigens have been identified such as CEA, CA19-9, CA-50, CA72-4, etc. Due to the high heterogeneity of tumor tissues, the existing TA cannot be expressed on all of the colon cancer tissues. Now, the original paradigm that focused mAb research on antigens expressed on the membrane of tumor cells is being expanded.Up to now, many techniques have been found to screen the tumor antigens. Monoclonal antibody was described firstly in 1975 by Kohler and Milstein. It is an immunological technique to produce antibodies against a single epitope. Due to the higher purity, stronger specificity and lower cross-reaction, monoclonal antibodies have been applied in each field of life science. Monoclonal antibody therapy has emerged as an important therapeutic modality for cancer. From the 1990s, with the development of molecular biological techniques, a series of methods have been established to screen tumor antigens recognized by CD4+ or CD8+ T cells, that include peptide elute method, serological analysis of recombinant cDNA expression libraries and so on. But mAb still holds the leading status for its advantageous application in clinical diagnosis and therapy. An important limitation of the mouse-derived mAbs was the induction of a human anti-mouse antibody (HAMA) response in the majority of patients. This resulted in rapid clearance of the antibody and reduced tumor targeting with subsequent dosing. To overcome this problem, strategies aimed to decrease the immunogenicity of the antibodies were developed. The chimeric mAbs were engineered by replacing the mouse constant domains with human constant domains. Afurther improvement came with the creation of humanized antibodies that are derived from human cells or genetically engineered mice in which murine immunoglobulin genes have been replaced with human antibody genes. Now the antibodies against colon cancer tissues, which include the conventional mAb and the recombinant genetic engineering antibodies, have applied to the clinic.In this study we established nine hybridoma cell strains(No.l~No.9) which can stably secrete mAbs against colon tumor-associated antigens We detected the isotype of these mAbs and found that the Ig isotype of mAb No.l, No.2, No.4, No.5, No.8, and No.9 was IgM, and that of No.3, No.6 and No.7 was IgGl. BALB/c mice were immunized with colon cancer cells and colon cancer tissues from clinic. The hybridoma cell lines were prepared by fusion of the spleen cells from the immunized mice with SP2/0 cells. Positive clones were screened by Rosette Assay and confirmed by indirect fluorescence staining and flow cytometry analysis. All mAbs recognized SW480 cells by FCM except for No.l while mAb No.l was strong positive in binding to COLO205 cells. We compared the two methods and found that the rosette assay not only can be used to screen out the positive antibodies to the membrane molecules on live cells but also is more sensitive than FCM.We applied the supernatants of hybridoma culture into immunohistochemistry. The paraffin sections were prepared from colon cancer cells, normal and tumor colon tissues from hospital. The primary results of immunohistochemistry indicated that clones No.2, No.6 and No.7 were negative in all kinds of sections. Clones No.3 was positive in the SW480 cell sections but negative in the colon cancer tissue sections. No.l, No.4 and No.9 were positive in most of sections including sections of colon cancer cells, normal tissues and colon cancer tissues. No.5 and No.8 were positive in 4 of 10 and 6 of 9 colon cancer tissue sections respectively, while negative in most of normal tissue sections.Based on the results of IHC using pathological sections, clones No.5 and No.8 were selected for further identification by tissue array including a large amount of colon cancer tissues, 16 kinds of other tumor tissues and 14 kinds of normal tissues. The positive percentages of mAbs No.5 and No.8 were much higher in colon cancer (54.9% and 65.0% respectively) than normal colon tissues (8.3% and 18.8% respectively). It was likely that the increase of...