Immunization Of Lymphocytes In Vitro And Monoclonal Antibody Preparation To Tumor-associated Glycosphingolipid Antigens

Abstract

Glycosphingolipids (GSL) are found in all vertebrate plasma membranes, usually asymmetrically located in the outer cell surface. They act as receptors of hormones, toxins, bacteria and other ligands (70). They are also surface antigens that participate in cell to cell recognition. In cancer tissues, it has been shown that there are certain characteristic changes in GSL biosynthesis and composition (14, 15, 20, 23, 54). These changes can be categorized into three groups: 1) incomplete sugar chain synthesis with accumulation of precursor compounds, 2) neosynthesis leading to the appearance of new glycolipids and 3) organizational rearrangement of glycolipids on the cell surface resulting in changes of their antigenic expression (16). Consequently, there are tumor-distinctive GSLs which have been named tumor-associated antigens (TAAs) (16). One class of such TAAs is the-heterophilic antigens such as Hanganutziu--Deicher (HD) antigens and Forssman GSL antigen (9, 18, 35). Hanganutziu-Deicher antibody was first noticed in the sera of patients who had received foreign sera of anti-toxoid and hence named "serum sickness" type antibody (18). Later, it was found in patients suffering from other ailments who had not received any foreign sera and subsequently named Hanganutziu- Deicher antibody (34). The HD antigen was first purified from equine and bovine erythrocytes, and the immunodominant group was identified to be N-glycolyl-neuraminic acid (NeuGc) (22). Glycosphingolipids with NeuGc are normally present in most animal

species but absent in human and chicken (74). Hanganutziu-Deicher antibodies have been found in sera from patients with various human cancers such as melanoma, retinoblastoma, breast, colon and stomach carcinomas (23, 24, 44, 52, 54). Forssman GSL is another heterophile antigen which is absent or in a small amount in normal human tissues but it occurs at elevated amounts in cancers such as, gastrointestinal, biliary and lung carcinomas (17, 26, 35, 62, 73). The alterations in GSLs render them antigenic. Immune complexes of various gangliosides were detected in cancer patient sera (19). Some antibody classes to TAAs have been shown to suppress tumor growth (31, 48, 66). Other classes of TAA antibodies can also be used to deliver anti-cancer drugs by antibody-drug conjugates directed to the TAAs (5, 69). Since chicken do not express HD antigens, they have been used to prepare anti-HD sera (10, 43). Using chicken antiserum against HD3, Higashi and collaborators demonstrated HD antigen in various human cancer tissues (23). The antibodies in patient sera and chicken anti-HD sera were shown to recognize the terminal end of - the carbohydrate chains that is N-glycolylneuraminyl group (23). Anti-HD antibodies used so far have been of chicken origin. But this had two major problems. Different GSLs expressed in transformed cells were detected as HD antigens (10, 19). The chicken serum used showed variable affinity to different GSLs, hence it is of poor diagnostic use. And repeated use of foreign immunoglobulin for cancer long-term therapy, would lead to a patient mounting an immune response with production of antichicken immunoglobulins. This results in sensitization of patient

with foreign antibody with a risk that may lead to adverse reactions like fever and rashes (69). In addition, the efficiency of the immunotherapy is severely reduced since chicken antibodies are complexed with the patient anti-chicken immunoglobulins. Human monoclonal antibody against specific HD cancer antigen would be the ideal reagent for both immunodiagnosis and immunotherapy (1). Most frequently used methods to immortalize immunized B cells are transformation with Epstein-Barr virus (EBV) and polyethylene glycol (PEG) induced myeloma cell fusion. For human monoclonal antibody preparation, peripheral blood lymphocytes is the readily available B cell pool (2). The major limitation is the small number of lymphocytes that can be drawn from an individual, since deliberate immunization of human cannot be done with tumor antigens. For human monoclonal antibody preparation, it is also critical to use an immortalization procedure with high efficiency as well as least toxicity to cells. Therefore, an alternative source of lymphocytes producing ~ cancer GSL-specific antibodies is very vital. In this thesis, I describe, the standardization of an efficient cell fusion method, electrofusion, for a human lymphoblastoid B cell line. Then, lymphocytes from a cancer patient were in vitro restimulated with HD3 antigen and a hybridoma was made by electrofusion, that produced anti-HD3 antibody. However, the hybridoma continued to secrete HD3 monoclonal antibody for a short time. Next, J describe a modified method, more suitable for in vitro immunization of unprimed lymphocytes with one of tumor-

associated glycolipid, Forssman GSL. Rat monoclonal antibody to Forssman GSL was prepared and its specificity was determined.