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.
Publisher
University of Hokkaido, College of health sciences,