detection and identification of tumor antigen
tumor immunology
The question of whether immunosurveillance against tumors truly exists is still unsolved. Bacteria and viruses. which can be effectively recognized and controlled by the immune system, consist of a variety of foreign proteins
Tumor cells. on the other hand, differ only slightly from normal cells. Sometimes. truncated or mutated proteins are present, but otherwise the cells use normal biochemical pathways. Even if some proteins are expressed at levels higher than normal, or if fetal antigens are expressed. these are in most cases normal cellular proteins.
In many cases. if mutated proteins are present, only few peptides can be potentially recognized as foreign. A number of findings indicate that immunosurveillance against tumors is not one of the primary tasks of the immune system. Patients with severe immune defects, such as HIV. have an increased incidence of virus associated tumors, but do not exhibit an increase in the most common cancer types. Furthermore, nude mice that lack functional T cells do not develop tumors but rather die of infections. Nonetheless, the findings of experimental animal studies and clinical studies do justify hope that manipulation of the immune system will eventually result in effective immunotherapy strategies in the future.
A. Recognition of Tumor Antigens
Researchers have attempted to manufacture antibodies against tumor cells since monoclonal antibody engineering techniques were developed about 20 years ago.
This involves the immunization of mice with tumor cells. After repeated application. the splenic cells are isolated and fused with myeloma cells from an immortal" myeloma cell line by addition of polyethyleneglycol (PEG).
Due to an enzyme defect. unfused myeloma cells die when placed in a medium containing hypoxanthine , aminopterin, and thymidine (HAT medium) (1),
Only those cells that inherited immortality from the myeloma cells and HAT resistance from the splenic cells will survive. Apart from HAT resistance.
the fusion cells (hybridoma cells) also inherit antibody specificity from the splenic cells.
Hybridoma cells that produce antibodies against tumor cells are subjected to a process known as limiting dilution."
They are then repeatedly cloned to obtain a cell line arising from a single cell that produces antibodies of a single specificity (monoclonal cell line or hybridoma). 0n the other hand, Tcells are potentially able to recognize tumor antigens (2). After intracellular degradation. such antigens (e.g.. mutated proteins) can be presented to CDS cytotoxic T cells as MHC class I bound peptides.
The T cell response is HLA restricted. that is, it depends on whether the mutated tumor peptide fits in the antigen presenting site of the HM mole cule (see p. 56). Tumor cells are not effective antigen presenting cells because they lack important co stimulatory molecules
B. Identification of Tumor Antigens
T cell clones that specifically lyse target cells can be isolated by cloning techniques). In order to identify the DNA sequence of the corresponding tumor antigen. the total DNA of the tumor cells is prepared. separated into multiple small fragments, and inserted into vectors (1).
The fragment bearing vectors are then transduced to cells with the same HlA restriction as the tumor cells. Only those cells containing the relevant DNA fragment present it in the MHC molecules. They can therefore be recognized and lysed by the T cell clones.
An alternative method is the elution of peptide from tumor cells. In order to find out which of the peptides presented by a cell are tumor specific. all peptides in the MHC moleCule are first dissolved by brief acid treatment (2).
The dissolved peptides are separated by high performance liquid chromatography (HPLC). The individual peptide fractions are then incubated with 3 TAP deficient cell line .
The cell lines is not able to supply MHC molecules with peptides. Empty" MHC molecules on the cell membrane are unstable and degraded quickly. The external addition of exactly matching peptides stabilizes them, and the MHC molecules are then able to present the bound peptides. If one of the peptide is tumor specific, the corresponding presenting cell is lysed by the tumor specific T cell clone. Further characterization of the peptides iso lated from the MHC molecules can be achieved by mass spectrometry.
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