Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 8 to 10 amino acid residues in defined sequence
Reexamination Certificate
1995-02-15
2002-12-31
Ungar, Susan (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
8 to 10 amino acid residues in defined sequence
C530S300000, C530S350000, C530S822000, C514S002600
Reexamination Certificate
active
06500919
ABSTRACT:
The present invention is concerned with cancer treatment and diagnosis, especially with a melanoma associated antigen, epitopes thereof, vaccines against melanoma, tumor infiltrating T lymphocytes recognizing the antigen and diagnostics for the detection of melanoma and for the monitoring of vaccination.
Tumor cells may emancipate themselves from restrictive growth control by oncogene activation, and/or by the inactivation of tumor suppression genes. The course of tumor progression proceeds by a series of gradual, stepwise changes in different ‘unit characteristics’, i.e. phenotypic traits, many of which are known to be determined or at least influenced by the altered expression of defined oncogenes and/or tumor suppressive genes. Emancipation of the cell from immunological host restriction may follow multistep pathways similar to the emancipation from growth control.
A problem often encountered in cancer immunotherapy is the lack of immunogenicity of the tumor. This escape of the immune control system can be understood on basis of phenotype differences encountered in neoplastic cells (differences found in Burkitt's lymphoma cells according to Klein, G. and Boon, T., Curr. Opinion in Immunol. 5, 687-692, 1993):
decreased ability to process and present antigens;
decreased ability to stimulate autologous T cells;
complete downregulation of immunogenic proteins associated with transformed cells;
no or low expression of leukocyte adhesion molecules or other accessory molecules; and
selective downregulation of certain MHC class I and class II alleles.
MHC Class I/II antigens are often downregulated in solid tumors. This may affect all class I/II antigens, or only part of them. Viral and cellular peptides that can sensitize appropriate target cells for cytotoxic T lymphocyte mediated lysis may fail to do so when produced in cells with a low level of expression of MHC class I antigen. Cytotoxic sensitivity may be induced, at least in some cases by raising the level of MHC class I/II antigen expression by interferon &ggr; and tumor necrosis factor &agr;.
However, during the stepwise changes from normal to tumor tissue tumor-associated antigens appear. These antigens can be exposed through various mechanisms:
they can be molecules that are masked in some way during normal cell development, but where the neoplastic change induces removal of the masking protection for the immunosystem;
deletion of some molecules from the plasma membrane may alter the profile of adjacent molecules in a given membrane patch, and thus, in effect generate a new profile that might become immunogenic to the host;
a membrane alteration accompanying neoplastic transformation may expose new, previously hidden regions of a molecule, or may result in addition of new structural features to an existing molecule.
shedding and disintegration of tumor cells may expose the immune system to nuclear, nucleolar, or cytoplasmic components that are normally hidden in the cell.
The characteristics of tumor-associated antigens are very much dependent on the origin of the tumor carrying them. The existence of antigens associated with animal tumors was documented in the last century, and the antigenic character of human cancers has been well established, primarily through recent studies with monoclonal antibodies.
Attempts to isolate and chemically characterize these antigens have encountered serious difficulties, many having to do with a lack of reagents suitable for precipitation of the antigen-bearing molecules from a solution.
Like many other stimuli, the tumor-associated antigens activate not one but a whole set of defense mechanisms—both specific and unspecific, humoral and cellular. The dominant role in in vivo resistance to tumor growth is played by T lymphocytes. These cells recognize tumor-associated antigens presented to them by antigen presenting cells (APC's), and will be activated by this recognition, and upon activation and differentiation, attack and kill the tumor cells. A special class of these sort of lymphocytes is formed by the tumor infiltrating lymphocytes (TIL's) which can be found in solid tumors.
It has already been suggested (EP 147,689) to activate T lymphocytes with an antigenic substance linked to an insoluble carrier in vitro and then to administer these activated lymphocytes to a tumor patient.
Conventional chemotherapy is relatively ineffective in the treatment of patients with metastasic melanoma, and approximately 6000 patients die of this disease in the United States each year.
Rosenberg et al. (New Eng. J. Med. 319(25), 1676-1681, 1988) have shown the beneficial effect of immunotherapy with autologous TIL's and interleukin-2 (IL-2) in melanoma patients.
This therapy constitutes of resection of the tumor deposit, isolation of the TIL's, in vitro expansion of the TIL's and infusion into the patient under concurrent treatment of high and toxicity inducing doses of IL-2.
The TIL's used by Rosenberg are directed to and able to recognize melanoma-associated antigens.
It has been our goal to isolate such a melanoma-associated antigen in order to be able to use the antigen and/or its epitopes for the development of an immunotherapy for melanoma patients.
Melanoma antigens have already been described by Old, L. (1981) who identified 6 antigenic glycoproteins and 3 glycolipids occurring in 120 melanoma cell lines.
Also vaccines with melanoma antigens have been described: in U.S. Pat. Nos. 5,030,621 and 5,194,384 a polyvalent vaccine has been made by culturing melanoma cells and subsequent isolation of excreted melanoma-specific antigens from the culture medium.
Some specific antigens have already been proposed for therapy and diagnosis of melanoma type of cancer: the peptide p97 has been disclosed in U.S. Pat. Nos. 5,262,177 and 5,141,742, while a 35 kD protein has been mentioned in EP 529,007.
SUMMARY OF THE INVENTION
We now have found a melanoma-associated polypeptide, characterized in that it comprises the aminoacid sequence of SEQ ID NO: 2.
This melanocyte lineage-specific antigenic polypeptide (also mentioned gp100) is recognized by the monoclonal antibody NKI-beteb, which antibody has proven suitable for diagnostic purposes. The antigens recognized by this antibody are intracellular proteins of approximately 10 kd (gp 10) and 100 kd (gp100). The latter is also detectable in a culture medium of melanoma cells (Vennegoor, C. et al, Am. J. Pathol. 130, 179-192, 1988). It has also been found that the gp100 antigen reacts with other melanoma-specific antibodies such as HMB-50 (described by Vogel, A. M. and Esclamado, R. M., Cancer Res. 48, 1286-1294, 1988) or HMB-45 (described by Gown, A. M. et al., Am. J. Pathol. 123, 195-203, 1986). Since the proteins reacting with these monoclonal antibodies have been shown te be glycosylated in melanoma cells, differences have been found in mobility when analyzed by SDS-PAGE.
Although this gp100 antigen is predominantly expressed intracellularly, it has now been established that it is a suitable immunogenic antigen, because it has been demonstrated that these intracellular proteins can be processed and presented as peptides in the context of MHC molecules to cells of the immune system. In fact, tumor infiltrating lymphocytes derived from tumors of melanoma patients have been found which react with the antigen.
Therefore, the gp100 polypeptide is a potential target for cellular responses against carcinoma and thus a suitable subject for therapy and diagnosis in melanoma patients.
Gp100 is a type I transmembrane protein, which has a threonine-rich domain containing repetitive amino acid sequences present in the middle of the protein (amino acids 309-427). This threonine-rich domain, which may be subjected to extensive O-linked glycosylation, is preceded by a histidine-rich region (amino acids 182-313) and followed by a cysteine-rich domain (amino acids 475-566). Based on hydrophobicity plot analysis (Kyte, J. and Doolittle, R. F., 1982), a single transmembrane domain bordered by charged residues is present in the carboxy-terminal p
Adema Gosse Jan
Figdor Carl Gustav
Davis Minh-Tam
IntroGene B.V.
TraskBritt
Ungar Susan
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