Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-12-11
2002-01-15
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S091500, C435S091500, C435S091500, C530S300000, C530S328000, C436S518000
Reexamination Certificate
active
06338945
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to identification of bio-active molecules from a combinatorial library of oligopeptides attached to solid phase supports. The peptides attached to a single bead have essentially the same amino acid sequence. The synthesis history of each peptide bead may be recorded on each solid support in a code of inert molecular tags, such that beads of interest can be rapidly and efficiently decoded. A photocleavable crosslinker allows release of some of the oligopeptide by exposure to UV light. Molecular tags if present, remain covalently bound to the beads for post-assay analysis. The bioactive molecules may be screened in cytotoxic T lymphocyte screening assays.
BACKGROUND OF THE INVENTION
Cellular immunotherapy is emerging as a technologically and intellectually compelling anti-cancer treatment. The generation of an immune response against tumors has been demonstrated in several animal models and has been inferred from reports of spontaneous tumor regression in man (Stotter and Lotze, 1990, Cancer Cells; 2:44-55). Cytotoxic T-lymphocyte (CTL) responses can be directed against antigens specifically presented by tumor cells, both in vivo and in vitro, without the need for prior knowledge of the molecular mechanism by which the tumor arose. In animal models, established tumors can be eradicated by the adoptive transfer of T-cells that are specifically immune to the malignant cells (Buen et al., Immunol. Today; 15:11-15). Techniques of adoptive T-cell therapy have recently been applied to the treatment of human viral disease, but the application of similar T-cell therapy for human malignancy has been hindered in part by the lack of well defined tumor antigens recognizable by autochthonous T-cells. Many human progressive or metastatic cancers, such as disseminated malignant melanoma or metastatic renal cell carcinoma, are resistant to conventional therapies, including chemotherapy and radiotherapy. In these types of cancers, immunotherapy has been tried over the past 10 years and although its success rate has been relatively modest, it remains a promising alternative to the conventional therapies (Bergmann et al., 1990, Onkologie; 13:137).
In man, spontaneous destruction of melanoma cells occurs in 15% to 20% of primary lesions, indicating that host protective mechanisms which can selectively destroy melanoma cells are present (Bystryn et al., 1993, Heme. Onc. Annals. 1:301). Vaccine immunotherapy with crude or partially purified melanoma vaccines can prevent tumor growth in 50% to 100% of mice immunized to otherwise lethal doses of melanoma cells. The protection is specific, indicating it is mediated by immune mechanisms. The challenge is to devise vaccine strategies that will induce similar immunoprotective responses in man.
For immunotherapy to be improved, epitopes recognized by tumor-specific-CTLs must be identified. CTL epitopes are 8-10 amino acid peptides derived from cellular proteins that are endocytically processed and presented on the tumor cell surface by major histocompatability complex (MHC) class I and class II glycoproteins. MHC molecules are expressed in virtually all nucleated cells and the combination of peptide and MHC molecule is specifically recognized by the appropriate T-cell receptors (TCRs). T-cells in the presence of antigen presenting cells and their corresponding antigen proliferate and acquire potent cytolytic activity.
Identification of the antigens recognized by these tumor-specific CTLs is vital to the rational development of peptide-based anti-tumor vaccines. A common strategy in the search for tumor antigens is to isolate tumor-specific T-cells and attempt to identify the antigens recognized by the T-cells. In patients with cancer, specific CTLs have been often derived from lymphocytic infiltrates present at the tumor site (Weidmann et al., 1994, ° Cancer Immunol. Immunother. 39:1-14). These tumor infiltrating lymphocytes (TILs) are a unique cell population that can be traced back to sites of disease when they are labeled with indium and adoptively transferred.
Indeed, the presence of a large number of T-cells in tumors has been correlated with a prognostically favorable outcome in some cases (Whiteside and Parmiani, 1994, Cancer Immunol. Immunother. 39:15-21). Recently it was shown that implantation of polyurethane sponges containing irradiated tumor cells can efficiently trap anti-tumor CTLs (4-times greater than lymph fluid, 50-times greater than spleen or peripheral blood) (Woolley et al., 1995, Immunology, 84: 55-63). Following activation with T-cell cytokines in the presence of their appropriately presented recognition antigen, TILs proliferate in culture and acquire potent anti-tumor cytolytic properties (Weidmann et. al., 1994, supra). Thus, TILs are a convenient source of lymphocytes greatly enriched for cells with rumor cell specificity. Additionally, tumor-specific CTLs have been found in peripheral blood or malignant ascites of patients with cancer, indicating that a systemic response to the tumor may be present or that redistribution of CTLs from the tumor to the periphery might occur (Wallace et al., 1993, Cancer Res. 53:2358-2367). In either case; this is an attractive feature for the, immunotherapeutic treatment of metastatic or disseminated cancers.
The reasons why tumor cells may express tumor-specific antigens (TSAs) are beginning to be understood. For example, TSAs may be the result of the processes of carcinogenesis, which are generally thought to stem from damage to a large number of genes, some of which have a role in the molecular mechanisms regulating cell growth and division. This damage results in uncontrolled cellular proliferation that defines the transformed cell. Thus, possible origins of TSAs include self proteins (such as fetal antigens) oncogene a products (including fusion proteins), mutated tumor suppressor gene products, other -mutated cellular proteins, or foreign proteins such as viral gene products. Nonmutated cellular proteins may also be antigenic if they are expressed aberrantly (e.g., in an inappropriate subcellular compartment) or in supernormal quantities. Given the numerous steps of cellular transformation and sometimes bizarre genotypes observed in cancer cells, it could be argued that tumor cells are likely to contain many new antigens potentially recognizable by the immune system.
Reports of shared tumor antigens are frequent in the literature. In the case of melanoma, there is recent evidence that the same T-cell-defined tumor antigens are expressed by independent human melanoma suggesting that transformation-associated events may give rise to recurrent expression of the same tumor antigen in different tumors of related tissue and cellular origin (Sahasrabudhe et al., 1993, J. Immunol., 151:6302-6310; Shamamian et al., 1994, Cancer Immunol. Immunother., 39:73-83; Cox et al., 1994, Science 264:716; Peoples et al., 1993, J. Immunol., 151:5481-5491; Jerome et al., 1991, Cancer Res., 51:2908-2916; Morioke et al., 1994, J. Immunol., 153:5650-5658). Previous studies in animal models have, in contrast, suggested that most chemical and ultraviolet radiation-induced tumors are antigenically diverse and that tumor rejection antigen may be generated by random mutation (Srivastava et al., 1986, Proc. Natl. Acad. Sci. USA, 83:3407-3411). However, it is highly improbable that a completely random process would give rise to shared antigens even in very closely related tumors. This data supports the possibility that specific anti-tumor immunotherapies, such as vaccines, may be active against more than one form of cancer and that the same vaccine may be effective against independently derived tumors of the same type.
While isolation, expansion, and retransfusion of TILs is appealing, there are severe adverse cardiorespiratory and hemodynamic effects such as tachycardia, increases in cardiac index, systemic vascular resistance, and pulmonary artery diastolic pressure which appear within two hours post-infusion. These effects are similar to the physiologic changes seen in interl
Genzyme Corporation
Genzyme Corporation
Ponnaluri Padmashri
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