Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1999-01-07
2004-05-11
Wehbe′, Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S320100, C435S455000, C424S093200, C424S093210, C514S04400A
Reexamination Certificate
active
06734014
ABSTRACT:
BACKGROUND OF THE INVENTION
T cells mediate most forms of cellular immunity, including cell lympholysis, delayed type hypersensitivity (DTH), transplantation rejection, and allograft rejection. An introduction to T cells and cell mediated immunity is found in Paul (1993)
Fundamental Immunology, Third Edition
Raven Press, New York, N.Y. and the references cited therein.
Typical T cells do not respond to free antigenic peptides. Instead, T cells interact with a specialized set of cell surface proteins (the class I and class II major histocompatibility complexes, or MHC) which present antigens on the surface of cells. Cytotoxic T cells are induced to proliferate by specialized antigen presenting cells such as macrophage and dendritic cells which present antigenic peptides on their cellular surfaces in conjunction with MHC molecules. T cells are induced by these antigen presenting cells to recognize corresponding antigens expressed on MHC antigens on the surface of target cells. T cells destroy these target cells.
The T cell recognizes the antigen in the form of a polypeptide fragment bound to the MHC class I molecules on target cells, rather than the intact polypeptide itself. The polypeptide is endogenously synthesized by the cell, and a portion of the polypeptide is degraded into small peptide fragments in the cytoplasm. Some of these small peptides translocate into a pre-Golgi compartment and interact with class I heavy chains to facilitate proper folding and association with the subunit &bgr;2 microglobulin. The peptide-MHC class I complex is then routed to the cell surface for expression and potential recognition by specific T cells. Investigations of the crystal structure of the human MHC class I molecule HLA-A2.1 indicate that a peptide binding groove is created by the folding of the &agr;1 and &agr;2 domains of the class I heavy chain (Bjorkman et al., (1987)
Nature
329:506. Falk et al., (1991)
Nature
351:290 have developed an approach to characterize naturally processed peptides bound to class I molecules. Other investigators have successfully achieved direct amino acid sequencing of the more abundant antigenic peptides in various HPLC fractions by conventional automated sequencing of peptides eluted from class I molecules (Jardetzky, et al. (1991)
Nature
353:326 and mass spectrometry Hunt, et al.,
Science
225:1261 (1992). A review of the characterization of naturally processed peptides in MHC Class I is found in Rotzschke and Falk (1991)
Immunol. Today
12:447.
Target T cells recognizing antigenic peptides can be induced to differentiate and proliferate in response to antigen presenting cells bearing antigenic peptides in the context of MHC class I and class II complexes. There are differences in the antigenic peptides bound to MHC class I and class II molecules, but the two classes of bound peptides share common epitopes within the same protein which enable a T cell activated by an antigen presenting cell to recognize a corresponding MHC class I epitope. MHC class I molecules on target cells typically bind 9 amino acid antigenic peptides, while corresponding MHC class II-peptide complexes have greater heterogeneity in the size of the bound antigenic peptide.
The generation of target T cells with a desired specificity has been limited by the ability of investigators to discover appropriate peptides for loading onto MHC molecules, and by investigator's ability to load peptide antigens onto antigen presenting cells used to induce proliferation of the T cells. In the past, investigators have generated antigen presenting cells by stripping the antigenic peptides normally found on antigen presenting cells by chemical or thermal techniques, followed by a reloading of the cells with a desired antigenic peptide. This approach has had limited success, due to inefficiencies in antigen presenting cell peptide loading, and due to the limited length of time that the loaded antigenic peptides remain loaded on the antigen presenting cells. In addition, only a single peptide fragment of a protein is loaded onto the surface of the antigen presenting cell using typical methods; thus, peptides important for activation of T cells against a target cell can be overlooked. The present invention overcomes these and other problems.
SUMMARY OF THE INVENTION
The invention provides new methods of making recombinant antigen presenting dendritic cells (DCs), which have been very difficult to transduce using existing methods. These new methods are applicable to the transduction of DCs with any recombinant nucleic acid. Also provided are new ways of expressing antigenic peptides on MHC molecules on the surface of the dendritic cells. It was surprisingly discovered that these expressed antigenic peptides are processed and displayed on the surface of the dendritic cells in the context of class I and class II MHC. These recombinant cells expressing antigenic peptides were found to be competent to activate T-cells against target cells expressing selected antigens in vivo. This provides powerful new treatments for cancers and cellular infections, as well as a variety of diagnostic and cell screening assays.
Naturally occurring dendritic cells are antigen presenting cells which activate T cell proliferation against target cells. Target cells express antigenic peptides in the context of MHC class I molecules on the surface of the target cell. Dendritic cells express related antigenic peptides on class I and class II MHC molecules. In a preferred use of the invention, dendritic cells are transformed with a nucleic acid encoding a heterologous protein which has a peptide subsequence corresponding to an antigenic peptide expressed on the surface of a target cell (on an MHC class I receptor). Preferably, a full-length protein is expressed, and several processed subsequences subsequently presented by the dendritic cell.
Surprisingly, heterologous proteins are expressed in the dendritic cell, processed into fragments, and expressed on the surface of the dendritic cell in the context of MHC class I and II molecules, making the dendritic cells capable of activating T cell proliferation against a target cell expressing the corresponding antigen. It is further demonstrated herein that T-cells activated by the dendritic cells of the invention by the methods of the invention are effective against established tumors and metastasis, in vivo. Thus, the present invention provides powerful new anti-cancer therapies based upon immunizing a patient with a recombinant dendritic cell, and/or T cell activated by a recombinant dendritic cell.
The new methods of transforming dendritic cells and expressing antigenic peptides on the surface of the cell to make the dendritic cell competent for T cell activation, provide significant advantages over prior art methods of loading peptides onto dendritic cells, including broader antigen expression and more efficient MHC class I and class II peptide loading, and the ability to expand the population of desired DCs, e.g., in culture. The invention has diagnostic, therapeutic and drug discovery assay uses.
DCs can be transduced with essentially any nucleic acid using the techniques provided. In one preferred embodiment, nucleic acids encoding cytokines (e.g., GM-CSF, an interleukin (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, etc.), or cell receptor ligands (e.g., transferrin, c-kit, viral receptor ligands, cytokine receptors, and the like) are transduced into stem cells to produce recombinant DC.
In one class of embodiments, the invention provides methods of transducing dendritic cells with selected nucleic acids. In the methods, a hematopoietic stem cell, e.g., a human CD34
+
stem cell, is transduced with a selected nucleic acid, and the stem cell is then differentiated into a dendritic cell. Typically, the stem cell is differentiated in vitro using appropriate cytokines. For instance, mouse stem cells are differentiated into dendritic cells by incubating the stem cells in culture with murine GM-CSF. Typically, the concentration of
Hwu Patrick
Reeves Mark
Rosenberg Steven A.
The United States of America as represented by the Department of
Townsend and Townsend / and Crew LLP
Wehbe′ Anne M.
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