Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1998-04-06
2001-09-11
Salimi, Ali R. (Department: 1643)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S204100, C435S320100, C435S235100, C435S325000, C435S343200, C536S023400, C536S023200
Reexamination Certificate
active
06287569
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of immunology, vaccination and immunotherapy.
BACKGROUND OF THE INVENTION
The following is a discussion of the relevant art, none of which is admitted to be prior art to the appended claims.
DNA vaccination is a technique whereby somatic cells are transfected in vivo with DNA directing synthesis of a target antigen. Ulmer et al. disclose hereologous protection against influenza by injection of DNA encoding a viral protein (
Science
259:1745, 1993). Watanabe et al. disclose the induction of antibodies to a kappa variable region by gene immunization (
J. Immunol.
151:2871, 1993). The expressed protein either can be secreted by the transfected cell or processed inside the cell and presented in the context of class I major histocompatibility (MHC) antigens, which can be recognized by T cells. One of the pathways whereby polypeptides are processed into peptides involves intracellular proteolysis of the polypeptide into peptide fragments that ultimately bind MHC molecules. One major candidate process for this pathway is that of polyubiquitination.
Ubiquitination (“Ub”), an ATP-dependent process, constitutes a preliminary step of targeting a proteolytic substrate for its eventual degradation by the proteosome, a large multi-catalytic protease. Experiments in yeast and rabbit reticulocyte lysates indicate that at least two distinct determinants can dictate the rate of its degradation: one is the identity of N-terminal residue (N-end rule) and the other is presence of specific internal lysine residue where polyubiquitin is initiated (Bachmair, A., et al.
Science
234:179-186, 1986; Gonda, D. K., et al.
J. Biol. Chem.
264:16700-16712, 1989; Bachmair, A., et al.
Cell
56:1019-1032, 1989). N-terminal amino acids are largely classified into three different categories based upon their destabilizing potential and the half-life of a given protein varies significantly (from 2 min to >20h) depending on the identity of N-terminal amino acid (Bachmair, A., et al.,
Science
234:179-186, 1986; Gonda, D. K., et al.
J. Biol. Chem.
264:16700-16712, 1989). Studies have indicated that intracellular degradation of proteins is required for antigen presentation to T cells (Townsend, A., et al.
J. Exp. Med.
168:1211-1224, 1988; Townsend, A., et al.
Cell
42:457-67, 1985; Germain, R. N.
Cell
76:287-299, 1994).
Evidence that the Ub-mediated proteolytic pathway provides all of the substrates for the proteosome has remained inconclusive from temperature-sensitive UBEL mutant cells (Michalek, M. T., et al.
Nature
363:552-554, 1993; Cox, J. H., et al.
J. Immunol.
154:511-519, 1995). A recent study using pairs of N-end rule substrate proteins that varied in their intracellular stability provided evidence that the proteolytic turnover of endogenously synthesized proteins is not directly proportional to the generation of processed antigenic peptide/MHC class I complexes (Goth, S., et al.
J. Immunol.
157:1894-1904, 1996). Goth et al., used the sindbis virus polymerase as the N-terminal target of the Ub-dependent degradation pathway. Earlier studies had shown that sindbis virus polymerase is a natural substrate for the N-rule (de Groot et al.
Proc. Natl. Acad. Sci. USA,
88:8967, 1991).
Levy et al. (
Proc. Natl. Acad. Sci. USA
93:4907, 1996) disclose a fusion protein consisting of a 21-kDa mouse DHFR moiety, an ubiquitin protein, a variable residue, 165 residues of nsP4 (Sinbis virus RNA polymerase) and &bgr; gal that is useful in a method to produce equimolar amounts of two or more specific proteins in a cell.
Grant, E. P., et al. (
J. Immunol.
155:3750-3758, 1995) disclose that chimeric proteins comprising ubiquitin, a destabilizing amino acid, a lacI extension and &bgr; gal when loaded into LB27.4 cells (a B lymphoblastoid cell line) showed enhanced class I presentation compared to that of proteins with a stabilizing amino acid.
Bachmair et al. U.S. Pat. No. 5,496,721 disclose the use of genetic constructs that encode for ubiquitin fusion proteins with destabilizing amino acids at their N-termini.
A recent study found that ovalbumin (OVA) with methylated lysine groups which was resistant to ubiquitin-mediated degradation could still be presented via class I MHC, albeit at a reduced amount (Michalek, M. T., et al.
J. Immunol.
157:617-624, 1996). This indicates that there may be a ubiquitin-independent pathway for class I presentation of antigens to the immune system.
WO 94/17816 disclose methods for the use of inhibitors of the ubiquitin-dependent proteolytic pathway to reduce cytolytic immune responses.
SUMMARY OF THE INVENTION
The present invention concerns methods for generating a cellular immune response by the introduction into cells DNA vectors encoding antigens that have enhanced rates of degradation. Such vectors allow for the production of a chimeric immunogen (protein) in the cell in which they are introduced. A chimeric immunogen includes a protein processing signal and a protein which is the target for a cellular immune response. The protein processing signal brings about enhanced rates of degradation of the target protein. For example, a protein processing signal may include a removable leader linked to an intervening amino acid which is linked to a ubiquitin acceptor. The protein processing signal is further linked to the target protein. In the cytoplasm the removable leader is cleaved off by proteolytic enzymes present in the cytoplasm. This exposes at the N-terminus of the protein an intervening amino acid which acts to reduce the stability of the immunogen. The chimeric immunogen contains a ubiquitin acceptor which allows for the attachment of ubiquitin by enzymes present in the cytoplasm of the cell, thus targeting the protein for degradation via the ubiquitin-proteosome pathway. Other protein processing signals that utilize the intracellular proteosome pathway for degradation (with or without ubiquitin) are encompassed in the present invention.
Applicants have unexpectedly discovered that such vectors that encode for chimeric immunogens which have enhanced rates of degradation via the ubiquitin-proteosome pathway are able to generate an enhanced cellular immune response. In addition, the response is limited to the cellular branch of the immune system and does not include the production of antibodies to the immunogen.
The present invention also concerns such vectors able to generate specific cellular immune response.
The vectors and methods of the present invention are especially useful in stimulating an immune response that can reject cancer cells or cells infected with virus. This may be particularly useful in the prevention or delay of the onset of de novo or recurrent cancer or in the treatment of viral infections.
The present invention offers several advantages over prior art methods for generating an immune response. The cellular immune response is greater than that achieved by the introduction of naked plasmid DNA encoding a target antigen. In addition, the ability to induce a cellular cytotoxic immune response against cells that express an antigen without inducing antigen specific antibodies offers other advantages. In regard to antigens that are presented by tumor cells, the production of antibodies directed to these antigens have been hypothesized to inhibit cellular immune responses to such antigens. Also, such antibodies may effect the growth/survival of tumor cells expressing an antigen that is also a signal transducing receptor by acting as agonists of the receptor. In addition, antibodies may cause pathology when cross reactive with self antigens.
In a first aspect the invention features a method for generating in a patient a cellular immune response to a target protein or portion thereof comprising the step of introducing into cells of the patient a vector containing a nucleotide sequence encoding a chimeric immunogen comprising a protein processing signal and the target protein or portion thereof, so that the chimeric immunogen is made within the cells and subsequently processed such that the ta
Kipps Thomas J.
Wu Yunqi
Lyon & Lyon LLP
Salimi Ali R.
The Regents of the University of California
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