Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-07-28
2001-11-13
Priebe, Scott D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S370000, C435S325000, C424S093210, C424S900000
Reexamination Certificate
active
06316420
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to DNA vaccines and DNA vaccination, and more particularly, to DNA encoding cytokines and the use of same as DNA vaccines for inducing protective immunity against autoimmune diseases. Most particularly, the present invention relates to DNA encoding C-C chemokines and tumor necrosis factor alpha and the use of same for protective immunity against multiple sclerosis.
Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS) which, for many years and for a variety of experimental protocols, serves as a model for the human disease, multiple sclerosis (MS), a chronic degenerative disease marked by patchy destruction of the myelin that surrounds and insulates nerve fibers and mild to severe neural and muscular impairments, since in both diseases circulating leukocytes penetrate the blood brain barrier and damage myelin resulting in impaired nerve conduction and paralysis (1, 2).
Molecular biologic techniques were previously used to follow leukocyte trafficking to the site of inflammation at the CNS of EAE rats, and a model that characterizes this process as a sequential multi-step event was suggested (3).
At first, a very limited repertoire of T-cells, named “the primary influx” interact with their target antigen at the site of inflammation, leading to the activation of the blood brain barrier to express various adhesion molecules and thus to increase its permeability to circulating leukocytes (3, 4). Enhanced permeability of this barrier allows a non-selective influx of leukocytes, which are named “the secondary influx”. This influx correlates with disease onset (3, 5). Subsequently, antigen specific autoimmune T-cells either become anergic or undergo programmed cell death (apoptosis) leading to a remission in disease severity (6). Inhibition of the secondary influx, by either soluble peptide therapy or anti-adhesion molecule blockade effectively prevented, or even reversed, an ongoing disease even though the primary influx remained apparent at the site of inflammation (3-5, 7). Taken together these results not only suggest novel therapeutic strategies, but also emphasize the important role of the non-selective leukocyte influx to a site of inflammation.
Chemokines are chemoattractants that mediate leukocyte attraction and recruitment at the site of inflammation. As such, they are likely to be key mediators in the recruitment of the secondary influx of leukocytes at an inflamed target organ. This has motivated us to use the novel technology of naked DNA vaccination (8-17) and explore the therapeutic potential of anti-chemokine immunotherapy in EAE.
Based on the positions of the first two cysteines, the chemokines can be divided into four highly conserved but distinct supergene families C-C, C-X-C, C and the newly discovered C-X3-C (18, 19). The C-C family is primarily involved in the activation of endothelium and for chemoattraction of T cells and monocytes to the site of inflammation (20-32). The protective competence of anti-C-C chemokine based immunotherapy has been demonstrated by Karpus at al. who blocked EAE in mice by immunizing them with rabbit anti-mouse polyclonal antibodies against macrophage inflammatory protein-1 &agr; (MIP-1&agr;) (33), and very recently by Gong at al. who used an antagonist of monocyte chemoattractant protein 1 is (MCP-1) to inhibit arthritis in the MRL-
1
pr mouse model (34). In another study Berman at al. used in situ hybridization to demonstrate the dominant expression of MCP-1 in rat EAE brain (35).
In the course of reducing the present invention down to practice we have cloned each of the major C-C chemokines: MCP-1, MIP-1&agr;, macrophage inflammatory protein-1&bgr; (MIP-1&bgr;) and regulation on activation normal T expressed and secreted (RANTES) from EAE brains into an eukaryotic expression vector and determined their capacity to block EAE when used as vaccines.
Thus, during the course of EAE various proinflammatory cytokines and chemokines are produced at the site of inflammation (40, 53-55). The pivotal role of one of these proinflammatory cytokines; tumor necrosis factor alpha (TNF-&agr;), in EAE has been well characterized. TNF-&agr; is produced by activated T cells (mostly Th1) and macrophages, and its elevated expression at the site of inflammation occurs during the critical phase of disease (55), at the time when the ‘secondary influx’ of leukocytes is apparent (3). Except for a single recent study carried out in genetically modified animals (56), all investigators agree that TNF-&agr; contributes to the proinflammatory process in EAE and MS (57-71). Early studies have shown that IFN-&ggr; and TNF&agr; together exhibit a synergistic effect on enhancing expression of adhesion molecules on endothelial cells (61), and on eliciting the inflammatory process, which can be reversed by either anti-adhesion molecule immunotherapy (4), or by blocking TNF-&agr; (57-61). More recent studies have demonstrated that inhibition of TNF-&agr; activity by either neutralizing antibodies, or soluble TNF receptor therapy, effectively prevent, or even reverse EAE (62, 64, 66-71). Overexpression of TNF-&agr; at the CNS aggravated the disease (65), whereas genetically impaired expression of this gene inhibited disease development and progression (63).
A major disadvantage in treating chronic diseases with xenogenic neutralizing antibodies lies in their immunogenicity. This has motivated investigators to develop chimeric humanized antibodies (reviewed in 50), and monoclonal antibodies engineered with human Ig heavy and light chain yeast artificial chromosome (YAC) (51). However, following repeated immunization, these engineered antibodies do trigger an apparently allotypic response.
The therapeutic strategy of the present invention, is of advantage over the above methods since it resulted in the generation of immunity to autologous antigens.
There is thus a widely recognized need for, and it would be highly advantageous to have, methods and compositions enabling vaccination with DNA encoding cytokines, such as C-C chemokines and tumor necrosis factor alpha and the use of such vaccination for protective immunity against multiple sclerosis, devoid of the limitations associated with the use of neutralizing antibodies.
SUMMARY OF THE INVENTION
DNA vaccination represents a novel means of expressing antigen in vivo for the generation of both humoral and cellular immune responses. The present invention uses this technology to elicit protective immunity against autoimmune diseases as exemplified by the experimental autoimmune encephalomyelitis (EAE), a T cell mediated autoimmune disease of the central nervous system that serves as an experimental model for multiple sclerosis.
RT-PCR verified by Southern blotting and sequencing of PCR products of four different C-C chemokines: MIP-1&agr;, MCP-1, MIP-1&bgr; and RANTES was performed on brain samples from EAE rats to evaluate mRNA transcription at different stages of disease. Each PCR product was then used as a construct for naked DNA vaccination. The subsequent in vivo immune response to MIP-1&agr; or MCP-1 DNA vaccines prevented EAE, even if disease was induced two months after administration of naked DNA vaccines. In contrast, administration of the MIP-1&bgr; naked DNA significantly aggravated the disease. Generation of in vivo immune response to RANTES naked DNA had no notable effect on EAE. MIP-1&agr;, MCP-1 and MIP-1&bgr; mRNA transcription in EAE brains peaked at the onset of disease and declined during its remission, whereas RANTES transcription increased in EAE brains only following recovery. Immunization of CFA without the encephalitogenic epitope did not elicit the anti C-C chemokine regulatory response in DNA vaccinated rats. Thus, modulation of EAE with C-C chemokine DNA vaccines is dependent targeting chemokines that are highly transcribed at the site of inflammation at the onset of disease.
We further demonstrate herein that EAE rats display a significantly increased TNF-&agr; specific anti
Karin Nathan
Wildbaum Gizi
Youssef Sawsan
Beckerleg Anne Marie S.
Priebe Scott D.
Technion Research and Development Foundation Ltd.
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