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Title:  DNA cytokine vaccines and use of same for protective immunity against multiple sclerosis

United States Patent:  6,316,420

Inventors:  Karin; Nathan (Haifa, IL); Youssef; Sawsan (Rama Villag, IL); Wildbaum; Gizi (Kiriat-Yam, IL)

Assignee:  Technion Research and Development Foundation LTD. (Haifa, IL)

Appl. No.:  123485

Filed:  July 28, 1998

Abstract

A method for treating a mammal for inducing protective immunity against an autoimmune disease including the step of administering to the mammal a therapeutic composition including a recombinant construct including an isolated nucleic acid sequence encoding a cytokine, the nucleic acid sequence being operatively linked to one or more transcription control sequences. A method for treating a mammal for inducing protective immunity against an autoimmune disease including the steps of (a) removing cells of the mammal; (b) transducing the cells in vitro with a recombinant construct including an isolated nucleic acid sequence encoding a cytokine, the nucleic acid sequence being operatively linked to one or more transcription control sequences; and (c) reintroducing the transduced cells to the mammal. A pharmaceutical composition including (a) a recombinant construct including an isolated nucleic acid sequence encoding a cytokine, the nucleic acid sequence being operatively linked to one or more transcription control sequences; and (b) a pharmaceutically acceptable carrier. And an antibody raised against a cytokine expressed by cells transduced with a recombinant construct including an isolated nucleic acid sequence encoding the cytokine, the nucleic acid sequence being operatively linked to one or more transcription control sequences.

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.alpha., MCP-1, MIP-1.beta. 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.alpha. 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.beta. naked DNA significantly aggravated the disease. Generation of in vivo immune response to RANTES naked DNA had no notable effect on EAE. MIP-1.alpha., MCP-1 and MIP-1.beta. 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-.alpha. specific antibody titer as compared to rats immunized in hind foot pads with Complete Freund's Adjuvant (CFA) alone. A positive correlation in time course between the elevated expression TNF-.alpha. at the CNS and the production of anti-self antibodies to this proinflammatory cytokine was observed. This natural immunity to TNF-.alpha. could not block the development of disease. An administration of TNF-.alpha. naked DNA vaccine, even two months before active induction of disease, enhanced the development of in vivo immune response to self TNF-.alpha. and conferred EAE resistance. Immunization of CFA without the encephalitogenic epitope, even though induced a local inflammatory process, did not elicit the anti TNF-.alpha. regulatory response in DNA vaccinated rats. These anti-self antibodies were found capable of inhibiting the development of disease when transferred to other EAE rats. Thus, modulation of EAE with TNF-.alpha. vaccines is dependent targeting cytokine that are highly transcribed at the site of inflammation during the course of disease and therefore provides a tool by which the immune system is encouraged to elicit anti-self protective immunity to restrain its own harmful reactivity only when such a response is needed.

According to the present invention there is thus provided a method for treating a mammal for inducing protective immunity against an autoimmune disease, the method comprising the step of administering to the mammal a therapeutic composition including a recombinant construct including an isolated nucleic acid sequence encoding a cytokine, the nucleic acid sequence being operatively linked to one or more transcription control sequences.

According to the present invention there is further provided a method for treating a mammal for inducing protective immunity against an autoimmune disease, the method comprising the steps of (a) removing cells of the mammal; (b) transducing the cells in vitro with a recombinant construct including an isolated nucleic acid sequence encoding a cytokine, the nucleic acid sequence being operatively linked to one or more transcription control sequences; and (c) reintroducing the transduced cells to the mammal.

According to still further features in the described preferred embodiments the transduced cells are reintroduced to the mammal parenterally.

According to the present invention there is further provided a pharmaceutical composition comprising (a) a recombinant construct including an isolated nucleic acid sequence encoding a cytokine, the nucleic acid sequence being operatively linked to one or more transcription control sequences; and (b) a pharmaceutically acceptable carrier.

According to further features in preferred embodiments of the invention described below, the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous physiologically balanced solution, an artificial lipid-containing substrate, a natural lipid-containing substrate, an oil, an ester, a glycol, a virus and metal particles.

According to still further features ill the described preferred embodiments the composition is useful for treating an autoimmune disease.

According to still further features in the described preferred embodiments the composition is suitable for parenteral administration to a human.

According to still further features in the described preferred embodiments the pharmaceutically acceptable carrier comprises a delivery vehicle that delivers the nucleic acid sequences to the mammal.

According to still further features in the described preferred embodiments the delivery vehicle is selected from the group consisting of liposomes, micelles, and cells.

According to the present invention there is further provided an antibody raised against a cytokine expressed by cell s transduced with a recombinant construct including an isolated nucleic acid sequence encoding the cytokine, the nucleic acid sequence being operatively linked to one or more transcription control sequences.

According to further features in preferred embodiments of the invention described below, the autoimmune disease is multiple sclerosis.

According to still further features in the described preferred embodiments the cytokine is a chemokine or tumor necrosis factor alpha.

According to still further features in the described preferred embodiments the chemokine is a C-C chemokine.

According to still further features in the described preferred embodiments the C-C chemokine is selected from the group consisting of macrophage inflammatory protein-1.alpha. (MIP-1.alpha.), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-1.beta. (MIP-1.beta.) and regulation on activation normal T expressed and secreted (RANTES).

According to still further features in the described preferred embodiments the transcription control sequences are selected from the group consisting of RSV control sequences, CMV control sequences, retroviral LTR sequences, SV-40 control sequences and .beta.-actin control sequences.

According to still further features in the described preferred embodiments the recombinant construct is an eukaryotic expression vector.

According to still further features in the described preferred embodiments the recombinant construct is selected from the group consisting of pcDNA3, pcDNA3.1(+/-), pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pCI, pBK-RSV, pBK-CMV, pTRES and their derivatives.

According to still further features in the described preferred embodiments the mammal is selected from the group consisting of humans, dogs, cats, sheep, cattle, horses and pigs.

The present invention successfully addresses the shortcomings of the presently known configurations by providing novel means to combat the incurable and poorly treatable disease--multiple sclerosis--devoid of the limitations associated with protective immunity via administered antibodies.

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

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