Immunostimulatory nucleic acid molecules for activating...

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Reexamination Certificate

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Reexamination Certificate

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06429199

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to methods and products for activating dendritic cells. In particular, the invention relates to oligonucleotides which have a specific sequence including at least one unmethylated CpG dinucleotide which are useful for activating dendritic cells.
BACKGROUND OF THE INVENTION
In the 1970s, several investigators reported the binding of high molecular weight DNA to cell membranes (Lerner, R. A., et al., 1971
, Proc. Natl. Acad. Sci. USA
, 68:1212; Aggarwal, S. K., et al., 1975
, Proc. Natl. Acad. Sci. USA
, 72:928). In 1985, Bennett et al. presented the first evidence that DNA binding to lymphocytes is similar to a ligand receptor interaction; binding is saturable, competitive, and leads to DNA endocytosis and degradation into oligonucleotides (Bennett, R. M., et al.,
J. Clin. Invest
., 76:2182). Like DNA, oligodeoxyribonucleotides (ODNs) are able to enter cells in a saturable, sequence independent, and temperature and energy dependent fashion (reviewed in Jaroszewski, J. W., et al. and J. S. Cohen, 1991
, Advanced Drug Delivery Reviews
, 6:235; Akhtar, et al., 1992, in: “Gene Regulation: Biology of Antisense RNA and DNA,” R. P. Erickson, Eds, Raven Press, Ltd., New York, p. 133; and Zhao, et al., 1994, Blood, 84:3660). No receptor for DNA or ODN uptake has yet been cloned, and it is not yet clear whether ODN binding and cell uptake occurs through the same or a different mechanism from that of high molecular weight DNA.
Lymphocyte ODN uptake has been shown to be regulated by cell activation. Spleen cells stimulated with the B cell mitogen LPS had dramatically enhanced ODN uptake in the B cell population, while spleen cells treated with the T cell mitogen ConA showed enhanced ODN uptake by T but not B cells (Krieg, A. M., et al., 1991
, Antisense Research and Development
, 1:161).
Several polynucleotides have been extensively evaluated as biological response modifiers. Perhaps the best example is poly(IC) which is a potent inducer of interferon (IFN) production as well as a macrophage activator and inducer of NK activity (Talmadge, J. E., et al., 1985
, Cancer Res
., 45:1058; Wiltrout, et al., 1985
, J. Biol. Resp. Mod
., 4:512; Krown, S. E., 1986
, Sem. Oncol
., 13:207; and Ewel, C. H., et al., 1992, Canc. Res., 52:3005). It appears that this murine NK activation may be due solely to induction of IFN-&bgr; secretion (Ishikawa, R., and C. A. Biron, 1993
, J. Immunol
., 150:3713). This activation was specific for the ribose sugar since deoxyribose was ineffective. Its potent in vitro anti-tumor activity led to several clinical trials using poly(IC) complexed with poly-L-lysine and carboxymethylcellulose (to reduce degradation by RNAse) (Talmadge, et al., cited supra; Wiltrout, et al., cited supra; Krown, et al., cited supra, and Ewel, et al., cited supra). Unfortunately, toxic side effects has thus far prevented poly(IC) from becoming a useful therapeutic agent.
Guanine ribonucleotides substituted at the C8 position with either a bromine or a thiol group are B cell mitogens and may replace “B cell differentiation factors” (Feldbush, T. L., and Z. K. Ballas, 1985
, J. Immunol
., 134:3204; and Goodman, M. J., 1986
, J. Immunol
., 136:3335). 8-mercaptoguanosine and 8-bromoguanosine also can substitute for the cytokine requirement for the generation of MHC restricted CTL (Feldbush, T. L., cited supra), augment murine NK activity (Koo, G.C., et al., 1988
, J. Immunol
., 140:3249) and synergize with IL-2 in inducing murine LAK generation (Thompson, R. A., and Z. K. Ballas, 1990
, J. Immunol
., 145:3524). The NK and LAK augmenting activities of these C8-substituted guanosines appear to be due to their induction of IFN (Thompson, cited supra). Recently a 5′ triphosphorylated thymidine produced by a mycobacterium was found to be mitogenic for a subset of human &ggr;&dgr; T cells (Constant, P., et al., 1994
, Science
, 264:267). This report indicated the possibility that the immune system may have evolved ways to preferentially respond to microbial nucleic acids.
Several observations suggest that certain DNA structures may also have the potential to activate lymphocytes. For example, Bell, et al. reported that nucleosomal protein-DNA complexes (but not naked DNA) in spleen cell supernatants caused B cell proliferation and immunoglobulin secretion (Bell, D. A., et al., 1990
, J. Clin. Invest
., 85:1487). In other cases, naked DNA has been reported to have immune effects. For example, Messina, et al. have recently reported that 260-800 bp fragments of poly(bG).(dC) and poly(dG, dC) were mitogenic for B cells (Messina, J. P., et al., 1993
, Cell. Immunol
., 147:148). Tokunaga, et al. have reported that poly(dg, dc) induces the &ggr;-IFN and NK activity (Tokunaga, et al., 1988
, Jpn. J Cancer Res
., 79:682). Aside from such artificial homopolymer sequences, Pisetsky, et al. reported that pure mammalian DNA has no detectable immune effects, but that DNA from certain bacteria induces B cell activation and immunoglobulin secretion (Messina, et al., 1991
, J. Immunol
., 147:1759). Assuming that these data did not result from some unusual contaminant, these studies suggested that a particular structure or other characteristic of bacterial DNA renders it capable of triggering B cell activation. Investigations of microbacterial DNA sequences have demonstrated that ODN, which contains certain palindrome sequences can activate NK cells (Yamamoto, et al., 1992
, J. Immunol
., 148:4072; and Kuramoto, et al., 1992
, Jpn. J. Cancer Res
., 83:1128).
Several phosphorothioate modified ODN have been reported to induce in vitro or in vivo B cell stimulation (Tanaka, et al., 1992
, J. Exp. Med
., 175:597; Branda, R. S., et al., 1993
, Biochem. Pharmacol
., 45:2037; McIntyre, K., et al., 1993
, Antisense Res. Develop
., 3:309; and Pisetesky, et al., 1994
, Life Sciences
, 54:101). These reports do not suggest a common structure motif or sequence element in these ODN that might explain their effects.
Dendritic cells are considered to be the most potent professional antigen-presenting cells (APC) (Guery, J. C., et al., 1995
, J. Immunol
., 154:536). Dendritic cells capture antigen and present them as peptide fragments to T cells, stimulating T cell dependent immunity. These powerful APCs have been found in skin, blood, dense tissue, and mucosa, and spleen. Several studies have demonstrated that after human dendritic cells which are isolated from peripheral blood are presented peptide antigen they can be used to stimulate and expand antigen specific CD4+ and CD8+ T cells, in vitro and ex vivo (Engleman, E. G., 1997
, Cytotechnology
, 25:1). Several clinical trials are currently underway, based on these findings, using ex vivo manipulation of dendritic cells to generate specific anti-tumor dendritic cells for reimplantation. There has been a growing interest in using dendritic cells ex vivo as tumor or infectious disease vaccine adjuvants (Nestle OF, et al., “Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells”,
Nat Med
, 1998; 4: 328-332; Rosenberg S A, et al., “Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma”,
Nat Med
, 1998; 4:321-327; Hsu F J, et al., “Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells”,
Nat Med
, 1996; 2: 52-58; Tjoa BA, et al., “Evaluation of phase I/II clinical trials in prostate cancer with dendritic cells and PSMA peptides”,
Prostate
, 1998; 36: 39-44. Numerous animal models demonstrate conclusively that ex vivo generated DC pulsed with protein antigen can be successfully applied for the immunotherapy of cancer and infectious diseases. (Fields R C, et al., “Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo”,
Proc Natl Acad Sci, USA
, 1998; 95: 9482-9487; Okada H, et al., “Bone marrow-derived dendritic cells pulsed with a tumor-specific peptide elicit effective anti-tumor immunity against intrac

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