Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-04-22
2003-07-08
Shukla, Ram R. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S091100, C435S455000, C435S458000, C514S002600, C530S300000, C536S023100
Reexamination Certificate
active
06589940
ABSTRACT:
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
1. Technical Field
The present invention relates to immunomodulatory compositions comprising an immunostimulatory oligonucleotide sequence (ISS). The invention further relates to immunomodulatory compositions comprising an ISS in which at least one base has been substituted with a base modified by the addition to C-5 or C-6 on cytosine with an electron-withdrawing moiety. It also relates to the administration of the oligonucleotide sequences to modulate at least one immune response. The invention further relates to in vitro screening methods to identify oligonucleotides with potential immunomodulatory activity.
2. Background Art
The type of immune response generated to infection or other antigenic challenge can generally be distinguished by the subset of T helper (Th) cells involved in the response. The Th1 subset is responsible for classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs), whereas the Th2 subset functions more effectively as a helper for B-cell activation. The type of immune response to an antigen is generally determined by the cytokines produced by the cells responding to the antigen. Differences in the cytokines secreted by Th1 and Th2 cells are believed to reflect different biological functions of these two subsets.
The Th1 subset may be particularly suited to respond to viral infections and intracellular pathogens because it secretes IL-2 and IFN-&ggr;, which activate CTLs. The Th2 subset may be more suited to respond to free-living bacteria and helminthic parasites and may mediate allergic reactions, since IL-4 and IL-5 are known to induce IgE production and eosinophil activation, respectively. In general, Th1 and Th2 cells secrete distinct patterns of cytokines and so one type of response can moderate the activity of the other type of response. A shift in the Th1/Th2 balance can result in an allergic response, for example, or, alternatively, in an increased CTL response.
Immunization of a host animal against a particular antigen has been accomplished traditionally by repeatedly vaccinating the host with an immunogenic form of the antigen. While most current vaccines elicit effective humoral (antibody, or “Th2-type”) responses, they fail to elicit cellular responses (in particular, major histocompatibility complex (MHC) class I-restricted CTL, or “Th1-type” responses) which are generally absent or weak. For many infectious diseases, such as tuberculosis and malaria, Th2-type responses are of little protective value against infection. Moreover, antibody responses are inappropriate in certain indications, most notably in allergy where an antibody response can result in anaphylactic shock. Proposed vaccines using small peptides derived from the target antigen and other currently used antigenic agents that avoid use of potentially infective intact viral particles, do not always elicit the immune response necessary to achieve a therapeutic effect. The lack of a therapeutically effective human immunodeficiency virus (HIV) vaccine is an unfortunate example of this failure.
Protein-based vaccines typically induce Th2-type immune responses, characterized by high titers of neutralizing antibodies but without significant cell-mediated immunity. In contrast, intradermal delivery of “naked”, or uncomplexed, DNA encoding an antigen stimulates immune responses to the antigen with a Th1-type bias, characterized by the expansion of CD4
+
T cells producing IFN-&ggr; and cytotoxic CD8
+
T cells. Manickan et al. (1995)
J. Immunol.
155:250-265; Xiang et al. (1995)
Immunity
2:129-135; Raz et al. (1995)
Proc. Natl. Acad. Sci. USA
93:5141-5145; and Briode et al. (1997)
J. Allergy Clin. Immunol.
99:s129. Injection of antigen-encoding naked DNA reproducibly induces both humoral and cellular immune responses against the encoded antigens. Pardoll and Beckerleg (1995)
Immunity
3:165-169. DNA vaccines can provide a new approach to infectious disease prophylaxis. See, for instance, Dixon (1995)
Bio/Technology
13:420 and references cited therein.
Certain types of DNA, without being translated, have been shown to stimulate immune responses. Bacterial DNA induces anti-DNA antibodies in injected mice, as well as cytokine production by macrophage and natural killer (NK) cells. Pisetsky (1996)
J. Immunol.
156:421-423; Shimada et al. (1986)
Jpn. J. Cancer Res.
77:808-816; Yamamoto et al. (1992a)
Microbiol. Immunol.
36:983-897; and Cowdery et al. (1996)
J. Immunol.
156:4570-4575.
B cell and NK cell activation properties of bacterial DNA have been associated with short (6 base pair hexamer) sequences that include a central unmethylated CpG dinucleotide. Yamamoto et al. (1992a); and Krieg et al. (1995)
Nature
374:546-549. Oligonucleotides comprising a CpG sequence flanked by two 5′ purines and two 3′ pyrimidines have been shown to be most potent in B cell and NK cell stimulation. For example, when a variety of oligonucleotides comprising hexamers were tested for their ability to augment the NK cell activity of mouse spleen cells, the most immunogenic hexamers included AACGTT, AGCGCT, GACGTC. Yamamoto et al. (1992b)
J. Immunol.
148:4072-4076. In a study in which B cell activation was measured in response to oligonucleotides, the most stimulatory hexamer sequences (e.g., AACGTC, AACGTT, GACGTC, GACGTT) also matched the sequence of 5′-purine, purine, CG, pyrimidine, pyrimidine-3′. Krieg et al. (1995). However, as shown herein, this prototypical hexamer sequence is found in many oligonucleotides that are not immunostimulatory. Thus, the prototypical hexamer sequence proposed by Krieg et al. (1995) is not predictive of immunostimulatory activity.
Bacterial DNA stimulated macrophages to produce IL-12 and TNF-&agr;. These macrophage-produced cytokines were found to induce the production of IL-12 and IFN-&ggr; from splenocytes. Halpern et al. (1996)
Cell. Immunol.
167:72-78.
In vitro
treatment of splenocytes with either bacterial DNA or CpG containing oligonucleotides induced the production of IL-6, IL-12 and IFN-&ggr;. Klinman et al. (1996)
Proc. Natl. Acad. Sci. USA
93:2879-2883. Production of all of these cytokines is indicative of induction of a Th1-type immune response rather than a Th2-type response.
To date, no clear consensus has been reached on the sequences both necessary and sufficient of immune stimulation. A recent study which examined induction of NK activity in response to CpG containing-oligonucleotides suggested that the unmethylated CpG motif was necessary but not sufficient for oligonucleotide induction of NK lytic activity. Ballas et al. (1996)
J. Immunol.
157:1840-1845. Sequences flanking the CpG appeared to influence the immunostimulatory activity of an oligonucleotide. Immunostimulatory activity of immunostimulatory sequences appears to be independent of adenosine-methylation, and whether the nucleotide is single or double-stranded. See, for example, Tokunaga et al. (1989)
Microbiol. Immunol.
33:929; Tokunaga et al. (1992)
Microbiol. Immunol.
36:55-66; Yamamoto et al. (1992b); Messina et al. (1993)
Cell. Immunol.
147:148-157; and Sato et al. (1996)
Science
273:352-354. Oligonucleotide length also does not seem to be a factor, as double-stranded DNA 4 kb long (Sato et al. (1996)) or single-stranded DNA as short as 15 nucleotides in length (Ballas et al. (1996)) illicited immune responses; though if oligonucleotide length was reduced below 8 bases or if the DNA was methylated with CpG methylase, immunostimulatory activity was abolished. Krieg et al. (1995).
Allergic responses, including those of allergic asthma, are characterized by an early phase response, which occurs within seconds to minutes of allergen exposure and is characterized by cellular degranulation, and a late phase response, which occurs 4 to 24 hours later and is characterized by infiltration of eosinophils into the site of allergen exposure. Specifically, during the early phase of the alle
Dina Dino
Raz Eyal
Roman Mark
Dynavax Technologies Corporation
Morrison & Foerster / LLP
Shukla Ram R.
Zara Jane
LandOfFree
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