Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-01-20
2004-04-27
LeGuyader, John L. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S023100, C536S024310, C536S024330, C536S024500, C435S006120, C435S091100, C435S325000, C435S366000, C435S375000
Reexamination Certificate
active
06727230
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed towards methods for stimulating a localized immune response and for enhancing the efficacy of antiinfective and anticancer agents through local immune stimulation. This invention is further directed toward immunopotentiators comprising phosphorothioate oligonucleotide analogs which produce the desired immune stimulation.
BACKGROUND OF THE INVENTION
Developments in recombinant DNA technology and peptide synthesis have made possible the creation of a new generation of drugs. However, small peptides and other agents do not always invoke the immune response necessary for a therapeutic effect. Substances which increase cell-mediated and/or humoral response may be required in the formulation for efficacy. The potency of a variety of agents, particularly antiinfective and antitumor drugs, may be enhanced by stimulation of an immune response.
The cell-mediated immune response (“local immune response”) is produced by T-cells or thymus derived lymphocytes. T-cells are able to detect the presence of invading pathogens through a recognition system referred to as the T-cell antigen receptor. Upon detection of an antigen, T-cells direct the release of multiple T-cell lymphokines including the interleukin-2 family (IL-2). IL-2 is a T-cell growth factor which promotes the production of many more T-cells sensitive to the particular antigen. This production constitutes a clone of T-cells. The sensitized T-cells attach to cells containing the antigen. T-cells carry out a variety of regulatory and defense functions and play a central role in immunologic responses. When stimulated to produce a cell-mediated immune response, some T-cells respond by acting as killer cells, killing the host's own cells when these have become infected with virus and possibly when they become cancerous and therefore foreign. Some T-cells respond by stimulating B cells while other T-cells respond by suppressing immune responses.
The antibody or humoral immune response (“systemic immune response”) depends on the ability of B-cells, or bone marrow-derived lymphocytes, to recognize specific antigens. The mechanism by which B-cells recognize antigens and react to them is as follows. Each B cell has receptor sites for specific antigens on its surface. When an antigen attaches to the receptor site of a B-cell, the B-cell is stimulated to divide. The daughter cells become plasma cells which manufacture antibodies complementary to the attached antigen. Each plasma cell produces thousands of antibody molecules per minute which are released into the bloodstream. As the plasma cells die, others are produced, so that, once the body is exposed to a particular antigen, antibodies are produced against that antigen as long as the antigen is present in the body. Many B-cells appear to be regulated by the helper T-cells and suppressor T-cells. Helper T-cells appear to stimulate B-cells to produce antibodies against antigens, while suppressor T-cells inhibit antibody production by either preventing the B-cells from functioning or preventing the helper T-cells from stimulating the B-cells. Some B-cells, however, are T-cell independent and require no stimulation by the T-cells.
Immunopotentiators, such as adjuvants, are substances which are added to therapeutic or prophylactic agents, for example vaccines or antigens used for immunization, to stimulate the immune response. Adjuvants cause an accumulation of mononuclear cells, especially macrophages, at the site of injection. Macrophages involved in this first stage of the immune response take in the protein antigens and break them down into peptide fragments which are then exposed on the cell surface where they form a physical association with class II histocompatibility antigens. The T helper cells recognize only protein fragments associated with class II histocompatibility antigen, and not the free undegraded protein. Nonprotein antigens are similarly processed by macrophages or other antigen-presenting cells. The macrophages release monokines from the interleukin-1 family (IL-1) which stimulate the T helper cells to secrete IL-2. The actions of IL-1 and IL-2 result in the clonal expansion of T helper cells. The clonal expansion of T helper cells is followed by their interaction with B-cells, which in turn secrete antibody.
Administration of an adjuvant resulting in stimulation of IL-1 and other cytokines results in a complex spectrum of biological activities. In addition to being a primary immunostimulatory signal, IL-1 proteins have been linked with prostaglandin production, inflammation and induction of fever. IL-1 proteins have been shown to have multiple effects on cells involved in inflammation and wound healing and are known to stimulate proliferation of fibroblasts and attract cells involved in the inflammatory response.
Adjuvants encompass several broad classes including aluminum salts, surface-active agents, polyanions, bacterial derivatives, vehicles and slow-release materials. At present, most adjuvants have been found to stimulate macrophages at the site of action; however, certain adjuvants have been found to act as T-cell replacers enabling B-cells to respond to antigen in the absence of T-cells. An example of such an adjuvant is endotoxin, a B-cell mitogen.
Polynucleotides and other polyanions have been shown to cause release of cytokines. Also, bacterial DNA species have been reported to be mitogenic for lymphocytes in vitro. Furthermore, deoxyoligonucleotides (30-45 nucleotides in length) have been reported to induce interferons and enhance natural killer (NK) cell activity. Kuramoto et al. (1992) Jpn.
J. Cancer Res
. 83:1128-1131. Oligonucleotides that displayed NK-stimulating activity contained specific palindromic sequences and tended to be guanosine rich. Immune stimulation has also been reported for antisense oligomers that are complementary to the initiation sequence of HIV rev and to the mink cell focus-forming (MCF) envelope gene initiation region. Krieg et al. (1989)
J. Immunol
. 143:2448-2451; Branda et al. (1993)
Biochemical Pharmacology
45:2037-2043. The MCF sequence is an endogenous retroviral sequence found in mice. In a study designed to determine whether expression of these endogenous viral sequences suppresses lymphocyte activation (as expressed infectious retroviral sequences can), antisense oligonucleotides and analogs complementary to the MCF env gene AUG region were used to inhibit expression of MCF mRNA. This resulted in increased lymphocyte activation. However, this was believed to be a specific effect resulting from inhibition of the target gene, rather than an effect of oligonucleotides per se. In this case both phosphodiester and phosphorothioate oligonucleotides complementary to this target had the same effect, whereas antisense oligonucleotides to other retroviral targets and phosphorothioate control oligonucleotides had no effect. Krieg et al. (1989)
J. Immunol
. 143:2448-2451; Branda et al. (1993)
Biochemical Pharmacology
45:2037-2043. Branda et al. showed that an anti-rev phosphorothioate oligonucleotide analog is mitogenic in both mononuclear cells from murine spleens and human peripheral blood mononuclear cells. A concentration-dependent stimulation of immunoglobulin production was also observed in vitro and in vivo. This mitogenic effect was specific for B-cells. These effects on B-cells were believed to be specific to this anti-rev oligomer as oligonucleotides complementary to the gag-pol initiation site and the 3′ splice site of endogenous retroviral sequences were known not to be stimulatory (Krieg et al. (1989)
J. Immunol
. 143:2448-2451) and because another phosphorothioate oligonucleotide analog of similar size, targeted to the human p53 protein, did not exhibit the same effect. The data suggested that endogenous retroviruses may suppress lymphocyte activation and that antisense oligonucleotides specific for these inhibitory retroviruses may reverse this suppression and stimulate B-lymphocytes. Though Branda et al. speculated about the possibility that the immune
Glover Josephine M.
Hutcherson Stephen L.
Coley Pharmaceutical Group, Inc.
LeGuyader John L.
Schmidt M
Wolf Greenfield & Sacks P.C.
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