JP2002517181A - Nucleic acids provided to regulate cell activation - Google Patents

Abstract

  (57) [Summary] The present invention relates to nucleic acids, derivatives thereof and sequence-specific binding substances that inhibit the expression of the CD30 antigen and / or the ribosome S6 kinase pp90rsk3. The invention also relates to pharmaceutical compositions comprising said nucleic acids and to regulate the activation of cells, suppress and prevent immune dysregulation, reduce viral load, and eradicate CD30 + and / or pp90rsk3 + cells and their tumors For the use of the nucleic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleic acid that inhibits the expression of the CD30 antigen and / or the ribosomal S6 kinase pp90rsk3, a derivative thereof and a sequence-specific binding substance, a pharmaceutical composition containing the nucleic acid, and regulation of cell activation and immunomodulation. It relates to the use of said nucleic acids for inhibiting and preventing deficiencies, reducing viral load and eliminating CD30 ⁺ and / or pp90rsk3 ⁺ cells and their tumors.

[0002] Cells use complex networks of cell activation to respond to signals from the environment and ensure proper response. To accommodate a variety of seemingly endless physical and chemical effects, cells have, as they wish, a physiological pattern of specific responses through the activation of various regulatable cells. However, cellular activation is often dysregulated, and little is known about the environmental effects that cause it or the signals that deregulate cells.

[0003] The spectrum of diseases involving deregulated cell activation is wide and cells and derivatives of all tissues, such as epithelial cells in response to bacterial toxins, or chronic viral infections, chronic inflammatory and atopic diseases, For example, atopic dermatitis (neurodermi
tis)). Some of these diseases, such as atopic syndrome, are very frequent, affecting up to 15% of the population.

[0004] Because of the considerable diversity of dysregulation of cell activation, a great number of methods for selectively and cell-specific interruption of pathophysiological control loops have been developed. However, most of these methods have very limited success with use or are particularly systemic acting inhibitors such as corticosteroids or derivatives thereof (eg, for immunoregulation dysfunction), vitamin D3, cyclosporine, 1
When 5-deoxy-spergualine, an antibody or an immunosuppressive cytokine is used, considerable side effects are likely to occur (survey: Sigal and Dumont, Fundamental Immunology, 3rd ed., P.
p. 903-915, 1993; Ed .: WEPaul; Raven Press, New York). All of the above immunosuppressants generally non-selectively suppress the entire spectrum of immune cells, resulting in an increased risk of infection and malignancy. Also, the current lack of knowledge about the control loops of cell activation has hampered the development of new strategies.

[0005] The CD30 antigen has been assigned to the TNF / NGF receptor family due to sequence homology and is a phosphorylated 1 expressed by several lymphocyte subpopulations.
It is a membrane glycoprotein of 0.5 to 120 kDa (Schwab et al., Nature 299, 65-
67 (1982); WO93 / 10232; and Duerkop et al., Cell 68, 421-427, 1992). CD
The 30 antigen is not exclusive, but predominantly Th2 T cells, especially 85% CD
A subpopulation of activated CD45RO ⁺ “memory” T cells expressing the four antigens simultaneously (
15-20%) of the cells. Cells with a persistent viral infection and cells of many virus-associated tumors also express the CD30 antigen. High CD30
Expression is also found in malignant cells of Hodgkin lymphoma, so-called Reed-Sternberg cells, cells of the anaplastic large-cell lymphoma, and cells of fetal and mesenchymal germ cell tumors (Suster et al. al., Hum. Pa
thol. 29, 737-742, 1998).

[0006] The function of the CD30 molecule in normal immune cells has not yet been elucidated in detail (Romagnani, The Immunologist 6, 137-141, 1998). When CD30 cross-links to the cell surface, it increases many cell functions, such as T cell proliferation (Smith et al.,
Cell 73, 1349-1360, 1993), induction of the transcription factor NFκB (McDonald et al., Euro
p. J. Immunol. 25. 2870-2876, 1995), induction of HIV gene expression in HIV infected T cells (Biswas et al., Immunity 2, 587-596, 1995; Maggi et al., Im
munity 3, 251-255, 1995), increased intracellular calcium (Ellis et al., J. Imm
unol. 151, 2380-2389, 1993) and the onset of cell death (Gruss et al., Blood 83,
2045-2056, 1994).

[0007] Ribosomal S6 kinase (pp90rsk1, rsk2, rsk3) is a family of mitogen-activated protein kinases. They are expressed everywhere at various levels in cells of various tissues. The pp90rsk1 kinase is functionally downstream of MAP kinase in the Ras-Raf-MEK-MAP kinase signaling pathway (Sturgill et al., Nature 334, 715-718, 1988; Blenis,
Proc. Natl. Acad. Sci. USA 90, 5889-5892, 1993). This is especially the factor IκBα
And stimulates its degradation (Ghoda et al., J. Biol. Chem. 272, 2128
1-21288, 1997). In contrast to pp90rsk1 kinase, pp90rsk3 kinase has been little studied to date. Unlike pp90rsk1, autophosphorylation of pp90rsk3 is E
It is stimulated by growth factors rather than RK2 / MAP kinase. Nuclear translocation of the pp90rsk3 protein is observed after stimulation of the cells, suggesting that pp90rsk3 phosphorylates an unidentified nuclear protein (Zhao et al., M.
ol. Cell. Biol. 15, 4353-4363, 1995).

[0008] The use of nucleic acids in sense and antisense orientation to specifically inhibit gene expression is a method known to those skilled in the art. However, at present there is no rational strategy for producing such effective molecules based on known rules (Branch: "A good an
tisense molecule is hard to find ", TIBS 23, 45-50, 1998).
Nucleic acids having such functional properties cannot be forcibly derived from known DNA or cDNA.

In view of a wide variety of possible dysregulations of cell activation, there is a method for specifically inhibiting cells that is capable of interrupting the pathophysiological control loop of self-stimulation. Desired.

[0010] Surprisingly, certain nucleic acids (oligonucleotides) bind in vivo with RNA, gene or promoter DNA specific for CD30 and / or ribosomal S6 kinase pp90rsk3, and bind to the CD30 antigen and / or ribosomal S6 kinase pp90rsk3 ( Thereafter, it was discovered that the expression of “rsk3” was selectively and functionally suppressed. Furthermore, using these nucleic acids, CD30 and / or
Or it was found that cells expressing rsk3 could be excluded. In particular, the nucleic acids selectively inhibit the activation of CD30 ⁺ and / or pp90rsk3 ⁺ (hereinafter "rsk3 ⁺ ") cells, prevent the activation of these cells, and provide deregulated cellularity. Restores the production of the substance to its original state, suppresses activation-induced proliferation, suppresses virus production, and leads to cell death without appreciably damaging other cells or tissues.

[0011] Accordingly, the present invention relates to (1) a nucleic acid, a derivative thereof and a sequence-specific binding substance that inhibit the expression of the CD30 antigen or ribosomal S6 kinase pp90rsk3 ("rsk3") (hereinafter simply referred to as "nucleic acid"). (Where the nucleic acid is CD30 and / or pp90rsk)
(2) a pharmaceutical composition comprising one or more nucleic acids as defined in (1); (3) CD30 ⁺ and / or Use of the nucleic acid as defined in (1) for producing a medicament for modulating the functional activity of pp90rsk3 ⁺ cells; and (4) for producing a medicament for suppressing and eliminating the function of CD30 ⁺ and / or pp90rsk3 ⁺ cells. Of the nucleic acids defined in (1).

A “nucleic acid derivative” within the scope of the present invention is a nucleic acid that starts with a natural nucleic acid and has at least one modification in the nucleobase, sugar component or phosphate bond known to those skilled in the art. . In the following, such modifications are described in detail. According to the present invention, “sequence-specific binding substance” means nucleic acids and other substances that are characterized by selectively binding to a certain DNA and / or RNA sequence. This includes nucleic acid derivatives in which two or more modifications have been made and, therefore, are no longer exactly termed "nucleic acids" in the narrower sense. Preferred nucleic acids of the invention are those that specifically bind to oligonucleotides and oligonucleotide derivatives, especially CD30 and / or rsk3 mRNA, gene or promoter DNA. Preferably, this is: (a) an oligonucleotide that binds to CD30 mRNA, CD30 gene DNA or CD30 promoter DNA; (b) an oligonucleotide that binds to rsk3 mRNA, rsk3 gene DNA or rsk3 promoter DNA; (c) CD30 and rsk3 oligonucleotides that bind to mRNA or gene DNA; (d) oligonucleotides that are CD30 DNA and / or rsk3 DNA triple helix forming nucleic acids; or (e) oligonucleotides that are ribozymes that bind to CD30 and / or pp90rsk3 mRNA. It may be.

The preferred length of the oligonucleotide is between 8 and 20, especially between 9 and 15 bases. Particularly preferred in the present invention are single-stranded oligonucleotides having the same nucleotide sequence (sense direction) and single-stranded oligonucleotides having a complementary nucleotide sequence (antisense direction) compared to CD30 mRNA. These can efficiently inhibit the synthesis of the CD30 antigen (Example 1). Region bp1
40-170, bp200-250, bp265-300, bp520-540
, Bp 600 to 940, bp 1200 to 1270, bp 1745 to 1790, b
p1885-1920 or bp2985-3005 (base number in EMBL database accession number M83554; Duerkop et al., Cell 68, 421-4
27, 1992; oligonucleotides which bind to CD30 mRNA in FIG. 1 and SEQ ID NO: 1) are preferred. The oligonucleotides described in Table 1 were found to be particularly effective. However, any other oligonucleotide sequence derivable from a CD30 mRNA, gene or promoter sequence is also available.

[0014]

[Table 1] Table 1: CD30 oligonucleotide

[0015] Another preferred embodiment of the present invention includes an oligonucleotide that binds to an rsk3-specific mRNA, gene or promoter DNA. These oligonucleotides suppress the synthesis of rsk3 protein. Regions bp 100 to 160,
bp 1085 to 1125 or bp 1540 to 1565 (base pair number in EMBL database accession number X85106; FIG. 2 and SEQ ID NO: 3) r
Oligonucleotides that bind to sk3 mRNA are preferred. Examples of particularly useful oligonucleotides are listed in Table 2.

[0016]

[Table 2] Table 2: rsk3 oligonucleotide

[0017] Oligonucleotides in the antisense orientation bind to each RNA and effectively prevent translation of CD30 or rsk3 mRNA. The oligonucleotide in the sense direction is D
It was shown to strongly form the NA triple helix and very effectively repress transcription of CD30 or rsk3 RNA.

The oligonucleotides listed in Table 1 bind to CD30-specific DNA or RNA sequences, and the oligonucleotides listed in Table 2 bind to rsk3-specific DNA or RNA sequences. Oligonucleotide AS1 binds to both CD30 mRNA and rsk3 kinase mRNA, its translation and both proteins, rsk3 and C
Inhibits the function of D30. Oligonucleotides S1 and S1-short are CD3
0 gene DNA and rsk3 gene DNA, and both RNA species, CD3
0 Inhibits the transcription of mRNA and rsk3 mRNA. Oligonucleotide AS
1 and S1 or derivatives thereof are preferred for inhibiting rsk3 and CD30 and have excellent efficacy in use according to the invention described below.

The oligonucleotide may be a linear, unmodified DNA or RNA molecule or a DNA or RNA molecule modified by known methods (Agrawal and Iy
er, Pharmakol. Ther. 76, 151-160, 1997). Examples include cyclic oligonucleotides (Kool, J. Am. Chem. Soc. 113, 6265-6266, 1991), phosphorothioate derivatives, 2'-O-methyl RNA, morpholino-modified oligomers, methylphosphonates, PNA (“peptide nucleic acid”), N3 ′ → P5 ′ phosphoramidates or so-called “clamp oligonucleotides” (survey: Cohen, Adv. Pharmakol. 25, 319-339, 1993, Matteucci, Ciba
Found. Symp. 209, 5-14, 1997; Sun et al., Curr. Opin. Struct. Biol. 6,
327-333, 1996; Helene et al., Ciba Found. Symp. 209, 94-102, 1997). The oligonucleotide may also be transcribed from a viral or non-viral expression vector, ie, the nucleic acids of the invention may be in the form of a component of the vector.

[0020] In another aspect of the invention, the nucleic acid of the invention is CD30 and / or rsk
3 A form of a component of a ribozyme or other enzymatically active nucleic acid that exhibits specific binding to mRNA or DNA. Ribozymes whose specific binding region comprises one of the nucleotide sequences listed in Table 1 or Table 2 are preferred. However, the nucleic acids, oligonucleotides and ribozymes according to the present invention have the C
The exact sequences of D30 and rsk3 mRNAs are not represented.

The pharmaceutical composition of the present invention may further comprise, in addition to one or more nucleic acids of the present invention,
A pharmaceutically acceptable carrier and / or pharmaceutical adjuvant can be included. Pharmaceutical carriers for nucleic acids are preferably lipids, such as cationic or anionic liposomes or DOPE (dioleoylphosphatidylethanolamine), DC-Chol (3β- [N- (N ′, N′-dimethylaminoethane)) Carbamoyl] cholesterol) or DOTAP (1,2-dioloyloxy-3- (
Trimethylammonio) propane (1,2-dioloyloxy-3- (trimethylammonio) propa
ne)). Furthermore, lipid-DNA particles coupled with an appropriate ligand are suitable for tissue-specific nucleic acid transport. Viruses, such as adenoviral or artificial particles, that bind to (complex) nucleic acids and promote DNA transport into cells may be used. The nucleic acids according to the invention may also be transported without pharmacological carriers, for example by nucleic acid precipitation, electroporation, injection, particle bombardment ("gene gun").

Experiments have shown a wide variety of pharmacological effects on the nucleic acids according to the invention: CD30 ⁺ and / or rsk3 ⁺ cells have their specific cell functions and synthetic activities (eg, cytokine synthesis) associated with cell activation arrested or regulated in the presence of the nucleic acids of the invention (Example 2). However, it was found that the function of the CD30 ^- rsk3 ^- cells was not affected.

[0023] 2. For example, the induced constitutive cell activation characterized by NFκB activation is specifically suppressed in the presence of the nucleic acids of the invention (Example 3). Whether such cell activation is the result of a chemical or physical activity, whether it is the result of contact with a bacterium or virus or a component thereof, or is based on an unknown stimulus It doesn't matter. As a result of the application of the nucleic acids according to the invention, cells change their function and suppress cell type-specific cell activation, such as endothelial cell adhesion molecules or A20 in CD30 ⁺ cells. And mitochondrial dehydrogenase (or manganese superoxide dismutase). Thus, one use of the nucleic acids of the invention is to selectively functionally inhibit cell activation in CD30 ⁺ and / or rsk3 ⁺ cells without immediately altering the functional activity of other cells. It is. CD30 ⁺ and / or rs in the presence of the nucleic acids of the invention
k3 ⁺ (tumor) cells can be transitioned to a state that is susceptible to cell death induced by chemotherapy, cytokines or irradiation.

[0024] 3. In pre-existing immune dysregulation, physiological Th1 / Th2 T cell equilibrium is restored by specific functional suppression of CD30 ⁺ and / or rsk3 ^{+ in} the presence of the nucleic acids of the invention (which is the result of certain cytokines and other (Characterized by a change in the secretion of effector substances) (Example 2).

[0025] 4. Furthermore, it was found that CD30 ⁺ and / or rsk3 ⁺ cells selectively stopped growing in the presence of the oligonucleotide of the present invention (Example 4) and died (Example 5). Thus, one use of the nucleic acids of the present invention is in malignant and benign C
D30 ⁺ and / or pp90rsk3 ⁺ cell proliferation, tumor and tissue infiltration of such cells, inhibition and prevention (prevention) of microcolonies and metastasis of such cells, and prevention of tumor recurrence of such cells Is for Examples of such tumors are Hodgkin's lymphoma, anaplastic large cell lymphoma, T-cell leukemia, germ cell tumor and fetal cancer. Other applications include CD30 ⁺ and / or during purging, eg, of grafts, bone marrow biopsies, or from the blood of a human or pollen allergic patient, eg, suffering from an atopic disease (Example 7). rsk3
⁺ Includes eliminating cells.

[0026] 5. In the presence of the nucleic acids of the invention, stimulation of blood lymphocytes does not affect CD30 ⁺ and / or
Alternatively, it was found that the cells could not develop into pp90rsk3 ⁺ cells (Example 6). Nor can it induce cell overstimulation. The sustained massive secretion of IL-10, observed after repeated stimulation of lymphocytes with antigen, is suppressed in the presence of the nucleic acids of the invention, while IFN-gamma secretion is not affected. (Example 2). Thus, the nucleic acids of the invention can be used for the specific prevention of immune hyperstimulation associated with the development of CD30 ⁺ and / or rsk3 ⁺ cells without affecting other immune stimuli.
Applications of this kind are extraordinarily diverse; for example, prevent the accumulation of CD30 ⁺ lymphocytes in the blood of humans or pollen allergic patients suffering from atopic diseases, thereby suppressing the acute symptoms of the disease Applications.

[0027] 6. It was found that the production of many viruses in virus-infected cells was suppressed in the presence of the nucleic acid of the present invention. This type of application is preferred for cells with a persistent viral infection to suppress the production of this virus and reduce the viral load on the cells. Because a number of persistent viral infections are known, with a permanent or inducible production of infectious viral particles, the scope of this aspect of the invention is manifold. For example, use of the nucleic acids of the invention to suppress the production of human immunodeficiency virus (HIV), Epstein-Barr virus, hepatitis virus, human T leukemia viruses I and II, and to reduce viral load in infected cells As well as to reduce new infections to reduce the number of infected cells. For example, according to the reverse transcriptase assay, cell-free culture supernatants of blood lymphocytes from HIV-infected donors are free of nucleic acids in the presence of the nucleic acids of the invention, preferably the nucleic acids listed in Tables 1 and 2, Alternatively, it was found to contain 10 to 100-fold lower HIV titers than in the incubation containing "nonsense" nucleic acids. This indicates that the infected lymphocyte population releases the HI virus (virus production).
Is suppressed in the presence of the nucleic acid of the present invention. In addition, RT PCR showed that the number of copies of the HIV genome in this culture was reduced by a factor of 10 to 100, indicating that the viral load of this lymphocyte population was suppressed.

[0028] 7. In addition, rsk3-specific oligonucleotides are useful for mucin,
Preferably, it was found to inhibit Pseudomonas-aeruginosa-induced overproduction of MUC2. Preferably, the nucleic acids according to the invention are applied for diseases involving overproduction of mucin by respiratory epithelial cells of eg cystic fibrosis patients.

Thus, the present invention provides a method for producing a medicament suitable for modulating the functional activity of, for example, CD30 ⁺ and / or pp90rsk3 ⁺ cells and for inhibiting and eliminating CD30 ⁺ and / or pp90rsk3 ⁺ cells. It relates to the use of the nucleic acids of the invention or derivatives thereof. The "modulation of functional activity" and "functional suppression and elimination" of CD30 ⁺ and / or pp90rsk3 ⁺ cells of the present invention include the following parameters (disease):-suppression and inhibition of cell activation; -CD30 ⁺ and / or Pp90rsk3 ⁺ modulation of functional activity of the cells; - reduction in virus load and virus production;-CD30 ⁺ and / or Pp90rsk3 ⁺ cells development and growth inhibition;-CD30 ⁺ and / or Pp90rsk3 ⁺ cells Dysregulation of cell activation, preferably immune dysregulation, atopic response disease, chronic inflammatory disease, preferably colitis, suppression and prevention of graft rejection;-viruses, bacteria and parasites Treatment of insect infections; treatment and prevention of tumors of CD30 ⁺ and / or pp90rsk3 ⁺ cells;
-Functional suppression and elimination of CD30 ⁺ and / or pp90rsk3 ⁺ cells.

In particular, the nucleic acids according to the invention can be used, for example, for the following purposes: activation of cells and their dysregulation, immunoregulation, chronic inflammation, colitis, atopic reactions, neurodermatitis To inhibit and prevent autoimmune reactions, transplant rejection, systemic sclerosis; to reduce viral production and / or viral load of CD30 ⁺ and / or rsk3 ⁺ cells; CD30 ⁺ and / or rsk3 ⁺ To inhibit the development, proliferation, tumor formation and tissue penetration of cells; to inhibit the production of cellular material associated with the activation of cells of CD30 ⁺ and / or rsk3 ⁺ cells; to activate CD30 ⁺ and / or rsk3 ⁺ To alter the tissue balance between cells and other activated or resting cells, affecting the Th1 / Th2 balance of immune cells; in vivo and in vivo To eliminate CD30 ⁺ and / or rsk3 ⁺ cells in Toro tissues, body fluids, biopsates or grafts; To suppress cell activation of CD30 ⁺ and / or rsk3 ⁺ cells; CD30 ⁺ and / or rsk3 ⁺ to suppress overproduction of cellular material in the cell; CD30 ⁺ and / or rSK3 ⁺ to change the spectrum of synthetic cellular material in the cell; CD30 ⁺ and / or rSK3 ⁺ for inhibiting the growth of cells; CD30 ⁺ and / or rSK3 ⁺ cells to eliminate; CD30 ⁺ and / or rSK3 ⁺ to initiate cell death in the cells; CD30 ⁺ and / or rSK3 ⁺ to reduce viral load and virus production in cells; cell activation CD30 ⁺ and / or rsk3 ⁺ cells to prevent immunity; to affect the balance between immune cells To prevent and prevent immune dysregulation, atopic reactions, neurodermatitis, atopic dermatitis, autoimmune diseases, systemic sclerosis, chronic inflammatory diseases, preferably colitis; To treat chronic viral and bacterial infections; to suppress and prevent graft rejection (allograft rejection); chemical and physical effects, environmentally harmful substances, ionizing radiation, bacteria or viruses or inhibited cell activation in those components, as well as for the prevention; CD30 ⁺ and / or rSK3 ⁺ cell malignancies and for treating benign tumors of; CD30 ⁺ and / or rSK3 ⁺ suppresses tumor formation of cell To eliminate and prevent cells; to eliminate cells from body fluids or tissues; and to suppress mucin overproduction.

The present invention also relates to a method for treating the above-mentioned diseases, comprising administering the nucleic acid of the present invention. In particular, the present invention relates to a method for treating and preventing the above-mentioned diseases or conditions, which comprises the local or systemic administration of the nucleic acid of the present invention or a derivative thereof, preferably together with a suitable pharmacological carrier.

Thus, in topical application, for example, for atopic dermatitis, an ointment or lotion comprising one or more oligonucleotides of the invention, preferably oligonucleotide AS1, preferably in the range of 10 mM to 1 M. Applied one to three times a day during the treatment period until the acute symptoms cease, or for prophylaxis, one or more oligonucleotides of the invention in the range of 0.1 mM to 100 M, preferably An ointment or lotion containing the oligonucleotide AS1 is applied topically once a day. Alternatively, injection may be by injection into or near the rash, preferably in the form of wet injection using a solution containing the nucleic acid of the present invention in combination with a suitable pharmacological carrier.

For systemic treatment, such as systemic treatment of chronic intestinal inflammatory disease, the nucleic acids of the present invention, together with a suitable pharmacological carrier, are preferably administered 1-6 times per day, 100 μl per dose.
parenteral or oral administration at doses of g to 10 g and treating until the acute symptoms are suppressed. In order to prevent or maintain the condition, treatment with 1-2 doses of the nucleic acid of the invention per day is preferred. However, other modes of administration are also possible depending on the course of the disease. Thus, the nucleic acids of the invention, preferably as phosphorthioate oligonucleotides, are administered for 5-10 days at a dose of 0.05-0.5 mg / k of body weight.
It may be administered systemically as a continuous infusion of g / h. Another administration method includes continuous infusion over 2-8 days with oligonucleotides 0.2～6Mg / body surface area m ² / day. Continuous infusion may be associated with systemic immunomodulation and CD30 ⁺ and / or
Preferred for disseminated benign and malignant growth of pp90rsk3 ⁺ cells.

The present invention also relates to biological fluids and biopsates, for example, purging bone marrow transplants.
Ex vivo treatment method. In this method, the explant is incubated in the presence of a nucleic acid of the invention. The nucleic acid incubation is preferably performed at a dose of 10 μM to 10 mM for 1-2 days. A preferred nucleic acid for the present method is oligonucleotide AS1.

The following examples further illustrate the present invention.

EXAMPLES Example 1 Specific Suppression of CD30 and pp90rsk3 Expression in Jurkat Cells Jurkat cells (ATCC TIB-152) (CD30 ⁺ , pp90rsk3 ⁺ ) were prepared using the following oligos in parallel batches (batches AF): Density of 5 × 10 ⁴ cells / mL of RPMI 1640 culture medium (Gibco BRL) in 10% (v / v) fetal calf serum (Gibco BRL) in the presence of nucleotides (40 μM each), ₂ at 37 ° C., 5% CO 2 Incubated for days.

In the next Western blot analysis, CD30 (120 k
D), pp90rsk1 (90 kD), pp90rsk3 (90 kD), actin (48 kD) were detected: anti-CD30 antibody HRS4 (1: 2,000) (Coulter Immunotech; Cat No. 0705) anti-rsk1 antibody C-21 (1: 1,000) (Santa Cruz Biotechnology; Cat No. sc-231) Anti-rsk3 antibody C-20 (1: 1,000) (Santa Cruz Biotechnology; Cat No. sc-1431) Anti-actin antibody (1: 2,000) (Calbiochem, Cat No.CP01 )

Each band of the Western blot was measured with a densitometer, and the signal intensity of each band in Batch F was set to 1.

[Table 3] Table 3 nt not tested

Results 1. Oligonucleotides (SEQ ID NOs: 5, 9 and 10) suppress CD30 protein expression. 2. Oligonucleotide (SEQ ID NO: 15) did not affect CD30 expression
Suppresses sk3 expression. 3. Oligonucleotide (SEQ ID NO: 9) was used for CD3 without affecting pp90rsk3
0 expression is suppressed. 4. The activity of the tested oligonucleotides is specific since the expression of pp90rsk1 and actin does not change.

Example 2: In the presence of an oligonucleotide (SEQ ID NO: 5), lymphocytes secrete an altered spectrum of cytokines. Lymphocytes were isolated from peripheral blood of healthy donors by density gradient centrifugation and immobilized on an immobilized anti-CD3 antibody (OKT3, 1 μg / mL) and an anti-CD28 antibody (15E).
6,1 μg / mL) for 10 days (v / v
) 1 mL of RPMI1640 culture medium (Gibco BRL) of fetal bovine serum (Gibco BRL)
CD6 ⁺ cells were induced at a density of 10 ⁶ cells per cell. At the same time, the cells were incubated in a parallel batch with the following oligonucleotides (40 μM each) at 37 ° C., 5%
It was incubated in CO _2.

Then, using an ELISA test (Biotrend, Cat. No. CY-1IA35-K, CYTO 07), interleukin 10 (IL-10) and interferon-gamma (IFN-γ) in the cell-free culture supernatant were used. ) Was measured.

Table 4: Table 10: Concentration of IL-10 and IFN-γ in culture supernatant

Results The oligonucleotide (SEQ ID NO: 5) suppresses IL-10 secretion of in vitro stimulated hyperactivated lymphocytes but does not affect IFN-γ secretion.

This indicates that secretion of certain cytokines was selectively regulated, resulting in altered lymphocyte reactivity (specific immunomodulation).

Example 3 Oligonucleotide (SEQ ID NO: 5) Suppresses NFκB Activation in Lymphocytes Lymphocytes were isolated from peripheral blood of healthy donors as described in Example 2 and immobilized anti-CD28 antibody (15E6, 1 μg / mL) and anti-CD3 antibody (OKT3,1).
(g / mL) at 37 ° C., 5% CO ₂ for 2 days, then in parallel batches (AD) at a density of 10 ⁶ cells / ml in the presence of the following oligonucleotides (40 μM each): Incubated for a further 2 days:

According to the method described in Naumann and Scheidereit, EMBO J. 13, 4597-4607, 1994, the NFκB-binding double-stranded DNA sequence 5′AGTTGAGGGGAC
DNA binding activity of NFκB in cell extracts was measured by electrophoretic mobility shift assay (EMSA) using TTTCCCAGGC3 ′ (Santa Cruz Biotechnology, Cat. No. sc-2505; SEQ ID NO: 19). Anti-p65 antibody C-20G (
Santa Cruz Biotechnology, Cat. No. sc-372X) and anti-p50 antibody C-19 (
"Super shift" using Santa Cruz Biotechnology, Cat. No. sc-1190X)
And blocking peptides (Santa Cruz Biotechnology, Cat. No. sc-372P,
sc-1190P) to establish the identity of the DNA binding protein.

[0046]

Table 5: DNA binding of NFκB in EMSA analysis -No detectable DNA binding + DNA binding

Results The activated DNA-bound forms of p65 and p50NFκB were labeled with oligonucleotides (
(SEQ ID NO: 5) and the oligonucleotide (SEQ ID NO: 10).

Since the active form of NFκB is required in an essential step of cell activation, this result indicates that cell activation is a central step in the presence of oligonucleotides (SEQ ID NOs: 5 and 10). Indicates that it was interrupted.

Example 4: Selective suppression of proliferation of CD30 ⁺ and / or pp90rsk3 ⁺ cells Human normal fibroblasts were obtained from skin biopsies in 10% fetal calf serum (Gibco BRL) in DME medium (Gibco BRL). And cultured. Cell lines Jurkat, Molt-4 and Ra
ji cells were cultured in RPMI 1640 medium (Gibco BRL) containing 10% fetal bovine serum. The cells were placed in parallel batches (AE) at a density of 5 × 10 ⁴ cells / mL,
Incubated with the following oligonucleotides (40 μM each) for 4 days at 37 ° C., 5% CO ₂ :

On the fourth day, Cell Proliferation Kit II (XTT Test) (Boehringer Mannheim,
Cat. No. 1 465 015) was used to measure cell proliferation. The measured value of the batch E (absorbance A ₄₉₂ nm-A ₆₉₀ nm) was set to 1, and the measured values of the batches A to D were standardized correspondingly.

The expression of CD30 in the test cells was measured using the antibody HRS4 (Coulter Immunotech, Cat. No. 0).
705) by FACS analysis (FACScan, Becton Dickinson).
pp90rsk3 expression was determined by anti-rsk3 antibody C-20 (Santa Cruz Biotechnology, Cat.
sc-1431) was determined by Western blot analysis.

[0052]

Table 6: Proliferation of CD30 ⁺ and / or pp90rsk3 ⁺ cells in the presence of oligonucleotides

Results 1. Jurkat cells express the CD30 antigen very strongly on their surface, but Molt-4
Cells express very weakly. For Raji cells and dermal fibroblasts, CD30 expression on their cell surface was not measured. pp90rsk3, Jurkat, M
It is expressed in olt-4 and Raji cells, but not in dermal fibroblasts.

[0054] 2. The oligonucleotide (SEQ ID NO: 5) was CD30⁺And / or pp90rsk3⁺
Suppresses cell growth but reduces CD30^-And pp90rsk3^-Does not suppress cell growth
. Double positive cells are better suppressed than single positive cells.

[0055] 3. The oligonucleotide (SEQ ID NO: 9) exclusively inhibits the growth of CD30 ⁺ cells, but not CD30 ^- cells. This suppression is independent of whether pp90rsk3 is expressed.

[0056] 4. Regardless of whether CD30 is expressed, the oligonucleotide (SEQ ID NO: 15) inhibits the growth of pp90rsk3 ⁺ cells, but not pp90rsk3 ^- cells.

[0057] 5. Cells inhibited in growth by the oligonucleotide were susceptible to cell death, whereas cells that grew uninhibited showed no signs of increased frequency of cell death.

Example 5: Induction of apoptosis of Jurkat cells Jurkat cells (10 ⁵ cells / mL) were cultured in parallel batches (AD) at 37 ° C., 5% CO 2 in the presence of the following oligonucleotides (40 μM each): ₂ for 3 days, 10
% Fetal bovine serum was incubated in RPMI 1640 medium:

One of the earliest recognized features of apoptotic cells is the presence of phosphatidylserine, which gives rise to a high binding affinity for Annexin V on the cell surface. On the third day, to detect apoptotic cells, incubate with FITC-conjugated Annexin V (Coulter Immunotech, Cat. No. 2375)
Annexin was then performed by FACS analysis (FACScan, Becton Dickinson).
The number of V-binding cells was determined. Dead cells were marked by incubation with propidium iodide and excluded from measurement.

[0060]

[Table 7] Table 7

Results The oligonucleotides (SEQ ID NOs: 5 and 10) induce cell death by apoptosis in Jurkat cells. NS oligonucleotides do not exhibit this property.

Example 6: Oligonucleotide (SEQ ID NO: 5) is CD30 in lymphocyte preparation
⁺ Selectively inhibits cell formation. As described in Example 2, resting lymphocytes from peripheral blood of healthy donors were treated with immobilized anti-CD3 and anti-CD28 antibodies at a density of 10 ⁶ cells / mL of culture medium in parallel batches (AC). Was used to stimulate CD30 expression. At the same time, the cells were incubated for 4 days at 37 ° C., 5% CO ₂ in parallel batch in the presence of oligonucleotide (40 μM each). Batch D contains lymphocytes and oligonucleotides without antibody stimulation.

Next, in FACS analysis (FACScan, Becton Dickinson), anti-CD30
The number of CD30 ⁺ cells was measured using antibody HRS4 (Coulter Immunotech, Cat. No. 0705), and FITC anti-CD69 antibody (Coulter Immunotech, Cat. No. 1943)
The number of CD69 ⁺ cells was measured using a FITC anti-CD4 antibody (Coulter Immunot).
ech, Cat. No. 6602393) measures the number of CD4 ⁺ cells was used to measure the number of CD8 ⁺ cells using PE anti-CD8 antibody (Coulter Immunotech, the Cat. No. 0452).

In parallel with these batches, Cell Proliferation Kit II (XTT Test) (Boehrin
Ger Mannheim, Cat. No. 1 465 015) was used to measure proliferation and cell activation. The measured value of batch D (absorbance A ₄₉₂ nm-A ₆₉₀ nm) is set to 1, and batches A to C
Measurements were correspondingly standardized (stimulation index).

[0065]

Table 8: FACS analysis

[0066]

Table 9: Cell proliferation assay

Results The oligonucleotide (SEQ ID NO: 5) selectively inhibits cell proliferation and cell activation and prevents the production of CD30 ⁺ cells upon stimulation with anti-CD3 and anti-CD28 antibodies.

Cells with the initial activation marker, CD69, accumulate in the presence of SEQ ID NO: 5 but do not differentiate into CD30 ⁺ cells. The number of CD4 ⁺ and CD8 ⁺ cells does not change.

Example 7: Oligonucleotide (SEQ ID NO: 5) selectively eliminates CD30 ⁺ cells in activated lymphocyte cell populations. As described in Example 2, lymphocytes from peripheral blood of a healthy donor were immobilized in parallel batches at a density of 10 ⁶ cells / mL of culture medium with immobilized anti-CD3 and anti-CD28.
The antibodies were used to stimulate CD30 ⁺ lymphocytes for 2 days (batch BD)
). After CD30 ⁺ cells had formed in these batches, the batches were further incubated for 3-5 days in the presence of oligonucleotides (40 μM each).

The anti-CD30 antibody HRS4 (Coulter Immunotech, Cat. No. 0705) and FA
The number of CD30 ⁺ cells was determined using CS analysis (FACScan, Becton Dickinson).

[0071]

[Table 10] Table 10

Results 1. Oligonucleotide (SEQ ID NO: 5) is activated CD in activated lymphocyte population
Reducing the number of 30 ⁺ lymphocytes. 2. Even with continued stimulation for differentiation into CD30 ⁺ cells, specific elimination of CD30 ⁺ occurs.

[0073]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows an accession number M8355 in the EMBL database.
The known CD30 cDNA sequence is shown under No. 4. Corresponding RNA or Genome D
The preferred binding region for NA is underlined.

FIG. 2 shows an accession number X8510 in the EMBL database.
The known rsk3 cDNA sequence is shown under No. 6. Corresponding RNA or genomic DN
The preferred binding region for A is underlined.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 105 C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), JP, US

Claims (19)

[Claims] 1. The CD30 antigen or ribosomal S6 kinase pp90rsk3 (rsk
3) A nucleic acid, a derivative thereof, and a sequence-specific binding substance (nucleic acid) that inhibit the expression of 3). 2. The nucleic acid according to claim 1, which specifically binds to CD30 mRNA, gene or promoter DNA and / or rsk3 mRNA, gene or promoter DNA. 3. The nucleic acid according to claim 1, which is an oligonucleotide or an oligonucleotide derivative, especially having 8 to 20 bases. 4. The nucleic acid according to any one of claims 1 to 3, which is selected from the following nucleic acids: (a) a nucleic acid which binds to CD30 mRNA, CD30 gene DNA or CD30 promoter DNA. (B) a nucleic acid that binds to rsk3 mRNA, rsk3 gene DNA or rsk3 promoter DNA; (c) a nucleic acid that binds to CD30 mRNA and rsk3 mRNA; (d) a nucleic acid that is a CD30 DNA and / or rsk3 DNA triple helix forming nucleic acid; (E) a nucleic acid that is a ribozyme that binds to CD30 and / or pp90rsk3 mRNA. 5. CD30 mRNA, gene DNA or promoter DN
The nucleic acid according to any one of claims 1 to 4, comprising the subsequence of A or a sequence complementary to the subsequence of the CD30 mRNA, gene DNA or promoter DNA. 6. The subsequence comprises the region bp 140 to 170, bp of SEQ ID NO: 1.
200-250, bp265-300, bp520-540, bp600-94
0, bp 1200-1270, bp 1745-1790, bp 1885-192
The nucleic acid according to claim 5, which is a fragment derived from 0 or bp 2985 to 3005 and having a length of at least 8 consecutive bases. 7. The method according to claim 1, wherein the subsequence is the region bp 140 to 170, bp
The nucleic acid according to claim 5 or 6, which is a fragment derived from 200 to 250 or bp 265 to 300 and having a length of at least 9 contiguous bases. 8. The nucleic acid of claim 7, having one of the following sequences: 5′GACGCGCATCCCCGG3 ′; 5′GACGCACAACCCCGG3 ′; 5′GGCCGGACGTGAGGT3 ′; 5′CAGCGCGGCGAGGAG3 ′; 5′CCTGTGGAGAGGCTC3 ′; 5'ATGCGCGTCTC3 '; 5'CGCGTCCTCCTCGC3'; 5'GTCCGGCCCCGGGG3 '; or 5'TGGGCGCCGCGCGCT3'. 9. The method according to claim 1, which comprises a subsequence of rsk3 mRNA, gene DNA or promoter DNA, or a sequence complementary to a subsequence of rsk3 mRNA, gene DNA or promoter DNA. The nucleic acid according to clause or more. 10. The subsequence comprises the region bp 100 to 160, b of SEQ ID NO: 3.
10. The nucleic acid according to claim 9, which is a fragment derived from p1085-1125 or bp1540-1565 and having a length of at least 8 contiguous bases. 11. The subsequence is a fragment derived from the region bp 100 to 160 of SEQ ID NO: 3 and having a length of at least 9 consecutive bases.
Or the nucleic acid according to 10. 12. The nucleic acid of claim 11, which has one of the following sequences: 5′GACGCGCATCCCCGG3 ′; 5′CATCCCAGGCGCGGG3 ′; 5′CCGGGGATGCGCGTC3 ′; 5′ATGCGCGTC3 ′; 5′GGCCGGGGGACGCGC3 ′; 'GCCGCAGGGCCGGGG3'. 13. The nucleic acid according to claim 4, which binds to CD30 mRNA and rsk3 mRNA. 14. The nucleic acid of claim 13, having the sequence: 5′GACGCGCATCCCCGG3 ′. 15. The nucleic acid according to claim 4, which is a CD30 DNA and / or rsk3 DNA triple helix forming nucleic acid. 16. The sequence: 5'CCGGGGATGCGCGTCTC3 '; 5'ATGCGCGTC3'; 5'CGCGTCCTCCTCGC3 '; 5'GTCCGGCCCCGGGG3'; or 5'TGGGCGCCGCGCGCT3 '. 16. The nucleic acid according to claim 15, having the formula: 17. A pharmaceutical composition comprising one or more nucleic acids as defined in claims 1-16. 18. Use of a nucleic acid as defined in claims 1 to 16 for the manufacture of a medicament for modulating the functional activity of CD30 ⁺ and / or pp90rsk3 ⁺ cells. 19. Use of a nucleic acid as defined in claims 1 to 16 for the manufacture of an agent that functionally suppresses and eliminates CD30 ⁺ and / or pp90rsk3 ⁺ cells.