JPH08245414A - Composition and method for medical treatment of il-6 related disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical compositions for treatment of disorders relating to the increase in cytokine levels and therapy therefor and a pharmaceutical composition for leukin-6-related diseases. SOLUTION: This is a pharmaceutical composition for treatment of disorder characterized by the increase of interleukin-6 level. A pharmaceutically acceptable carrier includes an effective amount of the composition enough to decrease the interleukin-6 level. This composition is the steilized culture medium in which transformed B-cells secret immune modulator or a composition comprising the purified fraction having IL-6 inhibitory activity, and the production process thereof.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to methods and compositions for the treatment of various disorders characterized by elevated levels of IL-6 and is based on substances derived from transformed B cell lines.

[0002]

BACKGROUND OF THE INVENTION This application is directed to US application no. 0, filed January 27, 1995, entitled "Methods for treating myasthenia gravis".
No. 8 / 379,960, and US Application No. 9/1993, entitled "Compositions and Methods for Treatment of Cancer and Autoimmune Diseases," by Thomas M. Rodgers Jr. and T. Ronald Theodore. It is a partially pending application of 08 / 089,751.

Since the early 1970s, new cancer treatments have
It has been developed based on lymphokines and modifiers of lymphokine activity. Early researchers began to test the size-filtered fraction of cell supernatants and found that certain cytokines have the ability to influence tumor activity and act on the hematopoietic system. . For example, McDaniel, MC et al., Clin.Immunol.Immunopathol.,
91104 (1976); and Papermaster, BW et al., Clin. Im.
munol. Immunopathol., 5: 48-59 (1976). The methods used include size filtration as well as lyophilization, chromatographic separation, and extraction or reconstitution in various buffers. Studies were then conducted focusing on the identification of individual components of these supernatants. That is, for example, Papermaster, B. et al., Advances In I.
mmunopharmacology, pp. 507-511 (1981); Dunn, PA
Et al., J. Immunol. Methods 64: 71-83 (1983); Papermast.
er, BW et al., Cancer 45: 1248-1253 (1980).

Many cytokines are now purified, cloned and expressed in recombinant hosts. However, with some success in medical trials, effective doses are often toxic, so most recent efforts have focused on the search for inhibitors of cytokines.

[0005]

Accordingly, it is an object of the present invention to provide compositions and methods for the treatment of disorders associated with elevated cytokine levels.

It is a further object of the present invention that IL-
It is to provide compositions and methods for treating disorders associated with elevated 6 levels.

[0007]

The transformed B cell line secretes an inhibitor of IL-6 into human serum culture medium under controlled conditions. This cell culture medium, or IL
The purified fraction, which has -6 inhibitory activity, is associated with disorders characterized by elevated levels of IL-6, in particular certain types of cancer,
It can be administered to treat patients with autoimmune disorders, pain associated with these disorders, side effects resulting from chemotherapy, and wasting or other symptoms of certain viral diseases. A preferred cell line is RPMI 17 obtained from the American Type Culture Collection.
88, but other B cell lines can also be used as a source of active material.

The present invention provides a method for preparing a cytokine-containing composition from hematopoietic cells for administration to a subject, which method comprises the steps of: a) the cells on fresh medium. B) collecting the cell culture supernatant without filtration of the supernatant; and c) maintaining the supernatant until administration, wherein the maintenance supernatant is It is a composition containing a cytokine.

In a preferred embodiment, the culturing step comprises:
It further includes activating the production of cytokines by said cells.

In a preferred embodiment, the cultured cells are
It is an immunostimulatory cell.

In a preferred embodiment, the method of the present invention comprises:
This is a method in which the cultured cells are previously exposed to a cancer-related virus.

[0012] In a further preferred embodiment, the virus is Epstein-Barr virus.

In a preferred embodiment, the supernatant is about-
Maintained by flash freezing to temperatures below 40 ° C.

In a more preferred embodiment, the frozen supernatant is maintained for less than about 3 months.

In another preferred embodiment, the supernatant is
Not freeze-dried while maintained.

In another preferred embodiment, the supernatant is
Maintained without buffer.

In a preferred embodiment, the medium is human serum albumin.

In a preferred embodiment, the cells are IgM
It is a secretory cell.

In a preferred embodiment, the cells are RP
A cell line called MI1788 with an accession number CLL
It has been deposited with the American Type Culture Collection at 166.

Furthermore, the present invention also provides a cytokine-containing composition produced by the above method.

The present invention provides a composition for treating a disorder characterized by elevated IL-6, the composition comprising IL-6 in a pharmaceutically acceptable carrier.
Included in the composition is an amount effective to reduce levels. Here, the composition consists of a sterile culture medium in which transformed B cells secrete an immune modulator or a purified fraction thereof having IL-6 inhibitory activity.

In a preferred embodiment, the composition corresponds to a dosage of less than 50 ml of culture medium given twice daily.

In a preferred embodiment, the disorder is IL
A cancer characterized by elevated levels of -6.

[0024] In a preferred embodiment, the disorder is an autoimmune disorder.

In a preferred embodiment, the disorder is a symptom resulting from treatment with a chemotherapeutic agent.

In a preferred embodiment, the disorder is pain associated with cancer or an autoimmune disorder.

In a preferred embodiment, the disorders include: multiple myeloma (mm); prostate cancer; ovarian cancer; Castleman's disease; Kaposi's sarcoma (KS); monoclonal hypergammaglobulinemia; Mesangial proliferative glomerulonephritis; rheumatoid arthritis and other arthritis (RA); systemic lupus erythematosus (SLE); multiple sclerosis (MS); myasthenia gravis (MG); Guean-Barre syndrome (GB)
S); toxic shock syndrome; bacterial and viral meningitis; HTLV-1-related myelopathy (HAM); Alzheimer's disease; osteoporosis (osteoclast disease); cardiac myxoma and hypercalcemia; allergies; Nonclassifiable immunodeficiency; Anorexia Nervosa; Inflammatory Bowel Disease; Critical Disease Polyneuropathy; Acute pyelonephritis; Autoimmune hepatitis; Large cell lymphoma; Alveolar echinococcosis; Endometriosis; Atherosclerosis Sclerosis; psoriasis; human parvovirus infection;
Renal cell carcinoma; preeclampsia; cholangiocarcinoma; HIV; acoustic neuroma; hairy cell leukemia; necrotizing enteritis; ulcerative colitis; bronchopulmonary insufficiency; schizophrenia and affective disorder; retinoblastoma; subcerebral Pituitary tumor; acute pyelonephritis; thyroid cancer; paraganglioma; Candida albicans infection; giant cell arteritis; psoriasis; melanoma; chronic pancreatitis; carpal tunnel syndrome; interstitial cystitis: astrocytes Tumor; respiratory syncytial virus infection; liver transplantation: amenindiomas; gastric cancer; sarcoidosis; uterine cancer; large cell lung cancer; Helicobacter pylori infection; ductal breast cancer; granulomas of giant cell arteritis; colorectal cancer; Clinical chorioamnionitis; non-Hodgkin's lymphoma; thyrotoxicosis; osteosarcoma; malignant mesothelioma;
Typhoid fever; IgA nephropathy; acute pancreatitis; Fanconi anemia;
Paraneoplastic thrombocytosis; and cystic fibrosis.

[0028] In a preferred embodiment, in the above composition, the cell line is transformed by infection with Epstein-Barr virus or a part thereof.

In a preferred embodiment, in the above composition, the cell line is RPMI1788 deposited at the American Type Cell Culture under accession number CLL156.

In a preferred embodiment, the cell culture medium is human serum.

In a preferred embodiment, the cell culture medium is human albumin.

In a preferred embodiment, the composition is purified on the basis of molecular weight and ion exchange chromatography.

The therapeutic effects on cancer patients and autoimmune disorders will be described in Examples.

[0034]

BEST MODE FOR CARRYING OUT THE INVENTION

I. Pharmaceutical Composition As used herein, “pharmaceutical composition” includes cell secretion-containing compositions. This composition is produced by B lymphoid cells, induced by transforming factors such as infection with Epstein-Barr virus, and cultivated in human albumin or serum-based culture medium or synthetic medium and centrifugation of the cell culture medium. Obtained by separation or filtration or in purified form.

(Cells) The cells that can be cultured to produce the pharmaceutical composition are primarily hematopoietic cells, especially human B lymphoid cell lines transformed with Epstein-Barr virus. The most preferred cell line is the American Type Cell Culture Collection with accession number CLL156.
RPMI 1788, which has been previously exposed to Epstein-Barr virus. This cell line is Epstein-Barr virus nuclear antigen positive, as does approximately 60% of the human population. The cell culture medium containing secretions from these cells, referred to herein as "LK-200", the secretions from only one cell line, ATCC CLL156, are generally clinically tested.

Other cell lines which can be used are IB4, Ra
EBV transformed cell lines such as
rkett lymphoma cell line, and non-EB such as Rjab
V-infected B cell line. These cell lines are commercially available from ATCC or other sources (eg Tufts New England Medical Ce).
nter, Boston, Massachusetts).

Reagents that can be used for the transformation of (inducer) cells include viruses such as Epstein-Barr virus. Inducers can be used alone in some cases or in combination with transforming agents, including tumor necrosis factor (TNF), endotoxin, and other factors known to those of skill in the art. Generally, the cultured cells are in an amount effective to activate the cells, as measured by cytokines, immunoglobulin secretion, and / or other indicators such as amplification or modification of cell surface properties or markers. Be exposed to. In some cases, the cells are first exposed to a small amount to "prime" the cells, and then to the next dose to induce greater activation of the cells. As mentioned above, these techniques and materials are published in the literature. This inducer causes the medium to be decanted when it is replaced, i.e. either by adding cells in small increments or by filtering or centrifuging, and then adding fresh medium. Are removed from the cell culture medium when replaced with.

(Cell culture medium) The cells are preferably cultured in a medium that can be directly administered to a human patient without inducing a reaction in the medium. In a preferred embodiment, the medium is, for example, Bio Whittaker, Inc., Walkersville, MD or Gibc.
o Based on 2% AB human serum obtained from Life Technologies, Inc., Grand Island, NY, albumin
It can be replaced with serum just prior to collection of cell culture medium. Human serum is Iscove's Modified Dulbecco's Medium (IMDM) containing sodium bicarbonate at a concentration of 2% L-glutamine.
And in 25 mM HEPES. This IMD
M and L-glutamine can also be obtained from Bio Whittaker.

Serum source affects cytokine levels in the final product, which is relatively negligible. For example, Bio Whitta after cell removal
The level of IL-8 in ker serum medium (regardless of cell depletion method, called "supernatant") was 0.367 ± 0.
17 ng IL-8 / ml, and the level of IL-8 in Gibco serum medium was 0.184 ± 0.1 n
g IL-8 / ml; level of TNF in Bio Whittaker serum medium was 0.311 ± 0.24 ng
TNF / ml and TNF in Gibco serum medium
Level of 0.179 ± 0.07 ng TNF / ml
Met. The cells, to some extent, grow and secrete the active ingredient in albumin.

It has been shown that the blood group of the serum source can elicit a response in some patients with different blood groups. Therefore, it is preferred, but not critical, to utilize a serum source of the same blood type as the patient.

(Culture conditions) Cells were 1 × 10 ⁶ cells / m 2.
Incubate at 37 ° C until a minimum density of 1 is reached. The pH at the time of collection is about 7.00 ± 0.15.

For example, RPMI1 obtained from ATCC
788 cells were lysed in a 37 ° C. water bath and 2% glutamine and 2-5% normal human serum (Gibco)
Or containing 0.2 micron filtered HEPES supplemented with 10-25% human albumin (Gibco)
Iscov medium (GIBCO BRL Life Technologies, Inc., Gaithe
(obtained from rsburg, MD) at room temperature. 1 x 1 cell
0 ⁶ until a cell density of greater than cells / ml, T75
Incubate at 37 ° C. in a flask in a 5% CO ₂ atmosphere. The cells may then be removed by centrifugation or filtration at 800 xg for 5 minutes, and the total volume in a T150 flask is 0.2 x 10 ⁶ cells / m 2 in 200 ml medium.
It can be suspended at a density of l. The cells are then cultivated again until a density of 1 × 10 ⁶ cells / ml is reached and the process is repeated.

Then, the cells were mixed with 0.2 × 10 ⁶ cells / m 2.
In the initial cell density of l, until a density of at least 1 × 10 ⁶ cells / ml, flushed with 10% CO _2, and may be cultured in roller bottles sealed 500 ml.

(Analysis of cell surface phenotype and cytokine level) RPMI 1788 (LK200 herein)
, And other cell lines: IB
4, Raji, Bjab, and Mitogen, PHA
Peripheral blood cells (PBC) that are activated by exposure to
And control: 2% human serum medium not exposed to cells, 10% fetal calf serum not exposed to cells (F
CS) medium, and supernatant of T cell line OKT3. The results are shown in Tables 1 to 3. Table 1 shows that RPMI1788 expresses lambda light chain for only one week. RPMI 1788 cells labeled "ATCC" (probably not growing in vitro since the initial purchase of this line) are significantly positive for this antigen. For unknown reasons, the RPMI1788 strain does not express the "naive" CD45 isoform found in other strains.
However, this strain is reactive with Becton Dickinson HLe-1 mAb. According to Dr. Michel Streuli (DFCI, Boston), an expert in alternative splice variants of CD45, this strain expresses isoforms containing only B or the substances encoded by the B and C exons.
Regardless of the structure of this isoform, the importance of the variant is unclear.

[0045]

[Table 1]

The (+) and (-) marks are 10 respectively.
Expression of 0% and 0% is shown. RPMI1788 and IB4 cell lines are EBV + LCL. Raji is
Burkitt lymphoma, and Bjab is a non-EBV infected B cell line. All mAbs used in these FACS analyzes were FITC except anti-CD21.
It was a complex antibody. Unconjugated anti-CD21 mAb is a CD
21 anti-murine IgG-FI to detect surface expression
Used with TC secondary antibody.

[0047]

[Table 2]

* IL-1β, TNF, IL-6, IL
-10, IL-1RA, soluble IL-1 receptor type II (sIL-1RII), and IL-8 were assayed by RIA. IL-2, IL-4, GM-CS
F, and IFN-γ were assayed by ELISA using a kit from Endogen. IL-12 is
A similar assay was performed using a kit from R & D Systems.

[0049]

[Table 3]

*> Highest concentration used to generate the standard curve.

[0051]

[Table 4]

The results shown in Tables 2, 3 and 4 are L
K200 and RPMI1788 supernatants have moderate amounts of IL
It is described to contain -8 (unchanged below 135 pg / ml) and a modest amount of TNF (below 250 pg / ml). The supernatant from the Bjab strain contains a large amount of IL-10.
(920 pg / ml). This is one EBV non-infectious studied B cell line. The IB4 strain is
Type II IL-1 receptor (1,100 pg / m
l) shed. The Bjab strain has an appropriate amount (227
pg / ml) of IL-1RA.

[0053]

[Table 5]

* PBMC for this experiment (5/9/95)
The provider of this was Damien Sorce. For each condition tested, 12.5 × 10 ⁶ cells were incubated in a total volume of 5 ml in a 50 ml polypropylene centrifuge tube previously coated with human albumin (10
μg / ml × 1 hour). Cells were RPMI supplemented with 2% human AB serum and 4 μg / ml polymyxin B.
Cultured in medium. After 48 hours at 37 ° C, to ensure equilibration of secreted cytokines throughout the culture medium,
PBMCs were vortexed briefly and then pelleted by centrifugation. For future analysis of the supernatant,
Removed under sterile conditions and frozen. IL-1β,
TNF, IL-6, IL-10, IL-1RA, soluble IL-1 receptor type II (sIL-1RII), and IL-8 were assayed by RIA. IL-2,
IL-4, GM-CSF, and IFN-γ are Endoge
Assayed by ELISA using the kit from n. IL-12 was assayed similarly using a kit from R & D Systems. * Refers to the final dilution of culture medium.

[0055]

[Table 6]

* Refers to the final dilution of culture medium. **> highest concentration used to generate the standard curve.

[0057]

[Table 7]

As shown in Tables 5, 6, and 7, the various LK200 preparations alone did not elicit PBMCs producing adequate amounts of any cytokine, but low levels of anti-TCR mAB OKT3. The evoked effect at the concentration was strengthened. In particular, these preparations enhanced the production of IL-1β. This was more than 10 times the production induced by OKT3 alone. TNF production increased by 250%,
And the production of IL-8 was increased by 50%. The production of IL-10 was doubled. OKT3 alone produces 2 pg / ml of GM-CSF, and OKT3 + LK200 (1: 6
Dilution) increased to 19 pl / ml.

The 2% human AB serum used to generate LK200 itself induces high amounts of IL-6, IL-8, and IL-1RA, probably due to the FcR signal. Supernatants of LK200 and RPMI1788 significantly suppress this effect. Especially, the production of IL-6 is 80%.
Greater reduced. IL-8 production was reduced by approximately 40%.

These results indicate that the composition did not act only as an inhibitor, but was diluted to lower concentrations, probably as a result of inhibition or competitive interaction with other components of the composition. It is shown to be more effective in some cases.

(Pharmaceutically Acceptable Carrier) The pharmaceutical composition may consist of the culture medium alone, or deionized water or normal saline (0.15N NaCl).
Or it may be diluted with other physiological buffers. Care must be taken not to change the pH. The medium should not be diluted until just prior to administration to the patient.

Treatment of Cell Culture Medium In a preferred embodiment, cells are removed from the culture medium and a supernatant containing the biologically effective molecule is obtained. Cells can be removed by centrifugation or filtration. Typically, this medium is
Recovered from roller bottles by decantation, pipetting, filtration, or centrifugation, then placing the medium in a sterile syringe. This material can be further processed based on separation by molecular weight, chromatography on ion exchange resins, and other methods known to those of skill in the art, where the active fraction is due to inhibition of IL-6 activity. Is defined.

(Sterilization) By culturing cells under standard sterilization conditions and removing the cell culture medium under aseptic conditions,
Be sure to be sterile. Alternatively, the cell culture medium does not exclude molecules of less than 250,000 daltons, more preferably less than 150,000 daltons,
It can be purified by filtration, for example using a 0.25 micron filter.

The (preserved) supernatant, once removed from cell culture bottles in a 10% nitrogen atmosphere at a temperature of 37 ° C. and separated from the cells, lost any activity for at least 2-4 hours at room temperature. Can be maintained without. For long-term storage, culture medium in liquid nitrogen, on dry ice,
Or in an ultrafreezer, about -40 ℃ to -80 ℃
It must be frozen quickly at the temperature.

(Analysis of pharmaceutical composition) 45 sets of LK-2
00 was assayed for various cytokines and concentrations are given in the nanogram range per milliliter range, as shown in Table 2. All sets showed essentially no change from batch to batch. There is some direct relationship between cytokine levels and human serum concentrations in the medium.
Endotoxin levels in the supernatant were consistently 10 pg endotoxin / m
It is measured at 1 or less. Generally, levels up to 10 ng can be administered to patients.

II. Treatment Method (Disorders that can be treated) Interleukin-6 (IL-
6) is a pluripotent cytokine that affects a wide variety of different cell types. It is generally recognized as a B cell growth factor and was initially identified as an inducer of immunoglobulin secretion in Epstein-Barr virus (EBV) transformed CESS lymphoblastoid strains.
Although classified as an acute phase protein, its role exhibits many aspects and is still somewhat obscure. Apparently, this is an important cytokine believed to be deeply involved in some lesions.

The receptor for IL-6 is gp13O
Is. It is a transmembrane transduction receptor, which is located across the cell membrane and signals the cell once the ligand binds. There are other cytokines that share the gp13O receptor with IL-6 (leukocyte inhibitory factor (LIF), oncostatin M (OS
M), IL-11, and ciliary neurotropic factor (CNT)
F) is included).

Inhibitors of IL-6 are retinoic acid and several steroids (dexamethasone). Soluble gp13O was considered to be inhibitory to transient IL-6 activity. However, IL-6 and soluble g
Binding to p13O rekindles the pathological effects, especially osteoclastogenesis. In several diseases, such as multiple myeloma and Kaposi's sarcoma, IL-6 is believed to play an autocrine or paracrine role. In these cases, IL-6 is produced in both and then used by the same or in close proximity to cells to perpetuate the disease.

Pathologies associated with IL-6 include: multiple myeloma (mm); prostate cancer; ovarian cancer; Castleman's disease; Kaposi's sarcoma (KS); monoclonal hypergammaglobulinemia. Mesangial proliferative glomerulonephritis; rheumatoid arthritis and other arthritis (RA); systemic lupus erythematosus (SLE); multiple sclerosis (MS); myasthenia gravis (MG); Guian-Barre syndrome (GBS) );
Toxic shock syndrome; bacterial and viral meningitis; HTLV-1-related myelopathy (HAM); Alzheimer's disease; osteoporosis (osteoclast disease); cardiac myxoma and hypercalcemia. Additional pathologies that may be associated with IL-6 include: allergy; unclassified immunodeficiency; anorexia nervosa; inflammatory bowel disease; critical illness Critical illness polyneuromyopathy; acute pyelonephritis Autoimmune hepatitis; large cell lymphoma; alveolar echinococcosis; endometriosis; atherosclerosis; psoriasis; human parvovirus infection; renal cell carcinoma; preeclampsia; cholangiocarcinoma; HIV; acoustic neuroma; Hairy cell leukemia;
Necrotizing enteritis; Ulcerative colitis; Bronchopulmonary insufficiency; Schizophrenia and affective disorders; Retinoblastoma; Pituitary tumors; Acute pyelonephritis; Thyroid cancer; Paraganglioma; Candida albicans infection; Giant cell arteries Inflammation; psoriasis; melanoma; chronic pancreatitis; carpal tunnel syndrome; interstitial cystitis: astrocytoma; respiratory syncytial virus infection; liver transplantation: amenindiomas (Am
gastric cancer; sarcoidosis; uterine cancer; large cell lung cancer;
Helicobacter pylori infection; ductal breast cancer; giant cell arteritis granulomas; colorectal cancer; clinical chorioamnionitis; non-Hodgkin lymphoma; thyrotoxicosis; osteosarcoma; malignant mesothelioma; typhoid fever; IgA nephropathy; Acute pancreatitis; Fanconi anemia; Paraneoplastic thrombocytosis; and cystic fibrosis.

Dosage and Dosing Schedule Dosing and dosing schedules are very personal and include relief of clinical symptoms (eg, reduction of tumor volume, loss of bone pain, and other subjective or objective). Individual), and is optimized individually.

Cancer and Viral Diseases In treating cancer or viral infections, a number of different criteria can be used as an indication of efficacy. For example, the size of a tumor is
It can be monitored by standard tumor detection measures such as CAT scanning (Computerized Body Tomography), MRI (Magnetic Resonance Imaging), and Nuclear Medicine scans known to those skilled in the art. The progression and involvement of lesions caused by the virus or the overall anatomical relationship of the disease, which is used herein as an indicator of disease progression, depends on the particular lesion type depending on the particular disease. For each, it can be determined by standard methods known in the art (eg organ function or antibody titer). About the treatment of viral infections,
The effect can be measured as the degree and duration of symptoms reduced by the present invention. This includes measurements of decreased pathological and pathophysiological activity, such as increased liver function, increased bilirubin levels, hypertrophy of liver size,
And enlargement of the spleen may be included.

Reducing Chemotherapy Pain and Side Effects The composition is also effective in reducing side effects of chemotherapy and radiation therapy. Side effects that can be reduced include nausea, vomiting, and hair loss. Pain is also often reduced. Pain relief includes pain relief by reducing the size of the tumor or the space occupied by the lesion, and thus the compression of organ pressure and anatomical structures (ie, nerves, blood vessels, and other organs), and Includes pain relief that is not related to tumor size reduction or focus reduction (eg, bone pain and other pain relief that occurs before or when the tumor is significantly reduced in size) .

Treatment of Autoimmune Disorders Autoimmunity is described as a mount immune response against self-ingredients, which ultimately leads to pathogenic consequences. Diseases resulting from autoimmune responses appear widespread and variable clinically. One of the common factors common to many of these diseases overall is that the etiological factors, or events that trigger the generation of these aberrant responses, are unknown. Rheumatic diseases include a number of and broad spectrum various autoimmune diseases, such as rheumatoid arthritis, scleroderma, dermatomyositis, polymyositis, discoid lupus erythematosus, Sjogren's syndrome, And systemic lupus erythematosus. For most of this, the etiological and pathogenic mechanisms of these disorders remain unknown. A common theme in many of these diseases is the presence of significant amounts of antibodies that are immunoreactive with self-components. One example of such rheumatic disease is systemic lupus erythematosus. A feature common to many of these patients is the presence of high titers of autoantibodies in their serum. These autoantibodies are associated with myriad host components (eg, ribonucleoproteins (Sm, nR
NP, Ro, and La), DNA, RNA, histones, erythrocytes, and immunoglobulins, and other characterized or uncharacterized autoantigens)
Can be directional to. Myasthenia gravis is an etiologically unknown disease and is characterized by circulating antibodies to acetylcholine receptors. The disease is indicated by muscle weakness that is biased to the eyes and other head muscles. It tends to rock to a severe degree. There are no signs of neurological lesions. The disease is believed to have immunological principles, and anticholinergic antibodies that bind to the acetylcholine receptor are found in most patients with the disease. The patient may also receive anti-muscle antibodies. Anticholinergic antibodies effectively reduce many functional acetylcholine receptors. It is also believed that the immune activity of the cells to the receptor was found. the patient,
It presents with generalized weakness that can sway, most commonly with movement of voluntary muscles. Symptoms such as diplopia, ptosis, and aphasia are noted. Activity increases the weakness of the affected muscles. The effect may be indicated by a decrease in autoantibody levels, or a decrease in the severity of symptoms. For example, the effect may be demonstrated by recovery in large and proximal muscle dysfunction, reduced anti-acetylcholine activity, reduced anti-smooth muscle antibody levels, and dysphagia and aphasia recovery.

(Dose) The general administration method and dose schedule of LK-200 are as follows. Frozen LK-200 is thawed within 2 hours without overheating or mechanical stirring. Then 50
cc of LK-200 is mixed with 50-100 cc of normal saline. In some cases, depending on the disease or response, 100-200 cc of LK-200 is 50-10.
It can be mixed with 0 cc of normal sterile saline. Generally, 50 cc mixed with 50-100 cc of normal sterile saline is administered once daily for the first 10-14 days, then on a schedule of 3 times a week.
Depending on the situation, up to 200 cc of LK-200 mixed with 50-100 cc of normal sterile saline can be administered daily 4
It is administered once. This mixture is infused intravenously over 5 to 45 minutes, depending on the total volume to be administered. Lower rates may be used if the patient is at risk of cardiac decompensation.

As shown by the data in Tables 5-7 and preliminary clinical data, the dose of cell culture supernatant administered 3-5 times per week was 50 ml to 10 or 2
The effect is enhanced by decreasing to 5 ml,
There is a negative dose dependency of the inhibitor of IL-6 in the composition.

Intravenous administration can be performed from the peripheral vein by conventional intravenous injection. In some cases, the patient is Hieman
It has a dedicated inlet that can be used for injection such as a catheter. This includes certain subclavian transcatheter catheters used for chemotherapy and / or nutritional supervision or other applications that accept the use of these catheters. L
K-200 can also be administered by direct intra-arterial infusion or by intraperitoneal infusion. In some cases, portable infusion pumps are preferred.

The dosage can be varied from 1 to 4 times per day, or can be reduced every other day or up to 2 or 3 times per week. A typical dosage is 0.1c
between c / day and 100 cc / day, about 1 on average
0-50 cc is administered once or twice daily or every other day.

Of course, the dosages provided above are
It is based on the administration of cell culture medium. Therefore, when using the purified fraction, the dosage is adjusted appropriately.

The invention will be further understood by reference to the following non-limiting examples.

[0080]

【Example】

Example 1: Treatment of Cancer Patients Day 1-7: Day 1 in 100 ml normal saline.
Single dose of 10-50 ml LK200 for 15-30
It was administered intravenously over a period of minutes.

Day 8 and later: 10 to 50 ml of LK200
Was administered 2-3 times a week.

It has been found that any increase in dose is better achieved by increasing the number of doses per day, rather than increasing the dose per dose, if the tumor response is not sufficient. . No adverse effects were observed with the addition of medication.

Patient Status Upon administration of the first dose of LK200, the patient immediately received the following effects: about 2
Within 4-48 hours, the tumors detailed in Table 8 immediately shrank, in addition to pain relief; increased appetite; increased physical fitness, stopped physical exhaustion; increased good sleep.

Data from patients treated with LK200 are listed in Table 8. This section details the reduction of specific tumors or lesions associated with the listed diseases.
Tumors and lesions were measured by CAT scan, MRI, and visual inspection.

[0085]

[Table 8]

Patient data is supplemented below.

Patient A112 (Bone and Liver Metastatic Malignant Melanoma) Pre-treatment: Extensive Bone and Liver Disease Bone Severe Pain Fast Treatment: Painless standard therapeutic function, play with alkaline phosphatase and BUN One week after the elevation: Yes; creatinine remained essentially normal; sternal lesions resolved 60% (approximate); approximately 50% of the liver was cleared.

Results of the analysis: The family decided to stop treatment. The patient was considered to be in the tumor necrosis group. Dialysis was not attempted, as determined by the family.

[0089]

Patient V103 (Psoriasis): Slowly reduced discomfort to treatment; early response with reduced red and hardened macula; Palmer lesions disappeared; patient may stop cortisone treatment for several days , And currently use 25% of the original dose.

Patient V105 (Kaposi's sarcoma): One month after treatment, the largest lesions disappeared and the four still-associated lesions were reduced by 30%. The treatment was stopped after 1 month. All lesions disappeared.

Patient R101 (Kaposi's sarcoma): Previous record: HIV immunocompromised man; CD4 <25; previous treatment with interferon 3 times per week for 1 year I couldn't see it.

Standard treatment reduced large lesions to 40-50%; some flat skin after 8 weeks: 60% reduction in skin lesions.

Example 2: Treatment of patients with psoriasis Psoriasis keratinocytes (skin cells)
Produces IL-6 and responds to IL-6 (Grossman, R.
Et al., `` Interleukin-6 is expressed in high levels in
Psoriatic Skin and Stimulates Proliferation of Cu
ltured Keratinocytes. " Proc. Natl. Acad. Sci. USA
1986: 6367, 1989; Bergui, L., `` Interleukin-3 and
Interleukin-6 Synergistically Promote the Prolifer
ation of Malignant Plasma Cell Precursors in Multip
le Myeloma. ” J. Exper. Med. 170: 613, 1989). As shown below, administration of LK-200 to patients with psoriasis showed resolution of the condition. Disruption of IL-6 production and / or action could explain these observations.

LK-200 treatment was initiated as described above in patients with severe psoriasis receiving 20 grams of hydrocortisone topically per day. Since hydrocortisone has a number of undesirable side effects, it was highly desirable to reduce this dosage and increase the efficacy of the treatment.

After 4 weeks, the patient resolved about 30% of his psoriasis with only 5 grams of hydrocortisone per day.

A second patient with psoriasis associated with malignant myeloma was treated with LK-200. Psoriasis was unresponsive to conventional medical treatments.

Over the course of 4 weeks, the patient's IgG levels were 21 gm% to 15 gm%. Psoriasis resolved 50% in the first 2 or 3 weeks. The patient had significantly reduced bone pain. Patients stopped using anesthetics and other powerful analgesics.

Example 3: Treatment of patients with rheumatoid arthritis LK-200 suppresses the production of inflammatory cytokines. The erythrocyte sedimentation rate (ESR) of sequentially obtained patients with AIDS wasting syndrome dropped dramatically after the initiation of treatment with LK-200. Sedimentation rates in other patients receiving LK-200 treatment were also dramatically reduced regardless of the type of tumor or potential immune disease. Sedimentation rate (ESR)
Is a rough guide to the severity of ongoing inflammatory disorders. It is primarily determined by the plasma level of fibrinogen, a coagulation protein produced in the liver. Fibrinogen production is regulated by IL-6. Thus, suppression of IL-6 production or interference with its biological effects can lead to a sharp drop in ESR.

In view of these results, middle-aged women with severe rheumatoid arthritis who were not cured by conventional treatments of all kinds, including gold treatment and methotrexate treatment, were given LK-200. I was treated with. The patient had a sedimentation rate of 60 at the start of treatment. LK-20
0 was administered for 2 weeks.

Two weeks after treatment, the patient's sedimentation rate is 15-
It returned to the normal value around 20. She felt no further pain in her hands, and she was able to resume her housework.

Example 4: Treatment of a female patient with myasthenia gravis Patient: The patient is a 13 year old Caucasian female with a confirmed diagnosis of myasthenia gravis. She was extremely debilitated, sitting in a wheelchair, unable to raise her hand,
And I couldn't easily move any of my great peripheral muscle groups. She was strongly positive in the Hess test for ophthalmoplegia. She had extreme ptosis and moderate diplopia. This patient had a peak flow of 325 cc prior to initiation of treatment. She maintained a dose of 60 milligrams every 2 hours during the day and 180 milligrams of methinenone at bedtime.

(Method): sterilized and thawed LK-200 was
Patients were dosed intravenously over 20-45 minutes. Fifty
cc of LK-200 was mixed with 50 cc of normal sterile saline and prepared for infusion by peripheral intravenous injection. IV solution from Monday to Friday for 2 weeks,
Patients were dosed daily.

During the first two weeks, the patient had a gradual recovery in peripheral muscle strength and a slight reduction in eye drop. The diplopia is somewhat better. In addition, her waking mestinone dosage could decrease from 60 milligrams every 2 hours to sometimes 30 milligrams every 2 hours. This was disseminated.

Since pulse therapy may be effective in the treatment used for the treatment of myasthenia gravis, her regimen is changed to 100 cc L in 50 cc normal saline.
I decided to give her K-200 every other day for over 30 minutes. Within 7 days the patient had a marked response. She no longer took a nap, her muscles increased in strength, her diplopia disappeared completely, and she was able to stand up from her wheelchair and walk without assistance. Mestinone dosage could be reduced to 30 milligrams every 2 hours, and was occasionally withheld. Peak flow is initially 325 cc to 220 cc
Went down. Over the next 3-4 weeks, peak flow has risen steadily and is now above 350-400 cc. The patient now walked uncare, her Hess test was completely negative, and the initially observed muscle weakness resolved. She continued to recover steadily until the 6th week of treatment. The dose of LK-200 was then reduced to 100 cc on Monday, Wednesday, and Friday.

Further effects are shown by measurement of acetylcholine antibody and anti-smooth muscle antibody. 1 for LK-200
After treatment for 0 days, the level of acetylcholine receptor antibody was 3.9 nmol / L. After another 1 month of treatment, the level decreased to 3.0 nmol / L.

Modifications and variations of the method of the present invention will be apparent to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to fall within the scope of the claims.

[0108]

Therefore, the present invention provides a composition for treating disorders associated with elevated cytokine levels, a method for producing the same, and a method for treating the same. In addition, the present invention also provides a composition for treating disorders associated with elevated levels of IL-6, methods of making the same and methods of treating the same.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 38/00 ABJ A61K 37/02 ABD ABX ABF ACJ ABG ACV ABJ ADA ABX ADV ACJ 35/12 ACV ACV C07K 14/52 ADA C12N 5/10 ADV C12P 21/00 9281-4B C12N 5/00 B // (C12N 5/10 C12R 1:91) (C12P 21/00 C12R 1:91) (71) Applicant 595103946 3400 Peachtree Road N.M. E. FIG. , Suite 811, Atlan ta, Georgia 30326, United States of Americana

Claims (26)

[Claims] 1. A method for preparing a cytokine-containing composition from hematopoietic cells for administration to a subject, the method comprising the steps of: a) the cells on fresh medium. B) collecting the cell culture supernatant without filtration of the supernatant; and c) maintaining the supernatant until administration, wherein the maintenance supernatant is administered. It is a composition containing a cytokine. 2. The method of claim 1, wherein the culturing step further comprises activating the production of cytokines by the cells. 3. The cultured cells are immunostimulatory cells,
The method of claim 1. 4. The method of claim 1, wherein the cultured cells have been previously exposed to a cancer-associated virus. 5. The method of claim 4, wherein the virus is Epstein Barr virus. 6. The method of claim 1, wherein the supernatant is maintained by flash freezing to a temperature below about -40 ° C. 7. The method of claim 6, wherein the frozen supernatant is maintained for less than about 3 months. 8. The method of claim 1, wherein the supernatant is not lyophilized while being maintained. 9. The method of claim 1, wherein the supernatant is maintained buffer free. 10. The method of claim 1, wherein the medium is human serum albumin. 11. The method of claim 1, wherein the cells are IgM secreting cells. 12. The method of claim 1, wherein the cell is a cell line designated RPMI1788 and has been deposited with the American Type Culture Collection under accession number CLL166. 13. A cytokine-containing composition produced by the method according to claim 1. 14. A cytokine-containing composition produced by the method according to claim 5. 15. A composition for treating a disorder characterized by elevated IL-6, wherein the composition is in a pharmaceutically acceptable carrier in an amount effective to reduce IL-6 levels. 1. A composition comprising, wherein the composition consists of sterile culture medium in which transformed B cells secrete an immune modulator or a purified fraction thereof having IL-6 inhibitory activity. 16. The dosage is 50 m given twice daily.
The composition according to claim 15, which corresponds to an amount less than 1 culture medium. 17. The composition of claim 15, wherein the disorder is a cancer characterized by elevated levels of IL-6. 18. The composition of claim 15, wherein the disorder is an autoimmune disorder. 19. The composition of claim 15, wherein the disorder is a condition resulting from treatment with a chemotherapeutic agent. 20. The composition of claim 15, wherein the disorder is pain associated with cancer or an autoimmune disorder. 21. The composition of claim 15, wherein
The disorders are multiple myeloma (mm); prostate cancer; ovarian cancer; Castleman's disease; Kaposi's sarcoma (KS); monoclonal hypergammaglobulinemia; mesangial proliferative glomerulonephritis; rheumatoid arthritis and others. Arthritis (R
A); systemic lupus erythematosus (SLE); multiple sclerosis (M
S); myasthenia gravis (MG); Guyan-Barre syndrome (GBS); toxic shock syndrome; bacterial and viral meningitis; HTLV-1-related myelopathy (HA)
M); Alzheimer's disease; Osteoporosis (osteoclast disease);
Cardiac myxoma and hypercalcemia; allergies; unclassified immunodeficiency; anorexia nervosa; inflammatory bowel disease; polyneuropathy with critical illness; acute pyelonephritis; autoimmune hepatitis; large cell lymphoma; lungs Cystic echinococcosis; endometriosis; atherosclerosis; psoriasis; human parvovirus infection; renal cell carcinoma; preeclampsia; cholangiocarcinoma; HIV; acoustic neuroma; hairy cell leukemia; necrotic enteritis; ulcer Colitis; bronchopulmonary insufficiency; schizophrenia and affective disorders; retinoblastoma; pituitary tumors; acute pyelonephritis; thyroid cancer; paraganglioma; candida albicans infection; giant cell arteritis;
Psoriasis; melanoma; chronic pancreatitis; carpal tunnel syndrome; interstitial cystitis: astrocytoma; respiratory syncytial virus infection; liver transplantation: amenindiomas; gastric cancer; sarcomatosis; uterine cancer;
Large cell lung cancer; Helicobacter pylori infection; ductal breast cancer;
Giant cell arteritis granulomas; colorectal cancer; clinical chorioamnionitis; non-Hodgkin lymphoma; thyrotoxicosis; osteosarcoma; malignant mesothelioma; typhoid fever; IgA nephropathy; acute pancreatitis; Fanconi anemia; paraneoplastic A composition comprising: thrombocytosis; and cystic fibrosis. 22. The composition of claim 15, wherein the cell line is transformed by infection with Epstein-Barr virus or a portion thereof. 23. The cell line is Accession No. CLL156.
Deposited at American Type Cell Culture at
16. The composition of claim 15, which is I1788. 24. The composition of claim 15, wherein the cell culture medium is human serum. 25. The composition of claim 15, wherein the cell culture medium is human albumin. 26. The composition of claim 1, wherein the composition is purified based on molecular weight and ion exchange chromatography.
The composition according to 5.