WO1992008792A2 - PROCEDE D'AUGMENTATION ET D'ACTIVATION DES MONOCYTES ET DES NEUTROPHILES ET D'INDUCTION DE LA MATURATION DE CELLULES MYELOïDES A L'AIDES DE L'INTERLEUKINE-5 - Google Patents

PROCEDE D'AUGMENTATION ET D'ACTIVATION DES MONOCYTES ET DES NEUTROPHILES ET D'INDUCTION DE LA MATURATION DE CELLULES MYELOïDES A L'AIDES DE L'INTERLEUKINE-5 Download PDF

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WO1992008792A2
WO1992008792A2 PCT/US1991/008264 US9108264W WO9208792A2 WO 1992008792 A2 WO1992008792 A2 WO 1992008792A2 US 9108264 W US9108264 W US 9108264W WO 9208792 A2 WO9208792 A2 WO 9208792A2
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cells
mammal
human
neutrophils
monocytes
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WO1992008792A3 (fr
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Loretta A. Bober
Michael J. Grace
Chuan-Chu Chou
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Merck Sharp & Dohme LLC
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Schering Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5409—IL-5
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a method for increasing and activating monocytes and neutrophils and for inducing maturation of myeloid cells in mammals by administering an effective amount of interleukin-5 (IL-5) for this purpose to a mammal in need of such treatment.
  • Interleukin 5 IL-5
  • IL-5 is a lymphokine (stimulator of the immune system) that has a broad range of immune cell stimulation in the mouse as described by Arai et al., An ⁇ u. Rev. Biochem., ⁇ £: 783-836 (1990); Sanderson et al., Immunological Rev., H22: 29-50 (1988); Coffman et al., J. Immunol., 122.: 3685-3690 (1987); Bond et al., J.
  • mice IL-5 is disclosed as showing activity as a B-cell growth factor (II), as an enhancer of cytotoxic T-cell activity, as an eosinophil differentiation factor, and as an IgA stimulating factor.
  • II B-cell growth factor
  • BCGF II mouse B-cell Growth Factor II
  • TRF mouse T-cell replacing factor
  • EDF mouse Eosinophil Differentiation Factor
  • the human molecule of interleukin 5 has been cloned and found to retain 77% genetic homology with murine IL-5 (Azuma et al., Nucleic Acid Res., 14: 9149-9158 (1986) and Lee et al., Behring. I ⁇ st. Mitt., 22, 8-14 (1988)).
  • Human IL-5 has been referred to as an EDF (Lopez et al., J.Exp.Med., 167:219-224 (1988)).
  • Human IL-5 has not been shown to have BCGF II activity (Clutterbuck et al., Eur. J.
  • IL-5 was originally thought to be important only for the co- stimulation of activated B-cells, Swain et al., supra. It has also been shown to modulate the activity of T cells and eosinophils in mice, Takatsu et al., supra , and Sanderson et al., supra .
  • T cells and B cells act in later stages of an immune response. Eosinophils, while acting at earlier stages in an immune response, provide protection mainly against parasitic infections. It would be highly advantageous to have an agent which would increase and activate neutrophils and monocytes which act in the early stages of infection against bacterial and fungal cells, constituting the body's first line of defense against common infection.
  • neutrophils and monocytes which act in the early stages of infection against bacterial and fungal cells, constituting the body's first line of defense against common infection.
  • cells In the normal process of white blood cell hematopoeisis, cells originate in the bone marrow from a primitive immature cell known as a stem cell and differentiate through progressively more mature stages along different lineage pathways to arrive at a terminal state of differentiation as a mature monocyte, granulocyte, or lymphocyte.
  • a property of immature, undifferentiated cells is the ability to multiply rapidly.
  • cancer is a disorder of cell differentiation.
  • myeloid leukemias are disorders in which cells of monocytic and granulocytic lineages are blocked at an early stage of maturation and thus have not lost their proliferative capacity. Because these maturation arrested cells continue to proliferate, they give rise to a population of immature cancer cells resulting in a diagnosis of leukemia.
  • myeloid leukemia cells can be induced to differentiate into normal macrophages and granulocytes and that upon differentiation these cells lose their capacity to proliferate. This suggests that the induction of terminal differentiation by an agent would have usefulness as a therapy for myeloid leukemia.
  • IL-5 increases the neutrophil and monocyte count in mammals and stimulates differentiation and activation of neutrophils and monocytes. This is very surprising since activity on monocytes and neutrophils has neither been reported previously in human nor in mouse systems. In fact, no effect of IL-5 in any system has been reported on monocytoid or on non-eosinophilic cells of the granulocytic lineage. We have thus discovered that IL-5 may be administered to a mammal to increase and activate neutrophils and monocytes and provide increased host resistance to infection or to treat infection at a very early stage.
  • the mammal may be an immunocompromised host, e.g., any host susceptible to unwanted bacterial infection such as a patient having severe burns or ulcers, a host whose immune defenses are lowered because of radiation or chemotherapy in the treatment of cancer, and a host with a genetic or acquired immunodeficiency.
  • immunocompromised host e.g., any host susceptible to unwanted bacterial infection such as a patient having severe burns or ulcers, a host whose immune defenses are lowered because of radiation or chemotherapy in the treatment of cancer, and a host with a genetic or acquired immunodeficiency.
  • IL-5 induces the maturation of myeloid and monocytoid cells.
  • myeloid leukemia is associated with the proliferation of immature myeloid cells
  • IL-5 by progressing myeloid cells to their mature state should reduce their proliferation and provide a method for treating myeloid leukemia.
  • the mammals will be treated with IL-5 derived from a human source, i.e., human IL-5 such as human IL-5 produced recombinantly from mammalian cells, such as NS-1 or CHO cells.
  • human IL-5 such as human IL-5 produced recombinantly from mammalian cells, such as NS-1 or CHO cells.
  • the dosage for the mammals will be administered by subcutaneous intravenous injection or intravenous infusion and will be in an amount of about 0.1 microgram to about 1 milligram of IL-5 per kilogram of body weight per day.
  • the lL-5 is administered in an amount of about 2 to about 25 micrograms of IL-5 per kilogram of body weight per day.
  • the invention also involves a method for preparing recombinant human IL-5 comprising culturing a CHO cell line transfected with a vector comprising an SRalpha driven DNA sequence coding for IL-5, a human beta globin polyA signal sequence downstream of the IL-5 coding sequence, and an SV40 splice junction immediately upstream or downstream of the IL-5 coding sequence, under conditions in which the DNA sequence is expressed; and isolating the IL-5 protein from the culture.
  • the expression level of IL-5 is increased by dihydrofolate reductase mediated gene amplification.
  • the IL-5 expressed by the CHO cells is preferably purified using an immunoaffinity column, e.g., an affinity chromatography on a sepharose column coupled with the TRFK 5 monoclonal antibody to murine IL-5.
  • the vector preferably further comprises a consensus vertebrate translation initiator upstream of the 5' end of the IL-5 DNA sequence.
  • Figure 1 is a schematic illustration showing the construction of pSRShlL-5 as described further below.
  • Figure 2 is a schematic illustration showing the construction of pDSRGhlL-5 as described further below.
  • Figure 3 is a graphical representation showing the effect on the differentiation of HL-60 cells by human IL-5.
  • Figure 4 is a graphical representation showing the effect on the activation (phagocytic index) of HL-60 cells by human IL-5.
  • Figure 5 is a graphical representation showing the effect on the activation (phagocytic index) of U-937 cells by human IL-5.
  • Figure 6 is a graphical representation showing the stimulation of the hexose monophosphate shunt on U-937 cells by human IL-5.
  • Figure 7 is a graphical representation showing the activation (phagocytic index) of human peripheral blood neutrophils by human IL-5.
  • Figure 8 is a graphical representation showing the activation (phagocytic index) of human peripheral blood monocytes by human IL-5.
  • IL-5 any suitable IL-5 may be employed in the present invention.
  • Complementary DNAs (cDNAs) for IL-5 have recently been cloned and sequenced by a number of laboratories, e.g., Azuma et al., supra; Campbell et al., Proc Natl. Acad. Sci.USA, 4: 6629-6633 (1987); Tsujimoto et al., J. Biochem., 1Q£: 23-28 (1989); Lee et al., supra, Chou et al., FASEB Journal, 4(7): A2068 (1990); Tavernier et al., DNA, : 491-501 (1989); European Patent Application No.
  • IL-5 purified from various culture supematants, such as those disclosed in the Takatsu et al., McKenzie et al., Coffman et al., and Yamaguchi et al. articles cited above and in Grace et al., FASEB Journal, M ): A2169 (1990), may also be employed.
  • the disclosures of all the above articles and patent publications are incorporated herein by reference for their teachings of DNA and amino acid sequences and of methods of obtaining suitable IL-5 materials for use in the present invention.
  • the IL-5 used in the present invention will be a human IL-5, and most preferably it will be the human version with the sequence described in SEQ. NO. 1 below.
  • mammals e.g., humans, are administered an effective amount of an IL-5 to increase monocytes and/or granulocytes (which might be any of all of the following: polymorphonuclear cells, eosinophils and/or basophils).
  • an effective amount is defined as any amount of IL-5 that will enhance the phagocytic capacity of neutrophils or monocytes by at least 25 percent, preferably by at least 50%.
  • From about 0.1 micrograms to about 1 milligrams of IL-5, preferably human IL-5 (hlL-5), per kilogram of body weight per day is preferably administered. More preferably, mammals are administered about 2 to about 25 micrograms of hlL-5 per kilogram of body weight per day.
  • the amount, frequency and period of administration will vary depending upon factors such as the level of the neutrophil and monocyte count (e.g., the severity of the monocytopenia or granulocytopenia) and activity, age of the patient, nutrition, etc. Usually, the administration will be daily initially and it may continue periodically during the patient's lifetime. Dosage amount and frequency may be determined during initial screenings of neutrophil count and the magnitude of the effect of IL-5 upon the increase in neutrophil count. Dosage will be preferably aimed to increase the neutrophil and monocyte counts to an acceptable level, e.g., at least a 25% enhancement, to generate the desired biological effect, which will need to be determined individually for each patient depending on clinical circumstance.
  • an acceptable level e.g., at least a 25% enhancement
  • selective manipulations may be performed, such as the enhancement of one or more subpopulations of lymphocytes.
  • it may be useful to administer it in conjunction with other biologically and/or pharmaceutically active compounds.
  • it can be combined with other white cell increasing agents [especially human colony stimulating factors such as granulocyte-macrophage colony stimulating factor (GM-CSF) and/or granulocyte-colony stimulating factor (G-CSF)].
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • G-CSF granulocyte-colony stimulating factor
  • IL-1 and/or IL-3 and/or IL-7 for the purposes of increasing the total white blood cell and neutrophil count;
  • IL-2 in combination with IL-5 may produce selective enhancement of specific and useful T-cell functions, and
  • IL-5 in combination with IL-4 and/or IL-6 may be useful in specifically enhancing the function and/or numbers of normal and/or neoplastic B-celis.
  • IL-5 may also be useful in increasing the utility of chemotherapeut ⁇ c agents, including, but not necessarily limited to, alkylating agents, mitotic spindle poisons or antitumor antibiotics. By enhancing the number of white cells or the functional or differentiational status of specific subpopulations of cells, the efficacy of the aforementioned chemotherapeutic agents may be enhanced.
  • interferon-gamma or alpha especially human interferons such as interferon alpha2B
  • IL-5 may also be useful in increasing numbers and functions of certain white cell, particularly T-cell subsets.
  • antibodies to any of the aforementioned interleukins or interferons may need to be administered instead of the native molecules.
  • Administration of the dose can be intravenous, nasal, parenteral, oral, subcutaneous, intramuscular, topical, transdermal or any other acceptable method.
  • the IL-5 could be administered in any number of conventional dosage forms. Parenteral preparations include sterile solutions or suspensions.
  • Topical dosage forms can be creams, ointments, lotions, transdermal devices (e.g., of the conventional reservoir or matrix patch type) and the like.
  • formulations and pharmaceutical compositions contemplated by the above dosage forms can be prepared with conventional pharmaceutically acceptable excipients and additives, using conventional techniques.
  • the IL-5 is preferably administered via the intravenous route.
  • the solutions to be administered may be reconstituted lypholized powders and they may additionally contain preservatives, buffers, dispersants, etc.
  • IL-5 is reconstituted with 10 millimolar citrate buffer, e.g., with pH > 7.2, and preservative-free sterile water with the maximum concentration not to exceed 100 micrograms per milliliter and administered by continuous intravenous infusion or by intravenous injection.
  • the daily dose can be added to 5 ml of normal saline and the solution infused by mechanical pump or by gravity.
  • the activation of neutrophils and monocytes by IL-5 can be determined by the following test protocols. METHODS
  • A. Isolation of cells Whole blood is drawn from a human donor. The blood is defibrinated using glass beads. Red blood cells are removed by two sequential gravity sedimentation steps over 4% dextran- phosphate buffered saline. White blood cells are washed once and layered over a 61 % -76% discontinuous Percoll-phosphate buffered saline gradient. Cells are centrifuged at 1250 x g. The top layer banding cells are mononuclear cells; the bottom layer banding cells are neutrophilic cells. The top layer banding cells are used for the monocyte assays and the bottom layer banding cells for the neutrophil assays.
  • U-937 is a cell line (ATCC CRL 1593) established from malignant cells from a patient with diffuse histiocytic leukemia (Sunstrom, C. and Nilsson K. Int. J. Cancer, lZ:565-577 (1976)). This cell line exhibits characteristics that indicate it is an immature monocyte.
  • HL-60 is a cell line (ATCC CCL 240) established from a patient with acute pro myelocytic leukemia. These cells exhibit characteristics that indicate it is an immature neutrophil.
  • C. Yeast Phagocytosis Assav is a cell line (ATCC CCL 240) established from a patient with acute pro myelocytic leukemia. These cells exhibit characteristics that indicate it is an immature neutrophil.
  • HL-60 cells were routinely cultured in RPMI-1640 containing 10% heat-inactivated fetal bovine serum prior to assay.
  • cells were suspended in culture medium alone or containing between 0.1 ng/ml and 250 ng/ml IL-5 at 1x10 5 cells/mi and incubated at 37°C for 3 days ( Figure 4).
  • U-937 cells were routinely cultured in RPMI-1640 containing 10% heat-inactivated fetal bovine serum prior to assay.
  • cells were suspended in culture medium alone or containing between 2.5 ng/ml and 500 ng/ml IL-5 at 1x10 5 cells/ml and incubated at 37°C for 3 days ( Figure 5).
  • Isolated monocytes from one donor ( Figure 8) and neutrophils from three donors (indicated as PP, LD, and DP in Figure 7) were incubated in Iscove's media containing 10% heat-inactivated fetal bovine serum alone or containing between 2.5 ng/ml and 250 ng/ml IL-5 at 1x10 6 cells/ml and incubated for 4 hours at 37°C Following treatment, HL-60, U-937, monocyte, and neutrophil cells were used for performing phagocytic assay as follows.
  • the phagocytic assay was performed by adding 300 ml of human serum, 50 ml of heat- killed yeast particles (10 s organisms/ml) to 12 x 75 mm polypropylene tubes containing approximately 5 x 10 5 of the isolated, treated cells to be assayed (human peripheral blood, monocytes, neutrophils, HL-60 cell line or U-937 cell line). Incubation was carried out for 90 minutes in a gyrorotary water bath at 37°C. Following centrifugation, the cell suspension was stained with 0.4% trypan blue and 0.2% eosin Y in saline.
  • Ingested yeast remains colorless and uningested yeast stains purple allowing for accurate determination of avidity (number of yeast ingested) as well as percent phagocytic cells. Mean values obtained for these parameters were multiplied to calculate a phagocytic index. Data were analyzed by the Student's T test comparing values from IL-5 treated cells to that of control cells receiving vehicle alone. The results are shown in Figure 4 for HL-60 cells, Figure 5 for U-937 cells, Figure 7 for human neutrophils, and Figure 8 for human monocytes.
  • the U-937 cells were resuspended in 0.2 ml RPMI-1640 containing 2% heat-inactivated fetal bovine serum and distributed into 12 x 75 mm polypropylene test tubes.
  • 0.1 ml of a 2 mg/ml solution of nitroblue tetrazolium (NBT) in 2% RPMI and 0.1 ml of a 0.25 mM stock of freshly prepared phorbol 12-myristate 13-acetate (PMA) in 2% RPMI were added to the cells.
  • the cell suspension was incubated in a 37°C water bath for 30 minutes. After incubation, cells were centrifuged onto glass slides.e.g., using a Cytospin® slide centrifuge.
  • HL-60 cells were routinely cultured as described above in the Yeast Phagocytosis Assay. HL-60 cells were incubated for 3 days in culture media containing either no IL-5 or 250 ng/ml IL-5. After three days, the cells were spun down on microscope slides to be stained for eosinophil granules (Luxol Blue), monocyte esterase (alpha-naphthyl acetate esterase) or PMN esterase (AS-D chloroacetate esterase) as described below.
  • eosinophil granules Loxol Blue
  • monocyte esterase alpha-naphthyl acetate esterase
  • PMN esterase AS-D chloroacetate esterase
  • Figure 3 shows the results of the differential stains for media (-) or for 250 ng/ml IL-5 (+) containing media and subsequent staining or monocyte esterase (mono), PMN esterase (PMN) or eosinophil granules (EOS).
  • monocyte esterase monocyte esterase
  • PMN PMN esterase
  • EOS eosinophil granules
  • Citrate-Acetone-CH3 ⁇ H Fixative Dilute Citrate concentrate 1 :9 with deionized water. Add 18 ml Citrate solution, 27 ml Acetone, and 5 ml absolute CH 3 OH. Store at room temperature. Prepare daily.
  • Trizmal 6.3 1 :9 Dilute Trizmal 6.3 1 :9 with deionized water. Warm 50 ml dilute Trizmal 6.3 to 37°C and add with constant stirring contents of 1 capsule of the dye Fast Corinth V salt. When salt is completely dissolved add 2 ml of Naphthol AS-D Chloroacetate solution. The solution will appear quite turbid. Continue stirring 15-30 min. and add to coplin jar. Do not filter.
  • Citrate-Acetone-CH 3 ⁇ H Fixative Dilute Citrate concentrate 1 :9 with distilled/deionized water. Add 18 ml Citrate solution, 27 ml Acetone, and 5 ml absolute CH3OH. Store at room temperature. Prepare daily.
  • B-5 Solution (may be stored): 15 g Sodium Acetate 60g Mercuric Chloride 900 ml deionized water Lugol's Solution:
  • Figure 3 summarizes the effect of human IL-5 on the maturation of HL-60 cells.
  • the addition of as little as 2.5 ng/ml will induce premyelocytic HL-60 cells to differentiate to mature neutrophils and monocytes as well as eosinophils.
  • Mature monocytes are measured by specific staining for alpha-naphthyl esterase; mature neutrophils are specifically measured by AS-D chloroacetate esterase; mature eosinophils are specifically measured by luxol fast blue.
  • Figure 4 demonstrates that human IL-5 activates premyelocytic and premonocytic cell lines as mature phagocytes.
  • HL-60 cells can be induced to undergo Fc-mediated phagocytosis of heat-killed yeast cells by pre-incubation of the cells with human IL-5 between 1 and 100 ng/ml. That such phagocytosis is not only due to eosinophils is demonstrated in Figure 5 by the fact that premonocytic, not differentiatable to eosinophils, U-937 cells can also be induced to undergo Fc-mediated phagocytosis with at least 25 ng/ml human IL-5.
  • Figure 6 demonstrates that IL-5 also induces U-937 cells to become activated as measured by NBT reduction. NBT reduction to NB Foramazon is indicative of upregulated hexose monophosphate shunt activity to provide energy and reducing equivalents for catabolism.
  • Figures 7 and 8 demonstrate that human purified neutrophils and monocytes are also activated by in vitro dosing with human IL-5. Such activation is donor dependent, with a broad range of activation. Construction of the Human IL-5 Expression plasmid pDSRGhlL-5
  • plasmids pCDSRalphahlL-5, pDSRG, an pSRS have been described (Yokota et al., Proc Natl. Acad. Sci. USA, 21, 7388-7392, 1987, and ATCC designation numbers 68233 and 68234).
  • a human IL-5 cDNA variant was constructed by the polymerase chain reaction (PCR) using pCDSRalphahlL-5 as the template.
  • Oligonucleotide AB790 and AB791 as shown below and in SEQ. NOS. 2 and 3, respectively, were used as the 5'-end and the 3'-end primers, respectively:
  • This resultant cDNA from the PCR contains essentially the identical codin region as the parental cDNA and modifications in the non-coding regions as follows to facilitate expression plasmid construction and high level gen expression in eucaryotic cells.
  • a restriction site Sal I shown in bold letters
  • the translational initiator underlined was changed (as shown above) to conform to the consensus context of vertebrate mRNAs (Kozak, Nucleic Acids Res., 15. 8125-8148, 1987) except that in position +4 (SEQ. NO.
  • the expression vector pDSRG (ATCC 68233) was used to construct plasmid pDSRGhlL-5 as shown in Figure 2 by replacing the Hind ⁇ i/EcoR I fragment of pDSRG (containing the SRalpha promoter) with the small Hind III/EcoR I fragment of the pSRShlL-5 (containing the SRalpha promoter and the hlL-5 cDNA) as shown in Figure 2.
  • the final human IL-5 expression plasmid, pDSRGhlL-5 contains the following elements, counterclockwise from the Xba I site:
  • hlL-5 present in the plasmid is the same as in pCDSRalphalL-5 given in Yokota et al., Proc. Natl. Acad. Sci. USA, 21, 7388-7392, 1987.
  • CHO-dhfr Chinese hamster ovary cell mutants deficient in dihydrofolate reductase activity
  • CHO-dhfr mutants have an auxotrophic requirement for hypoxanthine, thymidine and praline.
  • Expression vectors incorporating a dhfr marker may be used to complement this mutation: selection is achieved by growing cells in the absence of the required media cofactors described above.
  • Gene amplification increase in copy number up to 1 ,000 X
  • MTX folate analog methotrexate
  • the plasmid (pDSRGhlL-5) having the coding sequence for dhfr and human IL-5 was constructed as described as above.
  • Transfection of pDSRGhlL-5 into the CHO-dhfr-cell line was carried out by the calcium phosphate precipitation method described by Chen and Okayama, BioTechniques, £, 632-637, 1988.
  • Transformants were selected in a selection medium (DMEM, Dulbecco's Modified Eagle's Medium) that lacks hypoxanthine and thymidine.
  • the survival clones were pooled and treated with stepwisely increased concentration of methotrexate in the following orders: 60 nM, 300 nM, and finally 1 ⁇ M.
  • a clone designated CHO-1 was chosen for the large scale propagation.
  • MBC Master Cell Bank
  • DMEM complete growth medium
  • fetal bovine serum fetal bovine serum
  • the cells were thawed and propagated in T flasks for 5-10 generations in the growth medium and the frozen procedures were repeated to constitute the Master Cell Bank (MCB).
  • hlL-5 production was carried out in roller bottles with 850 cm 2 in surface area.
  • To start production one frozen vial from the MCB was thawed and inoculated into a T-75 flask.
  • the culture was expanded in T- flasks and finally inoculated into roller bottles.
  • the cells are washed with the serum free medium (complete medium without the addition of serum) and subjected to two batch media changes for production of hlL-5. No methotrexate was used throughout the production process.
  • the conditioned media was collected and filtered to remove any cells that may be present.
  • the filtrate which contained crude hlL-5, was forwarded to the purification.
  • hlL-5 Purification of hlL-5 from the crude CHO hlL-5 media was carried out by performing an affinity chromatography on a sepharose column (Affi-gel 10, BioRad) coupled with the TRFK 5 monoclonal antibody (ATCC No. HB9897) to murine IL-5 (Coffman et al., Science, 215, 308-310 (1989) and Schumacher et al., J. Immunology, HI: 1576-1581 (1988)). After the crude material was loaded onto the column, the column was washed sequentially once with phosphate buffered saline (ph 7.4) (PBS), PBS plus 0.5 M NaCI, and once with PBS, to remove non-specifically bound proteins.
  • PBS phosphate buffered saline
  • hlL-5 was then eluted with 0.2 M glycine, pH 2.95 and immediately neutralized with 1M Tris-HCI, pH 8. The purity of the hlL-5 protein was examined first by SDS-polyacrylamide gel electrophoresis and then NH2-terminal sequencing. A uniform polypeptide sequence was consistently observed, indicating greater than 99% purity of the hlL-5 protein.
  • Lys Lys Tyr He Asp Gly Gin Lys Lys Lys Cys Gly Glu Glu Arg

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Abstract

Procédé d'augmentation et d'activation des monocytes et des neutrophiles, et induisant la maturation de cellules myéloïdes chez des mammifères par administration de IL-5. L'invention concerne également un procédé de production de IL-5.
PCT/US1991/008264 1990-11-16 1991-11-14 PROCEDE D'AUGMENTATION ET D'ACTIVATION DES MONOCYTES ET DES NEUTROPHILES ET D'INDUCTION DE LA MATURATION DE CELLULES MYELOïDES A L'AIDES DE L'INTERLEUKINE-5 Ceased WO1992008792A2 (fr)

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JP4501059A JPH06502644A (ja) 1990-11-16 1991-11-14 インターロイキン５による単球および好中球の増加および活性化法および骨髄細胞の成熟誘導法

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO1996030039A1 (fr) * 1995-03-24 1996-10-03 Schering Corporation Methode d'amelioration de la reponse d'anticorps a des antigenes specifiques avec l'interleukine-10
EP0901370A4 (fr) * 1996-05-07 1999-07-14 Cornell Res Foundation Inc Stimulation de la reponse immunitaire par de faibles doses d'interleukine -2 (il-2)

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AU711087B2 (en) * 1995-03-24 1999-10-07 Schering Corporation Method for enhancing the antibody response to specific antigens with interleukin-10
US6509313B1 (en) 1996-02-28 2003-01-21 Cornell Research Foundation, Inc. Stimulation of immune response with low doses of cytokines
EP0901370A4 (fr) * 1996-05-07 1999-07-14 Cornell Res Foundation Inc Stimulation de la reponse immunitaire par de faibles doses d'interleukine -2 (il-2)

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IE913984A1 (en) 1992-05-20
WO1992008792A3 (fr) 1992-07-23
AU9052591A (en) 1992-06-11
JPH06502644A (ja) 1994-03-24
EP0557420A1 (fr) 1993-09-01

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