EP0740552A1 - Verwendung von il-10 zur anregung der zytolischen aktivität von mononucleären zellen in peripherem blut - Google Patents

Verwendung von il-10 zur anregung der zytolischen aktivität von mononucleären zellen in peripherem blut

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Publication number
EP0740552A1
EP0740552A1 EP94904303A EP94904303A EP0740552A1 EP 0740552 A1 EP0740552 A1 EP 0740552A1 EP 94904303 A EP94904303 A EP 94904303A EP 94904303 A EP94904303 A EP 94904303A EP 0740552 A1 EP0740552 A1 EP 0740552A1
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Prior art keywords
cells
human
pbmcs
cell
ifn
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French (fr)
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Martin A. Schwarz
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Merck Sharp and Dohme Corp
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Schering Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • This invention relates to the use of Interleukin-10 (IL-10), formally cytokine synthesis inhibitory factor (CSIF), for adoptive immunotherapy in the treatment of neoplastic disorders (cancer) by stimulation of cytolytic activity of peripheral blood mononuclear cells (PBMC).
  • IL-10 Interleukin-10
  • CSF cytokine synthesis inhibitory factor
  • Immunologic approaches to cancer therapy are based on the notion that cancer cells have somehow evaded the body's defenses against aberrant or foreign cells and molecules, and that these defenses might be therapeutically stimulated to kill or inhibit the growth of the cancer cells, e.g., as discussed by Klein (Immunology, Wiley-Interscience, New York, 1982, pp. 623-648).
  • Klein Immunology, Wiley-Interscience, New York, 1982, pp. 623-648.
  • immune effectors can directly or indirectly inhibit tumor growth has led to renewed interest in this approach to cancer therapy [Heberman, Concepts Immunopath. 7 :96 (1985) (natural killer cells resist tumor growth); Rosenberg et al., Ann. Rev. Immunol.
  • Immune responsiveness to neoplasms is regulated by a variety of cell types and involves the actions of T-cell and monocyte-derived cytokines.
  • One immunologic approach that has shown clinical promise has been so-called “adoptive immunotherapy” using IL-2-activated killer cells [Rosenberg, supra-, Rosenberg, Sci. Am., pp. 62-69 (May 1990)].
  • IL-2 alone or in combination with more traditional chemotherapeutic agents appears to be effective in treating certain malignancies (e.g., renal cell carcinoma)
  • unfortunate toxic side effects such as vascular bed leakage and edema associated with administration of effective dosages of IL-2 have led some to suggest that the risks may outweigh the benefits [Cotran et al., J. Immunol. 759: 1882 (1987); Edwards et al, Cancer Res. 52:3425 (1992)].
  • vascular endothelial cells appear to be particularly sensitive to IL-2 toxicity, as evidenced by increased vascular permeability (i.e. vascular leak syndrome) and edema.
  • vascular permeability i.e. vascular leak syndrome
  • edema vascular permeability
  • One of the factors contributing to this pathology may be augmented adhesion of IL-2-activated T cells and neutrophils to endothelial monolayers, as has previously been noted in vitro [Edwards et al., supra', Damle et al, 138:1719 (1987)].
  • Adoptive immunotherapy is defined as the transfer to a tumor-bearing host of active immunologic reagents, such as cells with anti-tumor activity that can mediate, either directly or indirectly, anti-tumor effects.
  • Adoptive immunotherapy represents an attractive approach to the therapy of neoplastic disease. It should be noted that because active immunologic reagents are being transferred to the host, complete host immunocompetence is not required. Thus, the immuno- suppression generally associated with the tumor-bearing state does not represent a major problem to this therapeutic alternative.
  • adoptive immunotherapy can easily be combined with other therapies such as chemotherapy or radiation therapy. Also, in contrast to other therapies, immunosuppression is unlikely to result from this treatment. Patients undergoing chemotherapy or radiation therapy tend to be immunocompromised and will generally have a depleted supply of effector peripheral blood mononuclear cells (PBMCs) available for activation. A therapy that shows efficacy at low effector cell:target cell ratios would thus be particularly advantageous for such patients.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • IL-2 IL-2
  • IL-5 Recombinant human IL-5 [Nagasawa et al., Cell. Immunol. 133:311 (1991)], IL-7 [Stotter and Lotze, Arch. Surgery 725: 1525 (1991)] and IL-12 [Gately et al, J. Immunol. 747:874 (1991)] have been reported to stimulate cytolytic activity in human PBMCs.
  • Interleukin-10 (IL-10) originally described in mice as a cytokine synthesis inhibitory factor secreted by specific helper T-cell subsets, appears to modulate the differentiation of murine cytotoxic T-cells [Chen and Zlotnik, J. Immunol 147:52$ (1991)].
  • IL-4 can adversely affect the generation of LAK activity by IL-2 [Nagler et al., J. Immunol. 747 :2349 (1988)].
  • human PBMCs are cultured in the presence of both BL-2 and IL-4, the lysis of LAK-sensitive targets is greatly reduced [Spits et al., J. Immunol. 141:29 (1988)].
  • the PBMCs are pre-cultured in medium supplemented with IL-2 for 3 days before adding IL-4, however, augmented cytolytic activity results [Spits et al., supra].
  • IL-2-driven cytotoxicity by IL-4 can be abated when alpha- interferon ( ⁇ -IFN) or tumor necrosis factor-alpha (TNF- ⁇ )is included in the initial incubation mixture [Swisher et al., Cell. Immunol. 725:450 (1990)].
  • ⁇ -IFN alpha- interferon
  • TNF- ⁇ tumor necrosis factor-alpha
  • Kedar et al. [Cancer Immunol. Immunother. 55:63 (1992)] have recently indicated that sequential administration of IL-2 and ⁇ -IFN is an effective immunotherapeutic regimen for treatment of MCA-105 sarcomas and M109 carcinomas in murine tumor models.
  • the primary finding from this study was that sequential administration of cytokines appeared to have greater efficacy than concomitant administration of both cytokines.
  • the present invention fills these needs by providing methods for the use of IL-10 alone or in combination with IL-2 and/or ⁇ -IFN to augment cytolytic activity of PBMCs, especially LAK and NK cells. More particularly, this invention provides a method for treating cancer comprising administering an effective amount of IL-10-activated PBMCs to a patient afflicted with cancer, to cause regression of such cancer. Preferably administration of the activated PBMCs is accompanied and/or followed by administration of EL-10.
  • the IL-10 is administered in combination with an amount of IL-2 sufficient to augment LAK cell activation but not to cause toxic side effects attributable to the use of IL-2 alone.
  • the IL-10 is administered in combination with an amount of ⁇ -IFN sufficient to augment LAK cell activation.
  • the IL-10 is administered in combination with (a) an amount of IL-2 sufficient to augment LAK cell activation but not to cause toxic side effects attributable to the use of IL-2 alone and with (b) an amount of ⁇ -IFN sufficient to augment LAK cell activation.
  • This invention further provides a method for antagonizing blockade of IL-2-induced cytotoxicity by endogenous IL-4 comprising administering to a patient in need of such treatment an effective amount of IL-10.
  • This invention still further provides pharmaceutical compositions comprising IL-10 in combination with IL-2 and/or ⁇ -IFN, and a pharmaceutically acceptable carrier.
  • human IL-10, IL-2 and ⁇ -IFN are used in the foregoing methods and compositions, most preferably, recombinant human IL-10, IL-2 and ⁇ -IFN.
  • the present invention is an improvement over methods of the prior art which employ IL-2 to induce cytolytic activity in NK and LAK cells.
  • the present invention greatly reduces the toxic side effects that typically result from the use of IL-2 in such methods by eliminating the IL-2 completely, or greatly reducing the amount of IL-2 that must be used.
  • the term "adoptive immunotherapy” means therapy involving the transfer of activated functional immune cells to a patient.
  • these cells will comprise LAK and NK cells originating from the actual patient undergoing treatment.
  • regression is defined herein to mean a measurable decrease in the size of one or more tumors, as commonly measured in the art.
  • interleukin-10 or "IL-10” is defined as a protein which (a) has an amino acid sequence of mature (e.g., lacking a secretory leader sequence) IL-10 as disclosed in U.S. Patent Application Serial No. 07/917,806, filed July 20, 1992, which corresponds to International Application No. WO 91/00349, and (b) has biological activity that is common to native IL-10.
  • glycosylated e.g. produced in eukaryotic cells such as CHO cells
  • unglycosylated e.g., chemically synthesized or produced in E. col ⁇
  • EL- 10 suitable for use in the invention can be obtained from a number of sources. For example, it can be isolated from culture medium of activated cells secreting the protein. Additionally, the IL-10, or active fragments thereof can be chemically synthesized using standard techniques known in the art. See Merrifield, Science 255:341 (1986) and Atherton et al, Solid Phase Peptide Synthesis: A Practical Approach, 1989, I.R.L. Press, Oxford. Preferably, the protein or polypeptide is obtained by recombinant techniques using isolated nucleic acid encoding the IL-10 polypeptide.
  • Libraries are constructed from nucleic acid extracted from appropriate cells, see, for example, International Application Publication No. WO 91/00349, which discloses recombinant methods for making IL-10.
  • Useful gene sequences can be found, e.g., in various sequence databases, e..g fashion GenBank and BMPL or nucleic acid and PIR and Swiss-Prot for protein, c/o Intelligenetics, Mountain View, California, or the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wisconsin, which are incorporated herein by reference.
  • IL-10 is performed by either nucleic acid hybridization or immunological detection of the encoded protein, if an expression vector is used. Oligonucleotide probes based on the deposited sequences disclosed in International Application Publication No. WO 91/00349 are particularly useful. Oligonucleotide probes sequences can also be prepared from conserved regions of related genes in other species. Alternatively, degenerate probes based on the amino acid sequences of IL-10 can be used. Standard methods can be used to produce transformed prokaryotic, mammalian, yeast or insect cell lines which express large quantities of the polypeptide. Exemplary E. coli strains suitable for both expression and cloning include W3110 (ATCC Bi, 27325), X1776 (ATCC No. 31244). X2282, RRl (ATCC Mp/ 31343). Exemplary mammalian cell lines include COS-7 cells, mouse L cells and CHP cells. See Sambrook (1989) and Ausubel et al, 1987 supplements).
  • Various expression vectors can be used to express DNA encoding IL-10.
  • Conventional vectors used for expression of recombinant proteins in prokaryotic or eukaryotic cells may be used.
  • Preferred vectors include the pcD vectors described by Okayama et al, Mol. Cell. Biol. 5:280 (1983); and Takebe et al, Mol. Cell. Biol. 5:466 (1988).
  • Other SV40-based mammalian expression vectors include those disclosed in Kaufman et al, Mol. Cell. Biol.
  • the IL-10 can be produced in soluble form such as a secreted product of transformed or transfected yeast or mammalian cells.
  • the peptides can then be purified by standard procedures that are known in the art. For example, purification steps could include ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and the like. See Methods in Enzymology Purification Principles and Practices (Springer- Verlag, New York, 1982).
  • IL-10 may be produced in insoluble form such as aggregates or inclusion bodies.
  • the IL-10 in such a form is purified by standard procedures that are well known in the art. Examples of purification steps include separating the inclusion bodies from disrupted host cells by centrifugation, and then solubilizing the inclusion bodies with chaotropic agent and reducing agent so that the peptide assumes a biologically active conformation. For specifics of these procedures, see, e.g. Winkler et al, Biochemistry, 25:4041 (1986), Winkler et al, Bio/Technology 5:9923 (1985); Koths et al, and U.S. Patent No. 4,569,790.
  • the nucleotide sequences used to transfect the host cells can be modified according to standard techniques to make EL- 10 or fragments thereof with a variety of desired properties.
  • modified IL-10 can vary from the naturally-occurring sequences at the primary structure level, e.g., by amino acid, insertions, substitutions, deletions and fusions. These modifications can be used in a number of combinations to produce the final modified protein chain.
  • the amino acid sequence variants can be prepared with various objectives in mind, including increasing serum half-life, facilitating purification or preparation, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic use.
  • the amino acid sequence variants are usually predetermined variants not found in nature, although others may be post-translational variants, e.g., glycosylated variants or proteins which are conjugated to polyethylene glycol (PEG), etc. Such variants can be used in this invention as long as they retain the biological activity of IL-10.
  • human IL-10 is used for the treatment of humans, although viral IL-10 or IL-10 from some other mammalian species could possibly be used. Most preferably, the IL-10 used is recombinant human IL-10.
  • the preparation of human and mouse IL-10 has been described in International Application WO 91/00349.
  • the cloning and expression of viral IL-10 (BCRF1 protein) from Epstein Barr virus has been disclosed by Moore et al, Science 245:1230 (1990).
  • Recombinant human IL-10 is also an article of commerce, available for purchase e.g., from PreproTech, Inc., Rocky Hill, NJ.
  • Individuals suitable for treatment by the methods of this invention include any individual with a neoplastic disorder that would benefit from stimulation of PBMC cytolytic activity, especially LAK and NK cell activation.
  • exemplary cancer patients are described, e.g., in the patent and Scientific American paper of Rosenberg, supra.
  • Also suitable for treatment by the methods of this invention are individuals predisposed to elevations of endogenous IL-4 levels, such that the EL-4 blocks the IL-2 activation of cytolytic activity. In such individuals, the preferred treatment would involve pre-treatment with IL-10 prior to the administration of IL-2.
  • IL-2 and ⁇ -IFN for use in this invention are also available from commercial sources (e.g., EL-2 is available from Cetus, Corporation, Emeryville, CA and ⁇ -IFN is available from Schering Corp., Kenilworth, NJ).
  • Extra-corporeal activation of PBMCs preferably obtained by standard methods from a patient that is to be treated
  • administration of such cells are carried out essentially as described in the references of Rosenberg mentioned above, except that IL-10 together with reduced levels of IL-2 and/or ⁇ -IFN are used as described herein.
  • the number of activated PBMCs administered is in the range of about 10 6 to about 10 12 cells.
  • administration of such activated cells is accompanied and/or followed by administration of IL-10 as described herein.
  • PBMCs are activated by pre-treatment with EL-10 [e.g., by incubating for about three days at 37°C in the presence of 4 ng/ml (100 units/ml) or 40 ng/ml human IL-10]. Then, the cells are washed to remove free IL-10, and low levels of IL-2 (typically about 2 units/ml) are added.
  • EL-10 e.g., by incubating for about three days at 37°C in the presence of 4 ng/ml (100 units/ml) or 40 ng/ml human IL-10.
  • Administration of cytolytic cells activated by IL-10, alone or in combination with the other cytokines used herein, is preferably by intravenous infusion. This can be carried out, e.g., through a central venous catheter, into a large peripheral vein, or into the hepatic artery via a percutaneous catheter.
  • IL-10 is generally administered as a pharmaceutical composition comprising a pharmaceutical carrier and effective amount of IL-10 alone or in combination with IL-2 and/or ⁇ -IFN.
  • a pharmaceutical carrier can be any compatible non-toxic substance suitable for delivery of the invention to a patient.
  • compositions useful for parenteral administration of such drugs are well-known, e.g., see Remington's Pharmaceutical Science, 15th Ed. (Mack Publishing Company, Easton, PA, 1980).
  • compositions of the invention may be introduced into a patient's body by implantable or injectable drug delivery system, e.g., Urquhart et al, Ann. Rev. Pharmacol. Toxicol. 24:199 (1984); Lewis (Ed.), Controlled Release of Pesticides and Pharmaceuticals (Plenum Press, NY.1981); U.S. Patent No. 3,270,960; and the like.
  • Cytokine administration can be carried out by any of the well known routes of administration, including by intravenous, intraperitoneal and subcutaneous administration. Intravenous administration is preferred.
  • the compositions When administered parenterally, the compositions are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutical carrier.
  • a pharmaceutical carrier examples of such carriers are normal saline, Ringer's solution, dextrose solution, and Hank's solution.
  • Non-aqueous carriers such as fixed oils and ethyl oleate may also be used.
  • a preferred carrier is 5% dextrose/saline.
  • the carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • the IL-10 is preferably formulated in purified form substantially free of aggregates and other proteins at a concentration in the range of about 5 to 20 ⁇ g/ml.
  • compositions of this invention can also be delivered by standard gene therapy techniques, including e.g., direct DNA injection into tissues, the use of recombinant viral vectors and implantation of transfected cells. See, e.g., Rosenberg, J. Clin. Oncol. 70:180 (1992).
  • Co-administration of one or more of the therapeutic agents described herein can be concomitant (together with the administration of the IL-10) or sequential.
  • the IL-10 is administered prior to the administration of the IL-2.
  • Administration of ⁇ -IFN can be concomitant with the IL-10 and/or the IL-2 or sequential. All of the administered agents should be present in the patient at sufficient levels to be therapeutically effective in producing tumor regression.
  • the term "effective amount” means the amount of IL-10 sufficient to reduce or prevent side effects in adoptive immunotherapy and to at the same time promote LAK and NK cell cytolytic activity.
  • the effective amount of cytokine(s) needed for a particular patient may vary depending on such factors as the state and type of the neoplastic disease being treated, the overall health of the patient, methods of administration, the severity of the side effects, the amount and kinds of other drugs being used concurrently, and the like.
  • An amount of a cytokine "sufficient to augment LAK cell activation” is defined herein to mean an amount of a cytokine required to produce at least about a 25% increase in the level of cytolytic activity induced by IL-10 alone, in a cytolytic assay based on Daudi cells.
  • the increase will be at least about 50%, and most preferably, at least about 100%.
  • the IL-10 is administered in the maximally tolerable dose, from about 10 U/kg body weight per day to about 10 8 U/kg body weight per day.
  • IL-2 and ⁇ -IFN are also to be administered in the maximally tolerable dose (e.g., for the IL-2 dose: 10 5 U/kg body weight given intravenously every 8 hours in 50 ml of 0.9% saline with 5% albumin as a carrier; for the ⁇ -IFN dose: 10 6 U/kg body weight given intravenously every 8 hours in 0.9% saline with 5% albumin as a carrier).
  • dosing is to be adjusted by attending physician to fall within those limits determined to be tolerable for each patient individually.
  • the methods of this invention can also be used in conjunction with traditional approaches to the treatment of cancer, such as radiation therapy and chemotherapy, using traditional chemotherapeutic agents such as the Vinca alkaloids, platinum compounds and 5-fluorouracil.
  • chemotherapeutic agents such as the Vinca alkaloids, platinum compounds and 5-fluorouracil.
  • IL-10 alone or in combination with other cytokines elicits an increase in cytolytic activity toward human tumor targets. Since the efficacy of immunotherapy is in large part dependent upon tumor burden, it would be especially beneficial to employ the treatment methods described herein after the bulk of the primary tumor mass has been excised. An inflammatory reaction which typically occurs at the surgical site may also be beneficial to the therapeutic outcome.
  • IL-10 stimulates lymphokine activated killing (LAK) and natural killer (NK) activities in human peripheral blood mononuclear cells.
  • LAK lymphokine activated killing
  • NK natural killer
  • IL-10 driven cytolytic activity can be neutralized by rat monoclonal antibodies against IL-10.
  • IL-10 derived from CHO and E. coli expression systems display similar concentration response patterns in stimulation of LAK and NK activities and are thus biologically equivalent.
  • PBMCs treated with IL-10 and low concentrations of D -2 display LAK activities greater than observed with either cytokine alone.
  • PBMCs pre-treated with IL-10 for 2 days demonstrate increased cytolytic activity upon subsequent addition of IL-2.
  • IL-10 antagonizes the ability of IL-4 to inhibit
  • IL-10 plus IL-2 produces enhanced LAK cytolytic activity at low effector cell:target cell ratios.
  • endothelial cells cultured in the presence of IL-10 demonstrate an unimpaired response to exogenous factors (i.e., to ⁇ -IFN and to TNF- ⁇ ), whereas endothelial cells incubated in the presence of IL-2 were unresponsive due to IL-2 toxicity.
  • Recombinant human IL-10 (both E. coli- and CHO-derived) was produced by standard methods. Specific activities obtained following purification by standard methods were 4.1 x 10 7 (£. coli) and 2.1 x 10 7 units/mg (CHO), as determined by the MC-9 cell proliferation assay [Thompson-Snipes et al, J. Exp. Med. 775:507 (1991)]. Approximately 4 ng of essentially homogeneous D -10 had about 100 units of biological activity as thus defined.
  • a rat anti-human IL-10 monoclonal antibody designated
  • Peripheral blood was obtained by venipuncture from healthy adult donors using heparin or EDTA as an anticoagulant.
  • PBMCs were separated by a two-step protocol consisting of dextran sedimentation followed by centrifugation on FICOLL PAQUE® at 1250 rpm for 30 minutes.
  • the interface bands comprised primarily of lymphocytes and monocytes were collected and washed at least twice with RPMI containing 10% fetal calf serum (complete medium) (JRH Biosciences).
  • the plates were centrifuged for 5 minutes at 1000 rpm before incubation for 4 hours at 37°C in a humidified 5% CO2 atmosphere. After 4 hours the plates were centrifuged for 5 minutes at 500 x g. Supernatants were collected using a SKATRON ® harvester (Skatron Instruments) and counted in a gamma counter (LKB-Pha ⁇ nacia). Total lysis was determined by incubating 51 Cr-labelled targets with 1% SDS. Data were represented as the mean of triplicate determinations.
  • Percent lysis was calculated as follows: cpm released experimental - cpm spontaneous
  • % lysis x 100 cpm total lysis - cpm spontaneous
  • PBMCs isolated as described above were maintained at a concentration of 1 x 10 6 cells/ml in RPMI- 1640 containing 10% fetal calf serum supplemented with IL-10 or human IL-10 (CHO) at 37°C for 3 days unless otherwise specified. Cytolytic activity was determined as described above. b. Simultaneous incubation with EL- 10 and IL-2
  • PBMCs were incubated with 4 ng/ml IL-10 with or without human IL-2 (Genzyme) (2 or 20 U/ml) at 37°C for 3 days.
  • PBMCs peripheral blood mononuclear cells
  • Human IL-2 was added to a final concentration of 2 or 20 U/ml. After overnight incubation, LAK and NK cytolytic activities were determined.
  • PBMCs were incubated with 40 ng/ml IL-10 for 3 days in the presence of 2 ⁇ g/ml of an anti-IL-10 monoclonal antibody (19F1) or an isotypic control (rat IgG2a).
  • Cytolytic activity of LAK and NK cells can be operationally distinguished based on the tumor cell target used. Daudi cells, derived from a human Burkitt's lymphoma, are the traditional targets for activated LAK cell cytolytic activity. Cells from the K562 cell line, a human erythroleukemic cell line, are employed as specific targets for the cytolytic activity of activated NK cells. In these experiments human PBMCs were treated with various concentrations of IL-10 for 3 days. Cytolytic activity was determined in a standard 51 Cr release assay as described above. Results based on LAK activity are shown in Table 1 , in which standard errors are shown below the mean values.
  • the data of Table 1 show the effect of IL-10 on LAK activity in PBMCs obtained from six human donors. Although variability among the donors was evident, IL-10 induced a concentration-dependent increase in cytolytic capacity in all 6 donors. Statistically significant activity (p ⁇ 0.05) was observed at concentrations of IL-10 of 0.4 ng/ml or greater. It was also observed that donor PBMCs displaying a basal level of cytolytic activity (i.e., in the absence of cytokine) of 5% or less were most responsive to IL-10 at all effector cell:target cell ratios tested (data not shown). Similar results were obtained against other tumor target cells, including a human renal cell carcinoma line, two different human melanoma lines and a human colon carcinoma line. The renal carcinoma and melanoma cell lines have also been used by Rosenberg, and patients bearing such tumors have been treated in vivo by Rosenberg using adoptive immunotherapy with IL-2. In all cases, percent lysis of target cells was IL-10 concentration dependent.
  • IL-10 alone or in combination with IL-2 was found to elicit an increase in cytolytic activity against U937 cells (human histiocytoma), SW620 (human colon carcinoma), and SKBR3 cells (human breast carcinoma).
  • Basal NK cytolytic activity i.e., lysis of K562 targets in the absence of cytokines
  • NK activity can be further increased by cytokines such as IL-2 [Perussia, supra', Phillips and Lanier, J. Exp. Med 764:814 (1986)].
  • the effect of IL-10 on NK activity was evaluated in PBMCs from the same 6 donors mentioned above, in experiments run in parallel with the LAK assays. The results are shown in Table 2, in which standard errors are shown below the mean values.
  • IL-10 induced a significant dose-dependent enhancement of NK-cell mediated cytotoxicity in PBCMs from the donors. There was a statistically-significant increase in lytic activity at concentrations of EL- 10 fif 4 ng/ml or greater. As in the case of LAK activity, the effect of IL-10 on NK activity varied from donor to donor.
  • Endothelial cells cultured in the presence of IL-10 demonstrated an unimpaired response to exogenous cytokines ( ⁇ -IFN and TNF- ⁇ ), whereas parallel cultures exposed to unit-equivalent doses of IL-2 for the same incubation period lost the capacity to respond due to IL-2 toxicity.
  • Human PBMCs were incubated with submaximal stimulatory concentrations of IL-2 ( 2 or 20 U/ml) in the presence of IL-10, using an effector-to-target ratio of 5:1, with the results shown in Table 5 in which standard errors are shown beneath the mean values.
  • PBMCs co-incubated with IL-10 and ⁇ -IFN display a similar additive increase in lytic activity against Daudi, but not NK target cells. This is shown in Table 6, in which standard errors are shown beneath the mean values.
  • IL-5, GMCSF or ⁇ -IFN were no more effective than IL-10 alone (data not shown).
  • PBMCs were maintained in medium alone or medium supplemented with
  • IL-10 IL-10. After two days, IL-2 was added to a final concentration of 2 or 20 U/ml. Cytotoxic activity against Daudi cells was assessed following an additional overnight incubation. The results from a 5-donor pool are shown in Table 7, in which stardard errors of the mean are shown beneath the mean values.
  • PBMCs from six human donors were cultured in medium containing 20 U/ml IL-2 alone, 20 U/ml IL-2 plus 1000 U/ml IL-4, or 20 U/ml IL-2 plus 1000 U/ml IL-4 plus 4 ng/ml IL-10.
  • the results are shown in Table 8, in which standard errors of the mean are shown under the mean values.
  • Daudi targets in standard chromium release assay at an effecto ⁇ target cell ratio of 20:1. Data are represented as the mean of triplicate determinations.
  • Human peripyeral blood mononuclear cells isolated from normal donors were treated with 20 U/ml IL-2 for 3 days.
  • c Donor PBMCs were incubated with 20 U/ml IL-2 and 100 U/ml human
  • IL-4 for 3 days.
  • d Donor PBMCs were incubated with 20 U/ml IL-2, 1000 U ml human IL-4, and 4 ng/ml IL-10

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EP94904303A 1994-01-20 1994-01-20 Verwendung von il-10 zur anregung der zytolischen aktivität von mononucleären zellen in peripherem blut Withdrawn EP0740552A1 (de)

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CN94194863A CN1142186A (zh) 1994-01-20 1994-01-20 用白介素-10激活外周血单核细胞的细胞溶解活性
PCT/IB1994/000008 WO1995019780A1 (en) 1994-01-20 1994-01-20 Use of il-10 to stimulate peripheral blood mononuclear cell cytolytic activity

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JP2002532556A (ja) * 1998-12-22 2002-10-02 シェリング・コーポレーション Ｃ型肝炎ウイルス感染のインターロイキン−１０での処置
WO2002026265A2 (en) 2000-09-29 2002-04-04 Schering Corporation Pegylated interleukin-10
RU2322452C2 (ru) * 2006-03-27 2008-04-20 Михаил Николаевич Смирнов Иммуномодулирующая композиция
AU2007314501B2 (en) 2006-09-28 2013-05-23 Merck Sharp & Dohme Corp. Use of pegylated IL-10 to treat cancer
CA2692282A1 (en) * 2007-06-27 2008-12-31 Joslin Diabetes Center, Inc. Regulatory t cells in adipose tissue
RU2549702C2 (ru) 2008-12-17 2015-04-27 Мерк Шарп И Доум Корп., Получение и применение моно- и ди-пэг il-10
CN106913865A (zh) 2013-04-18 2017-07-04 阿尔莫生物科技股份有限公司 使用白细胞介素‑10治疗疾病和病症的方法
EP3010527B1 (de) 2013-06-17 2018-08-08 Armo Biosciences, Inc. Verfahren zur beurteilung der proteinidentität und -stabilität
CN105658232A (zh) 2013-08-30 2016-06-08 阿尔莫生物科技股份有限公司 使用白细胞介素-10治疗疾病和病症的方法
RU2016122957A (ru) 2013-11-11 2017-12-19 Армо Байосайенсиз, Инк. Способы применения интерлейкина-10 для лечения заболеваний и расстройств
WO2015187295A2 (en) 2014-06-02 2015-12-10 Armo Biosciences, Inc. Methods of lowering serum cholesterol
AU2015333827A1 (en) 2014-10-14 2017-04-20 Armo Biosciences, Inc. Interleukin-15 compositions and uses thereof
KR20170084033A (ko) 2014-10-22 2017-07-19 아르모 바이오사이언시스 인코포레이티드 질환 및 장애를 치료하기 위해 인터루킨-10을 사용하는 방법
WO2016126615A1 (en) 2015-02-03 2016-08-11 Armo Biosciences, Inc. Methods of using interleukin-10 for treating diseases and disorders
WO2016191587A1 (en) 2015-05-28 2016-12-01 Armo Biosciences, Inc. Pegylated interleukin-10 for use in treating cancer
WO2017035232A1 (en) 2015-08-25 2017-03-02 Armo Biosciences, Inc. Methods of using interleukin-10 for treating diseases and disorders
IL312238A (en) * 2021-10-25 2024-06-01 Ellennbe Gmbh A pharmaceutical preparation and a kit containing an immunomodulatory agent for the treatment of diseases

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DK0567586T3 (da) * 1991-01-16 1995-12-04 Schering Corp Anvendelse af interleukin-10 ved adoptiv immunterapi af cancer
KR100207766B1 (ko) * 1991-01-16 1999-07-15 둘락 노먼 씨. 신생물 질환 치료용 인터루킨-10 조성물
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AU5842594A (en) 1995-08-08
FI962813A0 (fi) 1996-07-11
AU707019B2 (en) 1999-07-01
JPH09508116A (ja) 1997-08-19
NO963029D0 (no) 1996-07-19
WO1995019780A1 (en) 1995-07-27
CN1142186A (zh) 1997-02-05
PL175343B1 (pl) 1998-12-31
FI962813A (fi) 1996-07-11
NO963029L (no) 1996-09-19

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