SK91696A3 - Use of il-10 to stimulate peripheral blood mononuclear cell cytolytic activity - Google Patents

Abstract

This invention relates to the use of IL-10, alone or in combination with IL-2 and/or alpha -IFN, to treat neoplastic disorders by stimulating cytolytic activity of peripheral blood mononuclear cells.

Description

Technical field

The invention relates to the use of interleukin-10 (IL-10) formally cytokine synthesis factor CSIF inhibitors for adoptive immunotherapy in the treatment of neoplastic diseases (cancer) by stimulating the cytolytic activity of peripheral blood mononuclear cells (PBMCs).

It is prior art

Immunological approaches to treating cancer are based on the recognition that cancer cells have thwarted the body's defense against various or foreign cells and molecules, and that this defense can be therapeutically encouraged to kill or prevent the growth of cancer cells, for example, as discussed. Klein (Immunology, Willey-Interscience, New York, pp. 623-648, 1982).

New insights that immune effectors can directly or indirectly thwart tumor growth have led to a renewed interest in cancer treatment (Heberman, Concepts).

Immunopath. 1: 96 (1985), (natural killer cells

1t prevent tumor growth); Ros.enberg et al. Ann. Rev. Immunol.

: 681 (1988) (use of IL-2 activated frog cells for the treatment of cancer); Ralf et al., J. Exp. Med. 167: 712 (1988) (macrophage destructive activity against tumors, supported by lymphokines); Tepper et al., Cell 57: 503 (1989) (IL-4 has a damaging effect on tumors); M. Cohen, Lymphokines and Tumor Immunity (lymphokines and tumor immunity), p. 237-253, S. Cohen, published Lymphokines and the Immune Response - lymphokines and immune response (CRC Press, Boca Raton, (1990)).

The ability of the immune response to neoplasms is controlled by a variety of cell types and involves the action of T cells and monocyte-derived cytokines. One of the immunological approaches that has been shown to be clinically promising was so-called. adoptive immunotherapy using IL-2 activated killer cells (Rosenberg, Sci. Am., pp. 62-69 (May 1990)). Although IL-2, alone or in combination with several traditional chemotherapeutic agents, appears to be effective in the treatment of certain malignancies (for example, kidney cell carcinoma), some unfortunate toxic side effects such as vascular leakage and swelling associated with the administration of effective doses of IL-2 suggesting that risks could outweigh utility (Cotran et al., J. Immunol. 139: 1882 (1987); Edwards et al. Cancer Res. 52: 3425 (1992)).

Human vascular endothelial cells are very sensitive to the toxicity of IL - 2, as evidenced by increased vascular permeability (i.e., vascular leakage syndrome) and swelling. One of the factors contributing to this pathology may be the consolidation of IL-2 activated T-cell adhesion and neutrophilic leukocytes on a monolayer vascular liner as previously observed in vitro (Edwards et al .; Damle et al. 138: 1779 ( 1987)).

An alternative approach to immunological treatment of neoplastic diseases is the adoptive transmission of immune cells. Adoptive immunotherapy is defined as the delivery to a subject with tumor-active immunological reagents, such as cells with antitumor activity, which may mediate or directly or indirectly antitumor effects. Adoptive immunotherapy is an attractive approach for neoplasm diseases. It should be noted that since active immunological reagents are transferred to an individual, the individual's immune ability is not required. Thus, immune suppression that is commonly associated with a tumor condition is not a major problem with this therapeutic alternative. Since an individual's immune ability is not needed, and may actually be beneficial to the effects of adoptive immune cell transfer, adoptive immunology can be readily combined with other therapies such as chemotherapy or radiation.

Patients undergoing chemotherapy or radiation tend to have lower immunity and will normally have a depleted supply of peripheral blood effector single nuclear cells (PBMCs) available for activation. In such patients, therapy showing efficacy at a low ratio of effector cells to target cells would be highly advantageous.

The ability of the immune response to neoplasms is controlled by a variety of cell types and involves the action of T cells and cytokines derived from monocytes. Adoptive immunotherapy using recombinant human cytokines has developed the administration of peripheral blood mononuclear cells (PBMCs) stimulated outside the body (Phillips et al., Clin. Oncol. 5: 1933: (1987); Perrusia, Curr. Opin. Immunol. 3: 49: ( 1991)) followed by administration of IL-2 (Rosenberg et al. J. Immunol. 138: 1779 (1985)). The extracorporeal processing of PBMCs produces lymphokine activated killer LAKs and natural killer NK cells that have cytolytic effects on various tumor cells.

It is reported (Nagasawa et al. Cell. Immunol. 133: 317 (1991) that human IL-5, IL-7 (Stotter and Lotze, Arch. Surgery 126: 1525 (1191)) and IL-12 (Gately and J. Immunol., 147: 874 (1991)) promote cytolytic activity in human PBMCs, and it appears that interleukin-10 (IL-10), originally described in mice as a cytokine synthesis inhibitor factor, secreted by specific T-cell helper substructures, modulates the differentiation of murine cytotoxic T-cells (Chen and Zlotnik, J. Immunol. 147: 528 (1991)) It has also been found that human PBMCs, incubated with supernatants derived from COS cells transfected with Human IL - 10 cDNAs lyse tumor cell targets in vitro T - cells corresponding to IL - 10 with increased cytolytic potential were identified as a CD 56 + phenotype indicative of NK cells.

Under certain circumstances, IL-4 may adversely affect the formation of LAK activity by IL-2 (Nagler et al. J. Immunol. 141: 2349 (1988). For example, when human PBMCs are cultured in the presence of both IL-2 and IL-2. 4, lysis is greatly reduced in LAK-sensitive targets (Spits et al. J. Immunol. 141: 29 (1988)) If PBMCs are pre-cultured in IL-2 supplemented media for 3 days prior to IL-4 addition, however, it results in cytolytic activity (Spits et al.)). In addition, IL-2-mediated cytotoxicity blockade by IL-4 can be reduced if alpha-interferon (α-IFN) or alpha-tumor necrosis factor (alpha TNF-α) (Swisher) is incorporated into the original incubation mixture. et al., Cell Immunol. 128: 450 (1990)).

Kedar et al. (Cancer Immunol. Immunother. 35: 63 (1992)) recently suggested that sequential administration of IL-2 and α-IFN is an effective immunotherapeutic regimen for the treatment of MCA-105 sarcomas and M109 carcinomas in murine tumor models. The primary finding from this study was that sequential administration of cytokines appears to be more effective than concomitant administration of both cytokines.

The feasibility and efficacy of adoptive immunotherapy as a method of treating a variety of diseases, particularly the treatment of neoplastic diseases (cancer) in humans, is described in U.S. Patent 4,690,915 (Rosenberg). As mentioned above, there is a lack of methods of performing such treatments that are not as toxic as methods that use IL2. Therapy that is effective at low effector to target cell ratios is also needed.

The invention fulfills these requirements by providing a method for administering IL-10 alone or in combination with IL-2 and / or α-IFN to enhance the cytolytic activity of PBMCs, particularly LAK and NK cells.

SUMMARY OF THE INVENTION

The method of treating cancer comprises administering an effective amount of IL-10 activated PBMCs to a subject afflicted with cancer to achieve cancer regression. Preferably, administration of activated PBMCs is accompanied and / or followed by administration of IL-10.

In one embodiment of the invention, IL-10 is administered in combination with an amount of IL-2 sufficient to increase activation of LAK cells but not causing toxic side effects attributable to the use of IL-2 alone.

In another embodiment, IL-10 is administered in combination with an amount of α-IFN sufficient to increase activation of LAK cells.

In yet another embodiment, IL-10 is administered in combination with (a) an amount of IL-2 sufficient to increase LAK cell activation but not causing toxic side effects attributed to the use of IL-2 alone, (b) the amount and

IFN sufficient to increase activation of LAK cells.

The invention further relates to a method of antagonizing the induced cytotoxicity block

IL-2 by endogenous IL-4, comprising administering to a patient in need of such treatment an effective amount of IL-10.

The invention also relates to a pharmaceutical composition comprising IL-10 in combination with IL-2 and / or α-IFN and a pharmaceutically acceptable carrier.

Preferably, human IL-10, IL-2 and α-IFN, most preferably recombinant IL-10, IL-2 and α-IFN are used in said methods and compositions.

The present invention is an improvement over prior art methods that use IL-2 to induce the cytolytic action of NK and LAK cells. The invention greatly reduces the toxic side effects that are a typical consequence of the use of IL-2 in such treatments, by completely eliminating or greatly reducing the amount of IL-2 that must be used.

Unless otherwise defined, the terms used herein have the same meaning as in the art to which the invention is directed.

As used herein, adoptive immunology means therapy involving the transfer of activated functional immune cells to a patient. These cells preferably include LAK and NK cells originating from the current patient being treated.

As used herein, the term regression is defined as a measurable reduction in size of at least one tumor, as commonly measured in the art.

As regards interleukin-10 or IL-10, it is defined herein as a protein having (a) an amino acid perfect sequence (i.e., lack of a secretory leader sequence) of IL-10, as set forth in U.S. Patent Application Serial No. 07 / 917,806 z

On July 20, 1992, which corresponds to the international application WO 91/003349 and (b) has a biological activity that is common to native IL-10. For the purposes of the invention, they are glycosylated (ie, formed in eukariotic cells such as CHO cells) and unglycosylated ( it is chemically synthesized or produced in E. coli IL-10 and can be used interchangeably. Also included are muteins and other analogs, including the BCRF1 protein (Epstein Barr virus viral IL-10), which has the biological activity of IL-10.

IL-10 suitable for use in the invention can be obtained from a variety of sources. For example, it may be isolated from a culture of activated cells that secrete the protein. Further, IL-10, or active fragments thereof, can be prepared by chemical synthesis using standard techniques known in the art (Merrifield, Science 233: 341 (1986) and

Altherton et al., Solid Phase Peptide Synthesis: A Practical Approach, 1989, I.R.L. Press, Oxford).

Preferably, the protein or polypeptide is obtained by a recombinant, using a coding polypeptide of the IL biology with binant acids, molecular biology being Sambrook et al. Molecular isolated nucleic

10. Common literature methods (e.g.

Spring Harbor, New York, (Publish 1ia) Current

Green / Woley, New Relevant to each technique described in

Cloning, A Laboratory Manual, Cold 2nd Edition 1989 and Ausubel et al.

Protocols

York (1987) and can obtain genomic polymerase or cDNA sequences in Molecular periodic by standard library techniques. Reactions (PCR) (e.g.

Biology, supplements)).

PCR Protocols: A Guide to Methods and Applications, 1990, Innis et al. (publisher!) Academic Press, New York.

Libraries are constructed from nucleic acid extracted from appropriate cells (e.g., International Publication No. WO 91/00349, which describes recombinant methods for preparing IL-10. Useful gene sequences can be found, for example, in various sequence databases, such as Gen Bank and BMPL, or nucleic acid and PIR and Swiss-Prot for protein, c / i Inteligenetics Mountain View, California, or the Genetic Computer Group, University of Wisconsin Biotechnology Center, Madison, Wi scons in.

Clones that contain sequences that encode human IL-10 are deposited at the American Type Culture Collection (ATCC), Rockville, Maryland under numbers 68191 and 68192. The identification of other clones that contain sequences encoding IL-10 is accomplished by hybridization. nucleic acid or immunological detection of the encoded protein when an expression vector is used. 01-nucleotide samples based on the deposited sequences disclosed in International Application No. WO 91/00349 are particularly useful. Oligonucleotide sample sequences can also be prepared from conserved related gene domains in other species. Alternatively, degenerate samples based on the IL-10 amino acid sequences may be used.

Standard methods can be used to produce transformed mammalian, yeast or insect cell lines that express a large amount of polypeptide. Exemplary E. coli strains suitable for both expression and cloning include W3110 / ATCC Bi, 2735, X1776 (TCC # 31244), X2282, EE1 (ATCC Mp / 31343). Exemplary mammalian cell lines include COS-7 cells, mouse L cells, and CHP cells (Sambrook (1989) and Ausubel et al., 1987 supplements).

Various expression vectors can be used to express the DNA encoding IL-10. Conventional vectors used to express recombinant proteins in precaryotic or eukaryotic cells may be used. Suitable vectors include the pcD vectors described by Okayama et al., Mol. Cell.

Biol. 3: 280 (183), and Takebe et al., Mol. Cell. Biol. 8: 466 (1988). Other VS40-based mammalian expression vectors include those disclosed by Kaufaman et al. (Mol. Cell. Biol. 1: 1304 (1982)) and those described in U.S. Patent 4,675,285. are particularly useful in monkey COS-7 cells (ATCC No. CRL 1651) as well as in other mammalian cells such as mouse L cells (Pouwels et al. (1989 supplements) Cloning Vectors: A Laboratory Manual, Elsevier, New York).

IL-10 can be produced in soluble form as a secreted product of transformed or transfected yeast or mammalian cells. The peptides can then be purified by standard procedures known in the art. For example, purification operations may include ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, and affinity chromatography (Methods of Enzymology Purification Principles and Practices, Springer, New York, 1982).

Alternatively, IL-10 can be produced in insoluble form as aggregates or inclusion bodies. In such form, IL-10 is purified by standard procedures well known in the art. Examples of purification operations include separating the inclusion bodies from the disrupted host cells by centrifugation and then dissolving the inclusion bodies with a chaotropic agent and a reducing agent so that the peptide assumes a biologically active arrangement. Details of these procedures are described, for example, by Winkler et al. (Biochemistre 25: 4041 (1986))

Winkler et al., (Bio / Technology 3: 9923 (1985), Koths et al. (U.S. Pat. No. 4,569,990).

The nucleotide sequence used to transfect the host cells can be modified according to standard techniques for generating IL-10 or fragments thereof with various desirable properties. Such modified IL-10 may differ from naturally occurring sequences at the level of the primary structure, i. j. amino acids, inserts, substitutions and fusions. The modification can be used in combinations to form the final modified chain of proteins.

Amino acid sequence variants can be prepared for a variety of purposes, including increasing serum half-life, facilitating purification or preparation, improving therapeutic efficacy, and reducing the severity or incidence of side effects in therapeutic use. Amino acid sequence variants are commonly predetermined, non-naturally occurring variants, although others may be translational variants, for example, glycosylated variants or proteins that are conjugated to polyethylene glycol (PEG). Such variants may be used in an embodiment of the invention as long as they retain the biological activity of IL-10.

Modification of polypeptide-encoding sequences can be readily accomplished by various techniques, such as site-directed mutagenesis (Gillman et al. Gene 8: 81 (1987). Most modifications are evaluated by routine screening in a suitable assay for desired properties. WO 91/00349 discloses a number of in vitro assays suitable for measuring IL-10 activity.

Human IL-10 is preferably used for the treatment of humans, although viral ILO-10 or IL 10 from some other mammalian species may be used. The most preferred use of IL-10 is recombinant human IL-10. The preparation of human and murine IL-10 has been described in WO 91/00349. The cloning and expression of viral IL - 10 (BCRF1 protein) from Epstein Barr virus has been described by Moore et al. (Science 248: 1230, 1990).

Recombinant human IL - 10 is also a commercial product of, for example, PreproTech., Inc., Rocky Hill, NJ.

Subjects suitable for treatment with the methods of the invention include all individuals with neoplasm disease in which the stimulation of the cytolytic activity of PBMCs, particularly LAK and NK cells, could be beneficial. Examples of cancer patients are given, for example, by Rosenberg's above-mentioned patent file and an article in the American scientific press. Suitable patients for treatment with the methods of the invention are, for example, individuals with an elevated endogenous IL-4 level, where IL-4 blocks activation of IL-2 cytolytic activity. In such subjects, appropriate treatment would include pretreatment of IL-10 prior to IL-2 administration.

The standard methods and techniques described for the production of IL-10 for use in the present invention can also be used to produce IL-2 and α-IFN. IL-2 and α-IFN are also commercially available (e.g., IL-2 is a commercial product of Cetus Corporation, Emeryville, CA and α-IFN a commercial product of Schering Corp., Kenilworth, NJ).

The extracorporeal activation of PBMCs (preferably obtained by standard methods from the patient to be treated) and the administration of such cells is carried out exclusively according to the above Rosenberger work, except that IL-10 together with reduced levels of IL-2 and / or α-IFN are used as described in this invention. The number of activated PBMCs administered is about 10 to about 10 cells. Preferably, but not necessarily, administration of such cells is accompanied and / or followed by administration of IL-10 as described herein.

In one embodiment, PBMCs are activated by IL-10 pretreatment (e.g., about three days incubation at 37 ° C in the presence of 4 ng / ml (100 units / ml) or 40 ng / ml human IL-10). The cells are then washed to remove free IL-10 and small amounts (typically about 2 units / ml) of IL-2 are added.

Administration of cytolytic cells activated by IL-10 alone or in combination with other cytokines used herein is preferably performed by intravenous infusion. This can be done, for example, by a central intravenous catheter into a large peripheral vessel, or into a hepatic artery by a percutaneous catheter.

IL-10 is conveniently administered as a pharmaceutical composition comprising a pharmaceutical carrier and an effective amount of IL-10 alone or in combination with IL-2 and / or α-IFN. The pharmaceutical carrier may be any non-toxic agent suitable for delivering the composition of the invention to a patient. Compositions suitable for parenteral administration of such drugs are well known (for example, Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, PA, 1980). Alternatively, the compositions of the invention may be introduced into a patient's body by a drug implantation or injection system (e.g., Urguhart et al., Ann. Rev. Pharmacol. Toxicol. 24: 199 (1984); Lewis (ed.) Controlled Release of Pesticides and Pharmaceuticals, Plenum). Press, NY (1981); U.S. Pat. No. 3,270,960).

Administration of cytokines is possible by well known routes, including intravenous, intraperitoneal and subcutaneous. Intravenous administration is preferred.

When formulated parenterally, the compositions are formulated into injectable unit doses (solutions, suspensions, emulsions) together with a pharmaceutical carrier. Examples of such carriers are normal saline, Ringer's solution, dextrose solution, and Hank's solution. Non-aqueous vehicles such as fixed ethyl oleate oils may also be used.

A preferred carrier is a 5% mixture of dextrose and brine. The carrier may contain a small amount of additives such as agents that facilitate isotonicity and chemical stability, for example, buffers and preservatives. Preferably, IL-10 is formulated in purified form without aggregates and other proteins at a concentration of about 5 to 20 µg / ml.

The compositions of the invention may also be delivered by standard gene therapy techniques, including, for example, direct injection of DNA into tissues, using recombinant viral vectors and implantation of transfected cells (e.g., Rosenberg, J. Clin. Oncol. 10: 180 (1992)).

Co-administration of one or more of the disclosed therapeutic agents may be co-administered (along with IL-10 administration) or sequential. For example, administration of IL-10 prior to administration of IL-2 is preferred. Administration of α-IFN may be concurrent with IL-10 and / or IL-2 or sequential. All agents administered should be in the patient's body at a level sufficient for the therapeutic effect of inducing tumor regression.

By an effective amount is meant an amount of IL-10 sufficient to reduce or prevent side effects in adoptive immunotherapy and at the same time promote the cytolytic activity of LAK and NK cells. The effective amount of cytokine required for a particular patient may vary depending upon such factors as the condition and type of neoplasm being treated, the general health of the patient, the route of administration, the severity of side effects, the amount and type of drugs co-administered.

The amount of cytokine sufficient to increase the activation of LAK cells is defined herein as the average amount of cytokines required to generate at least a 25% increase in the level of cytotoxic activity induced by IL-10 alone in a Daudi cell based cytolytic assay. An increase of at least about 50% and most preferably at least about 100% is preferred.

Preferably, IL-10 is administered at a maximum tolerable dose of from about 10 units per kilogram of weight (J / kg) per day up to about 10 units per kilogram of weight (J / kg) per day up to about 10 units per kilogram of weight ( J / kg) per day. Similarly, IL-2 and α-IFN should also be administered at the maximum tolerable dose (for example, for IL-2, the dose is: 10 J / kg body weight, administered intravenously every 8 hours in 50 ml of 0.9% brine with 5% albumin as carrier: for α-IFN, a dose of 10 ⁶ J / kg body weight administered intravenously every 8 hours in 50 ml of 0.9% saline with 5% albumin as carrier). As mentioned above, the dosage will be determined by the physician within the limits determined to be tolerable for each patient individually.

The methods of the invention may also be used in connection with traditional cancer treatments such as radiation and chemotherapy using traditional chemotherapeutic agents such as Vinea alkaloids, platinum compounds and 5-fluorouration1.

Treatment of human single peripheral blood IL-10 nuclear cells alone or in combination with other cytokines induces an increase in cytolytic activity against human target tumor cells. Since the efficacy of immunotherapy is largely dependent on tumor burden, it would be particularly beneficial to apply the methods of treatment described herein after excision of most of the primary tumor mass. The inflammatory reaction typically occurring at the surgical site can also be advantageously therapeutically prevented.

The invention is illustrated in more detail by the following non-limiting examples.

DETAILED DESCRIPTION OF THE INVENTION

Effect of IL - 10 on the cytolytic activity of human peripheral blood single nuclear cells.

The results of an in vitro study of the effect of producing cytolytic activity of human peripheral blood mononuclear cells by IL-10 are summarized as follows:

IL-10 stimulates the activity of lymphokine activated killing (LAK) and natural killer cells (NK) in human peripheral blood mononuclear cells. IL-10-directed cytolytic activity can be neutralized by rat anti-IL-10 monoclonal antibodies.

IL-10 derived from CHO and E. coli expression systems show similar concentration response patterns in stimulating LAK and NK activities and are therefore biologically equivalent.

3. PBMCs treated with IL-10 and low concentrations of IL-2 exhibit LAK activities greater than those observed with each cytokine alone.

4. PBMC pre-treated, IL-10 treated at two days showed increased cytolytic activity following subsequent addition of IL-2.

5. IL - 10, in turn, acts on the ability of IL - 4 to inhibit IL - 2 - induced LAK activity.

IL - 10 together with IL - 2 produce improved cytolytic activity of LAK at low effector cell to target cell ratios.

In addition to the effects observed on PBMCs, endothelial cells cultured in the presence of IL-10 were found to exhibit an incomparable response to exogenous factors (ie, gamma-IFN and TNF-α), with endothelial cells incubated in the presence of IL-2 being unresponsive to toxicity of IL - 2.

Material and methods

Recombinant Human Cytokines and Anti-IL-10 Antibodies Recombinant IL10 (derived from both E. coli and CHO) was prepared by standard methods. After purification by standard methods, activities of 4.1 x 10? (E.

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. 173: 507 (1991)). Approximately 4 ng of substantially homogeneous IL-10 has about 100 units of biological activity as defined herein.

A rat anti-human IL 10 monoclonal antibody, designated 19F1, was obtained from Dr. John Adams of DNAX Molecular Biology Palo Alto, CA.

Isolation of human peripheral mononuclear blood cells (PBMC)

When heparin or EDTA is used as an anti-clotting agent, peripheral blood is obtained from healthy adult donors. PBMCs are separated by a two-operation protocol consisting of dextran sedimentation followed by centrifugation on apparatus D

FICOL PAQUE at 1250 rpm. The interphase junctions, mainly composed of lymphocytes and monocytes, are concentrated and washed at least twice with RPMI containing 10% fetal calf serum (complete medium) (JRH Biosciences).

Cytotoxicity tests

Daudi target cells (LAK sensitive) and K5762 (NK sensitive) with the order numbers CCL 213 and CCL 243 are obtained from the American Type Tissue Collection tissue tissue. et al. (J. Immunol. 141: 29 (1998)). After the culture period, PBMCs are pooled, washed twice and used as effector cells in the β-Cr isotope release assay (Spits et al.). 5 x 10 ^ cells, labeled with the S ^ Cr isotope, are mixed with different amounts of effector cells (E / T = 20: 1; 5: 1 and 2: 1) in 100 μΐ 96-well V-bottom plates. For 1 hour incubation at 37 ° C in a humidified 5% carbon dioxide environment, the plates are centrifuged at 1,000 rpm for 5 minutes. After 4 hours, the plates are centrifuged at 500 x g for 5 minutes. Supernatants were collected by SKATRON® (Skatron Instruments) and counted in a gamma counter (LKB - Pharamcia). Total lysis is determined by incubating the isotope S 1 Cr labeled target cells with 1% SDS. Data are the average of 3 determinations. The percentage lysis is calculated as follows:

cpm released experimentally - cpm spontaneous% lysis = ----------------------------------------- --------- x 100 cpm total lysis - cpm spontaneous

Incubation of human-PBMC with cytokines and / Incubation with IL-10 alone

PBMCs isolated as described are 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 three days, unless otherwise noted. Cytolytic activity is determined as described above.

b) Co-incubation with IL-10 and IL-2

PBMCs are incubated for 3 days with 4 ng / ml IL-10 with or without human IL-2 (Genzyme) (2 or 20 J / ml) at 37 ° C.

(c) Gradual incubation with IL - 10 and IL - 2

The PBMCs are incubated for 2 days with 4 ng / ml IL-10 in complete medium. Human IL-2 is added to a final concentration of 3 or 20 J / ml. After overnight incubation, the cytolytic activities of LAK and NK are determined.

Effect of anti-IL-10 monoclonal antibodies on activation of IL-10 cells of LAK and NK cells.

PBMCs are incubated for three days with 4 ng / ml IL-10 in the presence of 2 µg / ml anti-IL-10 monoclonal antibody (19F1) or isotype control (rat IgG2a).

Effect of IL - 10 on the cytolytic activity of lymphokine activated killer cells and natural killer cells in human peripheral blood mononuclear cells.

The cytolytic activity of LAK and NK cells can be operatively differentiated based on the target tumor cells used. Traditional targets for the cytolytic activity of activated LAK cells are Daudi cells derived from human Burkitt's lymphoma. As specific targets for the cytolytic activity of activated NK cells, cells from the K562 cell line, the human erythroleukemic cell line, are used. In these experiments, human PMBCs were treated for three days at different concentrations of IL-10. Cytolytic activity was determined by the above standard assay of Cr3 release.

Results based on LAK activity are shown in Table I, where standard deviations are below the mean values.

Table I.

IL-10 lymphokine-activated PBMC stimulation (expressed as% lysis ^a )

Concentration of IL-10 (ng / ml)

donor 0 0.04 0.4 4 40 100 1 0 4.25 18.3 44 26.2 67.5 2 0 5.5 7.2 10 31.5 27.6 3 18 17.7 29.2 39.1 29.7 55.1 4 0 8.8 10.2 22.2 35.8 35.9 5 4.6 8.1 15.6 20.6 20.1 12.1 6 14.6 19.7 40.7 54.4 42.9 43.4 average 6.2 10.6 20.2 ^C 31.7 ^C 31,0 ^c 40.2 ^C ± 3.3 ± 2.6 ± 5.1 ± 6.8 ± 3.2 ± 8.0

^and Percent lysis of Daudi ® Cr targets in a standard chromium release assay at an effector to target cell ratio of 20: 1. Data are the mean of <three determinations.

b Human PBMCs, prepared from normal donors, are treated three days before IL-10 is assayed for cytolytic activity.

^c Significant difference between IL-10 treated and intermediate control at p <. 0.05 found by Student's t-test.

The data in Table I show the effect of IL-10 on LAK activity in PBMCs obtained from six human donors. Although diversity among donors is evident, IL - 10 induced a concentration - dependent increase in cytolytic activity in all donors. Statistically significant activity (p <0.05) is observed at IL-10 concentrations of 0.4 ng / ml or greater. It is also evident that the donor PBMCs with a baseline cytolytic activity level (i.e., in the absence of cytokine) of 5% or less, most suggest IL-10 at all effector cell to target cell ratios tested (data not shown).

Similar results were achieved against other target cells, including the human ladin cancer cell line, two different human melanoma lines, and the human colon cancer lines. Rosenberg and melanoma cancer cell lines were also used by Rosenberg, and patients with such tumors were treated with Rosenberg in vivo by adopting IL-2 adoptive immunolotherapy. In all cases, the percentage lysis of target cells was dependent on IL-10 concentration.

In further experiments, IL - 10 alone or in combination with IL - 2 was found to induce an increase in cytolytic activity against U937 (human histiocytoma), SW620 (human colon cancer) and SKBR3 (human breast cancer) cells. In experiments using HS294T (human melanoma) cells as targets, IL - 10 did not appear to elicit a response at the concentrations tested.

The basal cytolytic activity of NK (i.e. lysis of K562 target cells in the absence of cytokines) tends to be higher than the basal activity of LAK. NK activity may be further enhanced by cytokines such as IL-2 (Perussia, supra, Phillips and Lanier, J. Exp. Med. 164: 814 (1986)).

The effect of IL-10 on NK activity was evaluated in PBMCs of the six above-mentioned donors in experiments running in parallel to LAK assays. The results are shown in Table II, where standard deviations are below the mean values.

Table II.

Stimulation of NK activity (expressed as% lysis ^a ) in PBMC by IL-10)

Concentration of IL-10 (ng / ml)

donor 0 0.04 0.4 4 40 100 1 22.2 30.6 25.2 57.2 51.5 49.7 2 20.4 24.7 31.3 56.7 65.6 71.5 3 61.4 55.6 37.9 81.1 80.8 81.5 4 13.8 25.2 23.2 37.5 52.2 54.2 5 13.0 19.9 20.2 25.1 23.5 20.3 6 15.9 25.3 45.4 66.6 43.7 56.1 average 24.4 30.2 30.5 54,0 ^c 52.75 ^c 55.5 ^C ± 7.5 ± 5.2 ± 3.9 ± 8.2 ± 7.8 ± 8.5 and Percent lysis of targets Daudi ⁵ ^ Cr in standard release test chromium at the ratio of effector to by target cells 20: 1 Oda is are center of three destinations

Human PBMCs, prepared from normal donors, are treated three days with IL-10 prior to determining cytolytic activity.

^c Significant difference between IL-10 treated and moderate control at p <0.05 determined by Student's t-test.

As shown in Table II, IL-10 induces a significant dose-dependent improvement in NK cell-mediated cytotoxicity in donor PBMCs. There was a statistically significant increase in lytic activity at IL-10 concentrations of 4 ng / ml or greater. Similar to LAK activity, the effect of IL - 10 on NK was altered in various donors.

Comparison of IL - 2 effects with IL - 10 effects on endothelial cells

Attempts have been made to assess the viability of endothelial cell monolayer after exposure to IL-10.

Endothelial cells cultured in the presence of IL-10 show an incomparable response to exogenous cytokines (gamma IFN and INF-α), with parallel cultures exposed to unit-equivalent doses of IL-2 equally long incubation times lost the ability to respond due to toxicity of IL-10. second

Effect of anti-IL-10 monoclonal antibodies on activation of LAK and NK cells by IL-10

As is apparent from Tables III. and IV., where standard deviations are reported with mean values, the stimulation of cytolytic activity of LAK and NK 40 ng / ml IL-10 is reduced 3-fold (p <0.05) in the presence of 2 µg / ml anti-IL-10 monoclonal antibody 19F1. . Instead, the results generated using 2 µg / ml isotype control rat IgG2a control antibodies resulted in a level of cytolytic activity not statistically indistinguishable from the results obtained with 40 ng / ml IL-10 alone.

Table III.

Inhibition of lymphokine-activated killer cell cytolytic activity induced by IL -10 (expressed as percent lysis ^a ) by anti-IL- monoclonal antibodies

10th

Incubation conditions ^ ³

donor medium 40 ng / ml IL-10 IL - 10 + 19F1 IL-10 + IgG2α 1 5.6 23.5 15.7 28.2 2 4.7 21.3 11.0 17.5 3 5.4 42.5 0.0 35.8 4 2.1 42.0 12.8 26.5 5 4.7 19.1 0.0 11.65

Mean 4.5 ± 0.6 29.6 ± 5.3 7.9 ^C ± 3.3 23.9 ± 4.3 ^a Percent lysis of Daudi targets in a standard chromium release assay at an effector to target cell ratio of 20: 1. Data are the centers of three determinations.

^b Human peripheral blood mononuclear cells isolated from normal donors are treated for three days with 40 ng / ml IL 10 in the presence of 2 mg / ml 19F1 (anti-human IL-10) or rat IgG2a (isotypic control) before determining cytolytic activity.

^(c) Significant difference between IL - 10 processing and a

IL-10 in the presence of anti-IL-10 antibodies at p < 0.05 as determined by Student's t-test.

Table IV.

Neutralization of IL-10 induced destructive cell activity (expressed as percent lysis ^a ) by anti-IL-10 monclonal antibodies.

Incubation conditions ^

donor medium 40 ng / ml IL - 10 IL - 10 + 19F1 IL-10 + IgG2α 1 21.1 38.3 20.6 27.2 2 28.0 71.0 22.2 53.2 3 22.2 51.5 0.0 70.5 4 18.0 25.4 12.3 20.8 5 23.2 53.2 54.5 84.9 average 22.5 ± 1.6 47.9 ± 7.6 19, I ^c ± 6.6 51.3 ± 12.7 and percentage target lysis Daudi ^5l Cr standard chromium release test at effector ratio to target cells 20: 1. Data are the centers of three destinations.

^D Human peripheral blood mononuclear cells isolated from normal donors are treated for three days with 40 ng / ml IL 10 in the presence of 2 mg / ml 19F1 (anti-human IL-10) or rat IgG2a (isotypic control) before determining cytolytic activity.

^c Significant difference between IL-10 processing alone and IL-10 in the presence of anti-IL-10 antibodies at p < 0.05 as determined by Student's t-test.

Cytolytic activity induced by a combination of IL-10 and other cytokines a / Co-incubation of IL-10 and IL-2

Human PBMCs are incubated with sub-maximal stimulatory concentrations of IL-2 (2 or 20 U / ml) in the presence of IL 10 at a effector cell to target cell ratio of 5: 1, with the results shown in Table V. .

Table V.

Induction of lymphokine-activated cytolytic activity (expressed as percent lysis ^a ) in human peripheral blood mononuclear cells by co-incubation of IL-10 and IL-2

Incubation conditions ^

donor medium 2 For IL-2 4 ng of IL-10 IL-10 + IL-2 2 OU IL 1 0.0 3.3 3.7 15.9 24.6 2 2.6 7.6 3.8 23.5 41.0 3 6.1 5.0 13.3 17.7 11.4 4 3.3 50.1 51.6 68.7 80.0 5 2.4 9.4 12.8 29.3 45.7 6 9.9 28.9 16.0 38.8 67.1

Average 4.1 17.4 16.8 32.3 44.9 ^C ± 1.42 ± 7.6 ± 7.2 ± 7.2 ± 10.4

^and percent lysis of Daudi targets _51ς Γ in a standard chromium release assay at an effector to target cells 20: 1 The data are the mean of triplicate determination.

b Human peripheral blood mononuclear cells, isolated from normal donors, are processed three days before the determination of cytolytic activity of 4 ng / ml IL-10 and 2 J / ml IL-2. ^c No significant difference was found between cytolytic activity in PBMC of IL-10 IL-2 treated donors compared to 20 J / ml IL-2 alone at p < 0.05 by Student's t-test.

As shown in Table V., there was an additional increase in LAK activity (effector cell to target cell ratio of 5: 1) after co-incubation with 2 J / ml IL-2 and 4 ng / ml IL-10. greater than the activity with cytokine alone, approaching the level produced by a 10-fold higher concentration of IL-2. This result is statistically significant at p <0.05 as determined by the Student's test.

b) Co-incubation of IL-10 and α-IFN

PBMCs incubated together with IL-10 and α-IFN show a similar increase in lytic activity over Daudi cells but not over NK target cells. This can be seen from Table VI, where the standard deviations are below the mean values.

Table VI.

Induction of lymphokine-activated killer cells activity (expressed as percent lysis ^a ) in human peripheral blood mononuclear cells by co-incubation of IL-10 and α-IFN.

Incubation conditions ¹³

donor medium a-IFN IL-10 IL-10 &lt; + &gt; 1 4.4 7.9 17.8 35.7 2 1.0 11.7 15.4 32.0 3 1.6 12.3 5.5 26.4 Average 2,3 10.6 12.6 31.4 ± 1.0 ± 1.4 ± 3.8 ± 2.7 and percentage target lysis Daudi 51cr v standard test release of chromium at effector ratio to target cells 20: 1. Data are the center of three destinations. b Human single nuclear cells peripheral blood I isolated from no rmal donors are processed for three days for determination cytolytic activity 4 ng / ml IL - 10 and 100 J / ml and IFN. Observed cytotox differences activity evoked in vista PBMC from donors by IL - 10 and IFN v compared to

activities induced by IL-10 and α-IFN alone are statistically significant at p < 0.05 as determined by Student's t-test.

In contrast, co-incubations with IL-10 and IL-4, IL-5, GMCSF or gamma-IFN are not as effective as IL-10 alone (data not shown).

Subsequent incubation with IL - 10 and IL - 2

Using the method described, PBMCs are maintained in media alone or in IL-10 supplemented media. After two days, IL-2 is added to a final concentration of 2 or 20 J / ml. Cytotoxic activity against Daudi cells is assessed after further overnight incubation. The results from five donors are shown in Table VII, where standard deviations are below the mean values.

Table VII.

Induction of lymphokine-activated cytolytic activity (expressed as percent lysis ^a ) in human peripheral blood single nuclear cells by co-incubation with IL 10 followed by IL-2.

Pre-incubation conditions ^ ³

Donor IL-10 ^c IL-2 medium ^d IL-10 IL-2 ^e

1 29.7 21.1 44.8 116.0 2 5.5 18.3 12.2 20.7 3 14.7 58.1 74.5 100.0 4 26.2 59.5 54.3 81.9 5 4.8 28.2 22.5 40.6 average 16.2 37.0 41.7 71.8 ± 5.1 ± 9.0 ± 11.4 ± 17.9

^and Percentage lysis of Daudi 51ς cannon cells _{in a} standard chromium release assay at an effector to target cell ratio of 20: 1. Data are the center of three determinations.

b human peripheral blood mononuclear cells prepared from normal donors are maintained in medium supplemented with 4 ng / ml IL-10 for two days prior to the addition of 2 U / ml IL-2.

Cytolytic activity is detected after an additional overnight incubation.

^c PBMCs from donors were incubated for three days with IL-10.

^d PBMCs from donors were incubated in medium alone before IL-2 addition.

^e Donor PBMCs were incubated from 2 days with IL - 10 before the addition of 20 U of IL - 2nd

As from Table VII. shows that in groups where donor PBMCs were pre-treated with IL-10 prior to IL-2 addition, a 2-fold higher cytolytic activity (78.8 ±

17.9%) compared to the level seen in cultures maintained in complete medium only prior to IL - 2 addition (41.7% ± 11.4). The observed differences between IL-10 pretreated samples and IL-2 pretreated samples were statistically significant at p <0.14.

A similar pattern of improved cytolytic activity is evident in sequential incubations with IL-10 followed by incubations with α-IFN (data not shown).

IL - 2 - induced cytotoxicity block

IL-10 and IL-4.

In a medium containing 20 J / ml IL-2 alone, 20 J / ml IL-2 plus 1000 J / ml IL-4 or 20 J / ml IL plus 1000 J / ml IL-4 plus 4 ng / ml IL- 10 are cultured with PBMCs from six human donors. The results are shown in Table VIII, where the standard deviations are below the mean values.

Table VIII.

Antagonism of IL - 2 blockade of IL - 4 by lymphokine - activated cytological activity by IL - 10 (expressed as percent lysis ^a ) in human peripheral blood single nuclear cells.

donor medium IL-2 ^b IL- ^2c + IL-4 ^IL-2d + IL-4 + IL · 1 12.7 27.6 35.0 45.7 2 5.4 26.3 17.5 33.7 3 1.7 25.1 14.1 36.0 4 3.8 29.5 14.1 22.1 5 3.3 47.1 17.1 63.8 6 6.6 49.5 20.6 40.8

Diameter 5,5 ± 1.6

34.2 ± 4.5

19.7 ± 3.2

40.3 ± 5.7 ^a Percent lysis of Daudi 1 c _r target cells in a standard chromium release assay at a ratio of 20: 1 effector to target cells. Data are the center of three determinations, b Human peripheral blood mononuclear cells isolated from normal donors are processed for three days 20 J / ml IL-2.

^c PBMCs from donors were incubated for three days with 20

J / ml IL-2 and 100 J / ml human IL-4.

d PBMCs from donors were incubated with J / ml IL-2,

1000 J / ml human IL-4 and 4 ng / ml IL-10.

From Table VIII. it appears that approximately 34.2% of the lysis of the LAK-sensitive target cells after treatment with 20 J / ml IL-2 alone is apparent. If 1000 J / ml human IL-4 is incorporated at the beginning of the incubation period, the cytolytic activity is suppressed approximately twice. However, this suppression caused by IL - 4 was not observed when 4 ng / ml IL - 10 was added during the first 24 hours of incubation.

There is no significant difference between the results produced by IL-2 alone and those produced by all three cytokines together (p <0.05, as determined by the Student's test), indicating that antagonism of ILO-10 blockade is essentially complete.

Without departing from the spirit and scope of the invention, various modifications and variations of the invention are possible, as will be apparent to those skilled in the art. Specific embodiments are offered herein by way of example only, and the invention is limited only by the following claims.

Industrial usability

The use of interleukin-10 (IL-10), a formally attenuated cell synthesis factor, for the manufacture of pharmaceutical compositions for adoptive immunotherapy in the treatment of neoplastic diseases (cancer) by stimulating the cytolytic activity of peripheral blood mononuclear cells.

Claims (11)

Patent claims 1. Method of treatment cancer, y z n and s a t ý m that is served effective amount PBMC activated IL - 10 individuals suffering from cancer, for causing retreat such cancer. Second Method according to claim 1, you z n and s a by administering simultaneously or sequentially to an individual an effective amount of IL-10. 3. A method for the manufacture of a pharmaceutical composition for treating cancer, comprising admixing IL-10 activated PBMCs with a pharmaceutically acceptable carrier. The method of claims 1 to 3, characterized in that IL-10 is administered in combination (a) with an amount of IL-2 sufficient to increase LAK cell activation but not causing toxic side effects and / or (b) with an amount of IL-2. and IFN sufficient to increase activation of LAK cells. A pharmaceutical composition for the treatment of cancer, characterized in that it comprises IL-10 activated PBMC and a pharmaceutically acceptable carrier. The pharmaceutical composition for the treatment of cancer according to claim 5, further comprising IL-10 alone or in combination with IL-2 and / or α-IFN. Use of IL-10 activated PBMCs for the treatment of cancer. Use of IL-10 activated PBMCs for the manufacture of a medicament for the treatment of cancer. The use of claim 7 or 8, wherein the IL-10 activated PBMCs are for use with IL-10 alone or in combination with IL-2 and / or α-IFN. 10. A method of antagonizing IL2 induced cytotoxicity by endogenous IL-4, comprising administering to a patient in need thereof an effective amount of IL-10. The use of IL-10 for the manufacture of a medicament for antagonizing IL-2 induced blockade of cytotoxicity by endogenous IL-4. The method, pharmaceutical composition and use of claims 1 to 11, wherein the IL 10 is human IL-10.