JPH09508116A - Use of IL-10 to stimulate peripheral blood mononuclear cytolytic activity - Google Patents

Abstract

  (57) [Summary] The present invention relates to the use of IL-10 alone or in combination with IL-2 and / or α-IFN to treat neoplastic diseases by stimulating the cytolytic activity of peripheral blood mononuclear cells.

Description

Detailed Description of the Invention             Use of IL-10 to stimulate peripheral blood mononuclear cytolytic activity   The present invention, by stimulating the cytolytic activity of peripheral blood mononuclear cells (PBMC), a neoplastic disease ( For the use of adoptive immunotherapy in the treatment of cancer. Leukin-10 (IL-10), formally related to the use of cytokine synthesis inhibitor (CSIF) .                                Background of the Invention   Immunological approaches to the treatment of cancer include, for example, Klein (Immunology, Wiley-Int erscience, New York, 1982, pp.623-648). , Somehow defuse the body's defenses against abnormal or foreign cells and molecules Evasion, and these defenses, can block or inhibit the growth of cancer cells. It is based on the concept that it can be stimulated therapeutically. Immune effector The recent observation that tumor growth can be directly or indirectly inhibited has been associated with cancer treatment. Has led to new interest in this approach [Heberman, Concepts Immu nopath. 1:96 (1985) (natural killer cells resist tumor growth); Rosenberg Et al. Ann. Rev. Immunol. 4: 681 (1988) (use of IL-2-activated killer cells to t reat cancer); Ralf et al., J. Exp. Med 167: 712 (1988) (tumoricidal activity of ma. crophages stimulated by lymphokines); Tepper et al., Cell 57: 503 (1989) (IL-4 h as tumoricidal activity); M. Cohen, "lymphokines and Tumor Immunity], pp.2 37-253 S. Cohen, Lymphokines and the Immune Response (CRC Press, Boca Rat on, 1990)]].   Immune responsiveness to neoplasms is regulated by various cell types, and It is accompanied by the action of cytokines derived from T-cells and mononuclear cells. One showing clinical promise The immunological approach of the so-called "adoptive immunization method" using IL-2 activated killer cells [Rosenberg, supra, Sci. Am. pp.62-69 (May 1990)]. IL-2 alone or Combinations with more addictive chemotherapeutic agents have been associated with certain malignancies (e.g. , But is associated with the administration of an effective dose of IL-2, For unwanted toxic side effects such as vascular bed leakage and edema Suggest that risk outweighs profit [Cotran et al., J. Immunol. 139: 1882 ( 1987); Edwards et al., Cancer Res. 52: 3425 (1992)].   Human vascular endothelial cells have increased vascular permeability (i.e. vascular leak syndrome) and It appears to be particularly susceptible to IL-2 toxicity, as evidenced by edema. This disease One of the contributing factors to science, as previously noted in vitro [Edwards et al. , Supra; Damle et al., 138: 1779 (1987)], endothelial monolayers of IL-2-activated T cells and neutrophils. Can be an increase in adhesions to.   Another therapeutic approach to immunological treatment of neoplastic diseases is adoptive transfer of immune cells. It is on. Adoptive immunity mediates anti-tumor effects either directly or indirectly Tumor-bearing host with active immunological agents, such as cells with potential anti-tumor activity Is to be populated. Adoptive immunity is an attractive treatment for neoplastic diseases Show approach. Since the active immunological drug is transferred to the host, It should be noted that immunocompetence is not required. Therefore, one Immunosuppression, commonly associated with tumor-bearing conditions, poses a major challenge to this treatment choice. Not shown. The immunity of the host is not required, and in fact the effect of adoptive transfer of immune cells Adoptive immunization is not used for other purposes such as chemotherapy or radiation therapy because it can benefit the fruit. Can be easily combined with the above treatment methods. Also, in contrast to other treatments, immunosuppression Seems not to be the cause of this treatment.   Patients undergoing chemotherapy or radiation therapy may be immunocompromised (immunocomprom Peripheral blood mononuclear cell (PBMC) effects that tend to be ised) and available for activation The supply of tar is generally small. Therefore, in the ratio of low effector cells: target cells Efficient therapies would be particularly beneficial to such patients.   Immune responsiveness to neoplasms is regulated by various cell types, and T- It is accompanied by the action of cytokines derived from cells and monocytes. Recombinant human cytokine The adoptive immunization method used involves the administration of in vitro stimulated peripheral blood mononuclear cells (PBMC). Include [Phillips et al., J. Clin. Oncol. 5: 1933: (1987) ； Perussia, Curr.Opin.Immuno l.3: 49: (1991)], followed by IL-2 [Rosenberg et al., J. Immunol. 138: 1779 (198). Five)]. In vitro treatment of PBMC involves activation of activated lymphocyte-activated killer (LAK) cells and And activated natural killer (NK) cells with cytolytic activity against various tumor cells Generate a cell.   Recombinant human IL-5 [Nagasawa et al., Cell. Immunol. 133: 317 (1991)], IL-7 [Stotter And Lotze, Arch. Surgery 126: 1525 (1991)] and IL-12 [Gately et al., J. Immunol. .147: 874 (1991)] was reported to stimulate cytolytic activity in human PBMCs. Was. Interleukin-10 (IL-10) was initially identified as a specific helper T-cell in mice. Described as a cytokine synthesis inhibitor secreted by vesicle subsets. It appears to regulate the differentiation of mitotoxic T-cells [Chen and Zlotnik, J. Immuno. l 147: 528 (1991)]. Recovered from COS cells transfected with human IL-10 cDNA Human PBMCs incubated with incubated supernatant target tumor cells in vitro Was also found to dissolve. Responds to IL-10 with increased cytolytic potential Having T-cells were identified to be the CD56 + phenotype implying NK cells.   In some situations, IL-4 adversely affects the production of LAK activity by IL-2. Obtain [Nagler et al., J. Immunol. 141: 2349 (1988)]. For example, human PBMC has IL-2 and IL Lysis of LAK-sensitive targets is greatly reduced when cultured in the presence of both -4 [ Spits et al., J. Immunol. 141: 29 (1988)]. However, IL-2 was added to the PBMC before it was added. Cytolytic activity increases when pre-cultured in supplemented medium for 3 days [Spits et al. Above]. In addition, alpha interferon (alpha-IFN) or tumor necrosis factor alpha (TNF-alpha) , The cytotoxicity of IL-2 when included in the initial incubation mixture Inhibition by IL-4 can be reduced [Swisher et al., Cell. Immunol. 128: 450 (1990)].   Kedar et al. [Cancer Immunol. Immunother. 35:63 (1992)] recently reported IL-2 and α. -Continuous administration of IFN treats MCA-105 sarcoma and M109 cancer in a mouse tumor model It was shown to be an effective immunotherapy prescription for placement. The main findings of this research are Sequential administration of cytokines is more effective than simultaneous administration of both cytokines Was to have.   Therapies for the treatment of various diseases, especially neoplastic diseases (cancer) in humans The potential and efficacy of adoptive immunization as a formula has been demonstrated by Rosenberg et al., U.S. Pat. Described in No. 15. However, as already mentioned above, treatment with IL-2 alone There is a need for methods of performing treatments that are not as toxic as. Low percentage effectors -Cells: There is also a need for therapeutics that are effective with target cells.                                Summary of the Invention   The present invention is directed to the cytolytic activity of PBMC cells, in particular of LAK cells and NK cells. Use of IL-10 alone or in combination with IL-2 and / or α-IFN to increase sex These needs are met by providing a method of interlocking use.   More particularly, the present invention provides an effective amount of IL-10 activated PBMC to patients suffering from cancer. Method of treating cancer comprising administering to reverse the symptoms of such cancer I will provide a. Preferably the administration of activated PBMC is concomitant with the administration of IL-10 and / or Or later.   In one embodiment, IL-10 increases activation of LAK cells, but IL-2 alone. Administration in combination with IL-2 in an amount sufficient to prevent toxic side effects caused by single administration Given.   In another embodiment, IL-10 has an amount of α sufficient to increase LAK cell activation. -Administered in combination with IFN.   In yet another embodiment, IL-10 (a) increases LAK cell activation, but I A sufficient amount of IL-2 and (b) LA to cause no toxic side effects due to the single administration of L-2 It is administered in combination with α-IFN in an amount sufficient to increase activation of K cells.   The invention further provides a method of neutralizing the inhibition of IL-2-induced cytotoxicity by endogenous IL-4. Depending on the need for such treatment, an effective amount of IL-10 may be administered to the patient. A method is provided that includes:   Furthermore, the present invention relates to IL-2 and / or α-IFN, and a pharmaceutically acceptable carrier. Provided is a pharmaceutical composition containing IL-10 in combination with L.   Preferably human IL-10, IL-2 and α-IFN are present in the above methods and compositions. Most preferably recombinant human IL-10, IL-2 and α-IFN You.                             Detailed description of the invention   All references cited herein are therefore incorporated by reference. The whole is incorporated by reference.   The present invention uses IL-2 to induce cytolytic activity in NK and LAK cells. It is an improvement on the prior art methods used. The present invention typically resides in prior art methods. To eliminate the toxic side effects caused by the use of IL-2, completely eliminate IL-2, or Significantly reduced by greatly reducing the amount of IL-2 to be used.   Unless otherwise defined, various terms used in this specification are directed to the present invention. Has the same meaning as is well understood in the art.   As used herein, the term "adoptive immunity" refers to activated functional immunological immunity. Treatment of the bleb to the patient. Preferably these cells Includes LAK cells and NK cells that originate from the actual patient undergoing treatment.   The term "receding" refers to one or more, as routinely measured in the art. These are defined herein as meaning a measurable reduction in tumor size. Is meant   As used herein, "interleukin-10" or "IL-10" refers to (a) US Patent application No. 07 / 917,806, filed June 20, 1992 (corresponding to International Application No. WO 91/00349 ) Of mature (e.g., lacking a secretory leader sequence) IL-10 A protein having an amino acid sequence and (b) a biological activity common to natural IL-10. Is defined as having a protein. For the purposes of the present invention, glycosylation (eg IL-10, produced in eukaryotic cells such as CHO cells, and non-glycosyl IL-10 (e.g., chemically synthesized or produced in E. coli) is Valuable and can be used interchangeably. In addition, mutein BCRFI (Epstein B), which has the biological activity of IL-10, is also included. arr Virus viral IL-10) protein.   IL-10 suitable for use in the present invention can be obtained from many sources. example For example, IL-10 was isolated from the culture medium of activated cells secreting this protein. Can be Furthermore, IL-10, or an active fragment thereof, is not known in the art. It can be chemically synthesized using standard techniques known in the art. Merrifield, Science 233: 3 41 (1986) and Atherton et al., Solid Phase Peptide Synthesis: A Practical Appr. oach, 1989, I.R.L. See Press, Oxford.   Preferably the protein or polypeptide encodes an IL-10 polypeptide Obtained by recombinant technology using the isolated nucleic acid. General of molecular biology For example, Sambrook et al., Molecular Cloning, A laboratory Manual, Co. ld Spring Harbor, New York, 2nd Edition, 1989 and Ausubel et al. (eds.) Current Protocol s in Molecular Biology, Green / Woley, New York (1987 and special edition). Have been. Appropriate sequences are standardized from genomic or cDNA libraries Can be obtained using conventional techniques. Polymerase chain reaction (PCR) techniques may also be used. You. For example, PCR Protocols: A Guide to Methods and Applications, 1990, Innis (Eds.), Academic Press, New York, New York.   The library is constructed from nucleic acids extracted from the appropriate cells. For example, IL See International Application Publication No. WO 91/00349, which discloses a recombination method for producing -10. When. For example, various sequence databases (eg GenBank and BMPL or nuclei Swiss-Prot, c / o Intelligeneti for c acid and PIR, and proteins cs, Mountain View, California, or Genetics Computer Group, Universit y of Wisconsin Biotechnology Center, Madison, Wisconsin Useful Genetics Child sequences can be found and are incorporated herein by reference.   A clone containing the sequence encoding human IL-10 is an American type culture. Collection (ATCC), Rockville, Maryland with accession numbers 68191 and 68192. Was entrusted. The identification of other clones containing sequences encoding IL-10 is described in Expression Vectors. Nucleic acid hybridization or encoding protein exclusion Performed by any of the epidemiological detections. Opened in International Application Publication No. WO 91/00349 Oligonucleotide probes based on the deposited sequences shown are particularly useful. Oh Is the ligonucleotide probe sequence also a conserved region for related genes in other species? Can also be prepared. Alternatively, degenerate based on the amino acid sequence of IL-10 Probes may also be used.   Transformed prokaryotic cell line expressing large amount of polypeptide, transformed mammalian cell line , To produce a transformed yeast cell line, or a transformed insect cell line, Methods can be used. An exemplary E. coli suitable for both expression and cloning Strains are W3110 (ATCC Bi, 27325), X1776 (ATCC No. 31244), X2282, RR1 (ATCC Mp / 31343). including. Exemplary mammalian cell lines include COS-7 cells, mouse L cells and CHP cells. Including. See Sambrook (1989) and Ausubel et al., 1987 supplement.   Various expression vectors can be used to express DNA encoding IL-10 . Used to express recombinant proteins in prokaryotic or eukaryotic cells Customary vectors can be used. Preferred vectors are Okayama et al., Mol. .Cell.Biol 3: 280 (1983); and Takebe et al., Mol.Cell.Biol. 8: 466 (1988) Included pcD vector. Other SV40-based mammalian expression vectors are aufman et al., Mol. Cell. Biol. 2: 1304 (1982) and U.S. Pat.No. 4,675,285. Included vectors. These SV40-based vectors are available in COS-7 monkey cells ( ATCC No. CRL1651), and especially in other mammalian cells such as mouse L cells. Useful. Pouwels et al. (1989 and special edition) Cloning Vectors: A Laboratory Manual , Elsevier, New York.   IL-10 can be used to transform transformed or transfected yeast cells or mammalian cells. It can be produced in solubilized form, such as the secreted product of animal cells. Then this peptide Can be purified by standard procedures known in the art. For example, the purification process Ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis , Affinity chromatography and the like. Methods in Enzymolog y Purification Principles and Practices (Springer-Verlag, New York, 1982) See.   Alternatively, IL-10 is produced in an insolubilized form such as an aggregate or inclusion body. Can be done. Such forms of IL-10 are compatible with standard procedures well known in the art. Therefore, it is purified. An example of a purification process is by centrifugation from crushed host cells. Inclusion body separation, then inclusion body solubilization with chaotropic agents, and peptization It involves the reduction of a drug to give it a biologically active structure. Of these steps For details, see, for example, Winkler et al., Biochemistry, 25: 4041 (1986); Winkler et al. See Bio / Technology 3: 9923 (1985); Koths et al. And US Pat. No. 4,569,790. That.   The nucleotide sequences used to transfect host cells may vary. Standard for making IL-10 or fragments thereof with the desired properties of It can be modified according to the technique. Such modified IL-10 includes, for example, amino acids, insertions , Substitutions, deletions and fusions alter the naturally occurring sequence at the primary structural level. Let obtain. These modifications are often combined in order to produce the final modified protein chain. Can be used together.   Amino acid sequence variants can be prepared for a variety of purposes under consideration, with serum half-life , Facilitating purification or preparation, improving therapeutic effect, and healing. Includes reducing the severity or occurrence of side effects during therapeutic use. Amino acid sequence A variant is a predetermined variant that is not normally found in nature, but Post-mutants (e.g., glycosylation variants, or polyethylene glycol (PEG) To which it is bound, etc.). Such mutants are As long as the biological activity of L-10 is retained, it can be used in the present invention.   Modification of the sequence encoding the polypeptide is a site-specific mutation [Gillman et al., Gene 8: 81 (1987)] and can be easily achieved by various techniques. Most of the modifications It is evaluated by routine screening in an assay appropriate for the desired characteristics. example For example, International Application Publication No. WO 91/00349 describes a number of suitable assays for measuring IL-10 activity. An in vitro assay was described.   Preferably human IL-10 is used for the treatment of humans, but viral IL-10 or IL-10 from several other mammalian species could probably also be used. Most preferred The IL-10 used is recombinant human IL-10. Of human IL-10 and mouse IL-10 The preparation was described in International Application WO 91/00349. Epstein-Barr virus Cloning and expression of the native virus IL-10 (BCRF1 protein) is described by Moore et al., Sc. ience 248: 1230 (1990). Recombinant human IL-10 has also been described, for example, in Prepro It is a product that can be purchased commercially from Tech, Inc., Rocky Hill, NJ.   Individuals suitable for treatment by the methods of the invention have PBMC cytolytic activity, especially LAK cell activity. Any with neoplastic disease that would benefit from stimulation of sexual activation and NK cell activation Including individuals. Specific cancer patients are described, for example, in the above patent and Rosenberg's Scien. Described in a tific American paper. Also suitable for treatment by the method of the invention. The body is a patient who is pre-treated with elevated levels of endogenous IL-4, resulting in IL-4 Blocks IL-2 activation of cytolytic activity. In such individuals, A preferred treatment involves pre-treatment with IL-10 prior to IL-2 administration.   The described standard methods and techniques for making IL-10 for use in the present invention are: , It can also be used to make IL-2 and α-IFN. For use in the present invention IL-2 and α-IFN are also available from commercial sources (eg IL-2 is Cet Available from us, Corporation, Emeryville, CA, and α-IFN is Scherimg C orp., Kenilworth, NJ).   PBMC (preferably obtained by standard methods from the patient to be treated) Exoactivation and administration of such cells results in IL-10 at reduced levels of IL-2 and <RTIgt; / or </ RTI> except that it is used as described herein with α-IFN Is performed as described in the Rosenberg reference above. Activated P administered The number of BMC is about 10⁶Cells to about 10¹²Within the range of cells. Preferably not always required However, the administration of such activated cells will result in the administration of IL-10 as described herein. Administration is simultaneous and / or later.   In one embodiment, PBMCs have been previously treated with IL-10 (eg, 4 ng / ml (10 0 unit / ml) or 40 ng / ml of human IL-10 at 37 ° C for about 3 days. Beate) to activate. The cells were then depleted of free IL-10 To wash and add low levels of IL-2 (typically about 2 units / ml).   IL-10 alone or in combination with other cytokines used herein Administration of the reactivated cytolytic cells is preferably by intravenous injection. this child Is, for example, via a central venous catheter, into the great peripheral vein, or percutaneous catheter. It is performed in the hepatic artery via tel.   IL-10 is generally a pharmaceutical carrier and an effective amount of IL-10 alone or IL-2. And / or as a pharmaceutical composition comprising a combination with α-IFN. The pharmaceutical carrier is any compatible, non-toxic substance suitable for delivery of the invention to the patient. possible. Compositions useful for parenteral administration of such agents are well known and include, for example: Remington's Pharmaceutical Science, 15th Edition (Mack Publishing Company, Easton, P A, 1980). Alternatively, the composition of the invention is implantable or injectable. Efficient drug delivery system (eg Urquhart et al., Ann. Rev. Pharmacol. Toxicol. 24: 1). 99 (1984); Edited by Lewis, Controlled Release of Pesticides and Pharmaceuticals ( Plenum Press, NY, 1981); US Pat. No. 3,270,960, etc.) Can be entered.   Cytokine administration can be by any known route of administration, including intravenous administration. , Intraperitoneal and subcutaneous administration. Intravenous administration is preferred.   When administered parenterally, the composition is in unit dosage form with a pharmaceutical carrier. It is formulated in injectable form (solution, suspension, emulsion). Examples of such carriers With normal saline, Ringer's solution, dextrose solution and Hank's solution is there. Non-aqueous carriers such as fixed oils and ethyl oleate may also be used. Could be used. The preferred carrier is 5% dextrose / saline. . Carriers may be osmotic and chemically stable, eg buffers and preservatives. Small amounts of additives such as sex enhancing substances may be included. IL-10 is preferably Substantially free of aggregates and other proteins at concentrations ranging from about 5 to 20 μg / ml Formulated in a purified form.   The compositions of the present invention also include standard gene therapy techniques (e.g., direct DNA injection into tissues). Input, use of recombinant viral vectors and transplantation of transfected cells Including. ). For example, Rosenberg, J. Clin. Oncol. 10: 180 (1992 )checking.   Co-administration of one or more therapeutic agents described herein is concomitant (with administration of IL-10. ) Or continuous. Preferably IL-10 is administered prior to the administration of IL-2. α-IFN administration is myelinated or continuous to IL-10 and / or IL-2 . All doses at levels sufficient to be therapeutically effective in tumor regression Should be present in the patient.   The term "effective amount" as used herein reduces side effects in adoptive immunization. Or sufficient to block or at the same time the cytolytic activity of LAK and NK cells. By IL-10 is meant a sufficient capacity to promote sex. The services required for a particular patient The effective dose of itocaine depends on the condition and type of neoplastic disease being treated, the patient's overall Health, mode of administration, severity of side effects, amount of other drugs used at the same time and It may change depending on factors such as type.   The amount of cytokine "sufficient to increase activation of LAK cells" is -Based cytolytic activity induced by IL-10 alone Cytokines required to produce an increase in sex levels of at least about 25% As defined herein. Preferably, the increase is at least about 50% And most preferably at least about 100%.   Preferably, IL-10 is administered at a maximally tolerated dose of about 10 U / day. / kg body weight ~ 10⁸U / kg body weight. Similarly, IL-2 and α-IFN are also maximally tolerated (Eg, IL-2 dose: 10^FiveU / kg body weight as a carrier 50 ml of 0.9% saline with 5% albumin was given intravenously every 8 hours. The dose of α-IFN is: 10⁶U / Kg body weight has 5% albumin as carrier 0.9% saline, given intravenously every 8 hours). As mentioned earlier, The dosage should be adjusted by such physician and determined to be acceptable to the individual patient. Within the capacity limits.   The method of the invention also provides a habitual approach to cancer treatment (e.g., radiation therapy). Method of, and Vinca alkaloids, platinum compounds, and 5-fluorouracil Chemotherapy using such customary chemotherapeutic agents).   Human peripheral blood mononuclear cells with IL-10 alone or in combination with other cytokines Treatment leads to increased cytolytic activity against human tumor targets. The efficacy of immunotherapy , Depends in large part on the tumor burden, so large volumes of primary tumor It is particularly useful to use the treatment methods described herein after being ablated . Inflammatory reactions, which typically occur at the surgical site, may also be beneficial for therapeutic outcome.                                  Example   The following non-limiting examples are provided as illustrations of the invention.Effect of IL-10 on cytolytic activity in human peripheral blood mononuclear cells   IL-10 for in vitro generation of cytolytic activity in human peripheral blood mononuclear cells Studied the effect. The results can be summarized as follows.   1. IL-10 is a lymphokine-activated killer (LAK) in human peripheral blood mononuclear cells Stimulates activity and natural killer (NK) activity. IL-10's cytolytic activity , Can be neutralized by a rat monoclonal antibody against IL-10.   2. IL-10 derived from CHO expression system and E. coli expression system has LAK activity and NK activity. Show similar concentration response patterns upon stimulation and are therefore bioequivalent.   3. PBMC treated with IL-10 and low concentrations of IL-2 showed that either cytokine alone It shows higher LAK activity than that observed by itself alone.   4. PBMCs that had been treated with IL-10 for 2 days in advance had increased fines upon subsequent administration of IL-2. Shows cytolytic activity.   5. IL-10 weakens the ability of IL-4 to inhibit IL-2-induced LAK activity.   6. Addition of IL-2 to IL-10 increased the effector cells to target cells at a low ratio. It produces an enhanced LAK cytolytic activity.   In addition to the effects seen in PBMC, endothelial cells cultured in the presence of IL-10 are exogenous Show an undiminished response to factors (ie, γ-IFN and TNF-α) Was found, but endothelial cells incubated in the presence of IL-2 showed toxicity of IL-2. I didn't respond.Materials and methods Recombinant human cytokine and anti-IL-10 antibody   Recombinant human IL-10 (both E. coli and CHO) was prepared by standard methods. Generated. The specific activity obtained following purification by standard methods is based on the MC-9 cell growth assay. Essay [Thompson-Snipes et al., J. Exp. Med. 173: 507 (1991)] Sea urchin 4.1 × 10⁷Unit / mg (E.coli) and 2.1 x 10⁷Unit / mg (CHO) . Therefore, about 4 ng of essentially homogeneous IL-10 has about 100 units of biological activity. Stipulated.   A rat anti-human IL-10 monoclonal antibody called 19F1 was added to Molecular Biology. Obtained from Dr. John Abrams of the DNAX Institute, Palo Alto, CA.Isolation of human peripheral blood mononuclear cells (PBMC)   Peripheral blood was tested on healthy adult donors using heparin or EDTA as an anticoagulant. Obtained by venipuncture. PBMCs were dextran precipitated followed by FICOLL PAQU Boundary bands consisting mainly of lymphocytes and monocytes were collected, and 10% fetal calf Washed at least twice with RPMI (complete medium) containing serum (JRH Biosciences).Cytotoxicity assay   Daudi (LAK-sensitive) target cells and K562 (NK-sensitive) target cells were  Obtained from the Tissue Collection under accession numbers CCL 213 and CCL 243, respectively. Sp its et al. [J. Immunol. 141: 29 (1988)], as described by Daudi and K562. To⁵¹Labeled with Cr. After the culture period, PBMC cells were harvested, washed twice, and⁵¹Cr Used as effector cells in the release assay (Spits et al., Supra). V-shaped bottom In 100 μl of 96 well plate,⁵¹5 × 10 labeled with Cr label^ThreeTarget cells of Mix with varying numbers of effector cells (E / T = 20: 1; 5: 1 and 2: 1) Was. This plate is moistened with 5% CO₂Incubate at 37 ° C for 4 hours in atmosphere Prior to centrifugation at 1000 rpm for 5 minutes. After 4 hours, plate for 5 minutes, 500 × g And counted on a gamma counter (LKB-Pharmacia).⁵¹Marks labeled with Cr Total lysis of cells was determined by incubating the target with 1% SDS. data Was expressed as the average of three measurements.   Percent lysis is calculated as follows: Incubation of human PBMC with cytokines a. Incubation with IL-10 alone   PBMCs isolated as described above contained 10% fetal bovine serum unless otherwise specified, 1 x 10 in RPMI-1640 supplemented with IL-10 or human IL-10 (CHO)⁶Cell / ml concentration And maintained at 37 ° C for 3 days. Cytolytic activity was measured as described above. b. Co-incubation with IL-10 and IL-2   4 ng / ml IL-10 and PB with or without human IL-2 (Genzyme) (2 or 20 U / ml) MC were incubated at 37 ° C for 3 days. c. Continuous incubation with IL-10 and IL-2   PBMC were incubated with 4 ng / ml IL-10 in complete medium for 2 days. Human IL-2 Was added at a final concentration of 2 U / ml or 20 U / ml. After overnight incubation, LA K cell lysis activity and NK cell lysis activity were measured.Effect of anti-IL-10 monoclonal antibody on IL-10 activity of LAK and NK cells   2 μg / ml anti-IL-10 monoclonal antibody (19F1) or isotype Incubate with 40 ng / ml IL-10 in the presence of control (rat IgG2a) for 3 days did.Lymphokine-activated killer cells and natural killer in human peripheral blood mononuclear cells Effect of IL-10 on cytolytic activity of cells   The cytolytic activity of LAK and NK cells is based on the target tumor cells used , Can be distinguished operationally. Derived from human Burkitt's lymphoma Daudi cells are habitual targets for cytolytic activity of activated LAK cells . Cells derived from the K562 cell line are human erythroleukemia cell lines, which are activated NK cells. Used as a specific target for cytolytic activity. In these experiments, human PB MCs were treated with various concentrations of IL-10 for 3 days. Cytolytic activity above the standard^{Five 1} It was measured by Cr release assay.   The results based on LAK activity are shown in Table 1 and the standard error is shown below the mean value.   The data in Table 1 show the LAK activity of IL-10 in PBMCs obtained from 6 human donors. Shows the effect against. Although variability between donors is apparent, IL-10 was found to Induced a concentration-dependent increase in cell lysis capacity. Statistically significant activity Sex (p ≦ 0.05) was observed at concentrations of IL-10 above 0.4 ng / ml. 5% base level A donor showing the following cytolytic activity (ie, in the absence of cytokines) PBMC are most responsive to IL-10 at all effector cell: target cell ratios tested (Data not shown).   Other tumor target cells (human renal cell carcinoma line, two different human melanoma lines and Similar results were obtained (including colon cancer lines). Renal carcinoma cell line and black Tumor cell lines are also used by Rosenberg, and have such tumors Patients were in vivo by Rosenberg using an adoptive immunization with IL-2. I was treated. In all cases, the percent lysis of target cells was determined by the concentration of IL-10. Was dependent on.   In another experiment, IL-10 alone or in combination with IL-2 was used in U937 cells (human population). Lymphoma), SW620 (human colon cancer), and SKBR3 cells (human breast cancer) It was found to lead to an increase in deactivation. Use HS294 T cells (human melanoma) as a target In the experiment used, IL-10 did not appear to induce a response at the doses tested .   Basic NK cytolytic activity (i.e. lysis of K562 target in the absence of cytokines) Solution) tended to be higher than the basal LAK activity. NK activity is similar to that of IL-2. It could be further increased by tocaine [Perussia, supra; Phillips and Lanie. r, J. Exp. Med 164: 814 (1986)].   The effect of IL-10 on NK activity was demonstrated in PBMC from the same 6 donors described above. Evaluations and experiments were performed in equilibrium with the LAK assay. Table 2 shows the results. Standard error Is shown below the average value.   As is clear from Table 2, IL-10 is mediated by NK cells in donor-derived PBMCs. Induced a significant dose-dependent increase in cytotoxicity. Concentration of IL-10 above 4ng / ml There was a statistically significant increase in lytic activity with degrees. In case of LAK activation As such, the effects of IL-10 on NK activity varied among donors.Comparison of the effects of IL-2 and IL-10 on endothelial cells   Experiments were performed to assess the survival of endothelial cell monolayers after exposure to IL-10. IL- Endothelial cells cultured in the presence of 10 were exposed to exogenous cytokines (γ-IFN and TNF-α) Showed an undiminished response to the same unit over the same incubation period. Parallel cultures exposed to different doses of IL-2 lost their ability to respond due to the virulence of IL-2. Was.Effect of anti-IL-10 monoclonal antibody on IL-10 activation of LAK and NK cells   As shown in Tables 3 and 4, the mean values are shown with standard error of 40 ng / m The stimulation of LAK cell lytic activity and NK cell lytic activity by IL-10 was 2 μm, respectively. There was a 3-fold decrease (p ≦ 0.05) in the presence of g / ml of anti-IL-10 monoclonal antibody 19F1. Obtained using 2 μg / ml rat IgG2a isotype control antibody instead The results obtained were statistically distinct from those obtained using 40 ng / ml IL-10 alone. It resulted in a poor level of cytolytic activity. Cytolytic activity induced by combination with IL-10 and other cytokines a.IL-10 and IL-2 co-incubation   Human PBMCs were quantified in the presence of IL-10 using a 5: 1 effector to target ratio. Incubation was with submaximal stimulating concentrations of IL-2 (2 U / ml or 20 U / ml). The results are shown in Table 5, and the standard error is shown below the average value.   As shown in Table 5, simultaneous incubation of 2 U / ml IL-2 and 4 ng / ml IL-10 In LAK activity (effector cell to target cell ratio 5: 1) There was a dramatic increase. This activity level was found in either cytokine alone. Significantly higher than that produced, approaching levels produced with 10-fold higher concentrations of IL-2. Was. This result was statistically significant with p ≦ 0.05 measured by Student's t-test. It was my intention. b.IL-10 and α-IFN co-incubation   PBMCs co-incubated with IL-10 and α-IFN show cytolysis against Daudi It shows a similar additional increase in activity, but not NK target cells. This is a table 6 and in Table 6 the standard error is shown below the mean value.   Cytolytic activity induced by IL-10 or α-IFN alone in donor PBMC Observed in cytolytic activity induced by IL-10 and α-IFN compared to sex Differences were statistically significant at p <0.05 determined by Student's t-test .   Conversely, co-incubation with IL-10 and IL-4, IL-5, GMCSF or γ-IFN Was less effective than IL-10 alone (data not shown).IL-10 and IL-2 continuous incubation   Maintain PBMCs in medium alone or medium supplemented with IL-10 using the procedure above did. After 2 days, IL-2 was added at a final concentration of 2 U / ml or 20 U / ml. One more After overnight incubation, the cytotoxic activity on Daudi cells was evaluated. 5 The results obtained from the pool of human donors are shown in Table 7. In Table 7, the standard error of the average value Differences are shown below the mean.   As shown in Table 7, donor PBMCs were pretreated with IL-10 prior to the addition of IL-2. In cultures maintained in complete medium alone before addition of IL-2 About twice as high level (71.8 +/- 17.9%) compared to the observed level (41.7% + /-11.4) Was observed. Samples pre-treated with IL-10 and untreated with IL-10 The observed difference with the sham sample was statistically significant with p ≦ 0.14 .   A similar pattern of enhanced cytolytic activity was observed with IL-10 followed by α-IFN Seen in incubation (data not shown).Blockade of IL-2-induced cytotoxicity by IL-10 and IL-4   PBMCs from 6 human donors were added to a medium containing 20 U / ml IL-2 alone, 20 U / ml Medium containing IL-2 and 1000 U / ml IL-4, or 20 U / ml IL-2 and 1000 U / ml IL-4 It was cultured in a medium containing 4 ng / ml of IL-10. Table 8 shows the results. Table 8 Smell The standard error of the mean is shown below the mean.   The data in Table 8 show that after treatment with 20 U / ml IL-2 alone, approximately 34.2% of LAK sensitive targets were detected. Indicates that dissolution of was observed. Incubation period with 1000 U / ml human IL-4 When it was included from the beginning, the cytolytic ability was suppressed about 2-fold. However, the incu When 4 ng / ml IL-10 was added during the first 24 hours of basation, IL-4 No inhibition was observed.   Results with IL-2 alone and all three of these cytokines used together There is no significant difference between the results obtained by Antagonism by inhibition of IL-10 is essentially complete, as determined by p ≦ 0.05) Indicates that   Many modifications and variations of the present invention are intended to be obvious to those skilled in the art. And can be made without departing from the scope. Specific embodiments described herein Are provided by way of example only, and the invention is defined by the terms of the appended claims. Only limited.

Claims (1)

[Claims] 1. A method for the treatment of cancer, wherein an effective amount of IL-10-activated PBMC is administered to the cancer. Administering to a swollen individual to reverse such cancer. 2. Further, it includes simultaneous or sequential administration of an effective amount of IL-10 to said patient. The method of claim 1. 3. Comprising mixing IL-10 activated PBMC with a pharmaceutically acceptable carrier A method of manufacturing a pharmaceutical composition for the treatment of cancer. 4. The IL-10 is sufficient to increase the activation of (a) LAK cells, but with toxic side effects. Sufficient to increase the ineffective dose of IL-2 and / or (b) LAK cell activation 4. The method of any of claims 1-3, wherein the method is administered in combination with different amounts of alpha-IFN . 5. Treatment of cancer containing IL-10 activated PBMC and a pharmaceutically acceptable carrier A pharmaceutical composition for storage. 6. IL-10 alone or in combination with IL-2 and / or α-IFN The pharmaceutical composition according to claim 5, which comprises: 7. Use of IL-10 activated PBMCs to treat cancer. 8. Use of IL-10 activated PBMC for the manufacture of a medicament for the treatment of cancer. 9. The IL-10 activated PBMC comprises IL-10 alone or in combination with IL-2 and / or α-IFN Use according to any of claims 7 or 8, used in combination. 10. Endogenous IL-4 neutralizes IL-2 induced cytotoxic inhibition A method of treating the patient with an effective amount of IL-10 as needed for such treatment. Administering to the subject. 11. Endogenous IL-4 neutralizes IL-2 induced cytotoxic inhibition Use of IL-10 for the manufacture of a drug for. 12. The method according to any of claims 1 to 11, wherein the IL-10 is human IL-10. A pharmaceutically acceptable composition or use.