CN1142186A - Cytolytic activity of peripheral blood mononuclear cells activated by interleukin-10 - Google Patents

Abstract

The present invention relates to the use of IL-10, alone or in combination with IL-2 and/or α-IFN, to stimulate the cytolytic activity of peripheral blood mononuclear cells for the treatment of cancer.

Description

Cytolytic activity of peripheral blood mononuclear cells activated by interleukin-10

The present invention relates to the use of interleukin-10 (IL-10) in the form of cytokine synthesis inhibitory factor (CSIF) for neoplastic diseases (cancer) by stimulating the cytolytic activity of peripheral blood mononuclear cells (PBMC) on adoptive immunotherapy.

Background of the invention

Immunotherapy for cancer treatment is based on the concept that cancer cells escape the body's defenses against aberrant or foreign cells and molecules for reasons that are not well understood and that these defenses can be therapeutically inactivated To kill or inhibit the growth of cancer cells, such as Klein once discussed this problem (Immunology, Wiley-Interscience, 1982 pp.623-648). Results The recent observation that immune effectors can directly or indirectly inhibit the growth of tumors has revived interest in this method of treating tumors. [Heberman, Concepts Immunopath.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 treat cancerRalf et al., J.Exp.Med.167:712(1988)(tumoricidal activity of macrophages stimulated by lymphokines); Tepper et al., Cell 57:503(1989)(IL-4 has tumoricidal activity); M.Cohen, "Lymphokines and Tumor Immunity", pp. 237-253, in S. Cohened., Lymphokines and the Immune Response (CRC Press, Boca Raton, 1990)].

Immune responsiveness to tumors is regulated by many cell types and also includes the action of T-cell and monocyte-derived cytokines. A clinically promising immunotherapy approach is the so-called "acceptive immunotherapy" using IL-2-activated killer cells. [Rosenberg, Supra Rosenberg, Sci. Am, pp. 62-69 (May 1990)]. Although IL-2 alone or in combination with traditional chemotherapeutic drugs is very effective in treating some malignant tumors (such as renal cell carcinoma), it is accompanied by adverse toxic side effects brought about by the use of effective doses of IL-2 such as vascular layer Leakage and edema have led some to suggest that the dangers of this treatment outweigh its benefits. [Cotran et al., J Immunol. 139:1882 (1987); Edwards et al., Cancer Res. 52:3425 (1992)].

Human vascular endothelial cells are particularly sensitive to IL-2 toxicity, manifested by increased vascular permeability (ie, vascular leak syndrome) and edema. One of the possible factors responsible for this pathological response is the enhanced adhesion of IL-2-activated T cells and neutrophils to the monolayer of the vascular endothelium, as has been done previously in vitro. As demonstrated [Edwars et al., Supra; Damle et al., 138:1779 (1987)].

Another approach to immunotherapy for neoplastic diseases is the adoptive transfer of immune cells. The definition of adoptive immunotherapy is: the transfer or transplantation of active immune agents such as cells with anti-tumor activity that can directly or indirectly mediate anti-tumor effects into the host body bearing the tumor. Adoptive immunotherapy is an attractive approach to treat neoplastic diseases. It should be pointed out that since the active immune agent is transferred into the host, complete immune activity of the host is not required. Therefore, the immunosuppression usually caused by having a tumor is not a major obstacle in this type of immunotherapy. Since host immunocompetence is not required and, in fact, may be beneficial for adoptive transfer of immune cells, adoptive immunotherapy can be readily combined with other treatments such as chemotherapy and radiotherapy. And unlike other treatments, this therapy may not produce immunosuppressive effects.

Chemotherapy or radiotherapy in tumor patients will inevitably cause immune damage, and will often reduce the supply of peripheral blood mononuclear cells (PBMCs), effective effector cells for activating immune responses. Treatments that exhibit efficacy at low effector:target cell ratios would thus be of particular benefit to such patients.

Immunoreactivity to tumors is regulated by many cell types and includes the action of T-cell and monocyte-derived cytokines. Adoptive immunotherapy using recombinant human cytokines has involved in vitro activation of peripheral blood mononuclear cells (PBMCs) with the use of IL-2 [Rosenkey et al., J. Immol 138:1779 (1985)] [Philips et al., J.Clin.Oncd.5:1933:(1987); Perussia, Carr, Opion Immunol.3:49:(1991)] In vitro treatment of peripheral blood mononuclear cells produces activated lymphokine-activated killer cells (LAK) and activated natural killer cells (NK), both of which have cytolytic activity against various tumor cells.

Recombinant human interleukin-5 (IL-5) has been reported [Nagasawa et al., Cell. Immunol.133:317 (1991)], interleukin-7 (IL-7) [Stotter and Lotze, Arch.Surgery126:1525 (1991)] and interleukin-12 (IL-12) [Gately et al., J. Immunol. 147:874 (1991)] can activate the cytolytic activity of human peripheral blood mononuclear cells. Interleukin-10 (IL-10) was originally reported to be secreted by specific T helper cell subsets in mice as a cytokine suppressor, which can regulate the differentiation of mouse cytotoxic T cells [Chen and Zlotnik, J. Immunol 147:528 (1991)]. It was also found that incubation of human peripheral blood mononuclear cells with supernatant recovered from COS cells transfected with human IL-10 cDNA lysed tumor target cells in vitro. T cells with enhanced lytic potential in response to IL-10 were identified as a CD56+ phenotype, indicative of natural killer (NK) cells.

In some cases, IL-4 had the opposite effect on the production of LAK activity induced by IL-2. [Nagler et al., J. Immunol. 141:2349 (1988)]. For example, lysis of LAK-sensitive target cells was greatly reduced if human peripheral blood mononuclear cells were incubated with both IL-2 and IL-4 [Spits et al., J. Immunol. 141:29 (1988)]. However, if PBMCs were pre-cultured with IL-2-supplemented medium for 3 days before adding IL-4, the lytic activity of the cells was increased [Spits et al., Supra]. And if alpha-interferon (α-IFN) or tumor necrosis factor-alpha (TNF-α) was included in the initial incubation mixture, the blocking effect of IL-4 on I-2 pair-driven cytolytic activity was sufficient Attenuation [Swisher et al., Cell. Immunol. 128:450 (1990)].

Kedar et al [Cancer Immunol. Immunother.35:63 (1992)] recently pointed out that the sequential use of IL-2 and α-IFN is an effective immunotherapy modality in the treatment of mouse tumor models MCA-105 sarcoma and M10g carcinoma. The main finding of this study is that sequential administration of cytokines has a higher effect than simultaneous administration of cytokines.

The feasibility and effect of adoptive immunotherapy as a treatment model for various human diseases, especially tumors (cancers), has been described in Rosenberg's US Patent No. 4,690,915. However, as indicated above, there is a need for methods of administering such adoptive immunotherapy without the toxicity associated with IL-2 alone. There is also a need for a therapeutic modality that is effective at low effector:target cell ratios.

  Summary of Invention

The present invention fulfills these needs by providing a method for increasing the cytolytic activity of PBMCs, especially LAK and NK cells, by using IL-10 alone or in combination with IL-2 and/or in combination with α-IFN.

More particularly, the present invention provides a method for administering to cancer patients an effective dose of IL-10 to activate PBMCs and cause cancer regression. A preferred embodiment for activated PBMCs is simultaneous or subsequent use of IL-10.

In one embodiment, IL-10 and IL-2 are used in combination, and the amount of IL-2 is sufficient to increase the activity of LAK cells, but does not cause toxic side effects when used alone.

Another embodiment is the combination of IL-10 and α-IFN in an amount sufficient to increase the activity of LAK cells.

Yet another embodiment is that IL-10 is administered together with (a) IL-2 in an amount sufficient to increase LAK cell activity but not causing toxic side effects when used alone and (b) α-IFN in an amount sufficient to increase LAK cell activity use together.

The present invention further provides a method for antagonizing and blocking IL-2-induced cytotoxicity with endogenous interleukin-4 (IL-4), comprising administering an effective dose of IL-10 to such patients.

The present invention further provides a pharmaceutical composition comprising IL-10 combined with IL-2 and/or α-IFN and a pharmaceutically acceptable carrier.

Preferably, human IL-10, IL-2, and α-IFN are used in the aforementioned methods and compositions, and more preferably, recombinant human IL-10, IL-2, and α-IFN are used.

  Detailed Description of the Invention

All references cited herein are incorporated by reference in their entirety.

The present invention is a method for improving the prior art of using IL-2 to induce cytolytic activity of NK and LAK cells. The present invention greatly reduces the typical toxic side effects of methods using IL-2 by either completely eliminating IL-2 or greatly reducing the dose of IL-2 that must be used.

Unless otherwise defined, various terms used herein have the same meanings as commonly known terms in the art for guiding the present invention.

The term "acquired immunotherapy" as used here means the treatment of transplanting activated and functional immune cells into the patient. In a preferred embodiment, these cells include LAK and NK cells derived from the patient being treated.

The term "regression" as used herein means a measurable decrease in the volume of one or more tumors, as commonly measured in the art.

As used herein, "Interleukin-10" or "IL-10" is defined as a protein (a) that is a protein of IL-10 with a mature amino acid sequence (e.g., lacking a secretory leader sequence) on July 20, 1992 As disclosed on the patent U.S.Patent Application Serial No.07/917,806 filed on the 1st, and this patent is the same as International Application No.WO91/00349; (b) has the same biological activity as natural IL-10 . For the purposes of the present invention, IL-10 is irrespective of whether it is glycosylated (e.g. produced in eukaryotic cells such as CHO cells) or unglycosylated (e.g. chemically synthesized or produced in E. coli). are equivalent and can be used interchangeably. Also included are mutant proteins and other analogs, including the BCRF1 protein (African lymphoblastic virus viral IL-10), which also has the biological activity of IL-10.

There are different sources of IL-10 suitable for use in the present invention. For example, it can be isolated from activated medium secreting the protein. Alternatively, IL-10 or its active fragments can be chemically synthesized according to standard techniques well known in the art. See Mettifield, Science 233:341 (1986) and Atherton et al., (Solid Phase) A Practical Approach, 1989, I.R.L. Press, Oxford.

The preferred solution to obtain the protein or polypeptide is to use the isolated nucleic acid encoding IL-10 polypeptide to obtain it through recombinant technology. Books introducing general molecular biology methods include, for example, Sambrook et al., (Molecular Cloning) (A Laboratory Maunal), Cold Spring Harbor, New York, 2d ed., 1989, and By Ausbel et al., (eds) ( Current Protocols in Molecular Biology), Green/Woley, New York (1987 and periodic supplements). The correct sequence can be obtained from a genomic or cDNA library using standard techniques. It can also be obtained using polymerase chain reaction (PCR). See, e.g., (PCR Protocols): (A Guide to Methods and Applications), 1990, Innis et al., (Ed.) Academic Press. New York.

Libraries are constructed using nucleic acids obtained from suitable cells, see, for example, International Application Publication No. WO 91/00349 for the preparation of recombinant IL-10. Useful gene sequences can be found, for example, in various sequence databases such as GenBank.andBMPL or ncleic acid and PIR and Swiss-Prot for protein, c/oIntelligenetics, Mountain View, California, or the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wisconsin The foregoing is incorporated herein by reference.

Clones containing the sequence encoding human IL-10 have been deposited with the American Type Culture Collection (ATCC), Rockville, Martyland, under accession numbers 68191 and 68192. Identification of other clones with sequences encoding IL-10 can be detected by nucleic acid hybridization or by immunization against the protein encoded by the expression vector. Oligonucleotide probes based on the sequences deposited in the published International Application Publication No. WO91/00349 are particularly useful. Oligonucleotide probe sequences can also be prepared from conserved regions of genes associated with other species. Alternatively, denatured probes based on the amino acid sequence of IL-10 can also be used.

Prokaryotic, mammalian, yeast or insect cell lines can be transformed to express large amounts of the polypeptide using standard methods. Representative strains of Escherichia coli suitable for both expression and cloning include: W3110 (ATCCBi, 27325), X1776 (ATCC No.31244), X2282 RR1 (ATCCMp/31343), and representative mammalian cell strains include: COS-7 cell strain, Mouse L cells and CHP cells. See Sambrook (1989) and Ausubel et al., 1987 Supplement).

DNA encoding IL-10 can be expressed using various expression vectors. Commonly used vectors for expression of recombinant proteins in prokaryotic or eukaryotic cells can be used. Preferred vectors include the PCD vectors described by Okayama et al., Mol Cell. Biol. 3:280 (1983); and Takebe et al., Mol. Cell. Biol. 8:466 (1988). Other SV40-based vectors include those disclosed in: Kaufmen et al., Mol. Cell. Biol. 2:1304 (1982) and U.S. Patent No. 4,675,285. These SV40-based vectors are effective against monkey COS- 7 cells (ATCC No. CRL 1651) and other mammalian cells such as mouse L cells are particularly useful. See also Douwels et al., (1989 and Supplements) (Cloning Vectors): (A laboratory Manual) Elsevier, New York.

IL-10 can be produced in a soluble form, eg, as a secretion product, in transformed or transfected yeast or mammalian cells. Polypeptides can then be purified by standard methods well known in the art. For example, purification steps include ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and the like. See, Methods in Enzymology Purificaition Principles and Practiles (Springet-Vertag, New York, 1982).

Alternatively, IL-10 can also be produced in an insoluble form such as aggregates or inclusion bodies. This form of IL-10 can be purified by standard procedures well known in the art. Examples of purification steps include: separating inclusion bodies from disintegrated host cells by centrifugation, and then using solubilizing and reducing agents to dissolve the inclusion bodies to form the polypeptide into a biologically active configuration. Details of these steps are found in: e.g. Winklet et al., Biochemistry, 25:4041 (1986); Winklet et al., Bio/Technology 3:9923 (1985); Kiths et al., and U.S., Patent No. 4,569,790 .

The nucleotide sequence used to transfect host cells can be modified according to standard techniques to impart various desired properties to IL-10 or fragments thereof. Such modifications make IL-10 identical to the naturally occurring sequence at the primary structural level such as by amino acid insertions, substitutions, deletions and fusions. These modifications can be used in combination to obtain the final modified protein chain.

Amino acid sequence mutants can be prepared according to different objectives, including increasing serum half-life, facilitating purification and preparation, improving therapeutic effect, and reducing the occurrence and severity of toxic and side effects during treatment. Amino acid sequence mutants are predetermined mutants that do not exist in nature, although some other mutants may be post-translational mutants such as glycosylated mutants or proteins conjugated with polyethylene glycol (PEQ) etc. These mutants can be used in the present invention as long as they retain the biological activity of IL-10.

Modifications to sequences encoding polypeptides are readily accomplished by various techniques such as site-directed mutagenesis [Gillman et al., Gene 8:81 (1987)]. Most modifiers are evaluated by routine screening for desired characteristics in an appropriate assay system. For example, in International Application Publication No.WO91/00349, several in vitro detection methods for evaluating IL-10 activity have been introduced.

Preferably, human IL-10 is used for the treatment of human diseases, although viral IL-10 or IL-10 from other mammals may also be used. A more preferred approach is to use recombinant human IL-10. The production of human and mouse IL-10 is described in International Application WO91/00349. Cloning and expression of IL-10 (BCRF1 protein) from African lymphoma virus has been published by Moore et al., Science 248:1230 (1990). Recombinant human IL-10 is commercially available, for example, from Prepro.Tech, Inc., Rocky Hill, NJ.

Individuals suitable for the treatment method of the present invention, including any tumor patients, can benefit from the activation of PBMC cytolytic activity, especially the activation of LAK and NK cell activity. Representative cancer patients have been introduced, such as Rosenberg, Supra's articles on patents and (Scientific American) have been introduced. The treatment regimens of the present invention are also applicable to individuals who have been pretreated to increase endogenous IL-4 levels, such that IL-4 blocks the activation of cytolytic activity by IL-2. The preferred treatment regimen in such individuals is pretreatment with IL-10 prior to IL-2.

The standard methods and techniques described in this invention for the production of IL-10 can also be used for the production of IL-2 and α-IFN, which are also commercially available (e.g. IL-2 and α-IFN) -2 is available from Cetus, Corporation, Emeryville, CA, α-IFN is available from Schenng, Corp., Keniluorth, NJ).

In vitro activation of PBMCs (preferably obtained by standard methods from the patient to be treated) and The use of these cells was carried out essentially as described by Rosenberg mentioned above. The number of activated PBMCs used ranged from 10 ⁶ to 10 ¹² . Although not required, a preferred embodiment is the use or subsequent use of IL-10 as described herein with such activated cells.

In one embodiment, PBMCs are activated by pretreatment with IL-10 [for example, in the presence of 4ng/ml (100 units/ml) or 40ng/ml human IL-10, cultured at 37°C for about 3 days], and then, Cells are washed to remove free IL-10 and then low concentrations of IL-2 (typically about 2 units/m) are added.

The preferred regimen for lysing cells by 1L-10 alone or in combination with other cytokines used herein is intravenous injection. This can also be done, for example, through a central venous catheter into a large peripheral vein or through a percutaneous catheter into the hepatic artery.

IL-10 is usually used in the form of a pharmaceutical composition, which includes a pharmaceutical carrier and an effective dose of IL-10 alone or in combination with IL-2 and/or α-IFN. The pharmaceutical carrier can be any compatible non-toxic substance suitable for delivery of the pharmaceutical to the patient. Useful compositions for parenteral routes of such pharmaceuticals are well understood, see for example Remingtons Pharmaceutical Science 15th Ed.( Mack Publishing Company Easton, PA, 1980). Alternatively, the compositions of the present invention may be delivered to the patient via implanted or injected drug delivery systems. For example, Urquhert et al., Ann. Rev. Pharmacol. Toxicol. 24:99 (1984); Leuis (Ed.) (Controlled Release of Pesticides and Pharmaceuticals) (Plenum Press, NJ. 1981) U.S. Patent No. 3,270,960; .

Cytokines can be administered by any of the well-known routes of administration, including intravenous, peritoneal and subcutaneous administration. Intravenous administration is the most preferred route of use.

When administered parenterally, the composition is combined with a pharmaceutical carrier to form a unitary unit dose of injectable form (solution, suspension, emulsion). Examples of such vehicles are physiological saline, Ringer's solution, dextrose solution and Hank's solution. As non-aqueous carriers, fixed oils and ethyl oleate can be used. A preferred vehicle is 5% dextrose/saline solution. The carrier may contain minor amounts of additives to increase isotonicity and chemical stability such as buffers and preservatives, and the like. A preferred uniform form of IL-10 is a pure form substantially free of aggregates and other proteins, at a concentration of about 5-20 [mu]g/ml.

Compositions of the invention can also be delivered into a patient using standard gene therapy techniques, including, for example, direct DNA tissue injection, use of recombinant viral vectors, and implantation of transfected cells, see, e.g., Rosenberg, J. Clin. Oncol. 10 : 180 (1992).

The combination of one or more therapeutic agents described herein can be used simultaneously (with IL-10) or can be used sequentially. Preferably, IL-10 is used before IL-2. [alpha]-IFN can be used together or subsequently with IL-10 and/or IL-2. All agents are present in sufficient concentrations in the patient to be therapeutically effective to cause tumor regression.

The term "effective dose" as used herein means an amount of IL-10 sufficient to reduce or prevent side effects in adoptive immunotherapy while simultaneously increasing the cytolytic activity of LAK and NK cells. The effective dosage of cytokines required for a particular patient may vary according to factors such as the type and state of the tumor being treated, the patient's overall health, the type of therapy being used, the severity of side effects, and other drugs being used at the same time. vary with factors such as quantity and type.

The amount of cytokines "sufficient to increase the activity of LAK cells" is defined here as using Daudi cells as the cell lysis evaluation system, and the cell lysis activity produced by the required cell amount can be increased by at least 25% when compared with IL-10 alone, preferably A protocol is to increase to at least 50%, and a most preferred protocol is to increase to at least about 100%.

The preferred solution is to administer IL-10 with its maximum limiting dosage, from 10 to 10 ⁸ units per kilogram of body weight per day. Similarly, IL-2 and α-IFN administration can also use the maximum limit dosage (for example, the dosage for intravenous administration of IL-2 is: 10 ⁵ units per kilogram of body weight every 8 hours, with 50ml 0.4% normal saline and 5% white protein as a carrier; the dose of α-IFN is: intravenous administration of 10 ⁶ U per kg body weight every 8 hours, using 0.9% normal saline plus 5% albumin as a carrier). As mentioned earlier, the dose used is adjusted by the clinician according to the maximum limit that each individual patient can bear.

The methods of the present invention can also be used in conjunction with traditional methods of treating cancer, such as radiotherapy and chemotherapy using traditional chemotherapeutic drugs like vinblastine, platinum compounds, and 5-fluorouracil.

Treatment of human peripheral blood mononuclear cells with IL-10 alone or in combination with other cytokines elicited enhanced cytolytic activity against human tumor target cells. Because the efficacy of immunotherapy is largely dependent on the degree of tumor burden, it would be particularly beneficial to use the treatment approach described here after a large primary tumor mass has been resected. Inflammatory reactions, which often occur at the surgical site, may contribute to therapeutic efficacy.

Example

The following non-limiting examples will serve to illustrate the invention.

Effects of IL-10 on Cytolytic Activity of Human Peripheral Blood Mononuclear Cells

The effect of IL-10 on the cytolytic activity of human peripheral blood mononuclear cells in vitro was studied. The results are summarized below:

I. IL-10 stimulates the activity of lymphokine-activated killer cells (LAK) and natural killer cells (NK) in human peripheral blood mononuclear cells. Rat anti-IL-10 monoclonal antibody neutralizes IL-10-driven cytolytic activity.

2. IL-10 derived from CHO and E. coli expression systems showed the same concentration-response pattern in stimulating the activities of LAK and NK cells, so they were equivalent in the biological sense.

3. PBMCs treated with IL-10 and low-dose IL-2 exhibited more active LAK cells than any cytokine alone.

4. The lytic activity of PBMCs pretreated with IL-10 for 2 days and then added with IL-2 increased.

5. IL-4 inhibits the activity of LAK cells induced by IL-2, while IL-10 antagonizes this inhibitory effect.

6. IL-10 plus IL-2 produces increased LAK cell-to-cell lytic activity at low effector:target cell ratios.

In addition to the effects observed for PBMCs, it was found that the response of endothelial cells cultured in the presence of IL-10 to exogenous factors (ie, γ-IFN and TFN-α) was not affected, whereas in the presence of IL-2 Endothelial cells cultured in culture were unresponsive due to IL-2 toxicity.

Materials and Methods

Recombinant human cytokines and anti-IL-10 antibodies

Recombinant human IL-10 was produced by standard methods (from both E. coli and CHO). The specific activities of the products purified by standard methods for the proliferation detection of MC-9 cells are 4.1×10 ⁷ E.coli and 2.1×10 ⁷ (CHO)Units/mg [Thompson-Sripes et al., J.Exp.Med 173:507 (1991)], which is defined as approximately 4 ng of substantially pure IL-10 containing approximately 100 units of biological activity.

A rat anti-human IL-10 monoclonal antibody named 19F1 was obtained from Dr. John Abrams of the DNAX Institute of Molecular Biology in Palo Alto.CA.

Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)

Peripheral blood was obtained from healthy adult donors by venipuncture with heparin or EDTA as anticoagulant. PBMCs were isolated using a two-step method, including dextran precipitation and centrifugation on FICOLL PAQUE" at 1250rpm (rev/min) for 30 minutes. The interface zone, which mainly included lymphocytes and monocytes, was collected and washed with 10% RPMI medium (complete medium) with fetal bovine serum was washed at least twice (JRH Biosciences).

Cytotoxicity Assay

Daudi (LAK sensitive) and K562 (NK sensitive) target cells were obtained from the American Type Tissue Collection under accession Nos. CCL 213 and CCL 243, respectively. Daudi and K562 cells were labeled with ⁵¹ Cr by the method introduced by Spits et al. [J. Immunol 141: 29 (1989)]. After cultured, PBMCs were collected, washed twice, and then used as effector cells in a ⁵¹ Cr release detection system (Spits et al., Supra). Target cells labeled with 5×10 ^{3 51} Cr were mixed with different amounts of effector cells (E/T=20:1, 5:1, 2:1) in a volume of 100 μl in a V-bottom 96-well plate. The 96-well plate was centrifuged at 1000rpm for 5 minutes and then incubated for 4 hours in CO ₂ gas with a saturated humidity of 5%, and then centrifuged at 500xg for 5 minutes after 4 hours of cultivation. The supernatant was collected with a SKATRON (Skatron Instruments) harvester and counted with a gamma counter (LKB-Pharmacia). Total lysis was determined by incubating ⁵¹ Cr-labeled target cells with 1% SDS. Data represent the mean of three determinations.

The percentage of lysis was calculated according to the formula:

Incubation of human PBMCs with cytokines

a. IL-10 alone incubation

Unless otherwise specified, the above isolated PBMCs were cultured in RPMI-1640 medium supplemented with IL-10 or human IL-10 (CHO) containing 10% fetal bovine serum at a concentration of 1×10 ⁶ cells per ml at 37°C. Cultivate for 3 days. Cytolytic activity was determined as described above.

b. Simultaneous incubation with IL-10 and IL-2

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

c. Sequential incubation with IL-10 and IL-2

PBMCs were incubated with 4ng/ml IL-10 in complete medium for 2 days. Add IL-2 so that the final concentration is 2U/ml or 20U/ml. After overnight culture, the cytolytic activity of LAK and NK cells was determined.

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

PBMCs were incubated with 40ng/ml IL-10 for 3 days in the presence of 2μg/ml anti-IL-10 monoclonal antibody (19F1) or an isotype control (rat IgG2a).

Effect of IL-10 on 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 operationally differentiated depending on the tumor target cells used. Daudi cells derived from Burkitt's lymphoma are classical target cells for detecting the cytolytic activity of activated LAK cells. Cells derived from human erythroleukemia K562 cell line were used as specific target cells to detect cytolytic activity of NK cells. In these experiments human PBMCs were treated with various concentrations of IL-10 for 3 days and cytolytic activity was determined using the standard ⁵¹ Cr release assay described above.

The activity results for LAK are shown in Table 1, where the standard deviation is given below the mean.

Table 1: The stimulating effect of IL-10 on lymphokine-activated PBMCs (expressed as ^a by % of lysis)

IL-10 concentration (ng/ml) ^b

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

Mean 6.2 ^{10.6 20.2c} 31.7c ^{31.0c 40.2c}

±3.3 ±2.6 ±5.1 ±6.8 ±3.2 ±8.0

a. Percent lysis of ⁵¹ Cr-labeled Daudi target cells was obtained at a 20:1 effector-to-target ratio and using a standard chromium release assay. Data represent the mean of three determinations.

b. Human PBMCs obtained from normal donors were treated with IL-10 for 3 days before measuring cytolytic activity.

c. As determined by the Student's-t test, there is a significant difference between the IL-10 treatment group and the medium control group, P≤0.05.

The data in Table 1 show the effect of IL-10 on LAK activity in PBMCs obtained from 6 human donors. Although there were significant differences among the 6 donors, IL-10-induced increases in cytolytic capacity appeared to be concentration-dependent on IL-10 in all 6 donors. Statistically significant concentrations of IL-10 were 0.4 ng/ml or greater (P < 0.05). It was also observed that donor PBMCs exhibited a basal level of cytolytic activity (i.e., under cytokine-free conditions) with 5% or less cytolytic activity for IL in all effector-to-target cell ratios measured. -10 is the most responsive (data not attached)

Similar results were obtained for other tumor target cells, including human renal cell tumor lines, two different human melanoma lines and human colon carcinoma cell lines. Rosenberg has also used human renal cell tumor and melanoma cells, and has also done in vivo adoptive immunotherapy with IL-2 in patients with such tumors. In all experiments, the percent cytolysis of target cells was dependent on IL-10 concentration.

In other experiments, it was found that IL-10 alone or in combination with IL-2 also induced increased cytolytic activity against U937 (human histiocytoma); SW620 (human colon cancer) and SKBR3 cells (human breast cancer). In experiments using HS294T cells (human melanoma) as target cells, IL-10 did not appear to elicit a response at the doses tested.

The cytolytic activity of NK at the basic level (that is, the lysis of K562 target cells under the condition of no cytokines tends to be higher than that of the basic level of LAK cells. The activity of NK can be used with cytokines such as IL- 2 Further enhancements [Perussia, Supra; Philips and Lanier J. Exp. Med 164:814 (1986)].

In a parallel experiment to measure LAK cell activity, the effect of IL-10 on NK cell activity in PBMCs was determined using the same 6 donors described above. The results are shown in Table 2, where the standard deviation is given below the mean.

Table 2: IL-10 stimulates NK activity in PBMCs ( ^expressed as % of lysis)

Concentration of IL-10 (ng/ml) ^b

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 80.0 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

Mean 24.4 30.2 30.5 ^{54.0c 52.75c 55.5c}

±7.5 ±5.2 ±3.9 ±8.2 ±7.8 ±8.5

a. Percent lysis of ⁵¹ Cr-labeled Daudi target cells was obtained using a standard chromium release assay at a 20:1 ratio of effector cells to target cells. Data are the mean of three experiments.

b. Human PBMCs derived from normal donors were treated with IL-10 for 3 days before assaying for cytolytic activity.

c. There is a significant difference between the IL-10 treatment group and the medium control group by the Student's-t test, P≤0.05.

It is evident from Table 2 that IL-10 induces a significant dose-dependent enhancement of NK cell-mediated cytotoxicity from donor-derived PBMCs. Lysis was statistically significantly increased at IL-10 concentrations of 4 ng/ml or higher. As in the LAK activity assay, the effect of IL-10 on NK cell activity varied by donor.

Comparison of Effects of IL-2 and IL-10 on Endothelial Cells

An experiment was designed to measure the viability of endothelial cell monolayers in the presence of IL-10. The response of endothelial cells to exogenous cytokines (γ-IFN and TFN-α) was not weakened when cultured in the presence of IL-10, while parallel experiments were incubated with the same unit dose of IL-2 for the same time due to the presence of IL-2 Toxicity leads to the loss of the ability of cells to respond to exogenous cytokines.

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

Mean values with standard deviations are expressed as shown in Table 3 and Table 4, in the presence of 2 μg/ml IL-10 monoclonal antibody 19F1, the cytolysis of LAK and NK cells with 40 ng/ml IL-10, respectively Activation of activity was reduced 3-fold (P≤0.05). Results obtained with 2 μg/ml rat IgG2a isotype control antibody in place of anti-IL-10 monoclonal antibody produced levels of lytic activity in cells that were statistically incompatible with those obtained with 40 ng/ml IL-10 alone difference.

Table 3: Inhibition of IL-10-induced cytolytic activity of lymphokine-activated killer cells by anti-IL-10 monoclonal antibodies (expressed as % lysis) ^a

Culture condition ^b

Donor Medium 40ng/ml IL-10 IL-10

IL-10 +19F1 +IgG2a

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

Average 4.5±0.6 29.6±5.3 7.9c±3.3 23.9±4.3

a. Percent lysis of ⁵¹ Cr-labeled Daudi target cells was determined at a 20:1 effector:target cell ratio and using a standard chromium release assay. Data are the average of three determinations.

b. Human peripheral blood mononuclear cells obtained from normal donors were treated with 40 ng/ml IL- 10 processed for 3 days.

c. There is a significant difference between IL-10 alone treatment and anti-IL-10 monoclonal antibody treatment by Student's-t test, P≤0.05.

Table 4: Neutralizing effect of anti-IL-10 monoclonal antibody on IL-10-induced natural killer cell activity (expressed as a lysing effect % ⁾

Culture condition ^b

Donor Medium 40ng/ml IL-10 IL-10

IL-10 +19F1 +IgG2a

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 value 22.5±1.6 47.9±7.6 19.1c±6.6 51.3±12.7

a. Percent lysis of [ ^51Cr ]-labeled Daudi target cells was determined at a 20:1 effector:target cell ratio and by standard chromium release assays. Data are the average of three determinations.

b. Human peripheral blood mononuclear cells obtained from normal donors were treated with 40 ng/ml IL- 10 processed for 3 days.

c. There is a significant difference between IL-10 alone treatment and anti-IL-10 monoclonal antibody treatment by Student's-t test, P≤0.05.

Cytolytic activity induced by combined use of IL-10 and other cytokines

a. Simultaneous incubation of IL-10 and IL-2

Human PBMCs were incubated with IL-2 (2 or 20 U/ml) at the sub-maximum activation concentration in the presence of IL-10 at a 5:1 effector cell: target cell ratio. The results are shown in Table 5, and the average values are standard Difference.

Table 5: Cytolytic activity of lymphokine activation induced by simultaneous incubation with IL-10 and IL-2 in human peripheral blood mononuclear cells (expressed as % of lysis ^a )

Culture condition ^b

Donor Medium 2U IL-2 4ng IL-10 IL-10 20U IL-2

+IL-2

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 44.9c

±1.42 ±7.6 ±7.2 ±7.2 ±10.4

a. Percent lysis of [ ^51Cr ]-labeled Daudi target cells was determined at a 5:1 effector cell:target cell ratio and by a standard chromium release assay. Data are the average of three determinations.

b. Human peripheral blood mononuclear cells from normal donors were treated with 4ng/ml IL-10 and 2U/ml IL-2 for 3 days before measuring cytolytic activity.

c. Using the Student's-t test to determine that the donor PBMCs were treated with IL-10 and IL-2 compared with 20U/ml IL-2 single treatment, there was no significant difference in the cytolytic activity, P≤0.05.

As shown in Table 5, the combination of 2U/ml and 4ng/ml IL-10 resulted in an additional increase in LAK activity (effector cells: target cells = 5:1). This level of activity was significantly higher than that of either of the two cytokines alone. Activity levels produced by 10-fold higher IL-2 concentrations were achieved. The results were statistically significant by Studeat's-t test, P≤0.05.

b. Simultaneous incubation of IL-10 and α-IFN

Co-incubation of PBMCs with IL-10 and α-IFN showed a similar additional increase in the cytolytic activity of Daudi cells, but not NK target cells. The results are shown in Table 6, with standard deviations given below the mean.

Table 6: Induction of lymphokine-activated killer cell activity with combined use of IL-10 and α-IFN in human peripheral blood mononuclear cells (expressed as % of lysis ^a )

Culture condition ^b

Donor Medium α-IFN IL-10 IL-10+α-IFN

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

a. Percent lysis of [ ^51Cr ]-labeled Daudi target cells was determined at a 10:1 effector cell:target cell ratio and using a standard chromium release assay. Data are the average of three determinations.

b. Human peripheral blood mononuclear cells prepared from normal donors were treated with 4 ng/ml IL-10 and 100 U/ml α-IFN for 3 days before assaying for cytolytic activity.

c. As measured by the Student's-t test, the difference observed in the cytolytic activity of donor PBMCs induced by IL-10 and α-IFN combined with IL-10 or α-IFN alone is statistically significant Yes, P<0.05.

In contrast, none of IL-10 combined with IL-4, IL-5, GMCSF, or γ-IFN was more effective than IL-10 alone (results not shown).

Sequential incubation of IL-10 and IL-2

PBMCs were maintained in medium or medium supplemented with IL-10 as described above. Two days later, IL-2 was added to make the final concentration 2 or 20 U/ml. Cytotoxic activity against Daudi cells was determined after another overnight incubation. The combined results from the 5 donors are shown in Table 7, where the standard deviation is given below the mean.

Table 7: Induction of lymphokine-activated cytolytic activity in human peripheral blood mononuclear cells pretreated with IL-10 followed by IL-2 treatment (expressed as % lysis ^a )

Pre-incubation condition ^b

Donor Medium IL- ^10c IL- ^2d IL-10+IL- ^2e

1 29.7 21.1 44.8 116

2 5.5 18.3 12.2 20.7

3 14.7 58.1 74.5 100

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

a. Percent lysis of [ ^51Cr ]-labeled Daudi target cells was determined at a 5:1 effector cell:target cell ratio and by standard chromium release assays. Data are the average of three determinations.

b. Human peripheral blood mononuclear cells prepared from normal donors were maintained in medium supplemented with 4 ng/ml IL-10 for 2 days before adding 2 U/ml IL-2. Cytolytic activity was determined after an additional overnight incubation.

c. Donor PBMCs were incubated with IL-10 for 3 days.

d. Donor PBMCs were cultured in culture alone before adding IL-2.

e. Donor PBMCs were incubated with IL-10 for 2 days before adding 20U IL-2.

As shown in Table 7, the observed cytolytic activity (71.8±17.9%) was higher in the experimental group in which the donor PBMCs were pretreated with IL-10 before adding IL-2 The cytolytic activity (41.7±11.4) of the experimental group cultured in the complete medium increased about 2 times. The difference observed between samples pretreated with IL-10 and those not pretreated with IL-10 was statistically significant at P < 0.14.

A similar pattern of increased cytolytic activity was seen in sequential incubations with IL-10 followed by [alpha]-IFN (data not shown).

Blockade of IL-2-induced cytotoxicity by IL-10 and IL-4

PBMCs obtained from 6 human donors were cultured in culture medium containing: 20U/ml IL-2 alone, 20U/ml IL-2 plus 1000U/ml IL-4, 20U/ml IL-2 plus 1000U/ml IL-4 plus 4ng/ml IL-10. The results are shown in Table 8, with standard deviations given below the mean. Table 8: Effect of IL-10 on IL-4 on IL-2-induced lymphokine activation in human peripheral blood mononuclear cells

Antagonism of blockade of cytolytic activity ( ^expressed as % of lysisa)

Donor Medium IL-2 ^b IL-2 ^c + IL-2 ^d +

IL-4 IL-4+

IL-10

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 29.6 14.1 22.1

5 3.3 47.1 17.1 63.8

6 6.6 49.5 20.6 40.8

Average 5.5 34.2 19.7 40.3

±1.6 ±4.5 ±3.2 ±5.7

a. Percent lysis of [ ^51Cr ]-labeled Daudi target cells was determined at a 20:1 effector:target cell ratio and by a standard chromium release assay. Data are the average of three determinations.

b. Human peripheral blood mononuclear cells isolated from normal donors were treated with 20U/ml IL-2 for 3 days.

c. Donor PBMCs were treated with 20U/ml IL-2 and 100U/ml human IL-4 for 3 days.

d. Donor PBMCs were treated with 20U/ml IL-2, 1000U/ml human IL-4 and 4ng/ml IL-10.

As shown in Table 8, the lysis ratio of LAK-sensitive target cells was about 34.2% when treated with 20 U/ml IL-2 alone. When 1000 U/ml of IL-4 was added at the beginning of the incubation, the lytic capacity was inhibited about 2-fold. But if 4ng/ml IL-10 was added in the first 24 hours of culture, the inhibitory effect of IL-4 was lost.

There was no significant difference between the results of IL-2 alone and all three cytokines (Student's-t test, P≤0.05), indicating that the antagonism of IL-10 to the blocking effect of IL-4 is basically complete.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention. The specific embodiments described herein are given by way of example only, and the invention is to be limited only by the terms of the appended claims.

Claims (12)

CLAIMS 1. A method of treating cancer comprising administering to a cancer patient an effective amount of IL-10-activated PBMCs to cause regression of the cancer. 2. The method of claim 1, further comprising simultaneously or sequentially administering to said individual an effective amount of IL-10. 3. A method for producing a pharmaceutical composition for treating cancer, comprising mixing IL-10-activated PBMCs with a pharmacologically acceptable carrier. 4. The method according to any one of claims 1 to 3, wherein the administration of IL-10 is combined with (a) a sufficient amount of IL-2 capable of enhancing LAK cell activity without causing toxic side effects and/or (b) a sufficient amount of It can be combined with α-IFN that can increase the activation activity of LAK cells. 5. A pharmaceutical composition for treating cancer, comprising IL-10-activated PBMCs and a pharmacologically acceptable carrier. 6. The pharmaceutical composition according to claim 5, further comprising IL-10 alone or in combination with IL-2 and/or α-IFN. 7. The application of PBMCs activated by IL-10 in the treatment of cancer. 8. The application of PBMCs activated by IL-10 in the manufacture of medicaments for treating cancer. 9. The use according to claim 7 or 8, wherein IL-10 activated PBMCs are used together with IL-10 alone or together with IL-10, IL-2 and/or [alpha]-IFN. 10. A method of antagonizing the blocking effect of endogenous IL-4 on IL-2-induced cytotoxicity comprising administering to a patient in need of such treatment an effective amount of IL-10. 11. Use of IL-10 for the manufacture of a medicament for antagonizing the blocking effect of IL-4 on IL-2-induced cytotoxicity. 12. The method, pharmaceutical composition or use of any one of claims 1 to 11, wherein the IL-10 is human IL-10.