AU707019B2 - Use of IL-10 to stimulate peripheral blood mononuclear cell cytolytic activity - Google Patents

Description

WO 95/19780 PCT/IB94/00008 USE OF IL-10 TO STIMULATE PERIPHERAL

BLOOD

MONONUCLEAR CELL CYTOLYTIC ACfIVITY This invention relates to the use of Interleukin-10 formally cytokine synthesis inhibitory factor (CSIF), for adoptive immunotherapy in the treatment of neoplastic disorders (cancer) by stimulation of cytolytic activity of peripheral blood mononuclear cells (PBMC).

BACKGROUND OF THE INVENTION 1 0 Immunologic approaches to cancer therapy are based on the notion that cancer cells have somehow evaded the body's defenses against aberrant or foreign cells and molecules, and that these defenses might be therapeutically stimulated to kill or inhibit the growth of the cancer cells, as discussed by Klein (Immunology, Wiley-Interscience, New York, 1982, pp. 623-648). The recent observation that immune effectors can directly or indirectly inhibit tumor growth has led to renewed interest in this approach to cancer therapy [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 cancer); Ralf 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. Cohen, ed., Lymphokines and the Immune Response (CRC Press, Boca Raton, 1990)].

Immune responsiveness to neoplasms is regulated by a variety of cell types and involves the actions of T-cell and monocyte-derived cytokines. One immunologic approach that has shown clinical promise has been so-called "adoptive immunotherapy" using IL-2-activated killer cells [Rosenberg, CONFIRMATION COPY WO 95/19780 PCT/IB94/00008 2 supra; Rosenberg, Sci. Am., pp. 62-69 (May 1990)]. Although IL-2 alone or in combination with more traditional chemotherapeutic agents appears to be effective in treating certain malignancies renal cell carcinoma), unfortunate toxic side effects such as vascular bed leakage and edema associated with administration of effective dosages of IL-2 have led some to suggest that the risks may outweigh the benefits [Cotran et al., J. Immunol. 139:1882 (1987); Edwards et al., Cancer Res. 52:3425 (1992)].

Human vascular endothelial cells appear to be particularly sensitive to IL-2 toxicity, as evidenced by increased vascular permeability vascular leak syndrome) and edema. One of the factors contributing to this pathology may be augmented adhesion of IL-2-activated T cells and neutrophils to endothelial monolayers, as has previously been noted in vitro [Edwards et al., supra; Damle et al., 138:1779 (1987)].

An alternative therapeutic approach to the immunologic treatment of neoplastic disease is the adoptive transfer of immune cells. Adoptive immunotherapy is defined as the transfer to a tumor-bearing host of active immunologic reagents, such as cells with anti-tumor activity that can mediate, either directly or indirectly, anti-tumor effects. Adoptive immunotherapy represents an attractive approach to the therapy of neoplastic disease. It should be noted that because active immunologic reagents are being transferred to the host, complete host immunocompetence is not required. Thus, the immunosuppression generally associated with the tumor-bearing state does not represent a major problem to this therapeutic alternative.

Since host immunocompetence is not required, and in fact may be beneficial to the effects of the adoptive transfer of immune cells, adoptive immunotherapy can easily be combined with other therapies such as chemotherapy or radiation therapy. Also, in contrast to other therapies, immunosuppression is unlikely to result from this treatment.

WO 95/19780 PCT/IB94/00008 3 Patients undergoing chemotherapy or radiation therapy tend to be immunocompromised and will generally have a depleted supply of effector peripheral blood mononuclear cells (PBMCs) available for activation. A therapy that shows efficacy at low effector cell:target cell ratios would thus be particularly advantageous for such patients.

Immune responsiveness to neoplasms is regulated by a variety of cell types and involves the actions of T-cell and monocyte-derived cytokines. Adoptive immunotherapy using recombinant human cytokines has involved administration of peripheral blood mononuclear cells (PBMCs) stimulated extracorporeally [Phillips et al., J. Clin. Oncol. 5:1933:(1987); Perussia, Curr. Opin. Immunol. 3:49:(1991)] followed by administration of IL-2 [Rosenberg et al., J. Immunol. 138:1779 (1985)]. The extra-corporeal treatment of the PBMCs produces activated lymphokine-activated killer (LAK) cells and activated natural killer (NK) cells having cytolytic activity for various tumor cells.

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)] have been reported to stimulate cytolytic activity in human PBMCs.

(IL-10), originally described in mice as a cytokine synthesis inhibitory factor secreted by specific helper T-cell subsets, appears to modulate the differentiation of murine cytotoxic T-cells [Chen and Zlotnik, J. Immunol 147:528 (1991)]. It has also been found that human PBMCs incubated with supernatants recovered from COS cells transfected with the human cDNA lysed tumor cell targets in vitro. The T cells responding to IL-10 with increased cytolytic potential were 3 0 identified to be of a CD56+ phenotype, indicative of NK cells.

In some circumstances, IL-4 can adversely affect the generation of LAK activity by IL-2 [Nagler et al., J. Immunol.

141:2349 (1988)]. For example, if human PBMCs are cultured in 4 the presence of both IL-2 and IL-4, the lysis of LAK-sensitive targets is greatly reduced [Spits et al., J. Immunol. 141:29 (1988)].

If the PBMCs are pre-cultured in medium supplemented with IL-2 for 3 days before adding IL-4, however, augmented cytolytic activity results [Spits et al., supra]. Moreover, the blockade of IL-2-driven cytotoxicity by IL-4 can be abated when alphainterferon (a-IFN) or tumor necrosis factor-alpha (TNF-a )is included in the initial incubation mixture [Swisher et al., Cell.

Immunol. 128:450 (1990)].

1 0 Kedar et al., [Cancer Immunol. Immunother. 35:63 (1992)] have recently indicated that sequential administration of IL-2 and a-IFN is an effective immunotherapeutic regimen for treatment of MCA-105 sarcomas and M109 carcinomas in murine tumor models.

The primary finding from this study was that sequential S 15 administration of cytokines appeared to have greater efficacy than concomitant administration of both cytokines.

The feasibility and efficacy of adoptive immunotherapy as a treatment modality for various diseases, particularly for the treatment of neoplastic disease (cancer) in humans, have been described in U.S. Patent No. 4,690,915 to Rosenberg. As already noted above, however, there is a need for methods for carrying out such treatments that are not as toxic as those employing IL-2 alone. There is also a need for a therapy that is effective at low effector cell:target cell ratios.

25 SUMMARY OF THE INVENTION The present invention broadly describes methods for the use of IL-10 alone or in combination with IL-2 and/or a-IFN to augment cytolytic activity of PBMCs, especially LAK and NK cells.

In a first aspect, this invention provides a method for treating cancer comprising administering IL-10 activated PBMCs to an individual afflicted with cancer, in an amount effect to cause regression of such cancer, wherein said PBMCs have been activated with IL-10 in the absence of IL-2.

Preferably administration of the activated PBMCs is accompanied and/or followed by administration of In one embodiment, the IL-10 is administered in combination with an amount of IL-2 sufficient to augment LAK cell activation but not to cause toxic side effects attributable to the use of IL-2 alone.

In another embodiment the IL-10 is administered in combination with an amount of a-IFN sufficient to augment

LAK

1 0 cell activation.

In still another embodiment the IL-10 is administered in combination with an amount of IL-2 sufficient to augment

LAK

cell activation but not to cause toxic side effects attributable to the use of IL-2 alone and with an amount of IFN sufficient to :1 5 augment LAK cell activation.

In another aspect, this invention provides a method of stimulating cytolytic activity of PBMCs comprising incubating PBMCs in the presence of IL-10 alone.

SIn another aspect, this invention provides a pharmaceutical composition for treating cancer comprising IL-10 activated PBMCs and a pharmaceutically acceptable carrier, wherein said PBMCs have been activated with IL-1 in the absence of IL-2.

Preferably, human IL-10, IL-2 and a-IFN are used in the foregoing methods and compositions, most preferably, recombinant 25 human IL-10, IL-2 and a-IFN.

DESCRIPTION OF THE INVENTION All references cited herein are hereby incorporated in their entirety by reference.

The present invention is an improvement over methods of the prior art which employ IL-2 to induce cytolytic activity in NK and LAK cells. The present invention greatly reduces the toxic side effects that typically result from the use of IL-2 in 'such methods WO 95/19780 PCT/IB94/00008 6 by eliminating the IL-2 completely, or greatly reducing the amount of IL-2 that must be used.

Unless defined otherwise, the various terms used herein have the same meanings as are well understood in the art to which this invention is directed.

As used herein the term "adoptive immunotherapy" means therapy involving the transfer of activated functional immune cells to a patient. Preferably, these cells will comprise LAK and NK cells originating from the actual patient undergoing treatment.

The term "regression" is defined herein to mean a measurable decrease in the size of one or more tumors, as commonly measured in the art.

As used herein, "interleukin-10" or "IL-10" is defined as a protein which has an amino acid sequence of mature lacking a secretory leader sequence) IL-10 as disclosed in U.S.

Patent Application Serial No. 07/917,806, filed July 20, 1992, which corresponds to International Application No. WO 91/00349, and has biological activity that is common to native IL-10. For the purposes of this invention both glycosylated produced in eukaryotic cells such as CHO cells) and unglycosylated chemically synthesized or produced in E. coli) IL-10 are equivalent and can be used interchangeably. Also included are muteins and other analogs, including BCRF1 (Epstein Barr Virus viral protein, which possess the biological activity of IL-10 suitable for use in the invention can be obtained from a number of sources. For example, it can be isolated from culture medium of activated cells secreting the protein. Additionally, the or active fragments thereof can be chemically synthesized using standard techniques known in the art. See Merrifield, Science 233:341 (1986) and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, 1989, I.R.L. Press, Oxford.

WO 95/19780 PCT/IB94/00008 7 Preferably, the protein or polypeptide is obtained by recombinant techniques using isolated nucleic acid encoding the polypeptide. General methods of molecular biology are described, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York, 2d ed., 1989, and by Ausubel et al., (eds.) Current Protocols in Molecular Biology, Green/Woley, New York (1987 and periodic supplements). The appropriate sequences can be obtained using standard techniques from either genomic or cDNA libraries. Polymerase chain reaction 1 0 (PCR) techniques can be used. See, PCR Protocols: A Guide to Methods and Applications, 1990, Innis et al., Academic Press, New York, New York.

Libraries are constructed from nucleic acid extracted from appropriate cells, see, for example, International Application Publication No. WO 91/00349, which discloses recombinant methods for making IL-10. Useful gene sequences can be found, in various sequence databases, GenBank and BMPL or nucleic acid and PIR and Swiss-Prot for protein, c/o Intelligenetics, Mountain View, California, or the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wisconsin, which are incorporated herein by reference.

Clones comprising sequences that encode human IL-10 have been deposited with the American Type Culture Collection

(ATCC),

Rockville, Maryland, under Accession Nos. 68191 and 68192.

Identification of other clones harboring the sequences encoding is performed by either nucleic acid hybridization or immunological detection of the encoded protein, if an expression vector is used. Oligonucleotide probes based on the deposited sequences disclosed in International Application Publication No.

WO 91/00349 are particularly useful. Oligonucleotide probes sequences can also be prepared from conserved regions of related genes in other species. Alternatively, degenerate probes based on the amino acid sequences of IL-10 can be used.

WO 95/19780 PCT/IB94/00008 8 Standard methods can be used to produce transformed prokaryotic, mammalian, yeast or insect cell lines which express large quantities of the polypeptide. Exemplary E. coli strains suitable for both expression and cloning include W3110 (ATCC Bi, 27325), X1776 (ATCC No. 31244). X2282, RR1 (ATCC Mp/ 31343).

Exemplary mammalian cell lines include COS-7 cells, mouse L cells and CHP cells. See Sambrook (1989) and Ausubel et al., 1987 supplements).

Various expression vectors can be used to express DNA 1 0 encoding IL-10. Conventional vectors used for expression of recombinant proteins in prokaryotic or eukaryotic cells may be used. Preferred vectors include the pcD vectors described by Okayama et al., Mol. Cell. Biol. 3:280 (1983); and Takebe et al., Mol.

Cell. Biol. 8:466 (1988). Other SV40-based mammalian expression 1 5 vectors include those disclosed in Kaufman et al., Mol. Cell. Biol.

2:1304 (1982) and U.S. Patent No. 4,675,285. These vectors are particularly useful in COS-7 monkey cells (ATCC No. CRL 1651), as well as in other mammalian cells such as mouse L cells.

See also, Pouwels et al., (1989 and supplements) Cloning Vectors: A Laboratory Manual, Elsevier, New York.

The IL-10 can be produced in soluble form such as a secreted product of transformed or transfected yeast or mammalian cells.

The peptides can then be purified by standard procedures that are known in the art. For example, purification steps could include ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and the like.

See Methods in Enzymology Purification Principles and Practices (Springer-Verlag, New York, 1982).

Alternatively, IL-10 may be produced in insoluble form such as aggregates or inclusion bodies. The IL-10 in such a form is purified by standard procedures that are well known in the art.

Examples of purification steps include separating the inclusion bodies from disrupted host cells by centrifugation, and then WO 95/19780 PCT/IB94/00008 9 solubilizing the inclusion bodies with chaotropic agent and reducing agent so that the peptide assumes a biologically active conformation. For specifics of these procedures, see, e.g. Winkler et al., Biochemistry, 25:4041 (1986), Winkler et al., Bio/Technology 3:9923 (1985); Koths et al., and U.S. Patent No. 4,569,790.

The nucleotide sequences used to transfect the host cells can be modified according to standard techniques to make IL-10 or fragments thereof with a variety of desired properties. Such modified IL-10 can vary from the naturally-occurring sequences at 1 0 the primary structure level, by amino acid, insertions, substitutions, deletions and fusions. These modifications can be used in a number of combinations to produce the final modified protein chain.

The amino acid sequence variants can be prepared with various objectives in mind, including increasing serum half-life, facilitating purification or preparation, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic use. The amino acid sequence variants are usually predetermined variants not found in nature, although others may be post-translational variants, glycosylated variants or proteins which are conjugated to polyethylene glycol (PEG), etc. Such variants can be used in this invention as long as they retain the biological activity of Modifications of the sequences encoding the polypeptides may be readily accomplished by a variety of 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 the desired characteristics. For instance, International Application Publication No. WO 91/00349 describes a number of in vitro assays suitable for measuring IL-10 activity.

Preferably, human IL-10 is used for the treatment of humans, although viral IL-10 or IL-10 from some other mammalian species could possibly be used. Most preferably, the WO 95/19780 PCT/IB94/00008 used is recombinant human IL-10. The preparation of human and mouse IL-10 has been described in International Application WO 91/00349. The cloning and expression of viral (BCRF1 protein) from Epstein Barr virus has been disclosed by Moore et al., Science 248:1230 (1990). Recombinant human is also an article of commerce, available for purchase e.g., from PreproTech, Inc., Rocky Hill, NJ.

Individuals suitable for treatment by the methods of this invention include any individual with a neoplastic disorder that 1 0 would benefit from stimulation of PBMC cytolytic activity, especially LAK and NK cell activation. Exemplary cancer patients are described, in the patent and Scientific American paper of Rosenberg, supra. Also suitable for treatment by the methods of this invention are individuals predisposed to elevations of 1 5 endogenous IL-4 levels, such that the IL-4 blocks the IL-2 activation of cytolytic activity. In such individuals, the preferred treatment would involve pre-treatment with IL-10 prior to the administration of IL-2.

The standard methods and techniques described for making IL-10 for use in this invention can also be employed to make IL-2 and c-IFN. IL-2 and a-IFN for use in this invention are also available from commercial sources IL-2 is available from Cetus, Corporation, Emeryville, CA and a-IFN is available from Schering Corp., Kenilworth, NJ).

Extra-corporeal activation of PBMCs (preferably obtained by standard methods from a patient that is to be treated) and administration of such cells are carried out essentially as described in the references of Rosenberg mentioned above, except that together with reduced levels of IL-2 and/or a-IFN are used as described herein. The number of activated PBMCs administered is in the range of about 106 to about 1012 cells. Preferably, although not necessarily, administration of such activated cells is WO 95/19780 PCT/IB94/00008 11 accompanied and/or followed by administration of IL-10 as described herein.

In one embodiment, PBMCs are activated by pre-treatment with IL-10 by incubating for about three days at 37 0 C in the presence of 4 ng/ml (100 units/ml) or 40 ng/ml human Then, the cells are washed to remove free IL-10, and low levels of IL-2 (typically about 2 units/ml) are added.

Administration of cytolytic cells activated by IL-10, alone or in combination with the other cytokines used herein, is preferably by intravenous infusion. This can be carried out, through a central venous catheter, into a large peripheral vein, or into the hepatic artery via a percutaneous catheter.

is generally administered as a pharmaceutical composition comprising a pharmaceutical carrier and effective 1 5 amount of IL-10 alone or in combination with IL-2 and/or a-IFN.

A pharmaceutical carrier can be any compatible non-toxic substance suitable for delivery of the invention to a patient.

Compositions useful for parenteral administration of such drugs are well-known, see Remington's Pharmaceutical Science, 15th Ed.

(Mack Publishing Company, Easton, PA, 1980). Alternatively, compositions of the invention may be introduced into a patient's body by implantable or injectable drug delivery system, e.g., Urquhart et al., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984); Lewis Controlled Release of Pesticides and Pharmaceuticals (Plenum Press, NY,1981); U.S. Patent No. 3,270,960; and the like.

Cytokine administration can be carried out by any of the well known routes of administration, including by intravenous, intraperitoneal and subcutaneous administration. Intravenous administration is preferred.

When administered parenterally, the compositions are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutical carrier. Examples of such carriers are normal saline, Ringer's solution, dextrose WO 95/19780 PCT/IB94/00008 12 solution, and Hank's solution. Non-aqueous carriers such as fixed oils and ethyl oleate may also be used. A preferred carrier is dextrose/saline. The carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, buffers and preservatives. The IL-10 is preferably formulated in purified form substantially free of aggregates and other proteins at a concentration in the range of about 5 to gg/ml.

The compositions of this invention can also be delivered by 1 0 standard gene therapy techniques, including direct DNA injection into tissues, the use of recombinant viral vectors and implantation of transfected cells. See, Rosenberg, J. Clin. Oncol.

10:180 (1992).

Co-administration of one or more of the therapeutic agents 1 5 described herein can be concomitant (together with the administration of the IL-10) or sequential. Preferably, the IL-10 is administered prior to the administration of the EL-2.

Administration of a-IFN can be concomitant with the IL-10 and/or the IL-2 or sequential. All of the administered agents should be present in the patient at sufficient levels to be therapeutically effective in producing tumor regression.

As used herein the term "effective amount" means the amount of IL-10 sufficient to reduce or prevent side effects in adoptive immunotherapy and to at the same time promote LAK and NK cell cytolytic activity. The effective amount of cytokine(s) needed for a particular patient may vary depending on such factors as the state and type of the neoplastic disease being treated, the overall health of the patient, methods of administration, the severity of the side effects, the amount and 3 0 kinds of other drugs being used concurrently, and the like.

An amount of a cytokine "sufficient to augment LAK cell activation" is defined herein to mean an amount of a cytokine required to produce at least about a 25% increase in the level of WO 95/19780 PCT/IB94/00008 13 cytolytic activity induced by IL-10 alone, in a cytolytic assay based on Daudi cells. Preferably the increase will be at least about and most preferably, at least about 100%.

Preferably, the IL-10 is administered in the maximally tolerable dose, from about 10 U/kg body weight per day to about 108 U/kg body weight per day. Likewise, IL-2 and a-IFN are also to be administered in the maximally tolerable dose for the IL-2 dose: 105 U/kg body weight given intravenously every 8 hours in 50 ml of 0.9% saline with 5% albumin as a carrier; for the a-IFN dose: 106 U/kg body weight given intravenously every 8 hours in 0.9% saline with 5% albumin as a carrier). As previously stated, dosing is to be adjusted by attending physician to fall within those limits determined to be tolerable for each patient individually.

1 5 The methods of this invention can also be used in conjunction with traditional approaches to the treatment of cancer, such as radiation therapy and chemotherapy, using traditional chemotherapeutic agents such as the Vinca alkaloids, platinum compounds and Treatment of human peripheral blood mononuclear cells with alone or in combination with other cytokines elicits an increase in cytolytic activity toward human tumor targets. Since the efficacy of immunotherapy is in large part dependent upon tumor burden, it would be especially beneficial to employ the treatment methods described herein after the bulk of the primary tumor mass has been excised. An inflammatory reaction which typically occurs at the surgical site may also be beneficial to the therapeutic outcome.

EXAMPLE

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

WO 95/19780 PCT/IB94/00008 14 Effect of IL-10 on Cvtolytic Activity in Human Peripheral Blood Mononuclear Cells The effect of IL-10 on in vitro generation of cytolytic activity in human peripheral blood mononuclear cells was studied. Results can be summarized as follows: 1. IL-10 stimulates lymphokine activated killing (LAK) and natural killer (NK) activities in human peripheral blood mononuclear cells. IL-10 driven cytolytic activity can be neutralized by rat monoclonal antibodies against 2. IL-10 derived from CHO and E. coli expression systems display similar concentration response patterns in stimulation of LAK and NK activities and are thus biologically equivalent.

3. PBMCs treated with IL-10 and low concentrations 1 5 of IL-2 display LAK activities greater than observed with either cytokine alone.

4. PBMCs pre-treated with IL-10 for 2 days demonstrate increased cytolytic activity upon subsequent addition of IL-2.

5. IL-10 antagonizes the ability of IL-4 to inhibit IL-2-induced LAK activity.

6. IL-10 plus IL-2 produces enhanced LAK cytolytic activity at low effector cell:target cell ratios.

In addition to the effects observed on PBMCs, it was found that endothelial cells cultured in the presence of demonstrate an unimpaired response to exogenous factors to y-IFN and to TNF-a), whereas endothelial cells incubated in the presence of IL-2 were unresponsive due to IL-2 toxicity.

WO 95/19780 PCT/IB94/00008 Materials and Methods Recombinant Human Cytokines and Anti-IL-10 Antibodies Recombinant human IL-10 (both E. coli- and CHO-derived) was produced by standard methods. Specific activities obtained following purification by standard methods were 4.1 x 107 coli) and 2.1 x 107 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 essentially homogeneous had about 100 units of biological activity as thus defined.

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

Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) Peripheral blood was obtained by venipuncture from healthy 1 5 adult donors using heparin or EDTA as an anticoagulant. PBMCs were separated by a two-step protocol consisting of dextran sedimentation followed by centrifugation on FICOLL PAQUE® at 1250 rpm for 30 minutes. The interface bands comprised primarily of lymphocytes and monocytes were collected and washed at least twice with RPMI containing 10% fetal calf serum (complete medium) (JRH Biosciences).

Cytotoxicitv Assays Daudi (LAK-sensitive) and K562 (NK-sensitive) target cells were obtained from American Type Tissue Collection under accession Nos. CCL 213 and CCL 243, respectively. Daudi and K562 were labelled with 5 1 Cr as described by Spits et al., Immunol.

141:29 (1998)]. After the culture period, PBMCs were harvested, washed twice, and used as effector cells in a 5 1 Cr-release assay (Spits et al., supra). 5 X 103 5 1 Cr-labelled target cells were mixed WO 95/19780 PCT/IB94/00008 16 with varying numbers of effector cells (E/T 20:1; 5:1 and 2:1) in 100 tl in V-shaped bottom 96-well plates. The plates were centrifuged for 5 minutes at 1000 rpm before incubation for 4 hours at 370C in a humidified 5% CO 2 atmosphere. After 4 hours the plates were centrifuged for 5 minutes at 500 x g. Supernatants were collected using a SKATRON® harvestor (Skatron Instruments) and counted in a gamma counter (LKB-Pharmacia). Total lysis was determined by incubating 51Cr-labelled targets with 1% SDS. Data were represented as the mean of triplicate determinations.

1 0 Percent lysis was calculated as follows: cpm released experimental cpm spontaneous lysis x 100 cpm total lysis cpm spontaneous Incubation of Human PBMCs with Cytokines a. Incubation with IL-10 alone PBMCs isolated as described above were maintained at a concentration of 1 x 106 cells/ml in RPMI-1640 containing fetal calf serum supplemented with IL-10 or human IL-10 (CHO) at 370C for 3 days unless otherwise specified. 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 human IL-2 (Genzyme) (2 or 20 U/ml) at 37oC for 3 days.

c. Sequential incubation with IL-10 and IL-2 PBMCs were incubated with 4 ng/ml IL-10 in complete medium for 2 days. Human IL-2 was added to a final concentration WO 95/19780 PCT/IB94/00008 17 of 2 or 20 U/ml. After overnight incubation, LAK and NK cytolytic activities were determined.

Effect of Anti-IL-10 Monoclonal Antibodies on IL-10 Activation of LAK and NK Cells PBMCs were incubated with 40 ng/ml IL-10 for 3 days in the presence of 2 jg/ml of an anti-IL-10 monoclonal antibody (19F1) or an isotypic control (rat IgG2a).

Effect of IL-10 on Lvmphokine-Activated Killer Cell and Natural Killer Cell Cvtolvtic Activity 1 0 in Human Peripheral Blood Mononuclear Cells Cytolytic activity of LAK and NK cells can be operationally distinguished based on the tumor cell target used. Daudi cells, derived from a human Burkitt's lymphoma, are the traditional targets for activated LAK cell cytolytic activity. Cells from the K562 cell line, a human erythroleukemic cell line, are employed as specific targets for the cytolytic activity of activated NK cells. In these experiments human PBMCs were treated with various concentrations of IL-10 for 3 days. Cytolytic activity was determined in a standard 5 1 Cr release assay as described above.

Results based on LAK activity are shown in Table 1, in which standard errors are shown below the mean values.

WO 95/19780 PCT/IB94/00008 18 Table 1: Stimulation of Lymphokine-Activated PBMCs by (expressed as Lysisa) Concentration of IL-10 (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 20.2 c 31.7c 31.0c 40.2c ±3.3 +2.6 ±5.1 ±6.8 ±3.2 a Percent lysis of 51 Cr Daudi targets in standard chromium release assay at an effector to target ratio of 20:1. Data are represented as the mean of triplicate determinations.

b Human PBMCs prepared from normal donors were treated with for 3 days before determination of cytolytic activity.

c Significant difference between IL-10 treated and medium control at pK. 0.05 as determined by Student's t-test.

The data of Table 1 show the effect of IL-10 on LAK activity in PBMCs obtained from six human donors. Although variability among the donors was evident, IL-10 induced a concentration-dependent increase in cytolytic capacity in all 6 donors. Statistically significant activity (p 0.05) was observed at concentrations of IL-10 of 0.4 ng/ml or greater. It was also observed that donor PBMCs displaying a basal level of cytolytic activity in the absence of cytokine) of 5% or less were most responsive to IL-10 at all effector cell:target cell ratios tested (data not shown).

WO 95/19780 PCT/IB94/00008 19 Similar results were obtained against other tumor target cells, including a human renal cell carcinoma line, two different human melanoma lines and a human colon carcinoma line. The renal carcinoma and melanoma cell lines have also been used by Rosenberg, and patients bearing such tumors have been treated in vivo by Rosenberg using adoptive immunotherapy with IL-2.

In all cases, percent lysis of target cells was IL-10 concentration dependent.

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

1 5 Basal NK cytolytic activity lysis of K562 targets in the absence of cytokines) tends to be higher than basal LAK activity.

NK activity can be further increased 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 from the same 6 donors mentioned above, in experiments run in parallel with the LAK assays. The results are shown in Table 2, in which standard errors are shown below the mean values.

WO 95/19780 PCT/IB94/00008 Table 2: Stimulation of NK Activity lysisa) in PBMCs by (expressed as 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 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 c 59 71c ,5 ;c +5.2 +3.9 +8.2 +7.8 a Percent lysis of 5 1 Cr Daudi targets in standard chromium release assay at an effector to target ratio of 20:1. Data are represented as the 2 0 mean of triplicate determinations.

b Human PBMCs prepared from normal donors were treated with for 3 days before determination of cytolytic activity.

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

As is evident from Table 2, IL-10 induced a significant dosedependent enhancement of NK-cell mediated cytotoxicity in PBCMs from the donors. There was a statistically-significant increase in lytic activity at concentrations of IL-10 of 4 ng/ml or greater. As in the case of LAK activity, the effect of IL-10 on NK activity varied from donor to donor.

Comparison of Effects of IL-2 versus IL-10 on Endothelial Cells Experiments were conducted to evaluate endothelial cell monolayer viability following exposure to IL-10. Endothelial cells WO 95/19780 PCT/IB94/00008 21 cultured in the presence of IL-10 demonstrated an unimpaired response to exogenous cytokines (y-IFN and TNF-a), whereas parallel cultures exposed to unit-equivalent doses of IL-2 for the same incubation period lost the capacity to respond due to IL-2 toxicity.

Effect of Anti-IL-10 Monoclonal Antibodies on IL-10 Activation of LAK and NK Cells As shown in Tables 3 and 4, where mean values are shown with standard errors, the stimulation of LAK and NK cytolytic activities, respectively, by 40 ng/ml IL-10 were reduced 3-fold 0.05) in the presence of 2 jig/ml anti-IL-10 monoclonal antibody 19F1. Results produced using 2 gg/ml rat IgG2a isotypic control antibodies instead produced levels of cytolytic activity that were statistically indistinguishable from those obtained using 40 ng/ml IL-10 alone.

WO 95/19780 PCT/IB94/00008 Table 3: Inhibition of IL-10-Induced Lymphokine-Activated Killer Cell Cytolytic Activity (expressed as lysisa) by Monoclonal Antibodies Incubation Conditionsb Donor 1 2 3 4 medium 40ng/ml IL-10 19F1 +IgG2a 5.6 4.7 5.4 2.1 4.7 23.5 21.3 42.5 42.0 19.1 15.7 11.0 0.0 12.8 0.0 28.2 17.5 35.8 26.5 11.65 Mean 4.5±0.6 29.6+5.3 7.9c+3.3 23.9+4.3 a Percent lysis of [5 1 Cr] Daudi targets in standard chromium release assay at an effector:target ratio of 20:1. Data are represented as the mean of triplicate determinations.

b Human peripheral blood mononuclear cells isolated from normal donors were treated with 40 ng/ml IL-10 for 3 days in the presence of 2 mg/ml 19Fl(anti human IL-10) or rat IgG2a (isotypic control) before determination of cytolytic activity.

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

WO 95/19780 PCT/IB94/00008 23 Table 4: Neutralization of IL-10-Induced Natural Killer Cell Activity (expressed as lysisa) by Monoclonal Antibodies Incubation conditionsb Donor medium 40ng/ml 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 23.2 53.2 54.5 84.9 Mean 22.5+1.6 47.9+7.6 19.1c+6.6 51.3+12.7 apercent lysis of [51Cr] Daudi targets in standard chromium release assay at an effector:target cell ratio of 20:1. Data are represented as the mean of triplicate determinations.

bHuman peripheral blood mononuclear cells isolated from normal 2 5 donors were treated with 40 ng/ml IL-10 for 3 days in the presence of 2 mg/mll9Fl( anti human IL-10) or rat IgG2a (isotypic control) before determination of cytolytic activity.

CSignificant difference between IL-10 treatment alone and treatment in the presence of anti IL-10 antibodies at 0.05 as 3 0 determined by Student's t-test.

Cvtolvtic Activity Induced by Combinations of and Other Cytokines a. Simultaneous Incubation with TL-10 and IL-2 Human PBMCs were incubated with submaximal stimulatory concentrations of IL-2 2 or 20 U/ml) in the presence of WO 95/19780 PCT/IB94/00008 24 using an effector-to-target ratio of 5:1, with the results shown in Table 5 in which standard errors are shown beneath the mean values.

Table 5: Induction of Lymphokine-Activated Cytolytic Activity (expressed as lysisa) in Human Peripheral Blood Mononuclear Cells by Co-Incubation with and IL-2 Incubation Conditionsb Donor medium 2U IL-2 4ng IL-10 IL-10 20U IL-2 5 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 Mean 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] Daudi targets in standard chromium release assay at an effector:target cell ratio of 5:1. Data are represented as the mean of triplicate determinations.

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

c No significant difference between cytolytic activity in donor PBMCs treated with IL-10 and IL-2 compared to 20 U/ml IL-2 alone at p< 0.05 as determined by Student's -test.

WO 95/19780 PCT/IB94/00008 As shown in Table 5, there was an additive increase in LAK activity (effector cell:target cell ratio of 5:1) following co-incubation with 2 U/ml IL-2 and 4 ng/ml IL-10. This level of activity, which was significantly greater than that seen with either cytokine alone, approached the level produced using a 10-fold higher concentration of IL-2. This result was statistically significant at p< 0.05 as determined by the Student's t-test.

b. Simultaneous Incubation with IL-10 and a-IFN PBMCs co-incubated with IL-10 and a-IFN display a similar 1 0 additive increase in lytic activity against Daudi, but not NK target cells. This is shown in Table 6, in which standard errors are shown beneath the mean values.

Table 6: Induction of Lymphokine-Activated Killer Cell Activity 1 5 (expressed as lysisa) in Human Peripheral Blood Mononuclear Cells by Co-incubation with IL-10 and a-IFN Incubation Conditionsb Donor medium a-IFN IL-10 IL 10 +a-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 Mean 2.3 10.6 12.6 31.4 +1.4 ±3.8 +2.7 a Percent lysis of [5 1 Cr]Daudi targets in standard chromium release assay at an effector:target cell ratio of 10:1. Data are represented as the mean of 3 0 triplicate determinations.

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

WO 95/19780 PCT/IB94/00008 26 The differences observed in cytolytic activity induced in donor PBMCs by IL-10 and a-IFN compared to that induced by or a-IFN alone were statistically significant at p< 0.05 as determined by the Student's r-test.

In contrast, concomitant incubations with IL-10 and IL-4, GMCSF or y-IFN were no more effective than IL-10 alone (data not shown).

Sequential Incubations with IL-10 and IL-2 Using the procedures described above, PBMCs were maintained in medium alone or medium supplemented with After two days, IL-2 was added to a final concentration of 2 or 20 U/ml. Cytotoxic activity against Daudi cells was assessed following an additional overnight incubation. The results from a pool are shown in Table 7, in which stardard errors of the mean are shown beneath the mean values.

WO 95/19780 PCT/IB94/00008 27 Table 7: Induction of Lymphokine-Activated Cytolytic Activity (expressed as lysisa) in Human Peripheral Blood Mononuclear Cells by Pre-incubation with Followed by IL-2 Pre-Incubation Conditionsb 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 Mean 16.2 37.0 41.7 71.8 +5.1 +9.0 +11.4 +17.9 a Percent lysis of [51Cr] Daudi target cells in standard chromium release assay at an effector:target cell ratio of 5:1. Data are represented as the mean of triplicate determinations.

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

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

d Donor PBMCs were incubated in medium alone before adding IL-2.

e Donor PBMCs were incubated with IL-10 for 2 days before adding 20 U of IL-2 WO 95/19780 PCT/IB94/00008 28 As shown in Table 7, in the groups where donor PBMCs were pre-treated with IL-10 before addition of IL-2 an approximately 2-fold higher level of cytolytic activity was observed compared to the level observed in cultures maintained in complete medium only before adding IL-2 41.7% 11.4 The differences observed between samples pre-treated with IL-10 and those not treated with IL-10 were statistically significant at 0.14.

A similar pattern of enhanced cytolytic activity was seen in 1 0 sequential incubations with IL-10 followed by a-IFN (data not shown).

and IL-4 Blockage of IL-2-Induced Cvtotoxicity PBMCs from six human donors were cultured in medium containing 20 U/ml IL-2 alone, 20 U/ml IL-2 plus 1000 U/ml IL-4, or 20 U/ml IL-2 plus 1000 U/ml IL-4 plus 4 ng/ml IL-10. The results are shown in Table 8, in which standard errors of the mean are shown under the mean values.

WO 95/19780 PCT/IB94/00008 29 Table 8: Antagonism by IL-10 of the Blockade by IL-4 of IL-2-induced Lymphokine-Activated Cytolytic Activity (expressed as lysisa) in Human Peripheral Blood Mononuclear Cells Donor Medium IL-2b IL-2c+ IL-2 d IL-4 IL-4 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 3.3 47.1 17.1 63.8 6 6.6 49.5 20.6 40.8 Mean 5.5 34.2 19.7 40.3 ±1.6 ±4.5 ±3.2 ±5.7 a Percent lysis of [51 Cr] Daudi targets in standard chromium release assay at an effector:target cell ratio of 20:1. Data are represented as the mean of triplicate determinations.

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

CDonor PBMCs were incubated with 20 U/ml IL-2 and 100 U/ml human IL-4 for 3 days.

dDonor PBMCs were incubated with 20 U/ml IL-2, 1000 U ml human IL-4, and 4 ng/ml The data of Table 8 show that following treatment with U/ml IL-2 alone, about 34.2% lysis of LAK-sensitive targets was observed. When 1000 U/ml human IL-4 was included at the start of the incubation period, cytolytic capacity was suppressed approximately 2-fold. This suppression by IL-4 was not observed, however, if 4 ng/ml IL-10 was added during the first 24 hours of incubation.

WO 95/19780 PCT/IB94/00008 There was no significant difference between the results produced by IL-2 alone and by all three cytokines together (p.0.05, as determined by Student's t-test), indicating that antagonism by IL-10 of the blockage was essentially complete.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will become apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims.

Claims (15)

1. A method for treating cancer comprising administering IL-10-activated PBMCs to an individual afflicted with cancer, in an amount effective to cause regression of such cancer, wherein said PBMCs have been activated with IL-10 in the absence of IL-2.

2. The method of claim 1 which further comprises concomitant or subsequent administration of IL- IL-2 and/or a-IFN.

3. A method for the manufacture of a pharmaceutical composition for treating cancer comprising admixing IL-10-activated PBMCs with a pharmaceutically acceptable carrier, wherein said PBMCs have been activated with IL-10 in the absence of IL-2.

4. The method of claim 3 further comprising admixing IL-10, IL-2 and/or a-IFN with said PBMCs and said pharmaceutical carrier. A pharmaceutical composition for treating cancer comprising IL-10-activated PBMCs and a pharmaceutically acceptable carrier, wherein said PBMCs have been activated with IL-10 in the absence of IL-2.

6. The pharmaceutical composition of claim which further comprises IL-10, IL-2 and/or a-IFN.

7. The use of IL-10-activated PBMCs for treating cancer, wherein said PBMCs have been activated with IL-10 in the absence of IL-2.

8. The use of IL-10-activated PBMCs for the manufacture of a medicament for treating cancer, wherein said PBMCs have been activated with IL-10 in the absence of IL-2.

9. The use of claim 8 in which the activated PBMCs are used with IL-10, or IL-10 in combination with IL-2 and/or a-IFN. A method of stimulating cytolytic activity of PBMCs comprising incubating PBMCs with IL-10 in the absence of IL-2. A 11. The method of claim 10 wherein the PBMCs are j J:\Speci\300 399\300 349\30087.doc 21/04/99 7 If/;, 32 incubated with IL-10 alone.

12. The method of claim 10 wherein the PBMCs are simultaneously incubated with IL-10 and X-IFN.

13. The method of claim 10 wherein the PBMCs are obtained from a cancer patient.

14. The method, pharmaceutical composition or use of any one of claims 1 to 13 in which the IL-10 is human A method for treating cancer according to claim i, the method being substantially as described herein with reference to the Example.

16. A method for the manufacture of a pharmaceutical composition for treating cancer according to claim 3, the method being substantially as described herein with reference to the Example.

17. A pharmaceutical composition for treating cancer according to claim 5, the composition being substantially as described herein with reference to the Example.

18. The use of IL-10 activated PBMC's for treating cancer according to claim 7, the use being substantially as described herein with reference to the Example.

19. A method of stimulating cytolytic activity according to claim 10, the method being substantially as described herein with reference to the Example. Dated this 21st day of April 1999 SCHERING CORPORATION By their Patent Attorneys GRIFFITH HACK J:\Speci\3OO 399\300 349\30087.doc 21/04/99