CA2181230A1 - Use of il-10 to stimulate peripheral blood mononuclear cell cytolytic activity - Google Patents

Abstract

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

Description

2lst~3a WO 95/19780 ~ /~3 USF OF !T -10 TQ STIMUI ~TE pF.R~ . BT I OD
MONONUCI F~R (~1 T CYTOLYTIC ACIIVITY
This invention relates to the use of ln~orlp~ n-lo (IL-10), 5 formally cytokine synthesis inhibitory factor (CSIF), for adoptive ~ :' ,"~ in the treatment of neoplastic disorders (cancer) by stimulation of cyto~ytic activity of ~. ', ' l blood m~ ' cells (PBMC).
~.9CKGROUND OF T~ V~ON
Immunologic aL~ h~F to cancer therapy are based on the notion that cancer cells have somehow evaded the body's defenses against aberrant or foreign cells and mr~l~c~ s, and that these defenses might be therapeutically stimlllot~d to kill or inhibit the growth of the cancer cells, e.g., as discussed by Klein (Immunology, 15 Wiley-T~ , New York, 1982, pp. 623-648). The recent observation that irnmune effectors can directly or indirectly inhibit tumor growth has led to renewed interest in this approach to cancer therapy [Tl~b.,.,--~" Concepts ~r~ 96 (1985) (natural killer cells resist tumor growth); Rosenberg et al., Ann.
20 Rev. Immunol. 4:681 (1988) (use of IL-2-activated killer cells to treat cancer); Ralf et al., J. Exp. Med. 16~:712 (1988) (tumoricidal activity of Llla.,lopl-ag~s stirn~ d by Iymrhol ~s); Tepper et al., Cell 57:503 (1989) (IL~ has; ~l activity); M. Cohen, "Ly ,'-' - and Tumor Immunityn, pp. 237-253, in S. Cohen, ed., 25 LJ r 1~,l' and the Immune Response (CRC Press, Boca Raton, 1 990)] .
Immune responsiveness to r-~ ~'~ is regulated by a variety of cell types and involves the actions of T-cell and monocyte-derived cytokines. One -~og- approach that has 0 shown clinical promise has been so-called "adoptive p~" using IL-2-activated killer cells [Rosenb~rg, CONFIRM~ION COPY

WO 9!i/19780 2 1 8 1 2 3 0 r~
supra; Rosenberg, Sci. Am., pp. 62-69 (May 1990)]. Although ~L-2 alone or in c~mhir~ with rnore traditional chemot~ .lic agcnts appears to be effectivc in treating certain ~qli~r~r~ri~s (e.g., renal cell C~il.J.~I~), unfortunate to~ic side effects such as 5 vascular bed leakage and edema a~ - ~ with q~' ali~u of effectiYe dosages of IL-2 have led some to suggest that the risks rnay outweigh the benefits [Cotran et al., J. Immunol. 139:1882 (1987); Edwards et al., Cancer Res. 52:3425 (1992)].
Human vascular ~ ot~ cells appear lo be particularly 10 sensitive to IL-2 to~icity, as ~ by increased vascular p~ Rhility (i.e. vascular leak :,~ ' o.llc) and edema. One of tbe facto}s contributing to this pathology may ~e ~ m~nt~d adhesio~
of IL-2-activated T cells and neutrophils to . ' '~ nol.~
as has previously been noted in vitro [Edwards et al., supra; Darnle 15 et al., 138:1779 (1987)].
An alternative therapeutic approach to the ~ gic treatment of r~ ctir disease is the adoptive transfer of ir~mune cells. Adoptive .,.~ is defined as the transfer to a tumor-bearing host of active imrnunologic reagents, such as cells

2 0 with anti-tumor activity that can mediate, either directly or indirectly, anti-tumor effects. Adoptive -' ..~,~
represents an attractive approach to the therapy of ~~~r'~ctjr disease. It should be noted that because active ~loO-reagents are being transferred to the host, complete host 2 5 ~ is not required. Thus, the immuno-~u~ ,ssi/ generally acsoci~d vith the tumor-bearing state does not represent a major problem to this therapeutic alternative.
Since host ~~:, is not required, and in fact may be benef~cial to the effects of the adoptive transfer of immune cells,

3 0 adoptive ~t~ can easily be combined with other therapies such as ~ or radiation therapy. Also, in contrast to other therapies, immun~u~ sio~ is unlikely to result from this tre~trn~nt 218123~
WO 95/19780 r.

Patients undergoing chemotherapy or radiation therapy tend to be - , u,ll-sed and will generally have a depleted supply of effector pçrirher~l blood ~ ~ ' cells (PBMCs) available for actiYation. A therapy that shows efficacy at low S effector cell:target cell ratios would thus be pa t; ' ~y adv~nt~g-.,b~ for such patients.
Immune ,~ .,..css to r- ~pl~CmC is rcgulated by a variety of cell types and involves the actions of T-cell and monocyte-derived cytokines. Adoptive -' a~/~ using 10 rec-~mhin~nt human cytokines has involved ' ' b ~. of peripheral blood mononuclear cells (PBMCs) ~ e~tra-corporeally [Phillips et al., J. Clin. Oncol. 5:1933:(1987); Perussia~
Curr. Opin. Immunol. 3:49:(19gl)] followed by ~J~lliDtl of IL-2 [Rosenberg et al.. J. Immunol. 138:1779 (1985)]. The 15 extra-co~poreal treatment of the PBMCs produces activated ly~ ' --activated killer (LAK) cells and activated natural killer (NK) cells having cytolytic activity for various tumor cells.
}2~c~ human IL-5 [Nagasawa et al., Cell. Immunol.
133:317 (1991)], IL-7 IStotter and Lotze. Arch. Surgery 126:1525 20 (1991)] and IL-12 [Gately et al., J. Immunol. 147:874 (1991)] have been reported to stimulate cytolytic activity in human PBMCs.
Il-l~.l~,.,~i..-10 (IL-10), originally described in mice as a cytolcine synthesis inhibitory factor secreted by specific helper T-cell subsets, appears to modulate the differentiation of murine 25 cytotoxic T-cells [Chen and Zlotnik, J. Immunol 147:528 (199~)]. It has also been found that human PBMCs incubated with s~p~ recovered from COS cells ll.. f~,~t.,d with the h~man IL-10 cDNA Iysed tumor cell targets in vitro. The T cells leO~ ' ~ to IL-10 with increased cytolytic potential were 3 0 identified to be of a CD56~ r~ ~. indicative of NK cells.
In some ci~ nr~ 5. IL-4 can adversely affect the of LAK activity by IL-2 [Nagler a al., J. Immunol.
141:2349 (198g)]. For example, if human PBMCs are cultured in WO95119780 218 t 23~ P~

the presence of boA;h IL-2 U?Ad IL-4, the Iysis of LAK-sensitive t~Argets is greatly reduced [Spits et al.. J. Lrnmunol. 141:29 (1988)].
If the AA~BMCs are pre-cultured in medium s_rr~ ~ with IL-2 for 3 days before adding IL-4, however, ~..A~...AA -tA d cytolytic 5 activity results [Spits cr al., sl~pra]. Moreover, Ahe blockade of IL-2-driven cytoto~icity by IL-4 can be abated when alpha-interferon (-IFN) or tumor necrosis factor-alpha (TNF-a )is included in the initial incubation n~ibcture rSwisher a al., Cell.
ImmAunol. 128:450 (1990)~.
Kedar e~ al., [Cancer Immunol. T ' . 35:63 (1992)]
have recently indicated that sequential a1~i~ L-~Alion of IL-2 and -IFN is arA effective: C!~' aAp.,lAlic regimen for treatmAent of MCA-105 sarcomas and M109 carcinomas in rnurine tumor r~Aodels.
The primary finding from this study was that A~ , - I
15 &~ini~ ion of cytokines appeared to have greater efficacy than C~-C~ A~ administration of both cytokines.
The feasibility and efficacy of adoptive ' ~y as a treat~ent ~odality for various diseases, particularly for tkAe treatnJAent of ~ -tir disease (cancer) in hurnans, have been 20 described in U.S. Patent No. 4,690,915 to Rosenberg. As a~Aready noted above, however, there is a need for methods for carrying out such II~A~A~ IS tha~ are not as to~ic as those e..,~ i..g lL-2 alone. There is also a need for a therapy that is effective at low effector cell:target cell ratios.
2 5 SUMMARY OF l~F. lDWENTlON
The present invention fills these needs by providing metAbods for the use of DL-10 alone or in cnmhir~ti~ with IL-2 and/or -IFN to augment-cytolytic activity of PBMCs, especially LAK and NK cells.
3 0 More ~Jaulh,ula~ly, thAis invention provides a method for treating cancer c~ i..g ~iminict~riA~g an effective amount of IL-10-activated PBMCs to a patient aMicoed with cancer, to cause 2t~1230 WO 95/19780 r~
s of such cancer. Preferably a ~ rl;~r of the activated PBMCs is ~c~ d and/or followed by a~ r~ ;~
of IL-10.
In one Gll.bo,lilll~ t, the IL-10 is s~iminirt^red in S cr~'~;-sti~r with an amount of IL-2 sufficient to augment LAl~ cell activation but not to cause toxic side effects ' '^ to thc use of IL-2 alone.
In another G...ho/~ - "t the IL-10 is & ~ ;C~ ,d in combination with an amount of a-IFN sufficient to augment LAK
10 cell activation.
In still another c,.llbo,lil.lc..~ the IL-10 is P~ ,1 in c~ with (a) an amount of IL-2 sufficient to augment LAK
cell activation but not to cause to~cic side effects = ' - ' ' - to the use of ~iL-2 alone and with (b) an amount of ~-IFN sufficient to 15 augment LAK cell activation.
This invention further provides a method for antagonizing blockade of IL-2-induced ~;ylOlO~h,iLy by _ ~ -g ~ IL-4 c~....l.. i~;..~ s,iminict~nng to a patient in need of such treat~eGnt an effective amount of IL-10.
2 0 This invention still further provides F' ~ ~ ~
C~lmrOci~ionc comprising IL-10 in c~.l.ir~ with IL-2 and/or a-IFN, and a ~ t cslly acceptable carrier.
Preferably, human IL-10, IL-2 and a-IF~ are used in the foregoing methods and compositions, most preferably, ,.
human IL-10, ~L-2 and -IFN.
DF.SCRTPIION OFTITF. INVF.l~l~ON
All ,~r~ cited herein are hereby - ~OIdt~ in their entirety by l~re.~ 'P.
The present invention is an illl~JlV~ t over methods of the prior art which employ IL-2 to induce cytolytic activity i~ NK
and LAK cells. The present inventioD greatly reduces the to~ic side effects that typically result from the use of IL-2 in such methods WO 95/19780 2 1 8 ~ 2 3 0 by elimi~ ing the IL-2 completely, or greatly reducing the a~nount of IL-2 that must be used.
Unless defined otherwise, the Yarious terms used herein have the same meanings as are well ~ ~ D~ood in the art to which this 5 invention is directed.
As used herein the terr~ "adoptive - ,"~" means therapy inYolving the transfer of activated fynctional irnmune cells to a patient. ~ ably, tnese cells will comprise LAK and NK cells originating from the actual patient ~ ~Oillg treatment.
The term ".~ .ssi~ " is defined berein to mean a l~ decrease in Lhe size of one or more ~umors, as cornmonly measured in the art.
As used herein, " ' 10" or "IL-10~ is defined as a protein which (a) has ân amino acid sequence of mature (e.g., 15 lac~ing a secretory leader sequence) IL-10 as disclosed in U.S.
Patent Application Serial No. 07/917,806, filed July 20, 1992, which c~ r ~- to IntPrr "ti~ -I Application No. WO 91100349, and (b) has biological activity that is cornmon to native ~-10. For the purposes of this invention both glycosylated (e.g. produced in 20 eukaryotic cells such as CHO cells) and L,,l~Dyl~t~,~ (e.g., chernically Dyttlh_DiL~d or produced in E. coli) IL-10 are equivalent and can be used interch~n~sl~ly. Also included are muteins and other analogs, including BCRF1 (Epstein Barr Virus viral IL-10) protein, which possess the biological activity of IL-10.
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. ~ -lly, the IL-10, or active fragments thereof can be ~h~mjrsl~y Dy ' using standard techniques lcnown in the a~t. See Ml~rrifiPlA
3 0 Science 233:341 (1986) and Atherton et al., Solid Phase P~ptide Synthesis: A Practical Approach, 1989, I.R.L. Press, Oxford.

21~t~3~
WO 95119780 r~

Preferably, the protein or polypeptide is obtained by recnn^hin^nt techniques using isolated nucleic acid encoding the IL-10 polypeptide. General methods of r^nl~cl~lnr biology are ~csrr~h~l e.g., Sambrook c^t al., Molecular Cloning, A Laboratory 5 Man~al, Cold Spring Harbor, New York, 2d ed., 1989, and by Ausubel et al., (eds.) Current Protocols in Molecl ~ar Biology, Green/Woley, New York (1987 and periodic ` ~ rl~ ). The appropriate se~ s can be obtained using standard tPch^i~, ~
from either genomic or cDNA libraries. PolymerA~se chain reaction 10 (PCR) i ' ,_-- can be used. See, ~.g., PCR Pro~ocols: A Gldde ~o Methods and Applications, 1990, Innis et al., (Ed.), Acade~Anic Press.
New York, New York.
Libraries are cv..s~ ,d from nucleic acid e~tracted from a~.v~lirAle cells, see, for example, International Application 15 Publication No. WO 91/00349, which discloses ~
methods for making IL-I0. Useful gene sequences can be found, e.g., in various sequence dotnhncps~ e..g., GenBank and BMPL or nucleic acid and PIR and Swiss-Prot for protein, c/o T 'lie~n~tirS
Mountain View, California, or the Genetics Coraputer Group, 20 University of Wisconsin Bict~ logy Center, Madison, Wisconsin, which are il.cv.~v..lt~,d berein by reference.
Clones comprising sc t, -eE that encode human IL-I0 have been deposited with the American Type Culture Cnl!~-ti~ (ATCC), Rockville, Maryland, under Accession Nos. 68191 and 68192.
2 5 T~' ^ rntin^ of other clones harboring the sequences encoding IL-I0 is p~.r~ -~ by either nucleic acid 1l~l,. ;.1;, -~i or logjrnl detection of the encoded protein, if an expression vector is uscd. OligJ^~I_ '-; 'r1~ probes based on the deposited C'' t~' - c,r5 disclosed in T t ,~-^l Application Publication No.
30 WO 91/00349 are particularly useful. Olig~ r probes S~ll can also be prepared from ~c - ~,d regions of related genes in other species. Alt~ ,ly, dc~ e probes based on the amino acid s~ s of IL-10 can be used.

woss/1s780 ~ ~ 8 1 230 1~I/.D ,. ~
Standard methods ean be used to produee llal.orO~
liC, ~ n yeast or inseet eell lines whieh express large quantities of the pol~,tidc. F ,l ~ E. coli strairls suitable for both C;~ and clonirlg include W3110 (ATCC Bi, 27325), X1776 (ATCC No. 31244). X2282, RRl (ATCC Mpl 31343).
F~ n cell lines include COS-7 ce~ls, rnouse L eells and CHP cells. See Sambrook (1989) aDd Ausubel et al., 1987 supplements) .
Various expression vectors ean be used to e~cpress DNA
10 encoding IL-10. Converltional vectors used for C~IJIC ~ of c~ t proteins in prokaryotic or euL,u~o~ic cdls may be used. Preferred vectors include the pcD vectors described by Okayarna a al., Mol. Cell. Biol. 3:280 (1983); and Takebe ~ al., l~ol.
Cell. Biol. 8:466 (1988). Other SV40-based .--~ enpression 15 vectors include those disclosed in Kaufman et al., Mol. Cell. Biol.
2:1304 (1982) and U.S. Patent No. 4,675,285. These SV40-based vectors are p.uli~ulally useful in COS-7 ~onlcey cells (ATCC No. CRL
1651), as well as in otner ' cells such as mouse L cells.
See also, Pouwels et al., (1989 and --rr ' ) Cloning Vectors:
20 A La~oratory Manual, Elsevier, New York.
The IL-10 can be produced in soluble for~n such as a secreted product of transformed or ~ ,rc~t~,d yeast or r~ cells.
The peptides can then be purified by standard p.~ O that are known in the art. For e~ample, ~_ ' steps could include 2 5 sulfate yl.~ , ion e~change chro~tcO , hy, gel filtration, elc~ ph~l~,oio, affinity ~hl~ , and the like.
See Methods in Enzymology Purification Principles and Practices (Springer-Verlag, New York, 1982).
Alternatively, IL-10 may be produced in insoluble for~ such 30 as Ag ~,~ or inclusion bodies. The IL-10 in such a form is purified by standArd ~ ,s that are well knowD in the art.
E~tamples of pllrifir~inn steps include s.,~u ali~g the inclusion bodies from disrupted host eells by c.,..llir,~, ' ADd then WO 95/19780 P~

sol-l~ili7in~ the inclusion bodies with chaotropic agent and reducing agent so that the peptide assumes a ''~'~gi~lly active ~ r - For specifics of these ~ s, see, e.g. Winkler e~ al., Bi~ , 25:4041 (1986), Winlcler et al., Bio/Te '~~ y 5 3:9923 (1985); Koths et al., and U.S. Patent No. 4 569,790.
The r~ Potitiç sc~l. used to transfect the host cells can be modified according to standard t.orhni~, 5 to ma~e IL-10 or fragments thereof with a variety of desired p~ lics. Such modified IL-10 can vary from the naturally-occurring s~ s at 10 the primary structure level, e.g., by amino acid, insertions, ~u~liluL, - deletions and fusions. These _-r l-- can be used in a number of cnmhi~f;~ - to produce the final modified protein chain.
The annino acid sequence variants can be prepared with 1~ various ~h;c~ in mind, including increasing serum half-life, f~ ing p "~~ n~ or preparation, improving tt` , '~
efficacy, and lessening the severity or o . ~e of side effects during i' a~ t;c use. The arnino acid sequence variants are usually rr~tçrrni--~ variants not found in nature, although 2 0 others may be post-tr~ncl~tionsl variants, e.g., glycosylated variants or proteins which are c ;,~, ~ to polyethylene glycol (PEG), etc. Such variants can be used in this invention as long as they retain the biological activity of IL-I0.
Mn~lifirsti( - of the sequences encoding the polypeptides 25 may be readily ~rc~ ' by a variety of techniques, such as site-directed mutagenesis ~Gillman et al., Gene 8:81 (1987)]. Most ~..o.1;r;..~ . e are evaluated by routine screening in a suitable assay for the desired chal.lct~ Lcs. For instance, Trt~rr.~ti- -l ~rp1irsti Publication No. WO 91/00349 describes a number of in 3 0 vitro assays suitable for . r7~ IL-I0 activity.
Preferably, human IL- 10 is used for the treatment of humans, although viral IL-10 or IL-I0 from some other ;9n species could possibly be used. Most ~r~ .bly, the WO95/19780 ~ ~ ~ t ~3 P ~

~-10 used is l~CC~-- k;.~ t human IL-10. The ~c~uatiOl~ of human and mouse IL-10 has been described in Interrlational ArF'--' WO 91/00349. The cloning and c;~ ,;.al. of viral lL-10 (BCRF1 protein) from Epstein Barr virus has been disclosed 5 by Moore et al., Science 248:1230 (1990). RPCC~ human IL-10 is also an article of CUIIIIII~ C, 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 ~ ctic disorder that 10 would benefit frorn ' ( of PBMC cytolytic activity, especially LAK and NK cell activation. E~emplary cancer patients are A~cr~ihetl e.g.. in the patent and Scientific American paper of Rosenberg, supra. Also suitable foJ treatment by the methods of this inventioD are individuals predisposed to elevations of pnl~-g--)u IL-4 levels. such that the IL-4 blocks the IL-2 activation of cytolytic activity. In such individuals, the preferred treat~nent would involve pre-treatment with IL-10 prior to the ad., i~ .tion of IL-2.
The standard methods and ~ s d~srnhed for making IL-10 for use in this invention can also be ernployed to make IL-2 and a-lFN. IL-2 and a-IFN for use in tnis invention are also available from c~ ,;al sources (e.g., IL-2 is available from Cetus, Corporation, Emeryville, CA and a-lFN is available from Schering Corp., Kenilworth, NJ).
2 5 E~tra-corporeal activation of PBMCs (preferably obtained by standard methods from a patient that is to be treated) and of such cells are calried out essentially as described in the ef,l~ - of Rosenberg ~ ~~ above, except that IL-10 together with reduced levels of IL-2 and/or ~-IFN are used as described herein. The number of activated PBMCs ~ ;r~-~.d is in the range of about 106 to about 1012 cells. Preferably. although not necessarily, a.l.. i..i~t.~.lion of such activated cells is WO95119780 2 1 8 1 230 1~11~ ,~
.
1 1 .
~c~ d and/or followed by ~JIlulliDLldlion of IL-10 as described herein.
In one ~ bo~' t, PBMCs are activated by pre-treatment with IL-10 [e.g., by ir^ul-~in~ for about three days at 37C in the S presence of 4 ng/ml (100 units/ml) or 40 ng/ml human IL-10].
Then, the cells are washed to remove free IL-10, and low levels of IL-2 (typically about 2 units/ml) are added.
Administration of cytolytic cells activated by IL-10, alone or in c~ n~tion with the other cytokines used herein, is preferably by i~lla~. ~ infusion. This can be carried out, e.g., through a ceDtral venous catheter, into a large p. ,' l vein, or into the hepatic artery via a percutaneous catheter.
IL-10 is generally ?~1minict~red as a r~
composition comprising a p~ l carrier and effective amount of IL-10 alone or in c~ ; 3ticr with IL-2 and/or a~-IFN.
A r~ r~ 1 carrier can be any c r~ ~ non-to~ic substance suitable for delivery of the invention to a patient.
C- n~roCitionc useful for paTenteral ~ ' - of such drugs are well-known, e.g., see Reming~on's Phar ~rr~lt;<-~1~ Science, 15th Ed.
2 0 (Mack Publishing Company, Easton, PA, 1980). Alternatively, compositions of the invention may be iu/~. d into a pa!ient's body by implantable or injectable drug delivery system, e.g., Urquhart et al., Ann. Rev. F`h~u.l.dcol. Toxicol. 24:199 (1984); Lewis (Ed.), Controlled ~elease of Pesticides and Phur ~ tic~r~ (Plenum Press, NY,1981); U.S. Patent No. 3,270,960; and the like.
Cytokine â.ll~ iDIIaliOn can be carried out by any of the well known routes of a.lllliuiD(.~.lion, including by i~
intraperitoneal and cllbcut~ r- aJ. UuiD~ n~ e. -~-ariminictr~tion is preferred.
3 0 When ~ parenterally, the compositions are ' ~ in a unit dosage ill;e :''- form (solution, D~D}J~ S-, emulsion) in ~cs~ci~tion with a pha.lua~ carrier. Examples of such carriers are normal saline. Ringer's solution, dextrose wo95/19780 2 ~ 8 f ~
solution, and Han~'s solution. Non-aqueous carriers such as fi~ed oils and ethyl oleste may also be used. A preferred carrier is 5%
d~All oDe/ ~ - The carrier may contain minor amounts of additives such as ~ - s that enhance ;c- ` ~y and chemical 5 stability, e.g., buffers and l.llGDe~ V~ D. The IL-10 is ~Jlcr~,~ably f~rn ~ d in purified form s~bstAntiAlly free of ~g~lC~It~,D and other proteins at a ,~ ~ in the range of about 5 to 20 ~Lg/ml.
The c ,- rJnC of this invention can also be delivered by 10 standard gene therapy techniques, including e.g.. dircct DNA
injection into tissues, the use of .,c-....k;, -l viral vectors and imrl~ ( of L,,.,.Or~,.,~d cells. See, e.g., Rosenberg, 1. Clin. Oncol.
10:180 (1992).
Co-adl.Pi.l;Di~ ion of one or more of the ~ ul;r agents 15 described herein can be C----~ AIII (together with the ad.lli.li~l.,.lion of the IL-10) or s~q~l~n~i~l Pl~r~ ly, the IL-10 is ~,7~mini~t~red prior to the ad.llilliD~_~ion of the IL-2.
,~-7~minic~r7~ti of ~L-IFN can be ~ ~ . _ with the IL-10 and/or the IL-2 or s~. 1 All of the 5~ d agcnts should be 2 0 present in the patient at sufficient levels to be ' A~uiically effective in producing tumor l~gl~DDiO--.
As used herein the term "effective amount" means the amount of IL-10 sufficient to reduce or prevent side effects in adoptive ~ J and to at the same time promote LAK
25 and NK cell cytolytic activity. The effective amount of cytokine(s) needed for a particular patient may vary d~r ' ~ on such factors as the state and type of the n~ ctir disease being treated, the overall health of the patient, methods of administration, the severity of the side effects, the amount and 30 kinds of other drugs being used ~ 7~, 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 2 1 8 1 2 3 0 . ~ "i ~

cytolytie activity induced by IL-10 alone, in a cyto~ytic assay based on Daudi eells. Preferably the increase will be at least about 50%, and most preferably, at least about 100%.
Preferably, the IL-I0 is ^-~1minict~red in the maximally S tolerable dose, from about 10 U/kg body weight per day to about 108 Ulkg body weight per day. Li~ewise, IL-2 and a-LFN are also to be ~ ,d in the ma~cimally tolerable dose (e.g., for the IL-2 dose: 105 Ulkg body weight given i~ r every 8 hours in 50 rnl of 0.9% saline with 5% albumin as a carrier; for the 10 ~,x-IFN dose: 106 U/kg body weight given iL.,A", 1y every 8 hours in 0.9% saline with 5% albumin as a earrier). As l lc;v;ousl~, sUted, dosing is to be adjusted by attending physician to fall within those lirnits ~et~rmir^~ to be tolerable for each patient individually.
The methods of this invention can also be used in conjunction with traditional ~w~ uach~ s to the treatrnent of cancer, such as radiation therapy and cL ~thr~rapy, using tr,~1itiQnAl - agents such as the vinr~^a alkaloids, platinum compounds and 5-fluorouracil.
2 0 Treatment of human peripheral blood r- '~ cells with IL-10 alone or in c~ hirAtil - with other cytokines elicits an increase in cytolytic activity toward human tumor targets. Sinee the efficacy of ~ , is in large part ~I r '~^ t upon tumor burden, it would be especially beneficial to employ the 2 5 treatment methods described herein after the bulk of the primary tumor mass has been e~cised. An infl^"~ t~ reaction whieh typically occurs at the surgical site may also be benefieial to the therapeutic outcome.
F.X~MPT .F
3 0 The following non-limiting Example will serve to illustrate the present invention.

woss/ls7so ? 1 81 233 ~"~,~ ~

FffeCt of n-lo on Cytolytic Activity ;n 1' ~~ I Per~pheral Blood Mo..v -~e Cells The effect of IL-10 on in vitro generation of cytolytic activity in human peripheral blood ' eells was studied. Results 5 can be ~ ~ as follows:
1. IL-10 ~ es 1~ activated l~illing (LAK) and natural ~iller (NK) activities in human F ,' ~I blood ~' eells. IL-10 driven cytolytic activity ean be r _ 1i7~d by rat monoclonal an~ihorli~s against IL-10.
2. IL-10 derived from CHO and E coli c~,~, - ic systems display similar concentration response patterns in 5tim~ of LAK and NK activities and are thus hirllogir~ly equivalent.
3. PBMCs treated with IL-10 and low c~ ~ ~liVIIS
of IL-2 display LAK activities g}eater than observed with either cytokine alone.

4. PBMCs pre-treated with IL-10 for 2 days d ~Ir~ increased cytolytic activity upon s~l~~~, t addition of IL-2.
2 0 5. IL-I0 antagonizes tne ability of IL-4 to i~hibit IL-2-induced LAK activity.
6. IL-I0 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 2 5 that ~ l eells cultured in the presence of IL-10 dr---- an b..il..~iled response to ~oO factors (i.e., to y-IFN and to TNF-a), whereas . 'o~ l eells incubated in the presence of IL-2 were ~ r ~, due to IL-2 toxicity.

21812~Qi WO 95/19780 1 ` I '~;

~at~rjAls And M,othods R~rr,mhi~nt ~Tl~m~n Cytr,ki--- An~l Anti-lT-10 An~ihr.~ c T~- ' human IL-10 (both E. coli- and CHO-derived) wa produced by standard methods. Specific activities obtained

5 following purifir~ti~ by standard methods were 4.1 ~ 107 (E coli) and 2.1 ~ 107 units/mg (CHO), as ~ ' -d by the MC-9 cell ~,lulir~,.alion assay [T~ Snipes et al., J. E~p. Med 1~3:507 (1991)]. Ap~lu~Lhl~ ;ly 4 ng of essentially ~- O - - IL-10 had âbOut 100 units of ~ ogir~l âctivity as thus defined.
A rat ânti-human IL-10 monoclonal antibody APcis 19FI was obtained from Dr. John Abrams of the DNAX Institute of Molecular Biology, Palo Alto, CA.
T~n1~tion of ~TIlmAn Peri~herAl Blood MononuclPAr Cells (PBMCs) Peripheral blood was obtained by ~ c from heâlthy 15 adult donors using heparin or EDTA as an ~n~iroA~ t PBMCs were separated by a two-step protocol ~ of de~tran s~ ;on followed by ruOa~On on FICOLL PAQUE~ at 1250 rpm for 30 minutes. The interface bands , ised primarily of 1~ t~o and ml ~ yt,,s were collected and 20 wâshed ât least twice with RPMI c~ r 10% fetal calf serum (complete medium) (JRH Ric s).
Cytotoxirity ACCays Daudi (lAK-sensitive) and K562 (NK-sensitive) target cells were obtained from American Type Tissue CollPrfi: under 25 accession Nos. CCL 213 ând CCL 243, I~,o~c~ ly. Daudi and K562 were labelled with 5ICr as described by Spits et al., [J. Irnmunol.
141:29 (1998)]. After the culture period, PBMCs were harYested, washed twice, and used as effector ceUs in a slCr-release assay (Spits et al., s~pra). S X 103 slCr-labelled target cells were mi~ed 2181~
WO 95/19780 1 _1/~, I.'t with varying numbers of effector cells (E/T = 20:1; 5:1 and 2:1) in 100 ~LI in V-shaped bottom 96-well plates. The plates were eentrifuged for 5 minutes at 1000 rpm before h~ion for 4 hours at 370C in a ' '~-d 5% CO2 ~ ..;. After 4 hours 5 the plates were c.,..t ir-,gcd for 5 minutes at 500 ~ g. SL~J.
were collected using a SKATRON~!9 harvestor (Skatron I~
and counte~ in a Bamma counter (LKB-~Lall..~c;a). Total Iysis was d: i by ' ~ 5ICr-labelled targets with 1% SDS. Data were ~ ,s~..t~d as the mearl of triplicate dct~ ;ons.
Percent Iysis was c~lr~ d as follows:
cpm released P~r~ ' - epm spon-or- ~
% Iysis = ~i 100 cpm total Iysis - cpm Sr ^
Tr~llhotir,n of ~TIlmon PBMCs with Cytrkir~c a. Incubation with IL-10 alone PBMCs isolated as described above were ~~ ~ ~ at a c. - - of 1 x 106 colls/ml in RPMI-1640 c~ 10%
fetal ealf serum su~l' ' with IL-IO or human IL-IO (CHO) at 370C for 3 days unless other~vise speeified. Cytolytie activity was 2 0 ~lp~rmin~pd as deseribed above.
b. ~imll!t~-~ i.. .' -rn with IL-10 and IL-2 PBMCs were ineubated with 4 ng/ml IL-10 with or without human ~-2 (Genzyme) (2 or 20 U/rnl) at 370C for 3 days.
C. Se~ -1 .' -- with IL-10 and IL-2 2 5 PBMCs were incubated with 4 ng/ml IL-10 in complete medium for 2 days. ~uman IL-2 was added to a fin I c ~

2t8123 woss/ls7so r~.,~s of 2 or 20 Ulml~ After overnight incubation, LAK and NK cytolytic activities were d~t~rl~ir-1 FffeCt of Anti-n.-10 Monocln~Pl Antibodi~c on n-]o ActiY~tion of L~K nd NK ('~llc PBMCs were incubated with 40 nglml IL-10 for 3 days in the presence of 2 ,ug/ml of an anti-IL-10 monoclonal antibody (19FI) or an isotypic control (rat IgG2a).
Fffect of Il-10 on Lyl7u?hokin~-Antivated ~ill.or C~ll pn~l N~ r~ Pr Cell Cvtolytic Activity 0 jn ~llrr.~ Per~pher~l 8100d M,~. L ~i ~. C~llc Cytolytic activity of LAK and NK cells can be op~rP~i. 'ly icti~lg~ d based on the tumor cell target used. Daudi cells, derived from a human Burkitt's Iymphoma, are the traditional targets for activated LAK cell cytolytic activity. Cells from the K562 cell line, a human erythr~leul~mi~ cell line, are employed as specific targets for the cytolytic activity of activated NK cells. In these r~ human PBMCs were treated with various c.,~ of IL-I0 for 3 days. Cytolytic activity was d - ~' in a standard 5 I 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 95119780 2 ~ ~ ~' 2 ~ Q , ~

Table 1: Stim~ tinn of L ~ rir~ Activated PBMCs by IL-10 (e~pressed as $ Lysis~
C~ atiOn of IT-10 tn~trnl)b 5Dnn~r 0 0.04 0.4 4 40 100 04.25 18.3 44 26.2 67.5 7.2 10 315 27.6 318 17.7 29.2 39.1 29.7 55.1 8.8 10.2 22.2 35.8 35.9 1 0 5 4.6 8.1 15.6 20.6 20.1 12.1 614.6 19.7 40.7 ~4.4 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 Iy6is of 51 Cr Daudi ~argets in staDdard cbromium rdease assay at an effector to target ratio of 20:1. Data are represeDted as thc mean of triplicate ~: -b l~umam PsMcs prepared from Dormal donors were treated witb IL-I0 2 0 for 3 days before s~ of cytolytic activity.
c Significant difference between IL-lo uea;ed and medium control at p 0.05 as determined by Student's ~-test.
The data of Table 1 show thc effect of IL-10 on LAK
activity in PBMCs obtained from si~ human donors. Although variability among the donors was evident, IL-I0 induced a er '~' ~ increase in cytolytic capacity in all

6 donors. St~tictir~lly ci~ifin~t activity (p < 0.05) was observed at ~( - of IL-10 of 0.4 ng/ml or greater. It was also observed that donor PBMCs displaying a basal level of cytolytic activity (i.e., in the absence of cytokine) of 5% or less were most vc to IL-10 at all effector cell:target cell ratios tested (data not shown).

~ Wo95/l9780 ~181230 Similar results were obtained against other tumor target cells, including a human renal cell carcinoma line, two different hurnan -' - lines and a human colon c~.: line. Thc renal c~ and ~ cell lines have also been used by 5 Rosenberg, and patients bearing such tumors have been treated in vi~o by Rosenberg using adoptive ' apy with IL-2.
In all cases, percent Iysis of target cells was IL-10 cr-- ' alion dependent.
In s~l~litiq--~l C~ C~ i, IL-10 alone or in c~mh~
10 with IL-2 was found to elicit an increase in cytolytic activity against U937 cells (human llislioc~ylv~a)~ SW620 (human colon carcinoma), and SKBR3 cells (human breast c~u. ). In ,ty using HS294T cells (human m~ nl ) as targets, IL-I0 did not appear to elicit a response at the doses tested.
Basal NK cytolytic activity (i.e., Iysis 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. E~p. Med 164:814 (1986)~.
The effect of IL-I0 on NK activity was evaluated in PBMCs 2 0 from the same 6 donors ~ d above, in c,.~ run in parallel with the LAK assays. The results are show~ in Table 2, in which standard errors are shown below the mean values.

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Table 2: S~ .. of NK Activity (e~pressed as %
lysisa) in PBMCs by IL-I0 C~c :~ ba~ion of IT.-10 (n~/~ub Donor 0 0.04 0.4 4 40 100 22.2 30.6 252 57.2 515 49.7 2 20.4 24.7 313 56.7 65.6 715 I 0 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 25.1 235 20.3 6 15.9 25.3 45.4 66.6 43.7 56.1 15 Mean: 24.4 30.2 305 54.0c 52.75C 55.5c +75 +5.2 +3.9 +8.2 +7.8 +8.5 2 Percent lysis of 5ICr Daudi targets in standard chromium release assay at an effector to target ratio of 20:1. Data are represented as tbe 2 0 mean of triplicate b Human psMcs prepared from norrnal donors were treated with IL-I0 for 3 days before ~ of cytolytic activity.
c Significant difference between IL-lo treated and medium control at p<0.05 as daermined by Student's l-test.

As is evident from Table 2, IL-10 indllced a ci~njfir~r~t dose-d r ' L~ of NK-cell mediated CylOtv~ ily in PBCMs from the donors. There was a 5t~-ictir~11y_ci~ ~ ' increase in Iytic activity at ~ ~ ations of IL-I0 Q 4 ng/rnl or greater. As 3 0 in t~te case of LAK actiYity, the effect of IL-I0 on NK activity varied from donor to donor.
C.. "~li of P.ffects of IL-2 versus IL-I0 on En~lothelia! Cells F-r~ were clln~u: ' to evaluate ~ l cell monolayer viability following exposure to IL-10. ~~~ tl '~l cells WO95/19780 2 t cultured in the presence of IL-10 demonstrated an u~ d response to exogenous cytokines (y-lFN and TNF-), whereas parallel eultures e~posed to unit-equivalent doses of IL-2 for the same ~ period lost the capaeity to respond due to IL-2 S to~ieity.
Fffect of A~i-n-10 Monoelnn~l Antibo~ c Qn IT-IO ArtivP~ion of L~K a~ NK ~Pllc As shown in Tables 3 and 4, where mean values are shown with standard errors, the - '.~t;~ of LAK and NK eytolytie 10 activities, .~s~ti~,ly, by 40 ng/ml IL-10 were reduced 3-fold (p< 0.05) in the presence of 2 ~Lg/ml anti-IL-10 ~--r~
antibody l9F1. Results p~oduced using 2 ~Lglml rat IgG2a isotypic control antibodies instead produced levels of cytolytic activity that were st~tictir~lly indistinguishable from those obtained using 15 40 ng/ml IL-I0 alone.

:~5 2~ 3~
WO 95119780 r~

Table 3: Inhibition of IL-10-lnduced L~ Activated Killer Cell Cytolytic Activity (e~pressed as % Iysisa) by Anti-IL-10 Monoclonal Antibodies Incubation Conditionsb Dono~ medium 40ng/ml ~-10 IL-10 IL 10 + l9Fl +IgG2a 5.6 235 15.7 28.2 24.7 21.3 11.0 17.5 35A 42.5 0.0 35.8 1 S 4 2.1 42.0 12.8 26.5 54.7 19.1 0.0 11.65 Mean 4.5+0.6 29.6+5.3 7 9c+3 3 23.9+4.3 2 0 a Pacent Iysis of [5 1 Cr] Daudi targets in standard chromium release ~ssay at an rf~ 2,.1 ratio of 20:1. Data 3re represeDted as tDe mean of tripiicate ~ -b H~man peripheral blood -- cells isolated from normal donors were treated with 40 ng/ml IL-I0 for 3 days in tbe presence of 2 mglml 19Fl(anti hurDan IL-I0) or rat IgG2a ~isotypic control) before ~' of cytolytic activity.
c Significant differeDce between IL-I0 treatmeni aioDe and IL-I0 treat~nent in the prese~ce of anti IL-I0 antibodies at p~ 0.05 as determined by Student's I-test.

2~31~
WO 95/19780 P~ Jf Table 4: NP~-~rali~tion of IL-10-lnduced Natural Killer Cell Activity (expressed as % Iysisa) by Anti-lL-10 S Monoclonal ~ntihorliP5 Tn~ubation conditionsb 10 Donor medium 40ng/ml IL-10 ~-10 IL-IO + 19FI +IgG2a 21.1 38.3 20.6 27.2 2 28.0 71.0 22.2 53.2 1 5 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 Mean 22.5+1.6 47.9i7.6 19.1Ci6.6 51.3il2.7 aPercent Iysis of [S I Cr] Daudi targets in standard cbromium release ssay at an crr~ cell ratio of 20:1. Data are represented as tbe mean of triplicate :'~
bHuman peripheral blood ' cells isolated from normal donors were treated witb 40 ng/ml IL-I0 for 3 days in tbe presence of 2 mg/mll9FI( anti buman IL-I0) or rat IgG2a (isotypic control) before .~ D-inn of cylolytic actinity.
CSignificant difference between IL-I0 treatment alone and IL-I0 treatment in tbe presence of anti IL-I0 antibodies at p~ 0.05 as 3 0 determined by Student's I-test.
Cytolytic Activity Tnduced by CU~ ;Y~Ls of n.-lo a - ' Otb-r Cytokii~
a. Sim~ Tnr..h~tion with n. lo - I IT-2 3 5 Human PBMCs were incubated with ' -' slilllul~
~ ^ of IL-2 ( 2 or 20 U/ml) in th~ presence of IL-10, WO 9Sl19780 2 1 ~ 1 2 3 0 P~

using an effector-to-target ratio of 5:1, vith the results shown in Table S in which standard errors are shown beneath the mean values.
Table 5: Induction of Lymrh-~ir~-ActtYated Cytolytic Activity (e~pressed as % Iysis') in Humarl Peripheral Blood M~ ' Cells by Co-' ' ' with IL-10 and IL-2 Incubation Conditionsb Donor medium 2U L-2 4ng IL-10 ~-10 20U IL-2 + IL-2 0-03.3 3.7 15.9 24.6 22.6 7.6 3.8 23.5 41.0 36.1 5.0 13.3 17.7 llA
433 50.1 51.6 68.7 80.0 2 05 2.4 9.4 12.8 29.3 45.7 69.9 28.9 16.0 38.8 67.1 Mean 4.1 17.4 16.8 32.3 44 9C
il .42 +7.6 _7.2 _7.2 + 1 0.4 ~ Pcent Iysis of [5 ICr] Daudi targcts in standard chromium release assay at an ~,rf~t~ .. cell ratio of 5:1. Data are represented as the mean of triplicate ~ -b Human peripheral blood -' cells prepared from normal donors were treated with 4 ng/mllL-10 ard 2 U/ml IL-2 for 3 days be~ore ~' of cytolytic activity.
c No significant difference between cytoiytic activily in donor PBMCs treated with 11.-10 and IL-2 compared to 20 U/ml IL-2 done at p< 0.05 as determined by Student's ~-test.

~ woss~s7so 2~12~ P~:111L,1l As shown in Table 5, there v7as an additive increase in LAK
activity (effector cell:target cell ratio of 5:1) following co-ir~ruhq.~i-with 2 Ulml IL-2 and 4 ng/ml IL-10. This level of activity, which was cir~;r;~ y greate~ than that seen with either cytokinc alone, 5 a~ r ' - d the level produced using a 1 0-fold higher Cull~ rlliu~l of IL-2. This result was stqtic~ir-qlly ci~nifirqnt at p< 0.05 as ~1 l. "";, rd by the Student's t-test.
b. Sim~ c Inr~h~tion with n.-10 qrlr1 ~-IFN
PBMCs co-incubated with IL-I0 and ~-IFN display a similar 10 additive increase in Iytic 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 Ly ,L~;~--Activated Killer Cell Activity (expressed as % Iysisa) in Human Peripheral Blood M ntlllclpqr Cells by C~inr~L~qtinn with IL-I0 and ~-IFN
Incubation Conditioncb Donor medium cc-lFN IL-I0 ~IO+c~-IFN

4.4 7.9 17.8 35.7 21.0 11.7 15.4 32.0 31.6 12.3 5.5 26.4 2 5 Me~n 2.3 10.6 12.6 31.4 il.0 il.4 t3.8 _2.7 a Percent Iysis of [S l Cr]Daudi targets in standard chromium rdease assay at an crr~~ l cell ratio of 10:1. Data _re represented as the mean of 3 0 ttiplicate ~ ~;rq~iDr-b Human peripheral blood cells prepared from normal donorswere treated with 4 nglml IL-I0 and 103 U/ml ~-IFN for 3 days before n of cytolytic activity.

wo ss/ls7so 2 ~ 8 ~ 2 3 Q` r~

The dir~ e i observed in cytolytic activity induced in donor PBMCs by IL-I0 and a-IFN compared to that induced by ~-10 or -IFN alone were stP-jctirAlly ci~nifir~ at p< 0.05 as d----- ~ d by the Student's t-test.
In contrast, ~ -- ir~ubAti- - with IL-10 and IL~, IL-S, GMCSF or y-IFN were ~o more effective than IL-10 alone (data not shown).
SC~ A~1 Incubations with IL-10 and IL-2 Using the ~luccJ.,l~s described above, PBMCs were 1 0 ~- - ~ d in medium alone or medium -rL ~ ' ' with IL-10. After two days, IL-2 was added to a final ~ of 2 or 20 U/ml. Cytoto~ic activity against Daudi cells was assessed following an additional overnight i~ The results from a 5-donor pool are shown in Table 7, in which stardard errors of the 15 mean are shown beneath the mean values.

WO95119780 2 ~ ~ ~ 2 3 ~ P ~

Table 7: Induction of Lyn~rh~ Activated Cytolytic Activity (e~pressed as % lysisa) in Human r~ .~ l Blood M,~ Cells by Pre-incubation with IL-IO
S Followed by IL-2 Pre-lncubation C~
Donor medium L_loc IL-2d IL-lO+IL-2e 29.7 21.1 44.8 116 255 18.3 12.2 20.7 314.7 58.1 74.5 100 426.2 59.5 54.3 81.9 15 5 4.8 28.2 22.5 40.6 Mean 16.2 37.0 41.7 71.8 i5.1 i9.0 $11A il7.9 2 0 a PercenL Iysis of [5 I Cr] Daudi target cells in standard chromium release ~ssay at an ~ rF ~ l cell ratio of 5:1. Data are ' as the mean of triplicaLe d b Human periphal blood -' cells prepared from normal donors were mainIained in medium . with 4 ng/ml IL-I0 2 5 for 2 days before addition of 2 U/ml IL-2. Cytolytic activity was determined following additional overnight i- ~
c Donor PBMCs were incubated with IL-I0 for 3 days.
d Donor PBMCs were incubated in medium alone before adding IL-2.
e Donor PBMCs were incubated witb IL-I0 for 2 days before adding 20 U of L-2 .

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As shown in Table 7, in the groups where donor PBMCs were pre-treated with IL-10 before addition of L-2, an aL~lu~hl,dlGly 2-fold higher level of cytolytic activity was observed (71.8+1-17.9%), compared to the level observed in 5 cultures n~ - ~ in complete medium only before adding IL-2 ( 41.7% +/- 11.4 ). The dir~.~,nc~s observed between sar~ples pre-treated with IL-I0 and those not treated with IL-I0 wcre st~tie~ir~lly cignifir~nt at p~ 0.14.
A similar pattern of enhanced cytolytic activity was seen in 10 seu~ h~ .- c with IL-I0 followed by ~-lFN (data not shown).
T7. 1Q ~n~l T7-4 Blocka~e of Tl-2-lnduced Cytotoxicity PBMCs from si~ human donors were cultured in medium c, ~ 2û Ulrnl IL-2 alone, 20 Ulml IL-2 plus 1000 U/ml IL-4, 15 or 20 U/ml IL-2 plus IOQ0 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 show~ under the mean values.

218123~
WO 95/~9780 P~ r Table 8: A~ ,iDI., by IL-10 of the Blockade by IL-4 of IL-2-induced Lymphokine-Activated Cytolytic Activity 5(expressed as % Iysisa) in Human r. . ' l Blood M -- - ' - Cells Donor Medium IL-2b IL-2'+ IL-2d +
IL-4 ILA +

12.7 27.6 35.0 45.7 2 5.4 26.3 175 33.7 3 1.7 25.1 14.1 36.0 1 5 4 3.8 29.6 14.1 22.1 5 3.3 47.1 17.1 63.8 6 6.6 49.5 2Q.6 40.8 Mean 5.5 34.2 19.7 40.3 2 0 il.6 +4.5 i3.2 +5,7 n Percent Iysis of [51 Cr] Daudi targets in standard chromium release assay at an erf~Lu~ L cell ratio of 2û:l. Data are represented as the mean of triplicate d.~ ~
2 5 bHumaD peripyeral blood cells isolated from normal donors were treated with 2û U/ml D:,-2 for 3 days.
CDonûr PBMCs were incubated with 2û U/ml IL-2 and lûû U/ml human IL4 for 3 days.
dDonor PBMCs were incubated witb Zû U/ml IL-2. 1000 U rnl human 3 0 IL-4. and 4 ng/ml IL-IO
The data of Table 8 show that following treatment with 20 U/ml IL-2 alone, about 34.2% Iysis of LAK-sensitive targets was observed. When I OQ0 U/ml human IL-4 was included at the start 3 5 of the ' l period, cytolytic capacity was DUIU~ ,SSC~
a~lul~t~ly 2-fold. This DU~JIJICDS;- by IL-4 was not observed, however, if 4 ng/ml IL-I0 was added during the first 24 hours of incubation .

wo 95119780 2 t ~ t ~ 3 ~ 5 ~

There was no ci~njfjr~r`t d~rr~.~..cc between the results produced Iby IL-2 alone and by all three cytokines together (p< 0.05, as ~ by Student's t-test), ~ir~in~ that a..... L~b. by IL-lO of the blOckâge WâS cssentially complete.
Many _--r-_ ons 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 ~ L_-' describcd herein are offered by way of exarnple only, ând the invention is to be limited only by the terms of the appended 1 O claims.

Claims (12)

WHAT IS CLAIMED IS:

1. A method for treating cancer comprising administering an effective amount of IL-10-activated PBMCs to an individual afflicted with cancer, to cause regression of such cancer.

2. The method of claim 1 which further comprises concomitant or subsequent administration of an effective amount of IL-10 to said individual.

3. A method for the manufacture of a pharmaceutical composition for treating cancer comprising admixing IL-10-activated PBMCs with a pharmaceutically acceptable carrier.

4. The method of any one of claims 1 to 3 in which the IL-10 is administered in combination with (a) an amount of IL-2 sufficient to augment LAK cell activation but not to cause toxic side effects and/or (b) an amount of .alpha.-IFN sufficient to augment LAK
cell activation.

5. A pharmaceutical composition for treating cancer comprising IL-10-activated PBMCs and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5 which further comprises IL-10 alone or in combination with IL-2 and/or .alpha.-IFN.

7. The use of IL-10-activated PBMCs for treating cancer.

8. The use of IL-10-activated PBMCs for the manufacture of a medicament for treating cancer.

9. The use of either claim 7 or claim 8 in which the IL-10-activated PBMCs are used with IL-10, alone or in combination with IL-2 and/or .alpha.-IFN.

10. A method for antagonizing blockade of IL-2-induced cytotoxicity by endogenous IL-4 comprising administering an effective amount of IL-10 to a patient in need of such treatment.

11. The use of IL-10 for the manufacture of a medicament for antagonizing blockade of IL-2-induced cytotoxicity by endogenous IL-4.

12. The method, pharmaceutical composition or use of any one of claims 1 to 11 in which the IL-10 is human IL-10.