CA2109315A1 - Method of suppressing tumor formation in vivo by administering je/mlp-1 - Google Patents
Method of suppressing tumor formation in vivo by administering je/mlp-1Info
- Publication number
- CA2109315A1 CA2109315A1 CA002109315A CA2109315A CA2109315A1 CA 2109315 A1 CA2109315 A1 CA 2109315A1 CA 002109315 A CA002109315 A CA 002109315A CA 2109315 A CA2109315 A CA 2109315A CA 2109315 A1 CA2109315 A1 CA 2109315A1
- Authority
- CA
- Canada
- Prior art keywords
- cells
- mcp
- tumor
- cell
- administering
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/521—Chemokines
- C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
A method of suppressing tumor formation in a vertebrate by administering JE/MCP-1 is described. Also described are methods of treating localized complications of malignancies and methods of combatting parasitic infection by administering JE/MCP-1.
Description
WO92/2037~ PCl'tUS92/040~9 :
ME~K~ 0~ SUPPRESSING TUMOR FORMATION IN VIVO BY ADMINIS'~TING ~E/MLP-l .
_~ckground Cancer resultr when a vert~r-te'~ own cells become m~lignant. Healthy lndi~idual0 ~t any gi~en time carry potenti~lly malignant cells ln the~r ~ody, Th~se cells ~re generally recognized ~nt killod by the indl~iduals' ~mmune system. Howe~er, ~ome mal~gnant cells ~re not destroyed by the ~mmune ~ystem and proliferate into tumors.
Currently, there ~re not ~tequatP, and ~peciflc ~herapies ~o~r cancer. For ex~mple, ~urgicsl cxc~sion of tumors is not an effect~e ~ethod o~ tre~tment where the Gancer has ~etsstssized. In ~dtit~on, rsdiat~ on and chemotherapy o~ten kill normal cells in adtit~on to 15 cancerous cells.
Another problem is that che~therapeutic agents follow first-order kin~tics. AF a r~sultf a constant perce~tagc, r~ther than a constsnt nu~ber of cell~ are killed by a given applic~at~on of a chcmother~peutic agent.
20 Consequently, ~alignant cells, wh~ch could c~u~e A rel~pse in the dise~se, remain even ~hen a patent is dia~nosed as having complete clinical remission.
A method of suppresslng cancer that ~mploys the indi~idual' 5 own ~mmun;e system would be useful , 2~ Summary of the In~ention The present invention relates to Applicant's findin~
that expression of the JE/MCP-1 protein in malignant cells ` suppresses their ability to form tumors in ~i~o. Thus, "
W092/2037~ PCT/US92/040g9 210~ 2-the inYention comprises, in one embodiment, a method of suppressing tumor formation in a ~ertebrate by adminis-tering to the ~ertebrat¢ a therapeutically effecti~e ~mount of J~/MCP-l. The protein can be administered alone or as ~ ad~u~ant to surgery or cytotoxic ~hemotherapy.
The suppressive eff~ct o~ JE/MCP-l d~pends on the induction of the vertebrate' fi immune response, speci~ic-~lly the response oP ~onocytes. Thus, tn another emboti-ment, the inven~tion comprises B method of increasing a 10 ~ertebrate's monocyte-mediated tumoricidal acti~ity in ~ivo by administering to the vertebrate an effective ____ amount of JE/MCP-l, JE/MCP-l~can also be administer~d to treat localized complications of maIignancy. For example, JE/MCP l could 15 be used to inhibit malignant pleural cffusions or ~scites.
Therefore, in a further embodiment, the in~ention com-prises methods of inhibiting pleural effusion or a~cites in a vertebrAte by locally ~dministering JE/MCP-l to the anatomic spaces between the lung and the pleural membrane 20 or the stomach and the peritoneum.
In a further embodiment tu~or killing cells, such as tumor infiltrating lymphocytes (TIL cells~ are-genetically engineered to express tbe JE/MCP-l protein. The engineer- ;~
ed cells therefore can be administered to a ~ertebrate to 25 pro~ide a synergistic local tumor cell killing.
The presence of JE/NCP-l in~ o is accompanied by a~
local increase in the presence of eosinophils. Therefore, ~nother aspect of the sub~ect invention comprises methods of combatting a parasitic infection in a vertebrste animal 30 by administering to that ~ertebrate an effective amount of JE/MCP~
: :
W092/20372 PCT/US92/04~99 21~9~.5 A major ad~antage of using JE/MCP-l in treating cancer is th~t it employs the indiYidu~l' 6 own ~mmune system and therefore would have fewer side-effects than conventionsl chemotherapies. In addition, JE/MCP-l 5 ~t~mulates m~nocytes and, as 6uch, does not depend on a total immunologic response.
Brief Descri~tion of the Drawin~s ____________ __________________ _ .
Figure 1 ~s an sutoradiograph of polyacrylamide gel electxophoresis of cells of DUKX-Bll cells transfected 10 with pXM alone (OA-2, OB-2, and OA-10), pXM-JEl (lA-2.and lB-2), pXM-JE10 (lOA-2, 10B-2, and 10A-10), or pXM-hJE34 (hJEC-10 and hJEC-100).
Figure 2 is a graph plotting the growth rate of tumors. Each symbol represents a different mouse. A. CH0 15 cells_only , 10 OA-2 cells (open square); 10 OA-2 cells plus 107 hJE~-10 cells (closed square and clcsed circle);
OA-2 cells and 10 10A-10 cells (closed triangle snd open triangle); (identical results were obtained with 107 OA-2 cells and 107 hJEC-100 cells). B. CH0_and_HeLa 20 cells. 10 HeLa cells and 10 OA-2 cells (open square and open~c~rcle); 105 HeLa cells and 107 hJEC-10 cells (closed squsre and closed circle); 105 HeLa cells and 107 10A-10 cells (closed triangle ant open triangle); tidentical results were obtained with 10 HeLa cells and 107 hJEC-100 25 cells in 3 out of 4 animals).
Figure 3 is a photograph (magnification 400X) of hematoXylin and eosin-stainet cellular infiltrate elicited by A. OA-10, B. 10A-10 or C. hJEC-10 cells.
W092/20372 PCT/USg2/04099 21~3~5 -4-Figure 4 is a photograph (magnification 800X) of hematoxylin ~nd ~osin-stained cellular inflltxate elicited by hJEC-10 cells.
Detai~ed-Descri~tio--of-the-l-vention The pre~ent invention i based OD the finding that expression of ~he JE/MCP-l protein in malig~ant cell5 ~uppress~s their ab~ y to ~orm t~or~ in vivo The JE gene i9 a platelet-derived growth factor (PDGF)-inducible "competence" or "early response gene" -~
10 first identified in mouse 3T3 cells ~Cochran, B.H. Cell 33:93~-947 ~1983)). S~quence ~nt express~on analysis showed that unlike other early response genes, such ns c-myc, c-fos, or ¢-~un, the murine JE gene encodes a secreted glycoprotein w~th cytokine-like properties (Kawahara, R.S. J._Biol._Chem 264:679-682 ~1989); Rollins, B.J. Proc. Natl. Acat. Sci. USA 85:3738-3742 (1988)). The human homolog of mur$ne JE has been cloned, (Rollins, B.J.
Mol. Cell._Biol. 9:4687-4695 (1989)), and the predicted amino acid sequence of its protein is identical ~o that of 20 a monocyte chemoattractant, MCP-l (Yoshimura, T., J. Exp.
Med. 169:1449-~459 (1989) ~Yoshimura T. JE. FEBS Lett.
244:487-493 (1989)) al80 called MCAF (Furuta~i, Y. et al., Biochem. BiQ~hys. Res. Commun. 159:249-255 (1989);
Matsushims, K. et_al., J._Ex~. Med. 169:1485-1490 (1989) 25 and SMC-CF (~raves, D.T., et_al., Science 245:1490-14g3 (1989); Valente, A.J. et al. Biochem 27:4162-4168 (1988)~.
The JE/MCP-llprotein is structurally related to the members of a large, recently identified family of low molecular weight secreted proteins that appear to be W092/~0372 PCT/US92/04099 21~3~ ~
in~olved in the inflammatory response (Leonard, E.J. and T. Yoshimura Immunol._Totay 11:97-101 (1990); Rollins, B.J. et al., Mol. Cell._Blol. 9:4687~4695 (1989); Wolpe, S.D. ant A. Ce~ami, FASEB J. 3:2563-2573 (1989)). The genes for many of these prote~ns, ~ncluding human JE~MCP~l, are clustered on chromosome 17qll.2-12 (Donlon, T.A. et_al., Qenomics 6:548-553 (1990); Irving, S.G., et al., Nucleic Acids Res. 18:3261~3270 (1990); Rol.lins, B.J., et al., Genomics_(in press)) or mouse chromosome 11 10 (~ilson, S.D., et_al., J._Ex~__Med. 171:1301-1314 (1990)).
These genes are also relatet to the genes encoding another family of cytokines, whose members include the neutrophil activstor NAP-l/IL-8 (Pe~eri, P., e~_al., J._Exp.~Med.
167:1547-1559 (1988); Schroder, J.M. et 81. J. Immunol 139:3474 3483 (1987); Yoshimura, T. et al., Proc._Natl.
Acad. Sci. ~SA 84:9233-9237 (1987)), many of which cluster at 4ql2-21 (Griffin, C,A. et al., CYto~enet-cell-Genet 45 67-69 (1987); Luster, A.D. et_~l. Proc-_Natl-_A.ead-Sci. ~SA 84:2868-2871 (1987); Richmont, A. E~BO_J 7:2025-20 2033 (lg~8)).
JE/MCP-l exerts several effects specifically on monocytes. Both natural and recombinant JE/MCP~l are potent chemoattractan~ts for human monocytes in ~itro, (Matsushima, K. et_al. J._Ex~._Med. 169:148~ 1490 (1989);
2~ Yoshimura, T. et al. J._Ex~._Med. 169:1449 1459 (1989)) and purified recombinant JE/NCP-l can stimulate an in-crease in cytosolic free;clalcium and the respiratory burst in monocytes (Zachariae, C.O.C., et_al., J. Ex~._Med.
171:2177 2182 (l990); Rollins, B.J. et_al. Blood (in 30 press)). Purified natural JE/NCP-l has also been reported W~92/20372 PCT/US92/~0~
:,.
2iO~3~.S -6- ;~
;, :'~
to activate monocyte-mediated inhibition of tumor cell growth, but not tumor cell killing, in ~itro (Matsushima, K. et al. J. Ex~. Med. 169:1485-1490 (1989)).
The following demonstrates ~hat expre~s$on of the JE
gene $n mal ign~nt cells suppre6s~s their ab~lity to form tumors in vivo. This spp~rent phenotypic revers~on requires lnteraction with host ~acto~s in ~ivo, ~ince expression of JE/MCP-l toes not alter the transformet character of these cells ln vitro. Furthermore, the following shows that JEjMCP-l-expressing cells exert their effect in trans by their ability to suppress tumor form-ation when co-inJected with JE/MCP-l-non-expressing tumor cells.
In order to create malignant cells expressing 15 JE/MCP-l, the DHFR deletion mutant CHO cell line, D~XXB-ll ~G. Urlaub and L.A Chasin, Proc.~Natl._Acat._Sci. USA, 77:4216-4220 (1980)) was transfectet with the express~on vector pXM ~Y~ng, Y.-C. et_al. Cell 47:3-10 ~1986) con-taining a variety of JE cDNA species. High levels of 20 JE/NCP-l protein expression were achieved in stably transfected llnes by methotrexate (MTX)-induced DNA
ampll~ication. R.J. Kaufman, et_al., ENBO_J. 6:187-193 (1987).
~igure l show- JE/~CP l protein expression in in-25 dependently derived cell lines selected for resistance to2 or 10 uM MXT. There is no detectable JE/MCP-l protein secreted from cell lines transfected with pXM alone (cell lines OA~2 and OB-2; cell line OA-lO was derived from!
OA-2) or with pXM containing murine JE cDNA in the anti-30 sense orientation (lA-2 and lB-2). Considerable JE/MCP-l .
~ ' :
W092/20372 2 1 ~ 9 ~ ~ ~i Pcr/usg2/o4099 :
protein was secreted by cell lines transfected with murine JE cDNA in the sense orientation (lOA-2 and lOB-2; lOA-10 ~as derived ~rom 10~-2) and human JE cDNA (hJEC-10). Cell line hJEC-100, derived from hJEC-10 by selecting for resistance to 1~0 uM MTX, al80 6ecreted human JE/MCP-l proteln.
Murine JE cDNA directs CHO cells to ~ecrets a micro-he~erogeneous protein of Nr 27-39,000, similar to the natur~l protein and the protein expressed in a COS cell 10 expression system. Rollins, B.J. et_al., Proc. Matl.
Acad. Sci._USA 85:3738:3742 (1988)). Nearly half of the apparent Mr is due to O-linked glycosylation. Human JE/MCP-l proteins expressed in CHO cells are al90 similar to native and COS cell expressed JE/MCP-l~ (MrlS,OOO~ and 15 JE/NCP 1~ (Mr 11,000). (Rollins, B.J., et al~ Mol.~Cell.
Biol. 9:4687-4695 (1989) ____ _ The monocyte chemoattra¢tant actiYiti~s (MCA) secreted by these cell lines were tetermined as described in detail in Example 1. They were 1415 U/24hr/106 cells 20 (lOA-2), 1079 U/24hr/10 cells (lOB-2), 3008 ~/24hr/10 .
cells (lOA-10), 54 U/24hr/10 cells ~hJEC-10), and 692~
U/24hr/106 ce~ls (hJEC-100) JE/MCP-l-non-expressing cells secreted 10-30 U/24hr/106 cells. The increased MCA
secreted by murine JE/NCP-l expressing lines ~s due to 25 increa9ed JE/MCP-l protein in the medium, not to higher specific acti~ity. Murine JE/MCP-l appears to be more stable than human JE/MCP-l in culturel perhaps due to its more exte~si~e glycosylat`io~. (Rollins, B.J., et aI. MoI.
Cell. Biol. 9:4687-4695 ~1989)) _________ _ .
WOg2/20372 ; , PCT/US92/0~09~ .
~ 31~ -8-By several criteria, JE/MCP-l expression did not alter the tran6formed phenotype of CH0 cells in vitro.
T~ble I shows that ~hile doubling times of each of the independently tsr~ved cell lines ~ar~ed considerably, the a~erage doubling t~me of all the JE/MCP-l-~xpressing lines (25.1 hrs ~ 5.7 sd) WBS nearly identical to the a~erage doubling time of the JE/MCP-l-non-expressors (26.4 hrs +
5.7 sd~. Also, JE/MCP-l expression tid not alter the transfor~ed cellular morphology of the CH0 cells.
10 Finally, all cell lines formed colonies in soft agar.
,. ;~ `
.
wo g~/20372 2 1 ~ g 3 ~ ~ PCT/US92/0~099 9 i :.
TABLE 1. PROPERTIES OF TRANSFECTED CHINESE HAMSTER OVARY CELL
LINES.
Doubllng Cell JE Time So~t Agar No. Cell5 Tumor~/Animal Line cDNA (hours~ Colonies~2~nl~ted____InieCted__ JE/MQP-l_Non expressors OA-2 None 24.2 91 +. 14.0 2 x 107 1~
n n ~ n 1 x 10 2/2 n n n n 2 x 10 3/4 ;.
OA-10 None 20.4 32 ~ 1.9 1 x 10 2/2 :
OB-2 None 19.0 73 ~ ~.6 1 x 107 0/4 lA-2Antisense 30~7 75 ~ 14.5 8 x 106 2/2 lB-2Antisense 31.2 74 i 1.7 1 x 10 1/~
JE~MCP-l Ex~ressors __ ________ _______ lOA-2Murine 28.8 72 ~ 7.3 2 x 107 0/1 ~ n n n 1 x 10 0 / 2 n n ~ 1l 2 x 10 0/4 lOB-2Murine 17.8 86 i 5.1 1 x 107 0/2 10A-10Murine 30.0 129 * 6.8 1 x 10 0/2 hJEC-10 Hu~an 28.8 32 ~ 6.8 1 x 10 0/2 In ~ivo, howe~er, J!E~NCP-l expression led to a striki~g difference in behavior. Table 1 shows that all but one (OB-2) of~ the JE/MCP-l-non-expressing cell lines formed lar~e subeutaneous tumors that appeared within W0~2/20372 PCT/U$92/040~9 2 1 l 9 r3 ~ 5 10-three weeks of in~ection into nude mice. In contrast, all of the JE/MCP-l-expressing l~nes, including the human JE/MCP-l expressing lines, ormed no tumors for ss long as ten months after injection. At ~utopsy, there was no S microscopic evidence of residual tumor in the Rnimals that received JE/MCP-l-expresslng cells.
These observations suggestet the possibllity that JE/NCP-l-secreting cells attxacted monocytes to the site of tumor cell inJection and once there, secreted JE/MCP-l 10 protein induced monocyte tumoric~dal activity. To test this hypothesis, JE/MCP-l-expressing cells were mixed with 107 OA-2 cells, a num~er of cell6 that reproducibly let to tumor formation when in~ectet slone (see Table 1).
Figure 2A shows that co~n~ection of murine JE/~CP~
15 expressing cells ~lOA-10) with OA-2 cells completely suppressed tumor format~on in two animals. Co-in~ection of high-le~el human JE/MCP-l-expressing cells (hJEC-100) wlth OA-2 cells also completely suppressed tu~or form-ation. Co-injection of low-le~el human JE/MCP-l-20 expressing cells (hJEC-10) suppressed tumor formation for 8-10 days, after which tumors appeased. Presumably hJEC-10 cells exerted a suppressive effect transiently unt~l the proliferating OA-2 cell mass reached a size that enabled it to escape the e$fect.
JE/NCP-l-expressors and non-expressors were also co-in~ected with He~a cells to test whether JE/MCP-l expression could suppress tumor formation by another cell type~. Figure 2B shows that lOA-lO cells completely suppressed the formation of tumors by HeLa cells. As 30 above, co-injection with hJEC-10 celIs delayed tumor ~ .
.
W092/20372 PCT/USg2/04099 21t~31~
formation. In a separate experiment, 4 o~ 4 animals in~ectet with 107 HeLs cells formed tumors, while only 1 of 4 an~mals in~ectet with 107 NeLa cells plus 107 hJEC-100 cells formet tumors. Mice t~at displayed no tumor growth after receiving HeLa plu~ hJ~C-100 c~118 were then inJected with 107 HeLa cells ~lone. These mice developed large tumors withln 14 days, ~nd$cating that prior suppression of HeLa cell tumor growth in the presence of JE/MCP-l does not render mice immune to 10 rechallenge with HeLa cells.
Histolog~c examination of the tumors arising from co-~n~ected HeLa and OA-2 cells revealed a mixture of epithelioid HeLa cells and spindle-shaped CHO cells.
Examlnation of the tumors formed in animals that r~ceived 15 HeLa and hJEC-10 cells also demonstrated a mlxture ~of HeLs cells and CHO cells. The presence of some hJE5-10 cells in these tumors was con~irmed by Northern blot analysis in which expression of human JE mRNA could ~e detected.
After reaching a certain s~ze, the-e tumors may overwhelm ;20 the host response elicited by the low levels of human JE/MCP-l secreted by hJEC-10 cells. However, tumor growth still requires the presence of a malignant JE/NCP-l-non-expressing cell line, since hJEC-10 cells in~ected by themsel~es cannot form tumors ~Table 1). This suggests 25 again that the intrinsic ~rowth properties of the CHO
cells have not been altered by JE/MCP-l expression, and that the human JEjMCP-l expressors will proliferate in ~ivo if; they are protectet by an enlarging mass of malignant cells.
Figures~3 and 4 show the results of a histological exa=ination of the s i -e s o ~ CHO cell inoc~lation 24 hours , ' W092/20372 PCT/USg~/~4099 210~3~ ~
after cells.were in~ected. JE/MCP-l-non-expressors ~darkly stained, large cells irdicated by arrows in Figure 3A) induce the sppearance in ~he underlying connective tissue vf only a fzw cells, moct of which ~re neutrophlls, In contrast, CH0 cells exprosslng murlne JE/MCP-l (indi-cated by arrows in Figure 3B) elicited un ~bund~nt cellular infiltrate, At higher power (Figure 4A), it i9 ~pparent that this in~iltsate consisted prinarily of monocytes with a reproducibly signiflcant proportion of 10 eoslnoph~ls (arrows), usually 10-20%. As mlght be ex-pected, low-level human JE/MCP-l-expressing cells (hJEC-10) induced a qualitatlvely slmilar infiltrate (Figure 4B) that was intermediate in inten~ity between the non-expressors and the muri~e JE/MCP-l expressors (compare 15 Figure 3C to Figures 3A and 3B).
The result~ repre~ented by Figure 3 ~nd 4 demonstrate a predominantly monocytic in~lltrate at the site of tumor cell in;ection, suggesting that monocy~es mediate tumor growth suppression. The effect is probably not mediated 20 by T lymphocytes or by natur~l killer cells, since puri-fied reeombinant JE/MCP-l has no stimulatory effect on~
natural kille~ ceIls in vitro.
~ f monocytes are responsible for tumor suppression, there are se~eral possible mechanisms whereby JE/MCP-l-25 acti~ated monocytes might exert their effe¢ts. JE/MCP-~coul~ induce the expression of a soluble mediator of tumor cells lysis such as tumor necrosis factor (TNF). L.J. Old, Sc~ence, 230: ~30-632 ~i985). J.L. Urban et al., Proc.
~atl. Acsd. Sci. USA 83: S233-5237 (1986). Alternatively, ______________ __ __ __ 30 TNF coul~ be expressed and displayed in an acti~e form on the cell surface of acti~ated monocytes. M. Kriegler, et al., Cell 53: 45-52 (1988).
WO9~/20372 PCT/US92/04099 ;
21~3!~31~ :.
Interestingly, nn eosinophilic component to the inflammatory cell infiltrate was also observed. This may be either a direct effect of JE/MCP-l or, JE/MCP~l may induce the expression of another factor with eosinophil chemoattxactsnt properties, These re6ults point to a clinical role for infused JE/MCP-l in vertebrate animsls, such BS humans. For example, JE/MCP-l can be atministered to pat~ents wlth cancer. This would necessarily be limlted to patients 10 with low tumor loads, i.e. as an at~uYant to surg0ry or cytotoxic chemotherapy. Systemically in~uset JE/MCP-l would lead to a generalizet increase in the acti~ation state of a ~ertebrate's monocytes. There i5 some evidence that patients harboring malignancies ha~e depressed 15 monocyte function. W.G. Chaney et al ., Cell Molec. Genet, 5 15-27 (1986). E.S. Kleinerman et_al., Lancet ii:
1102-1105, (19~0). If the te~ect were cytokine basett rether than an inherent monocyte defect, JE/MCP-l infusion would correct the abnormality.
Alternatively, JE/MCP-l may prove useful in treating localized complications of malignancy, 6uch a8 pleural effus~ons or ascltes. Inst~lling JE/MCP-l into the invol~ed anatom~ic space ~e.g. the space between the lung and the pleural membrane or the space between the stomsch 25 and the peritoneum~ can lead to local monocyte accumu-lation and activation.
As a therapeutic, JEtNCP-l can be administered to vertebrate animals (i.e. animals having an immune system), including humans. The compounds of this invention can be 30 employed in admixture with conventional excipients, i.e., W09~/20372 PCT/US92/04099 2 1 0 ~ 3 ~
.
pharmaceutically accep~able organlo or inorganic carrier subst~nces suitable for parentsl or topical applic~tion.
Suitable pharmaceutical c~rriers include, but ~re not llmited to water, salt ~olutions, ~lcohols, p~ly~thylene glycols, gelatin, carbohydr~tes such ~5 lacto~e, ~mylo~e or starch, magneslum stearate, t~lc, silicic acid, vi~cous paraifin, perume oil, ~tty ncld esters, hydroxym~thyl-cellulose, polyvinyl pyrrolldone, etc. The ph~rmaceutical preparations can be sterlized and i~ desired, mixet w~th 10 auxiliary sgents, e.g., lubricant~, prcservatives, stabi-lizers, wetting agen~s, emul~ifiers, salts for in1uencing osmotic pre~sure, buffers, coloxing, flavoring and/or aromatic s~bstances ~nt the like which do not delete-riously react with the active compounds. They can also be 15 combined where desired with other active a~ents, e.g., enzyme ~nhibitors, to ~urther reduce metabolic degradation.
For parenteral application, pa~ticularly suitable are in~ectable, sterile solutions, preferably oily or ~queous 20 solutions, as well as suspensions, emulsions, or implants, ~ncluding suppo~itories. Ampoules are convenient unit dosa~es.
For topical application, there are employed as nonsprayable ~orms, ~iscous to semi-solid or solid forms 2~ comprising a oarrier compatible with topical application and having a dynamic viscosity preferably greater than water. Suitable formulations include but are not limi*ed to solutions, sùspensions, emulsions, creams, ointments, powders, liniments, salves, serosols, etc., which are, if 30 desired, sterilized or mixed with auxiliary agents, (e.g., .
WO~/20372 PCT/US92/04~99 3 ~. 5 preser~atives, sta~ilizers, wetting agent~, buffers of salts for influencing osmotic pres~ure, etc.). For topical application, also suitsbl~ are 4prayable aerosol preparations wherein the actl~e ingrediiont, preferably in combination with a ~olid or liquld lnort cisrrier material, i9 packaged in ~i squeeze bottle or in ~dmixture with a pre~isurized volatile, normally gaseou~ propell~nt, e.g., a Freon.
It will be appreclated that the actual preferred 10 amounts o~ JE/MCP-l in a ~peci~ic csi9e will vary according to thé speci~ic compound being utilized, the particular compositions formulatod, the mode of ~pplication, ant the particular situs ant organism being treated. Dosage~ for a given host can be detexminet using conventional consit-15 eratlons, (e.g., by means of an appropriate, conventionalpharmacological protocol.).
Alternatively, tumor killing cells, ~uch as tumox infiltrating lymphocytes ~TIL cells) could be genetically engineered to express the JE/MCP-l protei~. Tumor killing 20 cells engineered in this way;can pro~ide synergistic local tumor cell k~lling. The tumor kllllng cells could be ~
engineered in_~itro ant administered to the vertebrate or the e~mor killing cells could be engineered i__~i~o into the ~ertebrate's own supply~of tumor killing cells using 25 ~ethods which are known ln the art.
The present invention will now be illustrated by the following examples, which are not intended to be limi.ting in any,wRy. I I !
: ~ .
~ ' w~g2/20372 PCT/US92/04099 -16- ;
3 ~
EXAMPLES
E~am~le 1 In Vi~o_Tumoricidal Effec~ o~ JE/MCP
Cell culture. DUKXB-ll ¢ell5 (Url~ub, G. and L.A.
____________ Ch~sin, Proc. Natl._Ac~d._Sci._USA, 83:5233-5237 (1987)) (dihytrofolate reductAse (DHFR) ?~utant Chi~cs~ hamster o~ary (CH0) cc115) ~ere grown in th~ alpha mod~ficatlon of minimal essenti~l medlum (NEM-~ ) without rlbonucl~os~des and deoxyribonucleosldes, supplemented wlth 10~ bo~ine calf serum ~nd 10 ug/ml ~denosine, deoxyadeno#ine, ant lO thymidine (NEM-~/BCS/ATT)~ (Kaufman, R.J. et,_al. EMB0 J.
6: 187-lg3, (1989~). HeLa cells were grown in Dulbecco's modified Eagle' 5 medium (DMEM) supplemented with 10%
heat-inacti~ated calf serum ~nd antibiotics. ~
Tra-sfectlons-a-d-Methotrexate-(MTx2-sele~tio-, ..
15 DUKXB-ll cells were grown in 10 cm cul~ure d~?shes as described abo~e. At 60% confluence, medium was remo~ed and replacet with fresh M~M-~/BCS/AAT supplemented with 3 ug/ml polybrene and incubated overnight. (Chaney, W.G.~et al., Somat.-ce~ oleG~-Genet~ 12:237 244, (1986)) The 20 next day, each dish was given 3 ml fresh MEM-~/BCS/AAT
with 83 ng/ml plasmid D~A and 10 ug/ml pclybrene. Cells were incubated at 37C fox 6 hr with rocking every 90 min.
Nedium was then removed and replacet with 5 ml fresh MEM-~/BC~/AAT con~aining 30% DMS0 for exactly 4 min.
25 Medium;was remo~ed, the~cells wére washed!once with NEM-~, and given 10 ml of MEM-~/BCS/AA . Cells were incubated at 37C for 48 hr, then trypsinized and re-plated into 4 culture dishes (10 cm) in nucleoside- and deoxynucleo-side-iree MEM-o supplemented :ith 10'~ dialyzed BCS, and ' 21~39~
..
refed w~th this medium every 3 days. Two independent transfections were per~ormed uslng pXM (Yang et_al. Cell 47:3-10 ~1986); pXM-JE10 ~murine JE cDNA (Rolli~s, B.J., et al., Proc. Natl. Acad. Sci. ~SA 85:3738-3742 (1988)) ln _____ .__________________________ __ the sense orientation), pXM-JEl ~murine JE cDNA in the antisense orientation), ant pXM-hJE34 (human JE cDNA) (Rollins, B.J., et_al., Mol,_Cell._Biol. 9:4687~4695 (1989)). Colonies from each ~ndepentent transfection that grew in ribonucleoside~ and deoxyri~onucleo~ide-free metium were trypsinized and combined. Step-wii~e ~election in increasing concentrations o methotrexate (MTX) was carried out at the following le~els: 0.02 uM, 0.1 uM, O.S
uM, 2.0 u~, 10.0 uM, and 100.0 uM. At each conce~tration, survi~ing colonies were trypsinlzet and pooled.
PEotei _analysis. Confluent cell cultures were incubated in methionine-free NEM-~ with 2% dialyzed BCS
for 45 min, then changed to 0.5 ml of the same medium with 500 uCi [ 5S]methionine (DuPont ~EN, Boston, MA). Cells were incubated at 37-C for 4 hrs. after wh~ch the medlum was collected, made`l mM in phenylmethylsulfonyl fluori~e (PMSF), centrifuged to remo~e cells and tebris, and stored at -70C. Immune precipitatlons uslng anti-JE/MCP-l antiQerum were performed (Rollins, B.J., et_al., Mol.
Cell. Biol. 9:4687-4695 ~19B9))~ ant the results analyzed 2~ by electrophoresis through an SDS-containing 17% poly-acrylamide ~el.
Soft a~ar_colony formatio_ assay. Five thousand cells were suspended~in MEN-~ containing 10% dialyzed BCS, 0.3~ agarj and the appropriate concentration of MTX.
.
, WOg2/20372 PCT/US92/0~9 21~'~3~ ~ -18-While still molten, this suspension was distrlbuted on a gelled 4 ml underlayer of NEM-~ contsining 10~ dislyze~
BCS, 0.6~ a~ar, and the appropriate concentration of NTX
~n a 60 mm culture di~h. Cells were ~ed wlth 3 drops o~ -fresh medium every 5 days. After 14 da~s, colonie~
consisting of grQater th~n 50 cell5 were counted.
Mon~te_che_oattracta_t_acti~it~_lMCA~. Confluent monolayers of CH0 cells were incubated in ~erum-~ree NEM-~for 24 hr, after which the medium was ccntrlfuged to remo~e cells and debris, and the remainlng adherent cells were trypsinized and counted. Fre~h humsn periphe~al blood mononuclear cells were puri~ied from the blood of ;, volunteer donors by centrifugation on a cushion of Ficoll-Hypaque (Pharmacia, Piscataway, NJ). Cells ~t the interface were washed tw~ce in ~ey's bslanced salt solution (GBSS) with 2~ BSA, thon resuspended at 4 x 106 cells/ml in GBSS with 0.24 8SA, and MCA was messuret in a 48-well microchamber apparatus. (Falk, W.; et_al., J.
Immunol. Methods 33:239-247 (1980)). The concentration of MCA in CH0 cell medium was tefined as the reciprocal of~
the dilution showing half-maximal acti~ity. (Yoshimura, T., et al., J.~Ex~. Med. 169:1449-1459 (1989)).
Nude mouse inieetions. Cells were suspended in 0.2 ~__________ _ _______ ml PBS and injected subcutMneously into ~-week old male ' Swiss nu/nu mice. Mice were monitored daily for tumor growth., Tumor volume was derived ~y multiplyin~ the values of ~hree pe~pentlcular diameters.
W092/20372 ~ 1~ 9 3 ~ X PCT/USg2/04099 E~uivalents _________ Those ~killed in the art will recognize or be able to ascertain using no ~ore than routine exper~mentation, many equi~alents to the specific embotiments of the in~ention describe herein. Such equi~alents are intended to be encompassed by the following claims.
:
ME~K~ 0~ SUPPRESSING TUMOR FORMATION IN VIVO BY ADMINIS'~TING ~E/MLP-l .
_~ckground Cancer resultr when a vert~r-te'~ own cells become m~lignant. Healthy lndi~idual0 ~t any gi~en time carry potenti~lly malignant cells ln the~r ~ody, Th~se cells ~re generally recognized ~nt killod by the indl~iduals' ~mmune system. Howe~er, ~ome mal~gnant cells ~re not destroyed by the ~mmune ~ystem and proliferate into tumors.
Currently, there ~re not ~tequatP, and ~peciflc ~herapies ~o~r cancer. For ex~mple, ~urgicsl cxc~sion of tumors is not an effect~e ~ethod o~ tre~tment where the Gancer has ~etsstssized. In ~dtit~on, rsdiat~ on and chemotherapy o~ten kill normal cells in adtit~on to 15 cancerous cells.
Another problem is that che~therapeutic agents follow first-order kin~tics. AF a r~sultf a constant perce~tagc, r~ther than a constsnt nu~ber of cell~ are killed by a given applic~at~on of a chcmother~peutic agent.
20 Consequently, ~alignant cells, wh~ch could c~u~e A rel~pse in the dise~se, remain even ~hen a patent is dia~nosed as having complete clinical remission.
A method of suppresslng cancer that ~mploys the indi~idual' 5 own ~mmun;e system would be useful , 2~ Summary of the In~ention The present invention relates to Applicant's findin~
that expression of the JE/MCP-1 protein in malignant cells ` suppresses their ability to form tumors in ~i~o. Thus, "
W092/2037~ PCT/US92/040g9 210~ 2-the inYention comprises, in one embodiment, a method of suppressing tumor formation in a ~ertebrate by adminis-tering to the ~ertebrat¢ a therapeutically effecti~e ~mount of J~/MCP-l. The protein can be administered alone or as ~ ad~u~ant to surgery or cytotoxic ~hemotherapy.
The suppressive eff~ct o~ JE/MCP-l d~pends on the induction of the vertebrate' fi immune response, speci~ic-~lly the response oP ~onocytes. Thus, tn another emboti-ment, the inven~tion comprises B method of increasing a 10 ~ertebrate's monocyte-mediated tumoricidal acti~ity in ~ivo by administering to the vertebrate an effective ____ amount of JE/MCP-l, JE/MCP-l~can also be administer~d to treat localized complications of maIignancy. For example, JE/MCP l could 15 be used to inhibit malignant pleural cffusions or ~scites.
Therefore, in a further embodiment, the in~ention com-prises methods of inhibiting pleural effusion or a~cites in a vertebrAte by locally ~dministering JE/MCP-l to the anatomic spaces between the lung and the pleural membrane 20 or the stomach and the peritoneum.
In a further embodiment tu~or killing cells, such as tumor infiltrating lymphocytes (TIL cells~ are-genetically engineered to express tbe JE/MCP-l protein. The engineer- ;~
ed cells therefore can be administered to a ~ertebrate to 25 pro~ide a synergistic local tumor cell killing.
The presence of JE/NCP-l in~ o is accompanied by a~
local increase in the presence of eosinophils. Therefore, ~nother aspect of the sub~ect invention comprises methods of combatting a parasitic infection in a vertebrste animal 30 by administering to that ~ertebrate an effective amount of JE/MCP~
: :
W092/20372 PCT/US92/04~99 21~9~.5 A major ad~antage of using JE/MCP-l in treating cancer is th~t it employs the indiYidu~l' 6 own ~mmune system and therefore would have fewer side-effects than conventionsl chemotherapies. In addition, JE/MCP-l 5 ~t~mulates m~nocytes and, as 6uch, does not depend on a total immunologic response.
Brief Descri~tion of the Drawin~s ____________ __________________ _ .
Figure 1 ~s an sutoradiograph of polyacrylamide gel electxophoresis of cells of DUKX-Bll cells transfected 10 with pXM alone (OA-2, OB-2, and OA-10), pXM-JEl (lA-2.and lB-2), pXM-JE10 (lOA-2, 10B-2, and 10A-10), or pXM-hJE34 (hJEC-10 and hJEC-100).
Figure 2 is a graph plotting the growth rate of tumors. Each symbol represents a different mouse. A. CH0 15 cells_only , 10 OA-2 cells (open square); 10 OA-2 cells plus 107 hJE~-10 cells (closed square and clcsed circle);
OA-2 cells and 10 10A-10 cells (closed triangle snd open triangle); (identical results were obtained with 107 OA-2 cells and 107 hJEC-100 cells). B. CH0_and_HeLa 20 cells. 10 HeLa cells and 10 OA-2 cells (open square and open~c~rcle); 105 HeLa cells and 107 hJEC-10 cells (closed squsre and closed circle); 105 HeLa cells and 107 10A-10 cells (closed triangle ant open triangle); tidentical results were obtained with 10 HeLa cells and 107 hJEC-100 25 cells in 3 out of 4 animals).
Figure 3 is a photograph (magnification 400X) of hematoXylin and eosin-stainet cellular infiltrate elicited by A. OA-10, B. 10A-10 or C. hJEC-10 cells.
W092/20372 PCT/USg2/04099 21~3~5 -4-Figure 4 is a photograph (magnification 800X) of hematoxylin ~nd ~osin-stained cellular inflltxate elicited by hJEC-10 cells.
Detai~ed-Descri~tio--of-the-l-vention The pre~ent invention i based OD the finding that expression of ~he JE/MCP-l protein in malig~ant cell5 ~uppress~s their ab~ y to ~orm t~or~ in vivo The JE gene i9 a platelet-derived growth factor (PDGF)-inducible "competence" or "early response gene" -~
10 first identified in mouse 3T3 cells ~Cochran, B.H. Cell 33:93~-947 ~1983)). S~quence ~nt express~on analysis showed that unlike other early response genes, such ns c-myc, c-fos, or ¢-~un, the murine JE gene encodes a secreted glycoprotein w~th cytokine-like properties (Kawahara, R.S. J._Biol._Chem 264:679-682 ~1989); Rollins, B.J. Proc. Natl. Acat. Sci. USA 85:3738-3742 (1988)). The human homolog of mur$ne JE has been cloned, (Rollins, B.J.
Mol. Cell._Biol. 9:4687-4695 (1989)), and the predicted amino acid sequence of its protein is identical ~o that of 20 a monocyte chemoattractant, MCP-l (Yoshimura, T., J. Exp.
Med. 169:1449-~459 (1989) ~Yoshimura T. JE. FEBS Lett.
244:487-493 (1989)) al80 called MCAF (Furuta~i, Y. et al., Biochem. BiQ~hys. Res. Commun. 159:249-255 (1989);
Matsushims, K. et_al., J._Ex~. Med. 169:1485-1490 (1989) 25 and SMC-CF (~raves, D.T., et_al., Science 245:1490-14g3 (1989); Valente, A.J. et al. Biochem 27:4162-4168 (1988)~.
The JE/MCP-llprotein is structurally related to the members of a large, recently identified family of low molecular weight secreted proteins that appear to be W092/~0372 PCT/US92/04099 21~3~ ~
in~olved in the inflammatory response (Leonard, E.J. and T. Yoshimura Immunol._Totay 11:97-101 (1990); Rollins, B.J. et al., Mol. Cell._Blol. 9:4687~4695 (1989); Wolpe, S.D. ant A. Ce~ami, FASEB J. 3:2563-2573 (1989)). The genes for many of these prote~ns, ~ncluding human JE~MCP~l, are clustered on chromosome 17qll.2-12 (Donlon, T.A. et_al., Qenomics 6:548-553 (1990); Irving, S.G., et al., Nucleic Acids Res. 18:3261~3270 (1990); Rol.lins, B.J., et al., Genomics_(in press)) or mouse chromosome 11 10 (~ilson, S.D., et_al., J._Ex~__Med. 171:1301-1314 (1990)).
These genes are also relatet to the genes encoding another family of cytokines, whose members include the neutrophil activstor NAP-l/IL-8 (Pe~eri, P., e~_al., J._Exp.~Med.
167:1547-1559 (1988); Schroder, J.M. et 81. J. Immunol 139:3474 3483 (1987); Yoshimura, T. et al., Proc._Natl.
Acad. Sci. ~SA 84:9233-9237 (1987)), many of which cluster at 4ql2-21 (Griffin, C,A. et al., CYto~enet-cell-Genet 45 67-69 (1987); Luster, A.D. et_~l. Proc-_Natl-_A.ead-Sci. ~SA 84:2868-2871 (1987); Richmont, A. E~BO_J 7:2025-20 2033 (lg~8)).
JE/MCP-l exerts several effects specifically on monocytes. Both natural and recombinant JE/MCP~l are potent chemoattractan~ts for human monocytes in ~itro, (Matsushima, K. et_al. J._Ex~._Med. 169:148~ 1490 (1989);
2~ Yoshimura, T. et al. J._Ex~._Med. 169:1449 1459 (1989)) and purified recombinant JE/NCP-l can stimulate an in-crease in cytosolic free;clalcium and the respiratory burst in monocytes (Zachariae, C.O.C., et_al., J. Ex~._Med.
171:2177 2182 (l990); Rollins, B.J. et_al. Blood (in 30 press)). Purified natural JE/NCP-l has also been reported W~92/20372 PCT/US92/~0~
:,.
2iO~3~.S -6- ;~
;, :'~
to activate monocyte-mediated inhibition of tumor cell growth, but not tumor cell killing, in ~itro (Matsushima, K. et al. J. Ex~. Med. 169:1485-1490 (1989)).
The following demonstrates ~hat expre~s$on of the JE
gene $n mal ign~nt cells suppre6s~s their ab~lity to form tumors in vivo. This spp~rent phenotypic revers~on requires lnteraction with host ~acto~s in ~ivo, ~ince expression of JE/MCP-l toes not alter the transformet character of these cells ln vitro. Furthermore, the following shows that JEjMCP-l-expressing cells exert their effect in trans by their ability to suppress tumor form-ation when co-inJected with JE/MCP-l-non-expressing tumor cells.
In order to create malignant cells expressing 15 JE/MCP-l, the DHFR deletion mutant CHO cell line, D~XXB-ll ~G. Urlaub and L.A Chasin, Proc.~Natl._Acat._Sci. USA, 77:4216-4220 (1980)) was transfectet with the express~on vector pXM ~Y~ng, Y.-C. et_al. Cell 47:3-10 ~1986) con-taining a variety of JE cDNA species. High levels of 20 JE/NCP-l protein expression were achieved in stably transfected llnes by methotrexate (MTX)-induced DNA
ampll~ication. R.J. Kaufman, et_al., ENBO_J. 6:187-193 (1987).
~igure l show- JE/~CP l protein expression in in-25 dependently derived cell lines selected for resistance to2 or 10 uM MXT. There is no detectable JE/MCP-l protein secreted from cell lines transfected with pXM alone (cell lines OA~2 and OB-2; cell line OA-lO was derived from!
OA-2) or with pXM containing murine JE cDNA in the anti-30 sense orientation (lA-2 and lB-2). Considerable JE/MCP-l .
~ ' :
W092/20372 2 1 ~ 9 ~ ~ ~i Pcr/usg2/o4099 :
protein was secreted by cell lines transfected with murine JE cDNA in the sense orientation (lOA-2 and lOB-2; lOA-10 ~as derived ~rom 10~-2) and human JE cDNA (hJEC-10). Cell line hJEC-100, derived from hJEC-10 by selecting for resistance to 1~0 uM MTX, al80 6ecreted human JE/MCP-l proteln.
Murine JE cDNA directs CHO cells to ~ecrets a micro-he~erogeneous protein of Nr 27-39,000, similar to the natur~l protein and the protein expressed in a COS cell 10 expression system. Rollins, B.J. et_al., Proc. Matl.
Acad. Sci._USA 85:3738:3742 (1988)). Nearly half of the apparent Mr is due to O-linked glycosylation. Human JE/MCP-l proteins expressed in CHO cells are al90 similar to native and COS cell expressed JE/MCP-l~ (MrlS,OOO~ and 15 JE/NCP 1~ (Mr 11,000). (Rollins, B.J., et al~ Mol.~Cell.
Biol. 9:4687-4695 (1989) ____ _ The monocyte chemoattra¢tant actiYiti~s (MCA) secreted by these cell lines were tetermined as described in detail in Example 1. They were 1415 U/24hr/106 cells 20 (lOA-2), 1079 U/24hr/10 cells (lOB-2), 3008 ~/24hr/10 .
cells (lOA-10), 54 U/24hr/10 cells ~hJEC-10), and 692~
U/24hr/106 ce~ls (hJEC-100) JE/MCP-l-non-expressing cells secreted 10-30 U/24hr/106 cells. The increased MCA
secreted by murine JE/NCP-l expressing lines ~s due to 25 increa9ed JE/MCP-l protein in the medium, not to higher specific acti~ity. Murine JE/MCP-l appears to be more stable than human JE/MCP-l in culturel perhaps due to its more exte~si~e glycosylat`io~. (Rollins, B.J., et aI. MoI.
Cell. Biol. 9:4687-4695 ~1989)) _________ _ .
WOg2/20372 ; , PCT/US92/0~09~ .
~ 31~ -8-By several criteria, JE/MCP-l expression did not alter the tran6formed phenotype of CH0 cells in vitro.
T~ble I shows that ~hile doubling times of each of the independently tsr~ved cell lines ~ar~ed considerably, the a~erage doubling t~me of all the JE/MCP-l-~xpressing lines (25.1 hrs ~ 5.7 sd) WBS nearly identical to the a~erage doubling time of the JE/MCP-l-non-expressors (26.4 hrs +
5.7 sd~. Also, JE/MCP-l expression tid not alter the transfor~ed cellular morphology of the CH0 cells.
10 Finally, all cell lines formed colonies in soft agar.
,. ;~ `
.
wo g~/20372 2 1 ~ g 3 ~ ~ PCT/US92/0~099 9 i :.
TABLE 1. PROPERTIES OF TRANSFECTED CHINESE HAMSTER OVARY CELL
LINES.
Doubllng Cell JE Time So~t Agar No. Cell5 Tumor~/Animal Line cDNA (hours~ Colonies~2~nl~ted____InieCted__ JE/MQP-l_Non expressors OA-2 None 24.2 91 +. 14.0 2 x 107 1~
n n ~ n 1 x 10 2/2 n n n n 2 x 10 3/4 ;.
OA-10 None 20.4 32 ~ 1.9 1 x 10 2/2 :
OB-2 None 19.0 73 ~ ~.6 1 x 107 0/4 lA-2Antisense 30~7 75 ~ 14.5 8 x 106 2/2 lB-2Antisense 31.2 74 i 1.7 1 x 10 1/~
JE~MCP-l Ex~ressors __ ________ _______ lOA-2Murine 28.8 72 ~ 7.3 2 x 107 0/1 ~ n n n 1 x 10 0 / 2 n n ~ 1l 2 x 10 0/4 lOB-2Murine 17.8 86 i 5.1 1 x 107 0/2 10A-10Murine 30.0 129 * 6.8 1 x 10 0/2 hJEC-10 Hu~an 28.8 32 ~ 6.8 1 x 10 0/2 In ~ivo, howe~er, J!E~NCP-l expression led to a striki~g difference in behavior. Table 1 shows that all but one (OB-2) of~ the JE/MCP-l-non-expressing cell lines formed lar~e subeutaneous tumors that appeared within W0~2/20372 PCT/U$92/040~9 2 1 l 9 r3 ~ 5 10-three weeks of in~ection into nude mice. In contrast, all of the JE/MCP-l-expressing l~nes, including the human JE/MCP-l expressing lines, ormed no tumors for ss long as ten months after injection. At ~utopsy, there was no S microscopic evidence of residual tumor in the Rnimals that received JE/MCP-l-expresslng cells.
These observations suggestet the possibllity that JE/NCP-l-secreting cells attxacted monocytes to the site of tumor cell inJection and once there, secreted JE/MCP-l 10 protein induced monocyte tumoric~dal activity. To test this hypothesis, JE/MCP-l-expressing cells were mixed with 107 OA-2 cells, a num~er of cell6 that reproducibly let to tumor formation when in~ectet slone (see Table 1).
Figure 2A shows that co~n~ection of murine JE/~CP~
15 expressing cells ~lOA-10) with OA-2 cells completely suppressed tumor format~on in two animals. Co-in~ection of high-le~el human JE/MCP-l-expressing cells (hJEC-100) wlth OA-2 cells also completely suppressed tu~or form-ation. Co-injection of low-le~el human JE/MCP-l-20 expressing cells (hJEC-10) suppressed tumor formation for 8-10 days, after which tumors appeased. Presumably hJEC-10 cells exerted a suppressive effect transiently unt~l the proliferating OA-2 cell mass reached a size that enabled it to escape the e$fect.
JE/NCP-l-expressors and non-expressors were also co-in~ected with He~a cells to test whether JE/MCP-l expression could suppress tumor formation by another cell type~. Figure 2B shows that lOA-lO cells completely suppressed the formation of tumors by HeLa cells. As 30 above, co-injection with hJEC-10 celIs delayed tumor ~ .
.
W092/20372 PCT/USg2/04099 21t~31~
formation. In a separate experiment, 4 o~ 4 animals in~ectet with 107 HeLs cells formed tumors, while only 1 of 4 an~mals in~ectet with 107 NeLa cells plus 107 hJEC-100 cells formet tumors. Mice t~at displayed no tumor growth after receiving HeLa plu~ hJ~C-100 c~118 were then inJected with 107 HeLa cells ~lone. These mice developed large tumors withln 14 days, ~nd$cating that prior suppression of HeLa cell tumor growth in the presence of JE/MCP-l does not render mice immune to 10 rechallenge with HeLa cells.
Histolog~c examination of the tumors arising from co-~n~ected HeLa and OA-2 cells revealed a mixture of epithelioid HeLa cells and spindle-shaped CHO cells.
Examlnation of the tumors formed in animals that r~ceived 15 HeLa and hJEC-10 cells also demonstrated a mlxture ~of HeLs cells and CHO cells. The presence of some hJE5-10 cells in these tumors was con~irmed by Northern blot analysis in which expression of human JE mRNA could ~e detected.
After reaching a certain s~ze, the-e tumors may overwhelm ;20 the host response elicited by the low levels of human JE/MCP-l secreted by hJEC-10 cells. However, tumor growth still requires the presence of a malignant JE/NCP-l-non-expressing cell line, since hJEC-10 cells in~ected by themsel~es cannot form tumors ~Table 1). This suggests 25 again that the intrinsic ~rowth properties of the CHO
cells have not been altered by JE/MCP-l expression, and that the human JEjMCP-l expressors will proliferate in ~ivo if; they are protectet by an enlarging mass of malignant cells.
Figures~3 and 4 show the results of a histological exa=ination of the s i -e s o ~ CHO cell inoc~lation 24 hours , ' W092/20372 PCT/USg~/~4099 210~3~ ~
after cells.were in~ected. JE/MCP-l-non-expressors ~darkly stained, large cells irdicated by arrows in Figure 3A) induce the sppearance in ~he underlying connective tissue vf only a fzw cells, moct of which ~re neutrophlls, In contrast, CH0 cells exprosslng murlne JE/MCP-l (indi-cated by arrows in Figure 3B) elicited un ~bund~nt cellular infiltrate, At higher power (Figure 4A), it i9 ~pparent that this in~iltsate consisted prinarily of monocytes with a reproducibly signiflcant proportion of 10 eoslnoph~ls (arrows), usually 10-20%. As mlght be ex-pected, low-level human JE/MCP-l-expressing cells (hJEC-10) induced a qualitatlvely slmilar infiltrate (Figure 4B) that was intermediate in inten~ity between the non-expressors and the muri~e JE/MCP-l expressors (compare 15 Figure 3C to Figures 3A and 3B).
The result~ repre~ented by Figure 3 ~nd 4 demonstrate a predominantly monocytic in~lltrate at the site of tumor cell in;ection, suggesting that monocy~es mediate tumor growth suppression. The effect is probably not mediated 20 by T lymphocytes or by natur~l killer cells, since puri-fied reeombinant JE/MCP-l has no stimulatory effect on~
natural kille~ ceIls in vitro.
~ f monocytes are responsible for tumor suppression, there are se~eral possible mechanisms whereby JE/MCP-l-25 acti~ated monocytes might exert their effe¢ts. JE/MCP-~coul~ induce the expression of a soluble mediator of tumor cells lysis such as tumor necrosis factor (TNF). L.J. Old, Sc~ence, 230: ~30-632 ~i985). J.L. Urban et al., Proc.
~atl. Acsd. Sci. USA 83: S233-5237 (1986). Alternatively, ______________ __ __ __ 30 TNF coul~ be expressed and displayed in an acti~e form on the cell surface of acti~ated monocytes. M. Kriegler, et al., Cell 53: 45-52 (1988).
WO9~/20372 PCT/US92/04099 ;
21~3!~31~ :.
Interestingly, nn eosinophilic component to the inflammatory cell infiltrate was also observed. This may be either a direct effect of JE/MCP-l or, JE/MCP~l may induce the expression of another factor with eosinophil chemoattxactsnt properties, These re6ults point to a clinical role for infused JE/MCP-l in vertebrate animsls, such BS humans. For example, JE/MCP-l can be atministered to pat~ents wlth cancer. This would necessarily be limlted to patients 10 with low tumor loads, i.e. as an at~uYant to surg0ry or cytotoxic chemotherapy. Systemically in~uset JE/MCP-l would lead to a generalizet increase in the acti~ation state of a ~ertebrate's monocytes. There i5 some evidence that patients harboring malignancies ha~e depressed 15 monocyte function. W.G. Chaney et al ., Cell Molec. Genet, 5 15-27 (1986). E.S. Kleinerman et_al., Lancet ii:
1102-1105, (19~0). If the te~ect were cytokine basett rether than an inherent monocyte defect, JE/MCP-l infusion would correct the abnormality.
Alternatively, JE/MCP-l may prove useful in treating localized complications of malignancy, 6uch a8 pleural effus~ons or ascltes. Inst~lling JE/MCP-l into the invol~ed anatom~ic space ~e.g. the space between the lung and the pleural membrane or the space between the stomsch 25 and the peritoneum~ can lead to local monocyte accumu-lation and activation.
As a therapeutic, JEtNCP-l can be administered to vertebrate animals (i.e. animals having an immune system), including humans. The compounds of this invention can be 30 employed in admixture with conventional excipients, i.e., W09~/20372 PCT/US92/04099 2 1 0 ~ 3 ~
.
pharmaceutically accep~able organlo or inorganic carrier subst~nces suitable for parentsl or topical applic~tion.
Suitable pharmaceutical c~rriers include, but ~re not llmited to water, salt ~olutions, ~lcohols, p~ly~thylene glycols, gelatin, carbohydr~tes such ~5 lacto~e, ~mylo~e or starch, magneslum stearate, t~lc, silicic acid, vi~cous paraifin, perume oil, ~tty ncld esters, hydroxym~thyl-cellulose, polyvinyl pyrrolldone, etc. The ph~rmaceutical preparations can be sterlized and i~ desired, mixet w~th 10 auxiliary sgents, e.g., lubricant~, prcservatives, stabi-lizers, wetting agen~s, emul~ifiers, salts for in1uencing osmotic pre~sure, buffers, coloxing, flavoring and/or aromatic s~bstances ~nt the like which do not delete-riously react with the active compounds. They can also be 15 combined where desired with other active a~ents, e.g., enzyme ~nhibitors, to ~urther reduce metabolic degradation.
For parenteral application, pa~ticularly suitable are in~ectable, sterile solutions, preferably oily or ~queous 20 solutions, as well as suspensions, emulsions, or implants, ~ncluding suppo~itories. Ampoules are convenient unit dosa~es.
For topical application, there are employed as nonsprayable ~orms, ~iscous to semi-solid or solid forms 2~ comprising a oarrier compatible with topical application and having a dynamic viscosity preferably greater than water. Suitable formulations include but are not limi*ed to solutions, sùspensions, emulsions, creams, ointments, powders, liniments, salves, serosols, etc., which are, if 30 desired, sterilized or mixed with auxiliary agents, (e.g., .
WO~/20372 PCT/US92/04~99 3 ~. 5 preser~atives, sta~ilizers, wetting agent~, buffers of salts for influencing osmotic pres~ure, etc.). For topical application, also suitsbl~ are 4prayable aerosol preparations wherein the actl~e ingrediiont, preferably in combination with a ~olid or liquld lnort cisrrier material, i9 packaged in ~i squeeze bottle or in ~dmixture with a pre~isurized volatile, normally gaseou~ propell~nt, e.g., a Freon.
It will be appreclated that the actual preferred 10 amounts o~ JE/MCP-l in a ~peci~ic csi9e will vary according to thé speci~ic compound being utilized, the particular compositions formulatod, the mode of ~pplication, ant the particular situs ant organism being treated. Dosage~ for a given host can be detexminet using conventional consit-15 eratlons, (e.g., by means of an appropriate, conventionalpharmacological protocol.).
Alternatively, tumor killing cells, ~uch as tumox infiltrating lymphocytes ~TIL cells) could be genetically engineered to express the JE/MCP-l protei~. Tumor killing 20 cells engineered in this way;can pro~ide synergistic local tumor cell k~lling. The tumor kllllng cells could be ~
engineered in_~itro ant administered to the vertebrate or the e~mor killing cells could be engineered i__~i~o into the ~ertebrate's own supply~of tumor killing cells using 25 ~ethods which are known ln the art.
The present invention will now be illustrated by the following examples, which are not intended to be limi.ting in any,wRy. I I !
: ~ .
~ ' w~g2/20372 PCT/US92/04099 -16- ;
3 ~
EXAMPLES
E~am~le 1 In Vi~o_Tumoricidal Effec~ o~ JE/MCP
Cell culture. DUKXB-ll ¢ell5 (Url~ub, G. and L.A.
____________ Ch~sin, Proc. Natl._Ac~d._Sci._USA, 83:5233-5237 (1987)) (dihytrofolate reductAse (DHFR) ?~utant Chi~cs~ hamster o~ary (CH0) cc115) ~ere grown in th~ alpha mod~ficatlon of minimal essenti~l medlum (NEM-~ ) without rlbonucl~os~des and deoxyribonucleosldes, supplemented wlth 10~ bo~ine calf serum ~nd 10 ug/ml ~denosine, deoxyadeno#ine, ant lO thymidine (NEM-~/BCS/ATT)~ (Kaufman, R.J. et,_al. EMB0 J.
6: 187-lg3, (1989~). HeLa cells were grown in Dulbecco's modified Eagle' 5 medium (DMEM) supplemented with 10%
heat-inacti~ated calf serum ~nd antibiotics. ~
Tra-sfectlons-a-d-Methotrexate-(MTx2-sele~tio-, ..
15 DUKXB-ll cells were grown in 10 cm cul~ure d~?shes as described abo~e. At 60% confluence, medium was remo~ed and replacet with fresh M~M-~/BCS/AAT supplemented with 3 ug/ml polybrene and incubated overnight. (Chaney, W.G.~et al., Somat.-ce~ oleG~-Genet~ 12:237 244, (1986)) The 20 next day, each dish was given 3 ml fresh MEM-~/BCS/AAT
with 83 ng/ml plasmid D~A and 10 ug/ml pclybrene. Cells were incubated at 37C fox 6 hr with rocking every 90 min.
Nedium was then removed and replacet with 5 ml fresh MEM-~/BC~/AAT con~aining 30% DMS0 for exactly 4 min.
25 Medium;was remo~ed, the~cells wére washed!once with NEM-~, and given 10 ml of MEM-~/BCS/AA . Cells were incubated at 37C for 48 hr, then trypsinized and re-plated into 4 culture dishes (10 cm) in nucleoside- and deoxynucleo-side-iree MEM-o supplemented :ith 10'~ dialyzed BCS, and ' 21~39~
..
refed w~th this medium every 3 days. Two independent transfections were per~ormed uslng pXM (Yang et_al. Cell 47:3-10 ~1986); pXM-JE10 ~murine JE cDNA (Rolli~s, B.J., et al., Proc. Natl. Acad. Sci. ~SA 85:3738-3742 (1988)) ln _____ .__________________________ __ the sense orientation), pXM-JEl ~murine JE cDNA in the antisense orientation), ant pXM-hJE34 (human JE cDNA) (Rollins, B.J., et_al., Mol,_Cell._Biol. 9:4687~4695 (1989)). Colonies from each ~ndepentent transfection that grew in ribonucleoside~ and deoxyri~onucleo~ide-free metium were trypsinized and combined. Step-wii~e ~election in increasing concentrations o methotrexate (MTX) was carried out at the following le~els: 0.02 uM, 0.1 uM, O.S
uM, 2.0 u~, 10.0 uM, and 100.0 uM. At each conce~tration, survi~ing colonies were trypsinlzet and pooled.
PEotei _analysis. Confluent cell cultures were incubated in methionine-free NEM-~ with 2% dialyzed BCS
for 45 min, then changed to 0.5 ml of the same medium with 500 uCi [ 5S]methionine (DuPont ~EN, Boston, MA). Cells were incubated at 37-C for 4 hrs. after wh~ch the medlum was collected, made`l mM in phenylmethylsulfonyl fluori~e (PMSF), centrifuged to remo~e cells and tebris, and stored at -70C. Immune precipitatlons uslng anti-JE/MCP-l antiQerum were performed (Rollins, B.J., et_al., Mol.
Cell. Biol. 9:4687-4695 ~19B9))~ ant the results analyzed 2~ by electrophoresis through an SDS-containing 17% poly-acrylamide ~el.
Soft a~ar_colony formatio_ assay. Five thousand cells were suspended~in MEN-~ containing 10% dialyzed BCS, 0.3~ agarj and the appropriate concentration of MTX.
.
, WOg2/20372 PCT/US92/0~9 21~'~3~ ~ -18-While still molten, this suspension was distrlbuted on a gelled 4 ml underlayer of NEM-~ contsining 10~ dislyze~
BCS, 0.6~ a~ar, and the appropriate concentration of NTX
~n a 60 mm culture di~h. Cells were ~ed wlth 3 drops o~ -fresh medium every 5 days. After 14 da~s, colonie~
consisting of grQater th~n 50 cell5 were counted.
Mon~te_che_oattracta_t_acti~it~_lMCA~. Confluent monolayers of CH0 cells were incubated in ~erum-~ree NEM-~for 24 hr, after which the medium was ccntrlfuged to remo~e cells and debris, and the remainlng adherent cells were trypsinized and counted. Fre~h humsn periphe~al blood mononuclear cells were puri~ied from the blood of ;, volunteer donors by centrifugation on a cushion of Ficoll-Hypaque (Pharmacia, Piscataway, NJ). Cells ~t the interface were washed tw~ce in ~ey's bslanced salt solution (GBSS) with 2~ BSA, thon resuspended at 4 x 106 cells/ml in GBSS with 0.24 8SA, and MCA was messuret in a 48-well microchamber apparatus. (Falk, W.; et_al., J.
Immunol. Methods 33:239-247 (1980)). The concentration of MCA in CH0 cell medium was tefined as the reciprocal of~
the dilution showing half-maximal acti~ity. (Yoshimura, T., et al., J.~Ex~. Med. 169:1449-1459 (1989)).
Nude mouse inieetions. Cells were suspended in 0.2 ~__________ _ _______ ml PBS and injected subcutMneously into ~-week old male ' Swiss nu/nu mice. Mice were monitored daily for tumor growth., Tumor volume was derived ~y multiplyin~ the values of ~hree pe~pentlcular diameters.
W092/20372 ~ 1~ 9 3 ~ X PCT/USg2/04099 E~uivalents _________ Those ~killed in the art will recognize or be able to ascertain using no ~ore than routine exper~mentation, many equi~alents to the specific embotiments of the in~ention describe herein. Such equi~alents are intended to be encompassed by the following claims.
:
Claims (7)
1. A method of suppressing tumor formation in a verte-brate animal comprising administering to said verte-brate a therapeutically effective amount of JE/MCP-1.
2. A method of increasing monocyte mediated tumoricidal activity in a vertebrate animal comprising adminis-tering to said vertebrate an effective amount of JE/MCP-1.
3. A method of treating a localized side-effect of malignancy in a vertebrate animal comprising locally administering to the vertebrate a therapeutically effective amount of JE/MCP-1.
4. The method of Claim 3 wherein the side effect is selected from the group consisting of pleural effusions or ascites.
5. A method of suppressing tumor formation in a verte-brate comprising administering to said vertebrate tumor killing cells which express JE/MCP-1.
6. A method of Claim 5, wherein the tumor killing cells are tumor infiltrating lymphocytes.
7. A method of combatting a parasitic infection in a vertebrate animal comprising administering to the vertebrate animal an effective amount of JE/MCP-1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/701,515 US5179078A (en) | 1989-05-12 | 1991-05-16 | Method of suppressing tumor formation in vivo |
| US701,515 | 1991-05-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2109315A1 true CA2109315A1 (en) | 1992-11-17 |
Family
ID=24817687
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002109315A Abandoned CA2109315A1 (en) | 1991-05-16 | 1992-05-15 | Method of suppressing tumor formation in vivo by administering je/mlp-1 |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US5179078A (en) |
| EP (1) | EP0585337A1 (en) |
| JP (1) | JPH06508117A (en) |
| CA (1) | CA2109315A1 (en) |
| WO (1) | WO1992020372A1 (en) |
Families Citing this family (49)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6869924B1 (en) | 1989-01-31 | 2005-03-22 | The United States Of America As Represented By The Department Of Health And Human Services | Human derived monocyte attracting purified protein product useful in a method of treating infection and neoplasms in a human body, and the cloning of full length cDNA thereof |
| IL92937A0 (en) * | 1989-01-31 | 1990-09-17 | Us Health | Human derived monocyte attracting protein,pharmaceutical compositions comprising it and dna encoding it |
| US6767535B1 (en) * | 1989-05-12 | 2004-07-27 | Dana-Farber Cancer Institute, Inc. | Suppressing tumor formation using cells expressing JE/monocyte chemoattractant protein-1 |
| US5316921A (en) * | 1992-05-18 | 1994-05-31 | Genentech, Inc. | Single-chain hepatocyte growth factor variants |
| JPH07508420A (en) * | 1992-05-18 | 1995-09-21 | ジェネンテク，インコーポレイテッド | Hepatocyte growth factor variant |
| EP0749475A4 (en) * | 1992-08-26 | 1997-05-07 | Harvard College | Use of the cytokine ip-10 as an anti-tumor agent |
| US5413778A (en) * | 1992-10-05 | 1995-05-09 | The Regents Of The University Of Michigan | Labelled monocyte chemoattractant protein material and medical uses thereof |
| US5605671A (en) * | 1992-10-05 | 1997-02-25 | The Regents Of The University Of Michigan | Radiolabeled neutrophil activating peptides for imaging |
| WO1994009128A1 (en) * | 1992-10-22 | 1994-04-28 | Mallinckrodt Medical, Inc. | Therapeutic treatment for inhibiting vascular restenosis |
| JPH0782169A (en) * | 1993-09-13 | 1995-03-28 | Toray Ind Inc | Wound healing agent |
| US6451562B1 (en) | 1993-12-22 | 2002-09-17 | Human Genome Sciences, Inc. | Polypeptides encoding myeloid progenitor inhibitory factor-1 (MPIF-1) polynucleotides |
| US6488925B2 (en) | 1993-12-22 | 2002-12-03 | Human Genome Sciences, Inc. | Macrophage inflammatory protein-4 (MIP-4) polypeptides |
| US6001606A (en) * | 1994-03-08 | 1999-12-14 | Human Genome Sciences, Inc. | Polynucleotides encoding myeloid progenitor inhibitory factor-1 (MPIF-1) and polypeptides encoded thereby |
| US6811773B1 (en) | 1993-12-22 | 2004-11-02 | Human Genome Sciences, Inc. | Human monocyte colony inhibitory factor (M-CIF) polypeptides |
| US6495129B1 (en) | 1994-03-08 | 2002-12-17 | Human Genome Sciences, Inc. | Methods of inhibiting hematopoietic stem cells using human myeloid progenitor inhibitory factor-1 (MPIF-1) (Ckbeta-8/MIP-3) |
| GB9406463D0 (en) * | 1994-03-31 | 1994-05-25 | Medical Res Council | Cervical ripening |
| US6075124A (en) * | 1994-05-16 | 2000-06-13 | Human Genome Sciences, Inc. | Human chemotactin protein |
| US6100389A (en) * | 1995-04-21 | 2000-08-08 | Human Genome Sciences, Inc. | Polynucleotides encoding a human chemotactic protein |
| US5866373A (en) * | 1995-04-21 | 1999-02-02 | Human Genome Sciences, Inc. | Polynucleotide encoding a human chemotactic protein |
| US6391589B1 (en) | 1994-08-23 | 2002-05-21 | Human Genome Sciences, Inc. | Human chemokine beta-10 mutant polypeptides |
| US5981230A (en) * | 1994-08-23 | 1999-11-09 | Human Genome Sciences, Inc. | Polynucleotide encoding chemokine β-4 |
| US6174995B1 (en) | 1994-08-23 | 2001-01-16 | Haodong Li | Human chemokines, CKβ4 and CKβ10/MCP-4 |
| US6458349B1 (en) | 1995-06-02 | 2002-10-01 | Human Genome Sciences, Inc. | Chemokine β-4 polypeptides |
| GB9501683D0 (en) | 1995-01-27 | 1995-03-15 | Glaxo Group Ltd | Substances and their uses |
| US6737513B1 (en) | 1996-06-07 | 2004-05-18 | Icos Corporation | Macrophage derived chemokine (MDC) and chemokine analogs and assay to identify modulators of MDC activity, and therapeutic uses for same |
| US6498015B1 (en) | 1995-06-07 | 2002-12-24 | Icos Corporation | Methods of identifying agents that modulate the binding between MDC and an MDC receptor |
| US6320023B1 (en) | 1995-06-07 | 2001-11-20 | Icos Corporation | Macrophage derived chemokine |
| US7018627B1 (en) | 1995-06-07 | 2006-03-28 | Icos Corporation | Macrophage derived chemokine (MDC), MDC analogs, MDC inhibitor substances, and uses thereof |
| US6790947B1 (en) | 1995-06-07 | 2004-09-14 | Icos Corporation | Polynucleotides encoding macrophage derived chemokine |
| US5932703A (en) * | 1995-06-07 | 1999-08-03 | Icos Corporation | Macrophage derived chemokine and chemokine analogs |
| US5688927A (en) * | 1995-06-07 | 1997-11-18 | Icos Corporation | Macrophage derived chemokine |
| US6013252A (en) * | 1995-08-28 | 2000-01-11 | Toray Industries, Inc. | Method promoting conception by administering IL-8 or MCAF |
| WO1997012220A2 (en) * | 1995-09-15 | 1997-04-03 | Samir Chachoua | Method for the identification and therapeutic use of disease-associated organisms, elements and forces |
| US6290948B1 (en) * | 1996-05-14 | 2001-09-18 | Smithkline Beecham Corporation | Method of treating sepsis and ARDS using chamohine beta-10 |
| US6673915B1 (en) | 1996-09-30 | 2004-01-06 | General Hospital Corporation | Nucleic acid encoding monocyte chemotactic protein 4 |
| CN1213401A (en) * | 1996-11-15 | 1999-04-07 | 先进研究及技术学会 | Exodus chemokine materials and method |
| US6028169A (en) | 1997-03-31 | 2000-02-22 | Human Genome Sciences, Inc. | Chemokine β-6 antagonists |
| ES2187972T3 (en) * | 1997-04-04 | 2003-06-16 | Max Planck Gesellschaft | METHODS FOR THE MODULATION OF THE GROWTH OF COLATERAL ARTERIES AND / OR OTHER ARTERIES FROM PREEXISTENT ARTERIOLARY CONNECTIONS. |
| US7067117B1 (en) | 1997-09-11 | 2006-06-27 | Cambridge University Technical Services, Ltd. | Compounds and methods to inhibit or augment an inflammatory response |
| US6989435B2 (en) | 1997-09-11 | 2006-01-24 | Cambridge University Technical Services Ltd. | Compounds and methods to inhibit or augment an inflammatory response |
| US7238711B1 (en) | 1999-03-17 | 2007-07-03 | Cambridge University Technical Services Ltd. | Compounds and methods to inhibit or augment an inflammatory response |
| DE19959045A1 (en) * | 1999-12-07 | 2001-06-28 | Deutsches Krebsforsch | Adeno-associated virus vectors that express monocyte chemotactic protein-1, useful for gene therapy of cancers, promotion of angiogenesis and treatment of inflammation |
| ATE518884T1 (en) | 2002-05-01 | 2011-08-15 | Human Genome Sciences Inc | ANTIBODIES SPECIFICALLY BINDING TO CHEMOKIN BETA-4 |
| US7713521B2 (en) * | 2005-08-12 | 2010-05-11 | Schering Corporation | MCP1 fusions |
| EP2415783B1 (en) | 2006-10-16 | 2016-12-14 | Genelux Corporation | Modified vaccinia virus strains for use in a diagnostic and therapeutic method |
| WO2011088402A2 (en) | 2010-01-15 | 2011-07-21 | Memorial Sloan-Kettering Cancer Center | The use of entrained neutrophils to treat metastatic and micrometastatic disease in at risk patients |
| US8524217B2 (en) | 2010-05-11 | 2013-09-03 | Merck Sharp & Dohme Corp. | MCP1-Ig fusion variants |
| PE20140633A1 (en) | 2010-11-19 | 2014-05-30 | Eisai Randd Man Co Ltd | NEUTRALIZING ANTIBODIES ANTI-CCL20 |
| EP2697368B1 (en) | 2011-04-15 | 2019-06-05 | Genelux Corporation | Clonal strains of attenuated vaccinia viruses and methods of use thereof |
-
1991
- 1991-05-16 US US07/701,515 patent/US5179078A/en not_active Expired - Lifetime
-
1992
- 1992-05-15 CA CA002109315A patent/CA2109315A1/en not_active Abandoned
- 1992-05-15 JP JP4511005A patent/JPH06508117A/en active Pending
- 1992-05-15 EP EP92911702A patent/EP0585337A1/en not_active Withdrawn
- 1992-05-15 WO PCT/US1992/004099 patent/WO1992020372A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| US5179078A (en) | 1993-01-12 |
| EP0585337A1 (en) | 1994-03-09 |
| JPH06508117A (en) | 1994-09-14 |
| WO1992020372A1 (en) | 1992-11-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2109315A1 (en) | Method of suppressing tumor formation in vivo by administering je/mlp-1 | |
| Marriott et al. | Thalidomide and its analogues have distinct and opposing effects on TNF-α and TNFR2 during co-stimulation of both CD4+ and CD8+ T cells | |
| Stellato et al. | Cutting edge: expression of the CC chemokine receptor CCR3 in human airway epithelial cells | |
| EP0640143B1 (en) | Senescent cell derived inhibitors of dna synthesis | |
| Gregory et al. | Membrane Fas ligand activates innate immunity and terminates ocular immune privilege | |
| KR102188605B1 (en) | Methods modulating immunoregulatory effect of stem cells | |
| Parolaro | Presence and functional regulation of cannabinoid receptors in immune cells | |
| Gallily et al. | Protection against septic shock and suppression of tumor necrosis factor α and nitric oxide production by dexanabinol (HU-211), a nonpsychotropic cannabinoid | |
| Brynskov et al. | Cytokines (immunoinflammatory hormones) and their natural regulation in inflammatory bowel disease (Crohn’s disease and ulcerative colitis): a review | |
| CA1265444A (en) | Effect of human tumor necrosis factor and human interferon on human cancer cells and methods | |
| Gallicchio et al. | Modulation of murine pluripotential stem cell proliferation in vivo by lithium carbonate | |
| Do et al. | Granulocyte-macrophage and macrophage colony-stimulating factors activate intramacrophage killing of Leishmania mexicana amazonensis | |
| Bozza et al. | Thymosin α1 activates the TLR9/MyD88/IRF7-dependent murine cytomegalovirus sensing for induction of anti-viral responses in vivo | |
| Mayadas et al. | von Willebrand factor biosynthesis and partitioning between constitutive and regulated pathways of secretion after thrombin stimulation | |
| Arenzana-Seisdedos et al. | Autocrine secretion of tumor necrosis factor under the influence of interferon-gamma amplifies HLA-DR gene induction in human monocytes. | |
| Sarkhosh et al. | Immune cell proliferation is suppressed by the interferon‐γ‐induced indoleamine 2, 3‐dioxygenase expression of fibroblasts populated in collagen gel (FPCG) | |
| Devisscher et al. | Metallothioneins alter macrophage phenotype and represent novel therapeutic targets for acetaminophen-induced liver injury | |
| Frishman et al. | Tumor necrosis factor (TNF)—α—induced interleukin-8 in human blood cultures discriminates neutralization by the p55 and p75 TNF soluble receptors | |
| Shin et al. | Enhanced Osteoclastogenesis in 4‐1BB—Deficient Mice Caused by Reduced Interleukin‐10 | |
| Yan et al. | Autocrine action of IL-10 suppresses proinflammatory mediators and inflammation in the HSV-1-infected cornea | |
| Matsui et al. | Cooperative regulation of c-myc expression in differentiation of human promyelocytic leukemia induced by recombinant γ-interferon and 1, 25-dihydroxyvitamin D3 | |
| Lilly et al. | Differentiation and growth modulation of chronic myelogenous leukemia cells by bryostatin | |
| Deresinski | Granulocyte-macrophage colony-stimulating factor: potential therapeutic, immunological and antiretroviral effects in HIV infection | |
| US6767535B1 (en) | Suppressing tumor formation using cells expressing JE/monocyte chemoattractant protein-1 | |
| Han et al. | Ligands for peroxisome proliferator-activated receptor inhibit monocyte CCR2 expression stimulated by plasma lipoproteins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |