USOO5639653A United States Patent 19 11 Patent Number: 5,639,653 Bloom et al. 45 Date of Patent: Jun. 17, 1997

(54) METHOD FOR PROLIFERATING Wy2V82T 56) References Cited CELLS PUBLICATIONS 75) Inventors: Barry R. Bloom, Hastings-on-Hudson; Cuthbert, J.A. et al., FASEB Journal, vol. 4(7), p. A1747, Yoshimasa Tanaka, Bronx, both of abs, 309 1990. N.Y.; Shigetoshi Sano, Nishinomiya, Chakrabarti, R. et al., The Journal of Biological Chemistry, Japan vol. 266(19), pp. 12216-12222 1991. Tanaka, Y. et al., Nature, vol. 375(6527), pp. 155-158 May 73) Assignee: Albert Einstein College of Medicine 1995. of Yeshiva University, a Division of Burk, M.R. et al., Eur, J. Immunol., vol. 25(7), pp. Yeshiva Universtiy, Bronx, N.Y. 2052-2058 1995. Morita, C.T. et al., Immunity, vol. 3(4), pp. 495-507 Oct. 21 Appl. No.: 390,881 1995. Primary Examiner Michael G. Wityshyn 22 Filed: Feb. 17, 1995 Assistant Examiner-Kristin Larson Related U.S. Application Data Attorney, Agent, or Firm-Amster, Rothstein & Ebenstein 57 ABSTRACT 63 Continuation-in-part of Ser. No. 93,528, Jul. 19, 1993, abandoned. This invention is directed to a method for stimulating the proliferation of Vy2V62 T cells comprising contacting 51 Int. C. m. C12N 5/02; C12N 5/06; Vy2V82 T cells with a Vy2V82 T cell proliferation stimu A61K31/66; CO7F 9/02 lating amount of a compound selected from the group 52 U.S. Cl...... 514/102; 514/106; 514/134: consisting of a monoalkyl phosphate and an alkenyl pyro 558/155; 558/156; 435/375; 435/384 phosphate. 58 Field of Search ...... 435/240.2: 514/102, 514/106, 134; 558/155, 156 15 Claims, 17 Drawing Sheets U.S. Patent Jun. 17, 1997 Sheet 1 of 17 5,639,653

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30000 O.05 E Na 0.04 E (f)L C f) CN 2 20000 OC 0.05 f) L t 1. O 2L O.O2 22. is 10000 0.019 - O 1. O ar (CCCCIII (CCCCCCC(C) O.OO O 2O 40 6O 8O TUBE NUMBER (1 TUBE=12ml) FG 4A U.S. Patent Jun. 17, 1997 Sheet 9 of 17 5,639,653

40000 O.4 E N1 S 30000 0.35 (f) OO 2 He ! 20000 0.2 3. 2 9 aC O 10000 o 0.1 % . . . CC - : e - l O d P3 a simil O.O O 2O 40 6O 8O TUBE NUMBER (1 TUBE=12mL) FG. 4B U.S. Patent Jun. 17, 1997 Sheet 10 of 17 5,639,653

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... 1 1 1O 1 OO 1 OOO 1 OOOO ANTIGEN (uM)

F.G. 6B U.S. Patent Jun. 17, 1997 Sheet 17 of 17 5,639,653 5,639,653 1. 2 METHOD FOR PROLIFERATING Vy2V82T the yö T cell clone DG.SF68, which expresses Vy2V82, CELLS when cultured with crude reaction mixtures of the following alkylphosphates: methyl phosphate (solid square), monoet STATEMENT OF GOVERNMENT INTEREST hyl phosphate (solid circle), n-propyl phosphate (solid 5 triangle), n-butyl phosphate (solid diamond), amyl phos This application is a continuation-in-part of application phate (open square), isopropyl phosphate (open circle), Ser. No. 08/093,528, filed Jul 19, 1993, now abandoned the isoamyl phosphate (open triangle), and secbutyl phosphate contents of which is incorporated by reference herein. (open diamond). FIG. 1B depicts the selective stimulation of This invention was made with government support under yó T cell clone SD9 by monoethyl phosphate (MEP, solid Grant No. NTH AI07118. As such, the government has square), diethyl phosphate (DEP solid circle), and triethyl certain rights in this invention. 10 phosphate (TEP, solid triangle). FIG. 1C depicts the stimu lation of the yá T cell clone SD9 by monoethyl phosphate BACKGROUND OF THE INVENTION (MEP, solid square), its hydroxyl derivative (B-hydroxyethyl T cells are thymus-derived cells in the immune system phosphate, open circle), and its carboxyl derivative which mediate cellular immune reactions and regulate (phosphoglycolic acid, open triangle). The percentage of the immune response. T lymphocytes (T cells) are divided into 15 maximal proliferation is shown. two subgroups by their usage of T cell receptors. About FIGS. 2A and 2B depict the effect of MEP on primary 90-98% of T cells have a? receptors (a? T cells) which PBMC and the specificity in the response of y& T cells to recognize antigenic peptides bound to major histo monoalkyl phosphate ligands. FIG. 2A depicts the expan compatibility complex I or Imolecules. About 2-10% of T 20 sion by MEP of yöT cells in primary cultures of PBMC as cells have yá receptors (yö T cells). determined by two-color FACS analysis. The increase of yö A major human yöT cell subset expressing Vy2 and V82 T cells by MEP is comparable to that stimulated by M. germlines (i.e. Vy2V62 T cells) is found in human myco tuberculosis (M.tb) lysates. FIG. 2B depicts the two-color bacterial lesions (Modlin, R. L., et al. Nature 339:544-548 FACS analysis showing that yoT cells expanded in primary (1989)). Vy2Vö2T cells have been shown to expand acutely 25 culture by MEP exclusively expressed V82. The percentages in response to mycobacterial pathogens such as M. tuber in the quadrants of interest are shown. culosis and M. luprae, bacterial pathogens such as Gram FIGS. 3A and 3B depict the effect of MEP on yöT cell positive and Gram-negative bacteria, as well as parasites clones and heterogeneity in the response of Vy2/VS2T cel, such as Plasmodium vivax (Kabelitz, D., et al. J. Exp. Med. clones to monoalkyl phosphate ligands. FIG. 3A shows the 171:667-679 (1990); Panchamoorthy, G., et al. J. Immunol. 30 proliferation in response to MEP was restricted to y& T cell 147:3360-3369 (1991); De Libero, G., et al. J. Exp. Med. clones expressing the Vy2/V62 receptor. The stimulation 173:1311-1322 (1991); Hara, T., et al. J. Clin. Invest. indices for the response to phytohemagglutinin (positive 90:204-210 (1992); Goerlich, R., et al. Eur: J. Immunol. control) were greater than 6 for all clones, with a mean value 21:2613-2616 (1991); and Goodier, M., et al. Int. Immunol. of 47.3. FIG. 3B shows the cytolytic activity of primary yö 4:33-41 (1992)). In addition, certain hematopoietic tumor 35 T cells expanded by MEP on tumor cell lines K562 and cell lines such as Daudi and RPMI 8226 are specifically Daudi, with 30 M MEP for 7 days (solid circle), with 30 recognized and lysed by Vy2V82 T cells (Fisch, P., et al. uMMEP for days on cell lines depleted of V82 cells by BB3 Science 250:1269-1273 (1990); Selin, L. K., et al. Scand. J. antibody and magnetic beads (Dynal)(solid triangle), and Immunol. 36:107-117 (1992)). medium only (solid square). E/T represents the effector to Because Vy2V82 T cells recognize antigens which are 40 target ratio. expressed by a variety of diseases in humans, there exists a FIGS. 4A, 4B, and 4C depict the purification of natural need to selectively stimulate the proliferation of Vy2V82T mycobacterial antigens. FIG. 4A depicts the elution patterns cells to enhance the immune response against these diseases. of M. Smegmatis bioactive preparations on anion-exchange The present invention satisfies this need. column chromatography. Proliferative responses (solid 45 circle) and absorbance (open circle). Arrows indicate the SUMMARY OF THE INVENTION fractions where inorganic phosphate (Pi) and pyrophosphate The present invention provides a method for stimulating (PPi) derived from mycobacterial preparation are detected the proliferation of Vy2V82 T cells comprising contacting by ESI-MS/MS analysis, respectively (data not shown). Vy2V82 T cells with a VY2V82 T cell proliferation stimu FIG. 4B depicts rechromatography of the partially purified lating amount of a compound selected from the group 50 M. smegmatis antigens on a Q SEPHAROSE HP column. consisting of a monoalkyl phosphate, a hydroxy monoalkyl Proliferative responses (solid circle) and absorbance (open phosphate, a carboxy monoalkyl phosphate, a monoalkyl circle). FIG. 4C shows that the M. tuberculosis antigens pyrophosphate, an alkenyl pyrophosphate, a Y-monoalkyl were eluted at the same fractions on Q SEPHAROSE HP triphosphate, a Y-monoalkyl deoxnucleoside column chromatography. Proliferative responses (solid triphosphate, a Y-alkenyl , and a 55 circle) and absorbance (open circle). Y-alkenyl deoxynucleoside triphosphate. FIGS.5A, 5B, 5C, and 5D depict the ESI-MS/MS analy The method of the present invention may be used for sis of the mycobacterial antigen for human yo T cells and enhancing the immune response against a variety of diseases stimulation of yo T cell clone 12G12 by prenyl pyrophos which express antigens recognized by Vy2V82 T cells. By phates. FIG. 5A depicts the ESI-MS/MS analysis of the stimulating the proliferation of VY2V82T cells, it is believed 60 purified mycobacterial antigens containing phosphate resi that an enhanced immune response will be generated against dues. FIG.SB depicts the daughterion spectra of the 245ion the diseases which express antigens recognized by Vy2V82 species derived from the purified mycobacterial antigens. T cells, thereby effectively treating the disease. FIG. 5C depicts the authentic daughter ion spectra of syn BRIEF DESCRIPTION OF THE FIGURES thetic isopentenyl pyrophosphate. FIG. 5D depicts the pro 65 liferation of 12G12 in response to isoprene units coupled FIGS. 1A, 1B, and 1C depict y& T cell response to with pyrophosphate. Pyrophosphate derivatives exemplified monoalkyl phosphates. FIG. 1A depicts the proliferation of are isopentenol (solid circle), dimethylallyl alcohol (open 5,639,653 3 4 triangle), geraniol (solid triangle), farnesol (solid square), depend upon the route of administration, the pharmacoki geranylgeraniol (open circle), and isoamyl alcohol (open netic properties of the individual treated, the disease being Square). treated, as well as the results desired. FIGS. 6A, 6B, and 6C depict the recognition of NTP or The compound may be formulated with one or more dNTP y-derivatives by human yöT cell clone 12C12 and a pharmaceutically acceptable diluents or carriers. The carrier schematic diagram of isoprenoid pathways in bacterial and must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious mammalian cells. FIG. 6A depicts the stimulation by to the recipient thereof. Optionally, the compound may be y-monoethyl derivatives of: 2'-deoxythymidine triphosphate administered with any other ingredients which may be (2'-dTTP, solid circle); 2',3'-dideoxythymidine triphosphate therapeutic per se, and/or may be synergistic with the (2',3'-ddTTP, solid triangle); 2'- triphosphate 10 compounds of the present invention. These include chemo (2'-dUTP, solid square); 2'-deoxythymidine diphosphate (2'- therapeutic agents known to act against the particular dis dUDP, open circle); and 2'-deoxythymidine monophosphate eases. The concentration of the compound present in the (2'-dUMP, open triangle). FIG. 6B depicts the stimulation by formulation will depend upon the choice of carrier as well as Y-monoethyl derivatives of: triphosphate (UTP, solid the results desired. circle); triphosphate (XTP, solid triangle); 15 Examples of suitable pharmaceutical carriers include triphosphate (ITP, solid square); triphos lactose, sucrose, starch, talc, magnesium stearate, crystalline phate (GTP, open circle); triphosphate (CTP, open cellulose, methyl cellulose, carboxymethyl cellulose, triangle); and triphosphate (ATP, open square). glycerin, sodium alginate, gun arabic, powders, Saline, FIG. 6C represents a schematic diagram of isoprenoid water, among others. The choice of carrier will depend upon biosynthetic pathways, and the relationship between the 20 the route of administration. The formulations may conve intermediates and oncogene modifications. niently be presented in unit dosage and may be prepared by methods well-known in the pharmaceutical art, by bringing DETALED DESCRIPTION OF THE the active compound into association with a carrier or INVENTION diluent, as a suspension or solution, and optionally one or 25 more accessory ingredients, e.g. buffers, flavoring agents, The present invention provides a method for stimulating surface active agents, and the like. the proliferation of Vy2Vö2 T cells comprising contacting For intravenous, intramuscular, subcutaneous, or intrap Vy2V82 T cells with a Vy2V82 T cell proliferation stimu eritoneal administration, the compound is combined with a lating amount of a compound selected from the group sterile aqueous solution which is preferably isotonic with the consisting of a monoalkyl phosphate, a hydroxy monoalkyl 30 blood of the recipient. Such formulations may be prepared phosphate, a carboxy monoalkyl phosphate, a monoalkyl by dissolving solid active ingredient in water containing pyrophosphate, an alkenyl pyrophosphate, a Y-monoalkyl physiologically compatible substances such as sodium nucleoside triphosphate, a Y-monoalkyl deoxnucleoside chloride, glycine, and the like, and having a buffered pH triphosphate, a Y-alkenyl nucleoside triphosphate, and a compatible with physiological conditions to produce an Y-alkenyl deoxynucleoside triphosphate. 35 aqueous solution, and rendering said solution sterile. The The method of the present invention may be used for formulations may be presentin unit or multi-dose containers enhancing the immune response against a variety of diseases such as sealed ampoules or vials. which express antigens recognized by Vy2Vö2 T cells. By For oral administration, the formulation may be presented stimulating the proliferation of Vy2V82T celis, it is believed as capsules, tablets, powders, granules or a suspension, with that an enhanced immune response will be generated against conventional additives Such as lactose, mannitol, corn starch diseases which express antigens recognized by Vy2V82T or potato starch; with binders such as crystalline cellulose, cells, thereby effectively treating the diseases. Specific dis cellulose derivatives, acacia, corn starch or gelatins; with eases include but are not limited to leukemia, lymphoma, disintegrators such as corn starch, potato starch or sodium tuberculosis, leprosy, malaria, AIDS, rheumatoid arthritis, carboxymethyl-cellulose; and with lubricants such as talc or ulcerative colitis and anemia. A“Vy2V82T cell proliferation 45 magnesium stearate. stimulating amount' is an amount effective to induce the Formulations suitable for parenteral administration con proliferation of said Vy2V82 T cells. veniently comprise a sterile aqueous preparation of the The "contacting” may be effected by administering to a active compound which is preferably made isotonic. Prepa subject in need of such immune enhancement a Vy2V82T rations for injections may also be formulated by suspending cell proliferation stimulating amount of one or more of the 50 or emulsifying the compounds in non-aqueous solvent, such compounds above. Alternatively, biological fluid from the as vegetable oil, syntheticaliphatic acid glycerides, esters of subject may be removed from the subject, treated with a higher aliphatic acids or propylene glycol. Vy2V82 T cell proliferation stimulating amount of one or The monoalkyl phosphate useful in the method of the more of the compounds above, and reinfused back into the present invention has the formula ROPO(OH) wherein Ris Subject by standard techniques. "Biological fluid' includes 55 a C1-C4 straight or branched chain alkyl, and may include but is not limited to sera, cerebrospinal fluid, synovial fluid, but is not limited to monoalkylphosphates selected from the and preferably is sera. The subject may be any mammal, and group consisting of methyl phosphate, monoethyl is preferably a human or a cow. phosphate, n-propyl phosphate, n-butyl phosphate and iso The administration may be affected by means known to propyl phosphate. Preferably, the monoalkyl phosphate is those skilled in the art such as oral, rectal, topical, 60 monoethyl phosphate. intravenous, Subcutaneous, intramuscular, or intraperitoneal The hydroxy monoalkyl phosphate has the formula routes of administration. The dosage form and amount can HOROPO(OH) wherein R is a C1-C3 straight or branched be readily established by reference to known chemothera chain alkyl, and may include but is not limited to hydroxy peutic treatments of the diseases. In general, however, the monoalkylphosphates selected from the group consisting of dosage of the compound will be within the range of about 65 hydroxy methyl phosphate, 3-hydroxy ethyl phosphate, 0.01 g/kg to about 100 mg/kg, and preferably between Y-hydroxy propyl phosphate and hydroxy isopropyl phos about 1 ug/kg and about 10 mg/kg. The actual dose will phate. 5,639,653 5 6 The carboxy monoalkyl phosphate has the formula n-butyl adenosine-triphosphate, n-butyl 2-deoxythymidine HOOCROPO(OH), wherein R is a C1-C2 straight or triphosphate, n-butyl 2',3'-dideoxythymidine-triphosphate, branched chain alkyl, and may include but is not limited to and n-butyl 2'-deoxyuridine-triphosphate; and carboxy monoalkyl phosphates selected from the group isopropyl uridine-triphosphate, isopropyl xanthosine consisting of phosphoglycolic acid and phosphopropionic triphosphate, isopropyl inosine-triphosphate, isopropyl acid. The alkenyl phosphate has the formula ROPO(OH) guanosine-triphosphate, isopropyl cytosine-triphosphate, wherein R is a C2-C20 straight or branched chain alkenyl, isopropyl a denosine-triphosphate, isopropyl and may include but is not limited to alkenyl phosphates 2'-deoxythymidine-triphosphate, isopropyl 2',3'- selected from the group consisting of allyl phosphate, crotyl dide oxythymidine-triphosphate, and isopropyl phosphate, dimethylally phosphate, isopentenyl phosphate, 10 2'-deoxyuridine-triphosphate. geranyl phosphate, farnesyl phosphate, geranylgeranyl The y-alkenyl nucleoside triphosphate or Y-alkenyl deoxy phosphate, 3-methyl-2-pentenylphosphate, and 2-methyl-2- nucleoside triphosphate each has the formula R-NTP or R-dNTP wherein R is a C2-C20 straight or branched chain hexenyl phosphate. Preferably, the alkenyl phosphate is alkenyl, Nis a nucleoside selected from the group consisting dimethylallyl phosphate. of uridine, Xanthosine, inosine, guanosine, cytosine, and The monoalkyl pyrophosphate has the formula ROPO 5 adenosine, dN is a deoxynucleoside selected from the group (OH)OPO(OH), wherein Ris a C1-C4 straight or branched consisting of 2'-deoxythymidine, 2',3'-dideoxythymidine, chain alkyl, and may include but is not limited to methyl and 2'-deoxyuridine, and NP is triphosphate. pyrophosphate, monoethyl pyrophosphate, n-propyl The Y-monoalkyl nucleoside triphoshates or Y-monoalkyl pyrophosphate, n-butyl pyrophosphate and isopropyl pyro deoxynucleoside triphosphates may include but are not phosphate. Preferably, the monoalkylpyrophosphate is ethyl 20 limited to: pyrophosphate. allyl uridine-triphosphate, allyl Xanthosine-triphosphate, The alkenyl pyrophosphate has the formula ROPO(OH) ally inosine-triphosphate, allyl guanosine-triphosphate, OPO(OH), wherein R is a C2-C20 straight or branched allyl cytosine-triphosphate, allyl adenosine-triphosphate, chain alkenyl, and may include but is not limited to allyl allyl 2'-deoxythymidine-triphosphate, allyl 2',3'- pyrophosphate, crotyl pyrophosphate, dimethylallyl 25 dideoxythymidine-triphosphate, and allyl 2'-deoxyuridine pyrophosphate, isopentenyl pyrophosphate, geranyl triphosphate; pyrophosphate, farnesyl pyrophosphate, geranylgeranyl crotyl uridine-triphosphate, crotyl Xanthosine pyrophosphate, 3-methyl-2-pentenyl pyrophosphate, and triphosphate, crotyl inosine-triphosphate, croty guanosine 3-methyl-2-hexenyl pyrophosphate. Preferably, the alkenyl triphosphate, crotyl cytosine-triphosphate, crotyladenosine pyrophosphate is isopentenyl pyrophosphate or 3-methyl-2- 30 triphosphate, crotyl 2-deoxythymidine-triphosphate, crotyl hexenyl pyrophosphate. 2',3'-dideoxythymidine-triphosphate, and crotyl The Y-monoalkyl nucleoside triphosphate or Y-monoalkyl 2'-deoxyuridine-triphosphate; nucleoside deoxynucleoside triphosphate each has the for dimethylallyl uridine-triphosphate, dimethylallyl mula R-NTP or R-dNTP, respectively, wherein Ris a C1-C4 xanthosine-triphosphate, dimethylallyl inosine straight or branched chain alkyi, N is a nucleoside selected 35 triphosphate, dimethylally guanosine-triphosphate, dim from the group consisting of uridine, xanthosine, inosine, ethylallyl cytosine-triphosphate, dimethylallyl adenosine guanosine, cytosine, and adenosine, dN is a deoxynucleo triphosphate, dimethylallyl 2-deoxythymidine-triphosphate, side selected from the group consisting of dimethylallyl 2',3'-dideoxythymidine-triphosphate, and 2'-deoxythymidine, 2',3'-dideoxythymidine, and dimethylallyl 2-deoxyuridine-triphosphate; 2'-deoxyuridine, and NP is triphosphate. 40 isopentenyl uridine-triphosphate, isopentenylxanthosine The Y-monoalkyl nucleoside triphoshates or Y-monoalkyl triphosphate, isopentenylinosine-triphosphate, isopentenyl deoxynucleoside triphosphates may include but are not guanosine-triphosphate, isopentenyl cytosine-triphosphate, limited to: is opentenyl adenosine-triphosphate, isopentenyl methyl uridine-triphosphate, methyl Xanthosine 2'-deoxythymidine-triphosphate, isopentenyl 2',3'- triphosphate, methyl inosine-triphosphate, methyl 45 dideoxythymidine-triphosphate, and isopentenyl guanosine-triphosphate, methyl cytosine-triphosphate, 2'-deoxyuridine-triphosphate; methyl adenosine-triphosphate, methyl 2-deoxythymidine geranyl uridine-triphosphate, geranyl Xanthosine triphosphate, methyl 2',3'-dideoxythymidine-triphosphate, triphosphate, geranyl inosine-triphosphate, geranyl and methyl 2'-deoxyuridine-triphosphate; 50 guanosine-triphosphate, geranyl cytosine-triphosphate, monoethyl uridine-triphosphate, monoethyl xanthosine geranyladenosine-triphosphate, geranyl 2'-deoxythymidine triphosphate, monoethyl inosine-triphosphate, monoethyl triphosphate, geranyl 2',3'-dideoxythymidine-triphosphate, guanosine-triphosphate, monoethyl cytosine-triphosphate, and geranyi 2'-deoxyuridine-triphosphate; monoethyl adenosine-triphosphate, monoethyl farnesyl uridine-triphosphate, farnesyl xanthosine 2'-deoxythymidine-triphosphate, monoethyl 2',3'- 55 triphosphate, farnesyl inosine-triphosphate, farnesyl dideoxythymidine-triphosphate, and monoethyl guanosine-triphosphate, farnesyl cytosine-triphosphate, far 2'-deoxyuridine-triphosphate; nesyl adenosine-triphosphate, farnesyl 2'-deoxythymidine n-propyl uridine-triphosphate, n-propyl xanthosine triphosphate, farnesyl 2',3'-dideoxythymidine-triphosphate, triphosphate, n-propyl inosine-triphosphate, n-propyl and farneysl 2'-deoxyuridine-triphosphate; guanosine-triphosphate, n-propyl cytosine-triphosphate, 60 geranylgeranyl uridine-triphosphate, geranylgeranyl n-propyl adenosine-triphosphate, n-propyl Xanthosine-triphosphate, geranylgeranyl inosine 2'-deoxythymidine-triphosphate, n-propyl 2',3'- triphosphate, geranylgeranyl guanosine-triphosphate, gera dideoxythymidine-triphosphate, and n-propyl nylgeranyl cytosine-triphosphate, geranylgeranyl 2'-deoxyuridine-triphosphate; adenosine-triphosphate, geranylgeranyl 2'-deoxythymidine n-butyl uridine-triphosphate, n-butyl Xanthosine 65 triphosphate, geranylgeranyl 2',3'-dideoxythymidine triphosphate, n-butyl inosine-triphosphate, n-butyl triphosphate, and geranylgeranyl 2'-deoxyuridine guanosine-triphosphate, n-butyl cytosine-triphosphate, triphosphate; 5,639,653 7 8 3-methyl-2-pentenyl uridine-triphosphate, 3-methyl-2- irradiated allogenic PBMC per flat or round bottom well of pentenyl xanthosine-triphosphate, 3-methyl-2-pentenyl a 96-well plate. M. tuberculosis antigen was prepared by inosine-triphosphate, 3-methyl-2-pentenyl guanosine suspending 100mg of heat killed H37Ra (Difco) with 10 ml triphosphate, 3-methyl-2-pentenyl cytosine-triphosphate, of distilled water followed by sonication. For some 3-methyl-2-pentenyl adenosine-triphosphate, 3-methyl-2- experiments, crude reaction mixtures of alkyl phosphates pentenyl 2'-deoxythymidine-triphosphate, 3-methyl-2- were diluted to 1:200 for use. After 24-42 hours, the cells pentenyl 2',3'-dideoxythymidine-triphosphate, and were pulsed with H) (1 ICi/well; 1 Ci=37 GBq), 3-methyl-2-pentenyl 2-deoxyuridine-triphosphate; and harvested at 48 hours, and counted by liquid Scintilation. 2-methyl-2-hexenyl uridine-triphosphate, 2-methyl-2- For cytotoxicity assays, primary lymphocytes expanded for hexenyl xanthosine-triphosphate, 2-methyl-2-hexenyl 7 days with MEP (30 M) were added to Daudi or K562 inosine-triphosphate, 2-methyl-2-hexenyl guanosine 10 cells (2x10) labeled for 90 min with 100 uCi of 'Cr, and triphosphate, 2-methyl-2-hexenyl cytosine-triphosphate, chromium release was measured at 6 h. 2-methyl-2-hexenyl adenosine-triphosphate, 2-methyl-2- MEP-Induced Expansion and Flow Cytometric Analysis of hexenyl 2'-deoxythymidine-triphosphate, 2-methyl-2- yö T cells. PBMC from healthy donors were cultured at hexenyl 2',3'-dideoxythymidine-triphosphate, and 2-methyl 1.2x10 ml in Yssel's medium together with 30 IMMEP or 2-hexenyl 2-deoxyuridine-triphosphate. 15 M. tuberculosis lysate (1:30 dilution). On day 7, viable cells The present invention is described in the following were separated by Ficoll-Hypaque and then stained with Experimental Details Section which is set forth to aid in the antibodies and analyzed. Antibodies used included: understanding of the invention, and should not be construed phycoerythrin-conjugated anti-TCR Ylö-1, fluorescein to limit in any way the invention as defined in the claims isothiocyanate-conjugated anti-CD3 (Becton Dickinson), which follow thereafter. 20 anti-V82 chain (BB3), anti-Vy2 chain, and fluorescein Experimental Details Section isothiocyanate-conjugated-anti-mouse IgG antibody (Ortho I. Monoalkylphosphates Diagnostics System Laboratories, Webster, Tex.). Stained Preparation of Monoalkylphosphates. Alkyl phosphates cells were analyzed on a FACSCAN analyzer (Becton were prepared by reacting 1 gram of phosphorus pentoxide Dickinson). Controls with isotype-matched antibodies (Fluka) with 1 ml of the appropriate alcohol (provided 25 established the quadrants such that over 99% of cells are in below), as described in Kosolapoff, et al. Organophospho the double-negative region. rous Compounds, John Wiley & Sons, New York, pp. MEP and Other Monoalkyl Phosphates Stimulate yöT Cells. 220-277 (1950). The reaction was carried out by heating at Various alkyl phosphates were tested for their ability to 65° C. for 9 hours. 0.2 ml of water was added at 7 hours of stimulate the proliferation of the yöT cell clone, DG.SF68, the reaction. To obtain the purified alkyl phosphate, the 30 which expresses Vy2V82. The results are presented in FIG. reaction mixture was applied to an anion exchange HPLC 1A. All alkyl phosphates tested with four or fewer carbons (Dionex) using sodium hydroxide and sodium acetate as induced proliferation. One compound, MEP, was 50- to eluates. The eluted fraction was desalted by gel filtration 100-fold more bioactive than the other compounds. YöT cell chromatography using a BIOGEL P2 column (Biolad). To clones proliferated in a dose-dependent manner to HPLC verify the identity of the compound, fast atom bombardment 35 purified MEP, reaching half maximal proliferation at 2 M, mass spectral analyses were performed on a Finnigran but failed to proliferate when exposed to the diethyl or MAT-90 mass spectrometer (FAB-MS) in the negative ion triethyl derivatives (see FIG. 1B). Neither of T-cell clones mode. Diethyl phosphate (DEP) and triethyl phosphate (FIG. 3A) nor NK clones (data not shown) proliferated in (TEP) were purchased from KODAK and Aldrich, respec response to MEP. Modification of C-2 by addition of a tively. B-hydroxyethyl-phosphate was prepared from ethyl 40 hydroxy group or by conversion to a carboxyl group reduced ene oxide by the action of aqueous disodium hydrogen the specific activity of MEP by 100- to 200-fold (see FIG. phosphate as described in Kosolapoff, et al., supra, and 1C), whereas the addition of an amino group totally abol purified by anion-exchange chromatography (QAE-A25, ished biological activity (data not shown). Common three Pharmacia). Phosphoglycolic was purchased from Sigma. carbon metabolites such as B-glycerophosphate, The alkyl phosphates also may be synthesized using the 45 2-phosphoglycerate, 3-phosphoglycerate, and phospho procedures described in Popjak, G., et al. J. Biol. Chem. enolpyruvate were also inactive (data not shown). 237:56-61 (1962). The Popjak, G., et al. procedures are MEP Selectively Activates the Vy2/V82+ Subset of yöT cell preferred. Cells Through Cognate TCR Recognition. Previously, it has Derivation/Maintenance of T Cell Clones. All T cell clones been shown that ligand(s) of M. tuberculosis and other were derived by limiting dilution. DG clones were derived 50 mycobacterial species predominantly induce the expansion from the synovial fluid of a rheumatoid arthritis patient by of y&T cells from primary cultures of freshly isolated PBMC stimulation with M. tuberculosis. The 12G12 yoT cell clone (Modlin, R. L., et al. Nature (London).339:544-548 (1989); was derived from M. tuberculosis H37Ra-stimulated Ficoll/ Kabelitz, D., et al. J. Exp. Med. 171:667-679 (1990); Hypaque-purified peripheral blood mononuclear cells Panchamoorthy, G., et al. J. Immunol. 147:3360–3369 (PBMC) of a patient with tuberculoid leprosy, yö clones 55 (1991); De Libero, G., et al. J. Exp. Med. 173:1311-1322 SD9, 10E12, 4H1, SE3, S58, PS7, 4B6, 24B1,5C, JF1, and (1991); and Hacker, G., et al. Infect. Immun. 60:2753-2757 OBT cell clones were derived from M. tuberculosis stimu (1992)). A similar selective expansion of y& T cells was lated PBMC of a normal donor. YöT clones, HF.2, HD.109, observed after a 7 day period of exposure of PBMC from all CP2B2, HF.15, and HF41 were derived from PMBC with healthy donors tested (12 individuals) to MEP (FIG. 2A). phytohemagglutinin (PHA) (Morita, C. T., et al. Eur: J. To determine if MEP expands the same Vy2/V82" subset Immunol. 21:2999-3007 (1991); Spits, H., et al. J. Immunol. of y&T cells that is stimulated by mycobacteria, primary 8 147:1180-1188 (1991)), and yö T clones, T7A5, T7C6, T cells from normalindividuals were expanded by MEP, and T7C7, T5B9, MD16, MD21, MD22, and MD26 were simi their V gene expression was determined by two-color larly derived (Casorati, G., et al. J. Exp. Med. fluorescence-activated cell sorting (FACS) analysis. The 170:1521-1535 (1989)). 65 expanded Yö T cell population was restricted to those that Proliferation and Cytotoxic Assays. Assays were performed expressed the V82 gene (FIG. 2B); no expansion of Vö1 in triplicate using 4-10x10'yo or OBT cells plus 5-10x10' expressing yo T cells was detected. 5,639,653 To define the V gene requirement for activation further, a panel of y& T cell clones expressing a variety of Y and 8V TABLE 2 genes was screened for proliferation to MEP. Strikingly, responsiveness to MEP was restricted to clones expressing 8T Cells (% T Cells Vy2 in conjunction with V82 (FIG. 3A). Clones expressing Vy2 paired with V81 or clones expressing V82 paired with DONOR CONTROL MONOETHYTL PHOSPHATE VY genes other than Vy2 were not reactive to either MEP B.B. 5 35 P.S. 2 33 (FIG. 3A) or M. tuberculosis lysates (data not shown). PR. 8 72 The proliferation of y& T cell clones to MEP was abol A.B. 4. 42 ished by inclusion of anti-TCR-6 antibodies (Table 1 below), 10 which, together with the V gene specificity, suggests that the Healthy donors whose ages range from 35 to 53. yöTCR is directly involved in the recognition of MEP. An AIDS patient of 32 years old who has been treated with AZT for 9 months and whose CD418 ratio was 0.85. TABLE 1. II. Purification of Natural Mycobacterial Antigens 15 Mycobacteria M. smegmatis was cultured in Middlebrook The Monoethyl Phosphate (MEP) Response 7H9 broth (Difco) with 0.5% glycerol and 0.05%TWEEN is Blocked by Antibody to the 80 at 37° C. for 2 days, and the culture filtrate was applied Gamma Delta T Cell Receptor (TCR) to charcoal-Celite column chromatography (2.5x1.5 cm, Proliferative Fisher). The mycobacteria were again cultured in the clear Responses (cpm) eluate supplemented with 7H9, glycerol and TWEEN 80 20 under the same condition. This procedure was repeated 9 MEP Antibody SD9 12G12 times and 7 million units were obtained. One unit is defined None 1075 151 as the antigen concentration required for half-maximal pro -- None 9955 4578 liferative responses of yö T cell clone 12612, derived from -- Control mouse IgG 980 4760 the lymphocytes of a tuberculoid leprosy patient. To the Anti-MHC Class 6827 37.82 25 solution, 5 volumes of methanoi were added, and the clear Anti-MHC Class I 1018.7 5347 Supernatant was evaporated to dryness under the reduced -- Anti-TCR (V82) 929 95 pressure at room temperature. The residue was rehydrated in 1 L H2O and passed through a collodion membrane yö clones SD9 and 12G12 were stimulated with monoet (Schleicher-Schuell). hyl phosphate (MEP) (30 M) in the absence of antigen 30 The filtrate was aliquoted to 40 portions and applied to Q presenting cells and in the presence of the indicated anti SEPHAROSE HP column chromatography (2.5x8 cm) with body. Antibodies used were mouse IgG1 plus IgG2a (2 0-500 mM triethylammonium bicarbonate buffer, pH 7.5, ug/ml, Zymed) as a control, anti-HLA-A, B, C, E (W6/32, respectively. Active fractions from 40 column chromatog 1:1000; Sera-Lab. Crawley Down, Sussex, U.K.), anti raphy runs as described in the description to FIG. 4A were HLA-DR (MID3, 2 ug/ml; Sera-Lab), and anti-V82 (BB3, 35 combined and applied to cellulose (Sigma) flash column 1:2000). All antibodies were dialyzed to remove sodium chromatography with isopropanol:conc. NH4OH:HO azide before assay. The proliferative responses to MEP in the (7:2:1, viviv) to remove contaminants, then the presence of irradiated allogeneic PBMC was 947 (-MEP) active fractions were rechromatographed by Q Sepharose and 10430 (MEP) for SD9, and 204 (-MEP) and 6841 HP (2.5x8 cm). M. tuberculosis H37Ra (0.6 g. Difco) (MEP) for 12G12. Results shown are representative of 40 ligands were extracted with HO by sonication in a water three separate experiments, and are means of triplicate bath overnight at 4° C. and applied to charcoal-Celite values, with standard errors of less than 10%. column chromatography (2.5X1.5 cm). Vy2/V62-Bearing T Cells Expanded in Culture by The eluate was passed through a collodion membrane and Monoalkyl Phosphate Lyse Tumor Cells. As shown in FIG. applied to Q SEPHAROSE HP column chromatography 3B, MEP-stimulated Vy2/V62-bearing T cells have the 45 (2.5x8 cm) with 0-500mM triethylammonium bicarbonate capacity to lyse human transformed hematopoietic cells as buffer, pH 7.5. For phosphate assay (Ames, B. N. Methods illustrated for Daudi and K562 cells. Thus, monoalkyl Enzymol. 8:115-118 (1966)), 10 L samples were used in phosphate compounds appear to act as antigens or Superan FIGS.5A, 5B, and 5C, and 0.5,0.01 or 2 L samples were tigens that stimulate Vy2/V82-bearing T cells through their used for the proliferation assay, respectively. Proliferation TCR in a manner similar to that of mycobacterial ligands. 50 assays were performed in triplicate using 5x10" cloned yöT Monoalkyl Phosphate Expands yöT cells in Culture of HIV cells per round bottom well of a 96-well plate. After 36 Infected Peripheral Blood. The expansion of y&T cells from hours, the cells were pulsed with methyl-H) thymidine (1 primary in vitro culture of peripheral blood cells of three uCi/well), harvested at 48 hours, and counted by liquid healthy individuals and one HIV positive individual by Scintilation. monoethyl phosphate was examined, and the results are 55 Mass spectral analysis was performed on a AP-III triple presented in Table 2 below. yö T cells in peripheral blood quadrupole mass spectrometer (PE-SCIEX, Ontario, ranged from 2-10% of T cells. However, when cultivated in Canada) using the SCIEX-IonSpray interface with nitrogen vitro with monoethyl phosphate, they greatly increased in as the nebulizer gas. Flow injection analysis using 100% number up to 72%. Surprisingly, the effect of monoethyl methanol plus 0.1% trifluoroacetic acid at 10 L/min was phosphate was obtained on peripheral yo T cells which did 60 used to introduce the samples into the mass spectrometer. not have the CD4 molecule, which is the receptor for HIV. Collisional activated dissociation (CAD) was performed yö T cells remained sensitive to monoethyl phosphate in using argon as the collision gas at a collision gas thickness AIDS patients and may therefore functionally compensate of 1.7x10" molecules/cm and a collision energy of 30 eV. for the loss of CD4+ a? T cells that undergo decrease in Under these CAD conditions, parent ion scans of 79 amu number or paralysis from HTV infection. Increasing yo T 65 and daughterion spectra of the selected parention, m/z.245, cells from all of the subjects were of V8 positive subset and were obtained. Samples were diluted 1:50 using 0.2% of CD4-CD8-. NH4OH and 10 Laliquot was injected. 5,639,653 11 12 Of the three peaks shown in FIG. 5A, the 339 ion was identified as a hexose diphosphate (immunologically TABLE 3 inactive), and the 245 and 275 ions were identified as The Minimal Ligand Concentration (M) isopentenyl pyrophosphate and its hydroxymethyl Required for Half-Maximal Proliferative derivative, possibly derived from 5-diphosphomevalonic Response of 8 T cell Clone 12G12 acid, respectively, based on the daughter ion analysis (data Anchor not shown) and synthetic data. The 275 ion was the major Chain Structure Pit PPs Up pyrophosphorylated ion detected in a highly purified non Methyl- CH- 1800 8 15 peptide antigen preparation from M. fortutum (data not O Ethyl- CHCH- 240 5 3 shown), suggesting that this compound is also biologically 1-Propyl- CHCHCH- 1200 12 12 2-Propyl- (CH),CH- 1600 90 w active. To confirm that isopentenyl pyrophosphate is the 1-Butyl- CHCH2CHCH- 2000 100 bioactive molecule, the compound was synthesized as Allyl- CH=CHCH- 800 5 4 described below, and tested for its ability to stimulate Crotyl- CHCH=CHCH- 350 8 3 15 Dimethy- (CH3)C=CHCH- 30 10 0.3 Vy2V82-bearing T cells. The results establish that the iso allyl pentenyl pyrophosphate and related prenyl pyrophosphates Iso- CH=C(CH)CHCH- TOO 3 4. pentenyl existin mycobacteria as naturalligands for human VY2V82 Geranyl- H(CHC(CH)=CHCH)- 800 10 8 bearing T cells. Farnesyl- HCCHC(CH)=CHCH)- - 10 w Geranyl- H(CHC(CH)=CHCH)- - 10 o III. Synthesis of Phosphate, Pyrophosphate, NTP, and dNTP 20 geranyl Derivatives 3-Methyl- CHCHC(CH)=CHCH- --- 8 w 2-pentenyl Alkyl- and Alkenyl- Alcohols. The following alkyl- and 3-Methyl- CHCHCHC(CH)=CHCH- 0.3 - alkenyl- alcohols were obtained from Aldrich Chemical 2-hexenyl Company: ethanol, methanol, 1-propyl alcohol, 1-butyl alcohol, allyl alcohol, crotyl alcohol, dimethylallyl alcohol, 25 *P is phosphate and PP is pyrophosphate. isopentenyl alcohol, geraniol, farnesol, and geranylgeranyl alcohol. The 3 methyl-2-pentenyl and 3-methyl-2-hexenyl TABLE 4 alcohols were synthesized as described in Ogura, K., et al. The Minimal Ligand Concentration (IM) J. Am. Chem. Soc. 92:20-25 (1970). 30 Required for Half-Maximal Proliferative Alkyl and Alkenyl Derivatives of Phosphate and Pyrophos Response of 6 T cell Clone 12G12 phate. Dimethylallyl pyrophosphate, isopentenyl Ethyl Derivatives pyrophosphate, geranyl pyrophosphate, farnesyl of diniTP and NTP Concentration (M) pyrophosphate, and geranylgeranyl pyrophosphate were 35 2.TP 3 obtained from Aldrich Chemical Company. The alkyl and alkenyl derivatives of phosphate and pyrophosphate may 2. duP 3 UTP 3 also be prepared by allowing ditriethylammonium pyro XTP 4 phosphate to react with the indicated alcohol in the presence IP 2O 40 GTP 20 of trichloro-acetonitrile as described in Popjak, G., et al. J. CTP 2O Biol. Chem, 237:56-61 (1962), and purified by Q ATP 200 SEPHAROSE HP column chromatography (2.5x8 cm) with 0-500 mM triethylammonium bicarbonate buffer, pH 7.5. All publications mentioned hereinabove are hereby incor NTP and dNTP. (UTP), xanthosine 45 porated in their entirety. triphosphate (XTP), (ITP), guanosine While the foregoing invention has been described in some triphosphate (GTP), cytosine triphosphate (CTP), adenosine detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of the triphosphate (ATP), 2'-deoxythymidine triphosphate (2'- disclosure that various changes in form and detail can be dTTP), 2',3'-dideoxythymidine triphosphate (2',3'-ddTTP), made without departing from the true scope of the invention and 2'-deoxyuridine triphosphate (2'-dUTP) were obtained 50 in the appended claims. from Aldrich Chemical Company, What is claimed is: 1. A method for stimulating the proliferation of Vy2V82 NTP and dNTPY-Derivatives. NTP and dNTPY-Derivatives T cells in a mammal comprising administering to the man were prepared by allowing the triethylammonium salt of the mala Vy2V52T cell proliferation stimulating amount of a indicated NTP or dNTP to react with the corresponding 55 monoalkyl- or alkenyl-pyrophosphate having the formula alcohol as described in Eckstein, F, et al. Biochemistry ROPO(OH)OPO(OH) wherein R is a C1-C4 straight or 23:5225-5232 (1975) and Knorre, D. G., et al. FEBS Letters branched chain alkyl, or a C2-C20 straight or branched 70:105-108 (1976), and purified by anion exchange chro chain alkenyl. matography on a Q SEPHAROSE HP column (2.5x8 cm) 2. The method of claim 1, wherein the monoalkyl pyro 60 phosphate is selected from the group consisting of methyl with 0-500mM triethylammonium bicarbonate buffer, pH pyrophosphate, ethyl pyrophosphate, n-propy 7.5. pyrophosphate, n-butyl pyrophosphate and isopropyl pyro IV. Testing of Compounds phosphate. The compounds synthesized as described above were 3. The method of claim 2, wherein the monoalkyl pyro tested for their ability to stimulate Vy2V82-bearing T cells. 65 phosphate is ethyl pyrophosphate. The results are presented in FIGS. 5D, 6A, 6B, 6C, and 4. The method of claim 1, wherein the alkenyl pyrophos Tables 3 and 4 below. phate is selected from the group consisting of allyl 5,639,653 13 14 pyrophosphate, crotyl pyrophosphate, dimethylallyl 10. The method of claim 9, wherein the monoalkyl pyrophosphate, isopentenyl pyrophosphate, geranyl pyrophosphate is selected from the group consisting of pyrophosphate, farnesyl pyrophosphate, geranylgeranyl methyl pyrophosphate, ethyl pyrophosphate, n-propyl pyrophosphate, 3-methyl-2-pentenyl pyrophosphate, and pyrophosphate, n-butyl pyrophosphate and isopropyl pyro 3-methyl-2-hexenyl pyrophosphate. phosphate. 5. The method of claim 4, wherein the alkenyl pyrophos 11. The method of claim 10, wherein the monoalkyl phate is 3-methyl-2-hexenyl pyrophosphate. pyrophosphate is ethyl pyrophosphate. 6. The method of claim 1, wherein the mammal has a 12. The method of claim 9, wherein the alkenyl pyro disease expressing antigens recognized by Vy2V82T cells. phosphate is selected from the group consisting of allyl 7. The method of claim 6, wherein the disease is selected O pyrophosphate, crotyl pyrophosphate, dimethylallyl from the group consisting of leukemia, lymphoma, pyrophosphate, isopentenyl pyrophosphate, geranyl tuberculosis, leprosy, malaria, AIDS, rheumatoid arthritis, pyrophosphate, farnesyl pyrophosphate, geranylgeranyl ulcerative colitis and anemia. pyrophosphate, 3-methyl-2-pentenyl pyrophosphate, and 8. The method of claim. 6, wherein the route of adminis 3-methyl-2-hexenyl pyrophosphate. tration is oral, intravenous, intramuscular, Subcutaneous or 13. The method of claim 12, wherein the alkenyl pyro intraperitoneal administration. phosphate is 3-methyl-2-hexenyl pyrophosphate. 9. A method for stimulating the proliferation of Vy2V62 14. The method of claim 9, wherein the mammal has a T cells in a mammal comprising administering to the mam disease expressing antigens recognized by Vy2V82T cells. mal biological fluid from the mammal that has been treated 15. The method of claim 14, wherein the disease is with a Vy2V82T cell proliferation stimulating amount of a 20 selected from the group consisting of leukemia, lymphoma, monoalkyl- or alkenyl-pyrophosphate having the formula tuberculosis, leprosy, malaria, AIDS, rheumatoid arthritis, ROPO(OH)OPO(OH) wherein R is a C1-C4 straight or ulcerative colitis and anemia. branched chain alkyl, or a C2-C20 straight or branched chain alkenyl. ck :: * : *k