ENHANCEMENT OF THE CELLULAR IMMUNE RESPONSE

Abstract: DTH-Effector cells are primed with carbohydrate antigens and used to enhance the cellular immune response. Tumors have been inhibited by DTH-Effector cells primed with epiglycanin and with synthetic T and Tn antigens. Either the DTH-Effector cells, or these tumour-associated carbohydrate antigens directly, may be used for tumor prophylaxis and therapy.

Description: I Enhancement of the Cellular Immune Response BACKGROUND OF THE INVENTION Vertebrates have two basic immune responses: humoral cellular. Humoral immunity is provided by the special cl of cells produced by B lymphocytes. These cells prod antibodies which circulate in the blood and lymphatic flu On the other hand, cell mediated immunity is provided by T cells of the lymphatic system. The cellular immune response is particularly effect against fungi, parasites, intracellular viral infectio cancer cells and foreign matter, whereas the humoral respo primarily defends against the extracellular phases bacterial and viral infections. Containment of antigen at its point of entry accomplished by walling off the area by local inflammati Acute inflammation is characterized by the influx of pla proteins and polymorphonuclear leukocytes. Chro inflammation is characterized by the infiltration of lymphocytes and macrophages. When acute (antibody induc and chronic (T-cell induced) inflammations occur in the sk they are called immediate and delayed type hypersensitiv reactions respectively. ITH peaks at 24 hours, and subsi in 48 hours DTH appears in 24-48 hours and peaks at 48 hours. The subset of T cells involved in DTH reactions called here DTH-Effector cells. Epiglycanin (epi) is the major cell surface glycoprot produced by the mammary adenocarcinoma transplantable c line TA3Ha. Friberg, Jr., J.N.C.I., 48:1463 (197 Codington, et al., Cane. Res., 43:4373 (1983). The TA carcinoma cells are covered by a mucin-like glycoca composed mainly of epiglycanin. Codington, et al., J.N.C. 60:811 (1978); Miller, et al., J.N.C.I., 68:981 (1982). is mainly carbohydrate in composition, and expresses multi T and Tn determinants. T and Tn are general carcinom autoantigens. Springer, Science, 224:1198 (1984). Synthetic T and Tn antigens have been prepared, and use in DTH diagnostics. Le ieux, Ep Appl. 44,188. However, their therapeutic or prophylactic use has not been disclosed. Bretscher, Eur. J. Immunol., 9:311-316 (1979) culture DTH-Effector cells in vitro and injected them with th sensitizing antigen (burro erythrocytes) into the foot pad of mice. We have used cancer-associated, well characterize carbohydrate antigens to sensitize effector cells and hav taught a therapeutic use for them. It is known that tumors may be treated with a mixture o IL-2 and IL-2-activated killer cells. See Fortune, 16-2 (November 25, 1985) , reporting on the work of Steve Rosenberg. The problem with the Rosenberg work is that th therapy induces a population of cells known as LA (lymphokine activated killer) cells. LAK cells are no antigen-specific; therefore normal tissue may be attacked. Also, since the LAK cells are non-specific, relatively larg doses of IL-2 are required to induce the required anti-tumo activity. Finally, . LAK cells are not thought to confe memory to the immune system. In contrast, DTH effector cell are antigen specific; therefore after stimulation muc smaller quantities of lymphokine may be required to induc proliferation. Also because of their specificity, DT effector cells are less likely to attack self. Moreover, DT effector cells are thought to have memory. SUMMARY OF THE INVENTION We have found that the cell-mediated immune response t cancer cells is enhanced by parenteral administration o natural and synthetic cancer-associated carbohydrate antigen or by parenteral administration of DTH effector cells prime by such antigens. This enhancement is likely to b advantageous for both therapy and prophylaxis when the bod primary defense against a challenge is a cellular imm response, e.g., against tumors. The use of other speci carbohydrate antigens, including polysaccharides, glycolip and glycoproteins, may be useful in protecting a subj against other threats controlled by T cells. It is known t parasites, viruses, bacteria and fungi bear surf carbohydrate determinants. Lymphokines may be used in conjunction with the speci antigens or primed DTH-Effector cells of this invention enhance the specific CMI esponse. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows that epi-primed splenocytes induce loc DTH swellings in the presence of epi, while unprimed sple cells, irradiated tumor cells, and primed cells in t absence of antigem do not. Fig. 2 shows that -this reaction is specific to canc cell lines beariag ftae p-rimer as a surface antigen. Fig. 3 shows that the reaction was with the T alpha, beta and Tn determinants of epi. Fig. 4 shows that the phenotype of the ant carbohydrate DTH effector cells is Thy-1+, Lyt-1+, Lyt-2-. Fig. 5 shows systemic transfer of DTH by a DTH reacti to irradiated tumor cells after IV administration Effectors. Fig. 6 shows iaϊ-M-b±t±oia of tumor growth after loc transfer of DTH-Effector cells "mixed with tumor cells. Fig. 7 shows itrie importance of the priming dose. Fig. 8 shows 1hat at lower doses of antigen the cellul response predominated. * DETAILED DESCRIPTION OF THE INVENTION Example 1 Use of S-TAGS to Enhance Cellular Immune Response t Tumors Synthetic tumor-associated glycoproteins (S-TAGS) an other carbohydrate antigens are known in the art and may b prepared by any convenient technique. T and Tn antigens ar preferred. For synthetic methods, see Kaifu and Osawa Carbohydr. Res., 58:235 (1977); Ratcliffe, et al. , 93:3 (1981); Paulsen, et al., 104:195 (1982); Bencomo and Sinay 116:69 (1983). Soluble S-Tags or their aggregates are mixed with a adjuvant for intradermal, intramuscular or intraperitonea administration. It is conceivable that intravenou administration will also be successful. Groups of five mice at a time were primed with specifi doses of T-alpha (TF) , T-beta (asialo-GMl disaccharide) an Tn S-Tags. After 7-10 days, they were challenged with 3xl0 live TA3Ha cells. Markedly increased survival was noted i mice receiving about 1 ug of synthetic antigen. Thus, for T beta, the following results were observed (Table I) : Table I Dose Survival (days) 0.2 ug 20, 27, 27, 29, 31 1.0 ug 27, 38, 43, 43, 49 2.0 ug 16, 21, 24, 30 10 ug 20, 21, 28, 33 20 ug 21, 21, 28, 28 50 ug 16, 18, 31 100 ug 20, 20, 21, 27, 29 PBS 23, 27, 29 Norm 13, 13, 18, 27, 28 It should be noted that TA3Ha is very deadly. normal post-transplantation life expectancy of a mouse only 15-20 days. Example 2 Use of Cultured DTH Effector Cells as Tumor Inhibitor We obtained mouse primed DTH effector cells by following procedure. CAF1/J (Balb x A/J) mice from animal unit were immunized with 30 ug Epi (or 2 ug of synthetic antigen), in 50% complete Freund's adjuva intraperitoneally, boosted one week later with a simil injection. 7-10 days after the last injection the anima were sacrificed, their spleens removed and passed through nylon mesh to make a single cell suspension. The cells we washed and cultured in a costar well at 1.5 x 10 7 cells/8 (RPMI + P.S. + 10% FCS)/well. Each well received 10 ug e (or 1-2 ug of any of the synthetic antigens) . The cells we cultured at 37' C and 5% CO2 for 6 days and then harvested gently dissociating them from the plastic with a rubb policeman. The primed cells were then tested for D reactivity with a number of antigens, and for their abili to inhibit the growth of TA3Ha cell line in vivo. To obtain human primed DTH Effector cells, we would u a modification of the method of Rosenberg. Lymphocytes a removed from blood of a living subject by plasmapheresi The lymphocytes are cultured with a specific antigen, such cancer-associated glycoprotein (epiglycanin or a T or Tn TAG) or the combination of a specific antigen with IL- rather than with IL-2 alone. The human lymphocytes a cultured in suitable media for 6 days at a preferr concentration of about 10 6 cells/ml. The cells are washed remove antigen. The cells may then be returned to t patient, intravenously, for therapeutic or prophylact purposes. We have demonstrated that co—injection of a mammal wit epiglycanin-primed DTH Effector cells and antigen (irradiate tumor cells) induced a DTH reaction (Figure 1) . Spleen cells from mice primed and boosted with 30 ug and 20 ug of ep extracted and purified from TA3Ha ascites fluid and CF (intraperitoneally administered one and two weeks beforehand) were cultured at 1.5 x 10 7 cells/well with: (a) 10 u epi/well and (b) no antigen. In addition, unprimed splee cells were cultured with (c) 10 ug epi/well and (d) n antigen. Cultures were harvested on the 6th day and 10 cells were injected subcutaneously into the foot pad o unimmunized mice together with 10 6 irradiated TA3Ha cells. As a control (e) , 10 6 irradiated TA3Ha cells were injecte alone. All the DTH values are presented as net DT swellings, i.e., [swelling of primed cells + Ag.] - [swellin of primed cells alone] . The large di ferential response demonstrated that immunization with a cancer mucin can induc a DTH reaction to those, cancer cells which have the mucin a the cell surface. Subsequently, we showed that this reaction was tumor specific (Fig 2) . A DTH effector cell population wa generated as in Example I and tested with a) TA3Ha; b) Epi M (epi linked to a sepharose microsphere) ; c) mKSA; and d no antigen. All the DTH values are presented as net DT swellings, i.e., [swelling of primed cells + Ag.] - [swellin of primed cells alone] . The result was that the immunizatio with the epi mucin induced a specific DTH response to th mucin and to the cells which bear the mucin (TA3Ha) but no against those that do not bear the mucin (mKSA) . We also explored the antigenic specificity of the DT reaction in greater detail (Fig. 3) . 10 7 T cells were prime with epi and were co-injected with a series of antigen (TA3Ha, Tn, T-alpha, T-beta, Epi, Fucose ("Fuc") , HSA, BSA sepharose microspheres, and no antigen) . All solubl antigens were linked to sepharose microspheres and used at 1 ug/foot pad except epi which was used at 50 ug/foot pad. DTH results are net DTH measurements. The experiment sh that the DTH measured by the injection of epi reacts with carbohydrate determinants on S-Tags which are also know to epi determinants but not those carbohydrates determinants represented on epi. (eg. Fucose) . SUMMARY OF SPECIFICITY OF DTH RESPONSE IMMUNIZING Aσ: TRIGGERING_Acr.. Epi: + + + + - - T a : + + + + - - T B : + + + + - - Tn: + + + + - - None: - - ND ND ND To confirm that the reactions we were witnessing w indeed DTH reactions we designed an experiment to check the cell surface phenotype of DTH effector cells. (Fig. An effector cell population was prepared as before treated with a) no treatment; b) anti-Thy 1.2 antibod plus complement; c) anti-Lyt 1.2 plus complement; d) an Lyt 2.2 plus complement; and e) complement alone. antigen alone was also used as a control. All the val presented are net DTH. Treatment of the DTH effector cel with anti-Thy or anti-Lyt-1 antibodies with complem eliminate the DTH effector cell activity but treatment wi anti-Lyt-2 plus complement did not eliminate the DTH effec cell activity; thus, the phenotype of the specific ant carbohydrate DTH effector cells is Thy-1+, Lyt-1+, Lyt-2" the phenotypic pattern shown by other DTH effector cel reacting against proteins. See Fujiwara, J. Immuno 135:2187 (September, 1985). The protocol for this comparison was as follows. primed cell population was obtained, washed, counted, an divided into separate tubes for treatment. The cells wer gently pelleted and resuspended in Leibowitz media containin 1) anti Thy 1.2 antibody (at 1/1000 diln., obtained fro NEN) , 2) anti Lyt 1.2 antibody (at 1/1000 diln., obtaine from NEN) or 3) anti Lyt 2.2 antibody (at 1/1000 diln., obtained from NEN) at 2 x 10 7 cells/ml., and incubated at 4' for 45 min. The cells were then spun down and resuspended i guinea pig complement (1/10 diln.) in Leibowitz media at 2 10 7 cells/ml., and incubated for 30 in. in a 37'C wate bath. After incubation, cells were washed with Leib + 10 FCS and counted. A comparative study was undertaken to determine whethe the reaction was of the delayed type. DTH Effector cell were injected together with TA3Ha and the reaction wa compared with reactions in other mice injected with only th DTH Effector Cells or only the TA3Ha. It was found that th reaction peaked in 24 hours and had disappeared by 96 hours No DTH (emphasis on delayed) was found with the two controls As further confirmation that the reaction observed was DTH, a mononuclear infiltrate was observed the site o swelling. The foregoing experiments all related to local transfe of DTH. We also investigated the possibility that DT Effector cells would have a systemic effect. DTH effector cells were administered intravenously t the host animal. Four hours later, irradiated TA3Ha cell were injected into the footpad. At 6, 24, 48 and 72 hours the DTH reaction was measured (open boxes) and compared wit a negative control (plusses (Fig. 5) . The irradiated tumor cells used in the above-describe experiments will not proliferate. In the next experiments however, untreated tumor cells were employed. DTH primed cells were induced as described above. 5x primed cells injected IV plus 2.5 x 10 5 live tumor cells w injected s.c. into the foot pad. The control mice w injected with normal spleen cells plus tumour cells. foot pad swelling was typically measured at 12, 24, 48, 72 hours and every two days thereafter. The determine component of the swelling which was due to a DTH reaction, opposed to a combination of DTH and tumour growth, mice w injected with primed or normal cells plus irradiated tu cells. The swelling was attributed only to the DTH reactio Tumor growth was measured every 2 days starting 48 hou after the peak DTH swelling. The DTH and the tumor size we measured using calipers in order to estimate the increase foot pad thickness. Fig. 6 shows that the primed cells inhibited the grow of the TA3Ha tumor. (Open boxes, primed DTH effector ce plans TA3Ha; pluses, normal cells plus TA3Ha. Other experiments have shown that the nature of t reaction to the antigen is dependent on the dose of prim administered to the DTH-Effector cells. At lower dose cell-mediated immunity was predominant, while at high doses, the reverse was true. A dose dependence was also found when splenocytes are primed with Tn in culture (Fig. 7) . (Rising shading b shows net DTH; Falling shading bar shows standard deviation We have further shown that the DTH effector cel stimulated by epi will "home in" on a tumor and inhibit tum growth. DTH-Effector cells were primed with epi previously described. One day prior to intravenous injecti of the epi-pri ed DTH effector cells, the TA3Ha tumor w transplanted into the footpads of mice. Mice with tumo transplanted at the same time were used as controls and tum growth was determined by measuring the welling of t footpads (in mm) with calipers. TSYPF.PTMEWT 1 Day 6 r post iv pay g r post jv Systemic Transfer 10, 12, 12, 10, 15 50, 15, 80, 80, 50 Effector cells Control Animals 140, 25, 35 190, 200, 220 (3 mice) EXPERIMENT 2 Systemic Transfer of DTH Effector cells (4 mice) 10,20,15,10 55,20,15,20 100,140,80,60 300,360,3 Control Animals (4 mice) ^0,75,60,110 170,160,170 340,300,320, 500(3mic 110 340 As may be seen from the above data-, the IV-administered, epi pr±med DTH-Effector cells exerted a tumor-inhibitory effec a£ "the footpad .site of tumor transplantation. ^ £x?gn l _3_ Use of Epiglycanin as a Tumor Inhibitor Epiglycanin (epi) was extracted from the ascites flui of TA3Ha tumor-bearing mice. 10 Ascites from outbrec. ~TS3Ηa ip tumors was collected an cells were removed by centrifugation. The samples wer stored frozen. Before extraction, the ascites was thawed an i-ncubated at 37" C for 2 hours, and any aggregates wer removed by centrifugation. The freezing, thawing an 15 clotting procedure was performed twice before PNA extraction 25 - 40 ml. bed volume of PNA agarose (E. Y Laboratories) was washed with PBS, mixed with ascites a tumbled gently at 4'C for 24 - 48 hours. The slurry w poured into a column and extensively washed with PBS at ro temperature. Epi was eluted from PNA agarose usi galactose. The eluted epi-galactose mixture was th dialyzed against water to remove the galactose. The samp was lyophilized and stored at -20'C. in dessicator. Groups of five mice at a time were primed with specif doses of epi. After 2-3 days they were challenged with 3 10 3 live TA3Ha cells. Table 2 below shows the relationsh between the priming dose and survival. Table 2 Data calculated on day 49 i.e. these mice are still alive These mice were sacrificed on dav 88 to continue th experiment. Thus, the preferred priming dose was 1 ug, and thi dosage appeared to confer protective immunity against TA3H cells. In another experiment, it was determined that th humoral response was negligible at that dosage (Fig. 8) , suggesting that the tumor was inhibited by a mechanism othe than antibodies, possibly CMI. Mice (CAF1/J) were injecte with the indicated doses of epi i.p. in 50% CFA. Ten day later they were challenged with irradiated TA3Ha cells t determine the DTH reactivity (closed boxes) . Antibody to epi (open circles) was determined by ELISA. A summary of our experiences with Epi and T-alpha in tumor inhibition is given in Table 3 below: Table 3: In Vivo Protection; Number of animals alive at 4 weeks. Dose of Ag Epi la. Glu-BSA/HSA 0.1 ug 1/20 4/15 0/10 0/5 ug 5/20 7/15 0/10 1.0 ug 8/20 5/15 0/10 5.0 ug 8/15 6/15 0/10 10.0 ug 7/20 3/15 0/10 cont. 0/20 0/15 0/10 Since Glu-BSA/HSA presents a carbohydrate which is not expressed on the surface of the TA3Ha tumor cells, administration of that substance did not provide protection. Epi, which is the predominant surface carbohydrate antigen on TA3Ha cells, and T-alpha, which is one of the Epi determinants, did provide protection.

Claims: CLAIMS : 1. A method of priming DTH effector cells to enhance thei immunological activity which comprises cultivating DTH effecto cells in a medium containing a priming amount of a specifi antigen bearing a carbohydrate determinant. 2. The method of claim 1 in which the determinant is tumor associated. 3. The method of claim 1 in which the antigen is a mucin. 4. The method of claim 3 in which the antigen is epiglycanin. 5. The method of claim 1 in which the antigen is a glycoprotein 6. The method of claim 1 in which the antigen is a glycolipid. 7. The method of claim 1 in which the antigen bears a T-alph determinant. 8. The method of claim 1 in which the antigen bears a T-bet determinant. 9. The method of claim 1 which the antigen bears a T determinant. 10. The method of claim 1 wherein the antigen comprises synthetic carbohydrate hapten conjugated to an immunologica carrier. 11. The method of claim 10 wherein the hapten bears a tumor associated carbohydrate determinant. 12. The method of claim 11 wherein the determinant is selected from the group consisting of T-alpha, T-beta and Tn determinants. 13. The method of claim 1 in which the antigen is a polysaccharide. 14. The method of claim 1 in which the medium further comprises a lymphokine. 15. The method of claim 14 in which the lymphokine is IL-2. 16. The method of claim 1 in which the antigen is an irradiated tumor cell. 17. An essentially biologically pure culture of primed DTH effector cells. 18. An essentially biologically pure culture of primed DTH effector cells, said cells having been primed by a method according to any of claims 1-16. 19. A therapeutic composition comprising an effective amount of primed DTH effector cells in a pharmaceutically acceptable carrier. 20. A therapeutic composition comprising an effective amount of DTH effector cells, primed by a method according to any of claims 1-16, in a pharmaceutically acceptable carrier. 21. A method of enhancing cellular immunity in a mammal which comprises administering the therapeutic composition of claims 19 or 20. 22. A composition for enhancing cellular immunity in a mamm which comprises an effective amount of a specific antigen beari a carbohydrate determinant in a pharmaceutically acceptab carrier. 23. The composition of claim 22 in which the determinant tumor associated. 24. The composition of claim 22 in which the antigen is a muci 25. The composition of claim 24 in which the mucin epiglycanin. 26. The composition of claim 22 in which the determinant is alpha. 27. The composition of claim 22 in which the determinant is beta. 28. The composition of claim 22 in which the determinant is Tn. 29. The composition of any of claims 26, 27 or 28 in which t antigen comprises a synthetic hapten conjugated to immunological carrier, and the hapten. 30. A method of enhancing cellular immunity which compris administering the therapeutic composition of any of claims 22-2

Inventors: HENNINGSSON CARINA, LONGENECKER BRYAN MICHAEL, HENNINGSSON, CARINA, LONGENECKER, BRYAN, MICHAEL
Applicants: HENNINGSSON CARINA, LONGENECKER BRYAN MICHAEL, HENNINGSSON, CARINA, LONGENECKER, BRYAN, MICHAEL
Application year: 1987
Main classification: A61K35
IPC codes: A61K35, A61K38, A61K39, A61K45, A61P35, A61P37, C07K14, C12N5, C12R1