WO1990011764A1 - Active specific immunotherapy of adenocarcinomas producing immunosuppressive mucins - Google Patents

Abstract

Adenocarcinomas display and circulate immunosuppresive mucins such as epiglycanin. Pretreatment with cyclophosphamide or other suppressor T cell-inhibitory agents enhances the specific immune response to a synthetic glycoconjugate presenting the T-alpha hapten. Tumor-bearing mice received this tumor vaccine formulation were able to achieve approximately 90 % long-term survival (> 90 days). When mice were treated with the same vaccine formulation without cyclophosphamide, 25 % survival for the same length of time was observed. Many surviving mice had good delayed-type hypersensitivity responsiveness to the synthetic conjugate and among them a few also had good IgG antibody titer against the same conjugate. Upon further intraperitoneal challenge with 1 x 104 live Ta3-Ha tumor cells, approximately 30 % of the surviving animals which had been pretreated with the complete vaccine formulation were still able to sustain long-term survival or complete care. Circulating immunosuppressive mucins may also be removed by pheresis.

Description

ACTIVE SPECIFIC IMMONOTHERAPY OF ADENOCARCINOMAS PRODUCING IMMUNOSUPPRESSIVE MUCINS

Cross-Reference to Related Application

We hereby cross-reference the related application of B. Michael ongenecker and Carina Henningsson, entitled ENHANCEMENT OF THE CELLULAR IMMUNE RESPONSE, Ser. No. 07/222,390, filed July 22, 1988, the entire text of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Active specific tumor immunotherapy is an attempt to antigenically stimulate the host's endogenous anti- tumor immunity. This is typically done by immunizing the host with whole cells or extracts obtained from the host's own tumors, from other patient's tumors, or from established tumor cell lines. The immunizing agent often includes a microbial or chemical adjuvant for nonspecifically stimulating the reticuloendothelial system.

Rather than using whole cells or their lysates, one may resort to purified tumor-associated antigens. While such agents are more specific than antigenically complex cells or lysates, they may induce a more equivocal immune response in that fewer antigenic determinants are presented. Since tumor cells are heterogeneous, and undergo ixαmunological changes with time, it is uncertain whether at the time of intervention that all tumor cells will express the immunizing antigen. Vertebrates have two basic immune responses: humoral or cellular. Humoral immunity is provided by the special class of cells referred to as B lymphocytes. These cells produce antibodies which circulate in the blood and lymphatic fluid. On the other hand, cell mediated immunity is provided by the T cells of the immune system.

The cellular immune response is particularly effective against fungi, parasites, intracellular viral infections, cancer cells and foreign matter, whereas the humoral response primarily defends against the extracellular phases of bacterial and viral infections.

Containment of antigen at its point of entry is accomplished by walling off the area by local inflammation. Acute inflammation is characterized by the influx of plasma proteins and polymorphonuclear leukocytes. Chronic inflammation is characterized by the infiltration of T-lymphocytes and macrophages. When acute (antibody induced) and chronic (T-cell induced) inflammations occur in the skin, they are called immediate and delayed type hypersensitivity reactions respectively. ITH peaks at 24 hours, and subsides in 48 hours; DTfl appears in 24-48 hours and peaks at 48-72 hours. The subset of T cells involved in DTH reactions are called here DTH-Effector cells and have the CD4⁺ phenotype.

T-lymphocytes can also differentiate into a subset of T cells which have cytotoxic activity. These T cells can destroy target cells either directly or through the secretion of cytotoxic factors. It is believed by some that another subset of T-cells have a εuppreεsive or regulatory role. (They may, of course. be the same subset of T cells, but differently activated) . Most cytotoxic T cells and suppressor T cells have the CD8⁺ phenotype. Suppression may be antigen-specific, and it may affect either or both limbs of the immune system.

Mucins are molecules which originate in the mucous membranes of mammals and 'are characterized by a molecular weight in excess of 150,000 daltons, a carbohydrate content in excess of 70%, and the capacity to form chemical bonds with water to provide a mucilaginous or lubricating fluid. Several mucins, such as epiglycanin, have been associated with adenocarcinomas.

While mucins may be purified for use in active specific tumor immunotherapy, an alternative is the preparation of a synthetic antigen: a conjugate of numerous tumor-associated carbohydrate hapten molecules with a suitable carrier molecule.

Epiglycanin (epi) is the major cell surface glycoprotein (500,000 daltons) produced by the mammary adenocarcinoma transplantable cell line TA3Ha. Friberg, Jr., J.N.C.I., 48:1463 (1972); Codington, et al.. Cane. Res., 43:4373 (1983). It should be noted that TA3Ha is very deadly. The normal post- transplantation life expectancy of a mouse is only 15- 20 days. Moreover, it has been reported that TA3Ha is imrounoresistant.

The TA3Ha carcinoma cells are covered by a ucin- like glycocalix composed mainly of epiglycanin.

Codington, et al., J.N.C.I., 60:811 (1978); Miller, et al., J.N.C.I., 68:981 (1982). Epiglycanin resembles many human tumor-associated mucins. Rittenhouse, et al.. Lab. Med., 16: 556 (1985). Antigens which cross- react with epiglycanin have been found in human breast, lung, colon, and ovarian cancers. Codington, JNCI, 73: 1029 (1984).

Epi is mainly carbohydrate (75-80%) in composition, and expresses multiple T and Tn determinants. T and Tn are general carcinoma autoantigens. Springer, Science, 224:1198 (1984). T- alpha antigen, also known as the TF (Thomsen- Friedenreich) antigen, is the immediate precursor of the human blood group MN antigens. Tn, in turn, is the immediate precursor of the T-alpha antigens. Normally, T-alpha antigens are not accessible to the human immune system because they are masked by sialic acid. Friedenreich exposed the T-alpha antigen by treatment of red blood cells with neuraminidase, whereupon they were bound by anti-T antibodies of human sera.

Kim and Ohlenbruck determined that the immunodominant portion of the T antigen was the disaccharide beta-D-Gal-(l-3)-alpha-D-GalNac. Z. Immun-Forsch. 130:88-89 (1966). It was later established that in contrast to healthy tissues, certain adenocarcinomas presented T-alpha and Tn determinants in reactive, unmasked form. Springer, et al.. Cancer, 45: 2949-54 (1980). Both TF and Tn determinants are found in approximately 90% of human adenocarcinomas. Springer, Science, 224: 1198 (1984).

This T-alpha determinant has been prepared synthetically. Ratcliffe, et al.. Carbohydrate Res.,

93: 35-41 (1981); Lemieux, EP Patent 44,188. Example 11 in the latter reference describes the use of T-alpha hapten on an HSA carrier (at incorporations of 7, 12, 14 and 22 haptens per HSA molecule) to detect a delayed type hypersensitivity response. The use of such haptens in the production of anti-T-alpha monoclonal antibodies was not mentioned. A synthetic T-alpha hapten is also described by Kolar, U.S. 4,442,284.

Rahman and Longenecker, J. Immunol. 129: 2021-2024 (1982) used the natural form of the T-alpha antigen (neuraminidase-treated erythrocytes) for the production of monoclonal antibodies whose binding to these cells was competitively inhibited by synthetic T-alpha hapten. Thus, their use of synthetic T-alpha hapten was as a characterizing agent.

Asialo-GMl, a gangliotetraosyl cera ide with the structure Gal (beta 1-3) GalNac (beta 1-4) Gal (beta 1- 4) Glc (beta 1-1) ceramide, is found in brain tissue as part of the ganglioside GM1. The immunodominant portion of this molecule (the terminal disaccharide) , differs from that of TF by the substitution of a beta linkage (underlined) for an alpha linkage, and hence is referred to herein as T-beta (as distinct from T- alpha) . Lemieux, U.S. 4,137,401 discloses reaction conditions for linking a bridging arm to an aldose by a beta-D-anomeric glycosidic linkage. Synthetic T-beta haptens have been used in a number of immunological studies. Hoppner, et al., Vox-Sang., 48: 246-53 (1985) ; Rahman and Longenecker, supra: Longenecker, et al.. Int. J. Cancer, 33: 123-129 (1984).

Synthetic T-alpha, T-beta and Tn antigens have been used to stimulate anticancer T cell immunity. Henningsson, et al.. Cancer Immunol. Immunother. , 25: 231-41 (1987) , incorporated by reference herein. Cyclophosphamide (N, N-bis[2-cholorethyl]- tetrahydro-2H-l,3,2-oxazaphosphorine-2-amine-2-oxide) , a nitrogen mustard derivative, is a cytotoxic agent which causes cross-linking of DNA. It is most effective against rapidly dividing cells, hence its use in cancer chemotherapy. Since it also destroys lymphocyte cells, it is also useful as a immunosuppressive agent, indeed, it is one of the most potent im unodepressants known.

Although most chemotherapeutic agents suppress host immunity, it has been demonstrated that certain chemotherapeutic agents, under specific conditions, are able to augment host anti-tumor immunity. Berd and

Mastrangelo, Cancer Res., 48: 1671-75 (1988);

Mastrangelo, et al.. Seminars in Oncology, 13: 186-94

(1986). Campbell, et al., J. Immunol., 141: 3227

(November 1, 1988) reported that cyclophosphamide reduced the tumor burden in mice implanted with a murine B cell lymphoma, rendering the tumor more amenable to active specific immunotherapy with an anti- idiotype antibody vaccine. Nothing in the article suggests that the idiotype resembled any carbohydrate epitope of the lymphoma. No immunosuppressive mucins are known to be associated with lymphomas. See also Reissman, et al.. Cancer Immunol. Immunotherap., 28: 179-84 (1989) (leukemias) .

Mitchell, et al.. Cancer Res., 48: 5883 (October

15, 1988) treated melanoma patients with cyclophosphamide and, several days later, immunized them with a melanoma cell lysate. The value of cyclophosphamide pretreatment was unclear. While cyclophosphamide seemed to favor increases in circulating cytolytic lymphocyte precursors, it had no effect on concanavalin A-inducible suppressor T-cell levels, and "the patients who received cyclophosphamide here had no better frequency of clinical response than those given the lysate mixture alone." In any event, no immunosuppressive mucins are known to be associated with melanomas.

In some cancers, the tumors themselves seem to release immunosuppressive factors. The most striking example of this phenomenon is Hodgkin's disease, in which a small tumor in a single lymph node releases or induces the release of immunosuppressive factors that have a powerful effect on the entire cell-mediated immune system. Patients with Hodgkin's disease have a poor delayed hypersensitivity response and are abnormally sensitive to intracellular parasitic infections such as tuberculosis and herpes virus infections. Jessup, et al.. Cancer Res., 48: 1689 (1988) mentions that when lymphocytes from patients with colorectal carcinoma are incubated with carcinoembryonic antigen (CEA) , a factor is secreted that inhibits immune responses. It has not been recognized previously, however, that tumor immunosuppressive activity can be mediated by mucins. It has been reported that TA3Ha cells are immunoresistant; this is not the same as immunosuppressive.

SUMMARY OF THE INVENTION

We have discovered that mucins, including the epiglycanin of adenocarcino as and bovine sub axillary mucins, have an immunosuppressive effect on subsequent immune response to cross-reactive antigens. The present invention relates to the enhancement of the immune response to active specific adenocarcinoma tumor immunotherapy with antigens cross-reactive with adenocarcinoma tumor-associated mucins by pretreat ent with an agent which inhibits the immunosuppressive effect of the tumor-associated mucin. The preferred agent is cyclophosphamide.

BRIEF DESCRIPTION OF THE DRAWINGS

Fiσure 1. Shows the results of a second TA3Ha tumor challenge of four groups of mice which survived a previous TA3Ha implantation as a result of combined therapy with cyclophosphamide and a TFo-bearing natural or synthetic antigen in Ribi adjuvant. The ordinate is the percent survival; the abscissa, the number of days after challenge. The groups are as follows: (3) cyclophosphamide+Epi-Ribi (4 immunizations, subcutaneously) ; (4) TFα/KLH-Ribi (4 s.c); (5) cyclophosphamide+TFα/KLH-Ribi (4 s.c); (c) control mice.

Figure 2. Shows the comparison of an expanded set of treatments. The groups are as follows: (1) no CY, no immunization (i.e., control); (2) CY only (day 1); (3)

CY (day l)+TFα/KLH-Ribi (day 2)+CY(day 5)+TFα/KLH-Ribi (days 6, 10, 17); (.5) CY (day l)+TFα/KLH-Ribi (days 2, 6, 10, 17); (7) CY only (day 5); (8) CY (day 5), TFα/KLH-Ribi (days 6, 10, 14, 21); (9) CY (day 1)+CY (day 5) .

Figure 3. Shows the results of a local Winn assay of the ability of lymph node cells from mice surviving

TA3Ha implantations thanks to therapy with cyclophosphamide and a TFα-bearing antigen to adoptively transfer the ability to inhibit TA3Ha tumor growth to other mice. Tumor growth is shown by trend lines, marked as follows:

Marking Donor Recipient

filled circles CY+TFα/KLH-Ribi saline open circles same CY filled triangles CY on day 5 saline open triangles same CY filled squares normal saline open squares same CY

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

We have found that a synthetic conjugate of the T alpha hapten and a conventional carrier protein, keyhole limpet hemocyanin (KLH) emulsified in a conventional adjuvant, trehalose dimycloate and monophosphoryl lipid A (MPL) (available in combination form Ribi Immunochem. Research, Inc., Hamilton, Montana and referred to herein as "Ribi") , when administered subcutaneously into hosts bearing tumors which express the T-alpha epitope provided 25% long-term survival. When administration of this conjugate was preceded by treatment with cyclophosphamide, 50% survival was seen for hosts in which the tumor had been established for five days and 90% survival when the tumor had been established for only two days.

Moreover, we have found that lymph node cells obtained from mice that had been treated with cyclophosphamide and T-alpha-KLH-Ribi and had survived this active specific tumor immunotherapy were able to inhibit tumor growth completely in a Winn-type assay. The present invention is not limited to the use of any particular adjuvant. Other chemical and microbial adjuvants, such as CFA, SAF-1, MDP, BCG, liposomes, and Bordetella pertussis toxin may be used in place of Ribi. The tumor-associated hapten may be conjugated to other carrier proteins, such as tetanus or diphtheria toxoid, or retrovirus peptides (e.g., VP6 viral peptide) , rather than KLH, and the hapten/molecule-to- carrier molecule substitution ratio may be varied. Either natural or synthetic antigens which cross-react with the immunosuppressive mucin may be employed.

While the experimental example relates to therapy of a mammary adenocarcinoma in a mouse model, it is believed that the treatment method of the present invention is adaptable to other mammals including human subjects, and to treatment of other adenocarcinomas.

Synthetic tumor-associated glycoproteins (S-TAGS) and other carbohydrate antigens are known in the art and may be prepared by any convenient technique. T and Tn antigens are preferred. For synthetic methods, see

Kaifu and Osawa, Carbohydr. Res., 58:235 (1977);

Ratcliffe, et al.. Id. _ 93:35 (1981); Paulsen, et al., Id. _f 104:195 (1982); Bencomo and Sinay, Id.. 116:69

(1983) .

While the preferred antigen presents a T-alpha disaccharide epitope, a T-beta or Tn epitope might be provided instead. Also, the immunodominant carbohydrate epitopes of other blood group antigens and precursors might be presented. Moreover, anti- idiotype antibodies may be used in place of synthetic or natural antigens. The time interval between administration of the cyclophosphamide and administration of the synthetic tumor-associated glycoconjugate is not fixed, but is dependent on the time of onset and duration of action of the cyclophosphamide's inhibitory effect on suppressor T cell activity or on the induction of such activity by tumor-expressed mucins. The dosage of cyclophosphamide may be selected to increase the antigenic specificity of the anti-suppressor T cell activity effect.

In place of cyclophosphamide, another antagonist of immunosuppression may be employed, such as other oxazaphosphorines, cimetidine or an anti-(suppressor cell) or anti-(suppressor factor) monoclonal antibody. Numerous antibodies of these two types are offered for sale (see Linscott's Directory of Immunological and Biological Reagents, p. 10, 5th ed., 1988-89).

The present invention is not to be restricted on the basis of the present interpretation of the mechanism whereby cyclophosphamide or a similar agent exercises an im unopotentiating effect. An agent antagonizes the immunosuppressive effect of a tumor- associated mucin if it interacts with the mucin or the T cell so that the mucin no longer activates suppressor T cell activity, or if it interacts with a T cell so activated or its suppressor factors so as to diminish the suppressor activity induced by said mucin, or if it interacts with other components of the cellular immune system so as to render them less vulnerable to suppressor T cells activated by said mucin or to suppressor factors released by such cells. In another embodiment, a monoclonal antibody specific for an epitope of a tumor-associated, immunosuppressive mucin is attached to a suitable support to form an immunosorbent. Circulating tumor- associable immunosuppressive mucins recognized by the immunosorbent are removed from the patient's bloodstream by plasmapheresis. The immune response to the tumor, with or without further stimulating the immune system by active specific tumor immunotherapy, is thereby enhanced. (Lectins or other binding substances might be used in place of antibodies).

In a third embodiment, such a monoclonal antibody is administered to the patient, so that it complexes the circulating mucin and thereby hinders its adverse interaction with the cellular immune system.

MATERIALS AND METHODS

Animal: 10 week old pathogen free female CAF1/J mice purchased from Jackson Laboratory were used throughout the investigation.

Tumor cell line: TA3-Ha tumor cell line was originally provided by Dr. J. F. Codington (Mass. General Hospital, Boston, Mass). The tumor cells were grown in vivo by weekly passage (i.p.) in CAF1/J mice.

Synthetic tumor-associated glvcoconiugate fS-TAG. and control antigens: S-TAGs (3Gall->3GalNAcα-Ser-Gly- carrier) of Tα-KLH and Tα-HSA synthesized by Biomira, Inc., Edmonton, Alberta. KLH was purchased from Cal- Biochem and HSA was purchased from Sigma, St. Louis, MO. Hapten substitution ratios were 10-35:1 for HSA and 800-3,000:1 for KLH. Cyclophospha ide (CY) treatments: Cyclophosphamide purchased from Sigma was dissolved in sterile saline. Mice were injected intravenously with CY at a concentration of 100 mg/Kg per mouse.

Tumor vaccine formulation and protocol for active specific immunotherapy: Mice were first injected intraperitoneally with approximately 700 TA3-Ha tumor cells on day 0. The animals were divided into various groups of 8 mice and the following tumor vaccine formulation was administered. Group 1: control mice received CY treatment and no immunization; Group 2 mice received CY on day 1 only; Group 3: mice received CY treatment on day 1, followed by subsequent subcutaneous immunization of Tα-KLH-Ribi emulsion on day 2, 6, 10 and 17 ; Group : mice received subcutaneous immunization of Tα-KLH-Ribi emulsion on day 2, 6, 10 and 17. In a separate experiment, the above experimental set up was repeated with two additional groups of animal added. Group 5: CY treatment was given on day 5, followed by subsequent subcutaneous immunizations of TA-KLH-Ribi emulsion on day 6, 10, 14 and 21. Group 6: CY treatment given on day 5 only and received no further immunization.

Separate groups of tumor injected mice (also 8 per group) were set up for experimental controls. Group l: mice received CY treatment on day 1 followed by subsequent subcutaneous immunizations of KLH-Ribi emulsion on day 2, 6, 10 and 17; Group 2: mice received CY on day 1 followed by subsequent subcutaneous immunizations of only Ribi compound on day 2 , 6, 10 and 17. All animals were monitored for survival daily for 60 or more days. CY was administered intravenously at a volume of 0.2 ml. Tα-KLH was emulsified in 2.0 ml of Ribi compound and 0.2 ml of the emulsion was distributed equally among 2 subcutaneous sites in the upper belly and 1 site at the base of the tail (for day 2 immunization only) .

ELISA: Levels of anti-TFα antibodies (IgG and IgM) in sera of surviving mice were determined about 7 weeks after, initial tumor transplantation in an ELISA. Briefly, test and control sera were serially diluted in wells of microtiter plate coated with TFα-HSA at a concentration of 0.25 μg/well. Bound anti-TFα IgG and IgM antibodies was detected with horseradish peroxidase-conjugate goat anti-mouse IgG and IgM antibodies, respectively.

Footpad testing for DTH responses: DTH was evaluated by testing mice in the footpad with Tα-HSA (50μg) glycoconjugate on day 54 after initial tumor transplantation. Mice were injected in the right (test) or left (control) hind footpads with 30-50μl of antigen in sterile saline or, for a control, sterile saline alone. Just before and 24-48 hours after injection, footpad thickness was measured with a vernier caliper. The results were calculated as the increase in footpad thickness of glycoconjugate-in- sterile saline-injected pads at 24 or 48 hours after footpad challenge minus the increase in footpad thickness of sterile saline only-injected pads over the same time period.

Second challenge of surviving mice with TA3-H&- tumor: Four days after footpad testing, surviving animals were further challenged with 1 x 10⁴ TA3-Ha tumor cells intraperitoneally. Mice were monitored daily for survival over a period of at least 60 days.

Immunoassavs for the Measurement of Immunocompetence/Immunosuppressive in Cancer Patients

The DTH response is an immunological specific cell-mediated response which develops after the intradermal injection or topical application of a test antigen. Cancer patients can be tested for DTH reactivities toward (i) an autologous tumor antigen in the form of synthetic tumor associated glycoconjugate, e.g., TαHSA, and (ii) a neoantigen such as 2,4-dinitro- chlorobenzene (DNCB) . If the patient has been previously sensitized to the antigen, an inflammatory reaction characterized by induration will develop 24 to 48 hours later. Failure to respond to these antigens is indicative of immunosuppression in the patient.

Lymphocyte transformation is an extremely popular in vitro technique used to measure cellular immunocompetence. Small resting lymphocytes are exposed to a itogen (such as phytoheroagglutin and concanavalin A) and are transformed into large lymphoblastic cells. The simplest method for assaying lymphoproliferation upon exposure to the mitogens is tritiated thymidine ([³H] thymidine) incorporation. This measures the counts per minute (cpm) of tritiated thymidine incorporated into DNA for a standard number of cells. In addition to the mitogens mentioned, synthetic tumor-associated glycoconjunate of TαHSA can be used as an immunostimulant in the assay. A significantly lower Stimulation Index (net cpm/unstimulated cpm) is indicative of active immunosuppression. . The identification of surface markers for lymphocyte subpopulations and the development of specific monoclonal antibodies to these markers enable one to detect as well as quantify specific lymphocyte subpopulation (such as T-helper (OKT4) and T- suppressor (OKT8)) in cancer patients. Active immunosuppression induced by a suppressor T lymphocyte subpopulation can be revealed by measuring the T4 and T8 lymphocyte subpopulation in the patient.

Natural Killer (NK) cells are of lymphoid appearance. Their cytotoxic capabilities are not dependent on prior sensitization. Measurement of NK cell activity is usually done using a chromium release assay, in which cells that are to be tested for NK activity are incubated with chromium labeled K562 cells. After 3 to 4 hours, the supernatant from each test well is collected, and the amount of chromium released into the supernatant is measured. Cytotoxic T lymphocyte activities against TF antigen-carrying carcinomas also can be tested by a chromium release assay as described above.

Example l: Observation of Immunosuppression of Mucins

At least two mucins, epiglycanin and bovine submaxillary mucins, are able to suppress DTH effector cells (CD4⁺) .

For the experiment whose results are shown in Table 1 below, mice were first injected with various amounts of Epiglycanin or just saline as control. Six to seven days later all mice were immunized with 50 μg epiglycanin emulsified in complete Freund's adjuvant. Foot-pad testings were preformed 7 days after immunization and net foot-pad swellings were measured at 24 and 48 hours. It will be seen that pretreatment with epiglycanin reduced the degree of foot-pad swelling (a classic measure of DTH response) by 70-95%.

We have also shown that this immunosuppressive effect is adoptively transferred (see Table [1A] below) . Mice were immunized subcutaneously with 50 μg Epiglycanin-CFA immediately after cell transfer and were footpad-tested 7 days later with 30 μg of the immunizing antigen. An 83% reduction in footpad swelling was observed.

Table 1: Immunosuppressive Effect of Epiolvcanin on DTH

Rgg gnse in Mice

Net Foot-pad swelling** (MM)

Expt. Treat¬ Immuni¬ 24 hr 48 hr % Reduc¬ ment zation" tion

1 0.4 mL 50 μg Epi- 0.35 0.33 2 saline CFA 6-7 0.29 0.18 —

3 ' i.v. days later 0.26 0.21

1 100 μg 50 μg Epi- 0.06 0.00 81.1 2 Epi CFA 6-7 ND ND 3 i.v. days later 0.14 0.01 To.6

1 200 μg 50 μg Epi- 0.01 0.05 90.5 2 Epi CFA 6-7 0.03 0.00 94.3 3 i.v. days later 0.15 0.00 71.2

*~* Average of 3-5 mice, ^ Subcutaneously, multiple sites of injections

Table 1A: Immunosuppressive Effect of Epiglvcanin on

DTH Response in Mice

Net Foot-pad swelling*** (MM)

Cells 24 hr 48 hr % Reduction Treatments Transferred at 24 hrs.

Injected 6.4 x 10⁷ 0.37 0.35 0.4 mL spleen cells saline i.v.

Injected 6.4 x 10⁷ 0.05 0.07 83.0 200 μg Epi spleen cells in 0.4 L saline i.v.

** Average of 5 mice For the determination of the suppressor activity of bovine submaxillary mucins (BSM) , mice were first injected intravenously with 200 μg of sterile saline as control. Six days later, animals were divided into various groups and were immunized with 50 μg BSM emulsified either in complete Freund's adjuvant or Ribi compound. All animals were footpad tested for DTH responsiveness 7 days after immunization with BSM. As shown in Table 2 below, net swelling was depressed by 85-95%.

Table 2

T r e a t m e n t immunized with Immuni- 50μg BSM-Ribi or 0 hours 24 hours Net zation CFA (S.C.) R L R L Swelling

1. BSM-CFA Treated with 0.2ml 2.10 2.10 2.10 2.10 0 .00

2. BSM-CFA saline footpad 2.05 2.10 2.10 2.10 0 .05

3. BSM-CFA with BSM(50μg) 2.00 2.05 2.05 2.05 0 .05 -85% .1 D33+. 02

1. BSM-Ribi Treated with 200μg 2.05 2.10 2.15 2.10 0 .10

2. BSM-Ribi BSM i.v. footpad 2.05 2.05 2.10 2.10 0 .00

3. BSM-Ribi with BSM (50μg 2.10 2.10 2.10 2.10 0 .00 -95% .1 033+ . .06

1. BSM-CFA Treated with 2μg 2.05 2.05 2.35 2.00 0.30

2. BSM-CFA BSM i.v. footpad 2.05 2.05 2.35 2.10 0.25

3. BSM-CFA with BSM (50μg) 2.15 2.15 2.30 2.10 0.25 .23+.076

1. BSM-Ribi Treated with 0.2ml 2.05 2.05 2.55 2.00 0.50

2. BSM-Ribi saline footpad 2.05 2.05 2.60 2.05 0.55

3. BSM-Ribi With BSM (50μg) 2.05 2.00 3.00 2.00 0.95 .67+.25

Example 2: Cyclophosphamide Inhibition of Immunosuppressive Effect of Mucin

Mice were injected either with 0.4ml (containing either 200 μg or 100 μg Epiglycanin) Epiglycanin or sterile saline solution intravenously. Six days after initial injection, mice were treated intravenously with CY (lOO l/kg) or sterile saline solution as control, twenty four hours prior to immunization with 50 μg Epiglycanin emulsified in equal volume of complete Freund's adjuvant. Seven days after immunization, mice were foot-pad tested with 50μg Epiglycanin. As shown in Table 3, pretreatment with cyclophosphamide enhanced the immune response to epiglycanin in mice previously given an immunosuppressive dose.

0 hours 24 hours 48 hours L R L R Net LL RR fiet

1. 100 μg Epi 2.00 2.00 2.05 2.00 0.05 2.00 2.00 . 0.00

5 iv 1/10

2. saline 1/16 2.10 2.00 2.10 2.00 0.00 2.05 2.00 0.00 50 μg Epi

10 3. CFA 2. 00 2. 00 2.05 2.00 0.05 2.05 2.00 0.05

4. 2. 00 2. 00 2.05 1.95 0.05 2. 00 2.00 0.00 (-82.14%)* .03+.025 .0125+.025

15 1. 100 μg Epi 2. .00 2. ,00 2.50 2.00 0.50 2. 20 2.00 0.20

2. CY 2. .00 2. ,00 2.05 2.00 0.05 2. 05 2.00 1.05

3. 50 μg Epi 2. .00 2. .00 2.30 2.00 0.30 2. 20 2.00 0.20

4. CFA 2. .00 2. .00 2.15 2.00 0.15 2. 25 2.00 0.25 (+19%)** .25±.195 .175+.087

1. Saline (0.4ml) 2. .00 2. .05 2.10 2.00 0.10 2 .05 2.05 0.05

2. 50 μg Epi 2, .00 2. .00 2.30 2.00 0.30 2 .25 2.00 0.25

3. CFA 2 .00 2 .00 2.25 2.00 0.25 2 .15 2.00 0.15

30 4. 2 .00 2 .05 2.20 2.05 0.20 2 .15 2.00 0.15 .21+.085 .15+.08

Clone

3 355 1. 200 μg Epi 2. ,00 2.00 2.05 2.00 0.05 2.00 2.00 0.00

2. saline 2. 00 2.00 2.00 2.00 0.00 2.00 2.00 0.00

3. 50 μg Epi 2. .00 2.00 2.05 2.00 0.05 2.05 2.00 0.05

4. CFA 2. .10 2.05 2.15 2.00 0.05 2.10 2.00 0.00 (-82.14%)* .037+.025

1. 200 μg Epi 2, .05 2.00 2.35 2.00 0.35 2.25 2.00 0.20

2. saline 2. .15 2.10 2.20 2.10 0.05 2.10 2.10 0.00

3. 50 μg Epi 2. .10 2.05 2.55 2.00 0.45 2.40 2.00 0.30

50 4. CFA 2 .05 2.05 2.50 2.00 0.40 2.30 2.00 0.25

(+47.6%)** ,31±.179 1. 200 μg Epi 2.00 2.00 2.20 2.00 0.20 2.10 2.00 0.1

2. saline 2.00 2.05 2.25 2.00 0.25 2.15 2.00 0.1

3. 50 μg Epi 2.05 2.00 2.30 2.00 0.25 2.20 2.00 0.1

4. CFA 2.05 2.05 2.20 2.05 0.15 2.20 2.05 0.1

.21+.048

Example 3: Therapeutic Effect of Combined Treatment with Cyclophosphamide and T-Alpha Glvcoconiuoate

The sequential administration of cyclophosphamide and a T-alpha epitope/bearing synthetic glycoconjugate improved survival (Table 3) of mice challenged with Ta3Ha mouse mammary adenocarcinoma tumor cells, which express epiglycanin.

Table 3. Effect of combined treatment of CY and S-TAG on the development of TA3-Ha tumor in CAF1/J Mice

No . Of tumor-

M e d i a n f r e e

Group s urv iva 1 m ice/ -

No. Treatments time (άaγs) total

1 none 17-19 0/16

2 CY(day 1) only 18-21 0/16

3 CY(day 5) only 23 2/9

4 CY(day 1&5) only 26 1/9

5 CY(day 1) + KLH-Ribi 17 0/8

6 CY(day 1) + HSA-Ribi 20 0/8

7 CY(day 1) + Ribi 20 0/8

8 CY(day 1) + TαKLH-Ribi >100 14/17

9 CY(day 5) + TαKLH-Ribi 35 4/8

10 CY(day 1&5) + TαKLH-Ribi 46 4/8

11 TαKLH-Ribi only 20-22 4/16

12 TαKLH-Ribi + CY(day 5) 23 1/8

Four days after footpad testing, surviving animals from active specific immunotherapy were further challenged with 1 x 10⁴ TA3-Ha tumor cells intraperit- oneally. (This dose is greatly in excess of the LD50) . Mice were monitored for survival daily for 60 or more days.

The results are shown in Figure 1. It will be seen that the best surviving group was the one given both cyclophosphamide and T-alpha/KLH in Ribi adjuvant.

A more complex experimental comparison is shown in Figure 2. It will be seen here that administration of cyclosphamide alone had only an ephemeral effect on survival. Best results (group 5) were obtained with repeated courses of T-alpha/KLH in Ribi adjuvant after the initial treatment with cyclophosphamide.

Example 4: Adoptive Transfer of Tumor Resistance From Long-Ter Survivors of Active Specific Immunotherapy

Long term survivors from the active specific immunotherapy experiment were used in a local Winn assay to test whether their immune splenic and lymph node cells were able to inhibit tumor growth in vivo. Splenic and lymph node cells obtained from various treatment groups of mice were mixed with live TA3-Ha tumor cells at a effector:target cell ratio of 100:1 and were injected subcutaneously into the footpads of recipient mice pretreated either in cyclophosphamide (lOOmg.kg i.v.) or saline in the same manner. The footpad swelling was measured at 24-48 hours and every 2 days thereafter. Tumor size in the footpad was expressed in terms of net swelling of footpad thickness (mm) .

Lymph node cells obtained from mice (treated with CY and TA-KLH-Ribi immunizations) which survived inhibit tumor growth completely in a Winn type assay. Spleen cells, on the other hand, did not transfer immunity.

The present invention extends to the adoptive transfer of cell-mediated immunity to adenocarcinomas expressing immunosuppressive mucins by means of lymph mode cells obtained from subjects who responded favorably to the combined anti-immunosuppression, active specific immunotherapy taught herein. For a general protocol for adoptive immunotherapy, see Rosenberg, U.S. 4,690,915, and the previously referenced applicaiton of Longenecker and Henningsson.

It is contemplated that cyclophosphamide and/or a carbohydrate epitope-bearing antigen which is immunologically cross-reactive with a mucin having an immunosuppressive activity associated with an ademocarcinoma may be used in the manufacture of a composition for use in the treatment of an adenocarcinoma. It is further contemplated that an antibody or lectin which is specific for a circulating tumor-associated mucin having immunosuppressive activity may be be used in the manufacture of a absorbent composition for the treatment of the tumor by removal of the circulating mucin from the bloodstream by pheresis. These treatment modalities may be further supplemented by active specific immunotherapy or by adoptive transfer of immunity with donor lymph node cells.

Claims

WE CLAIM :

1. A method of inhibiting the growth of an adenocarcinoma tumor in a human or animal subject, said tumor being associated with a mucin having immunosuppressive activity, which comprises (a) administering to the subject an immune response potentiating amount of a cyclophosphamide, and (b) administering to the subject an i munogenically effective amount of a carbohydrate epitope-bearing antigen which is immunologically cross-reactive with a mucin having an immunosuppressive activity associated with such tumor.

2. The method of claim 1 wherein the antigen is a synthetic glycoconjugate.

3. The method of claim 1 in which the antigen is epiglycanin.

4. The method of claim 1 wherein both the tumor- associated mucin and the antigen are characterized by a T or Tn determinant.

5. The method of claim 1 wherein the synthetic glyconconjugate is a conjugate of a T-alpha hapten and a pharmaceutically acceptable, immunogenic protein carrier.

6. The method of claim 1 in which the tumor is a mammary adenocarcinoma.

7. A method of enhancing the responsiveness of a human or animal subject to active specific tumor immunotherapy, when said tumor is associated with a circulating mucin having immunosuppressive activity, which comprises (a) removing blood from the subject, (b) incubating the blood with a specific adsorbent for such mucin to obtain a blood fraction depleted of said mucin, and (c) returning the blood fraction to the patient's circulatory system.

8. The method of claim 7 wherein the adsorbent is an immobilized antibody which preferentially binds the mucin.

9. A method of treating an adenocarcimona tumor in a human or animal, where the tumor is associated with a circulating mucin having immunosuppressive activity, which comprises enhancing the susceptibility of the tumor to control by immunological means by the method of claim 7, and then treating the tumor by active specific immunotherapy with a vaccine comprising an antigen which immunologically cross- reacts with said tumor.

10. A method of inhibiting the growth of an adenocarcinoma tumor in a human or animal subject, said tumor being associated with a mucin having immunosuppressive activity, which comprises administering to the subject (a) an immune response potentiating amount of an agent which antagonizes said immunosuppressive activity, and (b) an immunogenically effective amount of a carbohydrate epitope- bearing antigen which is immunologically cross-reactive with said mucin.

11. The method of claim 1 wherein the antigen is a blood group antigen or precursor thereof, or is a synthetic glyconjugate bearing the immunodominant carbohydrate epitope of a blood group antigen or precursor thereof.

12. A method of inhibiting the growth of an adenocarcinoma tumor in a human or animal recipient which comprises providing lymph node cells of a donor, said donor characterized as a human or animal subject in whom an adenocarcinoma tumor had been inhibited by the method of claim 1, and administering said lymph node cells to the recipient under conditions suitable for adoptive transfer of immunity.

13. Use of cyclophosphamide in the manufacture of a composition for the treatment of an adenocarcinoma in a human or animal subject.

14. Use as in claim 13, wherein the adenocarcinoma is associated with an immunosuppressive mucin.

15. Use of a carbohydrate epitope-bearing antigen which is immunologically cross-reactive with a mucin associated with an adenocarcinoma tumor and having an immunosuppressive activity in the manufacture of a composition for the treatment of an adenocarcinoma tumor in a human or animal subject.

16. Use as in claim 15, wherein the human or animal subject has been pretreated with an immune response potentiating amount of cyclophosphamide.

17. Use of cyclophoshamide and a carbohydrate epitope-bearing antigen which is immunologically cross-reactive with a mucin associated with an adenocarcinoma tumor and having an immunosuppressive activity in the manufacture of a composition for the treatment of an adenocarcinoma tumor in a human or animal subject.