CA2013966C - Active specific immunotherapy of adenocarcinomas producing immunosuppressive mucins - Google Patents

Abstract

A synergistic composition is provided herein for inhibiting the growth of an adenocarcinoma tumour. The tumour is associated with a mucin having immunosuppressive activity. The composition comprises: an immune-response-potentiating amount of cyclophosphamide, and an immuno-genically-effective amount of a carbohydrate epitope-bearing antigen. Such antigen is immunologically cross-reactive with a mucin having immunosuppressive activity associated with the tumour.

Description

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_2013966 This application relates to active specific tumor immunotherapy.
Active specific tumor immunotherapy is an attempt antigenically to stimulate the endogenous antitumor immunity of a host. 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 non-specifically 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 immunological changes with time, it is uncertain whether, at the time of intervention, all tumor cells will express the immunizing antigen.

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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: p~Ti 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 suppressive or regulatory role. (They may, of course, /'""", ,.... , a _.

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., Canc. 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 immunoresistant.
The TA3Ha carcinoma cells are covered by a mucin-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-(1-3)-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 5 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 character-izing agent.
Asialo-GMl, a gangliotetraosyl ceramide with the structure Gal(beta 1-3)GalNac et 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).

5a Synthetic T-alpha, T-beta and Tn antigens have been E
used to stimulate anticancer T cell immunity. Henningsson ~a~~~~~

Cyclophosphamide (N, N-bis[2-cholorethyl]-tetrahydro-2H-1,3,2-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 immunodepressants 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 20139fi6 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, e.g., 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.

20139fi6 It has been discovered that mucins, including the epiglycanin of adenocarcinomas and bovine submaxillary 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 immuno-therapy with antigens cross-reactive with adenocarcinoma tumor-associated mucins by pretreatment with an agent which inhibits the immunosuppressive effect of the tumor-asso-ciated mucin. The preferred agent is cyclophosphamide.
Thus, by one broad aspect of the present invention, a synergistic composition is provided for inhibiting the growth of an adenocarcinoma tumour, such tumour being associated with a mucin having immunosuppressive activity, the composition comprising an immune-response-potentiating amount of cyclophosphamide, and an immunogenically-effec-tive amount of a carbohydrate epitope-bearing antigen, that antigen being immunologically cross-reactive with a mucin having immunosuppressive activity associated with the tumour.
By variants of such composition, the antigen may be a synthetic glyconconjugate, or epiglycanin.
By another variant thereof both the tumour-associated mucin and the antigen are characterized by a T or a Tn determinant.

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By yet another variant thereof, the synthetic glycon-conjugate is a conjugate of a T-alpha hapten and a pharma-ceutically-acceptable, immunogenic protein carrier.
By still another variant thereof, the tumour is a mammary adenocarcinoma.
By another aspect of this invention, mucin-depleted blood fraction is provided for imparting enhanced respon-siveness to active specific tumour immunotherapy, such tumour being associated with a circulating mucin having immunosuppressive activity, the mucin-depleted blood fraction having been incubated with a specific adsorbent for the mucin.
By one variant thereof, the adsorbent is an immobilized antibody which preferentially binds the mucin.
By yet another aspect of this invention, a combined therapeutic agent is provided for treating an adenocarci-noma tumour, the tumour being associated with a circulating mucin having immunosuppressive activity, the combined therapeutic agent comprising a mucin-depleted blood fraction, the blood having been incubated with a specific adsorbent for the mucin, and a vaccine comprising an antigen which immunologically cross-reacts with the tumour.
By still another aspect of this invention, a synergistic composition is provided for inhibiting the growth of an adenocarcinoma tumour, the tumour being associated with a mucin having immunosuppressive activity, .-..
io ''20 1 396 6 the synergistic composition comprising an immune-response-potentiating amount of an agent which antagonizes the immunosuppressive activity, and an immunogenically-effective amount of a carbohydrate epitope-bearing antigen which is immunologically-cross-reactive with the mucin.
By one variant thereof, the antigen is a blood group antigen or precursor thereof, or is a synthetic glycon-conjugate bearing the immunodominant carbohydrate epitope of a blood group antigen or precursor thereof.
By a still further aspect of this invention, lymph node cells adapted for inhibiting the growth of an adenocarcinoma tumour, the lymph node cells having been derived from a donor treated with a synergistic composition for inhibiting the growth of an adenocarcinoma tumour, the tumour being associated with a mucin having immunosuppressive activity, the synergistic composition comprising an immune-response-potentiating amount of cyclophosphamide, and an immunogenically-effective amount of a carbohydrate epitope-bearing antigen which is immunologically cross-reactive with a mucin having immunosuppressive activity associated with the tumour.
~c ,,_, _ '~ 201~g66 By still another aspect of this invention, the use is provided of a carbohydrate epitope-bearing antigen which is immunologically cross-reactive with a mucin associated with an adenocarcinoma tumour and having an immunosuppressive activity, in the manufacture of a composition for the treatment of an adenocarcinoma tumour on a patent previously treated with an immune response-potentiating amount of a cyclophosphamide.
By a still further aspect of this invention, the use is provided of cyclophosphamide and a carbohydrate epitope bearing antigen which is immunologically cross-reactive with a mucin associated with an adenocarcinoma tumour and having an immunosuppressive activity, in the manufacture of a composition for the treatment of an adenocarcinoma tumour.
In the accompanying drawings, Figure 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 TFa-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-Rbi (4 immunizations, subcutaneously); (4) TFa/KLH-Ribi (4 s.c.); (5) cyclophosphamide+TFa/KLH-Ribi (4 s.c.); (c) control mice.
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-' 20 1 396 fi lla 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 1)+TFa/KLH-Ribi (day 2)+CY (day 5)+TFa/KLH-Ribi (days 6, 10, 17) ; (7) CY only (day 5) ; (8) CY (day 5) , TFa/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 TFa-bearing antigen to transfer adoptively 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+TFa/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
It has been 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 ~r .
a 203966 llb established for five days and 90% survival when the tumor had been established for only two days.
Moreover, it has been 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, in its various aspects, is not limited to the use of any particular adjuvant. Other chemical and microbial adjuvants, e.g., 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, e.g., 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 preferred. For synthetic methods, see Kaifu and Osawa, Carbohydr. Res., 58:235 (1977); Ratcliffe et al, Id., 93:35 (1981); Paulsen ,,r., llc et al, Id., 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 cyclo-phosphamide 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 cyclophos-phamide'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, e.g., other oxazaphos phorines, 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).

v 2013966 lld The present invention, in its various aspects, is not to be restricted on the basis of the present interpretation of the mechanism whereby cyclophosphamide or a similar agent exercises an immunopotentiating 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 that 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 that mucin or to suppressor factors released by such cells.
.s l ~p..~g~6 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-s 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 anti.bodies).
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: l0 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 glycoconiuQate fS-TAG) and control antigens: S-TAGs (~QGall->3GalNAca-Ser-Gly-carrier) of Ta-KLH and Ta-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.

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CLrclophosphamic~e ICYI 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 Ta-KLI-i-Ribi emulsion on day 2, 6, 10 and 17; Group 4: mice received subcutaneous immunization of Ta-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 ~ . ~...i --a volume of 0.2 ml. Ta-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-TFa antibodies (IgG and IgM) in sera of surviving mice were determined about 7 weeks after initial tumor transplantation in an ELISA.
l0 Briefly, test and control sera were serially diluted in wells of microtiter plate coated with TFa-HSA at a concentration of 0.25 ug/well. Bound anti-TFa 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 Ta-HSA
(50~,g) glycoconjugate on day 54 after initial tumor transplantation. Mice were injected in the right (test) or left (control) hind footpads with 30-50~C1 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-Ha-tumor: Four days after footpad testing, surviving animals were further challenged with 1 x 104 TA3-Ha ~4~~.~~~
tumor cells intraperitoneally. Mice were monitored daily for survival over a period of at least 60 days.
Immunoassays for the Measurement of 5 ImmunocomgetenceLImmunosuQpressive 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 10 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., TaHSA, and (ii) a neoantigen such as 2,4-dinitro-chlorobenzene (DNCB). If the patient has been 15 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 mitogen (such as phytohemagglutin 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 TaHSA 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.

,,..,.,. , Examgle 1- 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 l0 amounts of Epiglycanin or just saline as control. Six to seven days later all mice were immunized with 50 ~,g 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 [lAJ
below). Mice were immunized subcutaneously with 50 ~g Epiglycanin-CFA immediately after cell transfer and were footpad-tested 7 days later with 30 ~tg of the immunizing antigen. An 83% reduction in footpad swelling was observed.

. ~ ~.~ _ Table 1- Immunosunpressive Effect of Epialvcanin on DTH
Response in Mice Net Foot-pad swelling**
(MM) Expt. Treat- Immuni- 24 hr 48 hr % Reduc-ment zation@ tion to 1 0.4 mL 50 ~.g 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 ~Cg 50 ~g Epi- 0.06 0.00 81.1 2 Epi CFA 6-7 ND ND _ 3 i.v. days later 0.14 0.01 70.6 1 200 ~g 50 /.1g 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 lA' Immunosuppressive Effect of Epicrlvcanin on DTH Response in Mice Net Foot-pad swelling***
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 ~cg Epi spleen cells in 0.4 mL
saline i.v.
** Average of 5 mice _ ~0~.~~~
,...

For the determination of the suppressor activity of bovine submaxillary mucins (BSM), mice were first injected intravenously with 200 ~g of sterile saline as control. Six days later, animals were divided into various groups and were immunized with 50 ~.g 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%.

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Table 2 5 T r a a t m a n t immunized with Immuni- 50~g BSM-Ribi or 0 rs 24 Net hou hours zation CFA (S. C.) R L R L Swelling 1. BSM-CFA Treated with 0.2m1 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(50~g) 2.00 2.05 2.05 2.05 0.05 -85% .033+.029 1. BSM-Ribi Treated with 20O~Cg 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 (50~tg 2.10 2.10 2.10 2.10 0.00 -95% .033+.066 1. BSM-CFA Treated with 2~Cg 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 (50~.g) 2.15 2.15 2.30 2.10 0.25 .23+.076 1. BSM-Ribi Treated with 0.2m1 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 (50~Cg) 2.05 2.00 3.00 2.00 0.95 .67+.25 Example 2: Cyclophosphamide Inhibition of Immunosup~ressive Effect of Mucin Mice were injected either with 0.4m1 (containing either 200 ~,g or 100 ~g Epiglycanin) Epiglycanin or sterile saline solution intravenously. Six days after initial injection, mice were treated intravenously with CY (100m1/kg) or sterile saline solution as control, twenty four hours prior to immunization with 50 ~Cg Epiglycanin emulsified in equal. volume of complete Freund's adjuvant. Seven days after immunization, mice were foot-pad tested with 50y~g Epiglycanin. As shown in Table 3, pretreatment with cyclophosphamide enhanced the immune response to epiglycanin in mice previously given an immunosuppressive dose.

202~~~
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0 hours 24 hours 48 hours treatment L R L R Net L R det 1. 100 ~.tg Epi 2.00 2.00 2.05 2.00 0.05 2.00 2.00 0.00 iv 1/ 10 2. saline 1/16 2.10 2.00 2.10 2.00 0.00 2.05 2.00 0.00 50 ~Cg Epi 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 .

1. 100 ~Cg 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 ~,t,g 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 (0.4m1) 2.00 2.05 2.10 2.00 0.10 2.05 2.05 0.05 2. 50 /~,g 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 4. 2.00 2.05 2.20 2.05 0.20 2.15 2.00 0.15 .2 1.085 .1 5.08 Clone 1. 200 ~g 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 ~.tg 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%)* 5 .037.02 1. 200 ~g 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 ~.tg Epi 2.10 2.05 2.55 2.00 0.45 2.40 2.00 0.30 4. CFA 2.05 2.05 2.50 2.00 0.40 2.30 2.00 0.25 (+47. 6%)** .31.1 79 1. 200 ~,g Epi 2.00 2.00 2.20 2.000.20 2.102.00 0.10 2. saline 2.00 2.05 2.25 2.000.25 2.152.00 0.15 3. 50 ~g Epi 2.05 2.00 2.30 2.000.25 2.202.00 0.15 4. CFA 2.05 2.05 2.20 2.050.15 2.202.05 0.15 .21+.048 Example 3- Thera peutic Effect of Treatment Combined with Cvclop hosphamide T-Algha Gl~ cocon-iuaate and 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 and S-TAG
of CY

on the development of TA3-Ha tumor in CAF1/J
Mice No . of tumor-M a d i a f r a n a Group survival mice/-No. Treatments time (days) 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) + TaKLH-Ribi >100 14/17 9 CY(day 5) + TaKLH-Ribi 35 4/8 10 CY(day 1&5) + TaKLH-Ribi 46 4/8 11 TaKLH-Ribi only 20-22 4/16 12 TaKLH-Ribi + CY(day 5) 23 1/8 Four days after f ootpad 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).

~J ',.i 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 Lonct-Term 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 (100mg.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 ,.~ ..~ 2 0 1 3 9 6 6 inhibit tumor growth completely in a Winn type assay.
Spleen cells, on the other hand, did not transfer immunity.
The present invention, in the various aspects, extends 5 to the adoptive transfer of cell-mediated immunity to adenocarcinomas expressing immunosuppressive mucins by means of lymph node cells obtained from subjects who responded favourably to the combined anti-immunosuppres-sion, active specific immunotherapy taught herein. For a 10 general protocol for adoptive immunotherapy, see Rosenberg, U.S. 4,690,915.

Claims (14)

1. A synergistic composition for inhibiting the growth of an adenocarcinoma tumour, said tumour being associated with a mucin having immunosuppressive activity, said composition comprising: (a) an immune-response-potentiating amount of cyclophosphamide; and (b) an immunogenically-effective amount of a carbohydrate epitope-bearing antigen, said antigen being immunologically cross-reactive with a mucin having immuno-suppressive activity associated with said tumour.

2. The composition of claim 1, wherein said antigen is a synthetic glyconconjugate.

3. The composition of claim 1, in which said antigen is epiglycanin.

4. The composition of claim 1, wherein both said tumour-associated mucin and said antigen are characterized by a T or a Tn determinant.

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

6. The composition of claim 1, in which said tumour is a mammary adenocarcinoma.

7. Mucin-depleted blood for imparting enhanced responsiveness to active specific tumour immunotherapy, when said tumour is associated with a circulating mucin having immunosuppressive activity, said blood having been incubated with a specific adsorbent for said mucin, thereby to obtain a blood fraction depleted of said mucin.

8. The blood of claim 7, wherein said adsorbent is an immobilized antibody which preferentially binds said mucin.

9. A combined therapeutic agent for treating an adenocarcinoma tumour, where said tumour is associated with a circulating mucin having immunosuppressive activity, said agent comprising: (a) mucin depleted blood, said blood having been incubated with a specific adsorbent for said mucin, thereby to obtain a blood fraction depleted of said mucin; and (b) a vaccine comprising an antigen which immunologically cross-reacts with said tumour.

10. A synergistic composition for inhibiting the growth of an adenocarcinoma tumour, said tumour being associated with a mucin having immunosuppressive activity, said composition comprising: (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 composition of claim 10, wherein said antigen is a blood group antigen or precursor thereof, or is a synthetic glyconconjugate bearing the immunodominant carbohydrate epitope of a blood group antigen or precursor thereof.

12. Lymph node cells adapted for inhibiting the growth of an adenocarcinoma tumour, said lymph node cells having been derived from a donor treated with a synergistic composition for inhibiting the growth of an adenocarcinoma tumour, said tumour being associated with a mucin having immunosuppressive activity, said composition comprising: (a) an immune-response-potentiating amount of cyclophosphamide; and (b) an immunogenically-effective amount of a carbohydrate epitope-bearing antigen which is immunologically cross-reactive with a mucin having immunosuppressive activity associated with said tumour.

13. Use of a carbohydrate epitope-bearing antigen which is immunologically cross-reactive with a mucin associated with an adenocarcinoma tumour and having an immunosuppressive activity, in the manufacture of a composition for the treatment of an adenocarcinoma tumour in a patent previously treated with an immune response-potentiating amount of a chclophosphamide.

14. Use of cyclophosphamide and a carbohydrate epitope-bearing antigen which is immunologically cross-reactive with a mucin associated with an adenocarcinoma tumour and having an immunosuppressive activity, in the manufacture of a composition for the treatment of an adenocarcinoma tumour.