WO1998046640A2 - Methods and compositions for inducing immunosuppression - Google Patents

Abstract

A proteinaceous composition isolated from the serum of a human having prostate cancer and having a molecular weight of approximately 105,000 to 112,000 daltons, as determined by SDS polyacrylamide gel electrophoresis, has the ability to induce immunosuppression in a culture of normal human peripheral blood lymphocytes (PBLs). This composition, and the polypeptide isolated therefrom, reduces the activation/proliferation response of normal donor PBLs, by inducing an alteration in the expression of the signal transducer, TCR-z. Detection and measurement of this immunosuppressive composition in a subject's PBLs can indicate immune dysfunction or a change in the immune status following treatment, e.g., chemotherapeutic treatment. This immunosuppressive composition or components isolated therefrom are useful in therapeutic methods for inducing immunosuppression, where desired. This composition is also useful to induce an immune response in a subject to the presence of the immmunosuppressive component on the surface of a cancer cell. Thus, this composition is useful in vaccines and as a diagnostic agent.

Description

METHODS AND COMPOSITIONS FOR INDUCING IMMUNOSUPPRESSION

Field of the Invention

The present invention relates to the identification, isolation and use of an immunosuppressive agent from sera of human prostate cancer patients, which agent induces immunosuppression.

Background of the Invention

The initiation of an immune response in a mammalian host, particularly a human, that efficiently eliminates any invading agent from the host, employs primarily two populations of cells: B lymphocytes, derived from the bone marrow, and T lymphocytes, derived from the bone marrow and thymus. B cells essentially recognize antigen in its intact, original form. In contrast, T cells recognize antigen fragments, or peptides, and are critical in the development of a cell-mediated immune (CMI) response. "T lymphocytes" or "T cells" include all subsets of lymphocytes which carry the T cell antigen receptor. These subsets include lymphocytes which are CD3+CD4+(αβ+); CD3+CD8+(αβ+); CD3+CD4-CD8-(γδ +). "NK cells" or "natural killer" cells refer to those lymphocytes that are CD3'CD56+, CD3 CD16+, CD56+, CD16+ and CD56+CD16+. The site of primary antigen recognition for T cells is the T cell receptor (TCR), which consists of a disulfide linked heterodimeric recognition unit, TCR α/β or TCR γ/δ. This multichain transmembrane receptor is noncovalently associated with the CD3 molecular complex, which itself consists of five noncovalently associated structures designated δ, γ, e, ( and η. The TCR ζ (zeta) subunit can exist in a variety of forms both hetero- and homodimeric, TCR ζ-ζ, TCR ζ-y and TCR -η.

Briefly summarized, the TCR participate in the immune response as follows: Antigens, including autoantigens, are processed by antigen-presenting cells (APC) and the resulting fragments are then associated with one of the cell surface proteins encoded by the Major Histocompatibility Complex (MHC). Antigen recognition is specific to the two types of MHC classes: CD4 positive T cells recognize antigen bound to MHC class II; and CD8 positive T cells recognize antigen bound to MHC class I. Tumor growth is controlled by CD8 cytotoxic cells that recognize the foreign antigen of the tumor within the context of the MHC class I. When the MHC/antigen fragment complex binds to a complementary TCR on the surface of a T cell, the T cell undergoes a chain of reactions involving a complicated series of signal transductions which induce cellular differentiation, activation and/or proliferation of a subpopulation of T cells that bear that particular TCR as well as the production of cytokines. Once activated, the cells have the capacity to regulate other cells of the immune system which display the processed antigen, and also the ability to destroy cells or tissues which carry epitopes of the recognized antigen. The ability of TCR to transduce signals to multiple biochemical cascades is thus a central event of T cell activation and immune response [see, e.g., Baniyash, M. et al, J. Biol. Chem.. 263:1822-1825 (1988); Klausner, R. et al, Clin. Cane. Res., 2:1825-1828 (1996); Collins, T. L. etal, J. Immunol.. 148:2159-2162 (1992); Beyers, A. D. et al, PNAS USA. 89:2945-2949 (1992)]. Many diseases are characterized by the presence of an impaired immune response (i.e., a T cell response altered from normal) and/or the development of progressive immunosuppression, for example, acquired immunodeficiency syndrome (AIDS), sepsis, leprosy, cytomegalovirus and parasitic infections, autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and lupus; as well as a variety of cancers. Undesirable immune responses are also seen in patients undergoing bone marrow and organ transplantation, causing post operative graft rejections. As one example, in tumor-bearing mammals the normal process of T cell activation is altered, resulting in the immunodysfύnctions characteristic of advanced malignancy, such as delayed type hypersensitivity, a decrease in targeted cytolytic ability, reduced target cell activation and a diminished proliferative response [Broder, S. etal, N. Engl. J.

Med.. 299: 1335-1341 (1978); Loeffler, C. et al, J, Immunol.. 149:949-956 (1992); Ghosh, P. et al, J. Natl. Cane. Instit.. 87: 1478-1483 (1995); Lai, P. et al, Clin. Cane. Res.. 2:161-173 (1996); Nakagomi, H. et al, Cane. Res _: 53 :5610-5612 (1993) and Kono, K. et al, Clin. Cane. Res._: 2:1825-1828 (1996)]. Alterations in a number of T cell associated signal transducing functions appear to be directly related to, or possibly the result of, an individual's diagnosis of cancer. For example, reductions in TCR-ζ chain expression has been hypothesized to directly correspond to poor patient prognosis and even patient death. This alteration in T cell activation and the poor response to antigen is a result of the inability of the T cells, upon antigen recognition, to respond appropriately. For example, in one study, a prostate cancer derived cell line co-cultured with normal peripheral blood lymphocytes (PBL) was found to restrict the ability of lymphocytes to respond to specific activation stimuli [W. Bolton et al, "Suppression of LL2R1 Expression Following CD3 Induced Activation in the Presence of Prostate Tumor Cells", International Symposium for Recent Advances in Diagnosis and Treatment of Prostate Cancer (abstract; September

21, 1995)].

Considerable evidence suggests that the TCR-ζ subunit has a key role in the process of cellular activation and hence in immune surveillance [Anderson et al, ∑. Immunol. 143:1899-1904 (1989) and Sussman et al, £e3i, 52:85-95 (1988)]. Evidence from studies performed with renal and colorectal cancer patients suggest that the decreased expression or even absence of TCR-( is responsible for the depressed immunofunction observed in patients with solid tumor malignancies. The absence of TCR-C expression might be the cause (or possibly the effect) of the impaired immune response observed in patients with solid tumor malignancies. In studies of colorectal cancer, T cells isolated from the tumors of patients with colorectal carcinoma expressed significantly less TCR-C than found in T cells isolated from their peripheral blood. In turn, the PBL from these patients expressed decreased levels of TCR-ζ when compared to the levels found in PBL from healthy controls. Where PBL from patients with lymph node involvement or distant organ metastases (Dukes stages C and D) had significantly less TCR-ζ than patients with localized disease (Dukes stages A and B), investigators concluded that the changes among the TCR associated signal transducing molecules were induced by unspecified tumor-associated factors [Matsuda et. al. Int. J. Cancer. 61:765-772 (1995)]. In another study, T lymphocytes from tumor-bearing mice expressed T cell antigen receptors that completely lacked the TCR-C chain [Mizoguchi et. al., Science. 258: 1795-1798 (1992)].

Similar alterations in the TCR-C chains of peripheral T lymphocytes (expression of the antigen TCR-C was considerably reduced or absent in patients with heavy tumor burden) in human cancer patients with no similar changes observed in normal patient controls [Finke et al, Cane. Res., 5.3:5613-5616 (1993)]. In addition, the progression of the malignancy correlates with the decrease in TCR-C expression [Zea et al, Clin. Cane. Res.. 1:1327-1335 (1995)]. Expression of the TCR-C chain was reported as significantly altered in peripheral blood in patients with prostate cancer (38% tested patients were positive for the receptor) vs. normal controls (82% tested positive for presence of the receptor) [W. Bolton et al, "Significant Alteration in TCR-C Expression in Peripheral Blood T Cells from Patients with Prostate Cancer", in Third Symposium of Impact of Cancer Biotechnology, Nice, France (October 21, 1996)]. Soluble immunosuppressor factors, such as circulating tumor antigens, immune complexes consisting of host antibody-tumor antigen, lipoproteins and acute phase reactants, have been found in serum, in malignant ascites, and in tumor-infiltrated tissue [See, e.g., Roth et. al., J. Immunol.. 130: 303-308 (1983); Hakim A. A., Cancer Immunol. Immunother.. 8: 1330 (1980); Hess, A. D. et al, Can. Res.. 40: 1842 (1980) and J. Immunol.. 130: 303-308 (1983)]. A few of these factors have been shown to markedly suppress mitogen-induced peripheral blood T cell proliferation and IL2 production [Maki, T. S. et al. J. Immunol., 136: 3298 (1986)]. HT29 factor, isolated from a colon cancer cell line for example, inhibits mitogen-induced peripheral blood T cell proliferation and cytokine production. However, inhibition of T cell proliferation was found to be reversible and was not due to a decline in cell viability [Ebert et al, ∑

Immunol.. 138: 2161-2168 (1987); Miescher et al, J. Immunol.. 136:1899-1907 (1986)]. Other immunosuppressive factors have been reported that include substances of 70 to 75 kd that were isolated from patients with colon carcinoma and esophageal cancer [Mohagheghpour et al, J. Immunol.. 132: 1350 (1979); and Remacle-Bonnet et al, J. Immunol.. 117: 1145 (1976); see, also, US Patent No. 5,556,763]. None of the above identified suppressor factors are associated with inducing an alteration in the expression of the signal transducer TCR-C-

There remains a need in the art for the identification of immunosuppressive agents, other than currently employed immunosuppression pharmaceutics, which suffer from well-known dosage and toxicity disadvantages for use in therapies requiring immunosuppression. There also remains a need in the art for other methods and compositions suitable to readily identify an immunosuppressed state of a diseased patient.

Summary of the Invention

In one aspect, the invention provides a proteinaceous composition isolated from the serum of a human having prostate cancer having a molecular weight of approximately 105,000 to 112,000 daltons, as determined by SDS polyacrylamide gel electrophoresis. This composition and components, such as polypeptides further purified therefrom, have the ability to induce immunosuppression in a culture of normal human peripheral blood lymphocytes (PBLs) by reducing the activation/proliferation response of the PBLs and inducing an alteration in the expression of the signal transducer, TCR-C. This composition is further characterized by its ability to suppress cell proliferation. In another aspect, the invention provides a method for providing an immunosuppressive proteinaceous agent by isolating from the peripheral blood of a patient having prostate cancer a biological material which bands on an SDS polyacrylamide gel at a molecular weight of between about 105 to about 112 kD, wherein said band is not present in the peripheral blood of normal patients. The method may further involve purifying the material by extracting the protein band from the gel, digesting it, separating the peptide fragments and sequencing the immunosuppressive polypeptide or fragment therefrom. In yet a further aspect, the invention provides an immunosuppressive composition produced by the method described above. The composition may be a polypeptide or fragment thereof having immunosuppressive activity.

In still a further aspect, the invention provides a therapeutic composition comprising the immunosuppressive compositions or components described above in a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method of inducing immunosuppression in a cell culture comprising contacting said cell culture with the immunosuppressive composition or components described above.

In yet another aspect, the invention provides a method of inducing immunosuppression in a mammal comprising administering to said mammal an effective amount of the immunosuppressive composition or component described above.

In still another aspect, the invention provides a method for monitoring the degree of immunosuppression in a patient comprising detecting the level of the immunosuppressive composition in PBLs of a patients before, during and after appropriate therapy and comparing such level to the level of a control subject..

Another aspect of the invention is a method for diagnosing the presence of occult cancer cells by detecting and measuring the level of the immunosuppressive composition described above in the peripheral blood of a patient. Still another aspect of the invention is a method for screening a test compound for use in treating immunosuppressed patients, comprising the steps of: (a) contacting the immunosuppressive composition or a component thereof as described above with a test compound; and (b) assessing the ability of the test compound to bind the composition, thereby blocking the immunosuppressive activity of the composition. Yet another aspect of the invention is a composition capable of inducing an immune response, preferably a protective immune response, to a cancer cell which carries on its cell surface, the immunosuppressive composition or component described above. This composition includes an effective immune response inducing amount of the immunosuppressive composition or component, and when administered, induces a response which blocks the immunosuppressive activity of the composition on the cell surface. Methods of administering this composition are also included.

Other aspects and advantages of the present invention will become apparent from the detailed description of the invention which follows.

Brief Description of the Drawings

Fig. 1 is a photograph of the SDS-PAGE gel illustrating the isolation of the unique 105-112kD band immunosuppressive composition of the present invention, from prostate cancer patient #4. The columns on the gel, from left to right are Col. 4: molecular weight standards: 205kD, 116kD, 97.4kD, 68kD, and 45kD; col 5: human serum from prostate cancer patient #4; col. 6: human serum from normal donor #2; col

7: human serum from prostate cancer patient #5 with low TCR-C levels; col. 8: human serum from normal donor #7.

Fig. 2 is a photograph of the native PAGE gel of the serum of cols. 5, 6, 7 and 8. These columns contain the same serum as identified in Fig. 1. Fig. 3 A represents the pattern of expression of the antigen, TCR-C, in normal donor A PBL, after culture for 48 hours in autologous serum.

Fig. 3B represents the pattern of expression of the antigen, TCR-C, in normal donor A PBL, after culture for 48 hours in the presence of prostate cancer serum obtained from patient X. Fig. 4 A represents the pattern of expression of the antigen, TCR-C, in normal donor B PBL, after culture for 48 hours in autologous serum.

Fig. 4B represents the pattern of expression of the antigen, TCR-C, in normal donor B PBL, after culture for 48 hours in the presence of prostate cancer serum obtained from patient X. Fig. 5 A represent the enhanced expression of the activation-associated antigen,

CD25, in normal donor A PBL after culture for 48 hours in autologous serum.

Fig. 5B represents the changes in the expression of the activation-associated antigen, CD25, in normal donor A PBL after culture for 48 hours in the presence of prostate cancer serum obtained from patient X. Fig. 6 A represents the enhanced expression of the activation-associated antigen, CD25, in normal donor B PBL after culture for 48 hours in autologous serum.

Fig. 6B represents the changes in the expression of the activation-associated antigen, CD25, in normal donor B PBL after culture for 48 hours in the presence of prostate cancer serum obtained from patient X. Fig. 7 A is a flow cytometry stain illustrating pattern of the dual expression of the antigens, TCR-C/CD25 in normal donor A PBL after 48 hours culture in autologous serum. This staining represents the normal response to an activation stimulus and demonstrates a significantly greater number of positive cells than can be seen in the sample cultured with patient serum. Fig. 7B is a flow cytometry stain illustrating the changes in the pattern of the dual expression of the antigens, TCR-C/CD25 in normal donor A PBL after 48 hours culture in the presence of prostate cancer serum obtained from a PC A patient with reduced ζ chain.

Fig. 7C is a flow cytometry stain illustrating pattern of the dual expression of the antigens, TCR-C/CD25 in normal donor B PBL after 48 hours culture in autologous serum. This staining represents the normal response to an activation stimulus and demonstrates a significantly greater number of positive cells than can be seen in the sample cultured with patient serum.

Fig. 7D is a flow cytometry stain illustrating the changes in the pattern of the dual expression of the antigens, TCR-C/CD25 in normal donor B PBL after 48 hours culture in the presence of prostate cancer serum obtained from a PCA patient with reduced ζ chain.

Fig. 8 is a graph illustrating the TCR-C expression in a population of normal donors with no known disease process (n= 60; •) and in prostate cancer patients (n =22; Δ).

Fig. 9A is a dual parameter histogram of TCR-C-RD1 vs. CD3-FITC staining of PBL from a representative normal donor .

Fig. 9B is a dual parameter histogram of TCR-C-RD1 vs. CD4ECD staining of PBL from a representative normal donor. Fig. 9C is a dual parameter histogram of TCR-C-RDl vs. CD8-PC5 staining of PBL from a representative normal donor.

Fig. 9D is a dual parameter histogram of TCR-C-RDl vs. CD16-FITC staining . of PBL fro a representative normal donor.

Fig. 9E is a dual parameter histogram of TCR-C-RDl vs. CD3-FITC staining of PBL from a representative prostate cancer patient.

Fig. 9F is a dual parameter histogram of TCR-C-RDl vs. CD4ECD staining of PBL from a representative prostate cancer patient.

Fig. 9G is a dual parameter histogram of TCR-C-RDl vs. CD8-PC5 staining of PBL from a representative prostate cancer patient. Fig. 9H is a dual parameter histogram of TCR-C-RDl vs. CD16-FITC staining of PBL from a representative prostate cancer patient.

Fig. 91 is a dual parameter histogram of TCR-C-RDl vs. CD3-FITC staining of PBL from a second, representative prostate cancer patient.

Fig. 9J is a dual parameter histogram of TCR-C-RDl vs. CD4ECD staining of PBL from a second, representative prostate cancer patient.

Fig. 9K is a dual parameter histogram of TCR-C-RDl vs. CD8-PC5 staining of PBL from a second, representative prostate cancer patient.

Fig. 9L is a dual parameter histogram of TCR-C-RDl vs. CD16-FITC staining of PBL from a second, representative prostate cancer patient. Fig. 10 is a bar graph of the mean values of TCR-C expression in prostate cancer patients vs. normal donors. Decreased expression of TCR-C i patient PBL was not confined to T Lymphocytes (CD3 + and CD4 + or CD8 +) but was reduced in patient natural killer (CD16 +) cells as well. Distribution of the PBL subset populations for both patient and normal populations were within the range of normal donors.

Fig. 11 A is a dual parameter histogram of PCNA-FITC vs. DNA staining of PBL from a normal donor.

Fig. 1 IB is a dual parameter histogram of PCNA-FITC vs. DNA staining of PBL from a second normal donor. Fig. 11C is a dual parameter histogram of PCNA-FITC vs. DNA staining of PBL from a third normal donor.

Fig. 12A is a dual parameter histogram of PCNA-FITC vs. DNA staining of PBL from a prostate cancer patient

Fig. 12B is a dual parameter histogram of PCNA-FITC vs. DNA staining of PBL from a second prostate cancer patient.

Fig. 12C is a dual parameter histogram of PCNA-FITC vs. DNA staining of PBL from a third prostate cancer patient.

Detailed Description of the Invention The present invention addresses the need in the art by providing a proteinaceous immunosuppressive composition, peptides and polypeptides purified therefrom. Although this composition was initially identified in and isolated from the peripheral blood of human prostate cancer patients, the invention also encompasses recombinant and synthetic products prepared from the polypeptides purified from this composition. This composition and/or the above components are useful in methods for the treatment, diagnosis and/or prevention of certain disease conditions or disorders.

Z 77?e Immunosuppressive Composition

The inventors isolated a new, heretofore unidentified proteinaceous composition from the serum of a human having prostate cancer (PCA), which has a molecular weight of approximately 105,000 to 112,000 daltons, as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) with a 4- 15% gradient. More preferably, the composition has a molecular weight of about 109 kD on the SDS-PAGE. This proteinaceous composition has not been found in serum from normal humans, and interestingly, is also absent from the serum and PBLs of subjects with advanced prostate cancer. See, e.g., the SDS-PAGE gels of Figs.1 and 2. Also encompassed by the present invention is a polypeptide or fragment thereof produced by further purification to homogeneity from the proteinaceous composition on the SDS-PAGE gels, which polypeptide or fragment thereof has immunosuppressive activity. As used herein, "fragment" of the polypeptide of the present invention refers to any sequence of amino acids found within the proteinaceous compositions which demonstrates in a suitable assay, immunosuppressive activity on normal PBLs. Fragments of the immunosuppressive polypeptide which share the same biological activity of the full-length protein as well as the DNA sequences which encode the polypeptide or fragments thereof may also be employed as the immunosuppressive composition. The full-length immunosuppressive polypeptide and biologically active fragments may be obtained by conventional molecular biology engineering methods. Such identification of the full length polypeptide and suitable biologically active fragments of the immunosuppressive composition for use in the methods and in a variety of therapeutic, vaccine, and diagnostic compositions of this invention involves only a minor amount of routine experimentation. Such a polypeptide or peptide of this invention may readily be obtained by isolating from the peripheral blood of a patient having prostate cancer the biological material described above, which bands on an SDS polyacrylamide gel at a molecular weight of between about 105 to about 112 kD, as described by the inventors. Further purification of the specific polypeptide may be accomplished by one of skill in the art with resort to known purification methodology generally used for protein separation, including, without limitation, extracting the protein/polypeptide from the band by, e.g., transferring the electrophoretically separated protein onto nitrocellulose, cutting out and digesting (i.e., salting out) the protein-containing region, separating protein fragments by centrifugation, dialysis, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse-phase chromatography, hydrophobic chromatography, chromatofocusing, electrophoresis, ultrafiltration, lyophilization, preparative isoelectric focusing, or chromatofocusing.. Sequencing of the fragments, and hence of the polypeptide is accomplished preferably using a tandem mass spectrometer. See, e.g., the procedure outlined in Example 3 below.

The resulting polypeptide or peptide sequences may then be prepared synthetically, or may be prepared using recombinant techniques or a combination of both techniques. Polynucleotide sequences encoding such polypeptides and peptides of this invention may also be obtained using similar known techniques. For example, a polynucleotide of the present invention encoding an immunosuppressive protein/polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA from cells as starting material. The present invention encompasses all such DNA sequences encoding an immunosuppressive polypeptide of this invention.

The polypeptide/peptide fragments are then tested for immunosuppressive activity in a variety of assays, including particularly administering them to normal cultures of PBLs, and observing the immunosuppressive effects described herein for the composition. See, e.g., the functional assays described in detail in Example 1 below.

This proteinaceous composition of the invention, and the polypeptides and fragments sequenced therefrom, induce immunosuppression in a culture of normal human peripheral blood lymphocytes (PBLs). As used herein "immunosuppression" refers to the suppression of a normal immunologic response in an animal, e.g., the behavior of B cells or T cells under normal conditions. Immunosuppression often accompanies tissue or organ transplants, tumor growth, or is the result of the use of physical or immunologic agents, or a disease process, and typical immunosuppressive events are well-known to those skilled in the art. The immunosuppressive effect of the composition on normal PBLs is demonstrated by one or more of the following events in the cell culture. The PBLs demonstrate a reversible reduction in the expression of the TCR C chain of T-cell receptors in the presence of the composition. Surprisingly, isolation and ex vivo culture of PBLs from prostate cancer patients caused the expression of TCR-C to be restored to the range observed in normal donors. The normal PBLs in the presence of the composition demonstrate a loss or reduced expression of activation markers. The PBLs also demonstrate a loss or reduced rate of proliferation associated antigen markers on the PBLs. In general, PBLs exposed to the composition demonstrate reduced activation or a reduced proliferative state or potential. By "activation" is meant the stimulation of the cells to behave in a certain manner, i.e., to proliferate in culture, to become cytotoxic, or to produce a substance, or to demonstrate the altered expression of cell-associated antigens.

As discussed in detail in Example 1 and reported in the figures identified therein, the inventors discovered an immunosuppressive composition in the serum of a patient with prostate cancer, which caused a decrease in the activation response of PBL isolated from normal, disease-free individuals. This decreased activation response, as measured by the expression of the interleukin 2 receptor (CD25), coincides with a diminished expression of the signal transducing molecule, TCR-C- The decrease in ζ expression indicates an alteration in the signal transduction pathway of these PBL, resulting in an inefficient and impaired response to activation stimuli. Briefly summarizing some results reported in Example 1, the normal range of TCR-C staining in normal subjects is 65 to 95 percent positive. TCR-C staining observed for the normal donors cultured in autologous serum equaled a mean value of 52.65 percent, in allogeneic serum, a mean value of 63 percent and when exposed to serum of a prostate cancer patient, a mean value of 28.35 percent. Similar changes were observed in the CD25 maker as described in Example 1, i.e., the range of expression for CD25 staining in normal donors was 75 to 100% positive, for the normal donors cultured in autologous serum about 79%, for normal donors in allogeneic serum, about 81% percent and for normal donors culture in prostate cancer patient serum, about 33.6%. In PBL isolated from 22 prostate cancer patients at various stages of the diseases, staining of TCR-C was found to had been significantly reduced (p< . 000001).

Mean percent positive of ζ in prostate cancer patients equaled 42.16 percent. In contrast, mean percent positive of ζ in normal donors equaled 82.4 percent. In addition, in a comparison of the initial C values of PBL from prostate cancer patients to the values obtained from PBL of those same donors isolated from whole blood, revealed a striking difference. In 8 out of 16 patients whose initial measure of TCR-C chain expression was significantly reduced, the expression of the ζ chain was recovered after 48 hour culture in serum-free control medium. The mean value of ζ in these patients equaled 31.9 when tested at zero hour. After removal from whole blood, the mean value of ζ in these same patients increased to 67.54%, indicating the presence of the immunosuppressive composition of this invention.

This immunosuppressive composition identified by the present invention, and once sequenced by conventional techniques has a variety of utilities, i.e., as a therapeutic immunosuppressive, as a vaccine agent, and as a diagnostic agent as described below. The composition also has utility in methods for developing ligands which bind to it and can themselves be useful in therapy and in assay methods.

IL_ Ligands to the Immunosuppressive Composition

Ligands developed to the immunosuppressive agent may be employed in diagnostic assays for detecting levels of the immunosuppressive protein in cells and tissues. As used herein "ligand" includes any protein or protein analogue which binds specifically to an appropriate epitope of the T cell receptor. Antibody also includes any protein or protein analogue which binds specifically to a TCR subunit protein, Fceγ, or a protein in the T-lymphocyte signal transduction pathway.

Such ligands to the immunosuppressive composition of the present invention may be developed by resort to conventional techniques. The 105-112 kD material, a polypeptide purified therefrom or fragments thereof, or cells expressing them can be used as immunogens to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies, as well as chimeric, single chain, and humanized antibodies, Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the immunosuppressive composition or its purified polypeptide can be obtained by direct injection of the polypeptides into an animal or by administering the composition to an animal, preferably a nonhuman. The antibody so obtained will then bind the immunosuppressive composition or polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can also be used to isolate the polypeptide from tissue expressing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique [G. Kohler and C. Milstein, Nature. 256:495-497 (1975)], the trioma technique, the human B-cell hybridoma technique [Kozbor et al., Immunology Today. 4:72 (1983)], and the EBV-hybridoma technique [Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pg. 77-96, Alan R.

Liss, Inc., (1985)].

Techniques described for the production of single chain antibodies [U.S. Patent No. 4,946,778] can also be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

This invention also encompasses ligands which bind to the 105-112 kD immunosuppressive composition of this invention, which are not antibodies. For example, genes encoding proteins for binding molecules to the immunosuppressive composition of this invention can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Such methods are described in many laboratory manuals such as, for instance, Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1, Chapter 5 (1991).

The above-described ligands or antibodies may be employed to isolate or to identify clones expressing the immunosuppressive composition or polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography. They may also be employed in diagnostic methods. III. Therapeutic Methods Employing the Composition

This immunosuppressive composition and the polypeptide or peptides purified therefrom are useful in therapeutic methods for obtaining an immune suppressed state in certain subjects in need thereof. The immunosuppressive composition or polypeptide may be formulated in a therapeutic composition and administered to a human or other mammalian subject to create a directed, controlled immunosuppressed state that would eliminate undesired immune responses. Such a therapeutic composition could replace the currently employed drugs such as cyclosporin. Conditions in which such treatment would be useful include pre- and post-transplant patients, who require management of transplant rejection or graft vs. host disease. Certain auto-immune disorders also require induced immune suppression for therapy.

Such disorders include, without limitation, multiple sclerosis, lupus, rheumatoid arthritis and Type 1 diabetes, among others. A state of immune suppression may also be required in therapies for other immune dysfunctions and disorders, such as cancer, tumors, and bone marrow depletion. For example, compositions of the present invention may mediate suppression of tumor growth if when provided to a mammalian subject, the administration results in either a decrease in the proliferation of tumor growth, and inhibition of the formation of metastases, a decrease in tumor burden, or a reversal of the course of cancer disease. Methods of employing the composition of this invention may involve both in vivo and ex vivo treatment of mammalian cells. For use in the various therapies described above, the composition or polypeptide of the present invention is desirably formulated with a suitable pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers may comprise excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically, these will be readily apparent to those of skill in the art. Suitable formulations containing the compositions of the present invention, for example, include aqueous solutions of the active compounds in water-soluble form such as water-soluble salts, or normal saline plus albumin. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oils, or synthetic fatty acids esters, for example, ethyl oleate or triglycerides. Aqueous injections suspensions may contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

Alternatively, such compositions may include conventional delivery systems into which protein/polypeptide of the invention is incorporated. In still another alternative, such therapeutic compositions may contain other active therapeutic ingredients useful to treat the subject's condition or disorder, e.g., chemotherapeutics.

These therapeutic compositions comprising the present invention may be administered in any manner known to the art. Typical modes of administration include by injection, infusion or implantation. Such therapeutic compositions of the present invention are administered to a mammalian subject in amounts effective to accomplish the immunosuppression desired for a particular patient. Thus, the amounts and dosages, as well as the frequency and routes of administration are factors to be determined by the attending physician, taking into account factors such as the patient's disorder, age, state of health, concurrent treatments, level of immunosuppression desired, etc. Repeated dosages may be administered. In general, the compositions are administered in an amount of at least about 10 mg/kg body weight. In most cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day. Preferably, in most cases, the administered dose is from about 10 mg/kg to about 1 mg/kg body weight, daily. Determinations of dosage and administration regimens are within the skill of the art, given the information provided herein.

IV. Methods of Inducing an Immune Response to Cancers

This immunosuppressive composition of the invention and the polypeptides/peptides derived therefrom may also be useful in vaccines for inducing an immune response, preferably a protective immune response, to cancer cells carrying the 105-112 kD immunosuppressive component of the invention on the cell surface. This immune response to the tumor antigen can cause regression of existing tumors and/or prevents the development of cancers. Among such cancers are prostate cancer, and possible colorectal and renal cancers. Still other cancer cells or tumors may also carry the 105-112 kD component on the surfaces of their cells.

In the event of such a cancer, and particularly where such a cancer is inheritable, the compositions of the present invention may be useful in preventing the occurrence of such cancers, or at least ameliorating some of the effects there of. In one embodiment, such a composition contains an effective amount of the 105-112 kD composition or a purified component thereof. As described above, the composition could be a proteinaceous composition isolated from the serum of a human having prostate cancer having a molecular weight of approximately 105,000 to 112,000 daltons, as determined by SDS polyacrylamide gel electrophoresis, and having the ability to induce immunosuppression in a culture of normal human PBLs. Alternatively, the composition could be a purified component thereof having a molecular weight of 109 kD; or a polypeptide or fragment thereof produced by extraction and sequencing. Desirably a composition for inducing an immune response to such cancer cells optionally contains conventional suitable vaccine adjuvants and pharmaceutically acceptable vaccine carrier.

In another embodiment, a vaccine composition of this invention may include recombinant virus vectors and DNA 'vaccine' compositions, e.g., expression vectors or naked DNA, containing polynucleotides encoding a polypeptide or peptide purified from the 105-112 kD immunosuppressive composition. Conventional genetic engineering techniques are employed to prepare the virus recombinants or naked DNA. See, for example, Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d edition, Cold Spring Harbor Laboratories, NY (1989); J. Cohen, Science. 259:1691- 1692 (March 19, 1993); E. Fynan et al, Proc. Natl. Acad. Sci.. 2Q: 11478-11482 (Dec.

1993); J. A. Wolff et al, Biotechniques. 11:474-485 (1991); International Patent Application PCT WO94/01139, published January 20, 1994, all incorporated by reference herein. Methods of administering these compositions, optionally with one or more selected chemokines, cytokines or combinations thereof, can induce an anti-tumor immune response. The administration of such a vaccine composition to a subject that has not yet developed a cancer or tumor cell carrying the immunosuppressive 105-112 kD component would permit the normal immune system to recognize the composition as "non-self, and develop a protective immune response, preferably a cytolytic or cytotoxic immune response that would destroy any cell carrying the composition on its cell surface. Thus, as a vaccine composition, the 105-112 kD component would operate to prime the immune system to mount an attack against a cancer cell, if after administration of the vaccine, such cancer cells would later develop in the patient. A vaccine composition according to this aspect of the invention may be administered to a human or veterinary patient, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle. A suitable vehicle is sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose.

Optionally, a composition of the invention may be formulated to contain other components, including, e.g. adjuvants, stabilizers, pH adjusters, preservatives and the like. Such components are well known to those of skill in the pharmaceutical art.

The vaccines are administered in an "effective amount", that is, an amount that is effective in a route of administration to provide sufficient levels of expression of the

105-112 kD component to provide a therapeutic benefit, i.e., protective immunity or tumor regression.

Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, subcutaneous, intradermal, rectal, oral and other parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the immunogen or the disease. The route of administration primarily will depend on the location and nature of the tumor being treated. Doses or effective amounts of the vaccine will depend primarily on factors such as the type of tumor, the age, weight and health of the subject, and may thus vary among animal or human subjects. For example, a prophylactically effective amount or dose of a virus vector composition according to this invention is that amount effective to induce a protective immune response without seriously negatively threatening the health of the subject. Depending on the type of vaccine composition employed, the dosages, routes of administration and need for booster vaccines are determined by the attending physician, and within the skill of the art.

Diagnostic Methods Employing the Composition and its Ligands

The presence of the immunosuppressive composition or polypeptide within the serum of a mammalian subject can indicate immune dysfunction and possibly the presence of a heretofore undiagnosed condition, such as advanced malignancy, particularly prostate cancer, but including other cancers as well, parasitic challenge, autoimmune disease, and acquired immune deficiency syndrome (AIDS), etc. The presence of this composition in diseases characterized by an impaired immune response such as AIDS, sepsis, ataxia-telangiectasia and leprosy as well as severe cytomegalo virus infections may be correlated with disease progression. In a particularly desirable embodiment, the invention provides a method for diagnosing the presence of occult cancer cells by detecting and measuring the level of the 105-112 kD immunosuppressive composition in the peripheral blood of a patient.

Additionally, the qualitative and quantitative identification of the immunosuppressive composition may be employed to monitor the effectiveness of a given therapy, e.g., a chemotherapeutic treatment, or the likelihood of a subject's response to such treatment.

A diagnostic assay in accordance with the invention for detecting expression of the 105-112kD immunosuppressive composition compared to normal control tissue samples may be used to detect the presence of a disease/disorder such as those above- recited. Assay techniques that can be used to determine levels of a protein in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Among these, ELISAs are frequently preferred. An ELISA assay initially comprises preparing an antibody specific to the 105-112kD immunosuppressive composition or a polypeptide derived therefrom, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds to the monoclonal antibody. The reporter antibody is attached to a detectable reagent such as a radioactive, fluorescent or enzymatic reagent. Alternatively, a competition assay may also be employed wherein antibodies specific to the immunosuppressive composition are attached to a solid support and the labeled composition or polypeptide and a sample derived from the host are passed over the solid support. The amount of detected label attached to the solid support can be correlated to a quantity of the immunosuppressive composition in the sample. Still other conventional diagnostic assay formats may be employed and selected for use by one of skill in the art.

Similarly, the immunosuppressive composition and/or its purified polypeptide may be employed in diagnostic assays to detect naturally occurring antibodies thereto in the serum of mammalian subjects, using conventional assays. The immunosuppressive composition can be used to isolate proteins which interact with it; and this interaction can be a target for interference. Inhibitors of protein-protein interactions between the immunosuppressive composition and other factors could lead to the development of pharmaceutical agents for the modulation of the immunosuppressive activity of this component when it naturally occurs, e.g., in certain cancers.

The present invention also encompasses diagnostic assay kits which contain suitable ligands as described above, and the immunosuppressive composition of the invention in suitable carriers, with conventional assay reagents, labels and detection apparatus. The following examples illustrate the identification and isolation of the immunosuppressive composition of the invention. These examples are illustrative only and do not limit the scope of the invention. Examples 1; Identification of an Immunosuppressive Composition

A. Biological Samples

Patient blood samples were obtained with informed consent from individuals with a histologically confirmed diagnosis of progressive prostatic carcinoma. Donor inclusion criteria included normal complete blood count, normal serum albumin, normal liver and renal function, Eastern Cooperative Oncology Group

(ECOG) performance status < 2, anticipated life expectancy of greater than 6 months and no history of recent infection or past history of autoimmune disease. Chemotherapy, radiation or cortico steroid treatment of patients was discontinued a minimum of 4 weeks prior to patient inclusion in the study . Serum was isolated from the whole blood by centrifugation and stored at about -70° C until used.

Peripheral blood lymphocytes (PBL) were obtained from blood samples drawn from healthy male donors over the age of thirty (mean age = 43 years).

B. Cell Preparation

Whole blood samples were collected using Vacutainers™ blood collection tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ ) containing EDTA/K3 anticoagulant. The blood was then stored for approximately 24 hours at room temperature. All patient samples were collected at Johns-Hopkins Hospital and shipped at room temperature in a temperature-controlled container to Coulter Corporation. PBL were sterilely harvested from the blood (EDTA/K3) of the donors using Ficoll-Paque ® reagents (Pharmacia Biotech,lnc, Uppsala, Sweden) according to the manufacturer's recommended procedure. Sample yield was then calculated and, in cases where cell yield permitted, a portion of cells was set aside for functional studies.

C. Functional Study PBL cell cultures from the normal donors were incubated in a humidified atmosphere at 37 °C and 5% C0₂ for a period of 48 hours. In one experiment, each PBL sample was divided into six portions:

(1) culture in control AIM-V medium at 2.5 X 10⁶ cells/ mL, w/ 10.0 % autologous serum;

(2) culture in AIM-V medium at 2.5 X 10⁶ cells/mL containing the activators: 0.125 μg/ml αCD3-5 aminodextran with 0.0625 μg/mL CD28 (Coulter Corporation, Miami, FL), w/ 10.0 % autologous serum;

(3) culture in control AIM-V medium at 2.5 X 10⁶ cells/ mL, w/ 10.0 % allogeneic serum;

(4) culture in AIM-V medium at 2.5 X 10⁶ cells/mL containing the activators: 0.125 μg/ml αCD3-5 aminodextran with 0.0625 μg/mL αCD28 (Coulter Corporation, Miami, FL), w/ 10.0 % allogeneic serum;

(5) culture in control AIM-V medium at 2.5 X 10⁶ cells/ mL, w/ 10.0 % prostate cancer patient serum; (6) culture in AIM-V medium at 2.5 X 10⁶ cells/mL containing the activators: 0.125 μg/ml αCD3-5 aminodextran with 0.0625 μg/mL αCD28 (Coulter Corporation, Miami, FL), w/ 10.0 % prostate cancer patient serum. In another experiment, samples were divided into two portions, each at 2.5 X 10⁶ cells/mL: one for culture in serum-free control medium and a second for culture in serum-free activation medium containing: 0.125 μg/mL CD3-dextran and 0.0625 μg/mL CD28 (Coulter Corporation, Miami, FL) in serum free AIM-V ® medium.

In each experiment, at harvest, PBL were removed from culture, counted, washed and stained with the appropriate antibodies for flow cytometric analysis. D. Flow Cytometric Analysis - Surface/internal Staining Protocol: Ficolled Samples

In one experiment, 1 X 10⁶ normal PBL in 100 μL of PBS were aliquoted into 12 X 75 mm tubes containing different four color antibody mixtures (CD4-ECD, CD25 or CD69-PC5; Coulter Corporation, Miami, FL). All samples were screened using the respective isotypic control labeled with the required fluorescent dye to detect background fluorescence. In addition, all samples were stained with a CD14/ CD45 (MO2-RD1/KC56FITC; Coulter Corporation) antibody mixture to ensure lymphocyte purity.

In another experiment, 100 μL of whole blood was aliquoted into 12 X 75 mm tubes containing different four-color CD3- or CD16-FITC/CD19-RD1/

CD4-ECD/CD8-PC5 antibody mixtures. Paired samples were stained with a mixture of isotypic controls labeled with the appropriate fluorescent dyes. In addition, all samples were stained with CD14-RD1/CD45-FITC as described above. Samples were mixed and incubated in the dark for 10 minutes. After incubation, the tubes were placed on the Multi-Q-Prep (Coulter Corporation) for simultaneous RBC lysis and cell fixation. Samples were then held at 4°C until analyzed by flow cytometry.

E. Flow Cytometry - Simultaneous Surface and Internal Staining Protocol: PBL

The following monoclonal antibodies and their respective isotype controls were utilized in this study: CD3-FITC, CD4-ECD, CD8-PC5, CD16-FITC,

CD14-RD1, CD45-FITC and TCRC-RD1 (Coulter Corporation). To simultaneously analyze both cell surface and internal marker expression, 200 μL of a solution containing 1 X 10⁶ WBC was aliquoted into tubes containing the different three color (FITC/ECD/PC5) surface marker antibody mixtures. Samples were then vortexed and kept in the dark for 10 minutes at room temperature. After incubation, the cells were washed in PBS (Coulter Corporation) and pelleted by centrifugation (5 minutes at 400g). The cells were then assayed for TCR ζ expression using a modification of a permeabilization protocol [Anderson et. al, J. Immunol., 143:1899-1904 (1989)] was used: after centrifugation, the cells were resuspended in 200 μL of a 500 μg/mL working solution of digitonin (Sigma Chemical Company, St. Louis, MO) in phosphate-buffered saline (PBS, Coulter Corporation) made immediately prior to use from a stock solution of 25 mg/mL digitonin in PBS containing as the internal marker antibody either TCR-C (monoclonal antibody, TCR-C, clone TIA-2, Coulter Corporation) conjugated to RD1 or the isotype control (MslgGl-RDl, Coulter Corporation). Samples were then mixed and incubated in the 1 dark for 10 minutes at room temperature. After incubation, cells were washed in PBS and centrifuged (300g, 5 minutes). The cells were resuspended in PBS and held at 4°C in the dark until screened by flow cytometry.

F. Functional Analysis

After 48 hour culture in either control or activation media, 1 X 10⁶ cells were aliquoted into 12 X 75 mm tubes containing different three-color antibody mixtures (CD4-ECD, CD25 or CD69-PC5, CD8FITC). All samples, where cell yield permitted, were stained with a CD 14/ CD45 antibody mixture to determine lymphocyte purity. Otherwise, all samples were processed as described above (Flow Cytometric Analysis).

G. Sample Analysis All blood samples were run on an EPICS® XL/MCL (Coulter

Corporation) flow cytometer equipped with an air-cooled 488 nm Argon laser. Green (FITC) fluorescence reflected by a 550 nm dichroic longpass filter and passed through a 525 nm bandpass filter. PE (phycoerythrin) emission was reflected by a 600 nm dichroic longpass filter and passed through a 575 nm bandpass filter. Orange (RD1) fluorescence was collected through a 575 nm bandpass. Red (ECD) fluorescence was reflected by a 645 nm dichroic longpass filter and passed through a 620 nm bandpass filter. Dark red fluorescence (PC5; phycoerythrin-cyanin 5.1) emission was transmitted through a 645 dichroic longpass filter and subsequently through a 675 nm bandpass filter. Data were displayed as dual-color dot plots to measure the distribution of the peripheral lymphocyte populations and the distribution of the TCR-C antigen within the various cell subsets. H Statistical Analysis.

Probability values for differences among mean responses were deteπnined using one-sided Student's t-tests. The relationships between CD3 and CD16 staining, and TCR-C expression and between donor age and ζ expression were tested using the Pearson product moment correlation coefficient method. Linear regression analysis was utilized to establish whether a relationship existed between the two variables of donor age and ζ expression in PBL. All statistical values were calculated using Microsoft Excel ® software, version 5.0. I. Results

As demonstrated in Fig. 10, no significant differences (p > 0.05) were found in lymphocyte subset distributions between the normal and prostate cancer patient whole blood samples using antibodies to the peripheral blood subset markers CD3, CD4, CD8, CD 16 and CD 19. Both donor populations displayed cell distributions within the normal ranges established for disease-free individuals: CD3 positivity averaged 81.5 for normal donors and 72.6 for patients, while CD16 positivity averaged 15.97 for normal donors and 19.15 for patients.

As illustrated in Fig. 8, the flow cytometric analyses to detect the expression of TCR-C in PBL obtained from prostate cancer patients in all clinical stages were compared to those obtained from control PBL of healthy male donors (n = 60).

Among the normal population only minimum variability in ζ expression was observed. The mean of total fraction positive for TCR-C staining was 81.6 % (standard deviation: 7.54 %; standard error of the mean (SEM): 0.97 %). The distribution of TCR-C within PBL of normal donors was consistent and closely approximated the percentage of T Cells and NK cells. However, the cancer patients showed much variability with a mean of

43.1 % (SEM: is 5.94 %) (p~ 0.000001); variation in ζ expression within the patient population ranged from a high of 85.5 % to the almost complete absence of the marker observed in 2 of the patients. The difference in the mean values of the two groups is statistically significant (Student's t test). A markedly reduced expression of TCR-C was observed in peripheral T cells and NK cells in 14 of 22 prostate cancer patients. No significant correlation was observed between TCR expression and prostate cancer antigen (PCA) patient serum antigen (PSA), age, clinical stage, Gleason grade or treatment history.

As illustrated in Figs. 9A through 9L, multicolor analysis in patients revealed that the expression of the TCR-C antigen was markedly reduced in all cell types examined, including CD3 (Figs. 9A, 9E and 91), CD4 (Figs. 9B, 9F, and 9J) and CD8

(Figs. 9C, 9G, and 9K) positive T cell subsets as well as in CD 16 positive NK cells (Figs. 9D, 9H and 9L). The mean fluorescent intensity (MFI) of TCR-C staining was significantly lower in patient samples (Figs. 9E-9L), averaging 3.41 (SEM = 0.398) than that observed in normal samples (Figs. 9A-9D) which averaged 6.73 (n=60, SEM = 0.37, p -0.000001).

The response of peripheral blood from prostate cancer patients and normal donors for T cell activation in the presence of activating reagents was measured using the expression of the "early" activation marker, CD69 as well as the "late" activation marker, CD25. Conjugation of the antibody to dextran results in cross-linking of the receptors, increasing cellular response. The addition of antibodies to CD28 provides the auxiliary stimulus that ensures cell activation and eventually cell proliferation. Patient PBL were considered to be reactive to the activation stimuli if the percent positive expression of CD25 was within 1 standard deviation of the normal mean. Response to the activation reagents (48 hour stimulation with αCD3 dextran and αCD28) in the patient samples was mixed as demonstrated in the following Table 1. In the table, percent positive values for all reagents were determined using an isotypic control according to standard procedures. Not all markers could be assessed due to insufficient cell yδeld upon harvest after 48 hour culture. Patient #7 was excluded due to insufficient blood sample. Table 1

Correlation with C at 0 hour: 0.5251 0.8618 0.4800 Functional alterations in patient peripheral T cells are evidenced by the muted response to activation stimuli, indicating that the normal sequence of signal transduction induced by activation has been altered or neutralized. Six patients in the study activated in a manner comparable to the normal donors. Four patients demonstrated a somewhat reduced response, just below the cutoff value, and five patients had a markedly reduced response to stimulation. Expression of CD25 in stimulated patient PBL averaged

41%; altogether, patients' response to activation stimuli was significantly less than that observed in normal donors (p<0.0006). Expression of CD25 positive cells in the normal donor population averaged 70.15% and ranged from 15.3 to 95.7 %.

The results may be observed in Figs. 3A, 3B, 4A, 4B, 5A, 5B and 7A-7D. Overall, the expression of CD69 in 48 hour-activated patient PBL averaged 28.1 % (SEM

= ± 4.97 %) whereas in the normal donors, expression of CD69 positive cells ranged from a low of 11.7 % to a high of 86.3 % and averaged 44%. Again, patient response to activation stimuli, as measured by this marker, was less than that observed in normal donors, although the differences in CD69 positive staining were less extreme (p< 0.020) than observed in CD25 reactivity. The appearance of the CD69 antigen is considered to be an early activation marker, reaching its maximum expression as early as 24 hours post mitogen stimulation. Despite this characterization, CD69 staining was included because the reactivity of the patient PBL to activation stimulus might be delayed, resulting in the appearance of an "early" activation marker at later times. Both CD69 and CD25 positivity in patient stimulated PBL was approximately 60%, or a little less than two-thirds, of that observed in normal donors. Neither the CD69 or CD25 expression observed was confined to either the CD4 or CD8 populations, but was usually evenly distributed within both. As the data indicate, not only do T cells from the majority of PCA patients have reduced TCR-C chain expression, but this reduction results in a significant (p<0.0006) decrease in activation potential, as measured by the expression of both early (CD693 and late (CD25) activation antigens.

Consistent with what had been reported in previous studies, TCR-C expression was reduced in activated PBL of normal donors. The average percent positive equaled 53 (SEM= ± 3.9), a decrease of approximately 30 percentage points from the mean value obtained at zero hour. Changes in TCR-C expression are, it appears, inversely affected by changes in CD69 or CD25 staining. In some donors the greater the activated response, the greater the decrease in TCR-C expression. However, this finding was not consistent for all subjects tested.

Reaction to activation stimulus among patient PBL was mixed: in 8 of 16 patients, TCR-C expression decreased in a manner similar to that observed in normal donors (which presupposes that the 0 hour expression of TCR-C in these patients' PBL fell within the range characteristic of normal donors.) L contrast, 5 patients improved after 48 hours in culture. In these patients, the average increase in expression equaled 41 percent (SEM= ± 3.35). The remaining 3 patients included in the testing showed no real change in TCR-C expression after 48 hours in activation medium. Overall, the average TCR-C chain expression in the patients equaled 41 percent (SEM: 3.35).

The cell proliferation associated marker, PCNA, is known to recognize the auxiliary protein to DNA polymerase and is essential for eukaryotic cell proliferation. Expression of PCNA is considered to reflect cellular proliferation. Expression of PCNA was variable among the PC A patients tested (Table 1 and Figs. 11 A through 11C), with an average value of 13 percent. Three of the fifteen patients tested exhibited PCNA values within the normal range. In contrast, twelve of fifteen patients had little or no PCNA. Overall, the difference between normal and patient PBL PCNA staining was significant (p~ 0.0002). Curiously, a number of patients with little or no PCNA expression were positive for the markers CD25 and CD69, indicating that the cells had been activated. PCNA expression among normal donors (Figs. 11 A-C) averaged 43.2 percent. Maximum expression of the marker usually occurred between 48 and 72 hours and was variable from donor to donor. Time constraints and concerns about patient PBL viability prevented the study from being carried out beyond 48 hours.

Summarizing the results of these experiments, the changes in TCR-C expression observed in the patients' cells were not due to the isolation of a separate negative population, but an overall "shift to the left," a significant loss in brightness implying a corresponding decrease in TCR-C antigen expression. Data indicate that the reduction in ζ chain affects all T Cells and NK cells. These observations imply that the two key effector cell populations that could mount an effective attack against the tumor and eliminate it from the host, had essentially been neutralized. The systemic nature of the reduction in TCR-C chain indicates that whatever the cause, its effect is pervasive. The tumor releases a factor capable of inducing downregulation of the immune system, and the dissemination of this factor is directly related to the invasiveness of the tumor. The data instead indicate no correlation between TCR-C chain expression and the extent of tumor invasion. The immunosuppressive factor likely circulates within the serum of a PCA patient, and, as this data indicate at least within one patient, such a factor alters the response of normal PBL to activating stimuli as well as decreasing the expression of TCR-C-

Subjecting patient PBL to activation stimuli allows the measurement of effector cell function. Removal of patient PBL from blood allows the TCR-C chain to return, identifying the presence of a systemic immunosuppressive factor, presumably secreted by the tumor.

Example 2: Isolation of the Immunosuppressive Factor by Gel Electrophoresis

Control cultures of PBL were set up with every activated culture sample and consisted of a suspension of cells from the same initial population, at the same cell concentration, in serum-free media minus the activating reagents. These cultures were incubated for the same duration under identical conditions as the activated cultures. Comparing the TCR-C staining patterns of these 48 hour control cultures from 0 hour normal donor PBL revealed few if any differences (p ~ 0.1645). Comparing 0 hour control cultures to 48 hour control cultures from PCA patients however, revealed a striking difference: in 8 of 16 patients whose initial TCR-C chain expression was significantly reduced, the expression of the TCR-C chain recovered after 48 hour culture in serum-free control medium. The mean value of in these patients equaled 31.9 % when initially tested, directly after Ficoll isolation. After 48 hours in culture, the mean value of TCR-C i these same patients increased to 67.5 %. Within these populations of patient PBL, there was no evidence of either activation or cell proliferation. It is unlikely, therefore, that a TCR-C-positive population of PBLs might have "grown out." Rather, it would seem that the reappearance of TCR-C is due to the dilution or removal of a patient (tumor)derived immunosuppressive factor that is capable of altering TCR-C chain expression. Normal PBL (n = 2 donors) were cultured in the presence of serum taken from a patient with a markedly decreased C expression (7.5 % positive). These cells were suspended in media containing the activating agents ( CD3 -aminodextran and αCD28) and patient serum at a concentration of 10%. Control PBL from the same normal donors were cultured in 10% allogeneic serum from an independent normal donor or in a separate culture with 10% autologous serum using the same medium containing the activating reagents. All other culture conditions were kept identical. As observed in Figs.5B, 6B, 7B and 7D, PBL cultured with patient serum demonstrated a significant decrease in CD25 expression: from 79 % and 81 % in PBL cultured of autologous and allogeneic serum respectively, to 34 % in the culture containing PCA patient serum. Even more noteworthy, TCR-C chain expression was sharply reduced in these cells when compared to PBL cultured in either allogeneic or autologous serum. TCR-C expression changed from 53 % and 63 % in PBL cultured in autologous or allogeneic serum respectively, to 29 % in the culture containing PCA patient serum. These data indicate that there was a tumor-derived or induced substance in the serum of these prostate cancer patients that effectively reduces TCR-C chain expression and alters activation response.

To isolate this factor, serum isolated from the blood samples prepared as described in Example 1, along with the molecular weight standards, were run according to the PHASTSystem® process (Pharmacia Biotech, Uppsala, Sweden) as recommended by the manufacturer. Essentially, the samples are diluted to 1.0 mg/ml in distilled water and then in sample buffer. Sample buffer consists of 10 mM Tris/HCl

(Trizma base, Sigma, St. Louis, MO), 1 mM EDTA ( Ethylenediaminetetraacetic acid, Sigma, St. Louis, MO ), 2.5% SDS (Sodium Dodecyl Sulfate, Bio-Rad Laboratories, Hercules, CA) and 0.01% BPB (Bromophenol Blue, Bio-Rad).

For molecular weight standard preparation, a high molecular weight standard 1 :20 was diluted in sample buffer. The sample was heated for about 5 minutes at about

100°C and cooled to room temperature. 5 % DL-Dithiothreitol (Sigma) was added, then the sample was heated for about 5 minutes at about 100°C and cooled to room temperature. The appropriate separation and development methods were programmed into the PhastSystem® process according to the manufacturer's recommended procedures. The staining solution consisted of one tablet of PhastGel Blue (Pharmacia) dissolved into 120 mL of methanol (Sigma). This was subsequently filtered. After the gels completed the staining process, they were air dried and scanned by using the Shimadzu CS 9000 U dual wavelength flying-spot scanner (Shimadzu Scientific Instruments,

Columbia, MD), following the manufacturer's recommended protocol.

The molecular weight was calculated as follows: the migration distance of the molecular weight standard proteins was measured and the Rf value was determined according to the following formula: Rf = Distance of the band from the origin/Distance from the origin to reference point

The same reference point was used to calculate every protein in the gel. The Rf value was plotted for the molecular weight standard against the logarithms of their molecular weight and the semi-logarithm regression curve was calculated. The Rf values for the sample proteins were calculated. The corresponding logarithms of the molecular weight was interpolated from the calibration curve. The band percent calculation was established as follows: the peak area for each band that has been integrated on the gel scan was totaled; and the individual peak area was divided by the total peak area then multiplied by 100 to give the band percentage.

As shown in the photographs of the gels in Figs. 1 and 2, the immunosuppressive composition of the present invention was identified and isolated as a protein of approximately 105,000 to 112,000 daltons. The first column, labeled Column 4 of Fig. 1 contains the molecular weight standards against which the molecular weights of the unknown serum-derived compounds from different donors are measured. Unique to the second column, labeled column 5, is a compound of 105,000 to 112,000 daltons. The immunosuppressive composition is more precisely located at a molecular weight of about 109kD. This compound was found only in the serum of prostate cancer patient #4 and was not evident in either of two normal donors or a different prostate cancer patient's serum. Example 3: Purification of the Immunosuppressive Polypeptide

To further purify the proteinaceous composition from the approximately 109kD band on the SDS-PAGE gel of Example 2 (See, Fig. 1), the following steps are taken. The electrophoretically separated protein of approximately 109kD band is cut from the gel and transferred onto nitrocellulose. The protein-containing region is cut out and digested by treatment with Staphylococcal V-8 protease. Alternatively, trypsin may be employed for this step. The resulting peptide fragment is then separated by reverse phase HPLC and sequenced in a gas sequenator.

Using another protocol, the peptide fragments following digestion are separated using capillary electrophoresis, which is connected through a microelectrospray ion source to a tandem mass spectrometer. The spectral pattern generated is compared to that generated for known protein sequences, and the sequence of amino acids is mapped. See, e.g., the procedures described in D. Figeys et al, Nature Biotech.. 14: 1580-83 (Nov. 1996); D. P. Hunt et al, Proc. Natl. Acad. Sci_r USA.. 83:6233-6237 (Sept. 1986); R. H. Aebersold et al, Proc. Natl. Acad. Sci_r USA, 84:6970-6974 (Oct. 1987); M. Wilm etal, Nature. 379:466-469 (Feb. 1, 1996), incorporated herein by reference.

This sequence of amino acids is then synthesized and tested for its immunosuppressive activity in the same manner as for the protein in Example 1.

All documents and publications referred to above are incorporated by reference herein. Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the composition and methods of the present invention are believed to be encompassed in the scope of the claims appended hereto.

Claims

WHAT IS CLAIMED IS:

1. A proteinaceous composition isolated from the serum of a human having prostate cancer having a molecular weight of approximately 105,000 to 112,000 daltons, as determined by SDS polyacrylamide gel electrophoresis, said composition having the ability to induce immunosuppression in a culture of normal human peripheral blood lymphocytes (PBLs).

2. The composition according to claim 1 wherein said immunosuppression is selected from the group consisting of :

(a) a reduction in the expression of the TCR ╬╢ chain of T-cell receptors in said PBLs;

(b) a loss or reduced expression of activation markers in said PBLs;

(c) a loss or reduced rate of proliferation associated antigen markers on said PBLs;

(d) reduced activation of said PBLs; and

(e) reduced proliferative state or potential of said PBLs.

3. The composition according to claim 1 wherein said molecular weight is 109 kd.

4. A method for providing an immunosuppressive proteinaceous agent comprising the step of: isolating from the peripheral blood of a patient having prostate cancer a biological material which bands on an SDS polyacrylamide gel at a molecular weight of between about 105 to about 112 kD, wherein said band is not present in the peripheral blood of normal patients.

5. The method according to claim 4 further comprising extracting protein from the band on said gel, digesting said protein and sequencing said protein.

6. An immunosuppressive composition produced by the method of claim 4.

7. The composition according to claim 4 which is a polypeptide or fragment thereof having immunosuppressive activity.

8. The composition according to claim 1 in a pharmaceutically acceptable carrier.

9. A composition which induces an immune response to cancer cells carrying a cell surface immunosuppressive protein comprising an effective amount of the immunosuppressive composition selected from the group consisting of:

(a) a proteinaceous composition isolated from the serum of a human having prostate cancer having a molecular weight of approximately 105,000 to 112,000 daltons, as determined by SDS polyacrylamide gel electrophoresis, said composition having the ability to induce immunosuppression in a culture of normal human peripheral blood lymphocytes (PBLs);

(b) a component of (a) having a molecular weight of 109 kD; and

(c) a polypeptide or fragment thereof produced by extraction from the composition of (a) and sequencing, which has immunosuppressive activity; an optional adjuvant and a pharmaceutically acceptable vaccine carrier.

10. A method for diagnosing the presence of occult cancer cells by detecting and measuring the level of the immunosuppressive composition of claim 1 in the peripheral blood of a patient.

11. A ligand which binds to, and blocks the immunosuppressive activity of, the immunosuppressive composition selected from the group consisting of:

(a) a proteinaceous composition isolated from the serum of a human having prostate cancer having a molecular weight of approximately 105,000 to 112,000 daltons, as determined by SDS polyacrylamide gel electrophoresis, said composition having the ability to induce immunosuppression in a culture of normal human peripheral blood lymphocytes (PBLs);

(b) a component of (a) having a molecular weight of 109 kD; and

(c) a polypeptide or fragment thereof produced by extraction from the composition of (a) and sequencing, which has immunosuppressive activity.