SERUM IMMUNOREGULATORY POLYPEPTIDE AND USES THEREFOR

Abstract: Previously unisolated serum immunoregulatory polypeptides (SIPs) having a molecular weight of approximately 14,000 to 31,000 are provided. The SIPs may be obtained from hemorrhagic serum of mammals and have been shown to activate suppressor T-cells and suppress lymphocyte proliferation and interleukin production. There is a significant homology between SIPs of different mammalian species. The SIPs have far reaching implications in allograft and transplant situations where suppression of the immune response and/or induction of tolerance are desirable, as well as in the treatment of auto-immune diseases.

Description: SERUM IMMUNOREGULATORY POLYPEPTIDE AND USES THEREFOR Background of the Invention Immunoregulatory agents are substances that regulate or modify the normal or aberrant immune response of a host animal. Such agents are extremely important in medical applications, specifically in diseases that are caused by an abnormal immune response, such as rheumatoid arthritis (RA) , systemic lupus erythrematosis (SLE) , AIDS, etc. They are also useful where it is desirable to suppress normal immune response, such as in organ transplants and other types of tissue or organ grafts. Various immunoregulatory agents are known in the art, but all have significant disadvantages. Some examples are cyclophospha ide, ethotrexate, 6-MP and cyclosporin A. These drugs suffer from various short¬ comings such as toxic side-effects and a narrow thera- peutic index that severely limits their therapeutic application. Immune response can be modified in many ways. One of the most desirable approaches is to influence or modulate the activity of suppressor T-cells. Suppressor T-cells are an important class of lymphocyte that have an important role in the regulation of immune response. These cells play an important role in the development of immuno-tolerance. Malfunction of these cells is believed to play a role in the pathogenesis of autoimmune diseases, such as RA and SLE. These diseases are believed to be caused by diminished suppressor T- cell activity and a resultant overproduction of auto antibodies. This situation may be reversed by activation of suppressor T-cells. In organ transplants, activation of suppressor T-cells, with the consequential suppression of the immune response, helps promote tolerance and prevent rejection of transplanted organs by the recipient's immune .system. Therefore, it would ;,bώ extremely advantageous to have an immunoregulatory agent that activates suppressor T-cells, thereby regulating immune response, but which has a wide therapeutic index and does not have toxic side-effects. The present invention satisfies this need and provides other related advantages. Summary of the Invention The present invention resides in the discovery of previously undescribed and unisolated relatively low molecular weight serum immunoregulatory polypeptides (SIPs) that are present in hemorrhagic mammalian blood. Different mammalian species appear to have different SIPs, and there may be more than one SIP in the blood of each mammalian species. The SIPs regulate the immune response in mammals and therefore have far reaching value in the treatment of many diseases, including allograft rejection, auto-immunity, graft versus host diseases, allergies, cancer and AIDS. One mechanism by which the SIPs function to regulate immune response is the activation of suppressor T-cells, resulting in suppression of lymphocyte proliferation. Additionally, the SIPs suppress production of ly phokines, including interleukin-2 (IL-2) . The SIPs of the present invention are low molecular weight compounds, having molecular weights of from approximately 14,000 to approximately 31,000, and can be obtained from mammalian hemorrhagic serum. Hemorrhagic blood is blood obtained from mammals following hemorrhage. Hemorrhagic serum is obtained by allowing hemorrhagic blood to clot, followed by centrifuging and subsequent removal of supernatants. Hemorrhagic serum shows immunoregulatory activity where control samples of normal serum do not. One SIP was obtained from hemorrhagic serum cf rats. The hemorrhagic ,serum was shown to suppress phytohemagglutinin (PHA) <. hduced proliferation of rat peripheral blood lymphocytes. The active fraction of the hemorrhagic serum was isolated, and activity of the serum showed that the active substance, the SIP, had a molecular weight of approximately 16,000. An SIP isolated from human hemorrhagic serum, and having similar biological activity, had a molecular weight of approximately 29,000. An SIP of the present invention was also shown to suppress the development of cell-mediated and humoral immunity in vivo SIP treated animals showed significantly lower cell-mediated and humoral immune response to EL4 cells than control animals. SIPs also suppress the proliferation of rat peripheral blood mononuclear cells (PBMC) . Human SIP was found to suppress PHA-induced proliferation of rat PBMCs. Similarly, rat SIP suppressed PHA-induced proliferation of mouse spleen cells. It thus appears that there is a substantial degree of homology between structure and function of SIP from different species. The implications of the SIPs of the present invention are far reaching and dramatic because they significantly affect the immune response. Regarding auto-immune diseases, suppressor T-cells ordinarily block the development of auto-antibodies in auto-immune diseases. There is substantial evidence indicating that auto-immune diseases such as SLE and RA involve a loss of suppressor T-cell activity. Activa- tion of suppressor T-cells using an SIP of the present invention, one mechanism by which the immune response is regulated, is useful in treating such diseases. It has been firmly established that suppressor T- cells monitor and regulate the immune response. Suppressor T-cells serve teleologically as an "off- switch" for the immune response and play an important role in establishing tolerance. For example, it has been shown that suppressor T-cells can replace response with non response at very low levels of antigen. IL-2, on the other hand, has an opposite effect. Current evidence suggests that exposure to antigen together with appropriate MHC causes unresponsiveness, and that a second signal, IL-2, is required for immunity. Thus, IL-2 converts a "tolerogenic" stimulus into an im- munogenic stimulus. By activating suppressor T-cells and decreasing the generation of IL-2, an SIP should promote the establishment of tolerance. This would make the SIPs of the present invention therapeutic in allograft rejection, graft versus host diseases, and in situations in which more subtle effects on the production of lymphokines may be important, such as in allergies or certain types of leukemias that are characterized by requiring IL-2 at certain stages. Regarding therapeutic applications, it is possible that SIPs may play a role in the pathogenesis of certain immunodeficiency diseases. Monoclonal antibodies against these SIPs may be derived and used to enhance the immune response in such situations. Preparing monoclonal antibodies directed against the SIPs also has diagnostic applications. Such antibodies would provide the means for measuring the blood levels of the SIPs, enabling the identification of subgroups of patients who are at particular risk of developing diseases of abnormal immune regulation or developing serious infections after trauma injury. The following detailed description demonstrates, by way of example, the principles and uses of the present invention. Detailed Description of the Invention The present invention is embodied in previously undescribed and unisolated serum immunoregulatory polypeptides (SIPs) of molecular weight approximately 14,000 to 31,000 that modulate the immune response in mammals, at least in part by activating suppressor T- cells. The SIPs also suppress lymphocyte proliferation and lymphokine production, including interleukin-2, in mammals. The SIPs of the present invention can be obtained from the hemorrhagic blood of various mammals, although rats and a brain-dead human were used for experimentation purposes. The SIPs of the present invention have dramatic potential for the effective treatment of a variety of conditions and diseases. For example, suppressor T- cells ordinarily block the development of auto- antibodies in auto-immune diseases, such as SLE and RA. The SIPs, by enhancing activation of these suppressor T- cells, could be useful in treating these diseases. Treatment can be accomplished by various methods. In vitro, the SIP need simply come into physical contact with the suppressor T-cells. In vivo, administration of the SIP may be by various mechanisms that function to bring the SIP into physical contact with the supressor T-cells, including but not limited to subccutaneous injection, intravenous injection, intraperitoneal injection and delivery via an appropriate pharmaceutical vehicle. The same is true for any cells that are to be treated with an SIP. Also, interleukin-2 has been known to convert a tolerogenic stimulus into an im unogenic stimulus. By activating suppressor T-cells and decreasing the generation of IL-2, the SIPs promotes tolerance, and would therefore have therapeutic use in allograft rejection cases, graft versus host diseases and other situations characterized by the requirement of IL-2 at various stages. As with suppressor T-cells, treatment can be effected in vitro or in vivo by suitable mechanism to bring the SIP into physical contact with the lymphocytes. By preparing monoclonal antibodies directed against the SIPs, which can be done using conventional techniques known in the art, the level of these SIPs in blood can be accurately measured using conventional methods. The SIPs are particularly advantageous because unlike other immunosuppressants, such as cyclosporin A, the SIPs are normal immunoregulatory proteins, and would be expected to have few significant side effects. The SIPs may be used to treat cells both in vitro and in vivo. In both cases, an effective amount of SIP must be used. For in vitro treatment, 1 ug of SIP per ml of cell medium is effective, but a lower concentration may also be used, depending on the concentration of cells in the medium. Experimentation can be used to determine the lowest effective limit for various cell concentrations. The upper limit for effective treatment is an amount that will not overwhelm the cell medium with too much activity, also determinable by experimentation. For in vivo treatment, .75 mg of SIP per 1 Kg of body weight of mammal I.P. has been found to be effective in mice, but the lowest effective limit may vary from mammal to mammal. This can be easily determined by experimentation. The upper effective limit is an amount of SIP, also determinable by experimentation, that will not cause so much biological activity in the animal so as to be harmful. The SIPs of the present invention were obtained from the hemorrhagic serum of rats and a human, but the serum of other mammals, including but not limited to rabbits, cats, dogs, horses, sheep, monkeys, etc. may be used. Hemorrhagic blood is blood obtained from mammals following hemorrhage. Hemorrhagic serum is obtained by allowing hemorrhagic blood to clot, followed by 1 centrifuging and subsequent removal of supernatants. Example 1 demonstrates how hemorrhagic serum, containing one of the SIPs of the present invention, was obtained from rats. In order to obtain hemorrhagic serum, 30% of the rats' total blood volume was bled from each animal over a period of ten minutes. The animals were permitted to recover for two hours, and additional blood samples were taken. These second blood samples constitute hemorrhagic blood, which has been produced by the animal's system after a major hemorrhage (in this case, loss of 30% of total blood volume) . Hemorrhagic serum was then obtained by clotting and removal of supernatants. Human hemorrhagic serum was similarly obtained from a brain-dead patient. It is important to note that hemorrhagic serum can be obtained using other parameters for percent of total blood volume bled and waiting time after blood loss before removing hemorrhagic blood. Although 30% of total blood volume was removed in Example 1, hemorrhagic blood can be obtained by removing as little as 1% or as much as 70% of total blood volume (more than 70% would kill the host animal prior to any opportunity to take a second blood sample) . Ten percent to forty percent is preferred. Similarly, although two hours elapsed after blood volume loss before removing hemorrhagic blood, as little as a 15 minute time period or as much as 72 hours after initial blood volume removal will also suffice. Thirty minutes to two hours is preferred. The particular method of isolation of the SIPs of the present invention from hemorrhagic blood is not critical. Isolation by chromatography is preferred, including gel elecrophoresis or isoelectric focusing (IEF) followed by eluting the SIPs from a gel. Other isolation techniques are known to those skilled in the art and may also be used. At the present time, the base sequences of the SIPs are not known. Once these, sequences are determined, however, the SIPs of the * present invention may be synthesized rather than isolated from blood. The fact that they are presently obtained from hemorrhagic blood is simply an identifying characteristic of the SIPs, and is not to be limiting. Those skilled in the art will appreciate that a synthesized SIP having substantially the same structure as an SIP generated by a mammal is within the scope of the present invention. The following examples serve to illustrate the present invention. EXA TMPLE 1 Obtaininσ of Hemorrhagic Serum The carotid artery o pentobarbital anesthetized rats is catheterized with PE 50 tubing. The tubing is tunneled through the back of the neck and secured with a jacket, tether and swivel apparatus. The rats are then placed in individual cages, given food and water ad libitum, and allowed to recuperate for a minimum of three days. The PHA-induced lymphocyte proliferation response of catheterized-recuperated animals is the same as in normal, uncatheterized control animals (1) . To produce hemorrhagic serum, a volume of blood approxi¬ mately equal to 30% of total blood volume, is bled first from each animal over a 10 minute period. Two hours later, the remaining blood is bled from each animal. Serum obtained from the second sample constitutes "hemorrhagic serum". Human hemorrhagic serum was similarly obtained from a brain-dead patient before the removal of various organs for transplantation. EXAMPLE 2 Effect of Hemorrhagic Serum on Mitogen- Induced Lymphocyte Proliferation Procedures as set forth in Example 1 were used to obtain hemorrhagic serum, but with different blood volume loss and waiting periods. When lymphocytes from normal rats (n = 28) were incubated with PHA (125 ug/ml) in the presence of pooled serum collected 2 hours after removal of 30% of total blood volume from three to five rats (hemorrhagic serum) , there was a 44% decrease in proliferative response as compared to that present when lymphocytes from the same animal were incubated in the presence of pooled normal serum: 10,084 ± 2108 cp vs. 17,973 + 2928 cpm, p .001. Similarly, lymphocyte proliferative response was reduced when the cells were incubated (1) in the presence of serum collected two hours after removal of 5% of total blood volume and (2) in the presence of serum collected 15 minutes after removal of 30% of total blood volume. (See Table 1) . Table 1 Group Hemorrhage 3H uptake P vol* Time** (cpmxl0 2 +SEM) Normal (Control) 87 ± 11 0.001 Hemorrhaged 5 120 39 + 1 Normal (Control) 116 + 4 Hemorrhaged 30 15 81 + 6 0.001 ♦Volume of hemorrhage, % of TBV **Minutes post hemorrhage when blood sample was taken EXAMPLE 3 Isolation of SIP from Rat Hemorrhagic Serum A serum immunoregulatory polypeptide was isolated from rat hemorrhagic serum by ammonium sulfate precipitation and purified sequentially by Sephadex G-75 chromatography, anion exchange HPLC, and reverse phase HPLC using a C18 column. Electrophoretically homogeneous SIP can be also obtained by the following procedures: (a) ammonium sulfate precipitation - DEAE Sephadex chromatography - Sephadex G-75 chromatography - reverse phase HPLC (C18) . (b) Amicon filtration - Anion exchange HPLC - Sephadex G-75 chromatography - reverse phase HPLC (C18) . Isolation of SIPs can be accomplished using various methods, with chromatography being preferred. \ \ EXAMPLE 4 Control Phvtohemagglutinin (PHA) Induced Lymphocyte Proliferation and Effect of SIP on PHA Induced Lymphocyte Proliferation Phytohemagglutinin (PHA) induced proliferation of rat peripheral blood lymphocytes is assessed by modifi¬ cation of the technique of Keller et al. (2) . Blood (approximately 7 ml) is collected from rats in hepari- nized syringes, diluted 1:6 with 0.9% saline, layered onto 70% Percoll. (Pharmacia, Piscataway, NJ) and centrifuged at 400 g for 30 minutes. The lymphocyte layers are harvested, washed 3 times with RPMI 1640 medium, supplemented with penicillin/streptomycin and adjusted to pH 7.3. Viability, as determined by trypan blue exclusion, is consistently greater than 95%. Lymphocyte purity is greater than 92% as determined by a non-specific esterase stain. The washed lymphocytes are adjusted to 2.5 x 10 6 cells/ml in RPMI 1640 medium supplemented with 5% pooled rat serum or 20% fetal calf serum, penicillin/strep¬ tomycin, at pH 7.3. Phytohemagglutinin (Difco, Detroit, MI) is added to the cell suspension to produce a final concentration of 125 ug/ml which we have previously found to produce maximal lymphocyte proliferation. Two fifth milliliter (200 ul) of the cell suspension (5 X 10 5 cells) is placed into each well of a flat bottom microliter plate (Falcon 3=040 icroliter plate, Oxnard, CA) . All cultures are performed at least in triplicate. The cultures are incubated at 37°C in a. humidified atmosphere at 5% C0 2 . After 2 days of incubation, 0.2 uCi of 5-(I 125 )-2-deoxyuridine (Amersham, Arlington Heights, IL) in 25 ul of 10~ 6 M 5-F-2-deoxyuridine (Sigma, St. Louis, MO) in RPMI 1640 is added to each culture. After 18 hours the cells are harvested onto glass fiber filter paper and radioactivity assayed in a gamma counter. Uptake of 125 I is calculated by deter- mining the mean of the triplicate cultures. The effect of SIPs on PHA-induced proliferation of peripheral blood mononuclear cells (PBMC) was studied in accordance with the techniques described above. The addition of rat SIP into rat PBMC cultures suppressed the proliferation of rat PBMC (control, 22362+2434; SIP, 13536+1645) . A substantial degree of homology in structure and function exists between SIPs of different species. Human SIP suppressed PHA-induced proliferation of rat PBMCs (Control, 3974+244; human SIP, 423+145). Rat SIP suppressed PHA-induced proliferation of mouse spleen cells (Control, 94633±_10390 ; rat SIP 65720+11808) . These results show a significant suppression, using an SIP, of the proliferation of rat peripheral blood mononucleus cells. Moreover, the results show substantial homology between SIPs of different species, including humans and rats, and rats and mice. This is of significant importance, as it could enable human lymphocyte proliferation to be controlled by an SIP obtained from the hemorrhagic serum of laboratory animals. EXAMPLE 5 Determination of Molecular Weight of SIP Obtained From Rat Hemorrhagic Serum The active fractions of SIP, i.e. fractions that suppressed PHA-induced lymphocyte proliferation in Example 4, obtained from anion exchange HPLC, which showed three bands corresponding to molecular weights of 14,000, 16,000 & 19,000, were further purified by reverse phase HPLC using a C18 column. SDS PAGE analysis of the peak activity fraction showed a single band corresponding to a molecular weight of approximately 16,000 with a possible variation of + 2,000. EXAMPLE 6 petermination of Molecular Weight of Human Hemorrhagic Serum SIP A human hemorrhage-induced serum immunoregulatory polypeptide with a molecular weight of 29,000 (also with a possible variation of + 2,000) was similarly isolated and purified from hemorrhagic serum obtained from a brain-dead patient in accordance with the procedures set forth in Examples 1-5. Minute amounts of this SIP were detected recently in normal blood suggesting that it may be a normal im¬ munoregulatory protein. Based on their molecular weight and biological activity, the SIPs of the present invention appear different from inhibitors of cell proliferation reported in the literature including contra-IL-2(6) , a 30-100 kd inhibitor of mitogen-induced lymphocyte proliferation found in synovial fluid of patients with rheumatoid arthritis (7) , a 70-kd factor which inhibits the production and action of IL-2 (8,9), a 25-kd inhibitor of IL-1 (10) , a 97-kd inhibitor of the response to IL-2 of an IL-2 dependent cell line (11) , suppressive factors in serum of patients after trauma and thermal injury (12-16) , and others (17-27) . EXAMPLE 7 Effect of SIPs on the Development of Immune Response to EL^ Cells a) J≥-^Cr Labeling of EL^ Cells Both the humoral and cellular assays of immune response are based upon the lysis of 51 Cr-labeled EL 4 cells. Labeling is carried out essentially according to the method of Canty and Wunderlich (3). To 1 X 10 8 EL 4 , cells in 20% fetal calf serum and Hank's minimum essential medium (HMEM) are added 60 uCi of 51 Cr. The mixture is incubated for 30 minutes in 20% C0 2 , at 37 °c. The labeled cells are washed three times in HMEM with fetal calf serum to remove unbound 51 Cr radiolabel and of b) Cel -me ia e Immilni v o EL4 Cells Cell-mediated immunity to EL4 cells is measured according to a previously reported procedure (4) . Briefly, spleen cells are obtained from EL 4 cell injected or saline injected Lewis rats. The cells are washed and resuspended in HMEM (20% fetal calf serum) to a concentration of 3 X 10 7 cells/ml. The spleen cell suspension (0.5ml) is mixed with 5 X 10 4 labeled EL 4 cells (0.5 ml) in a 35 mm petri dish (Falcon Plastics) and incubated for 3 hours at 37 n 'C in 10% C0 2 on a rocker platform making 8 reciprocations per minute. The amount of 5 ^C released from the EL4 cells is then determined. All assays are performed in triplicate. c) Humoral Immunity Humoral immunity to EL 4 cells is measured by a procedure we have reported previously (4) . Briefly, serum from Lewis rats is diluted in HMEM containing fetal calf serum (20%). A 0.2 ml portion of 51 Cr labeled EL 4 cell suspension (1.25 X 10 6 cells/ml) is mixed with 0.3 ml of a 1/6 dilution of rabbit complement (absorbed with 5 X 10 8 EL 4 cells/ml) and then added to 0.5 ml of serial dilutions of rat sera. The dilution giving 50% lysis of EL 4 cells is determined from the van Krogh equation (5) to be the titer of the serum. d) Effect of SIPs Effects of SIPs on the development of immune response to EL4 cells were studied. Mice were given 25 ug of SIP or saline (control), I.P., immediately before and 3 days after immunization with 1 x 10 8 EL 4 cells. Spleen lymphocytes and serum samples were harvested 7 days after immunization and were assayed for cell- mediated and humoral immunity according to a procedure we have reported previously (4) . Cell-mediated cyto- toxicity was assayed at a 300:1 effector:target cell IS ratio. Complement-dependent antibody mediated lysis of target cells was assayed t ( a 100 x dilution of serum. Results (Table 2) are expressed as mean + SEM. The data show that SIP treated animals have significantly lower cell-mediated as well as humoral immune response than the controls. II* REFERENCES 1. Abraham E, Chang Y-H: The effects of hemor¬ rhage on lymphocyte proliferation. Circ Shock 15:141-49, 1985. 2. Keller SE, Schleifer SJ, McKegney MP, et al: A simplified method for assessing phA induced stimulation of rat peripheral blood lympho¬ cytes. J Immunol Methods 51:287-91, 1982. 3. Canty TG, Wunderlich JR: Quantitative in vitro assay of cytotoxic cellular immunity. J Nat Cancer Inst. 45:761, 1970. 4. Chang Y-H: Adjuvant polyarthritis I: Incor¬ poration of quantitative measures of humoral and cellular immune response. J Pharmacol Exp Ther. 201:1-7, 1977. 5. Kabat EA, Mayer MM: Experimental immunochemis- try. Springfield Illinois, Charles C. Thomas, 1961, p 136. 6. Spiegel, J.P., Djew, Jy, Stock, N.I., Masur H., Gelmann E.P., Quinnan, G.v. , r. (1985) Sera from patients with acquired immunodefi¬ ciency syndrome inhibit production of inter¬ leukin-2 by normal lymphocytes. J.Clin.Invest. 75, 1957-1964. 7. Zembala, M. , Lemmel, E-M (1980) Inhibitory factor(s) of lymphoproliferation produced by synovial fluid mononuclear cells from rheuma¬ toid arthritis patients:the role of monocytes in suppression. J Immunol 125, 1087-1092. 8. Krakauer, T. (1985) A macrophage derived factor that inhibits the production and action on interleukin 2. J. Leukocyte Biol 3,429-440. 9. Miossec, P., Kashiwado, T. , and Ziff, X. (1987) Inhibitor of interleukin-2 in rheuma- toid synovial fluid. Arth & Rheum 30, 121. π 10. Arend, W.P., Joslin, F.G., Massoni, R.J. (1985) Effects of the immune complexes on production by human onocytes of interleukin 1 or an interleukin 1 inhibitor. J. Immunol 134. 3868-3875. 11. Fontana, A., Hengartner, H. , deTRibolet, N., Weber, E. (1984) Glioblasto a cells release interleukin 1 and factors inhibiting inter¬ leukin 1-mediated effects. J. Immunol. 132.1837-1844. 12. Ninneman, J.L. (1980) Immunosuppression following thermal injury through B cell activation of suppressor T cells. J Trauma 20,206-13. 13. Constantian, M.B., Menzoian, Nimberg, R.B., Schmid, K. , Mannick, J.A. (1977) Association of a circulating immunosuppressive polypeptide with operative and accidental trauma. Ann Surg 185.73-9. 14. Constantian, M.B. (1978) Association of sepsis with an immunosuppressive polypeptide in the serum of burn patients. Ann Surg i88 2 09-15. 15. Ninneman, J.L., Fisher, J.C. Wachtel, T.L. Thermal injury-associated immunosuppression occurrence and in vitro blocking effect of post-recovery serum. J Immunol. 122 , 1736-41. 16. Ninneman, J.L. , Condie, J.T., Davis, S.E., Crockett, R.A. (1982) Isolation of immunosup- pressive serum components following thermal injury. J Trauma. 22,834-44. 17. Aune, T.M., Webb, D.R. , Pierce, C.W. (1983) Purification and initial characterization of the lymphokine, soluble immune response suppressor. J Immunol. 131,2848-5 . 18. Thomas, D.W. , Roberts, W.K., Tal age, D.W. (1975) Regulation of the immune response: production of a soluble suppressor by immune spleen cells in vitro. J. Immunol. 114,1616- 21. 19. Namba, Y., Waksman, B.H. (1975) Regulatory substances produced by lymphocytes. I. Inhibitor of DNA synthesis in the rat. Inflammation 1,5-12. 20. Lee, S.C., Lucas, Z.J. (1977) Regulator factors produced by lymphocytes. II. Inhibi¬ tion of cellular DNA synthesis is associated with a factor inhibiting DNA poly erase-alpha activity. J Immunol. 118.88-95. 21. Chiba, K. , Nishimura, T. , Hashimoto, Y. (1985) Stimulated rat T cell derived inhibitory factor for cellular DNA synthesis (STIF) . J Immunol. 13_4,1019-27. 22. Namba, Y. , Waksman, B.H. (1985) Regulatory substances produced by lymphocytes II. Lymphotoxin in the rat. J Immunol. 1JJ5,1018- 14. 23. Greene, W.C., Fleisher, T.H. , Waldmann, T.A. (1981) Soluble suppressor supernatants elaborated by concanavalin A-activated human mononuclear cells. I. Characterization of a soluble suppressor of T cell proliferation. J Immunol. ' 12_6,1185-90. 24. Payan, D.G., Hess, CA. , Goetzl, E.J. (1984) Inhibition by somatostatin of the proliferation of T-lymphocytes and Molt-4 lymphoblasts, Immunology 84,433-41. 25. Payan, D.G., Goetzl, E.J. (1985) Modulation of ly phoctyes function by sensory neuropeptides. J Immunol. 135.783-875. 26. Ottaway, CA. , Greenberg, G.R. (1984) Interac¬ tion of VIP with mouse lymphocytes: specific binding and the modulation of itogen responses. J. Immunol. l_32 . ,417-24. 27. O'Dorisio, M.S., Wood, C.L., O'Dorisio, T.M. (1985) Vasoactive intestinal peptide and neuropeptide modulation of the immune response. J Immunol. 135.792s-98s. It is apparent from the foregoing that the SIPs of the present invention are extremely effective in regulating the immune response in mammals, as well as in diagnostic applications. Such properties have far reaching and wide therapeutic and diagnostic applications. Therefore, the above examples are given for illustrative purposes only, and the present invention is not to be limited in any way, except as by the appended claims.

Claims: ■ j* We claim: . • (1) A method of iso " lIating from the blood of a mammal a serum immunoregulatory polypeptide (SIP) having a molecular weight of from about 14,000 to about 31,000, and which modulates immune response in mammals, comprising: A. hemorrhaging a mammal by taking a first blood sample of at least approximately 1% of said mammal's total blood volume; B. waiting at least approximately 5 minutes; C taking a second blood sample, which constitutes hemorrhagic blood that contains said SIP, from said mammal; D. obtaining hemorrhagic serum from said hemorrhagic blood; and E. isolating said SIP from said hemorrhagic serum. (2) The method of Claim 1 wherein the blood sample in step (a) comprises between about 1% to about 70% of said mammal's total blood volume. (3) The method of Claim 1 wherein the blood sample in step (a) comprises between about 10% to about 40% of said mammal's total blood volume. (4) The method of Claim 1 wherein, in step (b) , the waiting period is between about 5 minutes to about 72 hours. (5) The method of Claim 1 wherein, in step (b) , the waiting period is between about 30 minutes to about 2 hours. (6) The method of C^ai 1 wherein the isolation in step (c) is accomplished usji g chromatography. ')' (7) A serum immunoregulatory polypeptide (SIP) having a molecular weight of from about 14,000 to about 31,000 and which suppresses lymphocyte proliferation in mammals. (8) The SIP of Claim 7 wherein the molecular weight is about 29,000. (9) The SIP of Claim 7 wherein the molecular weight is about 16,000. (10) A purified serum immunoregulatory polypeptide (SIP) having a molecular weight of from about 14,000 to about 31,000, isolated from hemorrhaged blood of a mammal, which suppresses lymphocyte proliferation in mammals. (11) The SIP of Claim 10 wherein said molecular weight is about 29,000. (12) The SIP of Claim 10 wherein said molecular weight is about 16,000. (13) A method of modulating immune response in a mammal by administering an immune response effective amount of the SIP of Claim 7 to said mammal. (14) A method of activating suppressor T-cells comprising treating said suppressor T-cells with an activating effective amount of the SIP of Claim 7. (15) A method of suppressing the generation of lymphokines by lymphocytes comprising treating said lymphocytes with a suppressing effective amount of the SIP of Claim 7. (16) A method of suppressing the production of Interleukin-2 by lymphocytes comprising treating said lymphocytes with a suppressing effective amount of the SIP of Claim 7. (17) A method of measuring the SIP of Claim 7 in body fluids using monoclonal antibodies directed toward said SIP. (18) A method of preparing monoclonal antibodies using the SIP of Claim 7 as an antigen. (19) A method of isolating from the blood of a mammal a serum immunoregulatory polypeptide (SIP) having a molecular weight of from about 14,000 to about 31,000 and which modulates immune response in mammals, comprising: (a) removing a sufficient amount of blood from said mammal to stimulate the production of said SIP; (b) waiting a sufficient amount of time for the level of said SIP in said blood to increase; (c) removing blood that contains said SIP from said mammal; and (d) isolating said SIP from said blood removed from said mammal that contains said SIP. (20) The method of Claim 19 wherein the sufficient amount of blood set forth in step (a) comprises between about 1% to about 70 % of said mammal's total blood volume. (21) The method of Claim 19 wherein the sufficient amount of blood set forth in step (a) comprises between about 10% to about 40% of said mammal's total blood volume. (22) The method of Claim 19 wherein the sufficient amount of time in step (b) is between about 5 minutes to about 72 hours. (23) The method of Claim 19 wherein the sufficient amount of time in step (b) is between about 30 minutes to about 2 hours. (24) The method of Claim 19 wherein the isolation in step (d) is accomplished using chromatography. (25) A serum immunoregulatory polypeptide (SIP) having a molecular weight of from about 14,000 to about 31,000 and which suppresses lymphocyte proliferation in mammals, said SIP having the identifying characteristic that it is generated in the blood of a mammal after a sufficient amount of blood is removed from said mammal and a sufficient period of time is permitted to elapse for the level of said SIP in said blood to increase.

Inventors: ABRAHAM EDWARD, CHANG YI-HAN, ABRAHAM, EDWARD, CHANG, YI-HAN
Applicants: ABRAHAM EDWARD, CHANG YI HAN, ABRAHAM, EDWARD, CHANG, YI-HAN
Application year: 1989
Main classification: C07K15
IPC codes: A61K38, A61P37, C07K1, C07K14, C07K16, C12P21, G01N33