PLASMIN-DEPLETION THERAPY

Abstract: This invention provides a method of increasing the therapeutic effect of an anti-cancer agent on cancerous cells comprising steps of: (a) reducing plasmins on the surface of the cancerous cells; and (b) contacting the therapeutic agent with cells resulted from step (a). This invention also provides a method of increasing the therapeutic effect of an anti-cancer agent on cancerous cells in a subject comprising steps of: (a) reducing plasmins on the surface of the cancerous cells in the subject; and (b) administering the anti-cancer agent to the subject. This invention also provides a compound comprising the fibrinolysis inhibitor unit linked to a plasminogen activating unit and the uses of such compound.

Description: P ASMIN-DEPLETION THERAPY This application claims priority of U.S. Provisional Application 60/023,592, filed August 14, 1996, the content of which is incorporated herein by reference. Background of the Invention In early stage of breast and colorectal cancer, cancer cells are confined within the area where it is originated (primary site) and form solid masses. Sometimes, cancer cells are spread to adjacent tissues and to the lymph nodes. Most common treatment for such early breast and colon cancer is surgery that removes local and regional cancer cells . Advanced cancer with metastases (cancer cells spread to distant organs, e.g. lung, liver, etc) is not curable (1-4) . Even in patients with operable tumors, surgery does not cure cancer in many cases. Because, at the time of surgery, cancer cells in a small number that are too low to be readily detected by clinicians are already migrated from primary sites and spread in the body (micrometastasis) in many cases. Micrometastatic cancer cells are eliminated by patient's defense systems. Cancer cells that escaped host defense systems cause the recurrence of cancer eventually. In breast and colon cancer patients, host defense systems often do not eliminate micrometastatic cancer cells efficiently. Within ten years of surgery, breast or colon cancer recurs in more than 50 % of patients (1-4) . To aid the elimination of cancer cells by host defense systems thus to reduce the rate of recurrence, various therapies (e.g. chemotherapy, monoclonal antibodies, radiation, etc) have been applied after surgery (postoperative adjuvant therapies) . The outcome of clinical trials of breast cancer and colon cancer shows that chemotherapies after surgery reduce the recurrence but the benefit is limited (1-4) . The failure of adjuvant chemotherapy in the eradication of micrometastatic breast and colon cancer cells may be due to drug resistance of cancer cells. Drug resistance, which describes cancer cells being resistant to the killing of chemotherapeutic drugs, is a major cause of the failure of chemotherapies (1-7) . Many characteristics of drug resistance are reported. Drug resistance could be either intrinsic or acquired during chemotherapies. Also drug resistance could be either resistant to a single drug or cross-resistant to various structurally unrelated drugs (multidrug resistance) . Drug resistance to a single drug could be overcome by treating patients with the combination of various chemotherapeutic drugs. Multidrug resistance cancer cells, on the other hand, pose serious problems in chemotherapies because they are resistant to most chemotherapeutic drugs thus cause the failure of chemotherapies (1,5-7). Cancer cells become drug resistant by engaging various mechanisms, for example, pumping out drugs through P- glycoprotein dependent or independent mechanism thus lower the amount of drug in cells to ineffective levels, altering the level of enzymes that metabolize drugs within cells, interfering with the action of drugs, and other mechanisms (5-11) . Since drug resistance is a major cause of the failure of chemotherapies, many efforts have been made to develop treatments that overcome drug resistance. In experimental therapies, for example, cyclosporin, cyclin adenosine monophosphate are shown to revert multidrug resistant cancer cells to sensitive cells (12-14) . A new mechanism by which breast cancer cells and colon cancer cells become resistant to chemotherapeutic drugs as well as to host defense systems (treatment resistance) is characterized by this invention. Through plasmin generated on their cell surface, cancer cells acquire and maintain treatment resistance. Subsequently, plasmin-depletion therapy that overcomes treatment resistance by inhibiting the generation of plasmin on cancer cells has been developed. Plasmin-depletion therapy blocks the generation of plasmin on cell surface by depleting the precursor of plasmin plasminogen. Plasmin occurs as its precursor plasminogen in the blood. Plasminogen is converted to plasmin by plasminogen activators such as urokinase, tissue plasminogen activators, streptokinase, staphylokinase, and others. On cancer cells, circulating plasminogen binds to its cell surface receptors and subsequently converted to plasmin primarily by urokinase (15-18) . By depleting circulating plasminogen, plasmin-depletion therapy deprives cancer cells of plasminogen thus blocks the generation of plasmin on cancer cells. Others also have shown that enhanced generation of plasmin on cancer cell surface facilitates the progress of cancer. In breast, colon, prostate, esophageal, gastric and other cancer, high urokinase activity in tumors that indicates increased generation of plasmin on cancer cells correlates to increased recurrence of cancer after curative surgery (19-27) . Plasmin activity on cancer cells is controlled by urokinase. Urokinase activity on cancer cells, in turn, is controlled by the expression urokinase receptors. ' Because urokinase in solution is unstable and has a low enzymatic activity. Urokinase is a protease that cleaves plasminogen to plasmin. Upon binding to its receptor on cell membrane, urokinase becomes stable with high enzymatic activity. In tissue culture, plasmin degrades the extracellular matrix of tumor tissues (15-18,28,29). The extracellular matrix confines cancer cells within tumor tissues. Plasmin, therefore, is suggested to mediate the invasion of cancer cells to the blood, to nearby tissues and to distant organs thus facilitate metastases (15,16,19,28,29). To prevent metastases, therefore, many treatments that block the generation of plasmin on cancer cell surface particularly by inhibiting urokinase activity have been developed. For example, urokinase inhibitors, monoclonal antibodies to urokinase, treatments blocking the binding of urokinase to its receptor on cell surface, or treatments inhibiting the expression of urokinase receptors have been used to block plasmin generation on cancer cells . Such treatments inhibiting urokinase activity are shown to be effective in the treatment of cancer in tissue culture and in animals in some cases (25-30) . The plasmin-depletion therapy also blocks the generation of plasmin on cancer cells. However, the plasmin-depletion therapy is applied to overcome treatment resistance of cancer cells thus to increase the efficacy of therapeutic agents rather than as a cancer therapy. Moreover, a completely new approach has been employed: the precursor of plasmin plasminogen is depleted instead of blocking urokinase activity. The data disclosed in this invention show that by converting plasminogen to plasmin, plasminogen activators render cancer cells treatment resistant. However, plasmin-depletion therapy engages plasminogen activators as therapeutic agents to deplete plasminogen in vitro as well as in vivo. The characteristics of plasminogen and plasmin allow plasminogen activators to deplete plasminogen in tissue culture and in vivo: plasminogen is continuously produced and inactivated in the blood, the amount of circulating plasminogen is constant, plasminogen is converted to plasmin by plasminogen activators, the half-life of plasminogen is 2.2 days, and the half-life of plasmin is less than one minute (15- 18,28,29). In tissue culture, plasminogen activators deplete plasminogen by converting plasminogen to plasmin that is inactivated immediately. In vivo, administration of excess plasminogen activators converts circulating plasminogen to plasmin that is inactivated within a minute. Since plasminogen is produced slowly, plasmin-depletion therapy will create a plasminogen- low period. During such period, cancer cells will be deprived of plasminogen consequently plasmin can not be generated on cancer cells. Cells may become treatment resistant briefly immediately after plasmin-depletion therapy due to excess plasmin generated. However, cells will revert to treatment sensitive during the plasminogen-depleted period because plasmin can not be generated. Although for different purposes, plasmin-depletion therapy has been already used in clinics in the treatment of the diseases caused by blood clots for example, ernboli, thrombi, stroke, heart attack, and other diseases (32-36) . Under normal physiological condition, plasmin dissolves blood clots thus allows blood to circulate freely (15) . When blood clots occur, the therapeutic agents of plasmin-depletion therapy plasminogen activators are administered to patients to generate excess plasmin thus to accelerate the dissolution of blood clots. In patients treated with plasminogen activators, plasminogen level is low for 24-48 hours after plasminogen activator therapies. During such period, plasmin could not be generated even in the presence of plasminogen activators (32,33). The reports demonstrate the feasibility of depleting circulating plasminogen in humans by plasmin-depletion therapy as well as the safety of the procedure . Plasmin dissolves blood clots by degrading its main component fibrin (fibrinolysis) . Although effectively dissolves blood clots, excess generation of plasmin occasionally causes systemic bleeding due to uncontrolled fibrinolysis. In the treatment of blood clots, since plasmin is the therapeutic agent and fibrinolysis is the therapeutic process, bleeding can not be prevented without decreasing the efficacy of the treatments. Instead of preventing bleeding, when bleeding occurs, patients are treated with plasma containing plasmin inhibitors . In the plasmin-depletion therapy, plasmin is a unnecessary byproduct and fibrinolysis is an unwanted adversary reaction causing the side effect bleeding. In the plasmin-depletion therapy, therefore, inhibition of plasmin activity and fibrinolysis will be beneficial without diminishing the effectiveness of the treatment. Plasmin activity and fibrinolysis can be readily blocked by the serum protease inhibitor such as α 2 -antiplasmin. c^ - antiplasmin inhibits fibrinolysis by inhibiting plasmin activity specifically as well as by interfering the binding of plasminogen to fibrin (37-39) . The binding of plasminogen to fibrin prior to its conversion is required for efficient plasmin-mediated fibrinolysis since plasmin generated on fibrin mediates fibrinolysis. α 2 -antiplasmin does not interfere the conversion of plasminogen to plasmin. Administered together with plasminogen activators, therefore α-.-antiplasmin will prevent fibrinolysis thus the side effect bleeding without decreasing the efficacy of the Plasmin-depletion therapy. Generation of excess plasmin occasionally causes uncontrolled fibrinolysis consequently bleeding. Plasmin has a short half-life. To prevent bleeding efficiently, therefore, the increase of the local concentration of fibrinolysis inhibitor at the site of plasmin generation and at the time of plasmin generation is required. Hence, the delivery of fibrinolysis inhibitor and plasmin generating agent plasminogen activator simultaneously to the same site is desirable. To be delivered in such manners, administration of plasminogen activator and fibrinolysis inhibitor as a single drug is preferable to the administration of the combination of two drugs. Moreover, administration of one drug with two functions will simplify the regimen of the plasmin-depletion therapy, will eliminate the complexity of clinical trials of the combination treatment of two drugs, and will lower the cost of the plasmin-depletion therapy in clinics. Considering these points, plasmin-depletion therapy drugs that are the hybrid of the plasminogen activator and fibrinolysis inhibitors are designed. Plasmin-depletion therapy drugs are designed to be a single chain polypeptide consisting of plasminogen activator domain and fibrinolysis inhibitory domain. Plasmin depletion therapy drugs can be modified by inserting the in vivo cleavage domain. The in vivo cleavage site is inserted between two functional domains to endow a single chain plasmin-depletion therapy drugs the ability to become two drugs, plasminogen activator and fibrinolysis inhibitor, in vivo. Although the cleavage is not essential for plasmin- depletion therapy drugs to be effective, it is preferred. Designed to be a potent plasminogen activator but a poor fibrinolytic agent, plasmin-depletion therapy drugs are expected to convert plasminogen to plasmin thus to deplete Plasminogen efficiently, thereby to enhance the sensitivity of tumor cells to therapeutic agents without causing bleeding. Summarv of the Invention A mechanism that induces as well as maintains the resistance of cancer cells to host defense systems and to chemotherapeutic drugs (treatment resistance) is characterized. The data disclosed in this invention show that through plasmin generated on cell surface, cancer cells regulate their sensitivity to anticancer agents and host defense systems. When plasmin on cell surface is high, cells become treatment resistant. In response to low plasmin, cancer cells become treatment sensitive. Moreover, cancer cells revert from resistant to sensitive and vice versa readily according to plasmin activity on cells. To overcome treatment resistance thus to increase the sensitivity of cancer cells to therapeutic agents, plasmin-depletion therapy that inhibits the generation of plasmin on cancer cells has been developed. Plasmin occurs as its precursor plasminogen in the blood. Plasminogen is converted to plasmin by plasminogen activators such as urokinase, tissue plasminogen activator, streptokinase, staphylokinase, etc. On cancer cells, plasmin is generated by binding of plasminogen to cell surface and subsequent conversion of plasminogen to plasmin (15-18) . In tissue culture, the source of plasminogen on cancer cells is plasminogen contained in plasma or serum added to culture medium. In vivo, the source of plasminogen on cancer cells is circulating plasminogen in the blood. To inhibit the generation of plasmin on cancer cells, plasmin- depletion therapy depletes the source of plasminogen on cancer cell surface plasminogen contained in culture medium or circulating plasminogen in vivo. As therapeutic agents, plasmin-depletion therapy utilizes plasminogen activators although plasminogen activators render cancer cells treatment resistant by converting plasminogen to plasmin. In tissue culture, plasminogen activators convert plasminogen contained in plasma or serum supplement to plasmin that is inactivated within a minute thus deplete plasminogen. In vivo, plasminogen activators convert circulating plasminogen to plasmin that, in turn, is inactivated within a minute. Meanwhile, plasminogen is produced slowly. Consequently, the level of plasminogen is low until plasminogen is replenished fully (15,32-36). The data disclosed in this invention show that plasmin- depletion therapy overcomes treatment resistance in tissue culture as well as in animals. In tissue culture, the therapy blocks the induction of treatment resistance induced by urokinase, reverts cancer cells that are already treatment resistant to treatment sensitive, and thus increases the sensitivity of cancer cells to chemotherapeutic drugs (doxorubicin and navelbine) as well as to host defense systems, (T cell cytotoxicity, T cell cytotoxicity mediated by bispecific antibody that binds to cancer cells and T cells, macrophage cytotoxicity, and macrophage- ediated antibody-dependent cellular cytotoxicity) . In vivo, plasmin-depletion therapy increases the efficiency of M79 antibody and doxorubicin eradicating HT29 cells transplanted in nude mice. Since plasmin-depletion therapy increases the sensitivity of cancer cells to chemotherapeutic drugs and to host defense systems, by combining together with chemotherapy or with immunotherapy, plasmin-depletion therapy could be applied (1) to reduce therapeutic dose of drugs, or (2) to enhance the efficacy of therapeutic agents. The former can be applied to abolish (or decrease substantially) toxic side effects of therapeutic drugs. The latter can be applied to enhance the efficiency of therapeutic agents at currently used doses . The therapeutic agents of the plasmin-depletion therapy, plasminogen activators, are already used in clinic to dissolve blood clots in the treatment of emboli, thrombi, stroke, heart attack, etc (32-36). The plasmin-depletion therapy, however, will provide a new use of plasminogen activators for the treatment of different diseases: i.e. plasminogen activators will be used to deplete circulating plasminogen thus to enhance the efficacy of therapeutic agents in the treatment of cancer. Moreover, in the treatment of cancer, the application of plasminogen activators can be modified as follows: The use of modified plasminogen activators. For efficient treatment of blood clotting, fibrin binding site of plasminogen activators is required because they must convert plasminogen to plasmin on fibrin (15) . In plasmin- depletion therapy, on the other hand, plasminogen activators without fibrin binding site is preferred because they will convert plasminogen to plasmin in solution. Plasmin generated in solution mediates fibrinolysis poorly thus will not cause bleeding. As a therapeutic agent of plasmin- depletion therapy, therefore, modified plasminogen activator fibrin binding site can be used. In addition, as the therapeutic agents of plasmin-depletion therapy, modified plasminogen activator with altered cell receptor binding site can be used to eliminate the unwanted functions of plasminogen activators. For example, in addition to activating plasminogen to plasmin, urokinase bound to its specific cell surface receptors mediates cellular effects. Some normal cells, for example macrophage, express urokinase receptor (47) . To prevent unwanted cellular effects of plasmin-depletion therapy engaging urokinase on such cells, urokinase can be modified further by altering the cell surface receptor binding site. Eventually, a portion of plasminogen activators containing catalytic site can be used. Such modified plasminogen activators can be constructed readily using molecular biology techniques. Administration of plasminogen activators together with inhibitors of plasmin. Excess activation of plasmin causes uncontrolled fibrinolysis leading to internal bleeding in some cases. In plasmin-depletion therapy, plasmin is a unnecessary byproduct and fibrinolysis is an unnecessary process. Combined administration of plasminogen activator and fibrinolysis inhibitors will inhibit fibronolysis thus prevent internal bleeding. Plasmin inhibitors are readily available (15, 37, 39, 48) . 3. The design of plasmin-depletion therapy drugs. For effective blocking of fibrinolysis, the accumulation of fibrinolysis inhibitor at the site as well as the time of plasminogen activator action is required. For such delivery, administration of one drug that could mediate plasmin generation as well as inhibit fibrinolysis simultaneously would be superior to the administration of the mixture of plasminogen activator and fibrinolysis inhibitor. Thus plasmin-depletion therapy drugs are designed. To be a single drug that is a potent plasminogen activator but a poor fibrinolytic agent, plasmin-depletion therapy drugs are designed to contain fibrinolysis inhibitor and plasminogen activator. Plasmin-depletion therapy drugs can be modified by inserting the cleavage sequence. The cleavage sequence contains a specific peptide bond that is cleaved by plasma proteases . By inserting the cleavage sequence between fibrinolysis inhibitor and plasminogen activator, Plasmin- depletion therapy drugs are expected to be cleaved specifically at the cleavage site by plasma proteases and become two drugs in vivo, fibrinolysis inhibitor and enzymatically active plasminogen activator. The cleavage is not essential for the drugs effectiveness, but is preferred. Plasmin-depletion therapy drugs, with or without the cleavage sequence, are expected to mediate its functions as follows. Being a plasminogen activator, Plasmin-depletion therapy drugs will activate plasminogen to plasmin, deplete plasminogen, and thus overcome treatment resistance. Being a fibrinolysis inhibitor and delivered to the site and the time of plasmin generation, fibrinolysis inhibitor will block fibrinolysis efficiently thus prevent bleeding. 4. Titration of streptokinase antibody prior to plasmin- depletion therapy. Due to previous streptococcal infections, cancer patients often have streptokinase antibody in their blood. In streptokinase antibody-positive patients, the efficacy of streptokinase will be reduced substantially because streptokinase will form immune complex with streptokinase antibody and the immune complex will be cleared rapidly by reticuloendothelial cells. Moreover, in patients with low titer streptokinase antibody, streptokinase will form small immune complex with streptokinase antibody, deposits on small vessels, and damages the vessels (49) . To avoid the treatment of streptokinase antibody-positive patients with streptokinase thus to prevent small vessel damage, the presence of streptokinase in patients can be pre-screened in plasmin-depletion therapy. Streptokinase antibody can be easily titrated. Others also have developed treatments that inhibit the generation of plasmin on cancer cells by blocking urokinase activity as cancer therapies preventing metastases (16,27,30,31). Although also inhibits the generation of plasmin, the use of plasmin-depletion therapy is to increase the sensitivity of cancer cells to therapeutic agents thus to increase the efficiency of therapies rather than as a cancer therapy. Furthermore, in plasmin-depletion therapy, completely new approach has been used to block the generation of plasmin on cancer cells: circulating plasminogen is depleted. Brief Description of the Figures FIG. 1. In response to urokinase, MCF7 cancer cells reduce their response to doxorubicin. MCF7 cells (3,000 cells in 0.1 ml) were cultured with 1.5% human serum either alone (O) or with 30 unit/ml urokinase (•) for two days, treated with doxorubicin at concentrations indicated for two days, and the thymidine uptake assay was performed (50) . FIG. 2-3. In response to urokinase, tumor cells increase the resistance to doxorubicin. 20,000 cells in 0.1 ml SKBR5 breast cancer cells (Figure 2) or HT29 colon cancer cells (Figure 3) were cultured with 1.5% human serum either alone (O) or with 30 units/ml urokinase (•) for two days, treated with doxorubicin at concentrations indicated for additional two days, and the LDH assay that measures lactate dehydrogenase released by dead cells was performed (51) . FIG. 4. Urokinase increases the resistance of cancer cells to navelbine . HT29 cells were cultured in the absence (O) or presence (•) of urokinase for two days, treated with navelbine for two days and the LDH assay was performed. FIG. 5. In response to urokinase, MCF7 cells but not HT29 or SKBR5 cells change their growth pattern. 20,000 cells in 0.1 ml HT29 colon carcinoma cells (A and B) , SKBR5 breast carcinoma cells (C and D) or MCF7 breast cancer cells (3,000 cells in 0.1 ml, E and F) were cultured with either 1.5% human serum alone (A, C, E) or with 30 units/ml urokinase (B, D, F) for two days. FIG. 6-7. Urokinase renders cancer cells resistant to macrophage cytotoxicity and macrophage-mediated ADCC. 20,000 cells in 0.1 ml HT29 (Figure 6) or SKBR5 (Figure 7) cells were cultured for two days with either 1.5% human serum alone (0,V) or with 30 units/ml urokinase ( , Υ) , incubated with murine PEC at numbers indicated for three days either alone (0,#) or with 5 ug/ml M79 antibody (V,T) and 4 -hour thymidine uptake assay was performed. PEC was obtained from Swiss mice treated with 1 ml thioglycollate for four days. FIG. 8. Plasmin increases the resistance of MCF7 cells to doxorubicin. MCF7 cells (3,000 cells in 0.1 ml) were cultured with 1.5% human serum in the absence or presence of 0.03 unit plasmin for two days, treated with 50 ng/ml doxorubicin for two days, and thymidine uptake assay was performed. - ; none, D; doxorubicin, P;plasmin; D+P; doxorubicin + plasmin. FIG. 9. Urokinase is ineffective in the absence of plasminogen. HT29 cells were cultured for two days with 1.5% normal human serum incubated at 37°C for 90 minutes either alone (O,*) or with 250 units/ml streptokinase (V,T) to deplete plasminogen in the absence (0,V) or presence (•,▼) 30 units/ml urokinase, added 30,000 PEC of thioglycollate- treated mice together with M79 antibody at concentrations indicated, and subsequently 4 -hour thymidine assay was performed three days later. FIG. 10. Urokinase induces the resistance only in the presence of plasminogen. HT29 cells were cultured for two days with 1.5% normal human serum incubated at 37°C for 90 minutes with either alone (V,T) or 250 unit/ml urokinase (0,#) , in the absence (0,V) or presence (•,▼) of 30 unit/ml urokinase. Subsequently, the sensitivity to doxorubicin was examined as described in Figure 1. FIG. 11. Addition of plasminogen to the culture depleted of plasminogen restores the ability of urokinase inducing the resistance of HT29 cells to doxorubicin. HT29 cells were cultured for two days with 1.5% normal human serum incubated with 250 unit/ml urokinase to deplete plasminogen, with (V,T) or without (O,*) addition of plasminogen, in the absence (0,V) or presence (•,▼) of 30 unit/ml urokinase, incubated with various doses of doxorubin for two days, and subsequently 4 -hour thymidine assay was performed. FIG. 12-13. Depletion of plasminogen by streptokinase increases the sensitivity of cancer cells to macrophages and ADCC. HT29 (Figure 12) or SKBR5 (Figure 13) cells were cultured with 1.5% human serum either control (O,*) or treated with streptokinase (V,T) for two days, added 30,000 PEC of thioglycollate treated mice either alone (Q,V) or with M79 antibody (•,▼), and 4 -hour thy idine assay was performed three days later. The numbers of PEC used in this experiment are too low to kill HT29 cells alone (see Figure 6) . FIG. 14. Depletion of plasminogen by streptokinase increases the sensitivity of HT29 cells to T cell cytotoxicity. HT29 cells are cultured for 18 hours either control human serum (O,*) or human serum treated with streptokinase to deplete plasminogen (V,T) added non-adherent peripheral blood lymphocytes (PBL) either alone (0,V) or with 10 ng/ml CD3/17- 1A antibody (#,Y), and 4-hour thymidine assay was performed 18 hours later. Nonadherent PBL were obtained by incubating PBL (5 x IO 6 cells/ml) of a healthy volunteer in tissue culture dishes at 37°C for one hour with 1% human serum and collecting nonadherent cells. FIG. 15. Depletion of plasminogen by streptokinase increases the efficacy of CD3-17-1A bispecific antibody. HT 29 cells cultured with either normal human serum (O) or human serum treated with streptokinase (•) for 18 hours, added 300,000 nonadherent PBL together with CD3/17-1A bispecific antibody at concentrations indicated. FIG. 16-17. Plasmin-depletion therapy blocks the induction of treatment resistance by exogenous urokinase . HT29 cells (Figure 16) or SKBR5 cells (Figure 17) were cultured in control medium containing plasminogen (0,#) or in the medium treated with urokinase-plasmin- depletion therapy (V,T) in the absence (0,V) or presence of 30 unit/ml urokinase (•,▼) for two days. Subsequently, cells were treated 5ug/ml M79 antibody and PEC at concentrations indicated for three days and the thymidine assay was performed. FIG. 18-21. Plasmin-depletion therapy reverts treatment resistant cells to sensitive. HT29 cells (Figure 18 and 20) or SKBR5 cells (Figure 19 and 21) were cultured for two days with 1.5% human serum with 30 unit/ml urokinase, washed, cultured again for 16 hours in the medium treated with plasmin- depletion therapy of urokinase (•) or control medium (O) treated with 30,000 PEC together with M79 antibody at concentrations indicated (Figure 18 and 19) for three days or with doxorubicin (Figure 20 and 21) for two days, and 4 -hour thymidine assay was performed. FIG. 22. TPA is effective as a therapeutic agent of plasmin-depletion therapy. HT29 cells were cultured for two days with 1.5% either human serum treated with 250 units/ml tPA (V,T) or control human serum (0,#) , treated with various doxorubicin for 2 days in the absence (0,V) or presence (#,Y) of 30 unit/ml urokinase, and the 4 -hour thymidine uptake assay was performed. FIG. 23. Plasmin-depletion therapy is effective in vivo. Swiss mice were treated with 0.2 ml either saline or 250 units streptokinase in saline intravenously twice at 30 minutes intervals. Two hours later, animals were bled. HT29 cells were cultured for two days with 1% either control mouse serum (O) or the serum of mice treated with streptokinase (•) cultured for three days with 45,000 PEC plus M79 antibody at the doses indicated for three days and 4 -hour thymidine assay was performed. FIG. 24. Plasminogen-depletion therapy increases the efficacy of M79 antibody in animals. Balb/C athymic nude mice were injected intraperitoneally with either 0.2 ml saline (O,*) or 150 units urokinase in 0.2 ml saline (V,T). Thirty minutes later, mice were injected with saline or urokinase twice more at 10 minutes intervals. Two hours after the first injection, 2 millions HT29 cells were inoculated intradermally into the abdomen. Mice were treated twice, 24 hours and 48 hours after tumor inoculation, with 0.2 ml saline (0,V) or 100 ug/ml M79 antibody in 0.2 ml saline (#,Y). On days indicated, the formation of tumors as well as the volume of tumors were assessed. Tumor volume=the length x the width x the height. FIG. 25. Plasmin-depletion therapy increases the efficacy of doxorubicin in vivo. One million HT29 cells were inoculated into the abdomen of nude mice. Twenty four hours later, animals were treated with either 150 unit urokinase (V,Y) or saline (0,#) twice at thirty minutes intervals. 24 hours later, animals were treated either with 0.2 ml saline (Q,V) or with 50 ug doxorubicin in 0.2 ml saline (•,▼) intravenously twice at 4 -hour interval. Tumors were measured as described in Figure 24 on the days indicated. Detailed Description of the Invention This invention provides a method of increasing the therapeutic effect of an anti-cancer agent on cancerous cells comprising steps of: (a) reducing plasmin on the surface of the cancerous cells; and (b) contacting the therapeutic agent with cells resulted from step (a) . This invention also provides a method of increasing the therapeutic effect of an anti-cancer agent on cancerous cells in a subject comprising steps of: (a) reducing plasmin on the surface of the cancerous cells in the subject; and (b) administering the anti -cancer agent to the subject . In an embodiment of the above methods, plasmin is reduced by depletion of plasminogen. In a further embodiment, the plasminogen is depleted by administering an effective amount of plas inogen-depleting substance to the subject to reduce the plasminogen from binding onto the surface of the cancerous cells. As used herein, a plasminogen-depleting substance are compound which is capable of reducing the amount of plasminogen in the body. In an embodiment, the plasminogen is depleted by administration to a subject a substance capable of activating the plasminogen. In a further embodiment, the substance is a plasminogen activator. The plasminogen activators include, but are not limited to tissue plasminogen activator, streptokinase, urokinase and staphylokinase . In another embodiment, the plasminogen-depleting substance is a polypeptide comprising the catalytic and plasminogen binding sites of a plasminogen activator. In a further embodiment, the substance is a portion of urokinase wherein the cellular receptor binding site is modified. In a separate embodiment, the fibrin binding site of the plasminogen activator is modified. This invention also provides proprietary drugs of Plasmin- depletion therapy. Plasmin-depletion therapy drugs comprises compounds which contain plasminogen activating unit and fibrinolysis inhibitor unit . As an example, at least one plasminogen activating unit is linked to at least one fibrinolysis inhibitor unit. For a preferred embodiment, the structure of Plasmin- depletion therapy drugs is as follows: NH 2 .fibrinolysis inhibitor-the cleavage sequence-plasminogen activating substance. The cancerous cells which may be used in this invention include, but are limited to cells from colon carcinoma, breast cancer, prostate cancer, ovarian cancer, stomach cancer and esophageal cancer. This invention also provides a method of increasing the therapeutic effect of an anti-cancer agent on cancerous cells in a subject comprising steps of: (a) pre-screening the subject to determine whether pre-exiting antibody which will interfere with the action of the plasmin-depleting substance; (b) reducing plasmin on the surface of the cancerous cells in the subject by administering the substance which will not be interfered by the pre-existing antibody; and (c) administering the anti-cancer agent to the subject . In an embodiment of the above method, the subject is screened with the presence of streptokinase antibody before step (a) and the subject will not be administered with streptokinase if the formation of the immune complex between streptokinase and the streptokinase antibody will affect the reduction of plasmin in the subject. This invention will be better understood from the examples which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter. Experimental Details Urokinase induces drug resistance of breast and colon cancer cells in tissue culture. Disseminated micrometastatic cancer cells in patients with high urokinase activity in tumor tissues are more resistant to host defense systems and to therapies than tumor cells in patients with low urokinase activity in tumor samples (19- 27) . Such poor response may be partly caused by tumor cells developing drug resistance in response to urokinase. Cancer cells are known to develop drug resistance by various mechanisms in vivo as well as in vitro (5-14). To see whether breast or colon cancer cells develop drug resistance in response to urokinase, the effect of urokinase on drug sensitivity of eight cancer cell lines was examined in tissue culture. The cells are four breast carcinoma cells (BT20, MCF7, SKBR5, and ZR-75-1) and four colon carcinoma cells (HT29, LS180, SW620 and SW1116) . SKBR5 cells are obtained from Dr. L. Old, Ludwig Institute, New York City, NY. Other cell lines are obtained from /American Type Culture Collection, Rockeville, MD. Tumor cells were cultured for two days either with 1.5 % human serum alone or with 30 unit/ml urokinase (Abbott, North Chicago, IL) and subsequently their response to a chemotherapeutic drug doxorubicin (Adria) was examined. Treated with urokinase, all eight cell lines reduce their response to doxorubicin. MCF7 breast cancer cells (Figure 1), SKBR5 cells (Figure 2), and HT29 colon cancer cells (Figure 3) are shown. It was examined next whether urokinase renders cancer cells resistant to another chemotherapeutic drug, navelbine (Burroughs Wellcome Co., Research Triangle Park NC) . Treated with urokinase, cancer cells increase their resistance to navelbine (Figure 4). The mechanism by which urokinase increases the resistance of cancer cells to drugs was examined. Not only reducing their response to doxorubicin, five cell lines (BT20, MCF7, ZR75- 1, SW620 and SW1116) alter their growth pattern in response to urokinase. Cultured with human serum, they grow as adherent monolayer cells. Treated with urokinase, they form multicellular spheroids (Figure 5E and 5F shows MCF7 cells) . Tumor cells in multicellular masses are shown to be less responsive to therapeutic agents due to poor penetrance of drugs to the inner part of the masses (52-55) . Reduced response of these five cell lines to doxorubicin may be partly due to urokinase inducing them to form multicellular spheroids thus to limit the access of doxorubicin to tumor cells. Reduced response of HT29 colon cancer cells, LS180 colon cancer cells and SKBR5 breast cancer cells can not be accounted for limited access of drugs to tumor cells. Because the growth pattern of untreated and urokinase- treated HT29, LS180 and SKBR5 cells is undistinguishable. In the presence or absence of urokinase, HT29 and LS180 cells grow as heterogenous cells loosely attached to culture flasks (Figure 5A and 5B shows HT29 cells) . Regardless of the treatment with urokinase, SKBR5 cells grow as single cells in suspension (Figure 5C and 5D) . Doxorubicin which is an anthracyclin and navelbine which is a vinca alkaloid are effective preferentially on proliferating cells. Reduced response of urokinase-treated cancer cells, therefore, may be due to their diminished proliferation. The data show that this is not the case. Cancer cells were cultured with 1.5 % human serum alone or together with 30 Units/ml of urokinase and their proliferation was assessed. Urokinase-treated cells proliferate as efficiently as untreated cells (Table 1 and figure 1) . Table 1. Effect: of urokinase on the proliferation of cells cell number x 1,000 Day 4 Day 7 MCF7 untreated 780(11) 2,000 (270: urokinase treated 980(20) 2,150 (360; HT29 untreated 540(10) 830 (130) urokinase treated 570 (9) 880 (45) SKBR5 untreated 250 (4) 370 (30) urokinase treated 270 (4) 380 (10) 200,000 cells/ml were cultured with 3% human serum in the absence or presence of 50 unit/ml urokinase. On days indicated, HT29 cells or SKBR5 cells were washed, suspended in 0.2 ml and counted. MCF7 cells were treated with 1% fresh serum for 24 hours on the days indicated, trypsinized, and counted using a hemocytometer. The cell numbers are shown with standard deviation. The difference is not significant at the p=0.05 in Student t-test. In response to urokinase, cancer cells reduce their response to chemotherapeutic drugs. The reduction is neither due to diminished proliferation of the cancer cells nor due to limited delivery of drugs to tumor cells. The data are interpreted that in response to urokinase, cancer cells reduce their response to chemotherapeutic drugs by acquiring drug resistance. In response to urokinase, breast and colon cancer cells develop the resistance to host defense systems . It was examined whether in response to urokinase, cancer cells acquire the resistance also to the host defense systems, macrophage cytotoxicity and macrophage-mediated antibody dependent cellular cytotoxicity (ADCC) . For the induction of ADCC, M79 antibody was used. M79 antibody reacts with human epithelial cell antigens and induces ADCC that kills tumor cells efficiently in tissue culture (56) . HT29 colon cancer cells and SKBR5 breast cancer cells were cultured for two days with 1.5% human serum either alone or with 30 unit/ml urokinase and their sensitivity to macrophage cytotoxicity was compared. Cancer cells were killed readily by mouse peritoneal macrophages (PEC). Treated with urokinase, cancer cells become resistant to macrophage cytotoxicity (Figure 6 and 7). Treated with urokinase, cancer cells become resistant also to macrophage- mediated ADCC induced by M79 antibody (Figure β and 7). Urokinase induces the resistance of cancer cells through the conversion of plasminogen to plasmin. The mechanism by which urokinase renders cancer cells resistant to host defense systems and to therapeutic drugs (treatment resistance) was examined next. A well known function of urokinase is the conversion of plasminogen to plasmin on cancer cell surface (15-20,28,29). It was examined whether urokinase mediates its effect through such pathway. Similarly to urokinase, plasmin increases the resistance of MCF7 cells to doxorubicin (Figure 8). The data shows that urokinase possibly induces the resistance indirectly through the conversion of plasminogen to plasmin. Plasmin, a serine protease, is produced as its precursor plasminogen. Plasminogen activators (urokinase, streptokinase, tissue plasminogen activators, etc.) convert plasminogen to plasmin (25-29) . If plasmin induced treatment resistance of cancer cells and urokinase induced the resistance indirectly by converting plasminogen to plasmin on cancer cell surface, urokinase should induce treatment resistance only in the presence of plasminogen the precursor of plasmin. Urokinase should not induce treatment resistance in the absence of plasminogen. The data show that this is the case. In culture supplemented with human plasma or human serum depleted of plasminogen, urokinase was unable to induce the resistance of HT29 cells to M79 antibody mediated ADCC and to doxorubicin (Figure 9 and 10) . Plasma or serum is the primary source of plasminogen in culture medium. Human plasma depleted of plasminogen was obtained from American Diagnostica (Greenwich, Conn.). Human serum depleted of plasminogen was obtained by treating normal human serum with streptokinase or urokinase. If indeed urokinase induced the resistance through the conversion of plasminogen to plasmin, addition of plasminogen to the culture depleted of plasminogen should restore the ability of urokinase inducing the resistance. HT29 human colon cancer cells were cultured in the medium depleted of plasminogen with or without addition of plasminogen. Urokinase was unable to induce the resistance of HT29 cells to doxorubicin in the culture depleted of plasminogen. Added plasminogen to the culture depleted of plasminogen, on the other hand, urokinase increases the resistance of HT29 cells to doxorubicin ( Figure 11 ) . Plasminogen restores the ability of urokinase inducing treatment resistance in the culture depleted of plasminogen. Urokinase induces treatment resistance only in the presence of plasminogen. The data demonstrate that urokinase induces treatment resistance indirectly by converting plasminogen to plasmin. The plasmin-depletion therapy overcomes the resistance induced by endogenous plasminogen activator. Generation of plasmin on cancer cells induces the resistance of cancer cells to therapeutic agents and host defense systems (Figure 1-4 and 6,7). It follows that blocking the generation of plasmin overcomes treatment resistance. Plasmin occurs as its precursor plasminogen in the blood. Upon binding to its receptor on cell surface or fibrin, plasminogen is converted to plasmin by plasminogen activators, for example, urokinase plasminogen activator (urokinase) , tissue plasminogen activator (tPA) , streptokinase, or others. Urokinase and tPA are physiological plasminogen activators. In tissue culture, plasmin on cancer cells is generated by plasminogen in the medium binding to its receptor on cancer cells and subsequently converted to plasmin primarily by urokinase (15-18). Thus the generation of plasmin on cancer cells could be blocked by depleting plasminogen in the medium, the source of plasminogen on cancer cells. Depleted of the source of plasminogen, cancer cells would be deprived of plasminogen on their cell surface. Deprived of the precursor plasminogen on their surface, cancer cells could not generate plasmin on its surface regardless of the presence of urokinase. Therefore, once plasminogen bound to cells prior to the therapy is converted to plasmin and plasmin is inactivated, cells would be free of plasmin. To deplete plasminogen in the medium thereby to inhibit the generation of plasmin on cancer cells, plasmin-depletion therapy is developed. Although plasminogen activators render cancer cells treatment resistant, plasmin-depletion therapy engages plasminogen activators as therapeutic agents. Depletion of plasminogen by plasminogen activators in tissue culture is feasible due to the half life of plasmin being less than one minute (15) . Treated culture medium with plasminogen activators, plasminogen will be converted to plasmin that, in turn, will be inactivated within a minutes. In the medium treated with plasmin- depletion therapy, plasmin could not be generated on cancer cells even if urokinase activity is high because its precursor plasminogen will be depleted. Consequently cells will be free of plasmin. The effect of plasmin-depletion therapy on treatment sensitivity of tumor cells was examined in tissue culture. Tumor cells such as HT29 cells are known to produce urokinase (57) . It was examined whether tumor endogenous urokinase induces treatment resistance and plasmin-depletion therapy overcomes such treatment resistance. HT29 cells were cultured either in the medium containing plasminogen or in the medium treated with plasmin-depletion therapy and their sensitivity to doxorubicin, macrophage cytotoxicity and M79 antibody-induced macrophage-mediated ADCC was compared. Cultured in the medium treated with plasmin-depletion therapy, cells increase their sensitivity to doxorubicin, macrophage cytotoxicity and ADCC greatly (Figure 9-11) . The observations show that endogenous urokinase renders cancer cells treatment resistant and plasmin-depletion therapy overcomes such resistance. The effect of plasmin-depletion therapy on treatment resistance induced by endogenous urokinase was examined further. Cancer cells were cultured in the medium depleted of plasminogen by plasmin-depletion therapy. Subsequently, their sensitivity to macrophage cytotoxicity and to ADCC were examined. In culture treated with plasmin-depletion therapy, HT29 cells (Figure 12) and SKBR5 cells (Figure 13) increased their sensitivity to macrophage and ADCC substantially. The data further show that endogenous urokinase induces treatment resistance and plasmin-depletion therapy overcomes such resistance. Endogenous urokinase induces the resistance of cancer cells to T cell cytotoxicity. It was examined next whether endogenous urokinase induces the resistance of cancer cells to other host defense systems and the resistance can be overcome by the plasmin-depletion therapy. The host defense systems examined are T cell cytotoxicity and T cell cytotoxicity mediated by CD3/17-1A T cell and tumor cell reactive bispecific antibody (CD3/17- 1A bispecific antibody). CD3/17-1A bispecific antibody (58) was kindly provided by Dr, G. Riethmuller, Munich, Germany. HT29 cells cultured in the medium treated with plasmin- depletion therapy are significantly more sensitive to T cell cytotoxicity and bispecific antibody-mediated T cell cytotoxicity than cells cultured in the medium containing plasminogen (Figure 14 and 15) . Endogenous urokinase induced the resistance of HT29 cells to T cell cytotoxicity as well as to bispecific antibody-mediated T cell cytotoxicity and the plasmin-depletion therapy overcomes the resistance. Plasmin-depletion therapy inhibits the induction of treatment resistance by exogenous urokinase. Having observed that plasmin-depletion therapy overcomes treatment resistance induced by tumor endogenous urokinase (Figure 10-15), it was examined next whether plasmin- depletion therapy blocks the induction of treatment resistance by exogenous urokinase. Cells were cultures in the medium treated with plasmin-depletion therapy or control medium containing plasminogen in the absence or presence of urokinase. In culture treated with plasmin-depletion therapy, urokinase was unable to render cancer cells treatment resistant: cancer cells treated with urokinase are killed by macrophages and M79 antibody-induced ADCC as efficiently as cancer cells untreated. On the other hand, urokinase increases the resistance of cancer cells signi icantly in control culture containing plasminogen (Figure 9,10,16,17). Plasmin-depletion therapy blocked the induction of treatment resistance by exogenous urokinase. Plasmin-depletion therapy reverts treatment resistance to treatment sensitive . It was examined whether the plasmin-depletion therapy could revert cancer cells that are already treatment resistant to sensitive. HT29 cells and SKBR5 cells were treated with urokinase for two days and subsequently cultured for 16 hours in the medium treated with the plasmin-depletion therapy or untreated medium. Cells cultured in the medium treated with the plasmin-depletion therapy were substantially more sensitive to therapeutic agents than cells cultured in the medium untreated. (Figure 18-21). Plasmin-depletion therapy reverted treatment resistant cancer cells induced by urokinase to treatment sensitive. Plasmin-depletion therapy engaging tPA overcomes treatment resistance . Since plasmin-depletion therapy is essentially the treatment of plasminogen activator, any plasminogen activators could be used as the therapeutic agent of plasmin-depletion therapy. In the previous experiments, it was observed that the plasmin-depletion therapy engaging streptokinase or urokinase overcomes treatment resistance (Figure 10-21). Here, it was examined whether plasmin-depletion therapy engaging tPA also overcomes treatment resistance. HT29 cells were cultured for two days with human serum either incubated alone or with tPA. Subsequently, the sensitivity to doxorubicin was examined. In the culture treated with tPA- plasmin-depletion therapy, cells become more sensitive to doxorubicin. In addition, urokinase was unable to induce treatment resistant in such culture. Urokinase induced treatment resistance in control culture ( Figure 22) . TPA- plasmin-depletion therapy reverts treatment resistant cells to sensitive, blocks the induction of treatment resistance by urokinase, and thus overcomes treatment resistance. Plasmin-depletion therapy is effective in vivo. The effectiveness of plasmin-depletion therapy in vivo was examined in animal models. It is feasible to deplete plasminogen in animals or in humans by plasminogen activator treatments because of the big difference of the half life of plasminogen and plasmin. The half life of plasminogen is approximately 2.2 days. Plasmin, on the other hand, is inactivated within one minutes (15) . When cancer patients are treated with the maximum tolerable amount of plasminogen activators, the majority of plasminogen in the body would be converted to plasmin that, in turn, would be inactivated within one minute. Since plasminogen is produced slowly, the level of plasminogen in the body would be low until it is replenished fully. In fact, it has been reported that in the blood of patients treated with plasmin-depletion therapy, the level of plasminogen is low and plasmin can not be generated in the presence of plasminogen activators for 24-48 hours (32,33). In cancer patients, during such plasminogen-low period, regardless of high urokinase activity in tumor tissues, plasmin will not be generated on cancer cells due to the lack of its precursor plasminogen, and thus treatment resistant will be overcome. To examine whether plasmin-depletion therapy is effective in vivo, mice were treated with the plasmin-depletion therapy and their serum was prepared. HT29 cells cultured with the serum of mice treated with the plasmin-depletion therapy were more sensitive to therapeutic agents than cancer cells cultured with the serum of control mice treated with saline (Figure 23) . Plasmin-depletion therapy was effective in vivo. Plasmin-depletion therapy increases the efficacy of M79 antibody in animals . It was examined whether the plasmin-depletion therapy increases the efficiency of M79 antibody eradicating HT20 cells transplanted in animals. Modified animal models that have been already used to examine the efficacy of 17-1A antibody have been used (59,60). 17-1A antibody is shown to be effective in colorectal cancer patients (4). HT29 cells were injected into the abdomen of nude mice intradermally. Mice bearing HT29 cells were treated with the plasmin- depletion therapy or saline as control. Twenty-four hours later, mice were treated with saline or M79 antibody twice at 24 hours intervals. HT29 cells form tumors in all control mice. Treated with plasmin-depletion therapy alone, tumors were formed in all animals but the volume of tumors were smaller than in control mice. Treated with M79 antibody, HT29 cells form tumors in all mice but the volume of tumors was smaller than in control mice. Treated with the combination of the plasmin-depletion therapy and M79 antibody, HT29 cells did not form tumors until 16 days after tumor inoculation (Figure 24). Plasmin-depletion therapy enhances the efficacy of M79 antibody in vivo tremendously. Plasmin-depletion therapy enhances the efficacy of doxorubicin in vivo. The effect of plasmin-depletion therapy on the efficacy of doxorubicin in vivo was examined. HT29 cells were inoculated into the abdomen of nude mice intradermally. Twenty-four hours later when tumor cells were settled on the skin, animals were treated with urokinase-plasmin-depletion therapy or saline as control. Twenty-four hours after the therapy, animals were treated with doxorubicin or saline, and the volume of tumors were measured) at intervals . To be able to see the effect of plasmin-depletion therapy, the suboptimum dose of doxorubicin was administered. In animals treated with plasmin-depletion therapy alone or doxorubicin alone, tumors were smaller than in control animals treated with saline. In animals treated with both plasmin-depletion therapy and doxorubicin, tumors were significantly smaller than in animals treated with either plasmin-depletion therapy alone or doxorubicin alone (Figure 25) . Plasmin- depletion therapy increases the efficacy of doxorubicin in vivo. Potential applications of plasmin-depletion therapy Plasmin-depletion therapy increases the susceptibility of cancer cells to therapeutic agents. The therapy, therefore, could be utilized either to reduce therapeutic dose of immunotherapeutic as well as chemotherapeutic drugs or to increase the efficiency of therapies. The former can be applied to chemotherapeutic drugs with toxic side effects. Treated together with plasmin-depletion therapy, therapeutic doses of drugs will be decreased thus toxic side effects will be abolished or decreased substantially. The latter can be applied to drugs without lasting toxic side effects. Administered together with plasmin-depletion therapy, drugs at currently used doses will eliminate more cancer cells. Plasmin-depletion therapy is proven to deplete plasminogen and to block plasmin generation in humans. The plasmin-depletion therapy is essentially plasminogen activator therapy that has been used already in clinics in the treatment of blood clotting (32-36) . Plasmin, under normal physiological condition, dissolves blood clots thus allows blood to circulate the body freely. Hence, plasminogen activators are administered to patients with blood clots (pulmonary embolism, coronary artery thrombosis, arteriovenous cannulae occlusion, etc.) to generate excess plasmin thus to accelerate the lysis of blood clots. Also after heart attack or stroke, patients are treated with plasminogen activators to generate excess plasmin, thus, to accelerate the lysis of blood clots. In patients treated with plasminogen activators, a large amount of plasmin is generated immediately after the treatment and subsequently decreased. Plasminogen activators injected into patients, after converting plasminogen to plasmin, are degraded within 20 minutes. The level of plasminogen decreases and remains low for 24-48 hours. During such plasminogen-depleted period, plasmin could not be generated regardless of the presence of plasminogen activator because of the lack of its precursor plasminogen (32,33). The reports demonstrate that plasmin-depletion therapy effectively deplete plasminogen and blocks plasmin generation in humans. Plasmin-depletion therapy is safe in humans . Plasmin-depletion therapy has been already used in clinics, which demonstrates that the depletion of plasminogen for short periods does not cause the lasting harm in humans. Furthermore, plasminogen "knockout" mice that are without plasminogen in their life time are shown to be healthy (61) . Plasminogen "knockout" mice are constructed by inactivating plasminogen gene using molecular biology techniques. Apparently, in the absence of plasminogen/plasmin, blood clotting is regulated effectively by alternative systems. The report not only further supports the safety of the plasmin-depletion therapy but also suggests that repeated or prolonged plasmin-depletion therapy is safe in patients. Plasmin-depletion therapy is proven to be safe in tumor- bearing animals . Since plasmin-depletion therapy is for cancer patients, its safety in tumor-bearing animals was examined. The side effect of plasmin-depletion therapy is occasional systemic bleeding that occurs immediately after treatment. It was examined whether plasmin-depletion therapy causes bleeding in animals bearing tumors. At the end of the experiments shown in Figures 24 and 25, animals bear various sizes of tumors that can be readily examined macroscopically . Such animals were treated with 150 unit urokinase twice at 30 minute-interval intravenously. Examined macroscopically immediately after treatment and daily thereafter for one week, none of tumors bled and animals were healthy. The health of animals can be assessed grossly by examining the fur, the mobility, cachexia, etc. Modification of plasminogen activators as well as of their application in plasmin-depletion therapy. In plasmin-depletion therapy, plasminogen activators will be engaged to deplete circulating plasminogen, inhibit the generation of plasmin on cancer cell surface, and thus overcome treatment resistant of cancer cells. Moreover, in plasmin-depletion therapy, plasminogen activators as well as its application can be modified as follows: 1. Use of modified plasminogen activators. In the treatment of blood clots, plasminogen activators are used to generate plasmin on clots. To dissolve blood clots, plasminogen activators bind to fibrin, convert plasminogen on fibrin to plasmin, and plasmin degrades blood clots (15) . Therefore, fibrin binding site of plasminogen activators is required in the treatment of blood clots. In the plasmin- depletion therapy, on the other hand, the role of plasminogen activators is to convert plasminogen to plasmin to deplete circulating plasminogen thus fibrin binding site of plasminogen activators is not required. Furthermore, plasminogen activators with modified fibrin binding site is preferred because it will be unable to bind to fibrin, thereby inefficiently mediate fibrinolysis consequently could not cause bleeding. In plasmin-depletion therapy, therefore, not only native plasminogen activators, but also active form plasminogen activators with modified fibrin binding site can be used. Plasminogen activators can be modified further by altering its cellular receptor binding sites for the elimination of unwanted functions of plasminogen activators. For example, urokinase mediates its biological effects upon binding to cell surface receptors (25-29) . Not only cancer cells, some normal cells, for example macrophage, express urokinase receptors (47) . By modifying cell surface binding site of urokinase, urokinase can not bind to cells, hence could not mediate the unwanted effect on normal cells. Moreover, a fragment of plasminogen activators containing only active catalytic site and plasminogen binding site can be used eventually. Such molecules can be readily constructed using molecular biology techniques . 2. Administration of plasminogen activators with inhibitors of plasmin. In the treatment of blood clots, the side effect of plasminogen activator treatment is internal bleeding due to excess plasmin activity causing uncontrolled fibrinolysis. Such bleeding can not be prevented in the treatment of blood clots, stroke or heart attack because plasmin is the therapeutic agent and fibrinolysis is the therapeutic process. When bleeding occurs, to stop bleeding, patients are treated with plasma that contains inhibitors of plasmin to inactivate plasmin (32-36). In the plasmin-depletion therapy of cancer, on the other hand, internal bleeding can be easily prevented without diminishing its therapeutic efficacy because plasmin is an unnecessary by-product, fibrinolysis is an undesirable process, and inhibitors of plasmin as well as fibrinolysis are available readily (15,36-39). By administering plasminogen activators and inhibitors of plasmin together, plasmin will be inactivated as soon as it is activated hence internal bleeding caused by plasmin can be prevented. Plasmin generated on fibrin mediates fibrinolysis. For efficient fibrinolysis, therefore, the binding of plasminogen to fibrin prior to its activation is required. Therefore, administration of the agents interfering the binding of plasminogen to fibrin together with plasminogen activating substances will also prevent fibrinolysis. 3. The design of Plasmin-depletion therapy drugs. Plasminogen activators as well as plasmin have a short half- life. For the efficient inhibition of fibrinolysis, therefore, the delivery of fibrinolysis inhibitor to the site of plasminogen activator delivery at the time of its action is required. For such delivery, administration of a drug with plasminogen activating capacity and fibrinolysis inhibitory capacity is preferred to the administration of the combination of two drugs. Considering these points, plasmin-depletion therapy drugs which are the hybrid of plasminogen activating substance and fibrinolysis inhibitor unit are designed. As used herein, a plasminogen activating unit is a compound capable of activating the plasminogen. After activation, the plasminogen will change from a proenzyme to an active enzyme. Such plasminogen activating unit may be the catalytic domain of tissue plasminogen activator or the catalytic domain of urokinase. Alternatively, such unit may be a streptokinase which is known to activate plasminogen without cleaving the molecule. The unit includes but is not limited to urokinase, tissue plasminogen activator, streptokinase, other plasminogen activator or their modified forms . As used herein, the fibrinolysis inhibitory unit means any compound capable of inhibiting fibrinolysis. Such compound include, but is not limited to alpha 2 -antiplasmin. Plasmin-depletion therapy drugs can be modified by adding the cleavage sequence derived from urokinase or tPA. Urokinase or tPA occurs as a single chain. In vivo, they are cleaved at the specific peptide bond by plasma proteases and become two chains. By inserting the cleavage sequence of urokinase or tPA between plasminogen activating unit and fibrinolysis inhibitory unit, a single chain plasmin- depletion therapy drugs are designed to becomes two drugs, plasminogen activator and fibrinolysis inhibitor, in patients . The sequence of the actions of the plasmin-depletion therapy drugs in cancer patients with or without the cleavage sequence is expected as follows. Injected as a single drug, plasminogen activator and fibrinolysis inhibitor will be delivered to the same site simultaneously. Injected plasmin-depletion therapy drug with the cleavage sequence, the drugs will be cleaved at the cleavage peptide bond by plasma proteases and become two drugs, active plasminogen activator and fibrinolysis inhibitor. Regardless of the cleavage, plasminogen activator will convert plasminogen to plasmin. Plasmin thus generated will be inactivated efficiently by fibrinolysis inhibitor delivered and accumulated at the site and at the time of plasmin generation. In addition or alternatively, fibrinolysis inhibitor can block fibrinolysis by interfering the binding of plasminogen to fibrin, thus facilitating the conversion of plasminogen to plasmin in solution not on fibrin. In such manners, plasmin-depletion therapy drugs are expected to convert plasminogen to plasmin efficiently, but poorly mediate fibrinolysis. Examples of two plasmin-depletion therapy drugs with the cleavage sequence (plasmin-depletion therapy drug-u and plasmin-depletion therapy drug-t) are as follows. Plasmin- depletion therapy drug-u is the hybrid of urokinase and 2 - antiplasmin. Plasmin-depletion therapy drug-t is the hybrid of tPA and 2 -antiplasmin . μ -antiplasmin is a plasma glyprotein that blocks fibrinolysis by inhibiting plasmin activity specifically as well as by interfering the binding of plasminogen to fibrin (37-39) . To prevent immune responses against plasmin-depletion therapy drugs, human plasminogen activators and human α 2 -antiplasmin are utilized. The DNA sequence, the gene structure, the amino acid sequence, and the functional domains of urokinase, tPA and α 2 -antiplasmin are well characterized (37-44). The information allows the design as well as the production of plasmin-depletion therapy drugs readily by applying the standard molecular biology techniques and conventional protein chemistry procedures. Plasmin-depletion therapy drugs can be produced by linking the entire urokinase, or tPA, to α 2 -antiplasmin. Alternately, their modified form can be used to eliminate their unnecessary effects. For example, the use of modified urokinase will eliminate unwanted cellular effects of urokinase on normal cells. Urokinase bound to its specific cell surface receptor mediates its cellular effects such as the modulation of the expression of cell surface integrins and others (64,65). By the use of urokinase containing modified cellular receptor binding region, plasmin-depletion therapy drug-u will be unable to bind to cells thus unable to mediate unwanted cellular effects of urokinase. An example of modified of urokinase is as follows. Prourokinase is an eyzymatically inactive single chain peptide consisting 411 amino acids. Prourokinase is cleaved at the site between 158th lysine and 159th isoleucine by plasma proteases and becomes active two chain urokinase. The peptide consisting amino acid from 1 to 158 has been designated as A chain of urokinase. The peptide consisting amino acid from 159 to 411 has been designated as B chain of urokinase. B chain has catalytic activity (40-42). To render the B chain enzymatically inactive, nine amino acids of A chain (a. a. 150-158) will be attached to the B chain. Attached nine amino acids of A chain, the B chain become the catalytic peptide containing the cleavage sequence. In patients, it will be cleaved at the cleavage site, the pepr-ide bond between 158 and 159 and become two peptides, a short peptide containing amino acid 150-158 and the peptide containing amino acid 159-411. It has been shown that truncated prourokinase consisting amino acid 150-411 preserves the catalytic activity and can be produced readily (62). The use of tPA containing modified non-catalytic domains, for example, will reduce the fibrinolytic capacity as well as prolong the circulation time of plasmin-depletion therapy drug-t. Unattached to fibrin, tPA is inefficient plasminogen activator. Upon binding to fibrin or by cleaved at the specific peptide bond, tPA becomes active enzyme. TPA binds to fibrin through its non-catalytic domains. By the use of modified tPA preserves its catalytic potential but altered non-catalytic domain, plasmin-depletion therapy drug-t will be unable to bind to fibrin thus its fibrinolytic capacity will be reduced. Furthermore, the circulation time of plasmin-depletion therapy drug-t can be prolonged by modifying non-catalytic domain. Intact tPA is cleared quickly in the liver. TPA binds to liver cells through the region in the non-catalytic domain (43-46) . By the use of modified tPA with modified region interacting with liver cells, plasmin-depletion therapy drug-t can not bind to liver cells thus its circulation time will be prolonged. An example of modified tPA is as follows. Unattached to fibrin, tPA is an inefficient single chain plasminogen activator comprising 527 amino acids. In vivo, tPA is cleaved at the arg275-Ile276 peptide bond by plasma proteases and becomes active two chain plasminogen activator (43-46) . Truncated tPA is designed to contain the entire catalytic domain and the cleavage sequence by attaching 13 amino acids of non-catalytic domain (amino acid from 263 to 275) to the catalytic sequence (the amino acid 276-527) . The binding site of tPA to fibrin and to liver cells is located in the non-catalytic region. Deleted of non- catalytic domains, truncated tPA would be unable to bind to fibrin consequently its fibrinolytic capacity would be reduced substantially. Unable to bind to liver cells, the circulation time of truncated tPA would be prolonged. The truncated tPA containing the amino acid 263-527 are shown to preserve the catalytic activity and can be produced readily (63) . An example of modified 2 -antiplasmin is as follows. 2 -antiplasmin comprises 452 amino acids. 2 -antiplasmin inhibits fibrinolysis by inhibiting plasmin activity specifically and by interfering the binding of plasminogen to fibrin. The C-terminal region of 2 -antiplasmin containing 137 amino acids inhibits plasmin activity and interferes the binding of plasminogen to fibrin (37-39) . To endow the necessary functions only, the truncated ? - antiplas m consisting the amino acid 316 to 452 will be used. An example of the method to produce Plasmin-depletion drugs is as follows: By applying the standard molecular biology techniques, the gene encoding urokinase, tPA or antiplasmin will be separately prepared. To construct plasmin-depletion therapy drug-u gene, urokinase gene will be linked to α 2 -antiplasmin gene. To construct plasmin-depletion therapy drug-t gene, tPA gene will be linked to 2 -antiplasmin gene. Plasmin- depletion therapy drug gene thus prepared will be expressed in appropriate host cells. Plasmin-depletion therapy drugs accumulated in host cells will be recovered and purified using standard procedures (37-44, 62, 63) . Briefly, (1) Preparation of all cytoplasmic RNA from cells producing desirable proteins. (2) Isolation of total polyadenylated mRNA using an oligo- dT column. (3) Fractionation of mRNA by their size using acid-urea agarose electrophoresis. (4) Selection of the fractions containing desired mRNA by in vitro translation of mRNA and subsequent treatment of the translated product with corresponding antibody. (5) Synthesis of double stranded cDNA using thus selected mRNA. (6) Attachment of poly-dC tail to cDNA. (7) Insertion of cDNA to the appropriate plasmid bearing phenotypic markers. (8) Transformation of bacteria with the plasmid prepared. (9) Culturing of transformed bacteria under the condition that allows the growth of transformed bacteria only. (10) Hybridization of bacterial DNA with various synthetic DNA oligemers complementary to the codons for desired protein. (11) Isolation of positive clones. (12) Digestion of the positive plasmid DNA with various restriction endonucleases and subcloning them to another plasmid. (13) Sequencing of the DNA of subclones and selection of DNA encoding desired amino acid sequence. (14) Prepared and modified genes will be tailored for the ligation. Subsequently, fibrinolysis inhibitor gene will be attached to the N-terminal of plasminogen activator gene. (15) Preparation and selection of the plasmid containing the DNA encoding the ligated plasmin-depletion therapy drug gene. The plasmid will be prepared and cloned using the method described above. The plasmids containing plasmin-depletion therapy drug gene will be tailored for insertion into the expression vector. (16) Transformation of prokryote or eukaryote host cells with the expression vector containing plasmin-depletion therapy drug gene. (17) Transformed host cells will be cultured under conditions which allow only transformed host cells to grow. Plasmin-depletion therapy drugs accumulated in the host cells will be recovered and purified. Briefly, transformed cells will be collected, disrupted by any standard methods, and centrifuged to collect a precipitate. Precipitate will be dissolved and subsequently purified by the combination of conventional purification procedures for recombinant protein, such as liquid chromatography, ion exchange chromatography, affinity and others. (18) The purity of plasmin-depletion therapy drug thus prepared and purified will be analyzed by an SDS-polyacrylamide gel electrophoresis. The essential functions of plasmin-depletion therapy drugs are : (1) the depletion of plasminogen by converting it to plasmin; and (2) the inhibition of fibrinolysis. The functions will be assessed in tissue culture and in vivo. The functions of plasmin-depletion therapy drugs will be assessed in vitro ad in vivo. In vitro assay Determination of plasminogen depletion. The capacity of plasmin-depletion therapy drugs depleting plasminogen from plasma will be assessed by incubating human plasma with plasmin-depletion therapy drugs or alone as negative control followed by measuring remaining plasminogen in the ELISA. Plasma will be also treated with urokinase or tPA as positive control. In the ELISA, antibodies react with human plasminogen will be used as antigens. It is expected that plasmin-depletion therapy drugs would convert plasminogen to plasmin thus deplete plasminogen. In the plasma treated with plasmin-depletion therapy drugs, therefore, plasminogen is expected to be absent or significantly reduced. The detection of plasminogen is not expected in the plasma treated with urokinase or with tPA. On the other hand, in the plasma incubated alone, the detection of substantial amount of plasminogen is expected. Determination of therapeutic efficacy. The therapeutic efficacy of plasmin-depletion therapy drugs will be assayed as described in Figures 9-22. Briefly, HT29 cells or SKBR5 cells will be cultured for two days with human plasma incubated either alone or with plasmin- depletion therapy drugs in the presence or absence of urokinase. As positive control, plasma treated with urokinase or with tPA will be used. Subsequently, their sensitivity to doxorubicin or to M79 antibody-mediated ADCC will be tested. It is expected that similarly to urokinase or tPA, plasmin-depletion therapy drugs converts plasminogen to plasmin thereby depletes plasminogen. Hence, as shown in Figures 9 and 10, tumor cells cultured with the human plasma incubated with plasmin-depletion therapy drugs will be more sensitive to doxorubicin and to ADCC than tumor cells cultured with plasma incubated alone. Furthermore, in the cultured treated with plasmin-depletion therapy drugs, it is expected that urokinase will be unable to induce treatment resistance. Determination of fibrinolysis inhibition. The ability of plasmin-depletion therapy drugs to inhibit fibrinolysis will be assessed in the standard clot lysis assay according to (63,66). The clot lysis assay measures fibrinolysis by measuring the turbidity of fibrin. Plasmin- depletion therapy drugs, urokinase, or tPA will be mixed with thrombin. Thrombin alone will be control. The mixture will be centrifuged together with fibrinogen and plasminogen and incubated to initiate clots and subsequent fibrinolysis. To assess fibrinolysis, the turbidity will be read using 340 mu m filter at intervals. In the samples treated with plasmin-depletion therapy drugs, because fibrinolysis is blocked by fibrinolysis inhibitor, the turbidity is expected to be high and comparable to that of the control samples incubated with thrombin alone without plasminogen activator. In the samples incubated with urokinase or tPA, because fibrinolysis will occurs, the turbidity is expected to be low. Determination of the cleavage. To examine whether modified plasmin-depletion therapy drugs containing the cleavage sequence are indeed cleaved to two chains by plasma protease, plasmin-depletion therapy drugs will be incubated alone or with trypsin. Urokinase or tPA will be treated similarly as control. Added trypsin inhibitor to stop trypsin proteolysis, the samples will be analyzed in the SDS gel electrophoresis. The detection of two peptides are expected in the all samples treated with trypsin. In the samples incubated without trypsin, on the hand, the detection of single chain is expected. In vivo assay. Determination of therapeutic efficacy. The efficacy of plasmin-depletion therapy drug in vivo will be assessed as described in Figure 24. Briefly, immunodeficient SCID mice or nude mice will be treated with saline, plasmin-depletion therapy drugs, urokinase or tPA. Subsequently, human colon cancer cells, breast cancer cells, prostate cancer cells, or other human solid cancer cells will be inoculated intradermally, treated with doxorubicin or saline and the formation of tumors will be assessed. It is expected that the formation of tumors will be delayed in mice treated with doxorubicin alone, plasmin-depletion therapy drugs alone, urokinase alone or tPA alone. Further delay of the formation of tumors is expected in animals treated with doxorubicin together with plasmin-depletion therapy drug, urokinase or tPA. The efficacy of plasmin-depletion therapy drugs will be also assessed in mice bearing tumors as described in Figure 25. Breast cancer cells, colon cancer cells, prostate cancer cells, or other human solid cancer cells will be inoculated to SCID or nude mice intradermally. Twenty-four hours later when tumors were settled on skin, mice will be treated with saline, plasmin-depletion therapy drugs, urokinase, or tPA. Two days later, mice will be treated with either 50 ug doxorubicin in 0.2ml saline or saline alone. The volume of tumors will be measured at intervals. It is expected that the size of tumors in mice treated with plasmin-depletion therapy drugs alone, urokinase alone, tPA alone or doxorubicin alone will be smaller than in mice treated with saline alone. In mice treated with doxorubicin together with plasmin-depletion therapy drugs, urokinase or tPA, the tumors are expected to be smaller than in mice treated with one drug alone. Determination of the side effect. Plasmin-depletion therapy drugs are designed to prevent the side effect of plasminogen activators systemic bleeding. To assess such function, the lethal dose (LD 0 ) of plasmin- depletion therapy drugs, urokinase, and tPA will be compared. The LD 50 of plasmin-depletion therapy drugs are expected to be significantly higher than the LD 50 of urokinase or tPA. 4. Titration of streptokinase antibody prior to the administration of plasminogen activators. Often, patients have various amounts of streptokinase antibody in the blood due to streptococcal infections. Injected into streptokinase antibody-positive patients, streptokinase will form the immune complex with streptokinase antibody consequently the treatment will be ineffective. To circumvent such problem, excess streptokinase was used in the treatment of blood clotting (33). In patients with high titer streptokinase antibody, streptokinase will form large immune complexes with streptokinase antibody, cleared rapidly by reticuloendothelial cells, and thus the efficacy of streptokinase will decreased substantially. In patients with low titer streptokinase antibody, streptokinase and streptokinase antibody will form small immune complexes, deposited on small blood vessel and cause the damage of the vessel (49) . Although the amount of streptokinase antibody in the blood can be easily titrated, the screening procedure of streptokinase antibody is not used in the treatment of blood clotting because patients have to be treated with plasminogen activators as soon as possible. In the plasmin- depletion therapy, on the other hand, the presence of streptokinase antibody in cancer patients can be pre- screened. The pre-screening will allow to administer streptokinase only to patients negative for streptokinase antibody thus increase the efficacy of streptokinase treatment as well as prevent the damage caused by small immune complex of streptokinase and streptokinase antibody. Patients positive for streptokinase antibody can be treated with other plasminogen activators, for example, tissue plasminogen activators, urokinase, or others. 5. Titration of effective dosage of an anti-cancer drug This invention also provides method of determining the effective dosage of an anti-cancer drug comprising steps of: (a) contacting cancerous cells with an appropriate amount of plasmin; (b) contacting the cells from step (a) with different amounts of the anti-cancer drug; and (c) determining a measurable effect of the drug to the cancerous cells, the amount which gives the effect is the effective amount. Such in vivo dosage mean to mimic the situation in a subject which contains plasminogen, the precursor of plasmin in the circulation. 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Claims: hat is claimed is : 1. A method of increasing the therapeutic effect of an anti-cancer agent on cancerous cells comprising steps of: (a) reducing plasmin on the surface of the cancerous cells in the presence of the anti-cancer agent; or (b) reducing plasmin on the surface of the cancerous cells and thereafter contacting the anti-cancer agent with the cells. 2. A method of increasing the therapeutic effect of an anti-cancer agent on cancerous cells in a subject comprising steps of: (a) reducing plasmin on the surface of the cancerous cells in the subject in the presence of the anti- caner agent; or (b) reducing plasmin on the surface of the cancerous cells in the subject and thereafter administering an effective amount of anti-cancer agent to the subject. 3. The method of claim 1 or 2, wherein the plasmin is reduced by depletion of plasminogen. 4. The method of claim 3, wherein the plasminogen is depleted by administering an effective amount of plasminogen-depleting substance to the subject to reduce the plasminogen from binding onto the surface of the cancerous cells. 5. The method of claim 4, wherein the plasminogen is depleted by administration to a subject a substance capable of activating the plasminogen. 6. The method of claim 5, wherein the substance is a plasminogen activator. 7. The method of claim 5, wherein the substance is a polypeptide comprising the plasminogen activating site of a plasminogen activator. 8. The method of claim 7, wherein the substance is tissue plasminogen activator, streptokinase, urokinase, staphylokinase or their modified forms. 9. The method of claim 8, wherein either the fibrin binding site or liver cell binding site of the tissue plasminogen activator is deleted or modified. 10. The method of claim 5, further comprising addition of a fibrinolysis inhibitor with the substance capable of activating the plasminogen. 11. The method of claim 10, wherein the fibrinolysis inhibitor comprises a plasmin inhibitor. 12. The method of claim 1 or 2, wherein the cancer is colon cancer, breast cancer, prostate cancer, ovarian cancer, stomach cancer or esophageal cancer. 13. The method of claim 2, wherein the subject is screened with the presence of streptokinase antibody before step (a) and the subject will not be administered with streptokinase if the formation of the immune complex between streptokinase and the streptokinase antibody will affect the reduction of plasmin in the subject. 14. A compound comprising at least one fibrinolysis inhibitor unit linked to at least one plasminogen activating unit. 15. The compound of claim 14, wherein the compound is a polypeptide. 16. The polypeptide of claim 15, wherein located between the fibrinolysis inhibitor unit and the plasminogen activating unit are linked by at least one cleavage site . 17. The polypeptide of claim 15, wherein the plasminogen activating unit has the sequence derived from tissue plasminogen activator, streptokinase, urokinase or staphylokinase . 18. The polypeptide of claim 15, wherein the fibrinolysis inhibitor unit has the sequence derived from alpha 2 - antiplasmin. 19. A pharmaceutical composition comprising effective amount of a compound having at least one fibrinolysis inhibitor unit linked to at least one plasminogen activating unit and a pharmaceutically acceptable carrier. 20. The composition of claim 19, wherein the fibrinolysis inhibitor unit and the plasminogen activating unit are linked by at least one cleavage site. 21. A method of increasing the therapeutic effect of an anti-cancer agent on cancerous cells comprising steps of: (a) contacting the cancerous cells with the pharmaceutical composition of claim 19 in the presence of effective amount of anti-cancer agent; or (b) contacting the cancerous cells with the pharmaceutical composition of claim 19 and subsequently contacting the cancerous cells with an effective amount of the therapeutic agent. 22. A method of increasing the therapeutic effect of an anti-cancer agent on cancerous cells in a subject comprising steps of: (a) administering the pharmaceutical composition of claim 19 to the subject in the presence of effective amount of anti-cancer agent; or (b) administering the pharmaceutical composition of claim 19 to the subject and subsequently administering an effect of anti-cancer agent to the subject.

Inventors: CHUN MYUNG HEE, HOFFMANN MICHAEL K, CHUN, MYUNG, HEE, HOFFMANN, MICHAEL, K.
Applicants: CHUN MYUNG HEE, HOFFMANN MICHAEL K, CHUN, MYUNG, HEE, HOFFMANN, MICHAEL, K.
Application year: 1997
Main classification: A01N37
IPC codes: A61K38, A61K45, A61P35, C07K14, C12N9