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EP0966197A1 - Therapie par appauvrissement de la plasmine - Google Patents

Therapie par appauvrissement de la plasmine

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Publication number
EP0966197A1
EP0966197A1 EP97937223A EP97937223A EP0966197A1 EP 0966197 A1 EP0966197 A1 EP 0966197A1 EP 97937223 A EP97937223 A EP 97937223A EP 97937223 A EP97937223 A EP 97937223A EP 0966197 A1 EP0966197 A1 EP 0966197A1
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EP
European Patent Office
Prior art keywords
plasmin
plasminogen
cells
urokinase
cancer
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EP97937223A
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German (de)
English (en)
Inventor
Myung Hee Chun
Michael K. Hoffmann
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Individual
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Individual
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A61K38/166—Streptokinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3153—Streptokinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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) .
  • 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) .
  • 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) .
  • 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.
  • plasminogen activators such as urokinase, tissue plasminogen activators, streptokinase, staphylokinase, and others.
  • plasminogen activators such as urokinase, tissue plasminogen activators, streptokinase, staphylokinase, and others.
  • circulating plasminogen binds to its cell surface receptors and subsequently converted to plasmin primarily by urokinase (15-18) .
  • plasmin-depletion therapy deprives cancer cells of
  • Plasmin activity on cancer cells is controlled by urokinase.
  • Urokinase activity on cancer cells is controlled by the expression urokinase receptors.
  • Urokinase is a protease that cleaves plasminogen to plasmin. Upon binding to its receptor on cell membrane, urokinase becomes stable with high enzymatic activity.
  • 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).
  • urokinase inhibitors for example, 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.
  • 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.
  • a completely new approach has been employed: the precursor of plasmin plasminogen is depleted instead of blocking urokinase activity.
  • plasminogen activators render cancer cells treatment resistant.
  • 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).
  • plasminogen activators deplete plasminogen by converting plasminogen to plasmin that is inactivated immediately.
  • 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.
  • 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) .
  • plasmin dissolves blood clots thus allows blood to circulate freely (15) .
  • 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.
  • plasminogen level is low for 24-48 hours after plasminogen activator therapies.
  • 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 .
  • plasmin is a unnecessary byproduct and fibrinolysis is an unwanted adversary reaction causing the side effect bleeding.
  • 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 ⁇ 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.
  • ⁇ 2 -antiplasmin does not interfere the conversion of plasminogen to plasmin.
  • ⁇ -.-antiplasmin Administered together with plasminogen activators, therefore ⁇ -.-antiplasmin will prevent fibrinolysis thus the side effect bleeding without decreasing the efficacy of the Plasmin-depletion therapy.
  • 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.
  • 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.
  • a mechanism that induces as well as maintains the resistance of cancer cells to host defense systems and to chemotherapeutic drugs 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) .
  • tissue culture the source of plasminogen on cancer cells is plasminogen contained in plasma or serum added to culture medium.
  • 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.
  • plasmin-depletion therapy utilizes plasminogen activators although plasminogen activators render cancer cells treatment resistant by converting plasminogen to plasmin.
  • plasminogen activators convert plasminogen contained in plasma or serum supplement to plasmin that is inactivated within a minute thus deplete plasminogen.
  • 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).
  • plasmin- depletion therapy overcomes treatment resistance in tissue culture as well as in animals.
  • 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) .
  • chemotherapeutic drugs doxorubicin and navelbine
  • 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
  • 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 .
  • 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 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.
  • plasminogen activators can be modified as follows:
  • fibrin binding site of plasminogen activators is required because they must convert plasminogen to plasmin on fibrin (15) .
  • 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.
  • modified plasminogen activator fibrin binding site can be used.
  • modified plasminogen activator with altered cell receptor binding site can be used to eliminate the unwanted functions of plasminogen activators.
  • urokinase bound to its specific cell surface receptors mediates cellular effects.
  • urokinase receptor (47) urokinase receptor 47) .
  • urokinase can be modified further by altering the cell surface receptor binding site.
  • plasminogen activators containing catalytic site can be used.
  • modified plasminogen activators can be constructed readily using molecular biology techniques.
  • Plasmin inhibitors are readily available (15, 37, 39, 48) .
  • plasmin-depletion therapy drugs are designed.
  • 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 .
  • 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.
  • 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.
  • streptokinase antibody 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) .
  • 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).
  • 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.
  • completely new approach has been used to block the generation of plasmin on cancer cells: circulating plasminogen is depleted.
  • 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) .
  • 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
  • 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.
  • 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
  • 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).
  • 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.
  • 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) .
  • plasmin is reduced by depletion of plasminogen.
  • 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.
  • a plasminogen-depleting substance are compound which is capable of reducing the amount of plasminogen in the body.
  • the plasminogen is depleted by administration to a subject a substance capable of activating the plasminogen.
  • the substance is a plasminogen activator.
  • the plasminogen activators include, but are not limited to tissue plasminogen activator, streptokinase, urokinase and staphylokinase .
  • the plasminogen-depleting substance is a polypeptide comprising the catalytic and plasminogen binding sites of a plasminogen activator.
  • the substance is a portion of urokinase wherein the cellular receptor binding site is modified.
  • 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 .
  • At least one plasminogen activating unit is linked to at least one fibrinolysis inhibitor unit.
  • Plasmin- depletion therapy drugs is as follows:
  • 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 .
  • 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.
  • 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
  • SKBR5 cells are obtained from Dr. L. Old, Ludwig Institute, New York City, NY. Other cell lines are obtained from /American Type Culture
  • 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
  • HT29 colon cancer cells Figure 3
  • 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) .
  • cancer cells 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.
  • breast and colon cancer cells develop the resistance to host defense systems .
  • ADCC antibody dependent cellular cytotoxicity
  • Urokinase induces the resistance of cancer cells through the conversion of plasminogen to plasmin.
  • 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.
  • 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.
  • 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.
  • plasminogen added 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.
  • Plasmin occurs as its precursor plasminogen in the blood.
  • plasminogen 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.
  • 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).
  • 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.
  • plasmin-depletion therapy 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.
  • 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.
  • Endogenous urokinase induces the resistance of cancer cells to T cell cytotoxicity.
  • 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, Kunststoff, 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.
  • 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.
  • 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.
  • 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 .
  • 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 .
  • plasmin-depletion therapy is essentially the treatment of plasminogen activator
  • any plasminogen activators could be used as the therapeutic agent of plasmin-depletion therapy.
  • the plasmin-depletion therapy engaging streptokinase or urokinase overcomes treatment resistance ( Figure 10-21).
  • 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.
  • 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.
  • plasminogen 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.
  • 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
  • Plasmin-depletion therapy increases the efficacy of M79 antibody in animals .
  • 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.
  • 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.
  • Plasmin-depletion therapy increases the susceptibility of cancer cells to therapeutic agents.
  • the therapy 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.
  • 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.
  • blood clots pulmonary embolism, coronary artery thrombosis, arteriovenous cannulae occlusion, etc.
  • patients are treated with plasminogen activators to generate excess plasmin, thus, to accelerate the lysis of blood clots.
  • 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.
  • 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. Hence, 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 .
  • 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.
  • 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.
  • 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.
  • plasminogen activators as well as its application can be modified as follows:
  • plasminogen activators are used to generate plasmin on 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.
  • fibrin binding site of plasminogen activators is required in the treatment of blood clots.
  • 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.
  • 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
  • plasminogen activators and inhibitors of plasmin 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • plasmin-depletion therapy drugs are expected to convert plasminogen to plasmin efficiently, but poorly mediate fibrinolysis.
  • 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 .
  • ⁇ -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) .
  • human plasminogen activators and human ⁇ 2 -antiplasmin are utilized.
  • the DNA sequence, the gene structure, the amino acid sequence, and the functional domains of urokinase, tPA and ⁇ 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 ⁇ 2 -antiplasmin. Alternately, their modified form can be used to eliminate their unnecessary effects.
  • 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).
  • 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.
  • tPA containing modified non-catalytic domains will reduce the fibrinolytic capacity as well as prolong the circulation time of plasmin-depletion therapy drug-t.
  • tPA Unattached to fibrin, tPA is inefficient plasminogen activator.
  • tPA 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.
  • 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.
  • 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.
  • modified tPA is as follows.
  • tPA is an inefficient single chain plasminogen activator comprising 527 amino acids.
  • 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.
  • 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) .
  • 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) .
  • the truncated ? - antiplas m consisting the amino acid 316 to 452 will be used.
  • the gene encoding urokinase, tPA or antiplasmin will be separately prepared.
  • urokinase gene will be linked to ⁇ 2 -antiplasmin 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,
  • 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.
  • 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.
  • plasmin-depletion therapy drugs The essential functions of plasmin-depletion therapy drugs are :
  • plasmin-depletion therapy drugs The functions of plasmin-depletion therapy drugs will be assessed in vitro ad in vivo.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mice bearing tumors 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.
  • 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.
  • 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.
  • 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.
  • streptokinase 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.
  • streptokinase and streptokinase antibody will form small immune complexes, deposited on small blood vessel and cause the damage of the vessel (49) .
  • 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.
  • 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.
  • Such in vivo dosage mean to mimic the situation in a subject which contains plasminogen, the precursor of plasmin in the circulation.
  • This invention provides method of determining the effective in vivo dosage of an anti-cancer drug comprising steps of: (a) contacting cancerous cells with an appropriate amount of plasminogen activator under conditions permitting the activation of plasminogen to plasmin, in medium which contains plasminogen; (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. In an embodiment, the measurable effect is cell death.
  • Physicians GenRx the complete drug reference. Alteplase, recombinant (0143), Streptokinase (2276) and urokinase (2417) (1996) .

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  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Cette invention porte sur une méthode visant à renforcer l'effet thérapeutique d'un agent anticancéreux sur des cellules cancéreuses. Cette méthode comporte les phases suivantes: (a), réduction de la plasmine à la surface des cellules cancéreuses et (b), mise en contact de l'agent thérapeutique avec les cellules obtenues dans la phase (a). Cette invention porte également sur une méthode visant à renforcer l'effet thérapeutique d'un agent anticancéreux sur les cellules cancéreuses d'un sujet, laquelle méthode comporte les phases suivantes: a), réduction de la plasmine à la surface des cellules cancéreuses du sujet et (b), administration de l'agent anticancéreux au sujet. Cette invention, qui concerne, de surcroît, un composé contenant un inhibiteur de la fibrinolyse lié à un activateur du plasminogène, a également trait aux applications de ce composé.
EP97937223A 1996-08-14 1997-08-13 Therapie par appauvrissement de la plasmine Withdrawn EP0966197A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US2359296P 1996-08-14 1996-08-14
US23592P 1996-08-14
PCT/US1997/014231 WO1998006264A1 (fr) 1996-08-14 1997-08-13 Therapie par appauvrissement de la plasmine

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EP0966197A1 true EP0966197A1 (fr) 1999-12-29

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EP97937223A Withdrawn EP0966197A1 (fr) 1996-08-14 1997-08-13 Therapie par appauvrissement de la plasmine

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EP (1) EP0966197A1 (fr)
JP (1) JP2001500849A (fr)
AU (1) AU3978697A (fr)
WO (1) WO1998006264A1 (fr)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4766111A (en) * 1985-10-31 1988-08-23 Trustees Of Boston University Lipids with plasmin inhibitory properties
ZA879286B (en) * 1986-12-16 1988-10-26 Smith Kline Rit New plasminogen activators
GB8827701D0 (en) * 1987-12-09 1988-12-29 Nippon Shinyaku Co Ltd Thrombolytic &c compositions
US4999194A (en) * 1988-01-14 1991-03-12 Collaborative Research, Inc. Two-chain urokinase plasminogen activators for treatment of thrombotic disease
FI895899A7 (fi) * 1988-12-12 1990-06-13 Yamanouchi Pharmaceutical Co Ltd Uudet trombolyyttiset proteiinit, menetelmä niiden valmistamiseksi ja näitä aktiiviaineina sisältävät lääkkeet
US5328996A (en) * 1989-03-29 1994-07-12 University Of Florida Research Foundation, Inc. Bacterial plasmin receptors as fibrinolytic agents

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9806264A1 *

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WO1998006264A1 (fr) 1998-02-19
AU3978697A (en) 1998-03-06
JP2001500849A (ja) 2001-01-23

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