KR100470535B1 - Immunoliposome conjugated with upa antibody - Google Patents

Abstract

The present invention relates to a selective drug delivery tool that targets cells expressing abundant urokinase type plasminogen activator (uPA) such as cancer cells or neovascular endothelial cells in cancer tissues. In addition to the cancer cell self-killing effect, when an anti-cancer agent is included inside an immunooliposome attached to an anti-uPA monoclonal antibody produced by a fusion cell of a splenic lymphocyte and myeloma cells In addition, by killing vascular endothelial cells to block the formation of neovascularization, the secondary effect of inducing cancer tissue necrosis can also be obtained.

Description

IMUUNOLIPOSOME CONJUGATED WITH UPA ANTIBODY}

The present invention is a selective drug delivery tool that targets cells expressing a lot of urokinase type plasminogen activator (uPA), such as neovascular endothelial cells in cancer cells or cancer tissues, and is a specific monoclonal antibody to uPA. It relates to an immunoliposome to which an anti-uPA antibody is attached.

Liposomes are artificial micelles composed of phospholipids, which can encapsulate various drugs therein. When the drug is contained in the liposomes, it is possible to prevent the drug from being lost by in vivo defense mechanisms before reaching the target when delivering the drug to specific cells or organs. In addition, by attaching an antibody capable of recognizing a specific cell surface molecule to liposomes, binding specificity between target cells and liposomes can be enhanced. This is what is called immunooliposomes.

When an antibody of an immunoliposome binds to a cell surface protein, endocytosis occurs, in which not only the protein but also the liposome enters the cell, so that the drug in the liposome enters the cell. Drugs can be selectively concentrated (Huwyler J., and Wu D., Pardridge WM Brain drug delivery of small molecules using immunoliposomes, Proc. Natl. Acad. Sci. USA 93: 14164-14169, 1996; Spragg D. , Alford DR, Greferath R., Larsen CE, Lee KD, Gurtner GC, Cybulsky MI, Tosi PF, Nicolau C., and Gimbrone Jr. MA Immunotargeting of liposomes to activated vascular endothelial cells: A strategy for site specific delivery in the cardiovascular system, Proc. Natl. Acad. Sci. USA 94: 8795-8800, 1997). The key to effectiveness in drug delivery in this manner is the specificity of the antibody. As an example, if one can obtain specific antibodies against substances that may be present only in malignant tumor cells, they may be effectively used for anticancer drug administration.

On the other hand, the high mortality rate due to malignant tumors is known to be a major cause in many cases due to invasion to the surrounding tissue and metastasis to distant organs. Invasion into surrounding lymph nodes and surrounding tissues is also important data for the treatment and prognosis of patients.

Depending on the type of cancer, the most basic treatment is surgery to remove the cancerous tissue, which may include chemotherapy, radiotherapy or immunotherapy. However, most of these therapies have significant toxicity not only to cancer cells but also to normal cells, so the treated patient must bear the side effects. Therefore, for better treatment, it is desirable to target only substances with a significant difference between normal cells and cancer cells and to effectively and selectively remove only cancer cells. Against this background, various proteases related to cancer cell 'invasion-metastasis' may be potential targets.

It is well known that infiltration and metastasis that characterize cancer cells are caused by cancer cells expressing more proteases than normal cells to break down the extracellular matrix (ECM). Such proteases include matrix metalloproteinases (MMPs) such as plasminogen activator, plasmin, and type IV collagenase (Andreasen PA, Egelund R.). ...., Petersen HH The plasminogen activation system in tumor growth, invasion, and metastasis Cell Mol Life Sci 57:.. 25-40, 2000; Mignatti P., Rifkin DB Plasminogen activators and matrix metalloproteinases in angiogenesis Enzyme Protein 49: 117-137, 1996; Vu TH, Werb Z. Matrix metalloproteinases: effectsors of development and normal physiology.Genes . Dev. 14: 2123-2133 ,. 2000).

The urokinase type plasminogen activator (uPA), a type of plasminogen activator, is a serine protease that makes plasminogen plasmin through limited proteolytic reactions. . 1 is a schematic diagram showing the process of releasing extracellular matrix (ECM) by generating plasmin after uPA is secreted and activated by inactive pro-uPA (or scuPA).

Evidence that uPA is involved in the invasion-metastasis of cancer cells is well known by several studies. In other words, the antibody of uPA or antisense RNA (Kook YH, John A., Zelent A., and Ossowski L. The effect of antisense inhibition of urokinase receptor in human squamous cell carcinoma, EMBO J. 13: 3983-3991, 1994; Ossowski L., Reich E. Antibodies to plasminogen activator inhibit human tumor metastasis, Cell 35: 611-619, 1983; Yu H., Schultz RM Relationship between secreted urokinase plasminogen activator activity and metastatic potential in murine B16 cells transfected with human urokinase sense and antisense genes, Cancer Res. 50: 7623-7633, 1990), several cancer tissues extracted from patients (lung cancer, colorectal cancer, breast cancer, melanoma) Or uPA expression levels of artificially produced cancer cell lines are higher than those of cancer cells or normal cells with low invasive-metastatic capacity (de Bruin AF, Grissioen.G., Verspaget HW, Verheijen JH, Dooijewaard G., et al.). vanden Ingh HF, Lamers CBHW Plasmi nogen activator profiles in neoplastic tissues of the human colon, Cancer Res. 48: 4520-4524, 1988; Markus G., Camiolo SM, Kohga S., Madeja JM, Mittelman A. Plasminogen activator secretion of human tumors in short-term organ culture, including a comparison of primary and metastatic tumors, Cancer Res. 43: 5517-5525, 1983). It is also reported that uPA is more distributed in the growth and infiltration of cancer cells (Skriver L., Larsson LI, Kielberg V., Nielsen LS, Andresen PB, Kristensen P., Danø K. Immunocytochemical localization of urokinase) -type plasminogen activator in Lewis Lung carcinoma, J. Cell Biol. 99: 752-757, 1984).

uPA is involved in the activation of MMPs by plasmin (Mackay AR, Corbitt RH, Hartzler JL) in addition to degrading ECM constituent proteins laminin, type IV collagen, etc. by plasmin production. , Thorgeirsson UP Basement membrane type IV collagen degradation: Evidence for the involvement of a proteolytic cascade independent of metalloproteinases, Cancer Res. 50: 5997-6001, 1990). In other words, the plasmin is released in an inactive form, MMPs are made active, these enzymes also contribute to cancer cell invasion-transition. Taken together, these studies may be important targets for anti-cancer or anti-electrification techniques that can differentiate cancer cells from normal cells to inhibit cancer cell invasion-metastasis. However, not all uPAs can be targeted, and uPAs that bind to cell surface receptors (uPA receptors, uPARs) and adhere to cell surfaces are good targets.

uPA is secreted into an inactive single chain uPA (scuPA) and activated into a double chain (two chain uPA; tcuPA) (Petersen LC, Lund LR, Nielsen LS, Danø K., Skriver L. One chain urokinase- type plasminogen activator from sarcoma cells is a proenzyme with little or no intrinsic activity, J. Biol. Chem. 263: 11189-11195, 1988; Skriver L., Nielsen LS, Stephens R., Danø K. Plasminogen activator released as inactive proenzyme from sarcoma virus transformed murine cells, Eur. J. Biochem. 124: 409-414, 1982), which involved uPA receptors on the cell surface (Ellis V., Scully MF, Kakkar, VV Plasminogen activation initiated by single-chain urokinase-type plasminogen activator, potentiation by U937 monocyte. J. Biol. Chem. 264: 2158-2188 1989), their binding does not interfere with enzymatic activity. It is speculated that not only uPA but also plasminogen may bind nearby to the cell surface, thereby accelerating plasmin production. In other words, it is possible to achieve the maximum effect by centralizing the substances involved in the production of plasmin at various stages on the cell surface. What is important at this point is the binding of uPA and receptors, which means that amplification of activation results in the production of large amounts of plasmin around the cancer cells, which in turn results in increased cancer cell invasion-transfer capacity (Danø K., Andreasen PA, Grøndahl-). Hansen K., Kristensen P., Nielsen LS Skriver L. Plasminogen activators, tissue degradation and cancer.Adv . Cancer Res . 44: 139 266, 1985; Hollas W., Blasi F., Boyd D. Role of the urokinase receptor in facilitating extracellular matrix invasion by cultured colon cancer, Cancer Res. 51: 3690-3695, 1991; Mignatti P., Rifkin DB Biology and biochemistry of proteinases in tumor invasion.Physiol . Rev. 73: 161-195, 1993; Werb Z. ECM and cell surface proteolysis: regulating cellular ecology. Cell 91: 439-442, 1997).

However, in the situation where a substantial amount of uPA is present in the state of binding to a cancer cell surface receptor, the activation of uPA bound to the cell surface receptor does not occur indefinitely, and a substance called plasminogen activator inhibitor (PAI) binds to uPA to activate its activity. Suppress The complex formed by PAI binding to the uPA bound to the receptor is introduced into the cell, and only the uPA and PAI are degraded in the cell, and only the receptor is released from the cell and recycled to the uPA.

Figure 2 is a schematic diagram showing the interaction between the uPA activation process and intracellular influx mechanism of the uPAR-uPA-PAI complex and α2-MR. Here, inactive uPA (scuPA) (A), when activated by binding to the cell surface uPAR (tcuPA) (B), the uPA inhibitor PAI binds to form a uPAR-uPA-PAI complex, α2-MR and The combined complex shows a circulating process (C) that enters the cytoplasm and degrades, with only the receptor reappearing on the cell surface. Immunoliposomes also bind to uPA with uPA antibody and undergo the same process as PAI (D, E), because this process can be induced with an anti-uPA antibody in addition to PAI (Andreasen PA). , Sottrup-Jensen L., Kj ller L., Nykj r A., Moestrup SK, Munch Petersen C. Receptor mediated endocytosis of plasminogen activators and activator: inhibitor complexes.FEBS Lett. 338: 239-245, 1994; Conese M. , Nykj r A., Petersen CM, Cremona O., Pardi R., Andreasen PA α2-Macroglobulin receptor: LDL receptor related protein (LRP) -dependent internalization of the urokinase receptor.J. Cell Biol . 131: 1609-1622, 1995; Cubellis MV, Andreasen PA, Ragno P., Mayer M., Danø K. Blasi F. Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor.Proc . Natl. Acad. Sci. USA 86: 4828-4832 1989; Ellis V., Wun TC, Behrendt N., R nne E. Danø K. Inhibition of receptor-bound urokinase by plasminogen activator inhibitors.J. Biol. Chem . 265: 9904- 9908, 1990; Nykjær A., Conese M., Christensen EI, Olson D., Cremona O., Gliemann J. Recycling of the urokinase receptor upon internalization of uPA: serpin complexes. EMBO J. 16: 2610-2620, 1997).

The behavior similar to the invasion-metastasis of cancer cells can be seen in normal vascular endothelial cell migration, blood cell migration and fertilization of placenta implantation process. to be. In general, when cancer cells grow to a size of about 1 mm in diameter, cancer cells located in the center of the tumor are less likely to obtain oxygen or nutrients than cells outside, and new blood vessels must be made to solve this problem (Bouck N. , Stellmach V., Hsu SC How tumors become angiogenic.Adv. Cancer Res. 69: 135-74, 1996; Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease.Nature Med . 1: 27-31, 1995; Risau W. Mechanisms of angiogenesis.Nature , 386: 671-674, 1997). At this time, the vascular endothelial cells proliferate from the surrounding microvascular vessels and move toward the cancer cells. The moving process, like cancer cells, decomposes surrounding tissues with protease to make space and move in the direction. Typical of these proteases are uPA and MMP (Mignatti P., Rifkin DB Plasminogen activators and matrix metalloproteinases in angiogenesis. Enzyme Protein . 49: 117-137, 1996).

As a result, uPA is expressed more than normal cells and plays an important role in invasion-metastasis characteristic of cancer cells, and also shows high uPA in endothelial cells involved in angiogenesis necessary for cancer cell proliferation (Hildenbrand R., Dilger I., Horlin A. Stutte HJ Urokinase plsminogen activator induces angiogenesis and tumor vessel invasion in breast cancer.Pathol.Res.Practic . 191: 403-409, 1995; Min HY, Doyle LV, Vitt CR, Zandonella L., Stratton Thomas JR, Shuman MA, Rosenberg S. Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice.Cancer Res . 56: 2428-2433, 1996) and others have been selected to target the circulatory process of the uPAR-uPA-PAI complex. It shows that the drug delivery is possible.

On the other hand, the most serious problem of the current anticancer drug therapy is the non-selective damage to the normal tissue as well as cancer tissue, which is a major obstacle of treatment. To overcome this, attempts have been made to deliver anticancer agents to cells by liposomes (Gregoriadis G. The carrier potential of liposomes in biology and medicine, N. Engl. J. Med . 295: 704-710, 1976; Pagano RE , Weinstein JN Interaction of liposomes with mammalian cells, Annu. Rev. Biophys.Bioeng . 7: 435-468, 1978; Poste G., Papahadjopoulos D. Drug-containing lipid vesicles render drug-resistant tumour cells sensitive to actinomycin D, Nature 261: 699-701, 1976). As mentioned earlier, liposomes are micelles that are made of phospholipids and have both hydrophilic (lipate and hydrophobic) groups. If you put it in the cell treatment, the drug will be delivered into the cell through fusion with similar cell membrane properties.

General liposomes are called nonspecific drug delivery methods because they can attach and fuse to any cell membrane without selectivity, but the specificity can be enhanced by attaching to the liposome surface membrane an antibody that can recognize only the surface material of a specific cell ( Martin, FJ, Hubbell WL, Papahadjopoulos D. Immunospecific targeting of liposomes to cells: a novel and efficient method for covalent attachment of Fab 'fragments via disulfide bonds.Biochemistry . 20: 4229-4238, 1981; Martin FJ, Papahadjopoulos D. Irreversible coupling of immunoglobulin fragments to preformed vesicles.An improved method for liposome targeting.Journal of Biological Chemistry.257 : 286-288, 1982). There are more uPAs on the surface of cancer cells than normal cells. If you have antibodies that can specifically bind to these substances, you can use immunoliposomes to selectively deliver anticancer drugs to cancer cells.

That is, since uPA plays a pivotal role in cancer cell invasion-metastasis process, it can be a representative and effective target of anti-cancer technique using immunoliposome for inhibiting cancer cell invasion and metastasis.

An object of the present invention is to provide a selective drug delivery (target selective drug delivery) to target cells that express a lot of uPA, such as neovascular endothelial cells in cancer cells or cancer tissues.

In order to achieve the above object, in the present invention, spleen lymphocytes and myeloma cells extracted from mice immunized with uPA (urokinase type plasminogen activator) Provided are a fusion cell, an anti-uPA monoclonal antibody against the antigen urokinase type plasminogen activator (uPA) produced thereby, and an immunooliposome to which the uPA monoclonal antibody is attached.

In the present invention, using uPA (urokinase type plasminogen activator) and the specific circulatory mechanism of its receptor to uPA to selectively deliver drugs to specific cells (such as cancer cells or neovascular endothelial cells in cancer tissues) with high uPA expression. An immunoliposome attached with a specific monoclonal anti-uPA antibody was prepared, and cancer cell killing was performed using the immunoliposome.

Liposomes are micelles formed of phospholipid molecules, which can be useful drug delivery means for delivering a specific drug to a target while reducing biotoxicity. Immunoliposomes are immunoliposomes that enhance the binding specificity between target cells and liposomes by attaching antibodies capable of recognizing specific cell surface substances to such liposomes. That is, the present invention is conceived from the fact that if an immunoliposome can be made to recognize an cancer cell-specific antibody by making an antibody against a cancer cell-specific surface material, an anticancer agent can be put therein and used as an in vitro or in vivo cancer cell killing means.

uPA is observed to be expressed more than normal cells in various malignant tumors. The expression of uPA is known to reflect cancer progression, metastasis, and recurrence. Endothelial cells also express much of this uPA. Inactive secreted uPA is a cell surface receptor (uPA receptor, uPAR), which is activated by binding to a plasminogen activator inhibitor (PAI) or an anti-uPA antibody, which is an active inhibitor, complexes with other endocytosis-related substances. After forming, enters the cell and shows a unique cyclic pattern that only uPA and PAI are degraded.

In the present invention, in order to use this specific mechanism of uPA for drug delivery, an immunoliposome attached with a specific monoclonal antibody to uPA was prepared to try to kill cancer cells. As a result, the immunoliposome prepared to contain the fluorescent sample was traced by confocal microscopy to confirm that the fluorescent material was introduced into the cells, and in the cytotoxicity test of the immunoliposome containing the anticancer agent, uPA expression on the cell surface was further increased. In many cells, the cell killing effect was confirmed to be higher.

By combining these results, it was confirmed that the immunoliposome prepared to bind to the cell surface uPA is a medium capable of selectively and effectively delivering drugs to cells that secrete much uPA, such as cancer cells.

Hereinafter, an anti-uPA monoclonal antibody (anti-uPA monoclonal antibody) according to the present invention, a fusion cell for producing the same, and an immunoliposome attached thereto and a manufacturing method thereof will be described in more detail.

(1) Preparation and identification of monoclonal anti-uPA antibody

Three injections of uPA into the abdominal cavity of BALB / c mice three weeks apart, three days after the last injection into the tail vein, the spleen of this immune mouse was sterilely extracted and the splenocytes were drained, Prepare spleen lymphocytes by removing them and isolating lymphocytes. Variant lines are removed from myeloma cell lines, fused using spleen lymphocytes of prepared immune mice with polyethylene glycol, and cultured in a 37 ° C., 5% CO ₂ incubator, and only fused cells are selected using HAT medium.

Antibody production was determined by ELISA on microtiter well plates pre-attached with uPA, and clones responding to uPA bound to cell surface receptors were selected by ELISA on plates pre-cultured with HEp3 cells. Specificity for uPA is verified by ELISA using uPA and tissue type plasminogen activator (tPA). As a result, all of the antibodies positive in ELISA responded only to uPA but not to tPA.

One clone from among the fusion cells producing-secreting specific antibodies to uPA is selected and injected into female BALB / c mouse intraperitoneally, and then, ascites is collected between 10 to 14 days to separate monoclonal antibodies. Flow cytometry confirmed that the monoclonal antibody (1E11) specifically recognized uPA. As a result, it was found that the monoclonal uPA antibody specifically binds to uPA molecules on the surface of cancer cells.

(2) Production and confirmation of immunoliposome

The purified antibody solution was treated by mixing with succinimidyl-S-acetylthioacetate as a linker, and then dialyzed. The liposome solution was added thereto and mixed slowly. Gel filtration is performed to separate only those bound to the antibody.

Immunoliposomes made with uPA antibodies bind to uPA bound to uPAR and enter into cells in the same manner as the intracellular introduction of uPAR-uPA-PAI complexes. To demonstrate drug delivery, cancer cells were treated with immunoliposomes containing fluorescent material, and then the change in position of the fluorescence was followed by confocal microscopy over time. As a result, it was found that the immunoliposome specifically binds to uPA bound to the uPAR on the cell surface, and 60 minutes after the immunoliposome treatment, the immunoliposome is introduced into the cytoplasm, spreads and reaches the nucleus. I could confirm that.

(3) Cytotoxicity assay

To confirm that uPA antibody immunoliposomes can be used for actual drug delivery, treatment of cancer cells with immunoliposome-containing immunoliposomes and free donovarubicin, in which an immunoliposome is added with an anticancer drug, donorrubicin, instead of a fluorescent substance Cytotoxicity was measured.

As a result, when treated with dounorubicin-containing immunoliposomes, it was found that the cells with more uPAR expression were killed more rapidly than the cells with less expression, and the immunoliposomes effectively killed cells with more uPA on the cell surface. there was. In addition, when treated with liposomes or immunoliposomes, the apoptosis rate was slower than that of dounorubicin.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these.

EXAMPLE

In this example, HEp3 (human squamous cell carcinoma cell line; storage of microbiology at Seoul National University College of Medicine) was used as a cancer cell line. These cells were treated with 10% fetal bovine serum (FBS) (GIBCO-BRL, Grand Island, NY), penicillin (100 U / ml, GIBCO-BRL) and streptomycin (50 g / ml, GIBCO-BRL). This was incubated at 37% with 5% CO ₂ DMEM (GIBCO-BRL).

1. Preparation of Monoclonal Anti-uPA Antibody

100 μg of uPA (Eurokinase, Green Cross) was injected intraperitoneally of BALB / c mice, followed by three injections at two week intervals, and used in the experiment three days after the last injection of the same antigen into the tail vein.

The spleens of the aseptically extracted immune mice were placed in DMEM (GIBCO-BRL) to drain the splenocytes, and the cells were suspended in 50 ml DMEM and centrifuged to recover. Lymphocytes were isolated by loosening these cells in 5 ml of 170 mM Tris, 145 mM NH ₄ Cl solution, and leaving red blood cells for 2 minutes at 37 ° C. The viable cell number was measured and finally 10 ⁸ spleen lymphocytes were prepared. Myeloma cell line was used P363Ag8V653 (V653) (stored in the Department of Microbiology, Seoul National University College of Medicine). V653 cells were incubated with RPMI 1640 (GIBCO-BRL) containing 20 μg / ml 8-azaguanine, 10% FBS, 50 μg / ml of gentamicin to remove mutants and were used 24 hours prior to use for cell fusion. The concentration was adjusted to 1 × 10 ⁵ cells / ml and cultured. On the day of cell fusion, 2 × 10 ⁷ cells were prepared by washing three times at 300 g for 5 minutes with RPMI 1640 solution without serum.

According to Kohler G., Milstein C. Continuous culture of fused cells secreting antibody predefined specificity.Nature , 256: 495-498, 1975, 2 × 10 ⁷ myeloma cells and 10 ⁸ splenic lymphocytes were prepared. After mixing in a 50 ml tube (cornical tube), washed with a medium, it was fused using 50% (w / v) polyethylene glycol (PEG 15000, BM) while maintaining 37 ℃. Immediately, the supernatant, which is the fusion reaction solution, was discarded by centrifuging at 300 × g for 5 minutes at room temperature, and the cells fused in 20 ml of DMEM containing 20% FBS and 50 µg / ml gentamicin were further suspended in 96 well-plate 4 Intestinal aliquots (50 μl / well) were cultured in a 37 ° C., 5% CO ₂ incubator.

HAT component (hypoxanthine 1.36 μg / ml, Amypox) designed by Littlefield (Littlefield JW.Selecion of hybrids from matings of fibroblasts in vitro and their presumed recombinants, Science 145: 709-710, 1964) to select only fused cells DMEM medium (HAT medium) containing 0.174 μg / ml of nofterin, 3.87 μg / ml of thymidine and 20% FBS, 50 μg / ml gentamicin was used.

Antibody production was primarily performed by ELISA on a microtiter well plate pre-attached with uPA by removing some of the culture supernatant by selecting only the wells with colonies of fusion cells larger than 2 mm in diameter. Secondly, clones producing antibodies that responded to uPA bound to cell surface receptors were pre-cultured HEp3 cells and were selected by ELISA without cell immobilization. Specificity for uPA was determined by uPA and tissue type plasminogen. validation with ELISA using an activator It was.

As a result, all the antibodies positive in the ELISA performed with the fusion cell culture supernatant responded only to uPA but not to tPA.

The 1E11 clone as an antibody-producing fusion cell was deposited with the KCLRF-BP-00056 dated June 5, 2002 to the Korea Cell Line Research Foundation, Cancer Research Institute, Seoul National University College of Medicine.

As such, the cells producing the antibody were transferred to 24-well plates and expanded. Among the monoclonal antibodies, IgG antibodies were purified by making the ascites with 1E11 clone, and once again confirmed by flow cytometry whether the monoclonal antibody specifically recognized uPA on the surface of cancer cells.

In other words, one clone (1E11) was selected from among the fusion cells producing-secreting specific antibodies against uPA and injected into 8-week-old female BALB / c mouse intraperitoneally. The fusion cells were washed in phosphate buffered saline, adjusted to 2 × 10 ⁷ cells / ml, and injected 0.5 ml intraperitoneally. After injection, ascites was collected between 10 and 14 days and centrifuged (400 x g, 10 minutes) to separate the supernatant. PH was adjusted by adding 1.0 M Tris-HCl (pH 8.0) to one tenth of the total volume to the ascites. The solution was passed through Protein G-Sepharose 4 Fast Flow (Amersham Pharmacia) to attach the antibody to the gel, followed by 0.1 M Tris-HCl (pH 8.0), 10 times the volume of the column. ) Was added, followed immediately by the same amount of 0.01 M Tris-HCl (pH 8.0). The antibody attached with 0.1 M glycine (pH 3.0) was eluted, and the presence or absence of the antibody in each fraction was confirmed by gel electrophoresis.

2. Cell surface uPA and uPAR expression analysis

To confirm that the monoclonal antibodies prepared above specifically recognize uPA on the surface of cancer cells, flowcytometry was performed as follows:

Monolayer cultured cancer cells were removed with trypsin-EDTA, washed with PBS and suspended in PBS containing 1% FBS and 0.1% sodium azide so that the cell number was 2 × 10 ⁶ cells / ml. I was. The purified uPA antibody (IgG, 2 mg / ml) was diluted (× 200), reacted at 4 ° C. for 1 hour, and washed three times with PBS. Fluorescein-isothiocyanate (FITC) -conjugated anti-mouse IgG (Capel) (× 400) was added and reacted at 4 ° C. for 1 hour, then washed three times with PBS and fixed with 1% paraformaldehyde to FAXcan 440 (Facscan). 440; Becton Dickinson) was used for flow cytometry. On the other hand, the expression of uPAR on the cell surface was performed using anti-human uPAR IgG (American Diagnostica).

Figure 3 is a graph showing the results of flow cytometry performed with monoclonal uPA antibody. Here, the monoclonal uPA antibody specifically binds to the uPA molecule on the cell surface, as compared to the control (black), which has a nonspecific reaction by the secondary antibody, and the peak (green) shown by the monoclonal uPA antibody is completely separated. It can be seen that. On the other hand, to determine the amount of uPAR that does not bind to uPA, the excess uPA was first treated with cancer cells, and then treated with uPA antibody and observed. Since no shift of the peak (red) occurs, it can be seen that most of the uPAR present on the cell surface binds to the uPA molecules already secreted from the cell.

3. Production of immunoliposomes

Phosphatidylcholine (Avanti polar lipid, USA), cholesterol (SIGMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine -N- [4- ( p-maleimido phenyl) butyramid] (Avanti polar lipid, USA) was mixed in a volume ratio of 62.5: 35: 2.5 mol% and the solvent was removed by evaporation.

The internalization assay of liposomes was performed using the fluorescent substance 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS) (SIGMA) in HBSE buffer (10 mM HEPES, 140 mM NaCl, 0.1 mM EDTA, pH). 7.5) was hydrated by stirring with 1 ml of a solution (40 mM) dissolved in water, and cytotoxicity assay (Daunorubicin; 10 mg / ml) was used as a cytotoxicity assay. The hydrated mixture was repeated four times freezing at −70 ° C. and thawing to room temperature. Extrusion was performed using a mini extrusion kit (Avant polar lipid, USA) in which a polycarbonate membrane having a pore size of 0.1 μm was inserted to form a single membrane structure.

The chemical manipulation of antibodies to make immunoliposomes was followed by Western et al. (Weston PD Devries JA Wrigglesworth R. Conjugation of enzymes to immunoglobulins using dimeleimides, Biochem. Biophys. Acta, 612: 40-49, 1980). That is, succinimidyl-S-acetylthioacetate (SATA, 2.7 mg /) is a linker that connects the antibody and the lipid to 1/100 (10 μl) of the purified antibody solution (1 mg / ml). Ml in DMF). The mixture was mixed slowly using a circulator for 30 minutes at room temperature, and then changed into a 50-100-fold volume of HBSE buffer for 18 to 24 hours using a dialysis cassette (2K MWCO, Pierce). Dialysis as it went. 100 ml of deacetylation solution (0.5 M hydroamine, 50 mM sodium phosphate, 25 mM EDTA, pH 7.0) was added to 1 ml of the antibody solution, and the mixture was slowly mixed at room temperature for 2 hours, followed by 1 M 2- [N 10 μl of morpholino] ethanesulfonic acid (MES, pH 6.5) was added to stop the reaction. 250 μl (1/4 Vol.) Of the prepared liposome solution was added to the antibody solution and slowly mixed at 4 ° C. for 18 to 24 hours. Gel filtration using Sepharose CL-4B resin (SIGMA) was performed to obtain only the antibody bound. Antibody concentrations were determined by quantitative gel scanning and lipid concentrations by phosphate assay. On the other hand, dounorubicin concentration was determined by measuring the absorbance (A ₄₇₀ ) with a spectrophotometer (spectrophotometer).

4. Intracellular migration tracking of immunoliposomes-Confocal Microscopy

To demonstrate that immunoliposomes made with uPA antibodies bind uPARs bound to uPAR as desired and enter the cells in the same manner as intracellular influx of uPAR-uPA-PAI complexes to deliver drugs contained in liposomes. Cancer cells were treated with immunoliposomes containing phosphorus 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), and the change in position of the fluorescence over time was analyzed by confocal microscopy (450 nm). Traced.

Specifically, the HEp3 cells cultured on a cover slip (2.4 cm in diameter) were reacted with a saturated concentration of immunoliposome solution containing HPTS for 30 minutes at 4 ° C, washed three times with cold PBS, and then washed at 37 ° C. Culture medium was added and left in a 37 ° C. incubator. After removing the cover slip for each time period and fixing for 20 minutes at room temperature of 4% paraformaldehyde, washed once with PBS-100 mM glycine and twice with PBS. Control staining stained nuclei with prophodium iodide (PI). On the other hand, anti-human uPAR IgG (American Diagnostica) and Tetramethyl-rhodamine isothiocyanate (TRITC) -conjugated anti-mouse IgG to confirm co-localization of uPA and uPAR (SIGMA) was used. Stained samples were observed with a confocal laser scanning microscope (Bio-Rad MRC 1024, equipped with a Zeiss Axio-plan microscope).

Figure 4 shows the change in fluorescence traced by confocal microscopy after treatment of cancer cells (HEp3) with a fluorescent substance-containing immunoliposome, (a) immediately after treatment, (b) 30 minutes after treatment, and (c ) Is the result after 60 minutes of treatment. Here, in (a) immediately after treatment, the green fluorescence is a uPA antibody-attached immunoliposome containing HPTS, and the red fluorescence is bound to the receptor as TRITC. You can see that. That is, it can be seen that the substances recognized by the uPA antibody and the uPAR antibody on the surface of the immunoliposome appear at the same position, which shows that the immunoliposome specifically binds to the uPA bound to the uPAR on the cell surface. On the other hand, until the initial 30 minutes after immunoliposome treatment (b) on the cell surface Although liposomes are present in the form of granules on the cell surface because granular green fluorescence is visible, after 60 minutes of leaving the immunoliposome-treated cells at 37 ° C., (c) no fluorescence was present in the form of granules on the cell membrane. It can be seen that it is introduced and spread into the cytoplasm and reaches into the nucleus. In Figure 4 the nucleus is stained with PI and looks red.

5. Cytotoxicity Test-Cancer Cell Killing Using Anti-Cancer-containing Immunoliposomes

To confirm that the uPA antibody immunoliposome can be used for actual drug delivery, the immunoliposome was used to kill cancer cells (HEp3 and SM-3) by adding an anticancer drug, doubinubisin, instead of a fluorescent substance. The used cancer cell line SM-3 (stored in the Department of Microbiology, College of Medicine, Seoul National University) is a clone obtained by transfecting uPAR cDNA in HEp3, and has more uPA on the cell surface because the cell surface uPAR is more than HEp3. These cells were treated with free dounorubicin and dounorubicin-containing immunoliposomes, respectively, to determine cytotoxicity.

Specifically, after 24 hours of incubating HEp3 cells to 1 × 10 ⁴ cells / well in a 96-well tissue culture plate, an immunoliposome solution and free dounorubicin were prepared based on the concentration of dounorubicin. Immunoliposomes and free donorubicin diluted 5 steps at a maximum of 45 μg / ml were added to the culture solution in three sets for each concentration and left in a 37 ° C. incubator for 24 hours. Cytotoxicity was used with XTT assay kit (BM, Indianapolis, IN). After 24 hours of treatment, DMEM media containing no phenol red and XTT solution were mixed 2: 1, 150 μl / well was added and reacted in a 37 ° C. incubator for 4 hours, followed by a microplate reader (Dynatech, Chantilly, VA), absorbance at 450 nm wavelength (A ₄₅₀ ) was measured to compare the average of three sets.

5 is a graph showing uPAR expression on the cell surface of two cancer cell lines (HEp3, SM-3). Black is contrast, green is HEp3, and pink is SM-3. Here, it can be seen that SM-3 expresses more uPAR than HEp3. The control is a reaction of only 2 ^nd Ab to the cells.

FIG. 6 is a graph showing cytotoxicity caused by dounorubicin-containing immunoliposome and free dounorubicin, and (a) is a case in which dounorubicin-containing immunoliposome is treated (b). ) Is the case of treatment with free dounorubicin. Here, when treated with dounorubicin, it can be observed that the two cells undergo similar killing process with increasing drug concentration, but when treated with dounorubicin-containing immunoliposome, the expression of uPAR is increased. The cells (SM-3) were killed more rapidly than the relatively few cells (HEp3), and it was found that the immunoliposomes effectively killed cells with more uPA on the cell surface.

FIG. 7 shows a process in which cancer cells are treated with three different methods of dounorubicin, namely, a free form (Daunorubicin), a liposome (Liposome), and an immunoliposome (Immunoliposome). Is a real-time picture of the trace, the number of hours elapsed after processing, and the arrow of dying cells. Display them consecutively. Here, it can be observed that the rate of apoptosis slows down when treated with liposomes or immunoliposomes, compared to the treatment with dounorubicin directly. In other words, when dounorubicin is directly treated, cells die through normal cell necrosis over two hours.However, when dounorubicin is added to liposomes or immunoliposomes, the cells undergo apoptosis. Dying was observed. In this case, blebbing of the cells takes place and the apoptotic body is active, and some cells start to die after 10 hours. In the case of immunoliposomes, cells take longer to die than liposomes. .

The present invention provides selective drug delivery using features such as overexpression of uPA and uPAR, uPA immobilization to the cell surface through cell surface binding, and internalization into cells by PAI or antibody binding. To develop an immunoliposome that can be, the key to such selective drug delivery is the specificity of the uPA antibody that can recognize the uPA bound to the receptor.

As shown in the above experimental results, the specificity of the uPA antibody was confirmed through ELISA, Western blot, flow cytometry, etc. of the fusion cell screening step to verify the specificity of the antibody. In addition, it was confirmed that the immunoliposome to which the uPA antibody was bound specifically binds to uPA bound to the cell surface receptor, and it was also confirmed that the uPA antibody was introduced into the cell over time. The efficacy of the uPA antibody immunoliposome was also confirmed by cytotoxicity tests measured using anticancer agents directly. In other words, when free dounorubicin was added to the cell medium, all the cell lines used had the same pattern regardless of the difference in the amount of uPA. It showed cytotoxicity, which reflects the indiscriminate cytotoxicity seen when injecting anticancer drugs into the human body. However, uPA antibody immunoliposomes were shown to effectively kill cancer cells despite much lower concentrations of dounorubicin. On the other hand, depending on the drug delivery method, there was a difference in not only the time but also the cell death. This is because dounorubicin simply passes through the cell membrane, whereas when treated in liposomes, various cell surface molecules It is thought to be due to cytotoxicity through intracellular binding and endocytosis.

As described above, the uPA antibody immunoliposome according to the present invention specifically binds to uPA bound to the cancer cell surface receptor, and it can be seen that the drug delivery effect on cancer cells varies depending on the number. Other previously tried immunoliposomes (Cerletti A., Drewe J., Fricker G., Eberle AN, Huwyler J. Endocytosis and transcytosis of an immunoliposome-based brain drug delivery system.J. Drug Target . 8: 435- 446, 2000; Nam SM, Kim HS, Ahn WS, Park YS Sterically stabilized anti-G (M3), anti-Le (x) immunoliposomes:... targeting to B16BL6, HRT-18 cancer cells Oncol Res 11: 9- 16, 1999; Park JW, Hong K., Kirpotin DB, Colbern G., Shalaby R., Baselga J., Shao Y., Nielsen UB, Marks JD, Moore JD, Moore D., Papahadjopoulos D., Benz CC Anti -HER2 immunoliposomes: Enhanced Efficacy Attributable to Targeted Delivery.Clin.Cancer Res . 8: 1172-1181, 2002) is designed to recognize substances that are highly expressed in, or only expressed in, cancer cells. Immunoliposomes using the uPA antibody according to the invention, when used as an anticancer agent as shown in the examples, (1) cancer cells themselves In addition to the effect, and (2) by blocking angiogenesis by killing the vascular endothelial cells it is an advantage can be obtained with the side-effect of inducing necrosis of tumor tissue.

1 is a schematic diagram showing the process of releasing extracellular matrix (ECM) by generating plasmin after uPA is secreted and activated by inactive pro-uPA (or scuPA).

Figure 2 is a schematic diagram showing the interaction between uPA activation process and intracellular influx mechanism of the uPAR-uPA-PAI complex and α2-MR,

Figure 3 is a graph showing the flow cytometry results performed with monoclonal uPA antibody,

Figure 4 shows the distribution of fluorescence traced by confocal microscopy after treatment of cancer cells (HEp3) with a fluorescent material-containing immunoliposome,

5 is a graph showing uPAR expression on the cell surface of two cancer cell lines (HEp3, SM-3),

Figure 6 is a graph showing the comparison of cytotoxicity (cytotoxicity) by dounorubicin-containing immunoliposome and free dounorubicin,

FIG. 7 shows the results of tracing the process of cell death after treatment of donorubicin with cancer cells in free form (Daunorubicin), in liposomes (Liposome), and in immunoliposomes (Immunoliposome). Picture.

Claims (5)

delete Fusion cells 1E11 (specified number KCLRF-BP-00056) of spleen lymphocytes and myeloma cells isolated from uPA-immunized mice, which produce an anti-uPA monoclonal antibody against the antigen uPA (urokinase type plasminogen activator). Monoclonal antibody against antigen uPA produced by the fusion cell of claim 2. Immunoliposomes attached with monoclonal antibodies against uPA produced by fusion cells of splenic lymphocytes and myeloma cells isolated from uPA immunized mice. The immunoliposome of Claim 4 which contains an anticancer agent inside.