KR20110030135A - Cancer targeting transferrin-polyethylene glycol-modified trail (tumor necrosis factor related apoptosis inducing ligand), preparation, and use therof - Google Patents

Abstract

PURPOSE: A method for manufacturing transferring-PEG-TRAIL complex is provided to ensure same or similar pharmacological activity and to enhance stability of drug. CONSTITUTION: A transferrin-PEG-TRAIL complex is obtained by binding N-terminal-specific transferring-PEG. The transferring is an AP-transferrin. The trail is a human TRAIL having 281 amino acid sequences. The TRAIL has an isoleucine zipper at the N-terminal. A method for preparing the complex comprises: a step of reacting transferring and PEG or transferring or PEG derivative under the presence of a reductant to shythesize transferring-PEG-aldehyde or transferrrin-PEG derivative-aldehyde complex; and a step of reducing the complex under the presence of reductant.

Description

Cancer targeting Transferrin-Polyethylene Glycol-modified TRAIL (Tumor Necrosis Factor Related Apoptosis Inducing Ligand), preparation, and use therof}

The present invention specifically relates to transferrin-polyethylene glycol (transferrin-polyethylene glycol, hereinafter transferrin-PEG) or transferrin-PEG derivative specifically at the N-terminus of a trail (Tumor Necrosis Factor-Related Apoptosis Inducing Ligand, TRAIL, or Apo2 ligand, Apo2L). The present invention relates to a transferrin-PEG-trail conjugate, a preparation method thereof, and a therapeutic agent for cancer and an autoimmune disease agent having enhanced effects in targeting, solubility, and stability using the conjugate.

Trail is a type of Tumor Necrosis Factor (TNF), a cell membrane protein involved in cell apoptosis. Among the proteins composed of 281 amino acids in total (Korean Patent Application No. 1997-7009578), the extracellular portion from arginine No. 114 to glycine No. 281 is known to affect cell self-extinction ( MH Kim, et al. BBRC 2004, 321, 930-935). In addition, three trail molecules exhibit a structurally bound trimeric structure, and it is known that the trail of the trimeric structure binds to a receptor involved in cell death and induces cell death (FC Kimberley, et al. Cell Research). 2004, 14, 359-372).

The most significant difference between the TNF superfamily, TNF and CD95L, and other trails is that they do not induce the death of normal tissue cells. Since proteins such as TNF and CD95L also induce cell death, various medical and pharmaceutical applications have been attempted. Proteins such as TNF and CD95L induce the death of cancer cells and overactivated immune cells and at the same time affect only normal cells. Trail, on the other hand, is known to induce self-killing of various types of cancer cells and overactivated immune cells and not affect normal cells. This is known to be due to the difference in expression of the trail receptor in each of the cells. Five types of trail receptors have been reported to date, and representative apoptosis related receptors include DR4 (TRAIL-R1) and DR5 (TRAIL-R2). Trail binding to these receptors causes cell death via activation of intracellular death-related domains and various signaling systems. In addition, there are three receptors, DcR1, DcR2, and OPG (osteoprotegerin), which are known to not be involved in cell death. Differences in the degree of expression of DR4 and DR5 involved in cell death in normal cells and cancer cells are known to be insignificant. On the other hand, the remaining three types of receptors that are not associated with cell death are well expressed in normal cells, but have been shown to have low or no expression in cancer cells. Thus, in the case of the trail, the binding of DcR1, DcR2, and OPG, which do not bind to death-related domains in normal cells, predominantly does not induce cell death, while DR4 and DR5, which bind to death domains in cancer cells and over-activated immune cells, do not. It is known that self-killing of cells by induction with is induced. Such selective apoptosis is considered to be a very useful property in the medical and pharmaceutical applications of the trail.

Cancer cells for which apoptosis is observed are reported to be colon cancer cells, glioma cancer cells, lung cancer cells, prostate cancer cells, brain tumors and multiple myeloma, and have shown excellent anticancer activity in animal experiments. Is reported. In addition, it is known to show superior anticancer efficacy than the use of trail alone as well as other anticancer drugs such as paclitaxel and doxorubicin and radiation therapy. Therefore, it is known that clinical trials on solid cancer using the excellent anticancer efficacy of these trails are in progress (Genentech / Amgen). In addition to advanced cancer, various approaches using trails have been attempted to induce the death of overactivated immune cells in the arthritis, which is a type of autoimmune disease, and to alleviate and treat the disease. In addition to protein therapy, various attempts have been made to gene therapy through delivery of trail genes. Therefore, the trail can be useful not only for the treatment of various carcinomas mentioned above, but also for the treatment of T-cell-derived autoimmune diseases such as Experimental Autoimmune Encephalomyelitis (EAE), rheumatoid arthritis and type 1 diabetes. have.

However, there are some problems with natural trails that must be addressed for successful application. The most representative problem is the low trimer formation rate of the natural trail. Trail monomers are not known to induce cell death because they do not bind to the trail receptors mentioned above. Therefore, various studies have been reported to improve the structure of the trimer of the trail and the trimer formation ratio. In natural trails, zinc ions are known to play an important role in the formation of trimers (S.H. Hymowitz, et al. Biochemistry 2000, 39, 633-640). In addition, approaches such as structural analysis using computers and development of modified trails using them have also been attempted (A.M. van der Sloot, et al. Protein Engineering, Design & Selection 2004, 17, 673-680). The introduction of a new amino acid sequence to help direct trimer formation is considered to be the most useful method for trail trimer formation, and leucine zipper arrangement and isoleucine zipper arrangement have been reported. Henning Walczak et al. Published the anticancer efficacy of a trimeric form of trail derivative in which the leucine zipper arrangement was introduced at the N-terminus of a natural trail in 1999 (H. Walczak, et al. Nature Medicine 1999, 5). , 157-163). In addition, Dai-Wu Seol et al. Have actively published reports on trail derivatives containing a new isoleucine zipper arrangement and excellent activity using the genes (MH Kim, et al. BBRC 2004, 321, 930-935). ).

Another problem in the clinical application of trails is the cytotoxicity seen in normal cells of some tissues. In most normal cells, cytotoxicity due to the expression of various trail receptors as described above is not expressed, but some hepatocytes and keratinocytes have been reported to be cytotoxic by trail (H. Yagita). , et al. Cancer Sci. 2004, 95, 777-783; M. Jo et al. Nature Medicine 2000, 6, 564-567; SJ Zheng, et al. J. Clin.Invest. 2004, 113, 58-64 ).

In addition to the general cytotoxicity of trails, the short half-life of trails in vivo is also considered to be a problem that must be addressed for successful clinical application of trails. Trails have different half-lives depending on the species of animals used in the experiments, and rodents have been reported to have half-lives within minutes and within approximately 30 minutes (H. Xiang, et al. Drug Metabolism and Disposition 2004, 32, 1230-1238). In particular, most trails are known to discharge rapidly through the kidneys. This short half-life is considered to be a drawback of pharmacological action by the trail, and simply explains the need for trails and trail derivatives with longer half-lives. In addition, the low solubility and solution stability of the trail is considered to be a problem to be improved.

Transferrin is a plasma protein involved in the transport of iron ions, a strong but reversibly glycoprotein that binds to iron ions. In vivo, the combined form of iron is less than 0.1% of the total iron, but it is the most important iron reservoir. The transferrin has a molecular weight of about 80 kDa and has two characteristic high affinity iron ion binding sites. APO-transferrin is a form of transferrin that is not bound to iron ions. Human transferrin is composed of a polypeptide chain containing 679 amino acids and forms two iron bond sites with alpha-helices and beta-sheets. Gomme PT. Et al, Drug Discov Today. 2005, 10, 267-273).

Polyethylene glycol (PEG) is a high molecular compound having a structure of HO-(-CH2CH2O-) n-H. Because of its high hydrophilicity, polyethylene glycol (PEG) can increase its solubility by binding to a pharmaceutical protein. Proper binding also increases the molecular weight of the bound protein while maintaining key biological functions such as enzymatic activity and receptor binding, thereby reducing kidney filtration, protecting the protein from cells and antibodies that recognize foreign antigens, Can also reduce the degradation of proteins. As such, the molecular weight range of PEG that can bind to protein is approximately 1,000 to 100,000, and when the molecular weight of PEG is 1,000 or more, it is known that the toxicity is quite low. Its molecular weight ranges from 1,000 to 6,000 and is distributed throughout the body and metabolized through the kidneys. In particular, PEG with a molecular weight of 40,000 is known to be distributed in organs including blood and liver, and metabolism occurs in the liver.

Medically and pharmacologically useful proteins administered through the parenteral route have antigenicity, and are generally poor in water solubility and have a short lifespan. U.S. Patent No. 4179337 discloses that the use of PEG-associated proteins, enzymes, and the like as a therapeutic agent can provide the advantages of PEG, such as reduced antigenicity, increased water solubility, increased body retention time, and the like. Since this patent, attempts have been made to overcome the shortcomings by combining bioactive proteins with PEG, for example ribonuclease and superoxide dismutase with PEG ( Veronese et al., 1985). In addition, US Pat. No. 4,766,106 and US Pat. No. 4,917,888 disclose binding of polymers containing PEG to proteins to increase the water solubility of the protein. US Pat. To reduce antigenicity and increase the duration of life in the body.

On the other hand, there are drawbacks to PEG and protein binding in addition to these advantages. That is, PEG usually binds covalently to one or more free lysine (lys) residues of the protein to be bound, wherein a portion of the surface portion of the protein that is directly related to the activity of the protein binds to PEG. If so, the site will no longer be able to perform biological functions, resulting in reduced protein activity. In addition, the binding of PEG and lysine residues usually occurs randomly, so that many kinds of PEG-protein conjugates exist as a mixture depending on the binding position, thus making the process of pure separation of the desired combination complicated and difficult. do.

Trails also present problems with PEG bonds similar to those mentioned above. Natural trails have 11 lysine residues, many of which are known to be involved in the interaction between the trail and the receptor or present in the active site (SG Hymowitz, et al. Molecular Cell 1999, 4, 563-571). . Therefore, the introduction of PEG molecules through the reaction with lysine residues may act as a very important inhibitor in the bioactivity of the trail. TNF, a trail superfamily, has also been reported to inhibit activation by binding PEG molecules to lysine residues (Y. Yamamoto, et al. Nature Biotechnology 2003, 21, 546-552; H. Shibata et al. Clin. Cancer Res. 2004, 10, 8293-8300).

Accordingly, the present inventors have found a method for preparing the molecular weight and reaction conditions of PEG, in which the transferrin-PEG specifically binds only to the N-terminus, but not other lysine residues of the trimeric trail, increasing cancer targetability and reducing hepatotoxicity. And the transferrin-PEG-trail conjugate with increased solubility and safety.

An object of the present invention is to provide a transferrin-PEG-trail or transferrin-PEG derivative-trail conjugate by selectively binding a transferrin-PEG or transferrin-PEG derivative only to the N-terminus of the trail.

The present invention also provides a method for preparing a transferrin-PEG-trail or transferrin-PEG derivative-trail conjugate selectively binding a transferrin-PEG or transferrin-PEG derivative to the N-terminus of the trail.

Another object of the present invention is to provide an anticancer and anti-inflammatory composition containing the transferrin-PEG-trail conjugate or transferrin-PEG derivative-trail conjugate as an active ingredient.

In order to achieve the above object, the present invention provides a trail derivative in which a transferrin-PEG or transferrin-PEG derivative is optionally covalently bonded only to the N-terminus of the trail.

The transferrin is preferably apo (APO) -transferrin.

When the transferrin protein binds to a receptor on the cell surface, it moves inside the cell in the form of a vesicle. When the pH of the endoplasmic reticulum is reduced by the hydrogen ion pump, iron ions are released from the transferrin and moved back out of the cell by the endocytological cycle. (He Q.Y. et al, Biochem J. 2000, 350, 909-915). In addition, transferrin receptors are highly expressed on the surface of tumor cells, and transferrins have a strong affinity with transferrin receptors overexpressed on the surface of tumor cells.

The advantages of drug-targeted therapies through transferrin and transferrin receptors are outlined below. (1) As it is a biological protein, it is not only good for human adaptation but also has biodegradable, nontoxic, and nonimmunogenic properties. (2), it can be applied to many drugs including small molecular weight drugs, peptides, proteins, nucleic acids, etc. (3) Because of the properties of targeting ligands that concentrate on tumor cells You can expect it and at the same time reduce side effects.

In addition, the PEG or PEG derivative may be selected from linear or branched (branched) form of the branch may be two or more multi-branch.

PEG derivatives include methoxypolyethylene glycol succinimidyl propionate (mPEG-SPA), methoxypolyethylene glycol N-hydroxysuccinimide (mPEG-NHS), methoxypolyethylene glycol Aldehydes (methoxyPEG aldehyde, mPEG-ALD), methoxypolyethylene glycol maleimide (methoxyPEG maleimide, mPEG-MAL) and multiple branched PEG derivatives.

The molecular weight of the PEG of the present invention may be selected in the range of 1000 to 40,000, preferably 10,000 to 40,000.

More preferably, the molecular weight of PEG is 9,000 to 11,000.

The trail of the present invention may be a recombinant trail obtained by a natural type or genetic modification, and may be a trail containing a zip amino acid sequence or end group that facilitates separation and purification to induce trimer formation of the trail. .

The trail is preferably a human trail having a 281 amino acid sequence. More preferably, the trail is a human trail having an amino acid sequence from alginine no. 114 to glycine no. 281.

In addition, the present invention,

Reacting transferrin and PEG under a reducing agent to synthesize a transferrin-PEG-aldehyde conjugate; And synthesizing the transferrin-PEG-trail conjugate by reacting the N-terminus of the trail dissolved in the buffer with the transferrin-PEG-aldehyde conjugate under a reducing agent. Provided, a method for preparing transferrin-PEG-trail conjugate.

The reducing agent is preferably NaCNBH ₃ .

The molecular weight of the transferrin dissolved in the buffer of the appropriate pH may be 1,000 to 40,000, preferably 10,000 to 40,000.

More preferably, the molecular weight of PEG may be selected from 9,000 to 11,000.

The reaction molar ratio of transferrin / PEG in the first step of the method of the present invention may be 2.5 to 12.5, more preferably 7.5 to 10, most preferably 9.

In addition, the reaction time of transferrin / PEG may be 1 hour to 10 hours, more preferably 4 hours to 5 hours and most preferably 5 hours.

The reaction molar ratio of transferrin-PEG / trail in the second step of the present invention is preferably selected from the range of 13 to 17, more preferably 15.

Method of the Invention According to one embodiment of the invention, by binding only the N-terminal of the trailing transferrin-PEG or transferrin-PEG derivative, the target of the drug is increased while maintaining the bioactivity of the trail itself, It is possible to provide a trail having improved stability, improved pharmacokinetic behavior through inhibition of renal excretion of a trail to which a transferrin-PEG or transferrin-PEG derivative is bound, and alleviated hepatotoxicity due to reduced absorption by hepatocytes. That is, by preparing an N-terminal specifically modified trail that does not react with the epsilon-amino group present in the side chains of lysine residues on the secondary and tertiary structures of the protein, the physiological activity of the trail itself is It is possible to drastically reduce hepatotoxicity, which is a side effect of the trail, by lowering the absorption and removal rate of drugs by the hepatocytes and the reticuloendothelial system.

The present invention also provides a composition for the prevention and treatment of cancer containing the transferrin-PEG- trail conjugate as an active ingredient.

The cancer may include glioma, prostate cancer, lung cancer, brain tumor, and multiple myeloma carcinoma.

In addition, the present invention provides a composition for the prevention and treatment of autoimmune diseases containing the transferrin-PEG- trail conjugate as an active ingredient.

The autoimmune disease may include meningitis, rheumatoid arthritis, type 1 diabetes, and the like.

The composition may consist of a trail to which an effective amount of the transferrin-PEG or transferrin-PEG derivative of the present invention is bound and a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier.

The composition of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions in a conventional manner.

Carriers, excipients, and diluents which may be included in the composition include tods, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate.

When formulating the composition may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. used in the general. The solid preparation for oral administration may be prepared by mixing at least one excipient, for example starch, with calcium carbonate, sucrose or lactose, gelatin, and the like in the extract. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories.

As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the effective dosage of the composition according to the present invention can be varied depending on the age, physical condition, weight, etc. of the patient, in general, it is possible to administer the effective dosage even once a week to 2 weeks . And it can be injected once a day or divided several times a day within the effective dosage range per day.

In the case of the transferrin-PEG-trail prepared according to the present invention, the cancer target of the drug is improved compared to the non-transferin-pegylated natural trail and the PEG-trail combined with PEG, and the anti-cancer effect by the trail is excellent. It can be very useful for treating various kinds of cancers by maximizing.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art to which the present invention pertains. Will be self-evident.

Example 1 Synthesis of Transferrin-PEG-ALD

The reaction picture of transferrin and di-PEG propionaldehyde (di-PEG-propionaldehyde, di-PEG-ALD, MW 3400, 5000, 10000 Shearwater, USA) used in this example is shown in FIG. 1 ml of transferrin (6 mg / ml in PB, pH 5.0) solution was added to the mPEG-ALD solution and mixed well. NaCNBH ₃ (Sigma, USA) was added to the final 20 mM as a reducing agent. The reaction proceeded at 4 ° C. for about 4-6 hours. The amount of transferrin added to di-PEG-ALD was in the range of 2.5 to 10.

Example 2 Isolation and Identification of Transferrin-PEG-ALD

Transferrin-PEG-ALD prepared in Example 1 was isolated and purified using size exclusion liquid chromatography ("SEC"). Sephacryl S-200 HR (Amersham Bioscience, Sweden) was used as a column, and the elution solution conditions were maintained at 1 ml / min flow rate with 50 mM acetic acid buffer added with 150 mM NaCl. Each peak was quantified using a UV absorbance 215 nm.

Figure 2 shows the results of size-exclusion liquid chromatography separated unreacted transferrin, transferrin-PEG-ALD and other by-products according to the reaction ratio of the transferrin and PEG-ALD and PEG molecular weight (3400, 5000, 10000).

FIG. 3 shows the results of analysis using a MALDI-TOF mass spectrometer to confirm PEG conjugation of each transferrin-PEG-ALD (3400, 5000, 10000) separated based on the size exclusion liquid chromatography. will be. The theoretical and measured molecular weight values of each transferrin-PEG-ALD (3400, 5000, 10000) are shown in Table 1.

Sample Theoretical molecular weight (Da) Measured Molecular Weight (Da) Transferrin 81000 81590.1 3.4k PEG-transferrin 84400 84749.1 5.0k PEG-transferrin 86000 87000.6 10k PEG-transferrin 91000 91446.2

To determine the PEG conjugation of each transferrin-PEG-ALD (3400, 5000, 10000) isolated based on size exclusion liquid chromatography, an 8% acrylamide electrophoretic gel was prepared to provide transferrin-PEG-ALD (3400, The molecular weight degree of 5000, 10000) was compared and analyzed using an electrophoretic marker (molecular weight range 50000 to 250000) (see FIG. 4).

Example 3 Synthesis of Transferrin-PEG-Trail

Add the transferrin-PEG-ALD solution to the diluted solution (TRAIL) at a concentration of 200 μg / ml (50 mM acetate buffer, pH 5.0) and mix well. At this time, add NaCNBH ₃ as a reducing agent. Add to the final 20 mM. The reaction proceeded at 4 ° C. for about 8-12 hours. The amount of transferrin-PEG-ALD added to TRAIL was 10-15 times.

Example 4 Isolation and Confirmation of Transferrin-PEG-TRAIL (PEG Molecular Weight 10000)

The transferrin-PEG-trail prepared in Example 3 was isolated and purified using size exclusion chromatography (elution solution is an acetate buffer of pH 6.5 containing 150 mM NaCl). However, the final synthesized and isolated transferrin-PEG-TRAIL was finally separated when synthesized using a molecular weight of 10000 PEG. This means that the molecular weight of PEG is very important for conjugating transferrin and TRAIL to each other, and that 10000 PEG should be used to connect transferrin and TRAIL.

 5 shows size exclusion chromatography of the final isolated transferrin-PEG-TRAIL (PEG molecular weight 10000).

SDS-PAGE was performed to confirm the final isolated transferrin-PEG-TRAIL (PEG molecular weight 10000), followed by immunostaining using western blotting (FIG. 6). A 0.75 mm thick 12% acrylamide gel was prepared and subjected to electrophoresis under the denatured condition.

Molecular weight markers (BioRad, Precision Plus Protein Standard ^™ ) were loaded with each sample and electrophoresis was performed at a current of 35 mA for 40-50 minutes. Thereafter, Western blotting immunostaining was performed to find transferrin using transferrin antibody.

Denature the transferrin-PEG-trail (PEG molecular weight 10000), and since TRAIL has a trimer structure, it is divided into one mono-trail-PEG (10000) -transferrin and two mono-trails (FIG. 7). Reference). As shown in FIG. 6, the mono-trail-PEG (10000) -transferrin was observed between 100 and 150 kDa (theoretical molecular weight 112 kDa), and it was found that it was detected by the transferrin antibody in the Western blotting experiment.

Example 5 Physiological Activity (Cell Necrosis Experiment) and Cancer Target Confirmation of Transferrin-PEG-TRAIL (PEG Molecular Weight 10000) (Transferrin Receptor Binding Experiment)

The physiological activity of the trail, 10000-PEG-trail and transferrin-PEG-trail (PEG molecular weight 10000), ie, cell necrosis experiment, was carried out using HCT116 cell line, a human colon cancer cell. RPMI-1640 (HCT 116 cell line) was used for culturing the cell line. For the experiment, each cell line was dispensed with 1 × 10 ⁴ cells in each well of a 96 well plate, and then the cells were stabilized by culturing for about 24 hours. After the incubation process, the control trail, 10000-PEG-trail and transferrin-PEG-trail (PEG molecular weight 10000) prepared by the above method, were prepared in each cell to have a final concentration of 0.1 ng / mL to 1 μg / mL. After administration, the cells were incubated for about 24 hours. After drug treatment, 20 μL of MTS solution (purchased from Promega) was added to each well, and the absorbance at 490 nm was measured about 2 hours later, and the cell viability was derived using this value ( See FIG. 8).

Figure 9 shows the binding behavior of the transferrin receptor on the surface of KB cancer cells. As shown in the figure, the binding of competitive ¹²⁵ I-transferrin (containing Fe) (radiolabeled transferrin) decreased with increasing concentration of the sample. It was also found that ¹²⁵ I-transferin-PEG-TRAIL (PEG molecular weight 10000) (containing Fe) had no significant decrease in activity even when conjugated with PEG 10000-TRAIL.

Example 6 Confirmation of Cancer Target of Transferrin-PEG-TRAIL (PEG Molecular Weight 10000) in Mouse

Trail, 10000-PEG-Trail and Transferrin-PEG-Trail (PEG Molecular Weight 10000) were used for ¹²⁵ I to radiolabel and cancer targets were measured to investigate biodistribution and cancer target. Cancer target measurement was performed after transplanting Sarcoma 180 (S-80) cancer cells into C57BL6 mice (20-25 g body weight) to make cancer-induced mice. Star radioactive substance content was measured using the γ-counter. As a result of measuring the substance content of each organ (cancer, blood, kidney, lung, spleen, liver) with each time (30 minutes, 2 hours, 12 hours), the amount of blood in the 10000-PEG-trail was higher than in the trail. And therefore more in cancer. Furthermore, in the case of transferrin-PEG-trail (PEG molecular weight 10000), the cancer targeting properties of transferrin showed better cancer targeting than the existing 10000-PEG-trail (see FIG. 10).

Figure 1 shows a schematic representation of the reaction structure of transferrin, PEG-ALD, trail.

Figure 2 shows the size exclusion chromatography separated unreacted transferrin, transferrin-PEG-ALD and other by-products according to the reaction ratio of the transferrin and PEG-ALD and the PEG-ALD molecular weight, respectively.

FIG. 3 shows MALDI-TOF mass spectrometry of each isolated transferrin-PEG-ALD based on FIG. 2.

Figure 4 shows the results of the electrophoresis of each isolated transferrin-PEG-ALD based on FIG.

FIG. 5 shows size exclusion chromatography of a transferrin-PEG-ALD (PEG molecular weight 10,000) separated from the trail and finally separated from the transferrin-PEG-ALD.

FIG. 6 shows electrophoresis and western blotting of the transferrin-PEG-trail (PEG molecular weight 10,000) separated based on FIG. 5.

7 is a graphical representation of the reaction structure when the trail having the trimer structure is denatured.

Figure 8 shows the physiological activity (cell necrosis experiment) in the human colon cancer cell line (HCT116) of transferrin-PEG- trail (PEG molecular weight 10,000).

Figure 9 shows the degree of binding of transferrin receptors in KB cancer cells of radioactivity ( ¹²⁵ I) labeled transferrin-PEG-trail (PEG molecular weight 10,000).

FIG. 10 shows cancer targets according to time zones by injecting Sarcoma 180 (S-80) cancer cells into C57BL6 mice to induce cancer and then injecting radioactivity ( ¹²⁵ I) labeled transferrin-PEG-trail (PEG molecular weight 10,000). will be.

Claims (17)

A transferrin-PEG-trail conjugate that specifically binds transferrin-PEG to the N-terminus of the trail. The transferrin-PEG-trail conjugate according to claim 1, wherein the transferrin is apo (APO) -transferrin. The transferrin-PEG-trail conjugate of claim 1 wherein the PEG comprises a derivative thereof. The transferrin-PEG-trail conjugate according to claim 1, wherein the PEG has a molecular weight selected from the range of 1000 to 40,000. The transferrin-PEG-trail conjugate of claim 1, wherein the trail is a human trail having a 281 amino acid sequence. The transferrin-PEG-trail conjugate of claim 1, wherein the trail comprises an isoleucine zipper at the N-terminus that aids trimer formation. Reacting transferrin and PEG or transferrin and PEG derivatives under a reducing agent to synthesize transferrin-PEG-aldehyde or transferrin-PEG derivative-aldehyde conjugate; And reacting the N-terminus of the trail dissolved in the buffer with the transferrin-PEG-aldehyde or transferrin-PEG derivative-aldehyde conjugate under a reducing agent to synthesize a transferrin-PEG-trail or transferrin-PEG derivative-trail conjugate. Including, transferrin-PEG- trail or transferrin-PEG derivative- trail conjugate preparation method. 8. The method of claim 7, wherein said transferrin is APO (transferrin). The method of claim 7, wherein the molecular weight of the PEG or PEG derivative is selected from the range of 1,000 to 40,000. 8. The method of claim 7, wherein said trail is a human trail having a 281 amino acid sequence. 8. The method of claim 7, wherein the reducing agent is NaCNBH ₃ . The method according to claim 7, wherein the reaction molar ratio of transferrin / PEG or transferrin / PEG derivative is selected in the range of 7.5 to 10. 8. The process according to claim 7, wherein the reaction molar ratio of transferrin-PEG / trail or transferrin-PEG derivative / trail is selected in the range of 13 to 17. The transferrin-PEG-trail conjugate according to any one of claims 1 to 6 is a composition for preventing and treating an effective cancer. 15. The composition of claim 14, wherein the cancer comprises colorectal cancer, glioma, prostate cancer, lung cancer, brain tumor and multiple myeloma carcinoma. A composition for the prevention and treatment of autoimmune diseases comprising the transferrin-PEG-trail conjugate of any one of claims 1 to 6 as an active ingredient. 17. The composition for preventing and treating autoimmune diseases according to claim 16, wherein the autoimmune diseases include meningitis, rheumatoid arthritis and type 1 diabetes.

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