JP3842304B2 - Cancer diagnosis and treatment - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a novel cancer diagnostic and therapeutic agent, and more specifically, an anti-MK obtained through a treatment process characterized by affinity purification of an antibody obtained by immunizing an animal with an MK protein. Protein antibody (hereinafter abbreviated as anti-MK antibody) and production method thereof, cancer diagnostic method and treatment method using the anti-MK antibody, cancer diagnostic agent containing anti-MK antibody, and cancer treatment Regarding drugs.
[0002]
[Background]
Various cancer-related antigens such as antigens specifically expressed in cancer cells, enzymes, or oncogene product proteins have been discovered so far, and antibodies prepared against each of these many antigens are used to detect cancer. Diagnosis and treatment have been performed and are being attempted. Representative antigens include α-fetoprotein and carcinoembryonic antigen (CEA), which have been used for many years as tumor markers. Many cancer-related antigens appearing or present in cancer cells, leukemia cells, and various cancer cells in various organs in the body will continue to be discovered through research efforts, and a combination of these new tumor markers. The results of cancer diagnosis and treatment are expected to improve.
[0003]
The present inventors previously found a new growth differentiation factor derived from mouse teratocarcinoma cells, named MK protein, Midkine, and cloned MK gene encoding Midkine (hereinafter abbreviated as MK). The findings were reported (Tomomura, M. et al, J. Biol. Chem., 265 , 10765-10770, 1990). This Midkine is a protein consisting of 118 amino acid residues, and subsequent studies revealed that MK has actions to induce neurite outgrowth, survival, and maturation of various neurons (Muramatsu, H. and Muramatsu, T., Biochem. Biophys. Res. Commun., 177 , 652-658, 1991).
[0004]
The inventors then cloned the human MK gene using the mouse MK gene (Tsutsui, J., Biochem. Biophys. Res. Commun., 176 , 792-797, 1991), and further transferred these genes to animal cells or It was expressed in E. coli and MK was successfully prepared, and a patent application was filed (Japanese Patent Application No. 3-195397).
[0005]
DISCLOSURE OF THE INVENTION
The present inventors immunized mice and rabbits with this MK to prepare antibodies, and further prepared a pure antibody by affinity purification of this crude antibody using the above-mentioned large amount of protein-expressed MK. However, using this anti-MK antibody, we tried histochemical staining of some cancer cells in addition to nerve cells, and added anti-MK antibody to the cancer cell culture system. Surprisingly, many of the cancer cells were stained with the anti-MK antibody, and in the cancer cell culture system, it was found that the proliferation of the cancer cells was considerably inhibited, and that MK is a cancer-related antigen. It came to clarify.
[0006]
The present invention has been made through extensive research based on such findings, and the object of the present invention is to provide a process characterized by affinity purification of antibodies obtained by immunizing animals with Midkine. The anti-MK antibody obtained through the above and a method for producing the antibody are provided. Another object of the present invention is to provide a method for diagnosing and treating cancer, which comprises using an anti-MK antibody. A further object of the present invention is to provide a cancer diagnostic agent and a cancer therapeutic agent comprising an anti-MK antibody.
[0007]
The anti-MK antibody of the present invention can be obtained, for example, by the following method. The MK antigen that is an immunogen can be purified from the culture supernatant of L cells transfected with the MK gene via various column operations. Alternatively, MK cDNA probes can be connected to various expression vectors, and MK proteins expressed in E. coli, Bacillus subtilis, yeast, etc. can be obtained through the same column operation. As animals to be immunized, mammals such as mice, rats, guinea pigs, hamsters, rabbits, goats, sheep, horses, cows and others are used. Anti-MK polyclonal antibodies can be obtained in the antiserum of these various animals immunized with MK. In addition, a hybridoma having the ability to produce an anti-MK monoclonal antibody can be produced by fusing the spleen cells of each immunized animal with a myeloma cell (myeloma cell) line. For immunization of animals, conventional methods can be used. For example, intraperitoneal, subcutaneous, etc. of various host animals, intramuscular, intradermal such as toes, intravascular, etc. with MK together with an appropriate adjuvant Inoculate. As an adjuvant, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi adjuvant, lipid A, silica, an immunostimulant, and the like can be used. After the initial immunization with a mixture of immunogen MK and adjuvant, booster immunization is repeated 2 to 10 times intraperitoneally, subcutaneously, intramuscularly, and intravascularly every 1 to 4 weeks thereafter, preferably every 1 to 2 weeks. I went. Also, myeloma cell lines used for fusion for hybridoma production include mouse NS-1 strain, SP-2 / 0 strain, X63-Ag8 strain, X63-6 / 5/3 strain, PAI strain, MPC-11 strain, etc. Mouse myeloma cell line, rat 210.RCY.Ag1.2.3 cell line, YB2 / 0 line, and the like.
[0008]
Blood can be collected from the animal immunized as described above to obtain antiserum, and affinity purification can be performed as follows to prepare an anti-MK antibody. For affinity purification, MK is coupled to a carrier such as agarose, Sephadex, Sepharose 4B (Pharmacia), Afigel 10 (Bio-Rad), various Toyopearls (Tosoh). As the antigen, MK itself or glutathione-S-transferase (GST) and MK fusion protein is used. After dialyzing the MK solution overnight with a coupling solution (containing 0.1 M NaHCO ₃ , pH 8.3, 0.5 M NaCl), a carrier such as BrCN-activated Sepharose 4B that is also washed with the coupling solution is added to the MK solution, Stir overnight. Next, after removing the supernatant, add about 0.1M ethanolamine (pH 8.0) and stir overnight at low temperature. Further, this MK coupling carrier suspension was transferred to a glass filter and washed with various buffers, and then the MK binding carrier was used for affinity purification of the anti-MK antibody. Apply anti-MK antiserum to a column packed with MK-bound Sepharose, wash with various buffers, elution fractionation with acidic buffer, neutralize immediately by adding Tris-HCl pH9.5 solution, affinity purified anti-MK An antibody solution was obtained.
[0009]
When the anti-MK antibody of the present invention is used as a diagnostic agent for cancer, it can be applied to cancer cells of humans and animals, particularly tissue pieces and cells removed from patients, or body fluids such as blood, thoracic fluid, and peritoneal fluid collected from patients. Using the anti-MK antibody of the present invention with radioisotope labeling, enzyme labeling, fluorescent labeling, etc., tissue staining using radioautography, enzyme antibody method, fluorescent antibody method, etc. Can be observed and detected. Alternatively, the MK antigen can be analyzed using an automatic cell analyzer or the like. MK antigens in cancer cell extracts or body fluids can be colorimetrically determined by methods such as RIA, EIA, and FIA. Furthermore, cancer lesions in a patient can be imaged in vivo with an anti-MK antibody labeled with a radioisotope such as ⁶⁷ Ga, ^99m Tc, ¹¹¹ In, ¹²⁵ I, and ¹³¹ I.
[0010]
On the other hand, when the anti-MK antibody of the present invention is used as a therapeutic agent for cancer, affinity-purified anti-MK polyclonal antibody, monoclonal antibody, or antibody fragment itself fragmented with an enzyme, and various anticancer agents, toxins, etc. The bound complex can be administered into the patient's body to provide anti-cancer therapy and prevent cancer growth.
[0011]
The pharmaceutical composition comprising the anti-MK antibody or anti-MK antibody-radioisotope conjugate of the present invention, anti-MK antibody-anticancer agent complex, anti-MK antibody-toxin complex as an active ingredient is prepared with various additives as necessary. It can be prepared by mixing. Examples of the additive include nonionic surfactants such as albumin, gelatin, dextran, polysorbate such as Tween 80, polyoxyethylene hydrogenated castor oil, fatty acid alcohol ester, polyglycol ether, phosphate buffered saline, various Examples thereof include saccharides such as amino acids, dextrose, mannitol, glucose, xylitol, lactose, sucrose, galactose, fructose, maltose, saccharose, sorbitol, and the like, and one or more of these can be used in combination.
[0012]
Furthermore, the pharmaceutical composition of the present invention includes a filler, a binder, a lubricant, a wetting agent, a disintegrating agent, an emulsion in addition to the above-mentioned examples as an additive such as a pharmaceutically acceptable carrier. And various substances that act as suspensions, preservatives, diluents, sweeteners, fragrances, and the like.
[0013]
Examples of these additives include corn starch, crystalline cellulose, gum arabic, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, tragacanth gum, gelatin, syrup, methylcellulose, carboxymethylcellulose, methylhydroxybenzoate, Examples include propylhydroxybenzoate, talc, magnesium stearate, inert polymers, water and mineral oil.
[0014]
The pharmaceutical composition of the present invention may be formulated so that the release rate of the active ingredient after administration is controlled as desired.
[0015]
In the case of parenteral administration such as intravenous infusion or injection, or intramuscular injection, for example, an effective amount of a complex such as anti-MK antibody, anti-MK antibody-radioisotope conjugate, anti-MK antibody-anticancer agent, etc. It can be dissolved in isotonic saline, sterile water or similar liquids and sealed in vials or ampoules.
[0016]
When a vial or ampoule is used, a freeze-dried product such as an anti-MK antibody, anti-MK antibody-anticancer agent or other complex may be prepared in the vial or ampoule in order to improve its stability. The content of the anti-MK antibody, anti-MK antibody-anticancer agent, etc. complex in the pharmaceutical composition of the present invention may be appropriately selected as necessary. For example, 0.1 μg to 50 mg per unit dosage form Can be about.
[0017]
【Example】
Hereinafter, the present invention will be described in more detail with reference examples and examples, but the present invention is not limited thereto.
Reference Example 1 Production of MK L cells transfected with the above MK gene (5 × 10 ⁶ per 10 cm dish) were cultured in serum-free medium in the presence of 40 μg / ml heparin, and 1 l of the culture supernatant was phenylsepharose CL. -4B column (2 x 12.7cm), heparin sepharose CL-6B column (1.5 x 4.5cm), purified to single band by SDS-PAGE (silver stain), concentrated to 10ml with Centricon 10, desalted As a result, 200 μg of MK was produced.
Reference Example 2 Production of GST-MK fusion protein
MK cDNA was introduced into a pG-EX-2TH vector having a glutathione S-transferase gene and infected with E. coli. The cells were collected from 1 l of the culture solution, and the cells were disrupted by sonication in 100 ml of 50 mM Tris-HCl, pH 7.5, 0.5% NP-40, 25% sucrose solution. The supernatant was obtained by centrifugation at 15,000 × g for 15 minutes. This supernatant was applied to glutathione-agarose (Sigma) (14 ml), washed, and eluted with 50 mM Tris-HCl, pH 9.6 containing 5 mM glutathione to obtain 60 mg of fusion protein.
Example 1 Preparation of anti-MK rabbit antiserum (1)
MK obtained in Reference Example 1 was immunized with rabbits (New Zealand White) four times every two weeks as follows. In the first immunization, 50 μg of MK was dissolved in 0.5 ml of PBS (containing 10 mg / ml of heparin), 0.5 ml of Freund's complete adjuvant was added, mixed well until emulsified, and injected into the footpad. In the second immunization, 0.5 ml of Freund's incomplete adjuvant was added to 0.5 ml of the same amount of MK (in heparin-containing PBS) as in the first, and the footpad was injected in the same manner as in the first immunization. In the third and fourth immunizations, 0.5 ml of Freund's incomplete adjuvant was added to 0.5 ml of MK (in heparin-containing PBS) in the same amount as in the first and second rounds. Were injected subcutaneously. Ten days after the final immunization, all the rabbits were collected and antiserum was prepared.
Example 2 Preparation of anti-MK rabbit antiserum (2)
Antiserum was prepared from rabbits and mouse MK obtained in Reference Example 2 in the same manner as in Example 1.
[0018]
Example 3 Production of affinity purified anti-MK antibody Coupling of GST-MK fusion protein and CNBr-activated Sepharose 4B
(1) 5 ml of the GST-MK fusion protein solution (protein concentration 4 mg / ml) obtained in Reference Example 2 was dialyzed overnight against a coupling buffer (containing 0.1 M NaHCO 3, pH 8.3, 0.5 M NaCl).
(2) 1 g of CNBr-activated Sepharose 4B (suspended in 1 mM HCl, pH 2.9, transferred to a glass filter, washed with 200 ml of 1 mM HCl, and finally washed with 100 ml of coupling buffer). And agitated on a miniplate shaker overnight (16-18 hours).
(3) The supernatant was removed, 5 ml of 0.1 M ethanolamine (pH 8.0) was added, and the mixture was stirred with a miniplate shaker at 4 ° C. overnight as in (2).
(4) Transfer (3) to a glass filter, wash with 200 ml of wash buffer A (0.1 M acetate buffer pH 4.0, containing 0.5 M NaCl), and then wash buffer B (0.1 M Tris HCl pH 8.0, 0.5 It was washed with 200 ml of M NaCl or a coupling buffer.
(5) (4) was repeated two more times.
(6) It was washed with about 100 ml of 0.1M glycine HCl pH2.7 for about 10 minutes without taking too much time. (7) Washed with 20 ml of 20 mM sodium phosphate buffer, pH 6.8, and suspended in the same buffer. When preserved, 1 mM Na-azide was added.
2. Affinity chromatography using GST-MK fusion protein-Sepharose column
(1) 2 ml of GST-MK fusion protein-Sepharose 4B was packed in a column.
(2) Equilibrated with 20 mM sodium phosphate buffer pH 6.8. When Na-azide was added, the column was washed 10 times.
(3) 3 ml of anti-MK rabbit serum was applied to the column.
(4) The column was washed with 20 mM sodium phosphate buffer, pH 6.8 (50 ml).
(5) Eluted with 0.1M glycine HCl pH2.7. The fractions were 2 ml each, and up to 10 bottles were taken.
At this time, 1 M Tris HCl pH9.5, 50 μl was added to each fraction (test tube) in advance, and the eluted one was immediately neutralized. The amount of protein was measured by absorption at OD280nm. Peaks are fractions 2 and 3, but protein was eluted up to fraction 6.
Collect fractions 2-6 (if you want a high IgG concentration, collect only fractions 2 and 3), add BSA to 1 mg / ml, and add phosphate buffered saline (PBS) Dialyzed thoroughly at 4 ° C.
[0019]
Example 4 ⁶⁷ Ga-deferoxamine mesylate (hereinafter abbreviated as DFO)-Production of anti-MK antibody (A) Production of DFO-anti-MK antibody-conjugate
Add 10 μl of 10% glutaraldehyde solution to 1.0 ml of a solution of DFO dissolved in PBS at a concentration of 2 × 10 ⁻⁵ M, stir at room temperature for 5 minutes, and add 1.3 mg / ml to the reaction mixture. 2 ml of an anti-MK antibody solution dissolved in PBS was added and stirred at 0-4 ° C. for 45 minutes. Next, 0.3 mg of sodium borohydride (NaBH ₄ ) was added, and the mixture was further stirred at 0 to 4 ° C. for 2 hours until the foam disappeared. Next, this reaction solution was subjected to gel filtration using a Sephadex G-50 column with PBS as an eluent, and 1.0 ml of eluate was fractionated per test tube. The absorbance at 280 nm of the collected eluate was measured, and the solution of three test tubes in which the protein fraction was eluted was collected to obtain a DFO-anti-MK antibody-conjugate.
(B) (Labeling with ⁶⁷ Ga)
10 μg of ⁶⁷ GaCl ₃ (400 μCi / ml) solution is mixed with 50 μl of the DFO-anti-MK antibody-conjugate solution (1.3 mg / ml as an antibody) obtained in the above section (A), and further 200 μl of PBS is added, followed by 30 at room temperature. Left for a minute. The obtained reaction solution was subjected to gel filtration on a Sephadex G-50 column using PBS as an eluent, and 30 eluates were collected at a rate of 1.0 ml per test tube. The radioactivity of each test tube was measured, and solutions of two test tubes with high radioactivity were collected to obtain ⁶⁷ Ga-DFO-anti-MK antibody.
[0020]
Example 5 Production of ¹¹¹ In-diethylenetriamine-penta-acetic acid (hereinafter abbreviated as DTPA) -anti-MK antibody (A) Production of DTPA-anti-MK antibody-conjugate
To 0.1 ml of 0.1 M sodium bicarbonate, 0.9 ml of a solution of anti-GP68 antibody dissolved in PBS at a concentration of 1.3 mg / ml was added, and further 250 times the molar amount of DTPA of anti-GP68 antibody was added and left at room temperature for 1 hour. . Next, this reaction solution was subjected to gel filtration with Sephadex G-50 using 0.01 M acetate buffer (pH 6.0), and 20 eluates were taken at 20 ml per test tube. The absorbance (280 nm) of the solution in each test tube was measured, and void fractions containing protein fractions with high absorbance were collected to obtain DTPA-anti-MK antibody-conjugate.
(B) (Labeling with ¹¹¹ In)
To 0.25 ml of the DTPA-anti-MK antibody-conjugate obtained in the above (A), 0.02 ml of ¹¹¹ InCl ₃ (250 μCi / ml) solution is mixed, and further, 0.23 ml of 0.01 M acetate buffer (pH 6.0). And left at room temperature for 30 minutes. The obtained reaction solution was subjected to gel filtration using Sephadex G-50 and PBS as an eluent, and 30 eluates were collected at a rate of 1.0 ml per test tube. The radioactivity of the solution in each test tube was measured, and the solutions in two test tubes with high radioactivity were collected to obtain ¹¹¹ In-DTPA-anti-MK antibody.
[0021]
Example 6 Production of ^99m Tc-DTPA-anti-MK antibody 0.25 ml of the DTPA-anti-MK antibody conjugate produced in Example 3 was added in 0.1 ml of 1 mg / ml tin chloride anhydrous saline solution at a concentration of 1 mg / ml. 0.1 ml of an ascorbic acid solution having a concentration and 0.1 ml of ^99m TcO4 (20 mCi / ml physiological saline) were added and left at room temperature for 30 minutes.
The resulting reaction solution was separated on a Sephadex G-50 column using PBS as the eluent, gel filtered, fractionated, and combined with the highly radioactive fraction to obtain ^99m Tc-DTPA-anti-MK antibody. It was.
[0022]
Example 7 Production of ⁶⁷ Ga-DFO-anti-MK Antibody F (ab ′) ₂ (A) (Production of F (ab ′) ₂ Fragment)
Dissolve 4 mg (25 nmol) of anti-MKIgG antibody in 1 ml of 0.1 M acetate buffer (pH 4.5) containing sodium chloride at a concentration of 0.1 M, add 2.5% pepsin (manufactured by Sigma) to this, Let stand for hours. The obtained reaction solution was subjected to gel filtration using a Sephadex G-50 column with pH 7.0 PBS as an eluent, and the eluted protein fraction was lyophilized to obtain an F (ab ′) ₂ fragment. 1.6 mg was obtained.
(B) (Production of DFO-anti-MK antibody F (ab ′) ₂ fragment-conjugate)
A DFO-anti-MK antibody F (ab ′) ₂ fragment-conjugate was produced in the same manner as in Example 3 (A).
(C) (labeled with ⁶⁷ Ga)
⁶⁷ Ga-DFO-anti-MKF (ab ′) ₂ fragment-conjugate was prepared in the same manner as in Example 3 (B).
[0023]
Example 8 Production of ¹²⁵ I-anti-MK antibody
Mix 100 μl of 1.3 mg / ml anti-MK antibody solution dissolved in PBS and 10 μl of ¹²⁵ I-NaI (74 MBq / ml), add 100 μl of PBS and 10 μl of chloramine-T (2 mg / ml) solution, and let stand at room temperature for 30 minutes. I put it. Next, 20 μl of 10 mg / ml sodium metabisulfate was added to stop the reaction. The obtained reaction solution was subjected to gel filtration using a Sephadex G-50 column with PBS as an eluent to obtain a ¹²⁵ I-anti-MK antibody fraction.
[0024]
Instead of ¹²⁵ I-NaI of Example 8 Example 9 ¹³¹ I- anti-MK antibody, except for using ¹³¹ I-NaI, the reaction was carried out and worked up in the same conditions as in Example 7, ¹³¹ I- Anti-MK antibody fraction was obtained.
[0025]
Experimental Example 1 Inhibition of growth of Wilms' tumor cells by anti-MK antibody Wilms' tumor cell culture
(1) Wilms' tumor cells (Japanese Cancer Research Resources Bank; abbreviated as JCRB) using McCoy5A medium containing 10% FCS (Nunc) and a 24-well tissue culture dish with 3 × 10 ⁴ cells / well I went.
(2) The next day, 0.1% FCS, ITS-containing F12 / DMEM medium (GLBCO, Ham'sF12 medium and DMEM were mixed at a ratio of 1: 1, 0.1% FCS, 10 μg / ml insulin, 10 μg / ml transferrin, Each well was washed once with 1 ml of 5 × 10 ⁻⁸ M sodium selenite), and 0.9 ml was added to each well.
(3) The purified anti-MK antibody was added with 0.1 ml / well of a control solution. Three wells were prepared for each experimental group. As a control, the serum of non-immunized rabbit was applied to a GST-MK fusion protein-Sepharose column, 1 mg / ml BSA was added to the fraction eluted with 0.1 M glycine buffer pH 2.7, and dialyzed against PBS Was used.
(4) After 8 hours, ³ H-labeled thymidine (hereinafter abbreviated as ³ H-TdR) was added at 2 μCi / well, and the culture was continued.
(5) after the addition of ³ H-TdR, it was measured respectively after 16 hours ³ H-TdR incorporation.
(6) 24 hours after (4) and further 48 hours later, ³ H-TdR was added to another well, and then the same operation as in (5) was performed.
2. ³ Measurement of H-TdR incorporation
(1) The supernatant of the medium in each well was aspirated with an aspirator or the like.
(2) Washed twice with 2 ml / well of cold PBS.
(3) 1 ml / well of acetic acid / methanol solution (acetic acid: methanol mixed at a ratio of 3: 1) was added and left at room temperature for 10 minutes.
(4) (3) was sucked out and washed with 2 ml / well of cold 10% trichloroacetic acid (TCA).
(5) 2 ml / well of cold 10% TCA was added and left at 4 ° C. for 15 minutes. This operation was repeated once more.
(6) Washed with 2 ml / well of distilled water.
(7) 0.2 μl NaOH was added at 300 μl / well and placed at 37 ° C. for 2 hours.
(8) Neutralized with 1N HCl 60 μl / well, transferred to a vial for each well, measured for radioactivity, and calculated the average value of 3 wells. The results are shown in FIG. Anti-MK antibody reduced Wilms' tumor cell growth to 30-40% of control.
[0026]
【The invention's effect】
According to the present invention, in addition to the fact that MK has the effect of maintaining the survival of nerve cells, it has been concluded that it works as a tumor growth factor of autocrine in cancer cells such as Wilms' tumor. The antibody is expected to detect a wide variety of cancer cells in which MK, which is a cancer-associated antigen, is expressed, and to inhibit the growth of many cancer cells in which MK is expressed.
[0027]
The present invention provides a cancer diagnostic method and therapeutic method characterized by using a newly produced anti-MK antibody, and a cancer diagnostic agent and cancer therapeutic agent comprising the anti-MK antibody.
[Brief description of the drawings]
FIG. 1 shows that growth of Wilms' tumor cells is blocked by anti-MK antibody.
Line 1 is a line to which a control reagent is added (control reagent obtained by applying non-immune rabbit serum to an affinity column in the same manner as MK immune rabbit serum is added), line 2 is a line with no antibody added, and line 3 is an anti-MK antibody added. It is a line.

Claims (2)

Includes anti-MK protein antibody obtained through a process characterized by affinity purification using MK protein carrier from serum obtained by immunizing animals (except humans) with MK protein (Midkine, MK) Wilms (Wilms') tumor of the therapeutic agent. The therapeutic agent for cancer according to claim 1 , wherein the animal is immunized by inoculating the animal with the MK protein together with heparin.

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