Immune conjugate having dendron conjugated to antibody and use thereof

Abstract

An immune conjugate according to the present invention provides a pharmaceutical composition, which can be used in the target-oriented drug treatment by conjugating a dendron allowing a plurality of drugs to be bound to a surface thereof and a target-directed antibody, and especially, can deliver high concentrations of drugs in a tumor-specific manner to exhibit a strong anticancer effect by conjugating a hydrophilic dendron, to which a plurality of anticancer drugs are bound, to an antibody.

Claims

1. An immunoconjugate comprising an antibody and a dendron, wherein the dendron has a thiol group at its center, wherein the dendron is conjugated to the cysteine residue present in a constant region or a variable region of a heavy or light chain of the antibody, and wherein an anti-cancer drug is conjugated to the dendron.

2. The immunoconjugate of claim 1, wherein the immunoconjugate further comprises a linker.

3. The immunoconjugate of claim 1, wherein the dendron is produced by reduction of a dendrimer having a cystamine core.

4. The immunoconjugate of claim 3, wherein the dendrimer is a polyamidoamine (PAMAM) dendrimer.

5. The immunoconjugate of claim 4, wherein the dendrimer is the polyamidoamine (PAMAM) dendrimer to which polyethylene glycol is conjugated.

6. The immunoconjugate of claim 3, wherein the dendrimer is the polyamidoamine (PAMAM) dendrimer to which polyethylene glycol is conjugated.

7. The immunoconjugate of claim 1, wherein the antibody is a tumor-specific antibody.

8. The immunoconjugate of claim 1, wherein the antibody is any one selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a Fv fragment, a Fab fragment, a F(ab).sub.2 fragment and a scFv fragment.

9. The immunoconjugate of claim 1, wherein the antibody is an antibody in which one or more residues of the constant region or variable region of the heavy or light chain are replaced by a cysteine residue, wherein the replaced cysteine residue is conjugated to the dendron.

10. The immunoconjugate according to claim 1, wherein the dendron is conjugated to the antibody via a linker.

11. A pharmaceutical composition for treating cancer, the composition comprising the immunoconjugate of claim 1 as an active ingredient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the reaction of doxorubicin with cis-aconitic anhydride to produce cis-aconityl doxorubicin.

(2) FIG. 2 shows chromatograms and mass spectra obtained by reversed-phase HPLC and MALDI-TOF mass spectrometry of the prepared cis-aconityl doxorubicin.

(3) FIG. 3 is a schematic diagram of a method for preparing the immunoconjugate according to the present invention using a dendrimer having a cystamine core.

(4) FIG. 4 shows a conjugate structure in which doxorubicin is bound to a polyethylene glycol-conjugated dendrimer.

(5) FIG. 5 is a chromatogram showing the detection of a dendron-antibody immunoconjugate at UV 280 nm and 490 nm after separating the antibody and the dendron-antibody immunoconjugate by size-exclusion HPLC.

(6) FIG. 6 is a chromatogram showing the concentration of doxorubicin contained in the dendron-antibody immunoconjugate by analyzing the amount of toxin doxorubicinone liberated after treating the dendron-antibody immunoconjugate with hydrochloric acid and methanol.

(7) FIG. 7 shows release rates of doxorubicin drug over time from the dendron-antibody conjugate in buffer at pH 4.5 and pH 7.4 at 37 C.

(8) FIG. 8 shows the tumor growth curves of the anticancer efficacy of the dendron-antibody conjugate (B12-Dendrimer Conjugates) in a mouse tumor model.

MODE FOR CARRYING OUT INVENTION

(9) Hereinafter, the present invention will be described in detail.

(10) However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1

(11) Preparation of Cis-Aconityl Doxorubicin

(12) Doxorubicin (ADEM injection solution) purchased from Dong-A Pharmaceutical Co., Ltd. was dissolved in dimethylformamide (DMF) containing 0.3% triethylamine (TEA) and reacted with cis-aconitic anhydride dissolved in the same solvent for 2 hours under light shielding (FIG. 1). Then, the reaction mixture was loaded on a cartridge filled with C-18 silica, and the unreacted material was removed using a solid-phase extraction method eluting with 80% methanol to separate the cis-aconityl doxorubicin. The methanol in the solution containing the separated cis-aconityl doxorubicin was removed by a vacuum centrifuge, followed by freeze-drying.

(13) The prepared cis-aconityl doxorubicin was injected into a DiKMA C-18 reversed-phase column and analyzed by UV light at 490 nm with 3% (NH.sub.4).sub.2CO.sub.3 and 1:2 (v/v) methanol. The mass value was also measured using a MALDI-TOF mass spectrometer. The mass value of cis-aconityl doxorubicin was confirmed to be 700.4 Da (FIG. 2).

Example 2

(14) Preparation of Dendron-Antibody Immunoconjugates

(15) Cis-aconityl doxorubicin was reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 15 min. PAMAM dendrimer with cystamine core was added to this solution and reacted for 24 hours. PAMAM dendrimers conjugated with doxorubicin were prepared by removing unreacted materials with a dialysis membrane having a molecular weight cut-off of 3,000. The disulfide bond of the PAMAM dendrimer was reduced using 1 M dithiothreitol (DTT) (FIG. 3).

(16) Separately, a dendron-antibody immunoconjugate was prepared using a dendrimer in which polyethylene glycol (PEG) was bonded to a part of amine group in the surface to reduce the toxicity of dendrimer and to secure solubility (FIG. 4). Table 1 below shows the number of PEG bonds and the number of doxorubicin bonds of the PEG-conjugated dendrimer used.

(17) TABLE-US-00001 TABLE 1 The number of PEG bonds and the particle size of the PEG conjugated dendrimer used PEG-dendrimer PEG bond No. Doxorubicin bond No. PEG-G2-DOX (DPG2) 4 5 PEG-G4-DOX (DPG4) 24 14 PEG-G5-DOX (DPG5) 32 22

(18) After the 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) dissolved in 0.1 M phosphate buffer (pH 8.0) was reacted with the cysteine group of the anti-claudin-4 antibody to activate its SH moiety, unreacted DTNB was removed using an ultrafiltration tube with a molecular weight cut-off of 30,000. A dendron-antibody immunoconjugate was prepared by mixing the DTNB-activated antibody and the doxorubicin-conjugated PAMAMA dendrimer for 3 hours.

(19) The prepared dendron-antibody immunoconjugates were analyzed by size-exclusion HPLC. Superose 6 10/300 GL (GE, USA) was used as a column, and 10 mM phosphate buffered saline (pH 7.0) was used as the mobile phase. The absorbance at 280 nm and 490 nm was used to detect the substance.

(20) As shown in the size-exclusion HPLC chromatogram of FIG. 5, the detection of the peak at 490 nm indicating the absorbance of doxorubicin confirmed the preparation of a dendron-antibody conjugate.

Example 3

(21) Measurement of Doxorubicin Content of Dendron-Antibody Conjugates

(22) To determine the amount of doxorubicin contained in the dendron-antibody conjugate, the amount of doxorubicinone released after treatment with 2.5 M hydrochloric acid and methanol was determined by reverse phase HPLC. Reversed phase HPLC was performed by using DiKMA Inspire C-18 reverse phase column (4.6250 mm, 5 micron) and mixture of 3% (NH.sub.4).sub.2CO.sub.3 and methanol 1:2 (v/v) as the mobile phase. The flow rate was 1 mL/min and detection was performed at UV 490 nm. Doxorubicin standards were used to generate calibration curves ranging from 5.0 to 200 g/mL, and the amount of doxorubicinone liberated from the dendron-antibody conjugate was determined using this calibration curve.

(23) The results are shown in FIG. 6.

(24) FIG. 6 is a HPLC chromatogram showing the detection of doxorubicin liberated from the doxorubicin standard product and the dendron-antibody conjugate, respectively. Quantification results indicated that on average 8 molecules of doxorubicin were bound per antibody molecule.

Example 4

(25) Test for Release of Doxorubicin Specific to Cancer Cell Environment from the Dendron-Antibody Conjugate

(26) Doxorubicin bound to the dendron-antibody conjugate is stably bound in neutral blood and has the property of selectively releasing it from the acidic condition of lysosomes/endosomes in tumor cells. Drug release experiments were performed on the dendron-antibody conjugates at pH 4.5 and pH 7.4, respectively, to identify cancer cell environment-specific drug release mechanisms. Each dendron-antibody conjugate in a dialysis bag (molecular weight cut-off 3,500 Da) was placed in a buffer solution of pH 4.5 and pH 7.4, respectively, and incubated at 37 C. A portion of the buffer solution outside of the dialysis bag was taken at predetermined times, and the amount of doxorubicin was measured by HPLC analysis. The results showed that doxorubicin release was less than 4% at pH 7.4, while the dendron G4-antibody conjugate (DPG4) showed a drug release rate of 38.9% for 48 hours at pH 4.5, and the dendron G5-antibody conjugate (DPG5) showed a drug release rate of 48.7%.

(27) The results are shown in FIG. 7.

(28) FIG. 7 shows the release rates of doxorubicin drug over time from a dendron-antibody conjugate in buffer at pH 4.5 and pH 7.4 at 37 degrees.

Example 5

(29) In Vitro Evaluation of Anti-Cancer Efficacy of the Dendron-Antibody Immunoconjugate

(30) In order to affirm the function of doxorubicin conjugated in dendron-antibody according to the present invention, cytotoxicity of B12 antibody alone or dendron-antibody conjugated with doxorubicin was confirmed. To confirm cytotoxicity, 110.sup.4 OVCAR-3 cells were placed in each well of a 96-well plate and cultured for 24 hours at 37.5 C. and 5% CO.sub.2. After 24 hours, the cell culture was removed and treated with B12 antibody alone, 1010000 ng/ml of the dendron-antibody conjugated with doxorubicin, and cultured for 24 hours or 48 hours. After incubation for 24 hours or 48 hours, the culture solution was removed and the PBS wash was performed twice. The WST reagent was mixed with the culture solution at a ratio of 1:10, and treated with 100 l/well. Absorbance was measured at 430 nm wavelength after left for 2 hours at 37.5 C. and 5% CO.sub.2.

(31) As a result, the cytotoxicity of B12 antibody alone did not show cytotoxicity, but the cytotoxicity was dose-dependent when cells were treated with the dendron-antibody conjugated with doxorubicin, indicating that the dendron-antibody conjugated with doxorubicin exhibited cytotoxic activity.

Example 6

(32) In Vivo Evaluation of Anticancer Efficacy of the Dendron-Antibody Conjugate

(33) The OVCAR-3 ovarian cancer cell line was mixed with matrigel and injected subcutaneously into Athymic nude immunodeficient mouse to observe tumor growth. After 77 days of tumor growth, treatment with the antibody (B12 antibody) and B12-Dendrimer Conjugates was initiated. Nine doses were administered intraperitoneally three times a week, and the tumor volume was measured and the efficacy of the anticancer therapies was evaluated and compared with the tumor growth curve.

(34) The anticancer efficacy was evaluated by using 5 mice per experimental group of the following three groups. The length of the tumor was measured using a digital caliper once or twice a week and the volume was calculated using a certain known equation.

(35) Experimental group 1. Human IgG control

(36) Experimental group 2. B12 antibody treatment (200 g/mouse)

(37) Experimental group 3. B12-Dendrimer Conjugates treatment (10 mg/kg)

(38) The results are shown in FIG. 8.

(39) As shown in the tumor growth graph of FIG. 8, normal tumor growth was observed in the human IgG control group. The B12-Dendrimer Conjugates treatment group showed not only a slow progress of tumor growth but also a marked decrease in tumor growth as compared with the control and B12 antibody treatment groups, indicating that the B12-Dendrimer Conjugate has an anticancer therapeutic effect.

INDUSTRIAL APPLICABILITY

(40) As described above, the immunoconjugate according to the present invention can be used for targeted drug therapy by conjugating a target-directed antibody with a dendron that can bind multiple drugs to its surface, and in particular, by bonding hydrophilic dendron conjugated with a plurality of anticancer drugs to the antibody. It is possible to provide a pharmaceutical composition capable of exhibiting a potent anticancer effect by delivering a high concentration of a tumor-specific drug, which is highly industrially applicable.