Aptamer-drug conjugate and use thereof

Abstract

A cancer targeted therapeutic agent includes a drug-linker-AS1411 structure. The drug may be selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), cytarabine, gemcitabine, maytansine, DM1, DM4, calicheamicin and a derivative thereof, doxorubicin, duocarmycin and a derivative thereof, pyrrolobenzodiazepine (PBD), SN-38, a-amanitin, or a tubulysin analog.

Claims

1. A cancer targeted therapeutic agent, comprising: a drug-linker-modified AS1411 structure, wherein the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), cytarabine, gemcitabine, maytansine, DM1 represented by Formula 1, DM4 represented by Formula 2, calicheamicin, acylated calicheamicin, doxorubicin, duocarmycin, pyrrolo-benzodiazepine (PBD), SN-38 represented by Formula 3, or α-ammantin; ##STR00012## and the linker is X-Y, wherein Y is selected from the group consisting of: maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-Val-Cit-PAB), maleimidocaproyl-glycine-phenylalanine-leucine-glycine (MG-Gly-Phe-Leu-Gly), hydrazone, disulfide, thioether, valine-citrulline, N-maleimdomethylcyclohexane-1-carboxylate (MCC), maleimidocaproyl, mercaptoacetamidocaproly, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and N-succinimidyl 4-(2-pyridylthio)pentanoate (SPDB), X is selected from the group consisting of: 5′-thiol-modifier C6, thiol-modifier C6 S—S, dithiolserinol, PC amino-modifier, 5′-amino-modifier C3, 5′-amino-modifier C6, 5′-amino-modifier C12, amino-modifier C2 dT, amino-modifier C6 dT, and S-Bz-thiol-modifier C6-dT, wherein X is linked to the modified AS1411 at a nucleotide residue of a 12 or 13 position or nucleotide residues of both 12 and 13 positions of the modified AS1411, and Y is bound to the drug; and the modified AS1411 has the sequence of SEQ ID NO: 1 with a modification that at least one of the nucleotide residue of the 12 and 13 position of the SEQ ID NO: 1 is substituted with uracil.

2. The cancer targeted therapeutic agent of claim 1, wherein the drug is monomethyl auristatin E (MMAE) or cytarabine.

3. The cancer targeted therapeutic agent of claim 1, wherein Y is maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-Val-Cit-PAB) or maleimidocaproyl-glycine-phenylalanine-leucine-glycine (MC-Gly-Phe-Leu-Gly), X is 5′-thiol-modifier C6, and Y has a side of maleimidocaproyl bound to the drug and the other side bound to X.

4. The cancer targeted therapeutic agent of claim 1, wherein the modified AS1411 has the sequence of SEQ ID NO: 9.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates Gel run data of HS-C6-T.sub.3-AS1411 and MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 and ESI-MS of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411.

(2) FIG. 2 illustrates Gel run data of HS-C6-T.sub.3-CRO and MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO and ESI-MS of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO.

(3) FIG. 3 illustrates MSI-MS of 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411.

(4) FIG. 4 illustrates ESI-MS of 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411.

(5) FIG. 5 illustrates MTT assay results of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 and MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO to A549 cell lines.

(6) FIG. 6 illustrates MTT assay results of citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 and citravin-(GLFG-MC-S-C6)-T.sub.3-CRO to Mv4-11 cell lines.

(7) FIG. 7 illustrates FDG PET images obtained before treatment of A549 lung cancer cell line-injected mice (a tumor with FDG intake in the left thigh region is observed).

(8) FIG. 8 illustrates FDG PET images obtained 30 days after treatment of A549 lung cancer cell line-injected mice with MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 (a decrease in FDG intake in the tumor is observed).

(9) FIG. 9 illustrates FDG PET images obtained before treatment of A549 lung cancer cell line-injected mice (a tumor with FDG intake in the left thigh region is observed).

(10) FIG. 10 illustrates FDG PET images obtained 30 days after treatment of A549 lung cancer cell line-injected mice with MMAE (an increase in FDG intake in the tumor is observed).

(11) FIG. 11 illustrates comparison of tumor sizes measured after treatment using MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 and MMAE, respectively, in A549 tumor model.

(12) FIG. 12 illustrates comparison of sizes of tumors excised 30 days after treatment using MMAE-(PAB-Cit-Val-MC-S-06)-T.sub.3-AS1411 and MMAE, respectively, in A549 tumor model.

(13) FIG. 13 illustrates FDG PET images obtained before treatment of Mv4-11 AML cell line-injected mice (a tumor with FDG intake in the left thigh region is observed).

(14) FIG. 14 illustrates FDG PET images obtained 30 days after treatment of Mv4-11 AML cell line-injected mice with citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 (a decrease in FDG intake in the tumor is observed).

(15) FIG. 15 illustrates FDG PET images obtained before treatment of Mv4-11 AML cell line-injected mice (a tumor with FDG intake in the left thigh region is observed).

(16) FIG. 16 illustrates FDG PET images obtained 30 days after treatment of Mv4-11 AML cell line-injected mice with MMAE (an increase in FDG intake in the tumor is observed).

(17) FIG. 17 illustrates A549 cell viability in each of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411 at different concentrations.

(18) FIG. 18 illustrates MTT assay results of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411 to A549 cell lines.

DETAILED DESCRIPTION

(19) Hereinafter, the present invention will be described in more detail with reference to the following embodiments.

(20) [GRO Aptamer]-Drug Conjugate Synthesis

Example 1

Synthesis of MMAE-(PAB-Cit-Val-MC-S-C6)-T.SUB.3.-AS1411 [AS1411-MMAE conjugate]

(21) By reacting maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonylmonomethyl auristatin E [MC-Val-Cit-PAB-MMAE] with HS-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 2) [HS-C6-T.sub.3-AS1411], monomethyl auristatin E-p-aminobenzoyloxycarbonyl-citrulline-valine-Mal-S-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 2) [MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411] was synthesized. In other words, RSS-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 2) [RSS-C6-T.sub.3-AS1411] was subjected to reductive reaction in the presence of DTT for about 3 hours, and the remaining DTT was removed by a centrifuge and replaced with an SB17 buffer solution, resulting in HS-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 2) [HS-C6-T.sub.3-AS1411]. After putting MC-Val-Cit-PAB-MMAE dissolved in a small amount of DMSO into the resultant product, the mixture was shaken overnight. Separation/purification were performed through reverse phase HPLC (Waters-Xbridge OST C18 10×50 mm, 65, TEAE/CAN buffer).

(22) ##STR00005##

(23) MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 was synthesized by reaction of MC-Val-Cit-PAB-MMAE and HS-06-T.sub.3-AS1411

(24) Synthesis of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 from HS-C6-T.sub.3-AS1411 was identified by Gel run, and a molecular weight of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 was determined through ESI-MS (FIG. 1). C.sub.364H.sub.479N.sub.120O.sub.202P.sub.29S [Cal. MW=10697.69, Obs. MW=10697.0]

Example 2

Synthesis of MMAE-(PAB-Cit-Val-MC-S-C6)-T.SUB.3.-CRO

(25) By reacting maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonylmonomethyl auristatin E [MC-Val-Cit-PAB-MMAE] with HS-C6-tttcctcctcctccttctcctcctcctcc (SEQ ID NO: 3) [HS-C6-T.sub.3-CRO], monomethyl auristatin E-p-aminobenzoyloxycarbonyl-citrulline-valine-Mal-S-C6-tttcctcctcctccttctcctcctcctcc (SEQ ID NO: 3) [MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO] was synthesized. In other words, RSS-C6-tttcctcctcctccttctcctcctcctcc (SEQ ID NO: 3) [RSS-C6-T.sub.3-CRO) was subjected to reductive reaction in the presence of DTT for about 3 hours, and the remaining DTT was removed by a centrifuge and replaced with an SB17 buffer solution, resulting in HS-C6-tttcctcctcctccttctcctcctcctcc (SEQ ID NO: 3) [HS-C6-T.sub.3-CRO). After putting MC-Val-Cit-PAB-MMAE dissolved in a small amount of DMSO into the resultant product, the mixture was shaken overnight. Separation/purification were performed through reverse phase HPLC (Waters-Xbridge OST C18 10×50 mm, 65, TEAE/CAN buffer).

(26) ##STR00006##

(27) MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO was synthesized by reaction of MC-Val-Cit-PAB-MMAE and HS-C6-T.sub.3-CRO

(28) Synthesis of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO from HS-C6-T.sub.3-CRO was identified by Gel run, and a molecular weight of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO was determined through ESI-MS (FIG. 2). C.sub.347H.sub.479N.sub.86O.sub.202P.sub.29S [Cal. MW=10018.13, Obs. MW=10017.28]

Example 3

Synthesis of 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).SUB.2.-AS1411 and 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411

(29) By reacting maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonylmonomethyl auristatin E [MC-Val-Cit-PAB-MMAE]with ggtggtggtggu (SEQ ID NO: 4)[5-N-(6-(3-thiopropanoyl)-aminohexyl)-3-acrylamido]u[5-N-(6-(3-thiopropanoyl)-aminohexyl)-3-acrylamido]gtggtggtggtgg (SEQ ID NO: 6)[12,13-(HS-C6).sub.2-AS1411], ggtggtggtggu (SEQ ID NO: 4)[5-N-(6-(3-monomethyl auristatin-p-aminobenzoyloxycarbonyl-citrulline-valine-Mal-thiopropanoyl)-aminohexyl)-3-acrylamido]u[5-N-(6-(3-monomethyl auristatin-p-aminobenzoyloxycarbonyl-citrulline-valine-Mal-thiopropanoyl)-aminohexyl)-3-acrylamido]gtggtggtggtgg (SEQ ID NO: 6)[12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411], and ggtggtggtggu (SEQ ID NO: 4)[5-N-(6-(3-monomethyl auristatin-p-aminobenzoyloxycarbonyl-citrulline-valine-Mal-thiopropanoyl)-aminohexyl)-3-acrylamido]ugtggtggtggtgg (SEQ ID NO: 5)][12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411] were synthesized. In other words, ggtggtggtggu (SEQ ID NO: 4)[5-N-(6-(3-benzoyl thiopropanoyl)-aminohexyl)-3-acrylamido]u[5-N-(6-(3-benzoyl thiopropanoyl)-aminohexyl)-3-acrylamido]gtggtggtggtgg (SEQ ID NO: 6)[12,13-(Bz-S-C6).sub.2-AS1411] was subjected to reductive reaction in the presence of DTT for about 3 hours, and the remaining DTT was removed by a centrifuge and replaced with an SB17 buffer solution, resulting in ggtggtggtggu (SEQ ID NO: 4) [5-N-(6-(3-thiopropanoyl)-aminohexyl)-3-acrylamido]u[5-N-(6-(3-thiopropanoyl)-aminohexyl)-3-acrylamido]gtggtggtggtgg (SEQ ID NO: 6) [12,13-(HS-C6).sub.2-AS1411]. After putting MC-Val-Cit-PAB-MMAE dissolved in a small amount of DMSO into the resultant product, the mixture was shaken overnight. Separation/purification were performed through reverse phase HPLC (Waters-Xbridge OST C18 10×50 mm, 65, TEAE/CAN buffer), thereby yielding 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411 and 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411.

(30) ##STR00007## ##STR00008##

(31) 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411 and 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6) were synthesized by reaction of MC-Val-Cit-PAB-MMAE and 12,13-(HS-C6).sub.2-AS1411

(32) Through ESI-MS, molecular weights of 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411 and 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 were determined (FIG. 3 and FIG. 4). 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411, C.sub.418H.sub.568N.sub.129O.sub.197P.sub.25S.sub.2 [Cal. MW=11390.21, Obs. MW=11390.0]; 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411, C.sub.350H.sub.463N.sub.118O.sub.182P.sub.25S.sub.2 [Cal. MW=10073.58, Obs. MW=10073.0]

Example 4

Synthesis of Citravin-(GLFG-MC-S-C6)-T.SUB.3.-AS1411 Conjugate

(33) By reacting maleimidocaproyl-(Gly-Phe-Leu-Gly (SEQ ID NO: 7))-citravin [MC-GFLG-citravin] with HS-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 2) [HS-C6-T3-AS1411], citravin-(Gly-Leu-Phe-Gly (SEQ ID NO: 8))-Mal-S-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 2) [Citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411] was synthesized. In other words, RSS-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 2) [RSS-C6-T.sub.3-AS141] was subjected to reductive reaction in the presence of DTT for about 3 hours, and the remaining DTT was removed by a centrifuge and replaced with an SB17 buffer solution. After putting Mal-GPLG-citravin dissolved in a small amount of DMSO into the resultant product, the mixture was shaken overnight. Separation/purification were performed through reverse phase HPLC (Waters-Xbridge OST C18 10×50 mm, 65, TEAE/CAN buffer), thereby yielding citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411. Through ESI-MS, a molecular weight of citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 was determined. C.sub.334H.sub.424N.sub.117O.sub.199P.sub.29S [Cal. MW=10191.91, Obs. MW=10190.88]

(34) ##STR00009##

(35) Citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 was synthesized by reaction of C-GFLG-citravin and HS-C6-T.sub.3-AS1411

Example 5

Synthesis of 12,13-(citravin-GLFG-MC-S-C6).SUB.2.-AS1411 Conjugate and 12 or 13-(citravin-GLFG-MC-S-C6)-AS1411 Conjugate

(36) By reacting maleimidocapryl-(Gly-Phe-Leu-Gly (SEQ ID NO: 7))-citravin [MC-GFLG-citravin] with ggtggtggtggu (SEQ ID NO: 4) [5-N-(6-(3-thiopropanoyl)-aminohexyl)-3-acrylamido]u[5-N-(6-(3-thiopropanoyl)-aminohexyl)-3-acrylamido]gtggtggtggtgg (SEQ ID NO: 6) [12,13-(HS-C6).sub.2-AS1411], ggtggtggtggu (SEQ ID NO: 4) [citravin-Gly-Leu-Phe-Gly (SEQ ID NO: 8)-Mal-thiopropanoyl)-aminohexyl)-3-acrylamido]u[citravin-Gly-Leu-Phe-Gly (SEQ ID NO: 8)-Mal-thiopropanoyl)-aminohexyl)-3-acrylamido]gtggtggtggtgg (SEQ ID NO: 6) [12,13-(citravin-GLFG-MC-S-C6).sub.2-AS1411] and ggtggtggtggu (SEQ ID NO: 4) [citravin-Gly-Leu-Phe-Gly (SEQ ID NO: 8)-Mal-thiopropanoyl)-aminohexyl)-3-acrylamido]ugtggtggtggtgg (SEQ ID NO: 5)][12 or 13-(citravin-GLFG-MC-S-C6)-AS1411] were synthesized. In other words, ggtggtggtggu (SEQ ID NO: 4) [5-N-(6-(3-benzoyl thiopropanoyl)-aminohexyl)-3-acrylamido]u[5-N-(6-(3-benzoyl thiopropanoyl)-aminohexyl)-3-acrylamido]gtggtggtggtgg (SEQ ID NO: 6) [12,13-(Bz-S-C6).sub.2-AS1411] was subjected to reductive reaction in the presence of DTT for about 3 hours, and the remaining DDT was removed by a centrifuge and replaced with an SB17 buffer solution, resulting in ggtggtggtggu (SEQ ID NO: 4) [5-N-(6-(3-thiopropanoyl)-aminohexyl)-3-acrylamido]u[5-N-(6-(3-thiopropanoyl)-aminohexyl)-3-acrylamido]gtggtggtggtgg (SEQ ID NO: 6) [12,13-(HS-C6).sub.2-AS1411]. After putting MC-GFLG-citravin dissolved in a small amount of DMSO into the resultant product, the mixture was shaken overnight. Purification was performed through reverse phase HPLC (Waters-Xbridge OST C18 10 X 50 mm, 65, TEAE/CAN buffer), thereby yielding 12,13-(citravin-GLFG-MC-S-C6).sub.2-AS1411 and 12 or 13 (citravin-GLFG-MC-S-C6)-AS1411. Through ESI-MS, molecular weights of each of the above products were determined: 12,13-(citravin-GLFG-MC-S-C6).sub.2-AS1411, C.sub.358H.sub.458N.sub.123O.sub.191P.sub.25S.sub.2 [Cal. MW=10378, 66 Obs. MW=10379.23]; 12 or 13 (citravin-GLFG-MC-S-C6)-AS1411, C.sub.320H.sub.408N.sub.115O.sub.179P.sub.25S.sub.2 [Cal. MW=9567.81, Obs. MW=9568.09]

(37) ##STR00010## ##STR00011##

(38) 12,13-(citravin-GLFG-MC-S-C6).sub.2-AS1411 and 12 or 13 (citravin-GLFG-MC-S-C6)-AS1411 were synthesized by reaction of MC-GFLG-citravin and 12,13-(HS-C6).sub.2-AS1411

Example 6

(39) In Vitro Efficacy Validation

MTT Assay of MMAE-(PAB-Cit-Val-MC-S-C6)-T.SUB.3.-AS1411 and MMAE-(PAB-Cit-Val-MC-S-C6)-T.SUB.3.-CRO to A549 Cell Lines

(40) With regard to A549 cell line as a lung cancer cell line over-expressing a nucleoline protein, cell inhibition efficacy of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 was verified in vitro by MTT assay. When comparing cell viability and cell proliferation of the cells through MTT assay of MMAE, AS1411, MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 and MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO, respectively, it was demonstrated that MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 has almost the same efficacy as MMAE. A549 cells (ATCC, IMDM+10% FBS) were seeded on a 96-well plate in a cell number of 2.5 to 5×10.sup.5 cells/well, which was determined by a cell test method to determine an appropriate cell concentration, followed by growing for 1 day. After heating each of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 and MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO at 95° C. for 5 minutes, the heated product was gradually cooled at room temperature and directly treated on each well at different concentrations. After incubating the treated A549 cells in 5% CO.sub.2 incubator for 72 hours, the incubated product was treated with 20 μL of a reagent solution for MTT assay (Cell Proliferation kit II, Roche) and incubated for different periods of time (10 min, 30 min, 1 hr). Thereafter, the final product was subjected to measurement of absorbance at 490 nm by an ELISA reader (FIG. 5).

Example 7

MTT Assay of Citravin-(GLFG-MC-S-C6)-T.SUB.3.-AS1411 and Citravin-(GLFG-MC-S-C6)-T.SUB.3.-CRO to Mv4-11 Cell Lines

(41) With regard to Mv4-11 cell line as a lung cancer cell line over-expressing a nucleoline protein, cell inhibition efficacy of citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 was verified in vitro by MTT assay. When comparing cell viability and cell proliferation of the cells through MTT assay of citravin, AS1411, citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 and citravin-(GLFG-MC-S-C6)-T.sub.3-CRO, respectively, it was demonstrated that citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 conjugate has almost the same efficacy as citravin. Mv4-11 cells (ATCC, IMDM+10% FBS) were seeded on a 96-well plate in a cell number of 2.5 to 5×10.sup.5 cells/well, which was determined by a cell test method to determine an appropriate cell concentration, followed by growing for 1 day. After heating each of citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 and citravin-(GLFG-MC-S-C6)-T.sub.3-CRO at 95° C. for 5 minutes, the heated product was gradually cooled at room temperature and directly treated on each well at different concentrations. After incubating the treated A549 cells in 5% CO.sub.2 incubator for 72 hours, the incubated product was treated with 20 μL of a reagent solution for MTT assay (Cell Proliferation kit II, Roche) and incubated for different periods of time (10 min, 30 min, 1 hr). Thereafter, the final product was subjected to measurement of absorbance at 490 nm by an ELISA reader (FIG. 6).

Example 8

(42) In Vivo Efficacy Validation

Validation of In Vivo Therapeutic Efficacy of MMAE-(PAB-Cit-Val-MC-S-C6)-T.SUB.3.-AS1411 on A549 Lung Cancer Cell Line-Injected Mice

(43) To A549 lung cancer cell line-injected mice, MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 and MMAE, respectively, were administered by IV injection for 30 days, followed by identifying tumor sizes through PET images. A549 lung cancer cells in a number of 6.1×10.sup.6 cells/ml were injected subcutaneously in the right thigh of each nude mouse. The tumor size was measured in 3 to 4 weeks after the injection and, when a diameter of the tumor reached 0.8 cm, microPET images were captured before treatment. For PET images, F-18 FDG 0.2 mCi was i.p. injected and then the images were captured (Siemens Inveon). After detection of FDG intake in the tumor from the images, MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 was i.v. injected in an experimental group of 5 mice 4 times by 5 day intervals (7.5 mg/kg, 0.5 mg/kg of MMAE). After 30 days from the start of treatment, microPET images were captured by the same method as used before treatment. After administration of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 to A549 lung cancer cell line-injected mice for 30 days through IV injection, it was identified from PET images that FDG intake in the tumor remarkably decreased (FIG. 7 and FIG. 8).

Example 9

(44) Validation of In Vivo Therapeutic Efficacy of MMAE on A549 Lung Cancer Cell Line-Injected Mice

(45) To A549 lung cancer cell-injected mice, MMAE was administered by IV injection for 30 days, followed by observation of PET images. A549 lung cancer cells in a number of 6.1×10.sup.6 cells/ml were injected subcutaneously in the right thigh of each nude mouse. The tumor size was measured in 3 to 4 weeks after the injection and, when a diameter of the tumor reached 0.8 cm, microPET images were captured before treatment. For PET images, F-18 FDG 0.2 mCi was i.p. injected and then the images were captured (Siemens Inveon). After detection of FDG intake in the tumor from the images before treatment, MMAE was i.v. injected in an experimental group of 5 mice 4 times by 5 day intervals (0.5 mg/kg). After 30 days from the start of treatment, microPET images were captured by the same method as used before treatment. Compared to before MMAE treatment, it was identified from FDG PET images that FDG intake in the tumor was increased even after treatment (FIG. 9 and FIG. 10).

Example 10

(46) Ex-Vivo Validation

(47) As compared to administration of MMAE alone, administration of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 to A549 lung cancer cell line-injected mice exhibited superior cancer inhibitory efficacy. As compared to administration of MMAE alone, it was determined that MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 inhibits 80% more cancer. After the start of treatment, sizes of tumors were measured in both of horizontal and vertical axes by 5 day intervals. 30 days after the treatment, tumors were excised from separate groups. As shown in the pictures below, each group was photographed and the tumor sizes were compared between MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 and MMAE (FIG. 11 and FIG. 12).

(48) As a result of verifying in vivo efficacies of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 and MMAE to A549 cell lines, it was demonstrated that MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 was superior over administration of MMAE alone.

Example 11

(49) In Vivo Efficacy Validation

Validation of In Vivo Therapeutic Efficacy of citravin-(GLFG-MC-S-C6)-T.SUB.3.-AS1411 on Mv4-11 AML Cell Line-Injected Mice

(50) To Mv4-11 AML cell line-injected mice, citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 and MMAE, respectively, were administered by IV injection for 30 days, followed by identifying tumor sizes through PET images. Mv4-11 AML cells in a number of 6.1×10.sup.6 cells/ml were injected subcutaneously in the right thigh of each nude mouse. The tumor size was measured in 3 to 4 weeks after the injection and, when a diameter of the tumor reached 0.8 cm, microPET images were captured before treatment. For PET images, F-18 FDG 0.2 mCi was i.p. injected and then the images were captured (Siemens Inveon). After detection of FDG intake in the tumor from the images, citravin-(GLFG-MC-S-C6)-T.sub.3-AS1411 was i.v. injected in an experimental group of 5 mice 4 times by 5 day intervals (7.5 mg/kg, 0.5 mg/kg of MMAE). After 30 days from the start of treatment, microPET images were captured by the same method as used before treatment (FIG. 13 and FIG. 14).

Example 12

(51) Validation of In Vivo Therapeutic Efficacy of MMAE on Mv4-11 AML Cell Line-Injected Mice

(52) To Mv4-11 AML cell line-injected mice, MMAE was administered by IV injection for 30 days, followed by observation of PET images. Mv4-11 AML cells in the number of 6.1×10.sup.6 cells/ml were injected subcutaneously in the right thigh of each nude mouse. The tumor size was measured in 3 to 4 weeks after the injection and, when a diameter of the tumor reached 0.8 cm, microPET images were captured before treatment. For PET images, F-18 FDG 0.2 mCi was i.p. injected and then the images were captured (Siemens Inveon). After detection of FDG intake in the tumor from the images before treatment, MMAE was i.v. injected in an experimental group of 5 mice 4 times by 5 day intervals (0.5 mg/kg). After 30 days from the start of treatment, microPET images were captured by the same method as used before treatment. Compared to before MMAE treatment, it was identified from FDG PET images that FDG intake in the tumor was increased even after treatment (FIG. 15 and FIG. 16).

Example 13

(53) In Vitro Efficacy Validation

MTT Assay of MMAE-(PAB-Cit-Val-MC-S-C6)-T.SUB.3.-AS1411, or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).SUB.2.-AS1411 to A549 Cell Lines

(54) With regard to A549 cell line as a lung cancer cell line over-expressing a nucleoline protein, cell inhibition efficacies of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411 were verified in vitro by MTT assay. When comparing cell viability and cell proliferation of the cells through MTT assay of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411, respectively, it was demonstrated that 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411 has superior efficacy over of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411. A549 cells (ATCC, IMDM+10% FBS) were seeded on a 96-well plate in a cell number of 2.5 to 5×10.sup.5 cells/well, which was determined by a cell test method to determine an appropriate cell concentration, followed by growing for 1 day. After heating each of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411 at 95° C. for 5 minutes, the heated product was gradually cooled at room temperature and directly treated on each well at different concentrations. After incubating the treated A549 cells in 5% CO.sub.2 incubator for 72 hours, the incubated product was treated with 20 μL of a reagent solution for MTT assay (Cell Proliferation kit II, Roche) and incubated for different periods of time (10 min, 30 min, 1 hr). Thereafter, the final product was subjected to measurement of absorbance at 490 nm by an ELISA reader (FIG. 17). In FIGS. 17, A, B, C and D are as follows.

(55) TABLE-US-00002 TABLE 1 MMAE-Linker-Aptamer conjugate A MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-CRO B MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411 C 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 D 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411

(56) FIG. 18 illustrates MTT assay results of MMAE-(PAB-Cit-Val-MC-S-C6)-T.sub.3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6).sub.2-AS1411 to the A549 cell lines.

(57) The AS1411-drug conjugate of the present invention can be usefully used as a cancer targeted therapeutic agent.