Adaptor-drug conjugates and uses thereof

Abstract

The present invention relates to a cancer targeting therapeutic agent comprising a drug-linker-AS1411 structure, wherein the drug can be selected from the group consisting of: monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), cytarabine, gemcitabine, maytansine, DM1, DM4, calicheamicin and derivatives thereof, doxorubicin, duocarmycin and derivatives thereof, pyrrolobenzodiazepine (PBD), SN-38, a-amanitin, and tubulysin analogs.

Description

Technical Field

[0001] This invention relates to an anti-nucleolin GRO aptamer toxin conjugate for the treatment of targeted cancers and a method for synthesizing the same, and more specifically, to an anti-nucleolin aptamer toxin conjugate and its use, which has proven in vitro / in vivo efficacy, thereby demonstrating excellent effects in cancer treatment. Background Technology

[0002] To date, numerous therapeutic agents incorporating anticancer drugs have been developed and proposed as drugs through clinical trials. However, how to select and effectively deliver materials with desired therapeutic effects, such as targeted anticancer drugs, to the desired site of onset remains a new area of ​​research. Anticancer drugs are typically used at approximately the maximum tolerated dose to provide clinical efficacy, and such anticancer drugs rapidly kill proliferating cells but cannot distinguish tumor cells or tumor tissue from normal cells. As mentioned above, chemotherapy has drawbacks, including a relatively low therapeutic index and therapeutic window due to non-tumor-specific systemic toxicity and cytotoxicity. Furthermore, because chemotherapy can lead to anticancer drug resistance during long-term treatment, there is an urgent need for new and improved therapies that kill cancer cells by precisely delivering cytotoxic drugs only to cancer cells.

[0003] Over the past 30 years, numerous attempts have been made to effectively deliver drugs to targets and increase efficacy. In this regard, attempts to conduct clinical trials of antibody-drug conjugates (ADCs), as combinations of antibodies and drugs, are considered to have a high probability of success compared to existing naked antibodies. In recent years, Scattle Genetics Inc. and Imunog Co. have successfully completed Phase 3 clinical trials of ADCs, obtained FDA approval, and launched them on the market, thus opening a new chapter for therapeutics. However, antibodies are typically composed of large proteins and present numerous challenges in quality control (QC), particularly regarding drug attachment sites, the amount of attached drug (1 to 6, average 3.5), and attachment positions. The target-directing properties of aptamers, comparable to antibodies, and the development of aptamer-drug conjugates consisting of aptamers and therapeutic agents have gained advantages, where the chemical reactions are readily performed, allowing for the desired tuning of the amount of therapeutic agent and drug attachment sites. Therefore, it is believed that leveraging the advantages of aptamers over antibodies could increase the likelihood of success.

[0004] Aptamer-drug conjugates (ApDCs) are highly toxic, and it is predicted that by attaching target-oriented aptamers to drugs (which is difficult in actual clinical applications) and by accurately delivering the conjugates only to cancer cells, therapeutic efficacy can be maximized without side effects.

[0005] In fact, the evaluation results of conventional ADC (antibody-drug conjugate) technologies developed to date reveal the technical difficulties and drawbacks of combining antibodies with drugs. The use of aptamers makes it easier and more effective to develop aptamer combination therapies, which are then intended for use as targeted anticancer drugs in medical treatment.

[0006] ADC technology (antibody-drug conjugate technology)

[0007] ADC technology focuses on drugs that target cancer cells specifically by maximizing the advantages of antibodies (specificity, non-toxicity in circulation, and pharmacokinetics). ADCs consist of three components: a monoclonal antibody, a drug, and a linker connecting the monoclonal antibody and the drug. ADC technology is a method of delivering drugs to tumor cells using antibodies that specifically bind to specific antigens expressed on the surface of cancer cells.

[0008] Antibody-addictive drugs (ADCs) typically enter cells when an antibody that specifically binds to the target binds to it. Once inside the cell, the ADC dissociates from the target, fuses with other vesicles within the cell, and then proceeds to the subsequent endosome-lysosome pathway. The linker is then cleaved by proteases in the acidic environment of the endosome, and the activated, “free” drug crosses the lysosomal membrane to move into the cytoplasm, where it binds to its molecular target, thereby inhibiting the cell cycle of tumor cells and killing them through apoptosis. The desired amount of drug can be passively diffused, actively transported, or expelled from the cell via dead cells. Here, when the expelled drug can enter surrounding cells during its crossing of the permeable cell membrane, it causes the so-called 'bystander cell killing' phenomenon and side effects on the patient.

[0009] Challenges (QC) in ADC Development

[0010] The development of ADCs (antibody-drug conjugates) began in the 1980s. Besides developing stable linkers for clinical practice in clinical trials, QC difficulties persist during ADC synthesis. In other words, when binding DM1 (drug) to an antibody (huJ591), if a mixture of single antibodies (huj591) conjugated to 1-7 DM1 drugs is obtained, these drugs cannot be isolated / purified. Therefore, ADCs conjugated to an average of 3.5 DM1 drugs are typically used in clinical trials.

[0011] Furthermore, the antibody (Tmab) has 88 lysine residues, to which the drug (DM1) is attached. Identifying which of these 88 lysine residues has a conjugate site for attaching an average of 3.5 units of drug (DM1) is not straightforward. Therefore, to determine the conjugate site, trypsin digestion and Asp-N protease digestion are performed, and the resulting fragments are analyzed by ESI-TOFMS. Comparative analysis of the results usually allows for the determination of the drug (DM1) conjugate site.

[0012] Furthermore, regarding ADC production, QC management is difficult because the average composition of the drug attached to the antibody varies from batch to batch. In other words, in the case of attaching DOTA to the antibody (juJ591), the MALDI-TOF MS spectra showed 5.0 DOTAs attached to the antibody in batch A, and 8.9 DOTAs attached to the antibody in batch B. In other batches, 6.0 and 6.2 DOTAs were attached. As mentioned above, ADC (antibody-drug conjugate) synthesis involves many unresolved issues, such as the difficulty of precise QC during drug assembly due to the properties of the antibody itself. Efforts are currently underway to address these issues in the ADC process.

[0013] Limitations of ADCs (antibody-drug conjugates):

[0014] The therapeutic effect of drugs can be achieved by delivering the drug (with antibodies binding to it in a targeted manner) to cancer cells. However, only 2% or less of the drug binds to the antibody and reaches the cancer cells, resulting in very low efficiency. Because clinically approved stable anticancer drugs such as doxorubicin are stable but have low efficacy, highly toxic agents, 100-1000 times more toxic than doxorubicin, are often used as the antibody-binding agents to address this problem.

[0015] aptamers

[0016] Aptamers are DNA or RNA oligonucleotides that can be used for diagnostics or targeted therapies. They possess antibody-like properties and bind highly selectively to biomarkers that cause diseases such as cancer. In other words, aptamers have unique three-dimensional structures that depend on the target material, and these structures bind very selectively and strongly to marker proteins (cancer-inducing proteins, biomarkers). Therefore, they can be novel biocapture materials capable of enabling targeted therapy, personalized diagnostic therapies, or missile-like therapies.

[0017] Comparison between aptamers and antibodies

[0018] As the most successful targeted therapies, 30 antibody-based targeted therapies have been approved by the FDA and are commercially available, while approximately 300 are in clinical trials. However, development costs are prohibitively high, and the market for these drugs has reached saturation. Therefore, leading pharmaceutical companies are turning to the development of novel therapeutics.

[0019] Antibodies are composed of proteins and still present challenges such as in vivo generation. However, aptamers can be discovered in vitro more quickly than antibodies and can be easily synthesized and modified, thus they are expected to serve as novel biocapture agents, known as "chemical antibodies".

[0020] Development Trends of Aptamer Therapeutic Agents

[0021] Eyetech Inc.'s first original aptamer therapeutic, 'Macugen,' received FDA approval in 2006 and entered the market under license from Pfizer. Macugen is a treatment for age-related macular degeneration (AMD), the cause of presbyopia-induced macular blindness. Macugen demonstrated therapeutic efficacy by inhibiting vascular endothelial growth factor (VEGF), which leads to abnormal blood vessel growth. Subsequently, as the possibilities for aptamer therapeutics increased, other manufacturers such as Merck Serono, Takeda, Pfizer, Elan, Eli Lilly, GlaxoSmithKline, and Ribomic have participated in the development of new aptamer drugs, resulting in approximately 10 aptamers currently in clinical trials.

[0022] Aptamers are often referred to as "chemical antibodies" because they exhibit higher target binding affinity and selectivity compared to antibodies. Furthermore, aptamers are produced through chemical synthesis, making them potentially more efficient in drug attachment and offering the advantage of very simple QC. If aptamers address the antibody problem of low target reach, they are expected to overcome the low efficiency of existing ADCs (antibody-drug conjugates).

[0023] Aptamer-drug conjugates are expected to have potential as a new therapeutic area; however, research using them for therapeutic development is still in its early stages. Recently, Weihong Tang's team at the University of Florida prepared an aptamer-drug conjugate (Sgc8c-Dox conjugate) by combining the anticancer drug doxorubicin (Dox) with an aptamer (Sgc8c), and then compared its anticancer efficacy with that of doxorubicin; however, no significant difference was found. High-purity Sgc8c-Dox conjugates could not be obtained due to the difficulty in separating / purifying the naked aptamer, Sgc8c, during synthesis, and both the Sgc8c-Dox conjugate and doxorubicin exhibited similar cytotoxic effects (20%).

[0024] GRO aptamers

[0025] Professor Paula J. Bate of the University of Louisville first synthesized the GRO (guanine-rich oligonucleotide) aptamer in 1990 and disclosed its mechanism of specific binding to nucleolin protein, which is highly expressed in cancer patients, thus raising the possibility of developing novel anticancer therapeutics. Currently, one of the GRO aptamers is being developed by Antisoma (UK) under the code AS1411, as a therapeutic for renal and non-small cell carcinoma (AML). Furthermore, a Phase II clinical trial for AML has recently been completed.

[0026] Typically, aptamers are very unstable in vivo, and in order to increase nuclease retention or circulation in vivo, aptamer formulations (PEG-aptamer-idT) prepared by PEGylation of idT (inverted dT) at the 3' to 5' positions are used in clinical trials.

[0027] The GRO aptamer possesses a unique G-quadruplex structure, making it highly stable and specifically binding to nucleolin, which is highly expressed in cancer cells. In other words, the GRO aptamer may interfere with the molecular interactions and functions of nucleolin in the nucleus, cytoplasm, and cell membrane, thereby inhibiting nucleolin expression and thus exerting anti-proliferative effects while promoting the expression of p53, a tumor suppressor protein, thereby inducing cancer cell necrosis.

[0028] Currently, AS1411 is a GRO aptamer that has completed Phase II clinical trials by Antisoma (UK) and has initiated Phase III clinical trials for non-small cell carcinoma and renal cell carcinoma. This is a very stable aptamer that specifically binds to nucleolin expressed in almost all cancers, thus exhibiting anti-cancer effects against a variety of cancers.

[0029] Current Research Issues and Prospects: Antisoma (UK) is currently developing AS1411, an anti-nucleolin aptamer drug, which is a GRO DNA aptamer with the sequence ggtggtggtggtggtgtggtggtg. It has completed a Phase II clinical trial as a treatment for renal cell carcinoma and non-small cell lung cancer. However, due to uncertainties regarding efficacy validation, Antisoma (UK) did not conduct a further Phase III clinical trial for non-small cell lung cancer, but instead transferred it to Advanced Cancer Therapeutics Inc. This company is currently preparing a Phase III clinical trial for ACT-GRO-777 (renamed AS1411).

[0030] Oligonucleotide drugs containing GRO aptamers, currently under development and in clinical trials, are rapidly degraded in vivo by nucleases abundant in plasma. In particular, injectable therapeutics are known to degrade even more rapidly without any chemical stabilization treatment. To modulate the degradation rate of therapeutics, oligonucleotides can be chemically modified, or oligonucleotide complexes can be formed with any suitable carrier, thereby reducing their degradation rate. Various chemical modifications, such as replacing the 2'F or 2'OMe group with the 2'-OH group of the ribose to resistant nucleases, and changing the phosphate backbone from PO to PS, have been successfully used in applications such as antisense oligonucleotides or siRNA. Macugen, the first aptamer-based therapeutic agent to receive PDA approval in 2004 and is now commercially available, has also been optimized since the discovery of the aptamer.

[0031] GRO aptamers also require nucleolin-targeting specificity, which is crucial for improving treatment efficacy. Furthermore, off-target effects, such as deterioration in efficacy if the aptamer is combined with different proteins other than nucleolin, should be minimized. The reason Antisoma (UK) struggled to advance to Phase III clinical trials after completing a Phase II trial of AS1411, the first GRO aptamer therapy, for non-small cell lung cancer (AML) is presumably not due to side effects or toxicity, but rather because high doses do not yield optimal anticancer effects. Summary of the Invention

[0032] [Technical Issues]

[0033] Therefore, the inventors synthesized an AS1411-drug conjugate by successfully conjugating a drug with AS1411, and found that the AS1411-drug conjugate has the ability of AS1411 to target nucleolin overexpressed in cancer cells, and is more effective in in vitro and in vivo cancer targeted therapy compared with the use of drugs that are only delivered to and attached to cancer cells.

[0034] [Technical Solution]

[0035] The cancer-targeted therapeutic agent of the present invention has the following drug (R)-linker (L)-AS1411 structure.

[0036] RL-ggtggtggtggttgtggtggtggtgg

[0037] Drug-linker-AS1411 conjugate

[0038] [RL-AS1411]

[0039] The location of the drug linker is particularly preferred to be at positions 12 and 13, or 12 or 13.

[0040]

[0041] In this article, the preferred drug R is methylaurestatin E (MMAE), methylaurestatin F (MMAF), cytarabine, gemcitabine, maytansine, DM1, DM4, cazithromycin and its derivatives, doxorubicin, pyrrolobenzodiazepine (PBD), SN-38, α-ammantin, tubulin lysin analogues, etc.

[0042]

[0043]

[0044]

[0045]

[0046] Where L is composed of X and Y [RYX-AS1411]

[0047] Y can be selected from the group consisting of maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-Val-Cit-PAB), hydrazone, peptide, disulfide, thioether, valine-citrulline, N-maleimide methylcyclohexane-1-carboxylate (MCC), maleimide hexanoyl, mercaptoacetamide hexanoyl, N-succinimide 4-(2-pyridyl dithio)valerate (SPP), SMCC, succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC), N-succinimide 4-(2-pyridyl dithio)valerate (SPDB), phosphodiester bonds, and nucleotides.

[0048] X can be selected from the group consisting of 5'-thiol-modifier C6, thiol-modifier C6S-S, dithiol serine, PC amino-modifier, 5'-amino-modifier C3, 5'-amino-modifier C6, 5'-amino-modifier C12, 5'-amino-modifier TEG, amino-modifier C2dT, amino-modifier C6dT, S-Bz-thiol-modifier C6-dT, phosphodiester bonds, and nucleotides.

[0049] [Beneficial Effects]

[0050] Compared with the use of drugs alone, the AS1411-drug conjugate of the present invention is more effective in targeted cancer therapy in vitro and in vivo. Attached Figure Description

[0051] Figure 1 Gel running data for HS-C6-T3-AS1411 and MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, as well as ESI-MS data for MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, are shown.

[0052] Figure 2 Gel running data for HS-C6-T3-CRO and MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO, as well as ESI-MS data for MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO, are shown.

[0053] Figure 3 The MSI-MS of 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 is shown.

[0054] Figure 4 ESI-MS of 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 is shown.

[0055] Figure 5 The results of MTT assays on the A549 cell line using MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 and MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO are shown.

[0056] Figure 6 The results of MTT assays on the Mv4-11 cell line are shown using citravin-(GLFG-MC-S-C6)-T3-AS1411 and citravin-(GLFG-MC-S-C6)-T3-CRO.

[0057] Figure 7 The image shows an FDG PET image obtained before treatment with A549 lung cancer cell line in mice (tumor with FDG uptake observed in the left thigh region).

[0058] Figure 8 The image shows an FDG PET image obtained 30 days after mice were injected with the A549 lung cancer cell line and treated with MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 (reduced FDG uptake was observed in the tumor).

[0059] Figure 9 The image shows an FDG PET image obtained before treatment with A549 lung cancer cell line in mice (tumor with FDG uptake observed in the left thigh region).

[0060] Figure 10 The image shows an FDG PET image obtained 30 days after A549 lung cancer cell line was injected into mice and treated with MMAE (increased FDG uptake was observed in the tumor).

[0061] Figure 11 The comparison shows the tumor size measured after treatment with MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 and MMAE in the A549 tumor model.

[0062] Figure 12 The size comparison of tumors resected 30 days after treatment with MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 and MMAE, respectively, is shown in the A549 tumor model.

[0063] Figure 13 FDG PET images obtained prior to treatment of mice injected with the Mv4-11AML cell line are shown (tumors with FDG uptake observed in the left thigh region).

[0064] Figure 14 The image shows an FDG PET image obtained 30 days after Mv4-11AML cell line mice were injected with limonene-(GLFG-MC-S-C6)-T3-AS1411 (reduced FDG uptake was observed in the tumor).

[0065] Figure 15 FDG PET images obtained prior to treatment of mice injected with the Mv4-11AML cell line are shown (tumors with FDG uptake observed in the left thigh region).

[0066] Figure 16The image shows an FDG PET image obtained 30 days after Mv4-11AML cell line mice were injected with MMAE (increased FDG uptake was observed in the tumor).

[0067] Figure 17 The viability of A549 cells under each of different concentrations of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 is shown.

[0068] Figure 18 The results of MTT assays for A549 cell lines are shown for MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411. Detailed Implementation

[0069] The present invention will now be described in more detail with reference to the following embodiments.

[0070] [Preferred Embodiment of the Invention]

[0071] [GRO Aptamer] - Synthesis of Drug Conjugates

[0072] Example 1

[0073] Synthesis of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 [AS1411-MMAE conjugate]

[0074] The methyl auratestatin E-p-aminobenzoyloxycarbonyl-citrulline-valine-mamma-benzoyloxycarbonyl-methyl auratestatin E [MC-Val-Cit-PAB-MMAE] was synthesized by reacting maleimide hexanoyl-valine-citrulline-valine-mamma-S-C6-tttggtggtggtggtgtggtggtggtgg[HS-C6-T3-AS1411]. In other words, RSS-C6-tttggtggtggtggtgtggtggtggtgg[RSS-C6-T3-AS1411] was reduced in the presence of DTT for approximately 3 hours. The remaining DTT was removed by centrifugation and replaced with SB17 buffer to generate HS-C6-tttggtggtggtggtggtgtggtggtggtgg[HS-C6-T3-AS1411]. MC-Val-Cit-PAB-MMAE dissolved in a small amount of DMSO was added to the generated product, and the mixture was shaken overnight. Separation / purification was performed by reversed-phase HPLC (Waters-Xbridge OST C18 10X 50mm, 65, TEAE / CAN buffer).

[0075]

[0076] MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 is synthesized by the reaction of MC-Val-Cit-PAB-MMAE and HS-C6-T3-AS1411.

[0077] The synthesis of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 from HS-C6-T3-AS1411 was identified by gel electrophoresis, and the molecular weight of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 was determined by ESI-MS. Figure 1 C 364 H 479 N 120 O 202 P 29 S[Calculated MW = 10697.69, Observed MW = 10697.0]

[0078] Example 2.

[0079] Synthesis of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO

[0080] The methylolactatine E-p-aminobenzoyloxycarbonyl-citrulline-valine-Mal-S-C6-tttcctcctcctcctctcctcctcctcctcc[MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO was synthesized by reacting maleimide hexanoyl-valine-citrulline-valine-Mal-S-C6-tttcctcctcctcctctcctcctcctcc[MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO]. In other words, RSS-C6-tttcctcctcctcctctcctcctcctcc[RSS-C6-T3-CRO] was reduced in the presence of DTT for approximately 3 hours. The remaining DTT was removed by centrifugation and replaced with SB17 buffer to generate HS-C6-tttcctcctcctcctctcctcctcctcc[HS-C6-T3-CRO]. MC-Val-Cit-PAB-MMAE dissolved in a small amount of DMSO was added to the generated product, and the mixture was shaken overnight. Separation / purification was performed by reversed-phase HPLC (Waters-Xbridge OSTC18 10X 50mm, 65, TEAE / CAN buffer).

[0081]

[0082] MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO is synthesized by the reaction of MC-Val-Cit-PAB-MMAE and HS-C6-T3-CRO.

[0083] The synthesis of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO from HS-C6-T3-CRO was identified by gel electrophoresis, and the molecular weight of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO was determined by ESI-MS. Figure 2 C 347 H 479 N 86 O 202 P 29 S[Calculated MW = 10018.13, Observed MW = 10017.28]

[0084] Example 3

[0085] Synthesis of 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 and 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411

[0086] The synthesis of [5-N-(6-(3-methylolactatine-p-aminobenzoyloxycarbonyl-citrulline-valine-Mal-thiopropionyl)-aminohexyl)-3-acrylamide] was achieved by reacting maleimide-hexanoyl-valine-citrulline-valine-p-aminobenzoyloxycarbonyl-methylolactatine E[MC-Val-Cit-PAB-MMAE] with [12,13-(HS-C6)2-AS1411]. ]u[5-N-(6-(3-methyloritamine-p-aminobenzoyloxycarbonyl-citrulline-valine-Mal-thiopropionyl)-aminohexyl)-3-acrylamide]gtggtggtggtgg[12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411]、ggtggtggtggu[5-N-(6-(3-methyloritamine-p-aminobenzoyloxycarbonyl-citrulline-valine-Mal-thiopropionyl)-aminohexyl)-3-acrylamide]ugtggtggtggtgg][12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411]. In other words, ggtggtggtggu[5-N-(6-(3-benzoylthiopropionyl)-aminohexyl)-3-acrylamide]u[5-N-(6-(3-benzoylthiopropionyl)-aminohexyl)-3-acrylamide]gtggtggtggtgg[12,13-(Bz-S-C6)2-AS1411] is reduced in the presence of DTT for about 3 hours. The remaining DTT is removed by centrifugation and replaced with SB17 buffer solution to generate ggtggtggtggu[5-N-(6-(3-thiopropionyl)-aminohexyl)-3-acrylamide]u[5-N-(6-(3-thiopropionyl)-aminohexyl)-3-acrylamide]gtggtggtggtgg[12,13-(HS-C6)2-AS1411]. After adding MC-Val-Cit-PAB-MMAE dissolved in a small amount of DMSO to the generated product, the mixture was shaken overnight. Separation / purification was performed by reversed-phase HPLC (Waters-Xbridge OST C18 10X 50mm, 65, TEAE / CAN buffer) to yield 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 and 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411.

[0087]

[0088] 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 and 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6) were synthesized by reacting MC-Val-Cit-PAB-MMAE with 12,13-(HS-C6)2-AS1411.

[0089] The molecular weights of 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 and 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 were determined by ESI-MS. Figure 3 and Figure 4 ). 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411,C 418 H 568 N 129 O 197 P 25 S2 [Calculated MW = 11390.21, Observed MW = 11390.0]; 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411, C 350 H 463 N 118 O 182 P 25 S2 [Calculated MW = 10073.58, Observed MW = 10073.0]

[0090] Example 4

[0091] Synthesis of limonene-(GLFG-MC-S-C6)-T3-AS1411 conjugate

[0092] Limonene-(Gly-Leu-Leu-Gly)-Mal-S-C6-tttggtggtggtggtgtggtggtggtgg[Limonene-(GLFG-MC-S-C6)-T3-AS1411] was synthesized by reacting maleimide hexanoyl-(Gly-Phe-Leu-Gly)-Mal-S-C6-tttggtggtggtggtgtggtggtggtgg[Limonene-(GLFG-MC-S-C6)-T3-AS1411]. In other words, RSS-C6-tttggtggtggtggtggtgtggtggtgg[RSS-C6-T3-AS141] was reduced in the presence of DTT for approximately 3 hours, the excess DTT was removed by centrifugation and replaced with SB17 buffer solution. After adding Mal-GPLG-limonin dissolved in a small amount of DMSO to the generated product, the mixture was shaken overnight. Limonin-(GLFG-MC-S-C6)-T3-AS1411 was separated / purified by reversed-phase HPLC (Waters-Xbridge OST C18 10X 50mm, 65, TEAE / CAN buffer). The molecular weight of limonin-(GLFG-MC-S-C6)-T3-AS1411 was determined by ESI-MS. 334 H 424 N 117 O 199 P 29 S[Calculated MW = 10191.91, Observed MW = 10190.88]

[0093]

[0094] Limonene-(GLFG-MC-S-C6)-T3-AS1411 is synthesized by the reaction of C-GFLG-limonene and HS-C6-T3-AS1411.

[0095] Example 5

[0096] Synthesis of 12,13-(limonin-GLFG-MC-S-C6)2-AS1411 conjugates and 12 or 13-(limonin-GLFG-MC-S-C6)-AS1411 conjugates

[0097] The [CMC-GFLG-Limonene] was synthesized by reacting maleimide-hexanoyl-(Gly-Phe-Leu-Gly)-limonene [MC-GFLG-Limonene] with ggtggtggtggu[5-N-(6-(3-thiopropionyl)-aminohexyl)-3-acrylamide]u[5-N-(6-(3-thiopropionyl)-aminohexyl)-3-acrylamide]gtggtggtggtgg[12,13-(HS-C6)2-AS1411]. [hexyl)-3-acrylamide]u[Limonene-Gly-Leu-Phe-Gly-Mal-thiopropionyl)-aminohexyl)-3-acrylamide]gtggtggtggtgg[12,13-(Limonene-GLFG-MC-S-C6)2-AS1411] and ggtggtggtggu[Limonene-Gly-Leu-Phe-Gly-Mal-thiopropionyl)-aminohexyl)-3-acrylamide]ugtggtggtggtgg][12 or 13-(Limonene-GLFG-MC-S-C6)-AS1411]. In other words, ggtggtggtggu[5-N-(6-(3-benzoylthiopropionyl)-aminohexyl)-3-acrylamide]u[5-N-(6-(3-benzoylthiopropionyl)-aminohexyl)-3-acrylamide]gtggtggtggtgg[12,13-(Bz-S-C6)2-AS1411] is reduced in the presence of DTT for about 3 hours. The remaining DTT is removed by centrifugation and replaced with SB17 buffer solution to generate ggtggtggtggu[5-N-(6-(3-thiopropionyl)-aminohexyl)-3-acrylamide]u[5-N-(6-(3-thiopropionyl)-aminohexyl)-3-acrylamide]gtggtggtggtgg[12,13-(HS-C6)2-AS1411]. After adding MC-GFLG-limonin dissolved in a small amount of DMSO to the generated product, the mixture was shaken overnight. Purification was performed by HPLC (Waters-Xbridge OST C18 10X 50mm, 65, TEAE / CAN buffer) to yield 12,13-(limonin-GLFG-MC-S-C6)2-AS1411 and 12 or 13(limonin-GLFG-MC-S-C6)-AS1411. The molecular weights of the above products were determined by ESI-MS: 12,13-(limonin-GLFG-MC-S-C6)2-AS1411, C 358 H 458 N 123 O 191 P 25S2 [calculated MW = 10378.66, observed MW = 10379.23]; 12 or 13 (Limonene-GLFG-MC-S-C6)-AS1411, C 320 H 408 N 115 O 179 P 25 S2 [Calculated MW = 9567.81, Observed MW = 9568.09]

[0098]

[0099] 12,13-(limonin-GLFG-MC-S-C6)2-AS1411 and 12 or 13(limonin-GLFG-MC-S-C6)-AS1411 are synthesized by the reaction of MC-GFLG-limonin and 12,13-(HS-C6)2-AS1411.

[0100] Example 6

[0101] In vitro efficacy verification

[0102] MTT assay of A549 cell line using MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 and MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO

[0103] For the A549 cell line, a lung cancer cell line overexpressing nucleolin protein, the cell-inhibitory efficacy of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 was verified in vitro by MTT assay. When cell viability and proliferation were compared using MTT assays of MMAE, AS1411, MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, and MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO, MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 demonstrated that it had almost the same efficacy as MMAE. A549 cells (ATCC, IMDM + 10% FBS) were cultured at (2.5-5) × 10⁻⁶ cells / cells. 5Cells were seeded in 96-well plates at a cell proliferation assay to determine the appropriate cell concentration, and then allowed to grow for 1 day. MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 and MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO were each heated at 95°C for 5 minutes, and the heated products were gradually cooled to room temperature and directly treated in each well at different concentrations. After incubating the treated A549 cells in a 5% CO2 incubator for 72 hours, the incubated products were treated with 20 μL of MTT assay (Cell Proliferation Kit II, Roche) reagent solution for different time periods (10 minutes, 30 minutes, 1 hour). The absorbance of the final product at 490 nm was then measured using an ELISA reader. Figure 5 ).

[0104] Example 7

[0105] MTT assay of Mv4-11 cell line by limonene-(GLFG-MC-S-C6)-T3-AS1411 and limonene-(GLFG-MC-S-C6)-T3-CRO

[0106] For the Mv4-11 cell line, a lung cancer cell line overexpressing nucleolin protein, the cell-inhibiting efficacy of limonin-(GLFG-MC-S-C6)-T3-AS1411 was verified in vitro by MTT assay. When cell viability and proliferation were compared with those of limonin, AS1411, limonin-(GLFG-MC-S-C6)-T3-AS1411, and limonin-(GLFG-MC-S-C6)-T3-CRO by MTT assay, the limonin-(GLFG-MC-S-C6)-T3-AS1411 conjugate demonstrated that it had almost the same efficacy as limonin. Mv4-11 cells (ATCC, IMDM + 10% FBS) were cultured at (2.5-5) × 10⁻⁶ cells / cells. 5Cells were seeded in 96-well plates at a cell proliferation assay to determine the appropriate cell concentration; the cells were then allowed to grow for 1 day. Limonene-(GLFG-MC-S-C6)-T3-AS1411 and Limonene-(GLFG-MC-S-C6)-T3-CRO were each heated at 95°C for 5 minutes, and the heated products were gradually cooled to room temperature and directly treated in each well at different concentrations. After incubating the treated A549 cells in a 5% CO2 incubator for 72 hours, the incubated products were treated with 20 μL of MTT assay (Cell Proliferation Kit II, Roche) reagent solution for different time periods (10 minutes, 30 minutes, 1 hour). The absorbance of the final product at 490 nm was then measured using an ELISA reader. Figure 6 ).

[0107] Example 8

[0108] In vivo efficacy verification

[0109] In vivo efficacy validation of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 injected into mice with A549 lung cancer cell line

[0110] For mice injected with the A549 lung cancer cell line, MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 and MMAE were administered intravenously for 30 days, respectively, followed by PET imaging to identify tumor size. Tumors with a cell count of 6.1 × 10⁻⁶ were used. 6 A549 lung cancer cells / ml were subcutaneously injected into the right thigh of each nude mouse. Tumor size was measured 3 to 4 weeks post-injection, and microPET images were captured before treatment when the tumor diameter reached 0.8 cm. For PET images, 0.2 mCi of F-18 FDG was injected intraperitoneally, followed by image capture (Siemens Inveon). After FDG uptake in the tumor was detected from the images, MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 (7.5 mg / kg, 0.5 mg / kg MMAE) was administered intravenously four times at 5-day intervals in the experimental group of 5 mice. MicroPET images were captured using the same method as before treatment 30 days after the start of treatment. A significant decrease in FDG uptake in the tumor was identified from PET images after 30 days of intravenous administration of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 to mice injected with the A549 lung cancer cell line. Figure 7 and Figure 8 ).

[0111] Example 9

[0112] In vivo efficacy validation of MMAE in mice injected with A549 lung cancer cell line

[0113] Mice injected with A549 lung cancer cells were given MMAE intravenously for 30 days, followed by PET image observation. The cell count was 6.1 × 10⁶. 6 A549 lung cancer cells / ml were subcutaneously injected into the right thigh of each nude mouse. Tumor size was measured 3–4 weeks post-injection, and microPET images were captured before treatment when the tumor diameter reached 0.8 cm. For PET images, 0.2 mCi of F-18 FDG was injected intraperitoneally, followed by image capture (Siemens Inveon). After FDG uptake in the tumor was detected from the pre-treatment images, MMAE (0.5 mg / kg) was administered intravenously four times at 5-day intervals in the experimental group of five mice. MicroPET images were captured 30 days after the start of treatment using the same method as before treatment. Compared to pre-MMAE treatment, FDG uptake in the tumor was even increased even after treatment, as identified from the FDG PET images. Figure 9 and Figure 10 ).

[0114] Example 10

[0115] In vitro verification

[0116] Compared with MMAE alone, administration of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 to mice injected with the A549 lung cancer cell line demonstrated superior cancer-suppressive efficacy. MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 inhibited more than 80% of cancer compared with MMAE alone. Tumor size was measured on both the x and y axes at 5-day intervals after treatment initiation. Tumors were excised from different groups 30 days post-treatment. As shown in the figures below, tumor size was photographed for each group, and comparisons were made between MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 and MMAE. Figure 11 and Figure 12 ).

[0117] As a result of validating the in vivo efficacy of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 and MMAE on the A549 cell line, MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 was demonstrated to be superior to MMAE alone.

[0118] Example 11

[0119] In vivo efficacy verification

[0120] In vivo efficacy validation of limonene-(GLFG-MC-S-C6)-T3-AS1411 in mice with Mv4-11AML cell line

[0121] Mv4-11AML cell line mice were injected with limonene-(GLFG-MC-S-C6)-T3-AS1411 and MMAE intravenously for 30 days, respectively, and tumor size was then identified by PET images. The tumors were 6.1 × 10⁻⁶. 6 Mv4-11AML cells / ml were subcutaneously injected into the right thigh of each nude mouse. Tumor size was measured 3–4 weeks post-injection, and microPET images were captured before treatment when the tumor diameter reached 0.8 cm. For PET images, 0.2 mCi of F-18FDG was injected intraperitoneally, followed by image capture (Siemens Inveon). After FDG uptake in the tumor was detected from the images, limonene-(GLFG-MC-S-C6)-T3-AS1411 (7.5 mg / kg, 0.5 mg / kg MMAE) was administered intravenously four times at 5-day intervals in the experimental group of 5 mice. MicroPET images were captured 30 days after the start of treatment using the same method as before treatment ( Figure 13 and Figure 14 ).

[0122] Example 12

[0123] In vivo efficacy validation of MMAE in mice injected with Mv4-11AML cell line

[0124] Mv4-11AML cell line mice were injected with MMAE intravenously for 30 days, followed by PET image observation. The cell line consisted of 6.1 × 10⁻⁶ cells. 6 Mv4-11AML cells / ml were subcutaneously injected into the right thigh of each nude mouse. Tumor size was measured 3–4 weeks post-injection, and microPET images were captured before treatment when the tumor diameter reached 0.8 cm. For PET images, 0.2 mCi of F-18FDG was injected intraperitoneally, followed by image capture (Siemens Inveon). After FDG uptake in the tumor was detected from pre-treatment images, MMAE (0.5 mg / kg) was administered intravenously four times at 5-day intervals in the experimental group of five mice. MicroPET images were captured 30 days after the start of treatment using the same method as pre-treatment. Compared to pre-MMAE treatment, FDG uptake in the tumor was even increased even after treatment, as identified from FDG PET images. Figure 15 and Figure 16 ).

[0125] Example 13

[0126] In vitro efficacy verification

[0127] MTT assay of A549 cell line using MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411

[0128] For the A549 cell line, a lung cancer cell line overexpressing nucleolin protein, the cell inhibitory efficacy of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 was verified in vitro by MTT assay. When cell viability and proliferation were compared using MTT assays of MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411, and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411, the superior efficacy of 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 over MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411 was demonstrated. A549 cells (ATCC, IMDM + 10% FBS) were cultured at (2.5–5) × 10⁻⁶ cells / cells. 5Cells were seeded in 96-well plates at a cell proliferation assay to determine the appropriate cell concentration; the cells were then allowed to grow for 1 day. MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411, and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 were each heated at 95°C for 5 minutes, and the heated products were gradually cooled to room temperature and directly treated in each well at different concentrations. After incubating the treated A549 cells in a 5% CO2 incubator for 72 hours, the incubated products were treated with 20 μL of MTT assay (Cell Proliferation Kit II, Roche) reagent solution for different time periods (10 minutes, 30 minutes, 1 hour). Subsequently, the absorbance of the final product at 490 nm was measured using an ELISA reader. Figure 17 ).exist Figure 17 In the given list, A, B, C, and D are as follows.

[0129] [Table 1]

[0130] A <![CDATA[MMAE-(PAB-Cit-Val-MC-S-C6)-T3-CRO]]> B <![CDATA[MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411]]> C 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411 D <![CDATA[12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411]]>

[0131] Figure 18 The results of MTT assays for A549 cell lines using MMAE-(PAB-Cit-Val-MC-S-C6)-T3-AS1411, 12 or 13-(MMAE-PAB-Cit-Val-MC-S-C6)-AS1411, and 12,13-(MMAE-PAB-Cit-Val-MC-S-C6)2-AS1411 are shown.

[0132] [Industrial Applicability]

[0133] The AS1411-drug conjugate of the present invention can be effectively used as a targeted cancer therapy agent.

Claims

1. A cancer-targeted therapeutic agent comprising a drug-linker-AS1411 structure, The drug mentioned is methylaurestatin E (MMAE). The linker is composed of XY, where Y is maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-Val-Cit-PAB), and X is a 5'-thiol-modifying agent C6, wherein X is bound to AS1411 and Y is bound to the drug. The connector is connected at both positions 12 and 13 of AS1411. The AS1411 has a sequence in which the portion of the amino acid sequence SEQ ID NO.1 that is connected to the linker described above is replaced with uracil.

Images