WO2021234550A1 - 신규 뉴클레오린-결합 펩타이드 및 이의 용도 - Google Patents
신규 뉴클레오린-결합 펩타이드 및 이의 용도 Download PDFInfo
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- WO2021234550A1 WO2021234550A1 PCT/IB2021/054235 IB2021054235W WO2021234550A1 WO 2021234550 A1 WO2021234550 A1 WO 2021234550A1 IB 2021054235 W IB2021054235 W IB 2021054235W WO 2021234550 A1 WO2021234550 A1 WO 2021234550A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Definitions
- novel nucleolin-binding peptides and uses thereof Technical Field
- the present invention relates to novel peptides that specifically bind to nucleolins, and more particularly, to A peptide represented by an amino acid sequence and a conjugate comprising the peptide and an anticancer agent, a fusion peptide comprising the peptide and a cell-penetrating peptide, a composition for diagnosing cancer comprising the peptide, and a composition for preventing or treating cancer comprising the peptide or conjugate is about BACKGROUND ART
- cancer-specific ligands as pharmaceutical carriers enables tissue or cell specific delivery of chemotherapeutic agents relative to that achieved by conventional drugs, thereby reducing systemic toxicity (Allen TM.
- Nanoparticles and antibodies have been widely studied in clinical cancer diagnosis and treatment (Yao VJ, et al. J Control Release. 2016; 240: 267-8Q. Detection of cancer in individual patients at an early stage) Nanoparticles and antibodies that can simultaneously diagnose and treat (theragnostic) nanoparticles and antibodies have great potential in the field of personalized medicine because they can deliver anticancer drugs over a long period of time to monitor and enhance the therapeutic effect. 2021/234550 - 01/162021/054235 (Palmieri D, et al. Proc Natl Acad Sci US A. 2015; 112: 9418-23).
- Nanoparticles are promising drug carrier systems, but present limitations in practical applications due to circulatory instability, inadequate tissue distribution and cytotoxicity (Sukhanova A, et al. Nanoscale Res Lett. 2018; 13:4 sh.
- therapeutic antibodies are
- it has limitations in slow delivery and spread to tumor tissue due to its large size (Epenetos AA, et al. Cancer Res. 1986; 46: 3183-91).
- cancer-specific peptides can be used (Mori T. Curr Pharm Des. 2004; 10: 2335-43 ⁇ 4.
- the hottide ligand is easy to mass synthesize, low immunogenicity, non-toxic.
- paclitaxel (PTX)-conjugated AGM improved PTX accumulation in cancer cells, and inhibited breast cancer and colorectal cancer cells more effectively than PTX alone treatment at in i//minute D.
- PTX-conjugated AGM treatment promoted the specific localization of PTX in tumor tissues, thereby increasing cytotoxicity in a xenograft model of breast cancer, and enhancing drug accumulation and anticancer efficacy.
- nucleolin (NCL) was a target protein of AGM through pull-down analysis and LC-MS/MS. NCL is the most abundant protein in phosphorus (nucleolus).
- nucleolin It is to provide a peptide that specifically binds to 1 ⁇ 1 (the 1_). Another object of the present invention is to provide a conjugate comprising the peptide and an anticancer agent. Another object of the present invention is to provide two or more multimers (1111
- Another object of the present invention is the peptide and the cell-penetrating peptide (Cell Penetrating Peptide; CPP)-conjugated fusion hypotide and the multimer and cell permeability It is to provide a fusion peptide to which the 2021/234550 -01/162021/054235 peptide is bound.
- CPP Cell Penetrating Peptide
- Another object of the present invention is to provide a composition for diagnosing cancer comprising the peptide or multimer, a method for diagnosing cancer, use of the peptide or multimer for diagnosing cancer, and the use of the peptide or multimer for manufacturing a medicament for diagnosing cancer is to do
- Another object of the present invention is the composition for preventing or treating cancer comprising the peptide, the conjugate or the multimer, the method for preventing or treating cancer, the use of the peptide, the conjugate or the multimer for preventing or treating cancer, and the prevention or treatment of cancer
- the present invention is to provide for the use of said peptide, conjugate or multimer for the manufacture of a medicament for treatment.
- NCL nucleolin
- the present invention also provides a conjugate comprising the peptide and an anticancer agent.
- the present invention also provides a multimer (multimer) comprising the peptide.
- the present invention also provides a conjugate comprising the multimer and an anticancer agent.
- the present invention also provides a fusion peptide in which the Hyt!tide and Cell Penetrating Peptide (CPP) are combined.
- the present invention also provides a fusion peptide in which the multimer and the cell-penetrating peptide are combined.
- the present invention also provides a composition for diagnosing cancer comprising the peptide or multimer, a method for diagnosing cancer, use of the peptide or multimer for diagnosing cancer, and the use of the peptide or multimer for manufacturing a medicament for diagnosing cancer.
- the present invention also provides a composition for preventing or treating cancer comprising the peptide, the conjugate or multimer, a method for preventing or treating cancer, the use of the peptide, the conjugate or multimer for preventing or treating cancer, and preventing or treating cancer
- a composition for preventing or treating cancer comprising the peptide, the conjugate or multimer, a method for preventing or treating cancer, the use of the peptide, the conjugate or multimer for preventing or treating cancer, and preventing or treating cancer
- the use of said peptide, conjugate or multimer for the manufacture of a medicament is provided.
- 1 is a view of 0! ⁇ /1-330 and de ( trivalent conjugated body ((:011 '119 6) according to the present invention) 2021/234550 - 01/162021/054235 It is a diagram showing the structure of a monomer (AGM-330m), a dimer (AGM-330d), and a tetramer (AGM-330t).
- 2 is a diagram showing the structure of an AGM-330-CPP fusion haftide in which the AGM-330-PEG conjugate and Cell Penetrating Peptide (CPP) are combined according to the present invention.
- Figure 3 is a schematic diagram illustrating the steps of peptide library synthesis and screening: 1, a combination 0B0C library is constructed by performing a split-mix synthesis method; 2, approximately ⁇ 2, 600, 000 library synthesis; 3, incubating the 0B0C library with cancer cells in a C0 2 incubator; 4, beads carrying ligands with affinity for cell surface molecules are covered with cells; 5, retake the positive beads with a pipette under an inverted microscope; 6, the sequence of the positive bead was measured by Edman microsequencing; 7, Cancer-specific peptide ligand candidates identified from 0B0C combinatorial peptide library screening. 4 shows the screening and MAP synthesis of the 0B0C library for cancer-specific ligands.
- Figure 4A shows the binding specificity of the peptides for cancer cell lines and normal cells
- Figure 4B is the result of a whole-cell binding assay showing the cell binding specificity of 8 selected beads, a number of breast cancer and colorectal cancer cells and normal breast and Colorectal cells were resuspended at 10 6 cells/ml and incubated with beads. All experiments were repeated three times, and the scale bar was 200Mm.
- Figure 4C shows the binding specificity determined by confocal imaging of immunofluorescence of FITC-labeled AGM-330, AGM-331 and AGM-332, the nucleus was stained with DAP (blue), and the scale bar was 5 () Mm. am.
- Figure 4D shows AGM- 2021/234550 ?01/162021/054235
- FIG. 4E shows the synthesis procedure of AGM-330, the reagents and conditions are as follows: (i) Fmoc-Lys- (Fmoc) -OH, piperidine, DMF; (ii) Fmoc-Lys-(Fmoc)-OH, piperidine, DMF; (iii) RHGAMVYLK-OH, piperidine; (iv) piperidine, DMF.
- 4F is a FACS analysis result showing the specificity of AGM-330 against cancer cells among a large number of breast and colorectal cancer cells and normal breast and colorectal cells.
- the bar graph represents the mean ⁇ SD, and one-way ANOVA with Dunnett's multiple. Statistical analysis was performed by comparison. *, ** and *** indicate p ⁇ 0.05, P ⁇ 0.01 and / 7 ⁇ 0.001, respectively.
- 5 is a result of in vivo stability evaluation of peptides by AGM-330 (AGM-330m, AGM-330d, AGM-330t).
- the figure shows the results of Coomassie brilliant blue staining of the protein eluted from the affinity column after separation by 10% SDS-PAGE, Lane 1: protein marker; Lane 2: total lysate before biotin pull-down assay; Lane 3: flow-through after incubation with AGM-330-biotin; Lane 4-6: bead washing fraction; Lane 7-8: elution of AGM-330 binding proteins.
- Red asterisk indicates that LC-MS/MS analysis was performed after the protein band was cleaved by in-gel digestion.
- 7C is a result of confirming NCL as an AGM-330 binding partner by searching a protein database of peptides detected by MS.
- FIG. 7D is a list of genes obtained from LC-MS/MS analysis, and the confidence score represents the mascot score of the identified protein.
- Fig. is an immunoblotting analysis result of proteins eluted from biotin pull-down using an anti-NCL antibody, and arrows indicate the presence of NCL.
- Figure 7F shows the analysis results of the NCL domain binding to AGM-330.
- AGM-330 (20nM-2.5MM) were incubated with immobilized NCL and analyzed on a Biacore T-200 instrument.
- 9 is a direct interaction analysis result between AGM-330 and purified NCL by biotin pull-down analysis. Immunoblotting analysis of the eluted protein forms biotin pull-down using an anti-NCL antibody. Lane 1: protein marker; Lane 2: input of purified NCL; Lane 3: flow through after incubated with AGM-330-biotin; Lane 4- 6: bead washing fraction; Lane 7-8: elution of AGM-330 binding proteins. Arrows indicate the presence of NCL. 10 is an affinity test for AGM-330 and other JSA variants.
- Cancer cells (1 x 10 4 cells s/well) were seeded on 96-well plates and cultured for 24 hours, and then the cells were treated with 13 peptides linked to increasing concentrations of biotin for 2 hours. Then, avidin-HRP was attached through high affinity avidin-biotin interaction, and the TMB substrate was converted to blue color through HRP enzyme. Then, the reaction was terminated by adding acid, and the wells were read at 450 mm, and the affinity of 13 species was evaluated by EUSA. 11 shows the binding specificity of in vitro and in i//i/o AGM-330. 2021/234550 -01/162021/054235 FIGS.
- 11A and 11B are fluorescence confocal microscopy results of multiple cancer and normal cells, MCF-10A, MCF-7, MDA-MB-231 (FIG. 11A) or CCD -18Co, HT-29, HCT-116 (Fig. 11 B) cells were incubated with 5jjmol/l AGM-330-FITC for 30 min at 37 °C, NCL was stained with primary anti-NCL antibody, and AGM The binding of -330 to NCL was revealed using an anti-mouse secondary antibody linked to Alexa Fluor 594, cells were fixed with 4% paraformaldehyde, and nuclei were stained with DAPI.
- FIG. 11C is the result of analyzing the distribution of NCL in various subcellular fractions in MCF-10A, MCF-7 and MDA-MB-231 cells. Plasma membrane, cytosol, and nuclear NCL were immunoblotted using an anti-NCL antibody. was rafted. 11D shows that AGM-330 binding to the cell membrane is inhibited by knockdown of NCL using siRNA.
- AGM-330-FITC is FITC-conjugated AGM-330, and the scale bar is 20jjm.
- Figure 11 E is the result of analyzing the distribution of NCL in various subcellular fractions in MCF-10A, MCF-7 and MDA-MB-231 cells after siNCLI treatment. was used for immunoblotting.
- 11 F is a schematic diagram of the experimental protocol, inoculated with 1:1 mix of Matrigel and 1 x 10 6 MDA-MB-231 -luc cells into the mammary gland fat pad of anesthetized 6-week-old male NPGä mice, and inoculated with tumor cells. Then, when the tumor volume reached approximately 100 mm 3 , free Alexa680 or AGM-330-Alexa680 was injected into the tail vein.
- 11 H is 30 minutes and 1 hour after intravenous injection of 10 nmol free Alexa680 (FIG. 11G) or 10 nmol AGM-330-Alexa680 (FIG. 11H) in MDA-MB-231-luc tumor-bearing mice,
- 111 and 11J are graphs showing the mean radiative efficiency of Alexa680-associated fluorescence from live imaging of three animals in tumors (FIG. 111) and other organs (FIG. 11), the bar graph representing the mean ⁇ SD and one Statistical analysis was performed by -way ANOVA with Dunnett's multiple comparison. *, ** and *** indicate P ⁇ 0.05, P ⁇ 0.01 and / 7 ⁇ 0.001, respectively.
- MDA-MB-231 cells were transfected with non-target scrambled siRNA (Scr) or NCL target siNCLI, siNCL2 or siNCL3, 24 hours later, gene silencing efficiency was determined by real-time PCR. Based on mRNA levels, siNCl_1 was chosen for further experiments as it exhibited remarkable efficiency in target gene knockdown.
- the bar graph represents the mean ⁇ SD, and statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison. *, ** and *** indicate P ⁇ 0.05, P ⁇ 0.01 and / 7 ⁇ 0.001, respectively.
- 13 shows the expression profile and functional study results of NCL in breast cancer and colorectal cancer.
- 13A shows oncogenic breast carcinoma observed in the Curtis dataset and the Alon dataset. 2021/234550 ?01/162021/054235 or with colorectal adenocarcinoma patients As a significant correlation between the two, the Oncomine dataset repository (www.oncomine.org) was used.
- 13B is a Kaplan-Meier survival analysis performed in breast and colorectal cancer patients based on NCL expression in two independent cohorts (Bertucci and Sveen).
- 13C and 13D show immunohistochemistry for NCL in normal tissue (FIG. 13C) and cancer tissue (FIG.
- 13E-FIG. 13H show the cell viability of cancer cells and normal cells after treatment with the indicated amounts of anti-NCL antibody or IgG in serum-free medium for 24 hours, MCF-10A (FIG. 13E), MDA- Cell viability of MB-231 (FIG. 13F), CCD-18Co (FIG. 13G) and HCT-116 (FIG. 1H) was measured by WST assay, and apoptotic cells were visualized by staining Annexin V + cells. became Cells were neutralized with anti-NCL antibody and cultured in serum-free medium for 24 hours, and apoptotic cells were determined by FACS analysis (FIG. 131).
- the bar graph represents the mean ⁇ SD rule, and statistical analysis was performed by one-way AN OVA with Dunnett's multiple comparison. *, ** and *** indicate P ⁇ 0.05, P ⁇ 0.01 and / 7 ⁇ 0.001, respectively.
- 14 shows the in vivo tumor suppression efficacy of AGM-330t-PTX.
- Figure HA shows the results of inhibition of proliferation by AGM-330t-PTX treatment, determined by WST assay for 48 hours in a number of breast cancer and colorectal cancer cells and normal cells.
- FIG. 14E show the effects of AGM-330t-PTX treatment evaluated in a breast cancer xenograft model
- FIG. 14C shows the primary tumor volume measured twice a week until the day of sacrifice, buoy
- (mm 3 ) (length (mm)) x (width (mm)) 2 X 0.5 was calculated by the formula.
- 14D shows that all primary tumors were isolated on the day of sacrifice, and FIG. 14E shows the results of evaluation of primary tumor weight.
- 14F-FIG. 141 shows immunohistochemical sections of representative tumors using Ki-67 (FIG. 14F, FIG. 14H) and TUNTA_ (FIG. 14G, FIG.
- ALB albumin
- GOT glutamic oxaloacetic transaminase
- GTP glutamate pyruvate transaminase
- TP-PS total protein/protein scan
- UA uric acid
- 15F shows body weight of xenograft mice after 3 weeks of different treatments. It shows the change of 2021/234550 ?01/162021/054235.
- FIG. 16 is a result of confirming the anticancer effect using AGM-330d-PTX, and FIG. 16A shows the structure of PTX-conjugated AGM-330d.
- 16C and 16D are the results of tumor growth monitoring through whole-body bioluminescence imaging. Growing MDA-MB-231 cells expressing firefly luciferase were injected into the mammary gland fat pad of mice.
- FIG. 16C The therapeutic effect of AGM-330d-PTX was evaluated by bioluminescence imaging ( FIG. 16C ), and bioluminescence intensity was shown every 5 days in 5 different mouse groups ( FIG. 16D ).
- 16E-FIG. 16G are the results of evaluating the therapeutic effect of AGM-330d-PTX treatment in a breast cancer xenograft model. On the day of sacrifice, all primary tumors were isolated and their volume was evaluated (FIG. 16E-FIG. 16F), the primary tumor weight was measured twice per week until the day of sacrifice ( FIG. 16G ). Primary tumor volume was calculated with the following formula: buoy
- (mm 3 ) (length (mm)) x (width (mm)) 2 X 0.5.
- 16H is a graph showing body weight changes after indicated 3 weeks of different treatments in xenograft mice. Bar graphs represent mean ⁇ SD, statistical analysis was performed by one-way ANOVA wi ⁇ 1 Dunnett's multiple comparison, *, ** and *** are each 2021/234550 ?01/162021/054235
- 17 is a view showing the synthesis process of the AGM-330t-mCPP fusion peptide.
- 18 shows the results of HPLC and MS analysis of AGM-330t-mCPP fusion peptide and AGM-330t and maleimide-CPP.
- 19 is a graph showing the cytotoxic effect of AGM-330t, mCPP and AGM-330t-mCPP fusion peptides.
- Figure 20 shows the apoptosis effect of AGM-330t, mCPP and AGM-330t-mCPP fusion peptide, apoptotic cells were measured by FACS analysis.
- the bar graph represents the mean ⁇ SD, and statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison. *** indicates P ⁇ 0.001, NS means Not Significant.
- 21 is a diagram showing the apoptosis-inducing effect according to the concentration of the AGM-330t-mCPP fusion peptide. 22 is a graph showing the cytotoxic effect of each concentration of AGM-330m-mCPR AGM-330d-dCPP and AGM-330t-tCPP fusion peptides.
- a library of 2,600,000 peptides was synthesized using a one-bead-one-compound (OBOC) combination method together with a multiple-antigen-peptide (MAP) synthesis method. Then, therefrom, the new cancer-specific peptide ligands AGM-330, AGM-331, AGM-332, AGM-333, AGM-334, AGM-335, AGM-336, AGM- 337 was identified (see FIG. 3 ) and in vitro LHC//? It was confirmed that the peptide ligands specifically bind to cancer cells in vitro) and in vivo ( ⁇ 1//1/1/ ⁇ ).
- nucleolin is a target protein of AGM-330, AGM-331, AGM-332, AGM-333, AGM-334, AGM-335, AGM-336, AGM-337, and paclitaxel (PTX)
- PTX paclitaxel
- the amino acid mutation is in the amino acid sequence of SEQ ID NO: 1,
- the amino acid sequence containing the mutation may be characterized in that it is selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 15 and SEQ ID NO: 20, but is not limited thereto (Table 2).
- peptide or "AGM peptide ligand” refers to a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20, or refers to one or more peptides among them.
- 2021/234550 ?01/162021/054235 In the present invention, The id may be characterized in that the nucleolin specifically binds to (1).
- the 61 ⁇ /1 peptide specifically 61 ⁇ /1-330, binds specifically to cancer cells, but weakly or not at all to normal breast and colorectal cells.
- the peptide is cancer-specific and makes it a major candidate for the development of cancer-targeting peptides.
- 61 ⁇ /1 peptide treatment was more effective in tumors than in the heart, spleen, lung or brain. 2021/234550 ?01/162021/054235 showed more accumulation.
- the present invention with the AGM peptide - [CH 2 -CH 2 -0]-°
- PEG polyethylene glycol
- the polyethylene glycol chain is used as a concept including not only the general polyethylene glycol chain having the structure of Formula 1 but also derivatives thereof, and as the derivative of the polyethylene glycol chain, an amino having the structure of Formula 2
- Polyethylene glycol carboxylic acid amino polyethylene glycol carboxylic acid
- the polyethylene glycol chain may be characterized as having 2 to 24 ethylene glycol units, preferably 4 to 20 units, more preferably 6 to 18 units, and most preferably 6 to 12 units. It may be characterized as having two ethylene glycol units, but is not limited thereto. the polyethylene It may be characterized in that it is connected through a linker with the id.
- the 61 ⁇ /1 peptide-de conjugate may have the form of the following Structural Formula 1, but is not limited thereto. 6! ⁇ /1 -Nee-de (Structural Formula 1) above Structural Formula refers to a polyethylene glycol chain, and knee refers to a linker.
- the linker preferably a single bond nieun (direct bond), -. ⁇ alkyl, - ⁇ 0 2021/234550 ?01/162021/054235 alkylene, and -0 - may be selected from the group consisting of, but not limited thereto,
- -[(-) with the carboxy group of the amino acid at the (:-terminal) may include, but are not limited to.
- a drug a functional group for conjugating Nii ( ⁇ 11 ⁇ :1) : 1 is 131 no
- the functional group include, but are not limited to, carboxyl 00 ratio, amine (1 ⁇ 11 ) and thiol ( ⁇ 1) groups.
- 61 ⁇ /1-300 and ode 61 ⁇ /1-330 which is a conjugate of 3 - 61 ⁇ /1 of a conjugate of 61 ⁇ /1 in 61 ⁇ /1 peptides
- 11) with lysine and cysteine introduced at the end may have the same form as Structural Formula 5.
- the drug is an agent exhibiting a pharmacological effect, specifically, a chemotherapeutic agent, a toxin, micro or a radioactive isotope.
- the chemotherapeutic agent may be, for example, a cytotoxic agent or an immunosuppressive agent. specifically microtubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors, or chemotherapeutic agents that can function as deintercalators. 2021/234550 ?01/162021/054235 there. It may also include an immunomodulatory compound, an anticancer agent and an antiviral agent, or a combination thereof.
- the drug may be an anti-cancer agent
- the anti-cancer agent is a taxol such as paclitaxel or docetaxel, and a taxane, maytansinoid, auristatin, aminopterin, Actinomycin, bleomycin, thalisomycin, camptothecin, N8-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethyl sulfonyl hydrazide, esperamicin, etoposide, 6- Mercaptopurine, dolastatin, tricotecen, calicheamicin, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase ( L-asparaginase), mercaptopurine, thioguanine, hydroxyurea, cytar
- 62 ( ⁇ 3
- 6 -330 - in a human breast cancer xenograft mouse model, 6 -330 - ) ( 61//1-330 - when treated with a de-conjugate and paclitaxel (conjugate with a ligament) ) (Compared to -treated group) showed a 60% and 46% reduction in the volume and weight of the tumor (see Fig. 14 [ to Fig. 14 d ].
- the efficient targeting ability of 6-330 to cancer cells and tissues provides a promising method in the delivery of anticancer agents for cancer treatment. do.
- Paclitaxel [ ⁇ 3X61; [Ji] is considered one of the most promising cancer chemotherapy drugs, and has been tested against many different human malignancies.
- the 61 ⁇ /1 ht!tide or 61 ⁇ /1 httide-de conjugate may be characterized in that it is linked to a drug, preferably an anticancer agent, via a linker (knee) or directly linked. .
- the linker (knee) may be cleavable.
- the cleavable linker allows the drug to be released from the antibody in a form cleavable under intracellular conditions, that is, through cleavage of the linker in the intracellular environment.
- the linker may be cleaved by a cleaving agent present in the intracellular environment, for example, lysosomes or endosomes, and may be cleaved by, for example, intracellular peptidase or protease enzymes such as lysosomal or endosomal proteases. It may be a peptide linker. In general, the peptide linker has a length of at least two or more amino acids.
- the cleaving agent may include cathepsin 6 and cathepsin da plasmin, and hydrolyze the peptide to release the drug into the target cell.
- the peptide linker can be cleaved by the thiol-dependent protease cathepsin-8, which is overexpressed in cancer tissues, for example, ⁇ -1_ae or ( ⁇ /-geun-1_ae-this linker can be used) .
- the peptide linker is, for example, cleavable by an intracellular protease, and is a ⁇ 3 ⁇ -[ linker or 2021/234550 ?01/162021/054235 there.
- the cleavable linker is ⁇ ) ⁇ sensitive, which may be sensitive to hydrolysis at certain ⁇ ) ⁇ values.
- pH sensitive linkers can be hydrolyzed under acidic conditions.
- acid labile linkers that can be hydrolysed in lysosomes, such as hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide ((-3(:i1 [3171 6), orthoesters, acetals, ketals, and the like.
- the linker may be cleaved under reducing conditions, for example Various disulfide bonds can be formed using pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha - (2-pyridyl-dithio)toluene).
- the linker may be, for example, a non-cleavable linker, and the drug is released through only one step of antibody hydrolysis, for example, to produce an amino acid-linker-drug conjugate.
- This type of linker may be a thioether group or a maleimidocaproyl group ⁇ 3 ⁇ 0
- the conjugate of id- £ (3 may be characterized in that it is directly linked to a drug, preferably an anticancer agent, through a non-covalent bond or a covalent bond, but is not limited thereto.
- the non-covalent bonds are, for example, hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, table-pi interactions and cation-pi interactions. It may be at least one selected from the group consisting of.
- the present invention includes two or more of the 61 ⁇ /1 peptide, or 61 ⁇ /1 peptide-de conjugate will be.
- the id, peptide-conjugates preferably 2 to 6, more preferably id, It may be characterized as comprising an id-de conjugate, but is not limited thereto.
- the dimer (a) among the multimers may be characterized in that two 61 ⁇ /1 peptides, or 61 ⁇ /1 peptide-de conjugates are linked to each other by a linker (1_ 2 ).
- dimer is connected to each other by an additional linker (1_ 3 ), but is not limited thereto.
- additional linker (1_ 3 )
- a hexamer 1163111a
- an octamer (0 3171a)
- more multimers may be linked in a similar manner.
- the dimers and tetramers of the 61 ⁇ /1 peptide-de conjugate according to the present invention may have the forms shown in Structural Formulas 7 and 8, but are not limited thereto. 2021/234550 1 ⁇ (:1 ⁇ 2021/054235
- the linkers L 2 and u may be the same or different from each other, and may be independently cleavable or non-cleavable.
- the linkers U and U are independently a single bond (direct bond), an amino acid or a derivative thereof, Q- C 20 alkyl, Q- C 20 alkylene, -S-, -NH- and -0-. It may be selected from, but not limited to, preferably an amino acid or a derivative thereof, more preferably lysine (Lys, K) or arginine (Arg, R) having two or more amine groups, but is limited thereto no.
- lysine (Lys, K) may be used for the linkers L 2 and U, and the amine group (NH 2 ) of the lysine may be linked with de in a form similar to a peptide bond such as -C-CONH-, but , but is not limited thereto.
- NH 2 amine group
- 2021/234550 -01/162021/054235 at one end of the linker of the 61 ⁇ /1 peptide or the 61 ⁇ /1 peptide-de conjugate, there is a functional group ( ⁇ ) for conjugating another substance, preferably a drug ( ). 11 ⁇ :mi011 no
- the structural formulas for the functional group bonded to the drug 119 to 1_ 3 in 1_2, Structure 8 of 7 can be introduced.
- functional groups include, but are not limited to, amine (1 ⁇ 11) and thiol ( ⁇ ) groups, and lysine 0 ⁇ having an amine (1 ⁇ 11), and/or thiol group.
- Cysteine [Gun, ⁇ is introduced, but is not limited thereto.
- 61 ⁇ /1-330 bimi 61 ⁇ /1-33001) and 61 ⁇ /1- 330 1-geun113111la ( 01 ⁇ /1-3301:) are shown in Table 3 and It may have the same structure, but is not limited thereto.
- 61 ⁇ /1-33a (Same as ⁇ 6 83 ⁇ 4 administration, mouse body weight was not affected by 61 ⁇ /1-33001- administration (Fig. 16b.
- the cancer specificity of 61/1/1-33 can enhance the efficacy of )(, increasing the concentration of therapeutic drugs in tumor tissues).
- the present invention provides a multimer containing two or more 61 ⁇ /1 peptide-de conjugates (1111
- the multimer-drug conjugate is The description of the id-drug conjugate or the 61 ⁇ /1 peptide-de(]-drug conjugate may be applied mutatis mutandis, and accordingly, each linker or drug, etc. may be used as a 61 ⁇ /1 peptide-drug conjugate or 61 ⁇ /1 peptide- It is interpreted the same as the definition in the de-drug conjugate.
- the present invention provides an AGM peptide-PEG conjugate o, cell-permeable heptide (Cell Penetrating Peptide; CPP) to an AGM hypotide-PEG-CPP fusion peptide.
- CPP Cell Penetrating Peptide
- CPP Cell Penetrating Peptide
- signal peptide signal peptide
- H!tides are either protein-permeable domains (
- the fusion peptide of the present invention uses a cell-permeable peptide, and the cell-permeable peptide is not particularly limited as long as it has the characteristics of entering cells by a cell internalization mechanism, but preferably the cell-permeable peptides listed in Table 4 below. Or it can be used by selecting from the group consisting of variants thereof.
- the cell-permeable peptide may be characterized in that it is selected from the group consisting of the cell-permeable peptides listed in Table 5 or variants thereof, and the preparation and properties of the peptides in Table 5 below are described in Korean Patent No. 10- See No. 1169030.
- the cell-penetrating peptide may include the amino acid sequence of SEQ ID NO: 48.
- the AGM peptide-PEG conjugate and the cell-penetrating peptide may be linked through a linker.
- it may be characterized in that it is linked through a maleimide-carboxy bifunctional linker, but is not limited thereto.
- the present invention relates to a multimer-CPP fusion peptide in which a multimer comprising two or more AGM peptide-PEG conjugates and a cell penetrating peptide (CPP) are combined.
- the multimer-[fusion peptide is 61 ⁇ /1 peptide-de-[fusion 2021/234550 ?01/162021/054235
- each linker or cell-penetrating peptide is interpreted the same as the definition in the 61 ⁇ /1 peptide-de-fusion peptide.
- the present invention provides the 61 ⁇ /1 peptide, 61 ⁇ /1 peptide-de conjugate, 61 ⁇ /1 peptide-de(]-drug conjugate, multimer, multimer-drug conjugate, 61 ⁇ /1 Peptide- £ ( 3 - fusion peptide or multimer- relates to a composition for diagnosing cancer comprising a fusion peptide.
- the present invention provides the 61 ⁇ /1 peptide, 61 ⁇ /1 peptide- de conjugate, 61 ⁇ /1 Peptide-de(]-drug conjugate, multimer, multimer-drug conjugate, 61 ⁇ /1 peptide-de-[ fusion peptide or multimer-[ relates to a method for diagnosing cancer using a fusion peptide.
- the 61 ⁇ /1 HA!tide, 61 ⁇ /1 peptide-de-conjugate, 61 ⁇ /1 peptide-de-drug conjugate, multimer, multimer-drug conjugate, 61 ⁇ / for cancer diagnosis 1 peptide- £ ( 3 - fusion peptide or multimer- relates to the use of the fusion peptide.
- the present invention provides the 61 ⁇ /1 peptide, 61 ⁇ /1 peptide-de conjugate for the manufacture of a medicament for diagnosis of cancer. , 61 ⁇ /1 peptide-de(]-drug conjugate, multimer, multimer-drug conjugate, 61 ⁇ /1 peptide- £ ( 3 -[ fusion peptide or multimer-[ fusion peptide.
- the diagnostic composition of the present invention it is possible to predict whether the onset or the possibility of onset of cancer is leukemia, myeloproliferative disease, lymphoma, breast cancer, liver cancer, stomach cancer, ovarian cancer, cervical cancer, glial cancer It may be carcinoma, colorectal cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, gastric adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, melanoma, squamous cell carcinoma, hematopoietic cancer, kidney cancer, head and neck cancer, etc., but is not limited thereto.
- the term "diagnosis” refers to determining the susceptibility of a subject to a specific disease or disorder ( 5 or 1), determining whether the subject currently has a specific disease or disorder, or having a specific disease or disorder. Determining the subject's prognosis (e.g., identification of a pre-metastatic or metastatic cancer state, determining the staging of cancer, or determining the responsiveness of the cancer to treatment) monitoring the state of an object to provide information about it). For the purposes of the present invention, the diagnosis is to determine whether or not the disease is onset or the possibility (risk) of the disease.
- the present invention provides the 61 ⁇ /1 peptide, 61 ⁇ /1 peptide-de conjugate, 61 ⁇ /1 peptide-de(]-drug conjugate, multimer, multimer-drug conjugate, 61 ⁇ /1 It relates to a composition for preventing or treating cancer comprising a peptide-£(3-fusion peptide or multimer-fusion peptide).
- the present invention provides the 61 ⁇ /1 peptide, 61 ⁇ /1 peptide-de conjugate, 61 ⁇ /1 peptide-de(]-drug conjugate, multimer, multimer-drug conjugate, 61 ⁇ /1 peptide-de-[ fusion peptide or multimer-[ fusion peptide to the subject 2021/234550 - It relates to a cancer prevention or treatment method comprising the step of administering 01/162021/054235.
- the present invention provides the 61 ⁇ /1 peptide, 61 ⁇ /1 peptide-de-conjugate, 61 ⁇ /1 peptide-de-drug conjugate, multimer, multimer-drug conjugate, 61 ⁇ /1 peptide- £ ( 3 - fusion peptide or multimer- relates to the use of the fusion peptide.
- the present invention provides the 61 ⁇ /1 peptide, 61 ⁇ for the manufacture of a medicament for the prevention or treatment of cancer.
- Cancers or carcinomas that can be treated with the composition of the present invention are not particularly limited, and may be solid cancers and nucleolin-related cancers such as leukemia.
- examples of such cancers include leukemia, myeloproliferative disease, lymphoma, breast cancer, liver cancer, stomach cancer, ovarian cancer, cervical cancer, glioma cancer, colorectal cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, gastric adenocarcinoma, uterine cancer), bladder cancer, thyroid cancer, It may be melanoma, squamous cell carcinoma, hematopoietic cancers, kidney cancer, or head and neck cancers, but is not limited thereto.
- cancer and “tumor” are used interchangeably and refer to or mean a physiological condition in a mammal that is typically characterized by unregulated cell growth/proliferation.
- prevention refers to the composition comprising the peptide or conjugate. 2021/234550 ?01/162021/054235 It refers to any action that suppresses or delays cancer by administration.
- treatment refers to the 61 ⁇ /1 peptide, 61 ⁇ /1 peptide-de-conjugate, 61 ⁇ /1 peptide- £ ( 3 -drug conjugate, multimer, multimer-drug conjugate, 61 ⁇ /1 Peptide-de-[fusion peptide or multimer- refers to any action in which the symptoms of cancer are improved or cured by administration of the composition containing the fusion peptide.
- composition for preventing or treating cancer is a pharmaceutically effective amount of the 61 ⁇ /1 peptide, 61 ⁇ /1 peptide-de-conjugate, 61 ⁇ /1 peptide- £ ( 3 -drug conjugate, multimer, multimer-drug conjugate,
- the id-de-[ fusion peptide or multimer-[ fusion peptide may be included alone, or may include one or more pharmaceutically acceptable carriers, excipients or diluents.
- the pharmaceutically effective amount refers to an amount sufficient to prevent, improve, and treat symptoms of cancer.
- pharmaceutically acceptable means that it is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans.
- Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
- fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
- Suitable pharmaceutically acceptable carriers and agents include
- composition of the present invention comprises 61 ⁇ /1 peptide, 61 ⁇ /1 peptide-de-conjugate, 61 ⁇ /1 peptide-de-drug conjugate, multimer, multimer-drug conjugate , 61 ⁇ /1 peptide-de-[ fusion peptide or multimer- may include one or more known active ingredients having a cancer therapeutic effect together with the fusion peptide.
- the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal other than a human.
- Formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
- the composition of the present invention can be administered through several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient depends on several factors such as the route of administration, the patient's age, sex, weight, and the severity of the patient. may be appropriately selected according to the present invention, and the composition for preventing or treating cancer according to the present invention may be administered in combination with a known compound having an effect of preventing, improving or treating symptoms of cancer.
- the present invention will be described in more detail through examples.
- Example 1 Materials and Methods 2021/234550 -01/162021/054235
- Example 1 -1 OBOC library synthesis
- the OBOC library was synthesized on solid-phase TentaGel MB NH2 resin (Rapp Polymere GmbH, Tubingen, Gemiany). A "split-mix" synthesis method was performed to construct combinatorial OBOC libraries, each containing a random library of millions of beads/ligands. 5 g of Tentagel MB NH2 resins (200
- the ligand on the surface of the bead is 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and N- hydroxybenzotriazole (HOBt) ⁇ GL Biochem, Shanghai, China)/N,N , - diisopropylcarbodiimide (DIC) (GL Biochem) standard solid-state peptide synthesis technique using coupling was synthesized by Coupling completion was confirmed by the ninhydrin test. Beads were stored in 70% ethanol at 4 °C until use.
- Example 1-2 Ethics and cell culture All studies related to human tissue were pre-approved by the Research Ethics Review Board (IRB) of the Gwangju Institute of Science and Technology (#20191008- BR-48-03) -02).
- MCF-7 Human breast and colorectal cancer cell lines including MCF-7, MDA-MB-231, HT-29 and HCM 16 were obtained from the Korea Cell Line Bank.
- Jurkat T cells were also obtained from the Korea Cell Line Bank.
- the Luciferase-expressing MDA-MB-231 cancer cell line was obtained from Perkin Elmer (Perkin Elmer, Hopkinton, Mass.).
- Each cancer cell line contains RPMI1640 (Gibco, Waltham, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 0.1 mg/ml streptomycin (Gibco) and 100 units/ml penicillin (Gibco) and this V ⁇ VI (Gibco).
- Examples 1-3 OBOC library screening for cancer-specific ligands Before screening, beads were washed extensively with double distilled water and phosphate buffered saline (PBS; Welgene Inc., RepubHc of Korea). Cancer cells and normal cells were isolated from the culture dish using trypsin/Ti TTA (Gibco) and washed with the corresponding culture medium. It was resuspended at 10 6 cells/ml and incubated with 0B0C beads in a Petri dish and shaking (60rpm) in a 37°C humidified C0 2 incubator. Beads bound by cells appeared under the microscope as rosettes with a central bead covered with one or more cell layers.
- PBS phosphate buffered saline
- Example 1-6 Serum Stability Test Preheated The peptide stock solution (10 Ah) was diluted 10-fold and incubated for 0, 3, 6, 9 and 24 hours at 37° (:). Peptide from the group was included as a control. After denaturing serum protein with urea (Niyi) at a final concentration of 3! ⁇ /1 for 10 minutes at 4°(:), the final concentration 2021/234550 ?01/162021/054235
- Fluorescence-activated cell-sorting (FACS) analysis was used to confirm the binding of FITC-hatffle. 1 ⁇ ( ; -Peptide storage solution It was prepared by dissolving.
- HCT-116 cells were seeded at a ratio of 10 5 [ratio 15/ ⁇ 11 to 6- ⁇ 1 micro-roots containing 3
- Control cells were treated similarly except for peptides.
- the prepared cells were analyzed on a FACScanto ll (BD Biosciences) flow cytometer. FACS data were analyzed with FlowJo software (TreeStar).
- Example 1-8 Biotin pull-down assay Cell lysates (500
- Example 1-9 AGM-330 binding domain identification GFP-NCL (residues 1 -710), GFP-AN-NCL (residues 322-710) and GFP-AC-NCL (residues 1) from human NCL cDNA clone (Addgene) -321), and subcloned into the Xhol and BamHI sites of the pEGFP-C2 vector (Addgene). These vectors were transfected with Lipofectamine 2000 (1 nvitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. Biotinylated AGM-330 HA!Tide was added to streptavidin beads (Thermo Fisher Scientific).
- Triton X-100 1 mM marimastat (AdooQ Bioscience, Irvine, CA, USA)
- protease inhibitor NCL was prepared by lysing 1.0X10 9 Jurkat cells in a cocktail (Millipore, Billerica, MA, USA) at 4 °C for 1 h.
- Triton X-100, 1 mM marimastat (AdooQ Bioscience, Irvine, CA, USA), protease inhibitor cocktail (Millipore, Billerica, MA, USA).
- the adsorbed protein was eluted with 10 ml of the same buffer containing 1 M NaCl.
- the eluate was 50 mM Tris/HCI, pH 7.9, 5 mM MgCI 2 ,
- MDA-MB-231 -luc xenograft tissues were formalin-fixed and paraffin-embedded for immunofluorescence staining.
- Cells were seeded on poly-L-lysine and collagen I-coated cover glasses and fixed with 4% formalin.
- Tissue slides and cells were permeabilized with 0.1% Triton X-100 and blocked with 2% BSA (Sigma-Aldrich). Staining was performed as described above using primary anti-NC (1 : 200), anti-TUNEL (1 : 200) and anti-Ki-67 (1 : 500) antibodies. All nuclei were counterstained with DAPI. Immunofluorescence images were matched with H&E-stained images. 2021/234550 ?01/162021/054235 Example 1 -14: 5
- siRNA targeting NCL (NM_005381.3 in NCBI database) and scramble siRNA (scr) were purchased from Bioneer (Daejeon, Republic of Korea).
- siRNA transfection was performed using Lipofectamine 2000 (lnvitrogen) according to the manufacturer's protocol. Three different siRNA sequences were used and their efficiency was evaluated by real-time PCR.
- siNCLI (sense) 5 f -GAGCUAACCCUUAUCUGUA (dTdT)-3 , (SEQ ID NO: 21)
- First-strand cDNA was synthesized using a Synthesis Kit (Takara, Shiga, Japan), and one tenth of the cDNA was used in each PCR mixture together with Power SYBR® Green PCR Master Mix (Applied Biosystems).
- Real-time PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). Relative mRNA expression of selected genes was normalized to expression of p-actin and quantified using the ddCt method.
- the sequences of the PCR primers are listed in Table 6 below.
- Cancer and normal cell lines were seeded at 1X10 4 cells/well in 96-well plates containing 100 [jl of medium, and the plates were incubated overnight at 37 °C. After overnight incubation, cells were incubated for 24 h in the absence or presence of an anti-NCL antibody (Cell Signaling Technology, Beverly, MA, USA) overnight at 37 °C. After incubation, cell viability was evaluated by CellVia WST-1 assay (Young In Frontier, Seoul, Korea) according to the manufacturer's instructions.
- FITC Annexin V-fluorescein isothiocyanate
- PI propidium iodide
- Example 1-18 Cell proliferation assay Cancer cells and normal cells (1x10 4 cells/well) were seeded in 96-well plates. After incubation for 24 hours, cells were treated with increasing concentrations of AGM-330, PTX and AGM-330-PTX for 48 hours. Cell viability was evaluated by CellVia WST-1 assay (Young In Frontier) according to the manufacturer's instructions. The number of viable cells was measured at a wavelength of 450 nm using a Versa Max ELISA plate reader (Molecular Devices).
- Example 1-19 Tumor formation (11
- mice 100
- jl volume of 1X10 6 MDA-MB-231 -luc cells were inoculated into mammary fat pad (/7 5 ⁇ 6 for each groupO. After tumor cell inoculation, when the tumor volume reached approximately 100mm 3 , Mice were randomly divided into 5 groups: (i) control, (ii) low-dose paclitaxel (2 mg/kg), (iii) high-dose paclitaxel (1 Omg/kg), (iv) AGM-330 ( 16.68 mg/kg or 8.34 mg/kg) and (v) AGM-330-PTX (19.05 mg/kg or 10.71 mg/kg).Measure the tumor size twice a week and calculate the tumor volume using the following equation Buoy
- (mm 3 ) (length (mm)) x (width (mm)) 2 X 0.5.
- D-luciferin 150 mg/kg (Perkinti mer) was added to each.
- Mice were injected intraperitoneally, and an IVIS 100 imaging system (Xenogen, Corporation, Alameda, CA) was used for bioluminescence monitoring
- the mice were anesthetized with gaseous isofurane (BK Pharm, llsan, Korea) and placed in an imaging chamber. After 10 minutes, each animal was imaged with an exposure time of 1 minute.All bioluminescence image data were provided by Living Image software (version 4.5.2, PerkinElmer).Photons detected from the tumor were measured with an average light intensity (photon/sec).
- Average luminance values are quantitative data obtained from a region of intensity (ROI), including photons emitted by bioluminescent cells in a rectangular region allocated throughout the body of each mouse. Examples 1-20: Bioinformatics 2021/234550 ?01/162021/054235
- Kaplan-Meier plots were generated using the R2 platform and , group patients according to NCL expression level in breast tumor and colorectal tumor.
- Example 2 A0 [combination screening and 1 ⁇ /1 - synthesis method.
- AGM -330 was resynthesized on Tentagel MB NH 2 resin Cell growth bead analysis results ⁇ , AGM-330 beads were synthesized from human breast cancer cell lines (MCF7 and MDA-MB-231) and human colorectal cancer cell lines (HT-29 and HCT-116) (Fig. 4D), on the other hand, AGM-331 and AGM-332 were relatively non-specific, and were not detected in human normal breast cell line (MCF-10A) and human normal colorectal cell line (CCD-18CO).
- AGM-330 binds very weakly to MCF-10A or CCD-18CO cells or not at all, so it can be a good candidate for both imaging and therapeutic targeting agents. Because of its current low stability The use of peptides as therapeutics has been largely limited: peptides are degraded mainly by proteases and peptidases at / 1// (Bottger R, et al. PLoS One. 2017; 12: e017894 key.
- AGM-330 in the form of a MAP dendrimer was synthesized, which may exhibit increased stability due to the acquired resistance to protease and peptidase activity.
- Synthesis of AGM-330 (Fig. 4E) was performed to prepare lysine core-conjugated Wang resin (2,3), Fmoc-Cys (Trt) Wang resin (1) and Fmoc- in the presence of 20% piperidine and this VIF Lys(Fmoc)-OH.
- AGM-330(4) Fmoc- and Trt-protected AGM-330(4) was prepared by treatment with RHGAMVYU ⁇ -PEG12-0H in the presence of 20% piperidine.
- the Fmoc- and Trt- protecting groups of AGM-330(4) were rapidly removed by piperidine of this VIF to prepare AGM-330(5).
- the selected peptide ligand AGM-330 was synthesized in the form of dimers and tetramers I through MAP (Multiple Antigen Peptide) synthesis to improve in vivo stability and binding power. To evaluate whether or not to enhance peptide stability, the in vivo half-life of the peptide was analyzed.
- AGM-330m, AGM-330d, and AGM_330t 2mg/kg were injected through the tail vein of C57BL/6 mice, and the remaining peptide portion in the serum was immediately immobilized on the ELISA plate using an anti-AGM-330 antibody.
- Absorbance was measured at a wavelength of 450 nm using a Versa Max ELISA plate reader (Molecular Devices), and pharmacokinetics were measured by phoenix WinNonlin. Corporation, Mountain View, CA, USA) program.
- T 1/2 half-lives (T 1/2 ) of AGM-330m, AGM-330d, and AGM-330t were 0.42 ⁇ 0.33 hours, 1.82 ⁇ 0.36 hours, and 9.43 ⁇ 1.21 hours, respectively, and the maximum blood concentration time (T max ) was all 0.167 hours.
- the maximum blood concentrations (C max ) were 1.27 ⁇ 0.12 ijg/mL, 1.71 ⁇ 0.11 jjg/mL, and 1.875 ⁇ 0.67 Mg/mL, respectively.
- the area under the 2021/234550 -01/162021/054235 curve (AUC) was 0.64 ⁇ 0.09ijg/h/mL, 1.77 ⁇ 0.12ijg/h/mL, and 21.42 ⁇ 0.8lMg/h/mL, respectively (FIG. 5).
- AGM-330t tetrameric AGM-330 had higher stability than monomeric or dimeric peptides.
- AGM-330-FITC cancer cells treated with FITC-labeled AGM-330
- AGM-330-FITC was incubated with cells (1X10 5 ) in serum-free medium at 37 °C for 2 h to keep the conjugates intact.
- the intrinsic fluorescence heat-treated cells were compared and measured based on the change in mean fluorescence intensity (MFI) with respect to the untreated cells (FIG. 4F).
- AGM-330-FITC showed significant cancer cell binding to MCF-7, MDA-MB-231, HT-29 and HCT-116, which was a relative increase in MFI of treated cells compared to untreated cells.
- the conjugates showed significantly reduced binding to normal cells, including MCF-10A and CCD-18CO, whereas strong preferential binding to cancer cells.
- AGM-330 exhibits cytotoxicity
- several cancer cells including MCF-7, MDA-MB-231, HT-29, HCM 16 and MCF-10A and CCD-18Co IC 50 values were evaluated in normal cells.
- IC 50 values of AGM-330t and AGM-330d were greater than 100MM in all cell lines regardless of serum concentration (Fig. 6). This means that AGM-330 does not significantly affect the cell viability of cancer or normal cell lines.
- Example 3 Potential target of AGM-330 NCL, a potent regulator of cancer cell growth
- AGM-330 To identify the unknown target protein of AGM-330, AGM-330 from cell lysates of MDA-MB-231 cells using affinity column chromatography and mass spectrometry-based proteomic approaches. -Identified the interacting protein (FIG. 7A). After affinity column chromatography, several distinct protein bands (55-100 kDa) present only in the elution fraction were identified through SDS-PAGE analysis (FIG. 7B). Six proteins were identified using LC-MS/MS analysis, and peptide identification of these excised bands showed that one of the major protein bands at 100 kDa was characterized as nucleolin (NCL).
- NCL nucleolin
- NCL consists of 710 amino acids and 22 other peptide fragments identified by LC-MS/MS analysis were consistent with the amino acid sequence of NCL (total scores 881 and 24 for human NCL (accession number: gi189306) in the NCBI database) % sequence coverage) ( FIGS. 7C-D ).
- the concentration of NCL in the eluate from the AGM-330 affinity column was further confirmed by immunoblot analysis using an anti-NCL antibody (Fig. 70.
- proteomic studies showed that NCL interacts with AGM-330 suggest that it is a protein.
- Biotinylated AGM-330 (AGM-330-Biotin) was used as a bait to pull down the NCL mutant.
- Several deletion mutants of NCL were generated.
- Cell extracts were prepared from HEK293T cells transfected with expression vectors encoding wild-type and various NCL mutants, all fused to GFP, NCLK1-710), NCL2 (323-710) and NCL3 (1-322).
- AGM-330-Biotin pulled down wild-type NCL1 (1-710) and NCL3 (1-322), but not NCL2 (323-710) ( FIG. 7F ).
- NCL N-terminal region of NCL (1-322) is important for binding of AGM-330.
- purified NCL and AGM-330-Biotin were used for biotin pull-down analysis.
- the concentration of NCL in the elution fraction was confirmed by immunoblot analysis using an anti-NCL antibody (FIG. 9).
- Example 4 Identification of amino acid residues essential for the interaction of 61 ⁇ /1-330 and ⁇ :1_ Cancer cells (1 X 10 4 [Ratio 15/ ⁇ 11) 96- ⁇ Ratio 1
- the 13 peptides were treated with increasing concentrations for 2 hours. After that, 3 ⁇ /you ⁇
- the absorbance of 13 PEGylated peptides is shown in FIG. 10 .
- the four peptides of SEQ ID NOs: 9, 10, 11, and 14 showed absorbance similar to that of AGM-330.
- the four peptides of SEQ ID NOs: 12, 13, 15, and 20 showed improved absorbance than AGM-330.
- Substitution of methionine (M5) residue 5 with leucine or norleucine, substitution of tyrosine (Y7) residue 7 with phenylalanine, and insertion of repetitive leucine (L) and lysine (K) residues at the C-terminus enhanced binding strength. .
- the substitution of the hydrophobic amino acids of the M5 and Y7 residues or the insertion of L and K at the C-terminus can improve the binding force.
- the peptides of SEQ ID NOs: 16 to 19 showed reduced absorbance than AGM-330. Deletion of No. 1 arginine (RI) and No. 2 histidine (H2) residues, insertion of repetitive arginine (R) and histidine (H) residues at the N-terminus, and inversion sequence reduced binding force.
- the orientation of the sequences, including R1 and H2 should be preserved.
- Example 5 61 ⁇ /1-330 Specific Binding to Cancer Cells at / /I 0 and / /I 1 /0 as Cell Binding Assay for Different Cell Lines Using Fluorescence Microscopy for 61 ⁇ /1- The specificity of 330 was evaluated. majority Cells were cultured for 1 hour with -231, Bar-29 and Yes 6 and normal cell lines, 1 ⁇ /10 : -10 and (; Binding specificity was confirmed by post-confocal imaging.Cells were incubated with Dimi and counterstained for nuclei (blue fluorescence).Anti- !_ antibody binding to the ! ⁇ 1-terminal domain of !_ and 6! ⁇ /1-330 - 2021/234550 ?01/162021/054235
- NCL and AGM-330-FITC were confirmed (Fig. 11A-3 ⁇ 4.
- the difference in the expression level or location of NCL is related to the sensitivity of AGM-330 to cancer cells It was checked whether this exists.
- the subcellular fraction of cells was isolated, and the expression of NCL in the cytosol, nuclear and cell membrane extracts as well as total intracellular total NCL was analyzed by Western blotting.
- NCL expression was elevated in the membrane and cytoplasmic extracts of MCF-7, MDA-MB-231, HT-29 and HCT-116 cells compared to MCF-10A and CCD-18Co cells (Fig.
- Fig. 12A No significant differences in nuclear levels of NCL were observed in any cell line.
- membrane-expressing NCL is critical for AGM-330 binding to cancer cells.
- siRNAs targeting NCL siNCL1, siNCL2 and siNCL3 showed different efficacy in NCL-knockdown cells.
- AGM-330-Alexa680 tumor-bearing mice treated with Alexa680-labeled AGM-330
- Aleax680 compared to mice treated alone.
- the near-infrared fluorescent (NIRF) intensity of the AGM-330-Alexa680 fraction tumor was significantly higher than that of the control tumor: (control) 3.04 X 10 7 ⁇ 1.38 10 6 (p/sec/cm 2 /sr) )/(
- Contrast (AGM-330-Alexa680) 1.19 X 10 9 ⁇ 1.90 10 7 (p/sec/cm 2 /sr)/(ijW/cm 2 ), n 3, P ⁇ 0.001 (FIGS.
- Example 6 Positive correlation between NCL and cancer progression and anti-NCL antibody to confirm cell proliferation inhibitory effect through membrane NCL neutralization Based on the above Examples, NCL expression patterns in cancer cell lines were confirmed.
- Oncomine dataset repository www.oncomine.org
- NCL mRNA expression was significantly upregulated in breast carcinoma compared with normal breast tissue ( ⁇ 0.001, FIG. 13A).
- NCL expression was analyzed by immunohistochemistry (IHC) analysis.
- IHC immunohistochemistry
- 2021/234550 ?01/162021/054235 revealed an increase in the membrane and cytoplasmic regions of human breast and colorectal cancer tissues compared with these normal breast and colorectal tissues (Fig. 13(1-)), which The results of Fig. 1 1/ ⁇ -Mi and Western blotting (Fig. 1 1(;)) are consistent with the results.
- the up-regulated membrane interacts as a receptor for a ligand involved in the inhibition of cancer proliferation and apoptosis (Fig. 01). rain. 0% 3 . 2015; 6: 16253-7( ⁇ .
- Membrane expression was neutralized using monoclonal anti- ⁇ antibody in 1 ⁇ /10/ ⁇ -1 ⁇ / - 231 and 4 6 cells and [ kawa and 0g -18 (;0 cells).
- apoptotic assays were performed to investigate the cause of the cytotoxic effect of anti-antibodies on cancer cells.
- 1 ⁇ / / ⁇ -1 ⁇ / -231 and HCT-1 16 cells treated with antibodies at different concentrations for 24 hours were stained using Annexin V and Mi.
- the percentage of cancer cells in early and late apoptotic stages increased in an anti- 1 ⁇ 1 ⁇ that!_ antibody concentration-dependent manner ( FIG. 131 ).
- the present inventors confirmed that this overexpression in the membrane and cytoplasm of various types of cancer is associated with poor prognostic survival of cancer patients.
- the above results are important for the membrane part in cancer cells, especially in terms of the control of cancer cell proliferation and apoptosis.
- Example 7 Confirmation of effective cancer growth inhibitory effect of paclitaxel-conjugated AGM-330 in vitro and in mouse xenograft model A cancer-targeting ligand linked to a well-established chemotherapeutic agent by conjugating paclitaxel (PTX) with AGM-330 ( AGM-330-PTX) was obtained. PTX was orthogonal conjugated with a maleimide-carboxy bifunctional linker.
- PTX paclitaxel
- M-330t-PTX inhibits cancer cell proliferation
- IC 50 values in several cancer cell lines including MCF-7, MDA-MB-231, HT-29 and HCT-116 (Fig. 14A).
- MDA-MB-231 cells the IC 50 values for PTX alone and AGM-330t-PTX were 6.8 and 3.6 M M, respectively.
- the results were similar in all three cancer cell lines (MCF-7, HCT-116 and HT-23 ⁇ 4.
- MCF-10A cells the IC 50 values for PTX alone and AGM-330t-PTX were 22.1 and 35.3MM, respectively.
- AGM-330t-PTX was a potential in vivo therapeutic agent.
- the mammary gland fat pads of NPG mice were inoculated with MDA-MB-231-luc cells subcutaneously. After the tumor grew to 100 mm 3 , twice a week by tail vein injection, vehicle (PBS), AGM-330t (16.68 mg/kg), PTX (2 mg/kg and 10 mg/kg, respectively), AGM-330t-PTX
- 61/1-33 was tested for anticancer efficacy using silver 61 ⁇ /1-33001-[ ⁇ ) (Fig. 16/ ⁇ ).
- the 61//1-33001-treatment significantly reduced the tumor volume and weight compared to the ) (-treated group (Fig. 16 (3)).
- cell penetrating peptide Cell Penetrating Peptide
- Example 10 Confirmation of the effect of inducing / /I ⁇ 0 cancer cell apoptosis (3 (30 nights 0 5) of the bound fusion peptide)
- HCT-116, 1 ⁇ “-29 and ⁇ ; [-18[:0] Cells were stained using / ⁇ 6) ⁇ V and Mi.
- Fig. As a result, it was confirmed that the apoptosis-promoting protein increased in a concentration-dependent manner ( FIG. 21 ).
- Example 11 61 ⁇ /1-330 and 61 ⁇ /1-330 - 61//1-330 unfermented [?
- AGM-330m-mCPR AGM-33001-dCPP and 61 ⁇ /1-330 [??The value of 1 (; 50 of the fusion peptide) was 23.3, 5.47 and 3.791 ⁇ for colorectal cancer cell lines, respectively, and in bar-29 for 20.67, 3.75 and 3.3 1 ⁇ /1, respectively.
- the breast cancer cell line showed >20,4.208 and 3.802 ⁇ 1 for 1 -/, respectively, and >20, 3.843, and 3.49 1 ⁇ /1 for 0/ ⁇ - 231, respectively (FIG. 22).
- 6! ⁇ /1-330 -[ fusion It can be confirmed that the 2021/234550 -01/162021/054235 peptide has a cancer cell-specific inhibitory effect.
- ⁇ 50 values were 14.3, 4.25 and 3.15 1 ⁇ /1 for colon cancer cell line 1 ⁇ 116, respectively, and 14.2, 4.76 and 3.05! ⁇ for Bar-29, respectively.
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