Peptide ligand targeting carbonic anhydrase ix, peptide construct comprising the same, and uses thereof

Abstract

SOLUTION: To provide a peptide ligand that specifically binds to carbonic anhydrase IX (CAIX), a peptide construct comprising the same, and uses thereof.EFFECT: The CAIX-binding peptide ligand of the present invention includes D-amino acids, is stable in the body, and has high binding specificity to CAIX, and a linear or cyclic CAIX-binding peptide construct comprising the ligand can bind to CAIX with high affinity in the body, and is useful for diagnosis, prevention, suppression or treatment of diseases mediated by CAIX.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a peptide ligand that specifically binds to carbonic anhydrase IX (CAIX) and a peptide structure containing the same. Specifically, the present invention relates to a CAIX-binding peptide ligand that is specific for CAIX and contains D-amino acids to enhance stability, a linear or cyclic high-affinity CAIX-binding peptide structure containing the peptide ligand, and their use for the diagnosis, prevention, suppression, or treatment of diseases mediated by CAIX.

Background Art

[0002] Carbonic anhydrase (CA) is a zinc (Zn 2+ ) metalloenzyme that commonly exists in higher vertebrates including humans, and is an enzyme that catalyzes a reversible hydration reaction that converts carbon dioxide into hydrogen ions and bicarbonate ions (CO2 + H2O ⇔ HCO3 - + H + ). Such CAs have been shown to have 16 isozyme forms, and in humans, they are present in various tissues such as the gastrointestinal tract, reproductive tract, nervous system, kidney, lung, skin, and eyeball. CA isozymes are known to be involved in important physiological processes such as almost respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid (Thiry et al., TRENDS in Pharmacological Sciences, 27(11):566-573, 2006). Among the CA family, carbonic anhydrase IX (CAIX) is specifically expressed very restrictively in normal tissues, while it is abnormally overexpressed in the majority of solid tumors. This is mainly due to hypoxia-induced factor (HIF-1), a transcription factor, being induced by hypoxia that occurs mainly in the excessive growth of solid tumors, leading to strong transcriptional activation by HIF-1 (De Simone et al., Biochimica et Biophysica Acta, 1804:404-409, 2010; Thiry et al., ditto).

[0003] Tumor hypoxia results from the creation of an oxygen-lean environment as solid tumors grow at a rate exceeding the blood supply capacity provided by the host's vascular system. Even in a hypoxic microenvironment, solid tumors maintain continuous growth and proliferation through various genetic mutations. In such hypoxic tumor cells, anticancer chemotherapeutic agents are difficult to be transmitted through the blood, and in the case of radiotherapy, there is a lack of oxygen required for the cytotoxic effect of radiation-derived free radicals, showing enhanced resistance to anticancer chemotherapy and radiotherapy. Also, hypoxic tumor cells induce overexpression of CAIX on the cell surface, and lower the pH of the extracellular environment of tumor cells by CO2 hydration by the extracellular catalytic domain of CAIX. The acidic tumor microenvironment thus formed can promote tumor cell invasion and metastasis and inactivate pH-sensitive drugs (Thiry et al., ditto). Therefore, tumor hypoxia is generally known as a poor prognostic factor for cancer patients.

[0004] Recently, research has been actively carried out to inhibit tumor growth and proliferation associated with CAIX by targeting CAIX overexpressed in tumor cells or disrupting pH regulation by tumor cells by interfering with the catalytic activity of CAIX. These studies mainly focus on the development of monoclonal antibodies that bind to CAIX and small molecule inhibitors of the sulfonamide series. However, when targeting solid tumors, monoclonal antibodies with large molecular weights have the problem of difficult efficient delivery, and small molecule inhibitors of the sulfonamide series are relatively unstable in solution state, and there are aspects where their usefulness as drug compounds is limited. Therefore, there is a need to develop high-affinity, stable, novel CAIX-specific binders and inhibitors that are suitable for the pharmaceutical uses of cancer treatment, prevention, diagnosis, prognosis prediction, and imaging, including cancer treatment.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] One object of the present invention is to provide a stable CAIX-specific binder and inhibitor suitable for use in the treatment, prevention, diagnosis or prognosis prediction of cancer diseases. One object of the present invention is to provide a stable CAIX-specific binding peptide ligand. One object of the present invention is to provide a high-affinity CAIX-specific peptide structure containing one or more effectors or functional groups such as sulfonamide functional groups together with a CAIX-specific binding peptide ligand. A further object of the present invention is to provide a conjugate containing the peptide structure. A further object of the present invention is to provide a composition for the diagnosis, prevention or treatment of cancer, containing the peptide ligand, peptide structure or conjugate. A further object of the present invention is to provide a method for diagnosing cancer using the peptide ligand, peptide structure or conjugate. A further object of the present invention is to provide a method for treating cancer using the peptide ligand, peptide structure or conjugate. A further object of the present invention is to provide a method for predicting the prognosis after cancer treatment using the peptide ligand, peptide structure or conjugate.

Means for Solving the Problems

[0007] As a result of intensive research to achieve the above object, the present inventors have developed a novel CAIX-specific binder and inhibitor that contains D-amino acids, is stable, and can specifically bind to CAIX with high affinity.

[0008] In one aspect, the present invention provides a CAIX-specific peptide ligand containing any one amino acid sequence among SEQ ID NOs: 1 to 44. At least one of the constituent amino acids of the CAIX-specific peptide ligand consists of a D-amino acid and can be stably retained in the body. Among the constituent amino acids of the CAIX-specific peptide ligand, the lysine (Lys) residue can be substituted with a chemical functional group at the side chain ε-amino group. Non-limiting examples of the chemical functional group include pentafluorobenzoic acid or diphenolic acid.

[0009] In one aspect, the present invention provides a peptide structure comprising a sulfonamide functional group-containing amino acid residue directly or via a spacer linked to the CAIX-specific peptide ligand. The peptide structure may further comprise one or more other functional group-containing amino acid residues other than the sulfonamide functional group, and may have a cyclic or linear structure.

[0010] In one aspect, the present invention provides a CAIX-specific peptide structure having a cyclic structure of the following Chemical Formula 1.

[0011]

Chemical Formula

[0012] In the above formula, P is the CAIX-specific binding peptide of the present invention, F1 is glycine (Gly) or a sulfonamide functional group-containing amino acid residue, F2 is a sulfonamide functional group-containing amino acid residue, F3 is glycine (Gly) or a functional group-containing amino acid residue other than sulfonamide, n and m are each independently 0 or 1, F4 is the group of general formula -(S1) o -(F5) p -(S2) q -(F6) r -NH2, where S1 and S2 are each independently a spacer, F5 and F6 are each independently a functional group-containing amino acid residue other than sulfonamide, o, p, q and r each independently represent an integer from 0 to 6.

[0013] In one aspect, the present invention provides a CAIX-specific peptide structure having a linear structure of the following Chemical Formula 2.

[0014] [Chemical formula]

[0015] In the above formula, P is a CAIX - specific binding peptide of the present invention, F8 is a sulfonamide functional group - containing amino acid residue, F7, F9 and F 10 are each independently a functional group - containing amino acid residue other than sulfonamide, or when there are multiple F9s, at least one F9 is a sulfonamide functional group - containing amino acid residue, and the remaining F9s, F7 and F 10 are each independently a functional group - containing amino acid residue other than sulfonamide, S3 and S4 are each independently a spacer, s, t, u, v and w each independently represent an integer from 0 to 3.

[0016] In the CAIX - specific peptide structure, the sulfonamide functional group - containing amino acid residue can have, but is not limited to, the following structures.

[0017] [Chemical formula]

[0018] In the CAIX - specific peptide structure, the functional group - containing amino acid residue other than sulfonamide can be introduced through the side - chain ε - amino group of a lysine residue, and includes, for example, a chelator, a cycloalkane, biotin, glucoheptonic acid, 4 - (p - iodophenyl) butyric acid (IB), a fluorescent dye, or a cytotoxic agent.

[0019] In one aspect, the present invention provides a conjugate in which a fluorescent dye, a cytotoxic agent or a radioisotope is conjugated to the CAIX - specific peptide structure.

[0020] In one aspect, the present invention provides a pharmaceutical composition for diagnosing, preventing or treating cancer, comprising the CAIX-specific peptide structure or the conjugate. The cancer may be a cancer that expresses CAIX. The cancer may be selected from the group consisting of, but not limited to, liver cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, cancer near the anus, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic cancer, melanoma, thyroid cancer, astrocytoma and glioblastoma.

[0021] In one aspect, the present invention provides a method for diagnosing cancer, comprising administering the CAIX-specific peptide structure or the conjugate to an individual. In one aspect, the present invention provides a method for treating cancer, comprising administering the CAIX-specific peptide structure or the conjugate to an individual. In one aspect, the present invention provides a method for predicting the prognosis after cancer treatment, comprising administering the CAIX-specific peptide structure or the conjugate to an individual.

Advantages of the Invention

[0022] The present invention provides novel, highly stable and high-affinity CAIX binders and inhibitors that specifically bind to CAIX. The CAIX binders of the present invention contain a CAIX-specific binding peptide ligand containing one or more D-amino acids, exhibit excellent stability in vivo, and are useful for CAIX targeting. The present invention also provides a peptide construct in which one or more effectors or functional groups such as a sulfonamide functional group are introduced via the side chain of an amino acid residue into the CAIX-specific peptide ligand, thereby providing a CAIX binder with high binding affinity that is particularly useful for cancer imaging or diagnostic applications. The CAIX-specific peptide constructs of the present invention can also be conjugated with fluorescent dyes, cytotoxic agents or radioisotopes and used as effective drugs for cancer diagnosis, prevention or treatment.

Brief Description of the Drawings

[0023]

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Mode for Carrying Out the Invention

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The present invention can be variously modified and applied within the scope of the claims described below and the equivalent scope analyzed therefrom.

[0025] The CAIX-specific peptide ligand of the present invention The present invention provides a peptide ligand that specifically binds to CAIX. The CAIX-specific peptide ligand of the present invention can contain any one of the amino acid sequences of SEQ ID NOs: 1-44. The peptide ligand can contain D-amino acids or consist only of D-amino acids. Further, the peptide ligand can be substituted with one or more chemical functional groups, such as, but not limited to, pentafluorobenzoic acid or diphenolic acid, at the side-chain ε-amino group of lysine (Lys) residues among the constituent amino acid residues. The peptide ligand can also further contain one or more chemical modifications at the side-chain of phenylalanine (Phe) residues among the constituent amino acid residues. Such modifications include, but are not limited to, modification of phenylalanine residues to homophenylalanine residues, modification of phenyl groups to naphthalene groups, or substitution of phenyl groups with halo, amino or phenyl substituents.

[0026] The peptide ligand of the present invention may be produced such that amino acid residues having a specific sequence are bonded to each other to form a chain-like or cyclic molecule. The peptide ligand of the present invention can be produced by known peptide synthesis methods and is not particularly limited. In one embodiment, the peptide ligand of the present invention can be produced by repeating the peptide synthesis process in a solid single bead form until a peptide of a desired length and sequence is completed. The CAIX-specific peptide ligand of the present invention also includes its salt form.

[0027] The CAIX-specific peptide construct of the present invention The present invention provides a peptide structure containing a sulfonamide functional group-containing amino acid residue directly or linked via a spacer to a CAIX-specific peptide ligand. The peptide structure can further contain one or more other functional group-containing amino acid residues other than the sulfonamide functional group. Chemical functional groups including the sulfonamide functional group in the peptide structure of the present invention can be introduced via the side-chain of amino acid residues. The amino acid residue into which the functional group is introduced is preferably lysine.

[0028] The peptide structure of the present invention can contain one or two sulfonamide functional groups. The introduction of the sulfonamide functional group can be achieved through known synthetic reactions and is not particularly limited. In one embodiment of the present invention, the sulfonamide functional group can be introduced into the peptide structure of the present invention through a click chemistry reaction. Examples of preferred sulfonamide functional group-containing amino acid residues that can be introduced into the peptide structure of the present invention include, but are not limited to, the following structures.

[0029]

Chemical formula

[0030] In one embodiment, non-limiting examples of functional groups other than sulfonamide that can be introduced into the peptide structure of the present invention can include a chelator, a cycloalkane having 5 to 15 carbon atoms, biotin, glucoheptonic acid, 4-(p-iodophenyl)butyric acid (IB), a fluorescent dye, or a cytotoxic agent.

[0031] The chelate may be, for example, one or more selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid-2,2',2'',2'''-(ethane-1,2-diylbisnitrilo)tetraacetic acid (EDTA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N'''',N'''''-hexaacetic acid (HEHA), 2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N'''',N'''''-pentaacetic acid (PEPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), (1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) tetrasodium salt (DOTMA), 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA), and triethylenetetramine (TETA), but is not limited thereto.

[0032] The cycloalkane having 5 to 15 carbon atoms may be, for example, one or more selected from cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, adamantane, norbornane, isobornane, and tricyclodecane, but is not limited thereto.

[0033] The peptide construct of the present invention can also link a chemically functional group-containing amino acid residue such as a sulfonamide functional group to the CAIX-specific binding peptide ligand via a spacer. The spacer may be, for example, one or more selected from a polyethylene glycol (PEG) linker, glycine, sarcosine, and a peptide linker composed of 1 to 5 D-amino acids or L-amino acids, but is not limited thereto.

[0034] The peptide structure of the present invention can have a cyclic or linear structure. Preferably, the peptide structure of the present invention has a cyclic structure of the following Chemical Formula 1.

[0035] [Chemical Formula]

[0036] In the above formula, P, F1, F2, F3, F4, n, and m are as defined above. Without being bound by a particular theory, a cyclic structure such as the cyclic peptide structure of the present invention is less flexible than a linear peptide, so there is less entropy loss during target binding, the binding affinity is higher, and the binding specificity for the target is increased.

[0037] The peptide structure of the present invention specifically binds to CAIX, but shows high selectivity and does not bind to other homologous enzymes of CAIX (for example, carbonic anhydrase XII).

[0038] Conjugate The CAIX-specific peptide structure of the present invention can be conjugated directly or via a linker to a fluorescent dye, a cytotoxic agent, a radioisotope, etc. to form a conjugate. The conjugate can target CAIX and label cancer cells expressing CAIX with a fluorescent dye or a radioisotope, or effectively deliver drugs such as a radioisotope or a cytotoxic agent to the cancer, so it is useful for cancer diagnosis, prevention, or treatment applications.

[0039] In one embodiment, the linker may be one or more selected from, but not limited to, 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 4-(2-pyridyldithio)butyric acid N-hydroxysuccinimide ester (SPDB), and N-succinimidyl (4-iodo-acetyl)aminobenzoic acid (SIAB).

[0040] In one embodiment, non-limiting examples of the fluorescent dye can include one or more selected from near-infrared fluorescent dyes, fluorescein types, rhodamine types, Alexa Fluor, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), Texas Red, dansyl, Lissamine, cyanine (Cy), and phycoerythrin.

[0041] In one embodiment, the cytotoxic agent may be one or more selected from, but not limited to, toxins, chemotherapeutic agents, drug moieties, antibiotics, and nucleolytic enzymes.

[0042] In one embodiment, the radioisotope may be one or more selected from fluorine-18 (F-18), carbon-11 (C-11), carbon-14 (C-14), technetium-99m (Tc-99m), copper-64 (Cu-64), copper-67 (Cu-67), dysprosium-168 (Dy-168), bismuth-213 (Bi-213), samarium-153 (Sm-153), strontium-89 (St-89), strontium-90 (St-90), erbium-169 (Er-169), phosphorus-32 (P-32), palladium-103 (Pd-103), rhenium-186 (Re-186), rhenium-188 (Re-188), oxygen-15 (O-15), selenium-75 (Se-75), sodium-24 (Na-24), strontium-85 (Sr-85), lutetium-177 (Lu-177), yttrium-90 (Y-90), iodine-123 (I-123), iodine-125 (I-125), iodine-131 (I-131), iridium-192 (Ir-192), iridium-196 (Ir-196), ytterbium-166 (Yb-166), indium-111 (In-111), xenon-133 (Xe-133), nitrogen-13 (N-13), calcium-47 (Ca-47), cobalt-57 (Co-57), cobalt-60 (Co-60), chromium-51 (Cr-51), krypton-81 (Kr-81), potassium-42 (K-42), holmium-166 (Ho-166), gallium-67 (Ga-67), gallium-68 (Ga-68), actinium-225 (Ac-225), zirconium-89 (Zr-89), lead-212 (Pb-212), and astatine-211 (At-211).

[0043] Therapeutic administration and dosage form The present invention provides a pharmaceutical composition for the diagnosis, prevention or treatment of cancer, comprising a CAIX-specific peptide ligand, peptide structure or conjugate of the present invention. The cancer of the present invention is preferably a solid cancer. More preferably, the cancer of the present invention is a cancer that expresses CAIX. For example, the cancer of the present invention may be a solid cancer such as liver cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, cancer near the anus, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, melanoma, thyroid cancer, astrocytoma or glioblastoma, but is not limited thereto.

[0044] The subject to which the pharmaceutical composition for diagnosing, preventing or treating the cancer of the present invention is administered may be a mammal at risk of carcinogenesis, diagnosed with cancer, or undergoing cancer treatment. The mammal may be a human or a mammal other than a human.

[0045] The pharmaceutical composition for diagnosing, preventing or treating the cancer according to the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, purifications, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions by ordinary methods, and may contain appropriate carriers, excipients or diluents usually used in the manufacture of pharmaceutical compositions for formulation. Examples of the carrier, excipient or diluent include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. When formulating, it can be manufactured using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, surfactants and the like usually used in the pharmaceutical industry.

[0046] The preferred dosage of the pharmaceutical composition for preventing or treating cancer according to the present invention varies depending on the patient's condition, body weight, degree of disease, drug form, administration route, and duration, but can be appropriately selected by those skilled in the art. However, for a preferable effect, it can be administered at 0.0001 to 2,000 mg / kg per day, preferably at 0.001 to 2,000 mg / kg per day. The administration may be carried out once a day or divided into several times. However, the scope of the present invention is not limited by the said dosage.

[0047] The pharmaceutical composition for preventing or treating cancer according to the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes. The administration method can be, for example, oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine intima, or intracerebroventricular injection.

[0048] The present invention also provides a method for treating cancer, which includes administering the CAIX-specific peptide structure or conjugate of the present invention to an individual in need of cancer treatment. The CAIX-specific peptide structures and conjugates of the present invention can also be used for targeting and visualizing cancer for cancer diagnosis, or for administering to an individual who has received cancer treatment to predict or observe the treatment prognosis of the individual after cancer treatment.

Examples

[0049] Hereinafter, the present invention will be described in more detail through examples. These examples are merely for more specifically illustrating the present invention and are not provided for limiting the legitimate scope of rights of the present invention. It will be apparent to those skilled in the art that various modifications are possible within the scope of rights of the present invention.

[0050] Example 1. Peptide screening for binding to human carbonic anhydrase IX A peptide library was synthesized to obtain a peptide that specifically binds to the extracellular domain (ECD) of human carbonic anhydrase IX (hCAIX). The synthesized peptide library was screened, and the binding affinity and specificity for hCAIX ECD were evaluated for the selected peptides. The peptide screening process for hCAIX ECD is shown in Figure 1.

[0051] 1-1. Synthesis of Peptide Library Incorporated with Beads For the synthesis of the peptide library, a Random OBOC (combinatorial one-bead-one-compound) library was synthesized using TentaGel beads. TentaGel TM S-NH2 resin (Cat#NSD30902) was purchased from Rapp Polymere GmbH (Germany). TentaGel TMS-NH2 beads (polyethylene glycol-grafted polystyrene beads) were used to synthesize through the repeated split and mix process on an automatic synthesizer (Apex 396, AAPPTEC) using 18 types of D-amino acids excluding cysteine and methionine at each residue position. In particular, in the case of Fmoc (fluorenylmethyloxycarbonyl)-D-isoleucine-OH and Fmoc-D-glutamine-OH, 10 mol% of Fmoc-glycine-OH was added respectively to distinguish isotope residues during amino acid sequence analysis of the peptide. TentaGel was swollen in NMP (N-Methylpyrrolidone) solvent, and after attaching a photocleavable linker, a peptide library was synthesized on an automatic synthesizer. When introducing the Fmoc-ANP linker (3-(Fmoc-amino)-3-(2-nitrophenyl)propanoic acid, Cat#LSP308, AAPPTEC), N-acetylglycine (3 equivalents, Cat#A16300, Sigma-Aldrich) was added to adjust the loading ratio to 1 / 4, and it was reacted with TBTU (3 equivalents, Cat#12806, Sigma-Aldrich), DIPEA (7.5 equivalents, Cat#8.00894, Sigma-Aldrich). The solid beads were washed with NMP, reacted with a piperidine / NMP (1:4) solution for Fmoc protecting group removal, and then washed successively with NMP, DCM (dichloromethane), and NMP. Thereafter, Fmoc-Arg(pbf)-OH (3 equivalents, Cat#36404, GL Biochem) and Fmoc-PEG1-OH (3 equivalents, Cat#246201, ChemPep) were synthesized in the same way successively to form a linker. The beads with the linker attached for peptide library synthesis were divided into the same amount and placed into 18 wells of the automatic synthesizer RV (reaction vessel).Once one cycle of coupling and Fmoc protecting group removal was completed, the solid beads were kneaded into the CV (collector vessel), and then further divided equally into 18 RV wells to proceed with coupling and Fmoc protecting group removal, repeating the separation and mixing process until the desired peptide length was achieved.

[0052] 1 - 2. Synthesis of Peptide Library with Sulfonamide Functional Group Introduced After introducing an azido substituent into the peptide synthesized in Example 1 - 1 above, the 18 RV beads were collected in one tube and then subjected to a click reaction with 4 - ethynylbenzenesulfonamide (Figure 2). Specifically, under an argon (Ar) atmosphere, 1 equivalent of the beads with the peptide library attached, 4 - ethynylbenzenesulfonamide (3 equivalents), CuI (1 equivalent), tris(benzyltriazolylmethyl)amine (TBTA, 3 equivalents, Cat#T2993, TCI), and DIPEA (10 equivalents) were mixed in NMP solvent and allowed to proceed at room temperature for 12 hours. After completion of the reaction, the beads were washed with NMP and then with a 0.1 M sodium diethyldithiocarbamate (Sigma - Aldrich, Cat#D3506) / NMP solution to remove copper impurities. For removal of the protecting groups of the amino acid residues, the reaction was carried out with trifluoroacetic acid (95%, TFA, Cat#299537, Sigma - Aldrich), triple - distilled water (2.5%), and triisopropylsilane (2.5%, TIS, Cat#233781, Sigma - Aldrich) for 2 hours, followed by washing with DCM, drying in vacuo, and storing at 4°C in the dark. Figure 2 shows an embodiment of the library structure used for the click reaction to introduce a sulfonamide functional group into the peptide library and hCAIX screening.

[0053] Example 2. Selection of peptides binding to human CAIX 2 - 1. Primary Screening of Peptide Library After transferring 50 mg of the beads introduced with the peptide library synthesized in Example 1 above to 4 mL of Extract-Clean Filter Columns (Cat#211104, S*PURE, Singapore), 2 mL of pH 7.4 phosphate buffer solution (PBS) was added and sonicated with an ultrasonic cleaner (Cat#5210R-DTH, BRANSON, USA) to swell the beads. After replacing the PBS with 2 mL of blocking solution [10% FBS, 0.1% Tween 20 (Cat#69295-1601, Junsei, Japan) in pH 7.4 PBS], incubation was carried out at room temperature with a 360° shaker (Cat#M04-238-157, SCILOGEX, USA). After adding 60 nM of the hCAIX ECD (ACROBiosystems, Cat#CA9-H5226) solution attached with a fluorescent dye and further proceeding with the incubation, the solution was removed. After transferring the beads to a conical tube, they were diluted with 45 mL of PBST buffer solution containing 0.1% Tween 20 and then aliquoted. To each aliquoted solution, PBST buffer solution containing 0.1% Tween 20 was added for further dilution, and after attaching to the sample vessel of COPAS, screening was carried out. The screening was carried out at Excitation 640 nm and Emission 680 / 30BP, and approximately 5,000 beads with high fluorescence intensity were selected under the conditions of Enrichment mode, PMT690, and Gain 3.0. The above process was repeated 5 times to select a total of 25,000 positive beads (i.e., beads attached with peptides that bind to hCAIX ECD) used for the secondary screening (Figure 3).

[0054] 2-2. Secondary Screening of Peptide Library In Example 2-1, hCAIX ECD attached with a fluorescent dye was removed from approximately 25,000 positive beads secured in the primary screening. Thereafter, 1 mL of a blocking solution was added to the beads, and after carrying out a constant temperature treatment using a 360° shaker, the solution was removed. After adding a 250 nM solution of hCAIX ECD attached with a fluorescent dye and further carrying out a constant temperature treatment, the solution was removed. After transferring the beads to a conical tube, they were diluted with 45 mL of a PBST buffer solution containing 0.1% Tween20 and then aliquoted. To each aliquoted solution, a PBST solution containing 0.1% Tween20 was added for further dilution, and they were attached to a sample container of COPAS and carried out in two steps under the conditions of Excitation 640 nm and Emission 680 / 30BP. In the first step, approximately 1,000 beads with high fluorescence intensity were selected under the conditions of Enrichment mode, PMT630, and Gain 3.0. The 1,000 beads secured were diluted with triple distilled water and then the second COPAS selection was carried out using a 96-well plate under the conditions of Pure mode, PMT620, and Gain 3.0.

[0055] 2-3. Separation and analysis of peptides secured in the secondary screening Peptides were separated from the solid single beads obtained through the secondary screening of Example 2-2 through a photoreaction. Specifically, after sealing a 96-well plate containing the beads under an argon (Ar) atmosphere, a UVP crosslinking agent (Cat#849-30101-2, Analytikjena, Germany) was used to carry out a photoreaction for 10 minutes under the conditions of a wavelength of 365 nm and an irradiation dose of 9,000 μJ / cm 2 After opening the 96-well plate, it was concentrated at room temperature using an acid benchtop concentrator (Cat#7310042, LABCONCO, USA). Subsequently, the molecular weight and amino acid sequence of the peptide were analyzed based on the MS and MS / MS obtained using an Enhanced ultrafleXtreme MALDI-TOF / TOF mass spectrometer (Bruker, USA). Examples of each analysis result are shown in FIG. 4a (mass spectrometry result) and FIG. 4b (MS / MS sequence analysis result), respectively.

[0056] 2-4. Tertiary screening of the peptide library for candidate peptide selection Out of about 250 to 300 peptides having the amino acid sequences secured in Examples 2-3 above, about 50 were selected and synthesized using an Apex396 automatic synthesizer in the same manner as in Example 1 on TentaGel beads (0.08 mmole / g, 10 mg per peptide) attached with a photocleavable linker. Subsequently, the protecting groups of the amino acid residues were removed using a mixed solution of TFA (95%) / triple-distilled water (2.5%) / TIS (2.5%). Subsequently, for the tertiary COPAS screening for deriving the final peptide candidate substances, 1 mg each of TentaGel beads attached with about 50 different peptides (total 50 mg) were kneaded into 4 mL Extract-Clean Filter Columns, and then 2 mL of PBS was added and sonicated to swell the beads. Subsequently, with the final concentration of hCAIX ECD attached with a fluorescent dye maintained at 250 nM, the COPAS screening operation was carried out in the same manner as in the secondary peptide library screening. To ensure the reproducibility of the results, this process was repeated three times. After sequencing the peptides in the order of the MS and MS / MS data analysis results and the positive hit number, candidate substances were selected based on the top-ranked peptides.

[0057] Example 3. Synthesis and purification of peptide constructs containing the selected peptides 3-1. Synthesis of a biotin linker Automated ultrasonic peptide synthesizer (Liberty Blue TMA solid biotin linker was synthesized using an automated microwave peptide synthesizer (CEM Corporation). Specifically, after treating Rink Amide-ChemMatrixR Resin (0.45 mmole / g) swollen in NMP with a piperidine / NMP (v / v = 1:4, 0.1 M OxymaPure) mixed solution to remove the Fmoc protecting group, it was washed with NMP. Thereafter, after coupling by treating with Fmoc-Lys(mtt)-OH (5 equivalents), Oxyma Pure (5 equivalents), and diisopropylcarbodiimide (DIC, Cat#D0254, TCI) (10 equivalents) in an NMP solvent, the Fmoc protecting group was removed. The same process was repeated twice using Fmoc-PEG1-OH (5 eq) for linkage. The resin recovered from the ultrasonic peptide synthesizer was treated with a 1.5% TFA / 2.5% TIS / 96% DCM mixed solution to remove the 4-methyltrityl protecting group (Mtt). To introduce biotin, it was reacted with biotin-NHS (3 equivalents) and DIPEA (10 equivalents) in an NMP solvent at room temperature for 1 hour, and then washed successively with NMP and DCM and dried under vacuum. An exemplary reaction formula for the synthesis of the biotin linker is as follows.

[0058]

Chemical formula

[0059] 3-2. Synthesis of the selected peptide A peptide of the amino acid sequence selected using the biotin linker synthesized in Example 3-1 was synthesized. First, after removing the Fmoc protecting group with a piperidine / NMP (v / v = 1:4) mixed solution from the beads introduced with the biotin linker, washing was performed using NMP, DCM, and NMP in that order. After reacting with an amino acid with an Fmoc protecting group (3 equivalents), TBTU (3 equivalents), and DIPEA (7 equivalents) at room temperature, washing was performed with NMP. The above process was repeated until the peptide of the desired sequence was completed. In particular, for the synthesis of a cyclic peptide, Fmoc-E(OAll)-OH was first introduced into the biotin linker, and then the peptide ligation (cyclization) reaction was allowed to proceed.

[0060] 3-3. Synthesis and Purification of Linear Peptide Structures A sulfonamide, adamantane, cyclooctane, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or biotin functional group was introduced into the peptide synthesized in Example 3-2 to produce a linear peptide structure. The sulfonamide functional group was introduced by a click reaction of a peptide containing an azido substituent as described in Example 1-2. The introduction of adamantane, cyclooctane or DOTA functional groups proceeded by removing the Mtt or Dde protecting groups from the beads to which a peptide containing lysine protected with an Mtt or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) substituent was attached. The Mtt protecting group was removed by treatment with a mixed solution of 1.5% TFA / 2.5% TIS / 96% DCM, and the Dde protecting group was removed by reacting with Pd(PPh3)4 (1 equivalent) and 1,3-dimethylbarbituric acid (30 equivalents) in a DCM solvent under an argon (Ar) atmosphere. Thereafter, 1-adamantanecarboxylic acid (Cat#106399-25G, Sigma-Aldrich), cyclooctanecarboxylic acid (Cat#EN300-85433, Enamine) or DOTA-tris(tert-butyl ester) (2 equivalents), and TBTU (2 equivalents), DIPEA (5 equivalents) were reacted at room temperature in an NMP solvent for introduction. The biotin functional group was introduced by reacting with biotin-NHS (3 equivalents) and DIPEA (10 equivalents) at room temperature in an NMP solvent. After completion of the reaction, the beads were washed with NMP and DCM in sequence and dried under vacuum. An example of the reaction formula for introducing an adamantane functional group into the selected peptide is as follows.

[0061]

Chemical formula

[0062] 3-4. Synthesis and purification of cyclic peptide structures The cyclic peptide structure was formed through a cyclization reaction that forms a covalent bond between the carbonyl group of glutamic acid bound to the C-terminus of the amino acid and the amide group of the N-terminus of the amino acid in the peptide into which glutamic acid was introduced in Example 3-2 above. First, the allyl protection group removal reaction of glutamic acid was carried out with solid bead - shaped peptides. Under an argon (Ar) atmosphere, Pd(PPh3)4 (0.5 equivalent) and PhSiH3 (20 equivalents) were mixed in DCM solvent and then reacted at room temperature. The beads were washed successively with NMP and a 0.1 M sodium diethyldithiocarbamate / NMP solution to remove Pd impurities. After removing the Fmoc protection group with a piperidine / NMP (v / v = 1:4) mixed solution, the beads were washed successively with NMP, DCM, and NMP. The cyclization reaction proceeded at room temperature with PyAOP (3 equivalents, Cat#36813, GL Biochem) and DIPEA (10 equivalents). After the reaction was completed, the beads were washed successively with NMP and DCM and then dried under vacuum. An example of the cyclization reaction formula for the synthesis of cyclic peptides is as follows.

[0063] [Chemical formula]

[0064] 3 - 5. Separation and purification of the obtained linear or cyclic peptide structures The beads with the dried linear or cyclic peptides obtained in Examples 3 - 3 and 3 - 4 were treated with a 95% TFA / 2.5% TIS / 2.5% H2O mixed solution. The beads were removed using a filter, and the TFA mixed solution containing the peptide was collected in a conical tube. Then, most of the mixed solution was removed by blowing with nitrogen gas. Next, diethyl ether was added to precipitate the peptide, and after being placed in a centrifuge and rotated, the upper - layer diethyl ether and the small amount of remaining TFA mixed solution were removed. Then, the remaining solid - state peptide was dried under vacuum. The peptide cut with solid beads was dissolved in an ACN / H2O (1:1) mixed solution, and then insoluble foreign matters were removed using a filter (45μm syringe filter, Cat#DISMIC-3HP, Advantec, Japan). It was purified under the following conditions using a 1260 Infinity II LC system (Agilent): (1) The stationary phase was a Kromasil 100-5-C18 column (21.2×250mm, 5μm). (2) The mobile phase solvent was a mixed solution of triple-distilled water with 0.1% TFA added and ACN. (3) The flow rate was 15mL / min. (4) The detection wavelengths were 214 and 254nm. The purity and molecular weight of the purified peptide were measured using LC-MS (1260 infinity II, Infinity Lab LC / MSD, Agilent), and the data was analyzed using MNOVA (v.14.2.0, Mestrelab research, Spain). The stationary phase used was an Agilent Poroshell 120 EC-C18 column (4.6×50mm, 2.7μm), and the column temperature was maintained at 40°C. Also, as the mobile phase solvent, a mixed solution of triple-distilled water with 0.1% TFA added and ACN was used. The wavelength was 214 and 254nm, and the flow rate was maintained at 1mL / min. The purified peptide solution was used in powder form after removing the solvent using a freeze dryer. As a result, unique linear or cyclic peptide structures of the following Chemical Formulas 3 to 8 containing a peptide that specifically binds to human CAIX and having one or two sulfonamide functional groups were obtained.

[0065]

Chemical Formula

[0066]

Chemical Formula

[0067]

Chemical Formula

[0068]

Chem.

[0069]

Chem.

[0070]

Chem.

[0071] In Chemical Formulas 3 - 8, AA1 - AA7 represent amino acid residues, where lowercase letters indicate D - amino acids and uppercase letters indicate L - amino acids, and U means a D - amino acid that is modified or unnatural and has the following structure defined in this specification.

[0072]

Chem.

[0073] Example 4. Evaluation of the stability of peptide constructs Stability tests were conducted on the peptide constructs obtained in Example 3 above in serum and plasma. Solutions of the peptide constructs diluted to a final concentration of 50 μM in 1 mL of 100% pure human serum (Cat#S1, Merk) or plasma (Cat#70039.1, STEMCELL Technologies) [or 100% pure mouse serum (Sigma, Cat#S7273) or plasma (Rockland, Cat#D508-06-0050)] were aliquoted in 100 μL portions and then stored at 37°C. They were analyzed once a day for 7 days using LC-MS (1260 infinity II, Infinity Lab LC / MSD, Aglient) equipped with an Agilent Poroshell 120 EC-C18 column (4.6×50 mm, 2.7 μm). Before injecting the sample into the LC, 100 μL of ACN was added to the serum or plasma solution containing the peptide, and then it was centrifuged to precipitate the sediment. Only 10 μL of the supernatant was used for analysis.

[0074] Figures 5a and 5b, and Figures 6a and 6b respectively show the results of the stability tests of peptide constructs 35 and 48 in serum and plasma. Peptide construct 35 containing D-amino acids was completely retained without any degradation even after 6 days in serum and plasma. However, when all the amino acid residues of 35 were changed to the L-form, it was confirmed that it was completely degraded within 1 day in serum and within 3 days in plasma (Figures 5a and 5b). Similarly, peptide construct 48 containing D-amino acids was also shown to be completely retained without any degradation after 7 days in human or mouse serum and plasma. However, when all the amino acid residues of 48 were changed to the L-form, it was completely degraded within 1 day in serum and mostly degraded within 7 days in plasma (Figures 6a and 6b).

[0075] Example 5. Confirmation of the biological properties of peptide constructs 5-1. Analysis of binding affinity and binding kinetics for 5-1.hCAIX ECD The peptide constructs were analyzed for their binding affinity and binding kinetics to the hCAIX ECD. To confirm the specific binding selectivity to the hCAIX ECD, the binding kinetics of the peptide constructs were also analyzed to the extracellular domain of human carbonic anhydrase XII (hCAXII), another isoform of carbonic anhydrase. Binding affinity was measured using Bio-Layer Interferometry (BLI) technology. TM A system (Cat#45-5000, ForteBio, USA) and the advanced kinetics module of the BLItz Pro ver1.3 software were used. A streptavidin biosensor (Cat#18-5019, Sartorius, France) was used for all measurements. A protein concentration of 0 nM was used as a reference for nonspecific binding and background correction. First, 10X Octet kinetics buffer (Cat#18-1105, Sartorius) was diluted to 1X using DPBS (Biowest, Cat#L0615) and used as the analysis buffer. The following analysis buffer was dispensed at 200 μl / well into a 96-well, black, flat-bottom polypylene (Cat#655209, Greiner Bio-One, Austria), and hydration was carried out for 10 minutes with the streptavidin biosensor immersed in it. After attaching the biosensor to the equipment, the peptide was loaded at 1 μM concentration at 2200 rpm for 120 seconds to attach the peptide. After that, the protein was subjected to association and dissociation for 120 seconds at 2200 rpm for each concentration, and the kinetics data was analyzed using the global fitting function. D Values were calculated as the ratio of kd to ka, and kinetic parameters were obtained by baseline correction and fitting a 1:1 binding model. The results are shown in FIGS. 7a to 7d. The peptide construct showed significantly higher selectivity for hCAIX ECD compared to hCAXII ECD.

[0076] 5-2. Analysis of Binding Specificity to Target Cells To confirm whether the peptide construct specifically binds to target cells expressing CAIX, in vitro cell binding properties were analyzed. The human kidney cancer cell line SK-RC-52 expressing CAIX was purchased from Memorial Sloan Kettering Cancer Center (MSK, USA), and the human lung cancer cell line A549 expressing CAXII was purchased from the Korean Cell Line Bank (Cat#10185, KCLB, Korea).

[0077] Fluorescence-Activated Cell Sorting (FACS) Analysis The peptide construct used for the analysis was dissolved in DMSO to make a 1 mM solution and then diluted to the desired concentrations (10000, 100, 30, 10, 5 nM) using a DMEM mixture containing 1% FBS (FACS buffer solution). Two to three days before conducting the FACS analysis, 5×10 5 cells of the A549 cell line and 1-1.5×10 6 cells of the SK-RC-52 cell line were seeded. On the day of the experiment, the culture medium was removed from the T75 flask in which the A549 or SK-RC-52 cell line was cultured at 80% confluency, and the cells were washed once with 15 mL of PBS. After treating with 1 mL of trypsin-EDTA for 5 minutes in a Galaxy 170S CO2 incubator (37 °C, 5% CO2, humidified conditions), the trypsin-EDTA was neutralized with 9 mL of the culture medium. The neutralized cell mixture was placed in a 15 mL tube and centrifuged at 1000 RPM for 4 minutes using a VARISPIN 15R centrifuge. The supernatant of the centrifuged cell mixture was removed, and fresh culture medium was added to make 5×10 5After dilution to the number of cells, 100 μL was dispensed into a 96-well round plate (Cat#34096, SPL LIFE SCIENCE) per well. After rotating at 1000 RPM for 4 minutes using a VARISPIN 15R centrifuge, the supernatant was removed from each well, and 200 μL of the pre-prepared peptide construct solution was added. Then, it was cultured in a Galaxy 170S CO2 incubator at 37°C, 5% CO2, and humid conditions. After 1 hour, after rotating at 1000 RPM for 4 minutes using a VARISPIN 15R centrifuge, the supernatant was removed and washed with 200 μL of FACS buffer solution. After centrifugation (at 1000 RPM for 4 minutes) and removal of the wash solution, 200 μL of Streptavidin-Alexa fluor488 (Cat#S11223, Invitrogen, USA) diluted to a concentration of 1 μg / mL in FACS buffer solution was treated and cultured in a Galaxy170 S CO2 incubator at 37°C, 5% CO2, and humid conditions for 1 hour (for peptide constructs 80, 81, 82, the Streptavidin-Alexa fluor 488 treatment process was omitted). After VARISPIN 15R centrifugation (at 1000 RPM for 4 minutes) and removal of the supernatant, it was washed with 200 μL of FACS buffer solution. After an additional wash once in the same manner, 200 μL of FACS buffer solution was added to each well and mixed with the cells, and then transferred to a 5 mL round bottom test tube with a cell strainer (Cat#352235, CORNING, USA), and the fluorescence intensity of each cell was measured using a BD Accuri C6 Plus (BD Biosciences, Singapore). The measured data was analyzed using FlowJo 10.7.1 (BD biosciences, USA). The cell mixture after FACS analysis was centrifuged at 1000 RPM for 4 minutes using a VARISPIN 15R centrifuge, and then the supernatant was removed. 200 μL of Hoechst 33342 diluted to a concentration of 1 μg / mL in the FACS buffer solution was added to the cell mixture, and nuclear staining was performed at 4°C for 30 minutes. After centrifuging at 1000 RPM for 4 minutes using a VARISPIN 15R centrifuge, the supernatant was removed and the cells were washed twice with 200 μL of PBS. The cell mixture with 200 μL of PBS added was transferred to a confocal dish (Cat#100350, SPL LIFE SCIENCE), and fluorescence was observed using an Axio Observer 3 (ZEISS, Germany). The FACS analysis results using the SK-RC-52 cell line are shown in FIGS. 8a to 8d, and the FACS analysis results using the A549 cell line are shown in FIGS. 8e to 8h. From the FACS analysis results, it was confirmed that the peptide construct of the present invention binds to target cells expressing CAIX with high affinity and selectivity.

[0078] Immunofluorescence analysis (colocalization) The A549 cell line was seeded at a density of 5×10 4 cells, and the SK-RC-52 cell line was seeded at a density of 1×10 5Individual cells were seeded in a confocal dish and cultured for 2 days in a Galaxy 170 S CO2 incubator (37 °C, 5% CO2, humidified conditions). Subsequently, the M75-FITC antibody or peptide construct diluted in 2 mL of FACS buffer solution was added to each cell line such that the final concentrations were 1:200 (dilution ratio from the stock solution) and 1 μM, respectively. After treatment, the cells were cultured in the Galaxy 170 S CO2 incubator at 37 °C, 5% CO2, and humidified conditions for 1 hour. Next, the cells were washed twice with 3 mL of FACS buffer solution and then treated with streptavidin-Texas red and Hoechst 33342 diluted in 2 mL of FACS buffer solution such that the final concentrations were 1:50 (dilution ratio from the stock solution) and 0.5 μg / mL, respectively. The cells were then cultured in the Galaxy 170 S CO2 incubator at 37 °C, 5% CO2, and humidified conditions for 1 hour. Thereafter, the cells were washed twice with 3 mL of FACS buffer solution and once with PBS, and fluorescence was observed using an Axio Observer 3. Figure 9 shows the immunofluorescence analysis results of peptide construct No. 22. It was confirmed that peptide construct No. 22 specifically binds to the SK-RC-52 cell line, which expresses CAIX, similar to the M75 antibody.

[0079] Confocal microscopy imaging SK-RC-52 cell line 1×10 5Cells were seeded in a confocal dish and cultured for 2 days in a Galaxy 170 S CO2 incubator (37°C, 5% CO2, humidified conditions). Subsequently, the cell line was treated with a peptide construct diluted in 2 mL of FACS buffer solution to a final concentration of 100 nM, and then cultured in a Galaxy 170 S CO2 incubator at 37°C, 5% CO2, and humidified conditions for 1 hour. Then, after washing 3 times with 3 mL of FACS buffer solution, the cells were treated with Hoechst 33342 diluted in 2 mL of FACS buffer solution to a final concentration of 0.5 μg / mL, and reacted in a Galaxy 170 S CO2 incubator at 37°C, 5% CO2, and humidified conditions for 15 minutes. After washing 3 times with 3 mL of PBS, fluorescence was observed using a Leica SP5 confocal microscope. Figure 10 shows the Z-stack confocal microscopy analysis image of peptide construct No. 94 using the SK-RC-52 cell line. Peptide construct No. 94 was confirmed to show good binding to the CAIX ECD expressed from cells.

[0080] 5-3. Biodistribution analysis using experimental animal models Six-week-old female BALB / c nude mice (Orient Bio, Korea) were used, and 2×10 6 SK-RC-52 cells were injected subcutaneously into the upper left forelimb of the nude mice. After subcutaneous injection, the weight and tumor size were measured on days 13, 20, 27, and 35 (Figure 11a). The tumor size was measured using calipers [volume = (short axis (width)) 2 × (long axis (length)) / 2]. After xenografting the SK-RC-52 cell line, on the 36th day, 5 μM of the peptide construct was injected via tail vein injection in 200 μL (5% DMSO in saline solution) aliquots. After injection, the mice photographed at 24 hours were sacrificed by injecting an excessive amount of carbon dioxide, and then laparotomized to excise the tumor and organs (liver, kidney, spleen, lung, spleen, stomach, pancreas, heart), and ex vivo imaging was taken (Figure 11b). After performing ex vivo imaging on the tumors, livers, kidneys, pancreas, lungs, spleens, stomachs, and hearts of each group obtained from animal experiments, they were frozen in dry ice and stored at -80 °C until before the experiment. Before the experiment, after measuring the weight of each tumor, ice-cold homogenization buffer (40 mM EDTA, 6 mg / mL trypsin, PBS at pH 7.4 containing 1.6% Triton X-100 with a small amount of DNase 1) was placed in 100 μL of buffer per 100 mg of tissue weight, and homogenization was performed using FastPrep-24 5G (MP biomedicals, USA) for 5 minutes. The homogenized organs were dispensed with 100 μL of the homogenate into a 96-well black plate, and fluorescence was measured using an EnSpire Multimode Microplate Reader (PerkinElmer, USA) (Figure 11c). It was confirmed that the peptide construct specifically binds to tumors overexpressing CAIX compared to other organs.

[0081] 5-4. Analysis of the Anticancer Effect of the Peptide Construct-Drug Conjugate To confirm whether the conjugate obtained by conjugating a drug to the peptide construct exhibits an in vivo anticancer effect, an animal experiment was conducted. The SK-RC-52 cell line, a renal cancer cell overexpressing CAIX, was diluted to a cell count of 2×10 6 in PBS and injected subcutaneously into the right anterior side of 6-week-old female BALB / c nude mice (Orient Bio, Korea). The mice transplanted with tumor cells were weighed for 2-3 weeks to measure changes in the health of the mice, and the size of the tumor (short axis × short axis × long axis / 2) was measured using a Digimatic Caliper (Cat#500-151-30, Mitutoyo, Japan). When the tumor size reached approximately 100 mm 3When reaching, saline containing 1% DMSO and 2% EtOH was injected into the tail vein of the Control group, the Peptide treatment group with only the peptide structure injected into the tail vein (i.e., peptide structure No. 85), and the PDC1 group treated with the peptide structure and the drug (MMAE) conjugated via a linker (i.e., peptide structure No. 86 in the drug-conjugated form) by injecting into the tail vein. Each group was divided into three animals, and the Peptide treatment group was carried out on two animals. The administration concentration was 250 nmole / kg, and saline containing 1% DMSO and 2% EtOH at 200 μl was used as a vehicle for administration. The administration frequency was three times a week at two-day intervals for a total of seven times via the tail vein. Body weight and tumor size were measured once every 1 - 3 days. The results are shown in Figures 12a and 12b. As can be seen from Figures 12a and 12b, the peptide structure No. 86 in the drug-conjugated form with the MMAE drug conjugated via a linker did not induce a significant change in the body weight of the mice (Figure 12a), but showed an anti-cancer effect of significantly reducing the size of tumors overexpressing CAIX (Figure 12b).

[0082] 5 - 5. In vivo SPECT / CT imaging experiment of the peptide structure labeled with isotope ( 177 Lu) The isotope of the peptide structure was labeled to confirm the imaging diagnosis and therapeutic functionality. Peptide structure No. 95 was dissolved in DMSO to make a 10 mM stock solution, and then diluted with an ammonium acetate buffer solution at pH 5.5 to make a 1 mM peptide structure solution. The volume was reduced to 7 nmole with the 1 mM solution, and 1 mCi isotope ( 177After mixing with LuCl3, Eckert & Ziegler Radiopharma GmbH), an ammonium acetate buffer solution with a pH of 7.0 was added to adjust the pH to 5.5. Isotope labeling was carried out at 90 °C for 1 hour. After the labeling was completed, it was cooled to room temperature and then purified using a Sep-Pak column (Cat#186005125, Waters). The sample was loaded onto the activated Sep-Pak column (5 mL of ethanol, 5 mL of distilled water), washed with 5 mL of distilled water, and then eluted with 1.5 mL of ethanol. The received solution was purged with nitrogen and concentrated. The concentrated ethanol solution was diluted with saline before use. Six-week-old male BALB / c nude mice (Orient Bio, Seoul, Korea) were subcutaneously injected with 2×10 6 cells / 100 μL of the SK-RC-52 cell line, a human-derived renal cell carcinoma (RCC) cell line (Memorial Sloan-Kettering Cancer Center, MSK, New York, USA), into the lower part of the right anterior limb flank. 3 When the size of the tumor generated by the engraftment of the transplanted cancer cells SK-RC-52 reached an average of 100 mm 3 (80 - 100 mm ), group separation was performed based on the tumor size. The body weight and tumor size were measured twice a week. The tumor size was measured using Vernier Calipers (Mitutoyo, Kawasaki, Tumor volume Japan) to measure the short axis (width) and long axis (length) of the tumor, and then the tumor volume was calculated by substituting into the following formula. 3 Tumor volume (mm 2 ) = short axis 3 × long axis / 2 When the size of the tumor generated by transplanting the SK-RC-52 renal cell carcinoma cell line reached an average of 100 mm 3 (80 - 100 mm 177The 95th peptide structure labeled with Lu was administered once by the method of tail vein injection to mice, and SPECT / CT (Simens Inveon, software: Inveon Acquisition Workplace) was measured at 6 hours, 2 days, and 8 days respectively. For imaging, after anesthetizing the mice with respiratory anesthesia using 0.2% isoflurane in oxygen for about 3 - 5 minutes, after positioning them in the equipment, the anesthetic was continuously injected into the mice at 1.5 L / min, and measurements were taken for 50 minutes with SPECT and 7 minutes with CT, and the images were obtained with Inveon Research Workplace 4.2. The SPECT / CT video image photos obtained from the above were shown in Figure 13. Isotope 177 It was confirmed that the 95th peptide structure labeled with Lu remained bound to cancer cells SK - RC - 52 even after 8 days.

[0083] After that, the mice that had been measured by SPECT / CT until 8 days after sample injection were euthanized by inhaling carbon dioxide (CO2). After laparotomy, the tumors and organs (liver, kidney, spleen) were removed, and their weights were measured respectively, and the radiation dose for each organ was measured with a portable radioactivity measuring instrument (Inspector survey meter, INSPECTER(078 - 510)). The isotope measured by such a method 177 After converting the remaining amount of the 95th peptide structure labeled with Lu into the radioactivity intensity per unit weight, the relative radioactivity intensity of the remaining organs was calculated based on the tumor value and shown in Table 1 below.

[0084]

Table 1

[0085] 5 - 6. Analysis of the anti - cancer effect of the CAIX - targeted peptide (the 95th peptide structure) labeled with isotope ( 177 Lu) Peptide construct No. 95 was dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted with an ammonium acetate buffer solution at pH 5.5 to obtain a 1 mM peptide construct solution. The volume corresponding to 40 nmole in the 1 mM solution was reduced, and it was mixed with 14 mCi of the isotope ( 177 LuCl3, Eckert & Ziegler Radiopharma GmbH). After that, an ammonium acetate buffer solution at pH 7.0 was added to adjust the pH to 5.5. Isotope labeling was carried out at 90 °C for 1 hour. After the labeling was completed, it was cooled to room temperature and then purified using a 1200 Infinity LC system (Agilent) under the following conditions: (1) The stationary phase was an Agilent Poroshell 120 EC-C18 column (4.6 × 50 mm, 2.7 μm); (2) The mobile phase solvent was a mixture of triple-distilled water with 0.1% TFA and ACN; (3) The flow rate was 1 mL / min; (4) The detection wavelengths were 254 nm and 214 nm. 177 The chromatogram of the No. 95 target peptide construct labeled with Lu is shown in Figure 14. Obtained by purification 177 The Lu-labeled No. 95 target peptide construct was loaded onto an activated Sep-Pak column (5 mL of ethanol, 5 mL of distilled water), washed with 5 mL of distilled water to remove the organic solvent, and then eluted with 500 μL of ACN:H2O (1:1) and 700 μL of ethanol in sequence. The received solution was purged with nitrogen and concentrated. The concentrated ethanol solution was diluted with saline (EtOH 20%, DMSO 1%) for use.

[0086] The SK-RC-52 cell line, which is a kidney cancer cell overexpressing CAIX, was diluted to 2×10 6 cells in PBS and injected subcutaneously into the right anterior side of 6-week-old female BALB / c nude mice (Orient Bio, Korea). The mice transplanted with tumor cells were weighed for 2 - 3 weeks to measure the health changes of the mice, and the size of the tumor (short axis 2 × long axis / 2) was measured using a Digimatic Caliper (Cat#500-151-30, Mitutoyo, Japan). When the tumor size reached approximately 100 mm 3When reaching, six control group mice injected with saline as the vehicle and 177 were divided into six experimental group mice injected with approximately 500 μCi of the 95th target peptide structure labeled with Lu into the tail vein. The body weight and tumor size were measured three times a week. The results are shown in FIGS. 15a to 15c. As can be seen from FIGS. 15a to 15c, 177 the 95th target peptide structure labeled with Lu showed an anti-cancer effect of significantly reducing the size of tumors overexpressing CAIX (FIGS. 15a and 15c) while not inducing a significant change in the body weight of the mice (FIG. 15b).

[0087] The above description of the present invention is for illustrative purposes, and those with ordinary knowledge in the technical field to which the present invention pertains should understand that the scope of the rights of the present invention is not limited by the disclosed embodiments and the accompanying drawings, and various modifications can be made in other specific forms within the scope not deviating from the technical idea of the present invention.

[0088] Sequence listing

Table 2

Claims

Claim 1 A peptide ligand consisting of any one amino acid sequence among SEQ ID NOs: 1 to 44, wherein at least one of the constituent amino acids is composed of D - amino acid, and the lysine (Lys) residue among the constituent amino acids may be substituted with a chemical functional group from the side - chain ε - amino group, and a CAIX - specific peptide construct comprising a sulfonamide functional group - containing amino acid residue linked directly or via a spacer to the peptide ligand, wherein the CAIX - specific peptide construct is characterized by having the following linear structure, 【Chemical 1】 In the formula, P is the peptide ligand, F 8 is a sulfonamide functional group-containing amino acid residue, F 7 , F 9 and F 10 are each independently a functional group-containing amino acid residue other than a sulfonamide, or F 9 When there are a plurality of Fs, at least one F 9 is a sulfonamide functional group-containing amino acid residue, and the remaining Fs 9 and F 7 and F 10 are each independently a functional group-containing amino acid residue other than sulfonamide, the functional group other than the sulfonamide is a chelator, a cycloalkane having 5 to 15 carbon atoms, biotin, glucoheptonic acid, 4 - (p - iodophenyl) butyric acid (IB), a fluorescent dye, or a cytotoxic agent, S 3 and S 4 are each independently a spacer, s, t, u, v, and w each independently represent an integer from 0 to 3, CAIX - specific peptide construct. Claim 2 The CAIX - specific peptide construct according to claim 1, wherein the sulfonamide functional group - containing amino acid residue has the following structure. [Chemical 2] Claim 3 The CAIX - specific peptide construct according to claim 1, wherein the functional group - containing amino acid residue other than the sulfonamide is introduced through the side - chain ε - amino group of the lysine residue. Claim 4 The chelate is one or more selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid 2,2',2'',2'''-(ethane-1,2-diylbisnitrilo)tetraacetic acid (EDTA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N'''',N'''''-hexaacetic acid (HEHA), 2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid (PEPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), (1R,4R,7R,10R)-α,α',α'',α''' -tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) tetrasodium salt (DOTMA), 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA), and triethylenetetramine (TETA), and is characterized in that it is the CAIX-specific peptide construct according to claim 1.

5. The cycloalkane having 5 to 15 carbon atoms is one or more selected from cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, adamantane, norbornane, isobornane, and tricyclodecane, and is characterized in that it is the CAIX-specific peptide construct according to claim 1.

6. The spacer is one or more selected from a polyethylene glycol (PEG) linker, glycine, sarcosine, and a peptide linker composed of 1 to 5 D-amino acids or L-amino acids, and is characterized in that it is the CAIX-specific peptide construct according to claim 1.

7. The CAIX-specific peptide construct according to claim 1 is characterized by having any of the following structures. [Chemical Formula 3] [Chemical Formula 4] 【Chemical Formula 5】 (In the above, lowercase letters represent D-amino acids, uppercase letters represent L-amino acids, and the substituents represented by U are as defined below, respectively.) ​

8. A conjugate comprising the CAIX-specific peptide structure according to claim 1 or 7, directly or via a linker, bound to a fluorescent dye, a cytotoxic agent or a radioisotope.

9. The conjugate according to claim 8, characterized in that the linker is one or more selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 4-(2-pyridyldithio)butyric acid-N-hydroxysuccinimide ester (SPDB), and N-succinimidyl (4-iodo-acetyl)aminobenzoic acid (SIAB).

10. The conjugate according to claim 8, characterized in that the fluorescent dye is one or more selected from near-infrared fluorescent dyes, fluorescein types, rhodamine types, Alexa Fluor, 4,4-difluoro-4-boro-3a,4a-diaza-s-indacene (BODIPY), Texas Red, dansyl, Lissamine, cyanine (Cy), and phycoerythrin.

11. The conjugate according to claim 8, characterized in that the cytotoxic agent is one or more selected from toxins, chemotherapeutic agents, drug moieties, antibiotics, and nucleolytic enzymes.

12. The radioactive isotope is one or more selected from fluorine-18 (F-18), carbon-11 (C-11), carbon-14 (C-14), technetium-99m (Tc-99m), copper-64 (Cu-64), copper-67 (Cu-67), dysprosium-168 (Dy-168), bismuth-213 (Bi-213), samarium-153 (Sm-153), strontium-89 (Sr-89), strontium-90 (Sr-90), erbium-169 (Er-169), phosphorus-32 (P-32), palladium-103 (Pd-103), rhenium-186 (Re-186), rhenium-188 (Re-188), oxygen-15 (O-15), selenium-75 (Se-75), sodium-24 (Na-24), strontium-85 (Sr-85), lutetium-177 (Lu-177), yttrium-90 (Y-90), iodine-123 (I-123), iodine-125 (I-125), iodine-131 (I-131), iridium-192 (Ir-192), iridium-196 (Ir-196), ytterbium-166 (Yb-166), indium-111 (In-111), xenon-133 (Xe-133), nitrogen-13 (N-13), calcium-47 (Ca-47), cobalt-57 (Co-57), cobalt-60 (Co-60), chromium-51 (Cr-51), krypton-81 (Kr-81), potassium-42 (K-42), holmium-166 (Ho-166), gallium-67 (Ga-67), gallium-68 (Ga-68), actinium-225 (Ac-225), zirconium-89 (Zr-89), lead-212 (Pb-212), and astatine-211 (At-211), and is characterized by being one or more selected therefrom, the conjugate according to claim 8.

13. A pharmaceutical composition for the diagnosis, prevention or treatment of cancer, comprising the CAIX-specific peptide structure according to claim 1 or 7.

14. A pharmaceutical composition for the diagnosis, prevention or treatment of cancer, comprising the conjugate according to claim 8.

15. The cancer expresses carbonic anhydrase IX, and the pharmaceutical composition according to claim 13.

16. The pharmaceutical composition according to claim 13, wherein the cancer is liver cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, cancer near the anus, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, melanoma, thyroid cancer, astrocytoma or glioblastoma.

17. The pharmaceutical composition according to claim 14, wherein the cancer expresses carbonic anhydrase IX.

18. The pharmaceutical composition according to claim 14, wherein the cancer is liver cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, cancer near the anus, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, melanoma, thyroid cancer, astrocytoma or glioblastoma.

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