Peptide structures targeting carbonic anhydrous enzyme IX and their uses

Abstract

The present invention relates to peptide structures that specifically bind to carbonic anhydrase IX (CAIX) and their uses. The CAIX-binding peptide ligand within the peptide structures of the present invention contains a D-amino acid, is stable in the body, and has high binding specificity to CAIX. A cyclic CAIX-binding peptide structure containing this ligand can bind to CAIX with high affinity in the body, making it useful for the diagnosis, prevention, inhibition, or treatment of diseases mediated by CAIX.

Description

Technology Field

[0001] The present invention relates to a peptide structure that specifically binds to carbonic anhydrase IX (CAIX). Specifically, the present invention relates to a high-affinity CAIX-binding peptide structure comprising a CAIX-binding peptide ligand that is specific to CAIX and includes a D-amino acid to enhance stability, and to the use thereof for the diagnosis, prevention, inhibition, or treatment of diseases mediated by CAIX.

[0002] This research was conducted with funding from the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare, and with support from the National Drug Development Project of the National Drug Development Agency (Project ID: RS-2023-00218641).

[0003] This research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (RS-2023-00218641, Republic of Korea). Background Technology

[0004] Carbonic anhydrase (CA) is a compound found in zinc (Zn), which is commonly present in higher vertebrates, including humans. 2+ It is a metalloenzyme that catalyzes the reversible hydration reaction converting carbon dioxide into hydrogen ions and bicarbonate ions (CO2 + H2O ↔ HCO3 + H +Sixteen isozyme forms of these CAs have been identified, and in humans, they exist in various tissues including the gastrointestinal tract, reproductive tract, nervous system, kidneys, lungs, skin, and eyes. Most CA isozymes are known to be involved in important physiological processes such as 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).

[0005] Among the CA family, carbonic anhydrous enzyme IX (CAIX) is uniquely expressed very restrictively in normal tissues, whereas it is abnormally overexpressed in the majority of solid tumors; this is reported to be due to strong transcriptional activation by the transcription factor Hypoxia-Inducible Factor (HIF-1), which is induced by hypoxia caused by the excessive proliferation of solid tumors (De Simone et al., Biochimica et Biophysica Acta , 1804 :404-409, 2010; Thiry et al, ditto ).

[0006] Tumor hypoxia arises from the creation of an oxygen-deficient environment as solid tumors grow at a rate exceeding the blood supply capacity of the host vascular system. Even in a hypoxic microenvironment, solid tumors maintain continuous growth and proliferation through various genetic mutations. These hypoxic tumor cells exhibit increased resistance to chemotherapy and radiotherapy because it is difficult to deliver anticancer agents via the blood, and in the case of radiotherapy, there is a lack of oxygen necessary for the cytotoxic action of radiation-derived free radicals. Furthermore, hypoxic tumor cells induce the overexpression of CAIX on their cell surface, lowering the pH of the extracellular environment through CO2 hydration by the extracellular catalytic domain of CAIX. This resulting acidic tumor microenvironment can promote tumor cell invasion and metastasis and neutralize pH-sensitive agents (Thiry et al., ditto Therefore, tumor hypoxia is generally known as a poor prognostic factor for cancer patients.

[0007] Recently, active research has been conducted to inhibit the growth and proliferation of tumors associated with CAIX by targeting CAIX overexpressed in tumor cells or by disrupting the catalytic activity of CAIX to destroy pH regulation by tumor cells. These studies primarily focus on the development of monoclonal antibodies that bind to CAIX or small molecule inhibitors of the sulfonamide class. However, when targeting solid tumors, monoclonal antibodies with large molecular weights present difficulties in efficient delivery, and small molecule inhibitors of the sulfonamide class are relatively unstable in solution, which limits their usefulness as pharmaceutical compounds.

[0008] Therefore, there is a need to develop novel CAIX-specific binders and inhibitors with high affinity, stability, and suitability for the pharmaceutical use of cancer treatment, including the treatment, prevention, diagnosis, prognosis prediction, and imaging of cancer. Prior art literature

[0009] Thiry et al., TRENDS in Pharmacological Sciences, 27(11): 566-573, 2006De Simone et al., Biochimica et Biophysica Acta, 1804:404-409, 2010 The problem to be solved

[0010] One objective of the present invention is to provide stable CAIX-specific binders and inhibitors suitable for use in the treatment, prevention, diagnosis, or prognosis prediction of cancer diseases.

[0011] One object of the present invention is to provide a high-affinity CAIX-specific peptide structure comprising one or more effectors or functional groups, including a sulfonamide functional group, together with a CAIX-specific binding peptide ligand.

[0012] An additional objective of the present invention is to provide a conjugate comprising the peptide structure.

[0013] An additional objective of the present invention is to provide a composition for the diagnosis, prevention, or treatment of cancer comprising the peptide structure or conjugate.

[0014] An additional objective of the present invention is to provide a method for diagnosing cancer using the peptide structure or conjugate.

[0015] An additional objective of the present invention is to provide a method for treating cancer using the peptide structure or conjugate.

[0016] An additional objective of the present invention is to provide a method for predicting the prognosis after cancer treatment using the peptide structure or conjugate. means of solving the problem

[0017] As a result of diligent research to achieve the above objective, the inventors of the present application have developed novel CAIX-specific binders and inhibitors containing D-amino acids that are stable, highly affinity for CAIX, and capable of specifically binding to it.

[0018] In one aspect of the present invention, the present invention provides a peptide structure comprising an amino acid sequence of any one of SEQ ID NOs 1 to 29, wherein at least one of the constituent amino acids is a D-amino acid, and a lysine (Lys) residue among the constituent amino acids may be substituted with a chemical functional group at a side chain ε-amino group, or a 2,3-diaminopropionic acid (DAP) residue among the constituent amino acids may be substituted with a chemical functional group at a side chain β-amino group; and a sulfonamide functional group-containing amino acid residue connected to the peptide ligand directly or through a spacer, and having a structure of the following chemical formula 1.

[0019] [Chemical Formula 1]

[0020]

[0021] In the above formula,

[0022] P is the above-mentioned peptide ligand, and

[0023] F1, F2, and F3 are each independently sulfonamide functional group-containing amino acid residues, and

[0024] m is 0 or 1, and

[0025] F4 is the general formula -(F5) o -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is the basis, and here

[0026] F5 is a sulfonamide functional group-containing amino acid residue, and

[0027] S1 and S2 are each independently spacers, and

[0028] F6 and F7 are each independently amino acid residues containing functional groups other than sulfonamide, and

[0029] S3 is -NH2 or -OH, and

[0030] o is an integer of 0 or 1, and

[0031] p, q, r, and s each independently represent integers from 0 to 6.

[0032] Non-limiting examples of chemical functional groups that can be substituted at the side chain ε-amino group of the lysine (Lys) residue among the above constituent amino acids include 4-(p-iodophenyl)butyric acid (IB) or 2-(4-iodophenyl)acetic acid (sIB). Additionally, non-limiting examples of chemical functional groups that can be substituted at the side chain β-amino group of the 2,3-diaminopropionic acid (DAP) residue among the above constituent amino acids include α-D-galacturonic acid (Gal).

[0033] In the above CAIX-specific peptide structure, the sulfonamide functional group-containing amino acid residue may have the following structures, but is not limited to them.

[0034] or

[0035] In the above CAIX-specific peptide structure, functional groups other than sulfonamide may be introduced through the side chain ε-amino group of a lysine residue or through the side chain β-amino group of a 2,3-diaminopropionic acid (DAP) residue, and include, for example, a chelator, biotin, glucoheptonic acid, 4-(p-iodophenyl)butyric acid (IB), 2-(4-iodophenyl)acetic acid (sIB), zwitteric ion (SZW), α-D-galacturonic acid (Gal), a cycloalkane having 5 to 15 carbon atoms, a fluorescent dye, or a cytotoxic agent.

[0036] In one aspect, the present invention provides a conjugate comprising a peptide structure that is directly or through a linker coupled to a fluorescent dye, a cytotoxic agent, or a radioisotope to the CAIX-specific peptide structure.

[0037] In one aspect, the present invention provides a pharmaceutical composition for the diagnosis, prevention, or treatment of cancer comprising the peptide structure or conjugate. The cancer may be a cancer expressing carbonic anhydrous enzyme IX. The cancer may be selected from the group consisting of 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, prostatic cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renopelvic carcinoma, melanoma, thyroid cancer, astrocytoma, or glioblastoma, but is not limited thereto.

[0038] 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.

[0039] 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.

[0040] 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. Effects of the invention

[0041] The present invention provides novel high-affinity CAIX binders and inhibitors that specifically bind to CAIX while remaining stable. The CAIX binder of the present invention exhibits excellent stability in vivo by comprising a CAIX-specific binding peptide ligand containing one or more D-amino acids and is useful for CAIX targeting. Furthermore, the present invention provides a peptide structure in which one or more effectors or functional groups, including a sulfonamide functional group, are introduced through the side chain of an amino acid residue to the CAIX-specific peptide ligand, thereby providing a CAIX binder that is particularly useful for cancer imaging or diagnosis due to its high binding affinity for CAIX. The CAIX-specific peptide structure of the present invention can also be used as an effective agent for the diagnosis, prevention, or treatment of cancer by conjugating it with fluorescent dyes, cytotoxic agents, or radioisotopes. Brief explanation of the drawing

[0042] FIG. 1a is a peptide structure according to one embodiment of the present invention h CAIX extracellular domain (ECD) and human carbonic anhydrous enzyme XII ( h This is a graph showing the results of analyzing the binding affinity and binding kinetics characteristics for CAXII) ECD using the BLItz® system. FIG. 1b shows peptide structures 103 to 105 according to one embodiment of the present invention and peptide structures 103 to 105 labeled with lutetium-175 (Lu-175). h This is a graph showing the results of analyzing the binding affinity and binding kinetics characteristics of the CAIX extracellular domain (ECD) using the Octet®R8 system. FIG. 1c is a peptide structure 106 to 107 according to one embodiment of the present invention. h This is a graph showing the results of analyzing the binding affinity and binding kinetics characteristics of the CAIX extracellular domain (ECD) using the Octet®R8 system. FIG. 2a is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using SK-RC-52 cell lines of peptide structures 4 to 19 according to one embodiment of the present invention. FIG. 2b is a graph of the results of FACS analysis using SK-RC-52 cell lines of peptide structures 20 to 28, 61 to 66 and 71 according to one embodiment of the present invention. FIG. 2c is a graph of the results of FACS analysis using SK-RC-52 cell lines of peptide structures 72 to 87 according to one embodiment of the present invention. FIG. 2d is a graph of the results of FACS analysis using SK-RC-52 cell lines of peptide structures 88 to 102 according to one embodiment of the present invention. Figures 3a to 3m show the results of the biodistribution analysis of a peptide structure according to an embodiment of the present invention in mice xenografted with an SK-RC-52 cell line. FIGS. 3a to 3c show in vitro ( ex vivo This is a graph showing the captured image and the results of measuring fluorescence intensity. Figure 3d shows tumors and organs excised 24 hours after injection into mice, respectively, of the negative control (Dye) and seven peptide constructs (31, 33, 39, 40, 43, 45, and 46) that showed high tumor signals in the results of Figures 3a to 3c. ex vivo This is a figure showing the captured image and the graph of the measured fluorescence intensity. Figure 3e shows tumors and organs excised 72 hours after injection of peptide constructs 33, 39, and 40, respectively. ex vivo This is a figure showing the captured image and the graph of the measured fluorescence intensity. Figure 3f shows tumors and organs excised 72 hours after injection of peptide constructs 55, 56, 57, 58, 59, and 60, respectively. ex vivo This is a figure showing the captured image and the graph of the measured fluorescence intensity. Fig. 3g shows tumors and organs excised 5 days after injection of peptide constructs 58 and 60, respectively. ex vivo This is a figure showing the captured image and the graph of the measured fluorescence intensity. Figure 3h shows whole-body images of mice 24 hours after being injected with peptide constructs 61 and 62, respectively, and images of excised tumors and organs. ex vivo This is a figure showing the captured image and the graph of the measured fluorescence intensity. Figure 3i shows whole-body images of mice 24 hours after being injected with peptide constructs 63 and 64, respectively, and images of excised tumors and organs. ex vivo This is a figure showing the captured image and the graph of the measured fluorescence intensity. Figure 3j shows whole-body images of mice 24 hours after being injected with peptide constructs 65, 75, 80, 83, and 84, respectively, and images of excised tumors and organs. ex vivo This is a figure showing the captured image and the graph of the measured fluorescence intensity. Figure 3k shows whole-body images of mice 24 hours after being injected with peptide constructs 85, 87, 89, 90, and 91, respectively, and images of excised tumors and organs. ex vivo This is a figure showing the captured image and the graph of the measured fluorescence intensity. Figure 3L shows whole-body images of mice 72 hours after being injected with peptide constructs 63, 85, 87, 92, and 97, respectively, and images of excised tumors and organs. ex vivoThis is a figure showing the captured image and the graph of the measured fluorescence intensity. Figure 3m shows whole-body images of mice 72 hours after being injected with peptide constructs 98 and 99, respectively, and images of excised tumors and organs. ex vivo This is a figure showing the captured image and the graph of the measured fluorescence intensity. Fig. 4a is 177 This figure shows the results of measuring mouse SPECT / CT images after 1.5 hours, 1 day, 2 days, 3 days, 7 days, 8 days, or 10 days in mice injected with Lu-labeled peptide constructs 67, 68, 69, and 70, respectively. Arrows indicate tumors. Fig. 4b is 177 This figure shows a graph of the results of measuring changes in body weight in mice injected with Lu-labeled peptide structures 67, 68, 69, and 70, respectively. Fig. 4c is 177 This figure shows a graph of the results of measuring changes in tumor size (anticancer effect) in mice injected with Lu-labeled peptide structures 67, 68, 69, and 70, respectively. Fig. 5a is 177 This figure shows the results of measuring mouse SPECT / CT images after 1.5 hours, 1 day, 2 days, or 3 days in mice injected with Lu-labeled peptide constructs 103, 104, and 105, respectively. Arrows indicate tumors. Fig. 5b is 177 This figure shows a graph of the results of measuring changes in body weight in mice injected with Lu-labeled peptide structures 103, 104, and 105, respectively. Fig. 5c is 177 This figure shows a graph of the results of measuring changes in tumor size (anticancer effect) in mice injected with Lu-labeled peptide structures 103, 104, and 105, respectively. Figure 6a shows the HPLC analysis conditions for the synthesized material. Figure 6b shows the HPLC retention times and molecular weights of synthesized substances 1 to 28 observed using electrospray ionization mass spectrometry (MS). Figure 6c shows the HPLC retention times and molecular weights of synthesized substances 29 to 68 observed using electrospray ionization mass spectrometry (MS). Figure 6d shows the HPLC retention times and molecular weights of synthesized substances 69 to 107 observed using electrospray ionization mass spectrometry (MS). Specific details for implementing the invention

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Any method or material similar or equivalent to those described herein may be used in the practice or testing of the present invention.

[0044] The present invention allows for various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0045] CAIX-specific peptide structure of the present invention

[0046] The present invention provides a peptide structure comprising a sulfonamide functional group-containing amino acid residue connected to a CAIX-specific peptide ligand either directly or through a spacer.

[0047] The above CAIX-specific peptide ligand may include any one of the amino acid sequences 1 to 29. The above peptide ligand may include D-amino acids or may consist solely of D-amino acids. Additionally, the above peptide ligand may be substituted with one or more chemical functional groups, for example, 4-(p-iodophenyl)butyric acid (IB) or 2-(4-iodophenyl)acetic acid (sIB), but not limited thereto, at the side chain ε-amino group of a lysine (Lys) residue among the constituent amino acid residues. Additionally, the above peptide ligand may be substituted with one or more chemical functional groups, for example, α-D-galacturonic acid (Gal), but not limited thereto, at the side chain β-amino group of a 2,3-diaminopropionic acid (DAP) residue among the constituent amino acid residues.

[0048] The above peptide ligand may be prepared such that amino acid residues having a specific sequence are combined with each other to form part of a peptide structure. The above peptide ligand may be prepared by a known peptide synthesis method and is not particularly limited. In one embodiment, the above peptide ligand may be prepared by repeating the peptide synthesis process on a solid single bead until a peptide of a desired length and sequence is completed.

[0049] The above CAIX-specific peptide ligand may also include its salt form.

[0050] The introduction of a sulfonamide functional group into the sulfonamide functional group-containing amino acid residue of the above-described peptide structure 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.

[0051] or

[0052] The peptide structure may further include one or more other functional group-containing amino acid residues other than the sulfonamide functional group. The functional group other than the sulfonamide may be introduced through the side chain of the amino acid residue. The amino acid residue into which the functional group is introduced may preferably be lysine or 2,3-diaminopropionic acid (DAP).

[0053] In one embodiment, non-limiting examples of functional groups other than sulfonamide that can be introduced into the peptide structure of the present invention may include a chelator, biotin, glucoheptonic acid, 4-(p-iodophenyl)butyric acid (IB), 2-(4-iodophenyl)acetic acid (sIB), zwitteric ion (SZW), α-D-galacturonic acid (Gal), cycloalkane having 5 to 15 carbon atoms, fluorescent dye, or cytotoxic agent.

[0054] The above chelator is, for example, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazcyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid-2,2',2'',2'''-(ethane-1,2-diyldinitrilo)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-pentazacyclopentadecane-N,N',N'',N''',N'''-pentaacetic acid (PEPA), 1,4,7,10-tetraazcyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), (1R, 4R, 7R, 10R)-α, α', α'', α'''-tetramethyl-1,4,7,10-tetraazcyclododecane-1,4,7,10-tetraacetic acid)tetrasodium salt (DOTMA), 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA) and triethylenetetramine (TETA), It may be one or more selected from 1,4,7,10-tetraazcyclododecane-7-acetamide-1,4,10-triacetic acid (Pb specific chelator, PSC) and 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazcyclododec-1-yl]acetamide (DOTAM or TCMC), but is not limited thereto.

[0055] The above cycloalkanes having 5 to 15 carbon atoms may be one or more selected from, for example, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, adamantan, norbonane, isobonane, and tricyclodecane, but are not limited thereto.

[0056] The peptide structure of the present invention may also connect a chemical functional group-containing amino acid residue, such as a sulfonamide functional group, to a CAIX-specific binding peptide ligand through a spacer. The spacer may be, for example, one or more selected from polyethylene glycol (PEG) linkers, glycine, sarcosine, γ-carboxyglutamic acid, and peptide linkers composed of 1 to 8 D-amino acids or L-amino acids, but is not limited thereto.

[0057] The peptide structure of the present invention has the structure of Chemical Formula 1 below.

[0058] [Chemical Formula 1]

[0059]

[0060] In the above formula,

[0061] P, F1, F2, F3, F4 and n are each as defined above.

[0062] In one embodiment of the present invention, the peptide structure has the structure of the following chemical formula 2.

[0063] [Chemical Formula 2]

[0064]

[0065]

[0066] In the above formula,

[0067] R5 is the general formula -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is the basis, and

[0068] Here, S1, S2, S 3, F6, F7, p, q, r, and s are each as defined above, and

[0069] Lowercase letters represent D-amino acids, uppercase letters represent L-amino acids, and other substituents indicated by abbreviations in R1 to R4 and AA1 to AA5 are defined as follows.

[0070]

[0071] Although not bound by any specific theory, it is believed that cyclic structures, such as the cyclic peptide structure of the present invention, have less flexibility compared to linear peptides, resulting in less entropy loss upon target binding, higher binding affinity, and increased binding specificity to the target.

[0072] The peptide structure of the present invention exhibits high selectivity, specifically binding to CAIX but not to other isoenzymes of CAIX (e.g., carbonic anhydrous enzyme XII).

[0073] Conjugate

[0074] The CAIX-specific peptide structure of the present invention can form a conjugate by being directly or through a linker bound to a fluorescent dye, a cytotoxic agent, or a radioisotope. The conjugate can target CAIX and label cancers expressing CAIX with a fluorescent dye or a radioisotope, or effectively deliver drugs such as radioisotopes or cytotoxic agents to said cancers, making it useful for the diagnosis, prevention, or treatment of cancer.

[0075] In one embodiment, the linker may be 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-pyridylthio)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)aminobenzoate (SIAB), but is not limited thereto.

[0076] In one embodiment, non-limiting examples of the fluorescent dye may include one or more selected from near-infrared fluorescent dyes, fluorescein type, rhodamine type, Alexa Fluor, 4,4-difluoro-4-boro-3a, 4a-diaza-s-indacen (BODIPY), Texas Red, dansyl, lissamine, cyanine (Cy), and phycoerythrin.

[0077] In one embodiment, the cytotoxic agent may be one or more selected from toxins, chemotherapy agents, drug moiety, antibiotics, and nucleases, but is not limited thereto.

[0078] In one embodiment, the radioisotopes are Fluorine-18 (F-18), Carbon-11 (C-11), Carbon-14 (C-14), Techtenium-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), It may be one or more selected from 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), thorium-227 (Th-227), radium-223 (Ra-223), tin-117m (Sn-117m), lead-103 (Pb-103), terbium-161 (Tb-161), lead-212 (Pb-212), and astatine-211 (At-211).

[0079] Therapeutic administration and formulation

[0080] The present invention provides a pharmaceutical composition for the diagnosis, prevention, or treatment of cancer comprising the CAIX-specific peptide structure or conjugate of the present invention. Preferably, the cancer of the present invention is a solid tumor. More preferably, the cancer of the present invention is a cancer expressing CAIX. For example, the cancer of the present invention may be a solid tumor 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, prostatic cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renopelvic carcinoma, melanoma, thyroid cancer, astrocytoma, or glioblastoma, but is not limited thereto.

[0081] The subject to whom the pharmaceutical composition for the diagnosis, prevention, or treatment of cancer of the present invention is administered may be a mammal that is at risk of developing cancer, has been diagnosed with cancer, or has received cancer treatment. The mammal may be a human or a mammal other than a human.

[0082] The pharmaceutical composition for the diagnosis, prevention, or treatment of cancer according to the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external formulations, suppositories, and sterile injectable solutions according to conventional methods, and may include a suitable carrier, excipient, or diluent that is conventionally used in the manufacture of pharmaceutical compositions for formulation.

[0083] Various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used as the carrier, excipient, or diluent.

[0084] When formulating, it can be manufactured using diluents or excipients such as fillers, weights, binders, wetting agents, disintegrants, and surfactants commonly used in the pharmaceutical industry.

[0085] The preferred dosage of the pharmaceutical composition for the prevention or treatment of cancer according to the present invention may vary depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at a dose of 0.0001 to 2,000 mg / kg per day, preferably 0.001 to 2,000 mg / kg. The administration may be performed once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosage.

[0086] The pharmaceutical composition for the prevention or treatment of cancer according to the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes. The method of administration may be, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater, or intracerebroventricular injection.

[0087] The present invention also provides a method for treating cancer, comprising administering the CAIX-specific peptide structure or conjugate of the present invention to an individual requiring treatment for cancer.

[0088] The CAIX-specific peptide structure and conjugate of the present invention may also be used to diagnose cancer by targeting and imaging cancer, or to predict or observe the treatment prognosis of an individual after cancer treatment by administering it to the individual who has received cancer treatment.

[0089] The present invention will be described in more detail below through examples. These examples are provided solely to illustrate the invention more specifically and are not intended to limit the legitimate scope of the invention; it will be obvious to those skilled in the art that various modifications are possible within the scope of the invention.

[0090] Examples

[0091] Example 1. Synthesis and purification of a peptide structure containing selected peptides

[0092] 1-1. Synthesis of reagents inserted into peptide structures

[0093] N-succinimidyl 2,3:4,5:6,7-tri-O-isopropylidene-D-glycero-D-gulo-heptonate (6OH(Protected)-NHS) inserted into a peptide structure S1 ) and Fmoc-DAP(DOTA(Protected))-OH ( S2 ), Fmoc-DAP(Gal(Protected))-OH ( S3 ), and 3-(dimethylamino)propane-1-sulfonic acid ( S4 ), Fmoc-D-ADMA(pbf)-OH (Fmoc-r(dimet)-OH) ( S5 ) was synthesized directly and used. First S1For the synthesis, methyl 2,3:4,5:6,7-tri-O-isopropylidene-D-glycero-D-gulo-heptonate (1 equivalent) and lithium hydroxide monohydrate (2.5 equivalents) were added to a 50 mL round-bottom flask containing a magnetic stirrer bar, and then MeOH was added and the reaction was carried out at room temperature for 24 hours. After the reaction was complete, volatile substances and the solvent were removed from the reaction mixture using an evaporator. The reaction mixture was dissolved in a 10% aqueous citric acid solution and transferred to a separatory funnel, after which the organic matter was extracted using ethyl acetate. The extracted ethyl acetate solution was washed with a saturated aqueous solution of NaCl in a separatory funnel and then dried with sodium sulfate in an Erlenmeyer flask. Afterward, the sodium sulfate was removed from the ethyl acetate solution using filter paper, and the volatile solvent was removed using an evaporator. The reaction product was dissolved once again in dichloromethane (DCM), and the volatile solvent was removed using an evaporator.

[0094] The 2,3:4,5:6,7-tri-O-isopropylidene-D-glycero-D-gulo-heptonic acid (1 equivalent) synthesized in this way was combined with N-hydroxysuccinimide (NHS) and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC hydrochloride, 1.6 equivalents) in a 50 mL round-bottom flask containing a magnetic stirrer bar for the following reaction, and then DCM anhydrous was added and the reaction was carried out at room temperature for 24 hours. After the reaction was finished, the reaction mixture was dissolved in ethyl acetate, transferred to a separatory funnel, and washed with a saturated aqueous solution of NaCl. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate, followed by washing with a saturated aqueous solution of NaCl. The organic layer was collected in an Erlenmeyer flask and dried with anhydrous sodium sulfate. Subsequently, the sodium sulfate was removed from the organic solution using filter paper, and the volatile solvent was removed using an evaporator. The reaction product was dissolved in DCM once again, and after completely removing the volatile solvent using an evaporator, it was used. As described above S1 Examples of reactions that synthesize functional groups are as follows.

[0095] [Reaction Equation 1]

[0096]

[0097] Fmoc-DAP(DOTA(Protected))-OH ( S2 ) and Fmoc-DAP(Gal(Protected))-OH ( S3The synthesis of ) first dissolves unprotected Fmoc-DAP-OH (1 equivalent) in N,N-dimethylformamide (DMF), and then tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, DOTA-tris(tert-butyl ester), or 1,2:3,4-di-O-isopropylidene-aD-galacturonide (1.5 equivalents) A solution of 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (1.2 equivalents), N,N-Diisopropylethylamine (DIPEA) (3 equivalents), and activated compounds dissolved in DMF was mixed and reacted for 2 hours. Subsequently, the progress of the reaction was monitored using LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent), and the results were purified using a 1260 Infinity II LC system (Agilent) for use. The analytical instrument used was LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent), and MNOVA (v. 14.2.0, Mestrelab research, Data were analyzed using (Spain). An Agilent Poroshell 120 EC-C18 column (4.6 x 50 mm, 2.7 μm) was used as the stationary phase, and the column temperature was maintained at 40°C. In addition, trifluoroacetic acid (TFA) 0.A mixture of acetonitrile (ACN) containing 1% TFA and triple-distilled water containing 0.1% TFA was used, and the wavelengths were maintained at 214 and 254 nm and the flow rate at 1 mL / min. Purification was performed using a 1260 Infinity II LC system (Agilent) under the following conditions: (1) the stationary phase was a Kromasil 100-5-C18 column (21.2 X 250 mm, 5 μm), (2) the mobile phase solvent was a mixture of triple-distilled water and ACN with 0.1% TFA added, (3) the flow rate was 15 mL / min, and (4) the detection wavelengths were 214 and 254 nm. S2 and S3 The synthesis process of is as follows.

[0098] [Reaction Equation 2]

[0099]

[0100] S4 For the synthesis of [the substance], 1 g of 1,3-propanesultone (1,3-Propanesultone (Cat# P0324, TCI)) and 16 mL of 2 M dimethylamine in THF solution (Cat# D3948, TCI) were added to a 100 mL round-bottom flask and stirred at room temperature for 24 hours. The reaction mixture was concentrated using an evaporator, and the resulting gel-like substance was first precipitated using ethyl acetate, and the precipitate was separated using a nylon membrane filter. The separated precipitate was dissolved in a minimal amount of water, and the substance was recrystallized by adding DMF. After separation using a nylon membrane filter, the substance was washed with ethyl acetate and dried. S4 Examples of synthesis are as follows.

[0101] [Reaction Equation 3]

[0102]

[0103] S5 The synthesis process of is as follows.

[0104] Fmoc-D-Orn(boc)-OH (1 equivalent, Cat# BD131117, BLD Pharmatech), N, N-Dicyclohexylcarbodiimide (N, N-Dicyclohexylcarbodiimide, DCC, 1.5 equivalents, Cat# 36650, Sigma-Aldrich), 1-hydroxybenzotriazole (1-hydroxy benzotriazole, HOBt, 1.5 equivalents, Cat# CXZ010, Aapptec), 4-(Dimethylamino)pyridine (4-(Dimethylamino)pyridine, DMAP, 0.05 equivalents, Cat# 107700, Sigma-Aldrich) and DCM anhydrous were added to a round-bottom flask and stirred well, and reacted at room temperature for 3 hours. After confirming that sufficient precipitation had occurred, allyl alcohol (2 equivalents, Cat# 240532, Sigma-Aldrich) was added and the reaction was carried out at room temperature for 16 hours. After the reaction, most of the precipitate was filtered through a paper filter, and the resulting solution was concentrated using an evaporator. The gel-like substance was then completely dissolved in a small amount of ethyl acetate, and the solution was stored at 20°C for 16 hours to induce precipitation. The precipitate of the ethyl acetate solution containing the dissolved reactant was filtered through a paper filter, transferred to a separatory funnel, and 100 mL of ethyl acetate was added. The organic layer was washed once with a saturated sodium bicarbonate aqueous solution (Cat# S0341, Samjeon Soonyak Industrial Co., Ltd.), twice with triple distilled water, and once with a saturated sodium chloride aqueous solution (Cat# S0476, Samjeon Soonyak Industrial Co., Ltd.) using a separatory funnel. Anhydrous magnesium sulfate (Cat# M1806, Samjeon Soonyak Industrial Co., Ltd.) was added to the washed organic layer and dried, and then the solution obtained by filtering the precipitate through a paper filter was concentrated using an evaporator.The concentrated gel-form material was completely dissolved in a minimal amount of DCM, and half of the solution was purified using a column (40 x 210 mm) containing 200 g of silica gel (0.063–0.200 mm, Cat# 1.07734.1000, Sigma-Aldrich). An ethyl acetate : hexane (Cat# 1.07734.1000, Sigma-Aldrich) = 1 : 1 solution was used as the developing solvent. The remaining DCM solution was also purified in the same manner using a silica gel column. The reaction product, Fmoc-D-Orn(boc)-OAll, had an Rf of 0.59 (ethyl acetate : hexane = 1 : 1). The purified solution with this Rf was collected and concentrated using an evaporator to obtain Fmoc-D-Orn(boc)-OAll.

[0105] For the next reaction, to remove the Boc protecting group, Fmoc-D-Orn(boc)-OAll (1 equivalent) was placed in a round-bottom flask, and a TFA : DCM = 1 : 1 mixed solution was slowly poured in at room temperature, mixed, and stirred well for 1 hour. After the reaction, the solution was concentrated using an evaporator. The process of dissolving the concentrated material in DCM and concentrating it again using an evaporator was repeated 3 times. Then, the concentrated material was dissolved in DCM anhydrous (Cat# 270997, Sigma Aldrich), sealed, and stored in a freezer. The molecular weight and purity of the concentrated material were confirmed using LC-MS as in Example 1-1 above, and then used in the next reaction without any special purification process.

[0106] 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl isothiocyanate (2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl isothiocyanate, pbf-NCS), which is required for the next reaction, was first synthesized as follows.

[0107] A solution of tetrabutylammonium thiocyanate (Bu4NNCS, 5 equivalents, Cat# T1278, TCI) and 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl chloride (pbf-Cl, 1 equivalent, Cat# BD41320, BLD Pharmatech) dissolved in anhydrous DCM was added to a round-bottom flask, and after adding a reflux condenser, the reaction solution was refluxed (75°C) and reacted for 1 hour. The reaction product pbf-NCS [Rf=0.49 (hexane : DCM = 1 : 1)] was confirmed by TLC. After the reaction was complete, the concentrated gel-like substance was completely dissolved in a minimal amount of DCM and purified using a column (40 x 210 mm) containing 200 g (0.063–0.200 mm) of silica gel. The developing solvent used was ethyl acetate : hexane = 1 : 1. The purified solution with an Rf of 0.49 was collected and concentrated using an evaporator to obtain a clear liquid pbf-NCS. The procedure of this reaction is as follows.

[0108] [Reaction Equation 4]

[0109]

[0110] For the next reaction, the Fmoc-D-Orn-OAll (1 equivalent) synthesized above, pbf-NCS (1.2 equivalents), and DIPEA (10 equivalents) were dissolved in DCM in a round-bottom flask and reacted at room temperature for 1 hour. After the reaction, the solution was concentrated using an evaporator, and the concentrated material was completely dissolved in a small amount of DCM and then purified using a column (40 x 210 mm) containing 200 g (0.063-0.200 mm) of silica gel. Ethyl acetate : hexane = 1 : 1 was used as the developing solvent. The reaction product, Fmoc-D-Orn(SCN(pbf))-OAll, had an Rf of 0.62 (ethyl acetate : hexane = 1 : 1). The purified solution with the corresponding Rf was collected, concentrated using an evaporator, and used for the next reaction.

[0111] In a round-bottom flask, synthesized Fmoc-D-Orn(SCN(pbf))-OAll (1 equivalent) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 2 equivalents, Cat# E7750, Sigma-Aldrich) were dissolved in DCM, and 2 M dimethyl amine in THF (2 equivalents, Cat# 391956, Sigma-Aldrich) was added and reacted at room temperature for 10 minutes. After confirming the molecular weight and purity of the reaction mixture using the LC-MS of Example 1-1, the reaction mixture was concentrated using an evaporator once the reaction was complete. The concentrated substance was dissolved in a mixed solution of ACN and triple-distilled water (1:1) and filtered through a 45 μm syringe filter (Cat# DISMIC-3HP, Advantec), and purified using HPLC as in Example 1-1. The purified substance, Fmoc-D-ADMA(pbf)-OAll, was freeze-dried to obtain it in powder form.

[0112] Finally, under Ar, the synthesized Fmoc-D-ADMA(pbf)-OAll (1 equivalent), Pd(PPh3)4 (0.05 equivalents), and N-methyl aniline (3 equivalents, Cat# M29304, Sigma-Aldrich) in a glass vial were dissolved in anhydrous THF and reacted at room temperature for 1 hour. After confirming the molecular weight and purity of the reactants by LC-MS, the reactants were concentrated using an evaporator. The concentrated material was dissolved in a mixed solution of ACN / triple distilled water (1:1), filtered through a 45 μm syringe filter, and purified under the same HPLC conditions used above. The purified substance was freeze-dried to obtain Fmoc-D-ADMA(pbf)-OH (N-α-Fmoc-N,N-ω-dimethyl-N-ωÆ-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-D-arginine, Fmoc-r(dimet)-OH, S5 ) was obtained in powder form. S5 The entire synthesis process is as follows.

[0113] [Reaction Equation 5]

[0114]

[0115] 1-2. Synthesis of Solid-Phase Peptides

[0116] Solid-phase peptide chains were synthesized using an automated ultrasonic peptide synthesizer (Liberty Blue™ automated microwave peptide synthesizer, CEM Corporation) or an automated synthesizer (Apex 396, AAPPTEC). Two types of solid-phase resins were used for the synthesis, differing in the C-terminus of the resulting peptides. Rink Amide-ChemMatrix® Resin (0.41 mmole / g, Cat# 7-600-1310, Biotage), used for most materials, has a -CONH2 C-terminus, while 2-Chlorotrityl Chloride Resin has a -COOH C-terminus. Rink Amide-ChemMatrix® Resin can be used directly for synthesis, but 2-Chlorotrityl Chloride Resin (1.07 mmole / g, Cat# GLS210819-48101, BL BioChem) was used after mounting by dissolving the first amino acid (5 equivalents) and DIPEA (10 equivalents) in DCM solvent and reacting for 12 hours under an Ar atmosphere. In an automated ultrasonic peptide synthesizer, a resin swollen in N-methyl-2-pyrrolidone (NMP) was treated with Fmoc-amino acid (5 equivalents), Oxyma Pure (5 equivalents, Cat# cxz021, AApptec), and diisopropylcarbodiimide (DIC, Cat# D0254, TCI) (10 equivalents) in NMP solvent. After coupling in NMP solvent at 75°C for 5 minutes, the resin was washed 3 times with NMP, and the Fmoc protecting group was removed by reacting with a piperidine / NMP (v / v = 1:4, 0.1 M OxymaPure) mixed solution at 90°C for 1 minute. After repeating the Fmoc-amino acid synthesis process to connect the beads, the beads were collected from the synthesizer.Similarly, in an automated synthesizer (Apex 396, AAPPTEC), the resin swollen in NMP was treated with Fmoc-amino acid (5 equivalents), TBTU (5 equivalents), and DIPEA (10 equivalents) in NMP solvent and coupled at room temperature for 45 minutes. Afterward, the resin was washed with NMP and DCM, and the Fmoc protecting group was removed by reacting a piperidine / NMP (v / v = 1:4) mixed solution at room temperature for 10 minutes. After repeating the Fmoc-amino acid synthesis process to connect the groups, the beads were collected from the synthesizer. Furthermore, when the materials synthesized in Example 1-1 were used for solid-phase peptide synthesis, the reaction was performed directly manually rather than through instrumental synthesis, and the number of equivalents of the reagents used in the synthesis was reduced to 2 equivalents. In particular, for the synthesis of cyclic peptides, Fmoc-E(OAll)-OH (Cat#36613, GL Biochem) was first introduced, followed by a peptide linkage (cyclization) reaction.

[0117] 1-3. Synthesis of Structures and Functional Groups of Solid-Phase Peptides

[0118] 1-3-1. Cyclic reaction

[0119] The reaction to remove the allyl protecting group from glutamic acid of solid beads synthesized using an automated synthesizer was carried out. Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4, 1 equivalent, Cat# 216666, Sigma-Aldrich) and PhSiH3 (30 equivalents, Cat# 335150, Sigma-Aldrich) were dissolved in DCM solvent under an argon (Ar) atmosphere, placed in a filter-equipped cylinder containing solid beads, sealed with a lid, and reacted for 1 hour while suspended from a 180° shaker at room temperature. Afterward, the beads were washed 3 times with NMP, and then washed at least 5 times with a 0.1M sodium diethyldithiocarbamate (Cat# D3506, Sigma-Aldrich) in NMP solution to remove Pd impurities. Afterward, the beads were washed three times with NMP, and 3 mL of piperidine / NMP (v / v = 1:4) solution was added to remove the N-terminal Fmoc protecting group. The beads were then hung on a 180° shaker at room temperature for 5 minutes, after which the solution was emptied and refilled, and the mixture was reacted for 10 minutes. After the reaction was complete, the beads were washed three times each with NMP, DCM, and then NMP. The cyclization reaction of beads from which both Fmoc and OAll had been removed was carried out by adding an NMP solution mixed with (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (5 equivalents, Cat# 36813, GL Biochem) and DIPEA (10 equivalents), sealing the container with a lid, and then hanging it on a 180° shaker at room temperature for 12 hours. The solid beads after the reaction were washed three times with NMP and DCM and used for the next reaction. An example of a cyclization reaction scheme for the synthesis of cyclic peptides is shown below.

[0120] [Reaction Equation 6]

[0121]

[0122] 1-3-2. Synthesis of Functional Groups

[0123] After the cyclization reaction, the solid-phase peptide requiring the synthesis of a functional group undergoes the following reaction on the beads.

[0124] The introduction of Biotin, DOTA, PSC, TCMC, IB, and sIB functional groups was carried out by removing the Mtt or Dde protecting group from a bead equipped with a peptide containing lysine protected by a 4-methyltrityl (Mtt) or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) substituent. The Mtt protecting group was removed by treating with a mixed solution of 1.5% TFA / 2.5% TIPS / 96% DCM at room temperature for 5 minutes each, for a total of 6 times, and the Dde protecting group was removed by mixing hydroxylamine hydrochloride / imidazole (1.3:1, w / w) in NMP solvent and reacting at room temperature for 2 hours twice or for 12 hours once.Subsequently, on beads having amine functional groups from which the protecting group has been removed, Biotin-NHS ester (2 equivalents, Cat# 246201, SY Innovation) or DOTA-tris(tert-butyl ester) (2 equivalents) or DO2Atbu-mono amide mono acid (PSC fitted with 2 tert-butyl protecting groups, 2 equivalents, Cat# C134, Chematech) or S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane (TCMC-NSC, 2 equivalents, Cat# BM02751, A2B chem) or 4-(4-iodophenyl)butanoic acid (IB-OH, 2 equivalents, Cat# BD11901, BLD Pharmatech) or 2-(4-iodophenyl)acetic acid (SIB-OH, 2 equivalents, Cat# BD6173, BLD Pharmatech) was reacted with TBTU (2 equivalents) and DIPEA (10 equivalents) in NMP solvent at room temperature for 1 hour. After the reaction was complete, the beads were washed sequentially three times each with NMP and DCM and used for the next reaction.

[0125] The introduction of zwitteric ion (SZW) functional groups is performed using peptides containing lysine or DAP residues protected by Mtt or Dde. Following the method mentioned above, the Mtt or Dde protecting groups were removed to convert the N-terminus of the peptides synthesized on the beads into a deprotected amine form. Subsequently, 2-bromoacetic acid (30 equivalents, Cat# 17000, Sigma-Aldrich) and DIC (30 equivalents) were dissolved in DMF, added to the beads containing the deprotected amines, and reacted for 3 hours. After the reaction was complete, the beads were washed sequentially three times each with NMP and DCM. Then, the directly synthesized S4 After dissolving (30 equivalents) in triple distilled water, the mixture was placed in beads submerged in DMF along with DIPEA (30 equivalents) and reacted for 12 hours. After the reaction was complete, the beads were washed sequentially three times each with NMP and DCM and used for the next reaction.

[0126] Subsequently, a peptide containing an azido functional group on a solid-phase bead was synthesized by proceeding with a click reaction with 4-ethynylbenzenesulfonamide (Cat# E1130, TCI). Under an Ar atmosphere, a peptide containing an azido functional group on a solid-phase bead (1 equivalent), 4-ethynylbenzenesulfonamide (2 equivalents per azido functional group), CuI (1 equivalent per azido functional group), tris(benzyltriazolylmethyl)amine (TBTA, 2 equivalents per azido functional group, Cat# T2993, TCI), and DIPEA (10 equivalents) were mixed in NMP solvent and reacted at room temperature for 12 hours. After the reaction was complete, the beads were washed 3 times with NMP and at least 5 times with a 0.1 M sodium diethyldithiocarbamate in NMP solution to remove copper impurities. An example of removing an Mtt protecting group from a peptide and introducing an IB functional group and SFA is as follows.

[0127] [Reaction Equation 7]

[0128]

[0129] 1-4. Peptide cleavage, separation, and purification from solid-phase beads

[0130] Beads loaded with dried peptides were treated with a mixed solution of 95% TFA / 2.5% TIPS / 2.5% triple-distilled water at room temperature for 6 hours. The beads were removed using a filter, and the TFA mixed solution containing the peptides was collected in a conical tube and most of the mixed solution was removed by blowing nitrogen gas into it. Subsequently, diethyl ether was added to precipitate the peptides, and the mixture was placed in a centrifuge and spun at 3000 rpm for 5 minutes. After removing the supernatant diethyl ether and the small amount of remaining TFA mixed solution, the remaining solid peptides were vacuum dried.

[0131] The peptides cleaved from solid phase beads were dissolved in a mixed solution of ACN and triple-distilled water (1:1), and insoluble impurities were removed using a 45 μm syringe filter. The solution was then purified and analyzed using the system mentioned in Example 1-1. The solvent conditions and analysis results of the LC-MS are shown in Figures 6a to 6d. The purified peptide solution was desolvated using a freeze-dryer and used in powder form.

[0132] 1-5. Mounting 6OH functional groups on purified peptides

[0133] Purified peptides in powder form containing residues of Lys or DAP and 6OH(Protected)-NHS ( S1) (6 equivalents) and DIPEA (15 equivalents) were dissolved in DMF and reacted for 2 hours under magnetic stirring. After the reaction, impurities were removed using a 45 μm syringe filter, and the reaction was observed and purified using the same system mentioned in Example 1-1. The purified peptide solution was desolvated using a freeze-dryer and then converted into a powder. To remove the acetal protecting group of 6OH (Protected), a mixed solution of 95% (v / v) TFA, 2.5% (v / v), and 2.5% (v / v) TIS water was added to the purified peptide and reacted for 2 minutes. Care must be taken at this time, as exceeding 2 minutes will cause the 6OH group to detach from the Lys or DAP residues. Immediately after the reaction, a mixed solution of ACN and triple-distilled water (1:1) is added, and to neutralize the solution, trimethylamine (Cat# T0424, TCI) is slowly added in an amount equal to the amount of TFA solution added under ice water at 0°C. This solution is purified once again under the same conditions as above, and the obtained peptide solution can be obtained in powder form using a freeze dryer.

[0134] 1-6. Mounting fluorescent dyes (AF488, Cy5) on purified peptides

[0135] After first purifying the peptide requiring the introduction of a fluorescent dye, the fluorescent dye was loaded onto the peptide through click chemistry between the alkyne functional group contained in the peptide chain (introduced using Fmoc-propargyl-Gly-OH (Fmoc-Pra-OH, Cat# 21528, GL Biochem)) and the azido functional group contained in the fluorescent dye. Powdered peptide (1 equivalent), fluorescent dye Alexa Fluor 488 azide (AF488 azide, Cat# 1275, Click Chemistry Tools) or Sulfo-Cyanine5 azide (Cy5 azide, cat# E3330, Lumiprobe) (1 equivalent), 1 M CuSO4 aqueous solution (3 equivalents), and 1 M ascorbic acid (6 equivalents) were reacted under an Ar atmosphere for 1 hour. After the reaction, the progress of the reaction was checked using the LC-MS mentioned in Example 1-1. For the peptide containing the DOTA functional group, sodium diethyldithiocarbamate (5 equivalents) was added to remove the copper coordinated to DOTA, and after reacting for 20 minutes, the removal of copper was confirmed by LC-MS. The confirmed peptide was purified using the same system mentioned in Example 1-1. An example of a peptide equipped with a fluorescent dye is as follows.

[0136] [Reaction Equation 8]

[0137]

[0138] Through the synthesis process described above, peptide structures having sulfonamide functional groups that specifically bind to human CAIX were obtained as follows. The HPLC analysis conditions, retention times, and molecular weights observed using electrospray ionization mass spectrometry (ESI-MS) for each substance are shown in Figures 6a to 6d. The analytical instrument used was LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent), and the data were analyzed using MNOVA (v. 14.2.0, Mestrelab research, Spain). An Agilent Poroshell 120 EC-C18 column (4.6 X 50 mm, 2.7 μm) was used as the stationary phase, and the column temperature was maintained at 40°C. In addition, a mixture of acetonitrile (ACN) containing 0.1% TFA and triple distilled water containing 0.1% TFA was used as the mobile phase solvent, and the wavelengths were maintained at 214 and 254 nm and the flow rate at 1 mL / min.

[0139] [Chemical Formula 2]

[0140]

[0141]

[0142]

[0143] In the above chemical formula, R1 to R4 may include sulfonamides. AA1 to AA5 represent amino acid residues containing conventional amino acid residues or functional groups (Gal, IB, sIB, dimethyl), where lowercase letters represent D-amino acids and uppercase letters represent L-amino acids. U represents a modified or unnatural D-amino acid. Chemical formulas excluding conventional amino acid residues have the following structures defined herein.

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150] [Structure of Peptide Structure 1]

[0151]

[0152] [Structure of Peptide Structure 2]

[0153]

[0154] [Structure of Peptide Structure 3]

[0155]

[0156] [Structure of Peptide Structure 4]

[0157]

[0158] [Structure of Peptide Structure 5]

[0159]

[0160] [Structure of Peptide Structure 6]

[0161]

[0162] [Structure of Peptide Structure 7]

[0163]

[0164] [Structure of Peptide Structure 8]

[0165]

[0166] [Structure of Peptide Structure 9]

[0167]

[0168] [Structure of Peptide Structure 10]

[0169]

[0170] [Structure of Peptide Structure 11]

[0171]

[0172] [Structure of Peptide Structure 12]

[0173]

[0174] [Structure of Peptide Structure 13]

[0175]

[0176] [Structure of Peptide Structure 14]

[0177]

[0178] [Structure of Peptide Structure 15]

[0179]

[0180] [Structure of Peptide Structure 16]

[0181]

[0182] [Structure of Peptide Structure 17]

[0183]

[0184] [Structure of Peptide Structure 18]

[0185]

[0186] [Structure of Peptide Structure 19]

[0187]

[0188] [Structure of Peptide Structure 20]

[0189]

[0190] [Structure of Peptide Structure 21]

[0191]

[0192] [Structure of Peptide Structure 22]

[0193]

[0194] [Structure of Peptide Structure 23]

[0195]

[0196] [Structure of Peptide Structure 24]

[0197]

[0198] [Structure of Peptide Structure 25]

[0199]

[0200] [Structure of Peptide Structure 26]

[0201]

[0202] [Structure of Peptide Structure 27]

[0203]

[0204] [Structure of Peptide Structure 28]

[0205]

[0206] [Structure of Peptide Structure 29]

[0207]

[0208] [Structure of Peptide Structure 30]

[0209]

[0210] [Structure of Peptide Structure 31]

[0211]

[0212] [Structure of Peptide Structure 32]

[0213]

[0214] [Structure of Peptide Structure 33]

[0215]

[0216] [Structure of Peptide Structure 34]

[0217]

[0218] [Structure of Peptide Structure 35]

[0219]

[0220] [Structure of Peptide Structure 36]

[0221]

[0222] [Structure of Peptide Structure 37]

[0223]

[0224] [Structure of Peptide Structure 38]

[0225]

[0226] [Structure of Peptide Structure 39]

[0227]

[0228] [Structure of Peptide Structure 40]

[0229]

[0230] [Structure of Peptide Structure 41]

[0231]

[0232] [Structure of Peptide Structure 42]

[0233]

[0234] [Structure of Peptide Structure 43]

[0235]

[0236] [Structure of Peptide Structure 44]

[0237]

[0238] [Structure of Peptide Structure 45]

[0239]

[0240] [Structure of Peptide Structure 46]

[0241]

[0242] [Structure of Peptide Structure 47]

[0243]

[0244] [Structure of Peptide Structure 48]

[0245]

[0246] [Structure of Peptide Structure 49]

[0247]

[0248] [Structure of Peptide Structure 50]

[0249]

[0250] [Structure of Peptide Structure 51]

[0251]

[0252] [Structure of Peptide Structure 52]

[0253]

[0254] [Structure of Peptide Structure 53]

[0255]

[0256] [Structure of Peptide Structure 54]

[0257]

[0258] [Structure of Peptide Structure 55]

[0259]

[0260] [Structure of Peptide Structure 56]

[0261]

[0262] [Structure of Peptide Structure 57]

[0263]

[0264] [Structure of Peptide Structure 58]

[0265]

[0266] [Structure of Peptide Structure 59]

[0267]

[0268] [Structure of Peptide Structure 60]

[0269]

[0270] [Structure of Peptide Structure 61]

[0271]

[0272] [Structure of Peptide Structure 62]

[0273]

[0274] [Structure of Peptide Structure 63]

[0275]

[0276] [Structure of Peptide Structure 64]

[0277]

[0278] [Structure of Peptide Structure 65]

[0279]

[0280] [Structure of Peptide Structure 66]

[0281]

[0282] [Structure of Peptide Structure 67]

[0283]

[0284] [Structure of Peptide Structure 68]

[0285]

[0286] [Structure of Peptide Structure 69]

[0287]

[0288] [Structure of Peptide Structure 70]

[0289]

[0290] [Structure of Peptide Structure 71]

[0291]

[0292] [Structure of Peptide Structure 72]

[0293]

[0294] [Structure of Peptide Structure 73]

[0295]

[0296] [Structure of Peptide Structure 74]

[0297]

[0298] [Structure of Peptide Structure 75]

[0299]

[0300] [Structure of Peptide Structure 76]

[0301]

[0302] [Structure of Peptide Structure 77]

[0303]

[0304] [Structure of Peptide Structure 78]

[0305]

[0306] [Structure of Peptide Structure 79]

[0307]

[0308] [Structure of Peptide Structure 80]

[0309]

[0310] [Structure of Peptide Structure 81]

[0311]

[0312] [Structure of Peptide Structure 82]

[0313]

[0314] [Structure of Peptide Structure 83]

[0315]

[0316] [Structure of Peptide Structure 84]

[0317]

[0318] [Structure of Peptide Structure 85]

[0319]

[0320] [Structure of Peptide Structure 86]

[0321]

[0322] [Structure of Peptide Structure 87]

[0323]

[0324] [Structure of Peptide Structure 88]

[0325]

[0326] [Structure of Peptide Structure 89]

[0327]

[0328] [Structure of Peptide Structure 90]

[0329]

[0330] [Structure of Peptide Structure 91]

[0331]

[0332] [Structure of Peptide Structure 92]

[0333]

[0334] [Structure of Peptide Structure 93]

[0335]

[0336] [Structure of Peptide Structure 94]

[0337]

[0338] [Structure of Peptide Structure 95]

[0339]

[0340] [Structure of Peptide Structure 96]

[0341]

[0342] [Structure of Peptide Structure 97]

[0343]

[0344] [Structure of Peptide Structure 98]

[0345]

[0346] [Structure of Peptide Structure 99]

[0347]

[0348] [Structure of Peptide Structure 100]

[0349]

[0350] [Structure of Peptide Structure 101]

[0351]

[0352] [Structure of peptide structure 102]

[0353]

[0354] [Structure of peptide structure 103]

[0355]

[0356] [Structure of peptide structure 104]

[0357]

[0358] [Structure of Peptide Structure 105]

[0359]

[0360] [Structure of peptide structure 106]

[0361]

[0362] [Structure of peptide structure 107]

[0363]

[0364] 1-7. Labeling of Lutetium-175 (Lu-175)

[0365] h For binding affinity experiments for CAIX proteins, some peptide constructs containing DOTA linkers were labeled with lutetium-175 (Lu-175) instead of the isotope lutetium-177 (Lu-177).

[0366] The synthesized peptide construct was dissolved in DMSO at a concentration of 2 mM, and lutetium-175 (LuCl3, Cat#450960, Sigma-Aldrich) was dissolved in sodium acetate buffer solution (pH 5.5) at a concentration of 10 mM. After adding 3 equivalents of lutetium-175 to the peptide construct (1 equivalent) dissolved in DMSO, 500 μL of sodium acetate buffer solution (pH 5.5) was added, and the mixture was reacted in a thermoblock (Cat#B10-48, BLE) at 90 °C for 30 minutes. After the reaction was complete, the solution was cooled to room temperature and purified using a 1260 Infinity II LC system (Agilent) under the following conditions: (1) stationary phase was a Kromasil 100-5-C18 column (10 x 250 mm, 5 μm), (2) mobile phase solvent was a mixture of triple-distilled water with 0.1% acetic acid added and ACN, (3) flow rate was 8 mL / min, and (4) detection wavelengths were 214 and 254 nm. The lutetium-175-labeled peptide purification solution was freeze-dried to obtain a powder form.

[0367] Example 2. Confirmation of biological properties of the peptide structure

[0368] 2-1. Human carbonic anhydrous enzyme IX ( h Analysis of binding affinity and binding kinetics for the extracellular domain (ECD) of CAIX

[0369] Regarding peptide structures h The binding affinity and binding kinetics characteristics for CAIX ECD were analyzed. h To confirm specific binding selectivity for CAIX ECD, human carbonic anhydrous enzyme XII, another isoform of carbonic anhydrous enzyme ( h The binding kinetics characteristics of the peptide structure were also analyzed for the extracellular domain of CAXII.

[0370] 2-1-1. Analysis using BLITZ equipment

[0371] Binding affinity is based on the BLItz® system (Cat# 45-5000, Fort ) which is based on Bio-Layer Interferometry (BLI) technology. The advanced kinetics module of BLItz Pro ver1.3 software was used. A streptavidin biosensor (Cat# 18-5019, Sartorius) was used for all measurements. Additionally, a protein concentration of 0 nM was used as a reference to correct for non-specific binding and background. First, 10X Octet kinetics buffer (Cat# 18-1105, Sartorius) was prepared to 1X using DPBS (Cat# L0615, Biowest) and used as the analytical buffer solution. Next, the analytical buffer solution was dispensed at a rate of 200 μl / well into 96-well black, flat-bottom polypropylene (Cat# 655209, Greiner Bio-One), and the streptavidin biosensor was immersed in the solution for 10 minutes to perform hydration. Then, after attaching the biosensor to the instrument, the peptide was loaded at a concentration of 1 μM at 2200 rpm for 120 seconds to attach. Subsequently, protein association and dissociation were performed at each concentration at 2200 rpm for 120 seconds, and the kinetics data were analyzed using the global fitting function for K D The value was calculated as the ratio of kd to ka. In addition, the kinetic parameter was obtained by fitting a baseline correction and a 1:1 binding model.

[0372] The results are shown in Fig. 1a. The curves shown in each graph in Fig. 1a represent the experimental results obtained by varying the protein concentration. The protein concentration corresponding to each curve is indicated below the curve. For example, in Graph 2 shown in Fig. 1a, the curve indicated at the very top is h This shows the result value when the concentration of CAIX(nM) is 125 nM, and the graph displayed at the bottom is h This represents the result value when the concentration of CAIX(nM) is 50 nM. The peptide structures are h Compared to CAXII ECD h It showed significantly high selectivity for CAIX ECD.

[0373] 2-1-2. Analysis using Octet equipment

[0374] The Octet®R8 system (Octet BLI Discovery 13 software) is an instrument based on the same principle as the BLItz® system, and a protein (Cat# CA9-H82E3, ACRO Biosystems) was attached to a streptavidin biosensor (Cat# 18-5019, Sartorius) for analysis. The protein was loaded by diluting it to 300 nM in DPBS (Cat# L001-02, WELGENE) at 1000 rpm for 600 seconds, and subsequently, association and dissociation were performed for 300 seconds and 600 seconds, respectively, at 1000 rpm with the peptide (0.5% DMSO) diluted in DPBS at each concentration. It was confirmed that there was no non-specific binding of peptides to the biosensor at 100 nM of each peptide, and a peptide concentration of 0 nM was used as the reference for background correction. K D The values ​​were obtained in the same way as the BLItz® system, and the results are shown in Figs. 1b and 1c.

[0375] 2-2. Fluorescence-activated Cell Sorting (FACS) Analysis

[0376] In vitro cell binding characteristics were analyzed to determine whether the peptide construct specifically binds to target cells expressing CAIX. The human renal cancer cell line SK-RC-52 expressing CAIX was purchased from Memorial Sloan Kettering Cancer Center (MSK, USA).

[0377] The peptide structures attached with fluorescent dye AF488 and Cy5 were measured using a NanoPhotometer for accurate quantification. ® Absorbance was measured using (N60, IMPLEN), and concentration was calculated according to Lambert-Beer's law. The freeze-dried peptide constructs were dissolved in dimethyl sulfoxide (DMSO) to prepare a millimola (mM) solution, and then, using a NanoPhotometer ® The absorbance of 1.5 μL of 100-fold diluted PBS was measured using equipment at a wavelength of 495 nm for AF488 and 650 nM for Cy5. The molar extinction coefficient of the AF488 fluorescent dye used in the calculation was 73,000 L·mol -1 cm -1 And, the molar extinction coefficient of the Cy5 fluorescent dye is 271,000 L·mol -1 cm -1 The quantified peptide construct was dissolved in DMSO to make a 1 mM solution, and then diluted to 5 nM or 10 nM using a mixture of Dulbecco's Modified Eagle Medium (DMEM) containing 1% fetal bovine serum (FBS) (buffer solution for FACS).

[0378] SK-RC-52 cell line (T75 flask; 1.0 ~ 1.5 X 10⁶ 2–3 days before FACS analysis 6 Pieces, T175 flasks; 5.0 ~ 8.0 X 10⁻⁶ 6Cells were seeded into T75 or T175 flasks. On the day of the experiment, the culture medium was removed from flasks containing SK-RC-52 cells cultured at 80% confluency and washed twice with 10 mL of PBS buffer. 1 mL of Trypsin-EDTA was added in an incubator (37°C, 5% CO2, humid conditions, Galaxy 170 S) for 5 minutes, after which the Trypsin-EDTA was neutralized with 9 mL of culture medium. The neutralized cell mixture and an additional 10 mL of culture medium were added to the flask, and the collected cell mixture was placed in a 50 mL tube and centrifuged using a VARISPIN 15R centrifuge at 1000 rpm for 3 minutes. The supernatant of the centrifuged cell mixture was removed, and fresh culture medium was added at 5 x 10⁶ per 200 μL. 5 The cells were diluted to a cell count and dispensed in 200 μL aliquots into 96-well round plates (Cat# 34096, SPL LIFE SCIENCE). After centrifuging at 1000 RPM for 3 minutes using a VARISPIN 15R centrifuge, the supernatant was removed from each well, and 200 μL of a pre-prepared 5 or 10 nM peptide construct solution was added, followed by incubation in an incubator. After 1 hour, the cells were centrifuged at 1000 RPM for 3 minutes using a VARISPIN 15R centrifuge, the supernatant was removed, and the cells were washed with 200 μL of FACS buffer. After washing, 200 μL of FACS buffer was added to each well and mixed with the cells. The cells were then transferred to a 5 mL round-bottom tube equipped with a cell strainer (Cat# 352235, CORNING), and the fluorescence intensity of each cell was measured using a BD Accuri C6 Plus (BD Biosciences). The measured data were analyzed using FlowJo 10.7.1 (BD Biosciences).

[0379] The results of FACS analysis using the SK-RC-52 cell line are shown in Figures 2a to 2d. From the FACS analysis results, it was confirmed that the peptide structure of the present invention has a high affinity for target cells expressing CAIX.

[0380] 2-3. Biodistribution Analysis Using Experimental Animal Models

[0381] Using 6-week-old female BALB / c nude mice (Orient Bio or Hana Bio, Korea) 2 X 10 6 or 5 x 10 6 SK-RC-52 was injected subcutaneously into the upper right forelimb of nude mice. Tumor size was measured 2–3 times a week starting 5 days after injection. Tumor size was determined using a caliper [Volume = (Width)] 2 It was measured as [X (length) / 2].

[0382] Tumor size 90 ~ 120 mm after xenograftment with SK-RC-52 cell line 3 When the level was reached, 200 μL (1% DMSO in PBS) of the 10 μM peptide construct was injected via tail vein, and after 24 hours ex vivo Images were measured (Figs. 3a to 3c). The number of mice injected with each peptide structure was 2, and the peptide structures injected with 2 or more mice are as shown in Table 1.

[0383] Peptide number 33 35 36 37 40 42 43 Number of rats used 6 3 4 3 6 4 4

[0384] For the replication experiment, seven peptide structures (31, 33, 39, 40, 43, 45, 46) showing high tumor signals in the results of Figures 3a to 3c were each injected into three mice for 24 hours ex vivo Images were taken (Fig. 3d), and three types of peptide structures (33, 39, 40) were injected into four mice, each for 72 hours ex vivo An image was taken (Fig. 3e).

[0385] Six peptide constructs (55, 56, 57, 58, 59, 60) were administered by tail vein injection at 200 μL (1% DMSO in PBS) to two mice per construct, and after 72 hours ex vivo Biodistribution was confirmed by measuring images (Fig. 3f). For 58 and 60, which showed high fluorescence intensity among the six types, 10 μM and 200 μL (1% DMSO in PBS), respectively, were injected into three mice via tail vein, and on the 5th day ex vivo The biodistribution was confirmed by measuring the image (Fig. 3g).

[0386] Four peptide constructs (61, 62, 63, 64) were injected into five mice in each group at 200 μL (5% DMSO in PBS) via tail vein injection, and after 24 hours, whole-body fluorescence imaging was performed, and after the mice were sacrificed ex vivo The biodistribution map was confirmed by measuring the images (Figs. 3h, 3i).

[0387] For 63 and 64, which exhibited high fluorescence intensity in the tumor and low fluorescence intensity in the kidney, 14 types of advanced peptide constructs (65, 75, 80, 83, 84, 85, 87, 89, 90, 91, 92, 97, 98, 99) were injected into 5 mice in each group at a rate of 5 μM, 200 μL (5% DMSO in PBS) via tail vein injection; after 24 or 72 hours, whole-body fluorescence imaging was performed, and after sacrificing the mice ex vivo The biodistribution map was confirmed by measuring the images (Figs. 3j to 3m).

[0388] To verify biodistribution, mice were sacrificed by injecting carbon dioxide, then laparotomy was performed to remove tumors and organs (spleen, lungs, heart, liver, kidneys, stomach, pancreas, small intestine, large intestine). in vivo With the imaging equipment AMI HTX (Spectral Instruments Imaging) ex vivoImages were captured, and the obtained fluorescence intensities were presented as a bar graph including the deviation of each individual. The results were based on the area adjusted for the smallest tumor size (0.08 cm²). 2 Based on ), the same area (0.08 cm²) in the part with the strongest fluorescence intensity in the tumor, kidney, liver, spleen, lung, and heart 2 It was derived by applying ). This work was performed using Aura imaging software (Spectral imaging, USA), and the unit of fluorescence intensity is photons / s / cm 2 / sr. It was confirmed that some substances that specifically bind to tumors overexpressing CAIX compared to other organs remain in the tumor even after 72 hours.

[0389] 2-4. In vivo (177Lu-labeled peptide structure) in vivo ) SPECT / CT Imaging Experiment

[0390] To confirm the imaging diagnostic and therapeutic functionality of peptide constructs 67, 68, 69, 70, 103, 104, and 105, each peptide construct was prepared by labeling it with an isotope as follows. A 1 mM stock solution was prepared by dissolving each peptide construct in DMSO, and 30 mCi of the isotope diluted in 0.04 M hydrochloric acid solution ( 177LuCl3 (ITM Medical Isotopes GmbH) was completely evaporated from the solution via nitrogen purging. 200 μL of 0.2 M sodium acetate (pH 5.5) buffer solution was added to dissolve the isotope; subsequently, 7 mCi (approx. 50 μL) of the isotope was dispensed, mixed with 7 μL of peptide stock solution, and isotope labeling was performed at 90°C for 30 minutes. Once labeling of each substance was complete, the samples were cooled to room temperature and 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 x 50 mm, 2.7 μm), (2) the mobile phase solvent was a mixture of triple-distilled water and ACN, (3) the flow rate was 1 mL / min, and (4) the detection wavelengths were 254 nm and 214 nm.

[0391] purified 177 Lu-labeled peptide constructs were processed using a Sep-Pak C18 column (Cat# 186005125, Waters) to remove organic solvents. The purified peptide constructs were diluted 5-fold with distilled water, loaded onto an activated Sep-Pak column (5 mL ethanol, 5 mL distilled water), washed with 5 mL of distilled water to remove organic solvents, eluted with 300 μL of 60% ethanol solution, and diluted with saline solution for use.

[0392] The average size of tumors generated by transplanting the human kidney cancer cell line SK-RC-52 is 120 mm 3 200 μL of isotope corresponding to a radiation dose of 500 μCi when reaching the inner or outer area 177Lu-labeled peptide constructs were administered once via tail vein injection to mice, and SPECT / CT (Siemens Inveon, software: Inveon Acquisition Workplace) measurements were taken for constructs 67, 68, 69, and 70 at 1.5 hours, 1 day, 2 days, 3 days, 7 days, or 8 days and 10 days, and for constructs 103, 104, and 105 at 1.5 hours, 1 day, 2 days, and 3 days, respectively. For imaging, mice were respiratory anesthetized with 0.2% isoflurane in oxygen for 3–5 minutes, then the anesthetized mice were placed in the equipment and the anesthetic was controlled to be continuously infused at 1.5 L / min. SPECT was taken at 40 minutes and CT at 5 minutes, and images were acquired using Inveon Research Workplace 4.2.

[0393] The SPECT / CT images obtained above are shown in Figs. 4a and 5a. Isotope 177 It was confirmed that the Lu-labeled peptide construct binds to cancer cell SK-RC-52.

[0394] 2-5. Analysis of the anticancer effect of CAIX targeted peptides labeled with an isotope (177Lu)

[0395] Isotope 177 To confirm the anticancer effects of Lu-labeled peptide constructs 67, 68, 69, 70, 103, 104, and 105, 6-week-old female BALB / c nude mice (Orient Bio or Hana Bio, Korea) were used to 5 x 10 6 SK-RC-52 cells were injected subcutaneously into the upper right forelimb of nude mice. After cell injection, the average tumor size was 120 mm 3 Grouping was performed into groups of 4 or more to ensure internal and external classification. Tumor size was measured using a caliper [Volume = (Width)] 2 It was measured as [X (length) / 2].

[0396] A control group that was xenografted with SK-RC-52 cell lines, grouped, and then injected with saline, and 177 Approximately 500 μCi of each Lu-labeled peptide construct (67, 68, 69, 70, 103, 104, 105) was injected via tail vein injection, and body weight and tumor size were measured 2 to 3 times a week.

[0397] The results are shown in FIGS. 4b, 4c, 5b, and 5c, respectively. As can be seen in these figures, 177 Lu-labeled peptide constructs (67, 68, 69, 70, 103, 104, 105) exhibited an anticancer effect that significantly reduced the size of tumors overexpressing CAIX without causing serious changes in mouse body weight.

[0398] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the scope of the present invention is not limited by the disclosed embodiments and attached drawings, and that various modifications can be made to other specific forms within the scope of the technical concept of the present invention.

[0399] Sequence list

[0400] Seq ID No. Amino Acid Sequences 1 YRFRR 2 RFRR 3 RARR 4 YRARR 5 ARFRR 6 ARARR 7 RXRR (where, X=DAP(Gal)) 8 RXRR (where, X=Uk(IB)) 9 RNRR 10 AAAA 11 RERR 12 YRXRR (where, X=DAP(Gal)) 13 RX1RX2 (where, X1=DAP(Gal), X2=r(dimet)) 14 RX1X2R (where, X1=DAP(Gal), X2=r(dimet)) 15 YSXRR (where, X=DAP(Gal)) 16 YRXSR (where, X=DAP(Gal)) 17 YRXRS (where, X=DAP(Gal)) 18 SRXRR (where, X=DAP(Gal)) 19 XRXRR (where, X=DAP(Gal)) 20 XSXRR (where, X=DAP(Gal)) 21 RXRS (where, X=DAP(Gal)) 22 RXSR (where, X=DAP(Gal)) 23 SXRR (where X=DAP(Gal)) 24 X1X2RR (where, X1=R(dimet), X2=DAP(Gal)) 25 X1RX2RR (where, X1=DAP(Gal), X2=Uk(sIB)) 26 RXRR (where, X=Uk(sIB)) 27 ERXRR (where, X=DAP(Gal)) 28 SSSS 29 SXRS (where, X=DAP(Gal)) 30 SEX1EEX2 (where X1=Uk(DOTA), X2=K(Biotin)) 31 SEX1EEX2 (where, X1=Uk(DOTA), X2=Pra(AF488)) 32 SX1X2EEX3 (where, X1=Gla, X2=DAP(DOTA), X3=Pra(AF488)) 33 SEX1EEX2 (where, X1=DAP(DOTA), X2=Pra(AF488)) 34 SX1X2EX3X4 (where, X1=Gla, X2=DAP(DOTA), X3=DAP(SZW), X4=Pra(AF488)) 35 SEX1EEX2X3 (where, X1=DAP(DOTA), X2=DAP(SZW), X3=Pra(AF488)) 36 EX1X2GVPTLQ (where, X1=Gla, X2=Pra(AF488)) 37 EX1X2EGRGDK (where, X1=Gla, X2=Pra(AF488)) 38 EX1X2KLPVM (where, X1=Gla, X2=Pra(AF488)) 39 EX1X2EGKLPVM (where, X1=Gla, X2=Pra(AF488)) 40 SX1X2EX3X4 (where, X1=Gla, X2=DAP(DOTA), X3=DAP(SZW), X4=Pra(Cy5)) 41 SX1X2EX3 (where, X1=Gla, X2=DAP(DOTA), X3=Pra(Cy5)) 42 SX1X2EEX3 (where, X1=Gla, X2=DAP(DOTA), X3=Pra(Cy5)) 43 EX1X2EEX3 (where, X1=Gla, X2=DAP(DOTA), X3=Pra(Cy5)) 44 SX1X2EX3X4 (where, X1=Gla, X2=DAP(DOTA), X3=Uk(IB), X4=Pra(Cy5)) 45 EX1X2EX3X4 (where, X1=Gla, X2=DAP(DOTA), X3=Uk(IB), X4=Pra(Cy5)) 46 SX1X2X1X3X4 (where X1=Gla, X2=DAP(DOTA), X3=Uk(IB), X4=Pra(Cy5)) 47 EX1X2EX3X4 (where, X1=Gla, X2=DAP(DOTA), X3=DAP(6OH), X4=Pra(Cy5)) 48 SX1X2EVPTLQ (where, X1=Gla, X2=Pra(Cy5)) 49 SX1X2EERGDK (where, X1=Gla, X2=Pra(Cy5)) 50 EX1X2GVPTLQ (where, X1=Gla, X2=Pra(Cy5)) 51 EX1X2EGRGDK (where, X1=Gla, X2=Pra(Cy5)) 52 EX1X2KLPVM (where, X1=Gla, X2=Pra(Cy5)) 53 EX1X2EGKLPVM (where, X1=Gla, X2=Pra(Cy5)) 54 SEEX1EX2 (where, X1=Pra(Cy5), X2=DAP(SZW)) 55 SEEX1EX2X3 (where X1=Pra(Cy5), X2=DAP(SZW), X3=Uk(IB)) 56 SEEXE (where, X=Pra(Cy5)) 57 SEEX1EX2 (where, X1=DAP(DOTA), X2=DAP(SZW)) 58 SEEX1EX2X3 (where X1=DAP(DOTA), X2=DAP(SZW), X3=Uk(IB)) 59 SEEXE (where, X1=DAP(DOTA)) 60 EX1EX2 (where, X1=Pra(Cy5), X2=DAP(SZW)) 61 SEEX1EX2 (where, X1=Pra(Cy5), X2=Uk(sIB)) 62 X1EEX2 (where, X1=Pra(Cy5), X2=Uk(sIB)) 63 X1EEX2 (where, X1=Pra(Cy5), X2=DAP(SZW)) 64 X1EEE (where, X1=Pra(Cy5)) 65 EEX1EX2* (where, X1=Pra(Cy5), X2=Uk(sIB)) 66 EEX1X2* (where, X1=Pra(Cy5), X2=Uk(sIB)) 67 SEEX1EX2X3 (where X1=Pra(Cy5), X2=DAP(SZW), X3=Uk(sIB)) 68 EEX1EX2X3 (where X1=Pra(Cy5), X2=DAP(SZW), X3=Uk(sIB)) 69 SEEX1EX2* (where, X1=Pra(Cy5), X2=Uk(sIB)) 70 SSEXE (where, X=Pra(Cy5)) 71 SEEXE (where, X=DAP(PSC)) 72 SEEXE (where, X=DAP(TCMC)) 73 X1YRFRRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA)) 74 X1X2RFRRX2E (where, X1=Uk(SFA), X2=Aha(SFA)) 75 X1X2RARRX2E (where, X1=Uk(SFA), X2=Aha(SFA)) 76 XXRARRXE (where, X=Aha(SFA)) 77 XYRFRRXEX (where, X=Aha(SFA)) 78 X1YRARRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA)) 79 X1ARFRRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA)) 80 X1RFRRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA)) 81 X1ARARRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA)) 82 X1RARRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA)) 83 X1X2RX3RRX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3=DAP(Gal))) 84 X1X2RX3RRX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3=Uk(IB)) 85 X1X2RNRRX2E (where, X1=Uk(SFA), X2=Aha(SFA)) 86 X1X2AAAAX2E (where, X1=Uk(SFA), X2=Aha(SFA)) 87 X1X2RERRX2E (where, X1=Uk(SFA), X2=Aha(SFA)) 88 X1YRFRRX2E (where, X1=Uk(SFA), X2=Aha(SFA)) 89 XYRFRRXE (where, X=Uk(SFA)) 90 X1YRX2RRX3EX3 (where, X1=Uk(SFA), X2=DAP(Gal), X3=Aha(SFA)) 91 X1X2RX3RX4X2E (where X1=Uk(SFA), X2=Aha(SFA), X3=DAP(Gal), X4=r(dimet)) 92 X1X2RX3X4RX2E (where X1=Uk(SFA), X2=Aha(SFA), X3=DAP(Gal), 93 X1YSX2RRX3EX3 (where, X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA)) 94 X1YRX2SRX3EX3 (where X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA)) 95 X1YRX2RSX3EX3 (where X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA)) 96 X1SRX2RRX3EX3 (where X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA)) 97 X1X2RX2RRX3EX3 (where, X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA)) 98 X1X2SX2RRX3EX3 (where, X1=Uk(SFA), X2=DAP(Gal), X3=Aha(SFA)) 99 X1X2RX3RSX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3= DAP(Gal)) 100 X1X2RX3SRX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3= DAP(Gal)) 101 X1X2SX3RRX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3= DAP(Gal)) 102 X1X2X3X4RRX2E (where X1=Uk(SFA), X2=Aha(SFA), X3=R(dimet), X4=DAP(Gal)) 103 X1X2RX3RRX1E (where X1=Uk(SFA), X2=DAP(Gal), X3=Uk(sIB)) 104 X1X2RX3RRX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3=Uk(sIB)) 105 X1ERX2RRX3EX3 (where, X1=Uk(SFA), X2=DAP(Gal), X3=Aha(SFA)) 106 X1X2SSSSX2E (where, X1=Uk(SFA), X2=Aha(SFA)) 107 X1X2SX3RSX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3=DAP(Gal)) * It is a trityl resin synthetic material that has a C-terminal of -COOH. In the above sequence, the amino acids may be D-amino acids or L-amino acids. Additionally, the chemical structural formulas of the abbreviations used in each sequence are as follows. abbreviation Chemical structure formula DAP(Gal) 2-amino-3-((2S,3R,4S,5R,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanoic acid Uk(IB) N 6 -(4-(4-iodophenyl)butanoyl)-lysine Uk(DOTA) 2,2',2''-(10-(2-((5,6-diamino-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid Pra(AF488) 5-((6-(4-(2-amino-2-carboxyethyl)-1H-1,2,3-triazol-1-yl)hexyl)carbamoyl)-2-(6-amino-3-imino-4,5-disulfo-3H-xanthen-9-yl)benzoic acid DAP(DOTA) 2,2',2''-(10-(2-((2-amino-2-carboxyethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid DAP(SZW) 3-((2-((2-amino-2-carboxyethyl)amino)-2-oxoethyl)(metheyliumyl)(methyl)-λ 4 -azaneyl)propane-1-sulfonate Gla γ-carboxyglutamic acid Pra(Cy5) 1-(6-((3-(4-(2-amino-2-carboxyethyl)-1H-1,2,3-triazol-1-yl)propyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium-5-sulfonate Uk(sIB) N 6 -(2-(4-iodophenyl)acetyl)-D-lysine DAP(6OH) (R)-2-amino-3-((2R,3R,4S,5R,6R)-2,3,4,5,6,7-hexahydroxyheptanamido)propanoic acid DAP(PSC) (R)-2,2'-(4-(2-((2-amino-2-carboxyethyl)amino)-2-oxoethyl)-10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid DAP(TCMC) (2R)-2-amino-3-(3-(4-((1,4,7,10-tetrakis(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)propanoic acid r(dimet) (R)-2-amino-5-((amino(dimethyl-l4-azaneylidene)methyl)amino)pentanoic acid R(dimet) (S)-2-amino-5-((amino(dimethyl-l4-azaneylidene)methyl)amino)pentanoic acid PEG1 2-(2-(2-aminoethoxy)ethoxy)acetic acid PEG(6atom) 2-(2-aminoethoxy)acetic acid K(biotin) N6-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)-L-lysine Uk(SFA) (R)-2-amino-6-(4-(4-sulfamoylphenyl)-1H-1,2,3-triazol-1-yl)hexanoic acid Aha(SFA) (R)-2-amino-4-(4-(4-sulfamoylphenyl)-1H-1,2,3-triazol-1-yl)butanoic acid

[0401] [Structure of Sequence No. 7]

[0402]

[0403] [Structure of Sequence No. 8]

[0404]

[0405] [Structure of Sequence No. 12]

[0406]

[0407] [Structure of Sequence No. 13]

[0408]

[0409] [Structure of Sequence No. 14]

[0410]

[0411] [Structure of Sequence No. 15]

[0412]

[0413] [Structure of Sequence No. 16]

[0414]

[0415] [Structure of Sequence No. 17]

[0416]

[0417] [Structure of Sequence No. 18]

[0418]

[0419] [Structure of Sequence No. 19]

[0420]

[0421] [Structure of Sequence No. 20]

[0422]

[0423] [Structure of Sequence No. 21]

[0424]

[0425] [Structure of Sequence No. 22]

[0426]

[0427] [Structure of Sequence No. 23]

[0428]

[0429] [Structure of Sequence No. 24]

[0430]

[0431] [Structure of Sequence No. 25]

[0432]

[0433] [Structure of Sequence No. 26]

[0434]

[0435] [Structure of Sequence No. 27]

[0436]

[0437] [Structure of Sequence No. 29]

[0438]

[0439] [Structure of Sequence No. 30]

[0440]

[0441] [Structure of Sequence No. 31]

[0442]

[0443] [Structure of Sequence No. 32]

[0444]

[0445] [Structure of Sequence No. 33]

[0446]

[0447] [Structure of Sequence No. 34]

[0448]

[0449] [Structure of Sequence No. 35]

[0450]

[0451] [Structure of Sequence No. 36]

[0452]

[0453] [Structure of Sequence No. 37]

[0454]

[0455] [Structure of Sequence No. 38]

[0456]

[0457] [Structure of Sequence No. 39]

[0458]

[0459] [Structure of Sequence No. 40]

[0460]

[0461] [Structure of Sequence No. 41]

[0462]

[0463] [Structure of Sequence No. 42]

[0464]

[0465] [Structure of Sequence No. 43]

[0466]

[0467] [Structure of Sequence No. 44]

[0468]

[0469] [Structure of Sequence No. 45]

[0470]

[0471] [Structure of Sequence No. 46]

[0472]

[0473] [Structure of Sequence No. 47]

[0474]

[0475] [Structure of Sequence No. 48]

[0476]

[0477] [Structure of Sequence No. 49]

[0478]

[0479] [Structure of Sequence No. 50]

[0480]

[0481] [Structure of Sequence No. 51]

[0482]

[0483] [Structure of Sequence No. 52]

[0484]

[0485] [Structure of Sequence No. 53]

[0486]

[0487] [Structure of Sequence No. 54]

[0488]

[0489] [Structure of Sequence No. 55]

[0490]

[0491] [Structure of Sequence No. 56]

[0492]

[0493] [Structure of Sequence No. 57]

[0494]

[0495] [Structure of Sequence No. 58]

[0496]

[0497] [Structure of Sequence No. 59]

[0498]

[0499] [Structure of Sequence No. 60]

[0500]

[0501] [Structure of Sequence No. 61]

[0502]

[0503] [Structure of Sequence No. 62]

[0504]

[0505] [Structure of Sequence No. 63]

[0506]

[0507] [Structure of Sequence No. 64]

[0508]

[0509] [Structure of Sequence No. 65]

[0510]

[0511] [Structure of Sequence No. 66]

[0512]

[0513] [Structure of Sequence No. 67]

[0514]

[0515] [Structure of Sequence No. 68]

[0516]

[0517] [Structure of Sequence No. 69]

[0518]

[0519] [Structure of Sequence No. 70]

[0520]

[0521] [Structure of Sequence No. 71]

[0522]

[0523] [Structure of Sequence No. 72]

[0524]

[0525] [Structure of Sequence No. 73]

[0526]

[0527] [Structure of Sequence No. 74]

[0528]

[0529] [Structure of Sequence No. 75]

[0530]

[0531] [Structure of Sequence No. 76]

[0532]

[0533] [Structure of Sequence No. 77]

[0534]

[0535] [Structure of Sequence No. 78]

[0536]

[0537] [Structure of Sequence No. 79]

[0538]

[0539] [Structure of Sequence No. 80]

[0540]

[0541] [Structure of Sequence No. 81]

[0542]

[0543] [Structure of Sequence No. 82]

[0544]

[0545] [Structure of Sequence No. 83]

[0546]

[0547] [Structure of Sequence No. 84]

[0548]

[0549] [Structure of Sequence No. 85]

[0550]

[0551] [Structure of Sequence No. 86]

[0552]

[0553] [Structure of Sequence No. 87]

[0554]

[0555] [Structure of Sequence No. 88]

[0556]

[0557] [Structure of Sequence No. 89]

[0558]

[0559] [Structure of Sequence No. 90]

[0560]

[0561] [Structure of Sequence No. 91]

[0562]

[0563] [Structure of Sequence No. 92]

[0564]

[0565] [Structure of Sequence No. 93]

[0566]

[0567] [Structure of Sequence No. 94]

[0568]

[0569] [Structure of Sequence No. 95]

[0570]

[0571] [Structure of Sequence No. 96]

[0572]

[0573] [Structure of Sequence No. 97]

[0574]

[0575] [Structure of Sequence No. 98]

[0576]

[0577] [Structure of Sequence No. 99]

[0578]

[0579] [Structure of Sequence No. 100]

[0580]

[0581] [Structure of Sequence No. 101]

[0582]

[0583] [Structure of Sequence No. 102]

[0584]

[0585] [Structure of Sequence No. 103]

[0586]

[0587] [Structure of Sequence No. 104]

[0588]

[0589] [Structure of Sequence No. 105]

[0590]

[0591] [Structure of Sequence No. 106]

[0592]

[0593] [Structure of Sequence No. 107]

[0594]

Claims

Claim 1 A peptide ligand comprising any one of the amino acid sequences of SEQ ID NOs. 1 to 29, wherein at least one of the constituent amino acids is a D-amino acid, and the lysine (Lys) residue among the constituent amino acids may be substituted with a chemical functional group at the side chain ε-amino group, or the 2,3-diaminopropionic acid (DAP) residue among the constituent amino acids may be substituted with a chemical functional group at the side chain β-amino group; and a peptide structure comprising a sulfonamide functional group-containing amino acid residue connected to the peptide ligand directly or through a spacer, and having a structure of the following Chemical Formula 1. [Chemical Formula 1] In the above formula, P is the peptide ligand, F1, F2, and F3 are each independently sulfonamide functional group-containing amino acid residues, m is 0 or 1, and F4 is general formula -(F5) o -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is a group, where F5 is a sulfonamide functional group-containing amino acid residue, S1 and S2 are each independently spacers, F6 and F7 are each independently amino acid residues containing a non-sulfonamide functional group, S3 is -NH2 or -OH, o is an integer of 0 or 1, and p, q, r and s are each independently integers from 0 to 6. Claim 2 A peptide structure according to claim 1, characterized in that the chemical functional group that can be substituted at the side chain ε-amino group of the lysine (Lys) residue among the constituent amino acids is 4-(p-iodophenyl)butyric acid (IB) or 2-(4-iodophenyl)acetic acid (sIB). Claim 3 A peptide structure according to claim 1, characterized in that the chemical functional group that can be substituted at the side chain β-amino group of the 2,3-diaminopropionic acid (DAP) residue among the constituent amino acids is α-D-galacturonic acid (α-D-galacturonic acid, Gal). Claim 4 A peptide structure according to claim 1, characterized in that the sulfonamide functional group-containing amino acid residue has the following structure. or Claim 5 A peptide structure according to claim 1, characterized in that the peptide structure has the structure of the following chemical formula 2.[Chemical Formula 2] In the above formula, R5 is the general formula -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is a group, where S1, S2, S3, F6, F7, p, q, r, and s are each as defined in the first paragraph above, lowercase letters represent D-amino acids and uppercase letters represent L-amino acids, and other substituents indicated by abbreviations in R1 to R4 and AA1 to AA5 are each as defined below. Claim 6 A peptide structure according to claim 1, characterized in that the functional group other than the sulfonamide is introduced through the side chain ε-amino group of a lysine residue or through the side chain β-amino group of a 2,3-diaminopropionic acid (DAP) residue. Claim 7 A peptide structure according to claim 1, wherein the functional group other than the sulfonamide is a chelator, biotin, glucoheptonic acid, 4-(p-iodophenyl)butyric acid (IB), 2-(4-iodophenyl)acetic acid (sIB), zwitteric ion (SZW), α-D-galacturonic acid (Gal), a cycloalkane having 5 to 15 carbon atoms, a fluorescent dye, or a cytotoxic agent. Claim 8 In claim 7, the chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazcyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid-2,2',2'',2'''-(ethane-1,2-diyldinitrilo)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-pentazacyclopentadecane-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), triethylenetetramine (TETA), A peptide structure characterized by being selected from 1,4,7,10-tetraazacyclododecane-7-acetamide-1,4,10-triacetic acid (PSC) and 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide (DOTAM or TCMC). Claim 9 A peptide structure according to claim 1, wherein the spacer is selected from a polyethylene glycol (PEG) linker, glycine, sarcosine, γ-carboxyglutamic acid, and a peptide linker composed of 1 to 8 D-amino acids or L-amino acids. Claim 10 A peptide structure according to claim 1, characterized in that the peptide structure has the structure of the following chemical formula 2.[Chemical Formula 2] In the above, lowercase letters represent D-amino acids and uppercase letters represent L-amino acids, respectively, and the substituents indicated by abbreviations in R1 to R5 and AA1 to AA5 are each as defined below. Claim 11 A conjugate comprising a peptide structure of any one of claims 1 to 10, which is directly or through a linker linked to a fluorescent dye, a cytotoxic agent, or a radioisotope. Claim 12 A conjugate according to claim 11, wherein the linker is selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)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)aminobenzoate (SIAB). Claim 13 A conjugate according to claim 11, wherein the fluorescent dye is selected from near-infrared fluorescent dyes, fluorescein type, rhodamine type, Alexa Fluor, 4,4-difluoro-4-boro-3a, 4a-diaza-s-indacen (BODIPY), Texas Red, dansyl, lissamine, cyanine (Cy), and phycoerythrin. Claim 14 In paragraph 11, the radioisotopes are Fluorine-18 (F-18), Carbon-11 (C-11), Carbon-14 (C-14), Techtenium-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), A conjugate characterized by being selected from potassium-42 (K-42), holmium-166 (Ho-166), gallium-67 (Ga-67), gallium-68 (Ga-68), actinium-225 (Ac-225), zirconium-89 (Zr-89), thorium-227 (Th-227), radium-223 (Ra-223), tin-117m (Sn-117m), lead-103 (Pb-103), terbium-161 (Tb-161), lead-212 (Pb-212), and astatine-211 (At-211). Claim 15 A conjugate comprising a peptide structure selected from the group consisting of peptide structures 67 to 70 and 103 to 105, which is coupled to a radioisotope directly or through a linker. [Structure of peptide structure 67] [Structure of Peptide Structure 68] [Structure of Peptide Structure 69] [Structure of Peptide Structure 70] [Structure of peptide structure 103] [Structure of peptide structure 104] [Structure of Peptide Structure 105] Claim 16 A conjugate according to claim 15, characterized in that the radioisotope is selected from the group consisting of fluorine-18 (F-18), copper-64 (Cu-64), indium-111 (In-111), gallium-68 (Ga-68), actinium-225 (Ac-225), lead-212 (Pb-212), and lutetium-177 (Lu-177). Claim 17 delete Claim 18 A pharmaceutical composition for the diagnosis, prevention, or treatment of cancer comprising the conjugate of claim 11. Claim 19 delete Claim 20 delete Claim 21 A pharmaceutical composition for the diagnosis, prevention, or treatment of cancer according to claim 18, wherein the cancer expresses carbonic anhydrous enzyme IX. Claim 22 A pharmaceutical composition for the diagnosis, prevention, or treatment of cancer according to claim 18, wherein the cancer is liver cancer, lung cancer, colorectal cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, prostate cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, 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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