Peptide structure targeting carbonic dehydrogenase ix and use thereof

Abstract

The present invention relates to a peptide structure specifically binding to carbonic anhydrase IX (CAIX), and a use thereof. The CAIX-binding peptide ligand of the present invention contains D-amino acids, and thus has high binding specificity to CAIX while being stable in the body, and a linear or cyclic CAIX-binding peptide structure comprising same can bind to CAIX with high affinity in the body, and thus is effective in the diagnosis, prevention, suppression or treatment of diseases mediated by CAIX.

Description

Peptide structures targeting carbonic anhydrogenase IX and their uses

[0001] The present invention relates to a peptide construct that specifically binds to carbonic anhydrase IX (CAIX). Specifically, the present invention relates to a high-affinity CAIX-binding peptide construct comprising a CAIX-binding peptide ligand that is specific for CAIX and has increased stability by including a D-amino acid, and their use for diagnosing, preventing, inhibiting, or treating diseases mediated by CAIX.

[0002] This study was supported by the National New Drug Development Project of the National Drug Development Fund (NDP) funded by the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare (Project Number: 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).

[0004]

[0005] Carbonic anhydrase (CA) is a zinc (Zn) enzyme commonly found in higher vertebrates including humans. 2+ ) is a metalloenzyme that catalyzes the reversible hydration reaction that converts carbon dioxide into hydrogen ions and bicarbonate ions (CO2+H2O↔HCO3+H +) Sixteen isoenzyme forms of CA have been identified, and in humans, they are present in various tissues, including the gastrointestinal tract, reproductive tract, nervous system, kidney, lung, skin, and eye. Most CA isoenzymes are known to be involved in important physiological processes, such as respiration, calcification, acid-base balance, bone resorption, and formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid (Thiry et al., TRENDS in Pharmacological Sciences, 27(11): 566-573, 2006).

[0006] Among the CA family, carbonic anhydrous enzyme IX (CAIX) is unique in that it is expressed very restrictively in normal tissues, but is abnormally overexpressed in most solid tumors. This is mainly due to the strong transcriptional activation by hypoxia-inducible factor-1 (HIF-1), a transcription factor induced by hypoxia caused by excessive proliferation of solid tumors (De Simone et al., Biochimica et Biophysica Acta, 1804:404-409, 2010; Thiry et al, ditto).

[0007] Tumor hypoxia arises from the oxygen-poor environment created when solid tumors grow at a rate that exceeds the blood supply provided by the host vasculature. Even in this hypoxic microenvironment, solid tumors maintain sustained growth and proliferation through various genetic mutations. These hypoxic tumor cells exhibit increased resistance to chemotherapy and radiotherapy because chemotherapeutic agents are difficult to deliver through the bloodstream and because they lack the oxygen necessary for the cytotoxic effects of radiation-derived free radicals. Furthermore, hypoxic tumor cells induce the overexpression of CAIX on the cell surface, which lowers the pH of the extracellular environment of the tumor cells through CO2 hydration via the extracellular catalytic domain of CAIX. This acidic tumor microenvironment promotes tumor cell invasion and metastasis and can render pH-sensitive drugs ineffective (Thiry et al., ditto). Therefore, tumor hypoxia is generally recognized as a poor prognostic factor in cancer patients.

[0008] Recently, active research has been conducted to inhibit the growth and proliferation of CAIX-associated tumors by targeting CAIX overexpressed in tumor cells or by interfering with its catalytic activity, thereby disrupting tumor cell-mediated pH regulation. These studies primarily focus on the development of monoclonal antibodies or small-molecule sulfonamide inhibitors that bind to CAIX. However, when targeting solid tumors, large-molecular-weight monoclonal antibodies present challenges in efficient delivery, and small-molecule sulfonamide inhibitors are relatively unstable in solution, limiting their utility as pharmaceutical compounds.

[0009] Therefore, there is a need for the development of novel, high-affinity, stable, CAIX-specific binders and inhibitors suitable for pharmaceutical applications in cancer therapy, including treatment, prevention, diagnosis, prognosis, and imaging of cancer diseases.

[0010] [Prior Art Literature]

[0011] [Non-patent literature]

[0012] (Non-patent literature 1) Thiry et al., TRENDS in Pharmacological Sciences, 27(11): 566-573, 2006

[0013] (Non-patent Document 2) De Simone et al., Biochimica et Biophysica Acta, 1804:404-409, 2010

[0014]

[0015] One object of the present invention is to provide stable CAIX-specific binding agents and inhibitors suitable for use in the treatment, prevention, diagnosis or prognosis of cancer diseases.

[0016] One object of the present invention is to provide a high affinity CAIX-specific peptide construct comprising one or more effectors or functional groups, including a sulfonamide functional group, together with a CAIX-specific binding peptide ligand.

[0017] An additional object of the present invention is to provide a conjugate comprising the peptide structure.

[0018] An additional object of the present invention is to provide a composition for diagnosing, preventing or treating cancer comprising the peptide structure or conjugate.

[0019] An additional object of the present invention is to provide a method for diagnosing cancer using the peptide structure or conjugate.

[0020] An additional object of the present invention is to provide a method for treating cancer using the peptide structure or conjugate.

[0021] An additional object of the present invention is to provide a method for predicting prognosis after cancer treatment using the peptide structure or conjugate.

[0022]

[0023] To achieve the above purpose, as a result of extensive research, the inventors of the present application have developed a novel CAIX-specific binder and inhibitor that is stable and can bind specifically to CAIX with high affinity, including D-amino acid.

[0024] In one aspect of the present invention, the present invention provides a peptide structure comprising a peptide ligand 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 the 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 the side chain β-amino group; and a sulfonamide functional group-containing amino acid residue linked directly or through a spacer to the peptide ligand, and characterized in that the peptide structure has a structure represented by the following chemical formula 1.

[0025] [Chemical Formula 1]

[0026]

[0027] In the above formula,

[0028] P is the above peptide ligand,

[0029] F1, F2 and F3 are each independently a sulfonamide functional group-containing amino acid residue,

[0030] m is 0 or 1,

[0031] F4 is a general formula -(F5) o -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is the base, where

[0032] F5 is a sulfonamide functional group-containing amino acid residue,

[0033] S1 and S2 are each independently spacers,

[0034] F6 and F7 are each independently an amino acid residue containing a functional group other than sulfonamide,

[0035] S3 is -NH2 or -OH,

[0036] o is an integer of 0 or 1,

[0037] p, q, r and s each independently represent an integer from 0 to 6.

[0038] Non-limiting examples of chemical functional groups that can be substituted at the side chain ε-amino group of a lysine (Lys) residue among the above constituent amino acids include 4-(p-iodophenyl)butyric acid (IB) or 2-(4-iodophenyl)acetic acid (sIB). In addition, non-limiting examples of chemical functional groups that can be substituted at the side chain β-amino group of a 2,3-diaminopropionic acid (DAP) residue among the above constituent amino acids include α-D-galacturonic acid (Gal).

[0039]

[0040] In the above CAIX-specific peptide structure, the sulfonamide functional group-containing amino acid residues may have the following structures, but are not limited thereto.

[0041] or

[0042] In the above CAIX-specific peptide structure, functional groups other than sulfonamide can 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), a zwitteric ion (SZW), α-D-galacturonic acid (Gal), a cycloalkane having 5 to 15 carbon atoms, a fluorescent dye, or a cytotoxic agent.

[0043] In one aspect, the present invention provides a conjugate comprising a peptide structure linked directly or via a linker to a fluorescent dye, cytotoxic agent or radioisotope to the CAIX-specific peptide structure.

[0044] In one aspect, the present invention provides a pharmaceutical composition for diagnosing, preventing, or treating cancer, comprising the peptide structure or conjugate. The cancer may be a cancer expressing carbonic anhydrase IX. The cancer may be selected from the group consisting of liver cancer, lung cancer, colon 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, anal 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, but is not limited thereto.

[0045] In one aspect, the present invention provides a method for diagnosing cancer comprising administering to a subject the CAIX-specific peptide structure or the conjugate.

[0046] In one aspect, the present invention provides a method of treating cancer comprising administering to a subject the CAIX-specific peptide structure or the conjugate.

[0047] In one aspect, the present invention provides a method for predicting prognosis after cancer treatment comprising administering to a subject the CAIX-specific peptide structure or the conjugate.

[0048]

[0049] The present invention provides novel high-affinity CAIX binders and inhibitors that specifically bind to CAIX and are stable. The CAIX binders of the present invention exhibit excellent stability in vivo and are useful for CAIX targeting by including a CAIX-specific binding peptide ligand comprising one or more D-amino acids. In addition, the present invention provides a peptide construct in which one or more effectors or functional groups, including a sulfonamide functional group, are introduced into the CAIX-specific peptide ligand through the side chain of the amino acid residue, thereby providing a CAIX binder that is particularly useful for cancer imaging or diagnosis due to its high binding affinity to CAIX. The CAIX-specific peptide constructs of the present invention can also be used as effective agents for the diagnosis, prevention, or treatment of cancer by conjugating fluorescent dyes, cytotoxic agents, radioisotopes, or the like.

[0050]

[0051] Figure 1a is a graph showing the results of analyzing the binding affinity and binding kinetics characteristics of a peptide structure according to one embodiment of the present invention to the hCAIX extracellular domain (ECD) and human carbonic anhydrase XII (hCAXII) ECD using the BLItz® system.

[0052] FIG. 1b is a graph showing the results of analyzing the binding affinity and binding kinetics characteristics of peptide structures 103 to 105 and peptide structures 103 to 105 labeled with lutetium-175 (Lu-175) according to one embodiment of the present invention to the hCAIX extracellular domain (ECD) using the Octet®R8 system.

[0053] Figure 1c is a graph showing the results of analyzing the binding affinity and binding kinetics characteristics of peptide structures 106 to 107 according to one embodiment of the present invention to the hCAIX extracellular domain (ECD) using the Octet®R8 system.

[0054] FIG. 2a is a graph showing the results of fluorescence-activated cell sorting (FACS) analysis using SK-RC-52 cell line with peptide constructs 4 to 19 according to one embodiment of the present invention.

[0055] FIG. 2b is a graph showing the results of FACS analysis using SK-RC-52 cell line with peptide structures 20 to 28, 61 to 66 and 71 according to one embodiment of the present invention.

[0056] Figure 2c is a graph showing the results of FACS analysis using SK-RC-52 cell line of peptide structures 72 to 87 according to one embodiment of the present invention.

[0057] FIG. 2d is a graph showing the results of FACS analysis using SK-RC-52 cell line with peptide structures 88 to 102 according to one embodiment of the present invention.

[0058] Figures 3a to 3m show the results of biodistribution analysis of a peptide structure according to an embodiment of the present invention in mice into which the SK-RC-52 cell line was xenografted.

[0059] Figures 3a to 3c are graphs showing ex vivo images and fluorescence intensity measurements of tumors and organs extracted 24 hours after injection of peptide structures 29 to 54, respectively.

[0060] Figure 3d is a graph showing the ex vivo images and fluorescence intensity measurements of tumors and organs extracted 24 hours later from mice injected with each of the negative control (Dye) and seven peptide structures (31, 33, 39, 40, 43, 45, and 46) that showed high tumor signals in the results of Figures 3a to 3c.

[0061] Figure 3e is a graph showing the ex vivo images and fluorescence intensity measurements of tumors and organs extracted 72 hours after injection of peptide constructs 33, 39, and 40, respectively.

[0062] Figure 3f is a graph showing the ex vivo images and fluorescence intensity measurements of tumors and organs extracted 72 hours after injection of peptide constructs 55, 56, 57, 58, 59, and 60, respectively.

[0063] Figure 3g is a graph showing the ex vivo images and fluorescence intensity measurements of tumors and organs extracted 5 days after injection of peptide constructs 58 and 60, respectively.

[0064] Figure 3h is a graph showing the whole body images of mice 24 hours after injection of peptide structures 61 and 62, as well as ex vivo images of the extracted tumors and organs, and the results of measuring fluorescence intensity.

[0065] Figure 3i is a graph showing the whole body images of mice 24 hours after injection of peptide constructs 63 and 64, as well as ex vivo images of extracted tumors and organs, and the results of measuring fluorescence intensity.

[0066] Figure 3j is a graph showing the results of whole body images of mice 24 hours after injection of peptide structures 65, 75, 80, 83, and 84, ex vivo images of extracted tumors and organs, and fluorescence intensity measurements.

[0067] Figure 3k is a graph showing the whole body images of mice 24 hours after injection of peptide structures 85, 87, 89, 90, and 91, as well as ex vivo images of excised tumors and organs, and the results of measuring fluorescence intensity.

[0068] Figure 3l is a graph showing the whole body images of mice and ex vivo images of excised tumors and organs, and the results of measuring fluorescence intensity 72 hours after injection of peptide structures 63, 85, 87, 92, and 97, respectively.

[0069] Figure 3m is a graph showing the whole body images of mice 72 hours after injection of peptide structures 98 and 99, as well as ex vivo images of extracted tumors and organs, and the results of measuring fluorescence intensity.

[0070] Figure 4a is 177 Figure 1 shows mouse SPECT / CT imaging images taken 1.5 hours, 1 day, 2 days, 3 days, 7 days, 8 days, or 10 days after injection of Lu-labeled peptide constructs 67, 68, 69, and 70, respectively. Arrows indicate tumors.

[0071] Figure 4b is 177 This is a graph showing the results of measuring changes in body weight in mice injected with Lu-labeled peptide structures 67, 68, 69, and 70, respectively.

[0072] Figure 4c is 177This graph shows the results of measuring changes in tumor size (anticancer effect) in mice injected with Lu-labeled peptide structures 67, 68, 69, and 70, respectively.

[0073] Figure 5a is 177 Figure 1 shows SPECT / CT imaging images of mice injected with Lu-labeled peptide constructs 103, 104, and 105, respectively, at 1.5 hours, 1 day, 2 days, or 3 days after injection. Arrows indicate tumors.

[0074] Figure 5b is 177 This is a graph showing the results of measuring changes in body weight in mice injected with Lu-labeled peptide structures 103, 104, and 105, respectively.

[0075] Figure 5c is 177 This is a graph showing the results of measuring changes in tumor size (anticancer effect) in mice injected with Lu-labeled peptide structures 103, 104, and 105, respectively.

[0076] Figure 6a is a diagram showing HPLC analysis conditions for the synthesized material.

[0077] Figure 6b is a diagram showing the HPLC retention times of synthesized materials 1 to 28 and the molecular weights observed using electrospray ionization mass spectrometry (MS).

[0078] Figure 6c is a diagram showing the HPLC retention times of synthesized materials 29 to 68 and the molecular weights observed using electrospray ionization mass spectrometry (MS).

[0079] Figure 6d is a diagram showing the HPLC retention times of synthesized materials 69 to 107 and the molecular weights observed using electrospray ionization mass spectrometry (MS).

[0080]

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0082] The present invention is capable of various modifications and applications within the scope of the claims described below and equivalents interpreted therefrom.

[0083]

[0084] CAIX-specific peptide structure of the present invention

[0085] The present invention provides a peptide construct comprising a sulfonamide functional group-containing amino acid residue linked directly or via a spacer to a CAIX-specific peptide ligand.

[0086] The CAIX-specific peptide ligand may comprise any one of the amino acid sequences of SEQ ID NOS: 1 to 29. The peptide ligand may comprise a D-amino acid or may consist solely of D-amino acids. In addition, the peptide ligand may be substituted with one or more chemical functional groups, such as, but not limited to, 4-(p-iodophenyl)butyric acid (IB) or 2-(4-iodophenyl)acetic acid (sIB), at the side chain ε-amino group of a lysine (Lys) residue among the constituent amino acid residues. In addition, the peptide ligand may be substituted with one or more chemical functional groups, such as, but not limited to, α-D-galacturonic acid (Gal), at the side chain β-amino group of a 2,3-diaminopropionic acid (DAP) residue among the constituent amino acid residues.

[0087] The peptide ligand may be manufactured such that amino acid residues having a specific sequence are linked together to form a part of a peptide structure. The peptide ligand may be manufactured by a known peptide synthesis method, and is not particularly limited thereto. In one embodiment, the peptide ligand may be manufactured by repeating the peptide synthesis process on a single solid-phase bead until a peptide of the desired length and sequence is completed.

[0088] The above CAIX-specific peptide ligand may also include its salt form.

[0089] The introduction of the sulfonamide functional group into the amino acid residue containing the sulfonamide functional group of the above peptide structure can be accomplished through a known synthetic reaction 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.

[0090] or

[0091] The above peptide structure may further comprise one or more amino acid residues containing a functional group other than a sulfonamide functional group. The functional group other than the sulfonamide functional group 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).

[0092] In one embodiment, non-limiting examples of functional groups other than sulfonamides that may 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), a zwitteric ion (SZW), α-D-galacturonic acid (Gal), a cycloalkane having 5 to 15 carbon atoms, a fluorescent dye, or a cytotoxic agent.

[0093] The above chelators include, for example, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid-2,2',2'',2'''-(ethane-1,2-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-pentaazacyclopentadecane-N,N',N'',N''',N'''-pentaacetic acid (PEPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), (1R, 4R, 7R, 10R)-α, α', α'', α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)tetrasodium salt (DOTMA), 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA) and triethylenetetramine (TETA), It may be at least one selected from, but is not limited to, 1,4,7,10-tetraazacyclododecan-7-acetamide-1,4,10-triacetic acid (Pb specific chelator, PSC), 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide (DOTAM or TCMC).

[0094] The cycloalkane having 5 to 15 carbon atoms may be, for example, one or more selected from cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, adamantane, norbornane, isobornane, and tricyclodecane, but is not limited thereto.

[0095] The peptide construct of the present invention can also link a chemical functional group-containing amino acid residue, such as a sulfonamide functional group, to a CAIX-specific binding peptide ligand via a spacer. The spacer may be, but is not limited to, one or more 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.

[0096] The peptide structure of the present invention has a structure represented by the following chemical formula 1.

[0097] [Chemical Formula 1]

[0098]

[0099]

[0100] In the above formula,

[0101] P, F1, F2, F3, F4 and n are each as defined above.

[0102] In one embodiment of the present invention, the peptide structure has a structure represented by the following chemical formula 2.

[0103] [Chemical Formula 2]

[0104]

[0105]

[0106]

[0107] In the above formula,

[0108] R5 is a general formula -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is the flag,

[0109] Here S1, S2, S 3, F6, F7, p, q, r and s are as defined above, respectively,

[0110] 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 each defined below.

[0111]

[0112]

[0113] Although not bound by a specific theory, it is believed that cyclic structures, such as the cyclic peptide structure of the present invention, have less flexibility than linear peptides, and thus have less entropy loss upon target binding, resulting in higher binding affinity and increased binding specificity to the target.

[0114] The peptide construct of the present invention exhibits high selectivity, specifically binding to CAIX but not to other isoenzymes of CAIX (e.g., carbonic anhydrase XII).

[0115]

[0116] conjugate

[0117] The CAIX-specific peptide structure of the present invention can be combined directly or via a linker with a fluorescent dye, cytotoxic agent, or radioisotope to form a conjugate. The conjugate can target CAIX and label a cancer expressing CAIX with a fluorescent dye or radioisotope, or effectively deliver a drug such as a radioisotope or cytotoxic agent to the cancer, making it useful for the diagnosis, prevention, or treatment of cancer.

[0118] In one embodiment, the linker may be one or more selected from, but is not limited to, 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).

[0119] 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-indacene (BODIPY), Texas Red, dansyl, Lissamine, cyanine (Cy), and phycoerythrin.

[0120] In one embodiment, the cytotoxic agent may be one or more selected from, but is not limited to, a toxin, a chemotherapeutic agent, a drug moiety, an antibiotic, and a nucleolytic enzyme.

[0121] In one embodiment, the radioactive isotope is selected from the group consisting of 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 at least one 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).

[0122]

[0123] Therapeutic administration and formulation

[0124] The present invention provides a pharmaceutical composition for diagnosing, preventing or treating cancer, comprising the CAIX-specific peptide structure or conjugate of the present invention. Preferably, the cancer of the present invention is a solid cancer. More preferably, the cancer of the present invention is a cancer that expresses CAIX. For example, the cancer of the present invention may be a solid cancer such as liver cancer, lung cancer, colon 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, anal 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, but is not limited thereto.

[0125] The subject to whom the pharmaceutical composition of the present invention is administered for diagnosing, preventing, or treating cancer may be a mammal at risk of developing cancer, diagnosed with cancer, or undergoing cancer treatment. The mammal may be a human or a non-human mammal.

[0126] The pharmaceutical composition for diagnosing, preventing or treating cancer according to the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterile injection solutions, according to conventional methods, and may include appropriate carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

[0127] The carrier, excipient or diluent may include 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, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0128] When formulating, it can be manufactured using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants commonly used in the pharmaceutical industry.

[0129] The preferred dosage of the pharmaceutical composition for preventing or treating cancer according to the present invention varies depending on the patient's condition, body weight, severity of the disease, drug form, route of administration, and duration of administration, but can be appropriately selected by those skilled in the art. However, for desirable effects, the dosage may be 0.0001 to 2,000 mg / kg per day, preferably 0.001 to 2,000 mg / kg. The dosage may be administered once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosage.

[0130] The pharmaceutical composition for preventing or treating cancer according to the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. Administration methods include, for example, oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

[0131] The present invention also provides a method of treating cancer, comprising administering to a subject in need of such treatment a CAIX-specific peptide construct or conjugate of the present invention.

[0132] The CAIX-specific peptide constructs and conjugates of the present invention may also be used for diagnosing cancer by targeting and imaging cancer, or for predicting or monitoring the prognosis of a subject after cancer treatment by administering the constructs to a subject undergoing cancer treatment.

[0133] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various modifications are possible within the scope of the invention.

[0134]

[0135] Example

[0136]

[0137] Example 1. Synthesis and purification of peptide structures containing selected peptides

[0138] 1-1. Synthesis of reagents inserted into peptide structures

[0139] N-succinimidyl 2,3:4,5:6,7-tri-O-isopropylidene-D-glycero-D-gulo-heptonate (6OH(Protected)-NHS) (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) were synthesized and used directly. First, for the synthesis of S1, 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 equipped with a magnetic stirrer bar, followed by the addition of MeOH, and the mixture was reacted at room temperature for 24 h. After the reaction was completed, volatile substances and solvents were removed from the reaction product using an evaporator. The reaction mixture was dissolved in a 10% citric acid aqueous solution, transferred to a separatory funnel, and 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 over sodium sulfate in an Erlenmeyer flask. Then, 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 in dichloromethane (DCM) once again, and the volatile solvent was removed using an evaporator.

[0140] 2,3:4,5:6,7-tri-O-isopropylidene-D-glycero-D-gulo-heptonic acid (1 eq.) synthesized in this way was placed in a 50 mL round-bottom flask with a magnetic stirrer bar, N-hydroxysuccinimide (NHS) and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC hydrochloride, 1.6 eq.) together with DCM anhydrous, and the mixture was reacted at room temperature for 24 h. After the reaction was completed, the reaction mixture was dissolved in ethyl acetate, transferred to a separatory funnel, and washed with a saturated NaCl aqueous solution. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate and then with a saturated aqueous solution of NaCl. The organic layer was collected in an Erlenmeyer flask and dried over anhydrous sodium sulfate. Afterwards, sodium sulfate was removed from the organic solution using a filter paper, and the volatile solvent was removed using an evaporator. The reaction product was dissolved in DCM once again, and the volatile solvent was completely removed using an evaporator before use. An example of the reaction for synthesizing the S1 functional group as above is as follows.

[0141]

[0142] [Reaction Formula 1]

[0143]

[0144] The synthesis of Fmoc-DAP(DOTA(Protected))-OH (S2) and Fmoc-DAP(Gal(Protected))-OH (S3) was carried out by first dissolving Fmoc-DAP-OH (1 eq) without protecting group in N,N-dimethylformamide (DMF), and then adding 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. (1,2:3,4-Di-O-isopropylidene-a-D-galacturonide) (1.5 eq) was dissolved in 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate, TBTU) (1.2 eq), N, N-diisopropylethylamine (N, N-Diisopropylethylamine, DIPEA) (3 eq) and activated in DMF, and the mixture was reacted for 2 hours. Afterwards, the reaction progress was checked by LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent), and purified by 1260 Infinity II LC system (Agilent) and used. The analytical equipment used was LC-MS (1260 Infinity II, Infinity Data were analyzed using Lab LC / MSD, Agilent, and MNOVA (v. 14.2.0, Mestrelab research, Spain). The stationary phase was an Agilent Poroshell 120 EC-C18 column (4.6 X 50 mm, 2.7 μm), and the column temperature was maintained at 40°C.In addition, a mixture of acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) and 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 was 1 mL / min. And the 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 distilled water and ACN containing 0.1% TFA, (3) the flow rate was 15 mL / min, and (4) the detection wavelengths were 214 and 254 nm. The synthetic processes of S2 and S3 are as follows.

[0145]

[0146] [Reaction Formula 2]

[0147]

[0148]

[0149] For the synthesis of S4, 1 g of 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-bottomed flask and stirred at room temperature for 24 h. The reaction mixture was concentrated using an evaporator, and the resulting gel-like substance was first precipitated using ethyl acetate and separated using a nylon membrane filter. The separated precipitate was dissolved in a minimum amount of water, and the substance was recrystallized by adding DMF, separated using a nylon membrane filter, washed with ethyl acetate, and dried. An example of the synthesis of S4 is as follows.

[0150]

[0151] [Reaction Formula 3]

[0152]

[0153]

[0154] The synthesis process of S5 is as follows.

[0155] In a round bottom flask, Fmoc-D-Orn(boc)-OH (1 eq., Cat# BD131117, BLD Pharmatech), N,N-dicyclohexylcarbodiimide (DCC, 1.5 eq., Cat# 36650, Sigma-Aldrich), 1-hydroxybenzotriazole (HOBt, 1.5 eq., Cat# CXZ010, Aapptec), 4-(dimethylamino)pyridine (DMAP, 0.05 eq., Cat# 107700, Sigma-Aldrich) and DCM anhydrous were added and stirred well, and the mixture was 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 reacted at room temperature for 16 hours. After the reaction, most of the precipitate was filtered through a paper filter, and the obtained solution was concentrated using an evaporator. The gel-like substance was completely dissolved in a minimum amount of ethyl acetate, and then stored at 20℃ for 16 hours to cause precipitation. The precipitate of the ethyl acetate solution containing the reactant was filtered through a paper filter and transferred to a separatory funnel, and 100 mL of ethyl acetate was added. Using a separatory funnel, the organic layer was washed once with a saturated aqueous sodium bicarbonate solution (Cat# S0341, Samchun Pure Chemical Industry Co., Ltd.), twice with triple-distilled water, and once with a saturated aqueous sodium chloride solution (Cat# S0476, Samchun Pure Chemical Industry Co., Ltd.). The washed organic layer was dried by adding anhydrous magnesium sulfate (Cat# M1806, Samchun Pure Chemical Industry Co., Ltd.), and the precipitate was filtered through a paper filter. The solution was concentrated using an evaporator.The concentrated gel-like substance was completely dissolved in minimal DCM, and half of the solution was purified by a column (40 X 210 mm) containing 200 g of silica gel (0.063-0.200 mm, Cat# 1.07734.1000, Sigma-Aldrich). A 1:1 solution of ethyl acetate: hexane (Cat# 1.07734.1000, Sigma Aldrich) was used as a developing solvent. The remaining DCM solution was also purified by a silica gel column in the same manner. The reaction product Fmoc-D-Orn(boc)-OAll had an Rf of 0.59 (ethyl acetate: hexane = 1:1), and the purified solution of the corresponding Rf was collected and concentrated by an evaporator to obtain Fmoc-D-Orn(boc)-OAll.

[0156] Next, to remove the Boc protecting group by reaction, Fmoc-D-Orn(boc)-OAll (1 equivalent) was added to a round-bottom flask, and a TFA: DCM = 1:1 mixed solution was slowly poured at room temperature, mixed, stirred well, and reacted for 1 hour. After the reaction, the solution was concentrated using an evaporator. The concentrated material was dissolved in DCM and concentrated again using an evaporator, and the process was repeated three times. Then, the concentrated material was dissolved in DCM anhydrous (Cat# 270997, Sigma Aldrich), sealed, and stored in a freezer. The concentrated material was used in the next reaction without any special purification process after confirming the molecular weight and purity by LC-MS as in Example 1-1 above.

[0157] 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl isothiocyanate (pbf-NCS), required for the following reaction, was first synthesized as follows.

[0158] 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) in DCM anhydrous was added to a round-bottomed flask. A reflux condenser was added, and the reaction solution was refluxed (75°C) for 1 hour. The reaction product, pbf-NCS [Rf=0.49 (hexane: DCM=1:1)], was confirmed by TLC. After the reaction was completed, the concentrated gel-like material was completely dissolved in a minimum amount of DCM and purified by 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 Rf=0.49 was collected and concentrated using an evaporator to obtain pbf-NCS as a transparent liquid. The process of this reaction is as follows.

[0159]

[0160] [Reaction Formula 4]

[0161]

[0162]

[0163] For the next reaction, Fmoc-D-Orn-OAll (1 eq), pbf-NCS (1.2 eq), and DIPEA (10 eq) synthesized above were dissolved in DCM in a round-bottomed flask and reacted at room temperature for 1 hour. After the reaction, the product was concentrated using an evaporator, and the concentrated material was completely dissolved in a minimum amount of DCM and 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 a developing solvent. The reaction product Fmoc-D-Orn(SCN(pbf))-OAll had an Rf of 0.62 (ethyl acetate: hexane = 1: 1), and the purified solution of the corresponding Rf was collected, concentrated using an evaporator, and used in the next reaction.

[0164] In a round bottom flask, synthesized Fmoc-D-Orn(SCN(pbf))-OAll (1 eq) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 2 eq, Cat# E7750, Sigma-Aldrich) were dissolved in DCM and 2 M dimethyl amine in THF (2 eq, 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 by LC-MS of Example 1-1, the reaction was concentrated using an evaporator when the reaction was completed. The concentrated material was dissolved in a mixture of ACN / 3-distilled water (1:1), filtered through a 45 μm syringe filter (Cat# DISMIC-3HP, Advantec), and purified using HPLC as in Example 1-1. The purified material, Fmoc-D-ADMA(pbf)-OAll, was freeze-dried to obtain a powder form.

[0165] Finally, under Ar, synthesized Fmoc-D-ADMA(pbf)-OAll (1 eq), Pd(PPh3)4 (0.05 eq), and N-methyl aniline (3 eq, Cat# M29304, Sigma-Aldrich) were dissolved in THF anhydrous in a glass vial and reacted at room temperature for 1 h. After confirming the molecular weight and purity of the reactant by LC-MS, the reactant was concentrated using an evaporator. The concentrated material was dissolved in a mixture of ACN / tridistilled water (1:1), filtered through a 45 μm syringe filter, and purified under the same HPLC conditions used above. The purified material was lyophilized 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) in powder form. The entire synthetic process of S5 is as follows.

[0166]

[0167] [Reaction Formula 5]

[0168]

[0169]

[0170] 1-2. Synthesis of solid-phase peptides

[0171] Solid-phase peptide chains were synthesized using an automated ultrasonic peptide synthesizer (Liberty Blue™ automated microwave peptide synthesizer, CEM Corporation) or an automatic synthesizer (Apex 396, AAPPTEC). Two solid-phase resins were used for synthesis, which differ in the C-terminus of the synthesized peptides. Rink Amide-ChemMatrix® Resin (0.41 mmole / g, Cat# 7-600-1310, Biotage), which is used for most materials, has a C-terminus of -CONH2, while 2-Chlorotrityl Chloride Resin has a C-terminus of -COOH. 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 mounted by reacting the first amino acid (5 equivalents) and DIPEA (10 equivalents) in DCM solvent under Ar atmosphere for 12 hours before use. In an automated ultrasonic peptide synthesizer, the resin swollen in N-methyl-2-pyrrolidone (NMP) was treated with Fmoc-amino acid (5 equiv.), Oxyma Pure (5 equiv., Cat# cxz021, AApptec), and diisopropylcarbodiimide (DIC, Cat# D0254, TCI) (10 equiv.) in NMP solvent, coupled in NMP solvent at 75°C for 5 min, washed three times with NMP, and reacted with a mixture of piperidine / NMP (v / v = 1:4, 0.1 M OxymaPure) at 90°C for 1 min to remove the Fmoc protecting group. After repeated coupling of Fmoc-amino acid synthesis by the same process, the beads were collected from the synthesizer.Likewise, in an automatic 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. After washing with NMP and DCM, the Fmoc protecting group was removed by reacting with a mixed solution of piperidine / NMP (v / v=1:4) at room temperature for 10 minutes. After repeating the same process for Fmoc-amino acid synthesis, the beads were collected from the synthesizer. In addition, when the materials synthesized in Example 1-1 were used for solid-phase peptide synthesis, the number of equivalents of the reagents used in the synthesis was reduced to 2 equivalents by performing manual reaction directly instead of instrumental synthesis. In particular, for the synthesis of peptides with a cyclic structure, Fmoc-E(OAll)-OH (Cat#36613, GL Biochem) was first introduced, and then the peptide linkage (cyclization) reaction was performed.

[0172]

[0173] 1-3. Structure and functional group synthesis of solid-phase peptides

[0174] 1-3-1. Cyclization reaction

[0175] The allyl protecting group removal reaction of glutamic acid of the solid beads synthesized using an automatic synthesizer was performed. 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 and placed in a cylinder equipped with a filter containing the solid beads, sealed with a lid, and hung on a 180° shaker at room temperature for 1 hour. Afterwards, the beads were washed three times with NMP and then washed more than five times with a 0.1 M sodium diethyldithiocarbamate (Cat# D3506, Sigma-Aldrich) in NMP solution to remove Pd impurities. After washing three times with NMP, 3 mL of piperidine / NMP (v / v=1:4) solution was added to remove the Fmoc protecting group at the N-terminus, and the beads were suspended on a 180° shaker at room temperature for 5 minutes. After the reaction, the solution was emptied and refilled, and the beads were washed three times each with NMP, DCM, and then NMP again. The cyclization reaction of the beads from which both Fmoc and OAll were removed was performed by adding a solution of (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (5 equivalents, Cat# 36813, GL Biochem) and DIPEA (10 equivalents) in NMP, sealing the solution with a cap, and incubating the beads on a 180° shaker at room temperature for 12 h. The solid beads upon completion of the reaction were washed three times with NMP and DCM and then used in the next reaction. An example of the cyclization reaction scheme for the synthesis of a cyclic peptide is as follows.

[0176]

[0177] [Reaction Formula 6]

[0178]

[0179]

[0180] 1-3-2. Functional group synthesis

[0181] After the cyclization reaction, the solid-phase peptide requiring functional group synthesis undergoes the following reaction on the beads.

[0182] Introduction of biotin, DOTA, PSC, TCMC, IB, and sIB functional groups was performed by removing the Mtt or Dde protecting group from beads loaded with peptides containing lysine protected with 4-methyltrityl (Mtt) or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) substituents. 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 min each for a total of 6 times, and the Dde protecting group was removed by treating with a mixed solution of hydroxylamine hydrochloride / imidazole (1.3:1, w / w) in NMP solvent at room temperature for 2 h each twice, or once for 12 h.Afterwards, beads with amine functional groups from which the protecting groups were removed were conjugated with Biotin-NHS ester (2 eq., Cat# 246201, SY Innovation), or DOTA-tris(tert-butyl ester) (2 eq.), or DO2Atbu-mono amide mono acid (PSC with two tert-butyl protecting groups, 2 eq., Cat# C134, Chematech), or S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane (TCMC-NSC, 2 eq., 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 for 1 hour at room temperature. After completion of the reaction, the beads were washed sequentially with NMP and DCM three times each and used in the next reaction.

[0183] Introduction of zwitterionic (SZW) functional groups was performed using peptides containing lysine residues protected by Mtt or Dde or DAP residues. The N-terminus of the peptide synthesized on the beads was converted to an amine form with the Mtt or Dde protecting group removed as described above. 2-Bromoacetic acid (30 equivalents, Cat# 17000, Sigma-Aldrich) and DIC (30 equivalents) dissolved in DMF were added to the beads containing the deprotected amine and reacted for 3 hours. After completion of the reaction, the beads were sequentially washed three times each with NMP and DCM. The directly synthesized S4 (30 equivalents) was dissolved in deionized water and added to the beads immersed in DMF together with DIPEA (30 equivalents) and reacted for 12 hours. After the reaction is complete, the beads are washed sequentially three times each using NMP and DCM and used in the next reaction.

[0184] The sulfonamide (SFA) functional group was then synthesized by a click reaction of a peptide containing an azido functional group on solid beads with 4-ethynylbenzenesulfonamide (Cat# E1130, TCI). Under an Ar atmosphere, the peptide containing an azido functional group on solid beads (1 eq), 4-ethynylbenzenesulfonamide (2 eq per azido functional group), CuI (1 eq per azido functional group), tris(benzyltriazolylmethyl)amine (TBTA, 2 eq per azido functional group, Cat# T2993, TCI), and DIPEA (10 eq) were mixed in NMP solvent and reacted at room temperature for 12 h. After the reaction was completed, the beads were washed three times with NMP and five or more times with a 0.1 M sodium diethyldithiocarbamate in NMP solution to remove copper impurities. An example of removing the Mtt protecting group from a peptide and introducing the IB functional group and SFA is as follows.

[0185]

[0186] [Reaction Formula 7]

[0187]

[0188]

[0189] 1-4. Peptide cleavage and separation purification from solid-phase beads

[0190] The beads loaded with dried peptides were treated with a mixture of 95% TFA / 2.5% TIPS / 2.5% tertiary distilled water at room temperature for 6 hours. The beads were removed using a filter, and the TFA mixture containing the peptides was collected in a conical tube and blown with nitrogen gas to remove most of the mixture. Subsequently, diethyl ether was added to precipitate the peptides, and the tube was centrifuged at 3000 rpm for 5 minutes. After removing the diethyl ether from the upper layer and a small amount of the remaining TFA mixture, the remaining solid peptides were vacuum-dried.

[0191] The peptide cleaved from the solid phase beads was dissolved in a mixture of ACN and distilled water (1:1), and then insoluble impurities were removed using a 45 μm syringe filter. The resulting product was purified and analyzed using the system described in Example 1-1. The solvent conditions and analysis results of LC-MS are shown in Figures 6a to 6d. The purified peptide solution was used in powder form after removing the solvent using a freeze dryer.

[0192]

[0193] 1-5. Attaching a 6OH functional group to the purified peptide

[0194] In a glass vial, the purified peptide in powder form with Lys or DAP residues and 6OH(Protected)-NHS (S1) (6 equivalents) and DIPEA (15 equivalents) were dissolved in DMF and reacted for 2 hours with magnetic stirring. After the reaction, foreign substances were removed using a 45 μm syringe filter, and the degree of reaction was observed and purified using the same system mentioned in Example 1-1. The purified peptide solution was made into a powder form after removing the solvent using a freeze dryer. To remove the acetal protecting group of 6OH(Protected), a mixed solution of 95% (v / v) TFA, 2.5% (v / v), and TIS 2.5% (v / v) water was added to the purified peptide and reacted for 2 minutes. At this time, caution should be taken because 6OH is detached from the Lys or DAP residues if the reaction time exceeds 2 minutes. Immediately after the reaction, a mixture of ACN / 3-distilled water (1:1) is added, and to neutralize the solution, trimethylamine (Cat# T0424, TCI) is slowly added in the same amount as the TFA solution 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.

[0195]

[0196] 1-6. Attaching fluorescent dyes (AF488, Cy5) to purified peptides

[0197] The peptide requiring the introduction of a fluorescent dye was first purified, and then the fluorescent dye was loaded onto the peptide through click chemistry between the alkyne functional group (introduced using Fmoc-propargyl-Gly-OH (Fmoc-Pra-OH, Cat# 21528, GL Biochem)) contained in the peptide chain and the azido functional group contained in the fluorescent dye. Powdered peptide (1 eq), fluorescent dye Alexa Fluor 488 azide (AF488 azide, Cat# 1275, Click Chemistry Tools) or Sulfo-Cyanine5 azide (Cy5 azide, cat# E3330, Lumiprobe) (1 eq), 1 M CuSO4 aqueous solution (3 eq), and 1 M ascorbic acid (6 eq) were reacted for 1 h under Ar atmosphere. After the reaction, the progress of the reaction was confirmed by LC-MS as mentioned in Example 1-1, and the peptide containing the DOTA functional group was added with sodium diethyldithiocarbamate (5 equivalents) to remove the copper coordinated to DOTA, and after the reaction for 20 minutes, the removal of copper was confirmed by LC-MS. The confirmed peptide was purified by the same system mentioned in Example 1-1. An example of attaching a fluorescent dye to a peptide is as follows.

[0198]

[0199] [Reaction Formula 8]

[0200]

[0201]

[0202] Through the synthetic process described above, peptide structures having a sulfonamide functional group that specifically binds to human CAIX were obtained as follows, and the HPLC analysis conditions and retention time of each substance, and the molecular weight observed using electrospray ionization mass spectrometry (ESI-MS) are shown in Figures 6a to 6d. The analytical equipment 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). The stationary phase used was an Agilent Poroshell 120 EC-C18 column (4.6 X 50 mm, 2.7 μm), and the column temperature was maintained at 40°C. Additionally, a mixture of acetonitrile (ACN) containing 0.1% TFA and tertiary distilled water containing 0.1% TFA was used as the mobile phase solvent, and the wavelengths were 214 and 254 nm, and the flow rate was maintained at 1 mL / min.

[0203]

[0204] [Chemical Formula 2]

[0205]

[0206]

[0207]

[0208] In the above chemical formula, R1 to R4 may include sulfonamide. AA1 to AA5 represent common amino acid residues or amino acid residues containing functional groups (Gal, IB, sIB, dimethyl), and lowercase letters represent D-amino acids and uppercase letters represent L-amino acids, respectively. U represents a modified or unnatural D-amino acid. The chemical formula, excluding common amino acid residues, has the following structure defined herein.

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] [Structure of peptide structure 1]

[0218]

[0219] [Structure of peptide structure 2]

[0220]

[0221] [Structure of peptide structure 3]

[0222]

[0223] [Structure of peptide structure 4]

[0224]

[0225] [Structure of peptide structure 5]

[0226]

[0227] [Structure of peptide structure 6]

[0228]

[0229]

[0230] [Structure of peptide structure 7]

[0231]

[0232] [Structure of peptide structure 8]

[0233]

[0234] [Structure of peptide structure 9]

[0235]

[0236]

[0237] [Structure of peptide structure 10]

[0238]

[0239] [Structure of peptide structure 11]

[0240]

[0241] [Structure of peptide structure 12]

[0242]

[0243]

[0244] [Structure of peptide structure 13]

[0245]

[0246] [Structure of peptide structure 14]

[0247]

[0248] [Structure of peptide structure 15]

[0249]

[0250]

[0251] [Structure of peptide structure 16]

[0252]

[0253] [Structure of peptide structure 17]

[0254]

[0255] [Structure of peptide structure 18]

[0256]

[0257] [Structure of peptide structure 19]

[0258]

[0259] [Structure of peptide structure 20]

[0260]

[0261] [Structure of peptide structure 21]

[0262]

[0263] [Structure of peptide structure 22]

[0264]

[0265] [Structure of peptide structure 23]

[0266]

[0267] [Structure of peptide structure 24]

[0268]

[0269] [Structure of peptide structure 25]

[0270]

[0271] [Structure of peptide structure 26]

[0272]

[0273] [Structure of peptide structure 27]

[0274]

[0275] [Structure of peptide structure 28]

[0276]

[0277] [Structure of peptide structure 29]

[0278]

[0279] [Structure of peptide structure 30]

[0280]

[0281] [Structure of peptide structure 31]

[0282]

[0283] [Structure of peptide structure 32]

[0284]

[0285] [Structure of peptide structure 33]

[0286]

[0287] [Structure of peptide structure 34]

[0288]

[0289] [Structure of peptide structure 35]

[0290]

[0291] [Structure of peptide structure 36]

[0292]

[0293] [Structure of peptide structure 37]

[0294]

[0295] [Structure of peptide structure 38]

[0296]

[0297] [Structure of peptide structure 39]

[0298]

[0299] [Structure of peptide structure 40]

[0300]

[0301] [Structure of peptide structure 41]

[0302]

[0303] [Structure of peptide structure 42]

[0304]

[0305] [Structure of peptide structure 43]

[0306]

[0307] [Structure of peptide structure 44]

[0308]

[0309] [Structure of peptide structure 45]

[0310]

[0311] [Structure of peptide structure 46]

[0312]

[0313] [Structure of peptide structure 47]

[0314]

[0315] [Structure of peptide structure 48]

[0316]

[0317] [Structure of peptide structure 49]

[0318]

[0319] [Structure of peptide structure 50]

[0320]

[0321] [Structure of peptide structure 51]

[0322]

[0323] [Structure of peptide structure 52]

[0324]

[0325] [Structure of peptide structure 53]

[0326]

[0327] [Structure of peptide structure 54]

[0328]

[0329] [Structure of peptide structure 55]

[0330]

[0331] [Structure of peptide structure 56]

[0332]

[0333] [Structure of peptide structure 57]

[0334]

[0335] [Structure of peptide structure 58]

[0336]

[0337] [Structure of peptide structure 59]

[0338]

[0339] [Structure of peptide structure 60]

[0340]

[0341] [Structure of peptide structure 61]

[0342]

[0343] [Structure of peptide structure 62]

[0344]

[0345] [Structure of peptide structure 63]

[0346]

[0347] [Structure of peptide structure 64]

[0348]

[0349] [Structure of peptide structure 65]

[0350]

[0351] [Structure of peptide structure 66]

[0352]

[0353] [Structure of peptide structure 67]

[0354]

[0355] [Structure of peptide structure 68]

[0356]

[0357] [Structure of peptide structure 69]

[0358]

[0359] [Structure of peptide structure 70]

[0360]

[0361] [Structure of peptide structure 71]

[0362]

[0363] [Structure of peptide structure 72]

[0364]

[0365] [Structure of peptide structure 73]

[0366]

[0367] [Structure of peptide structure 74]

[0368]

[0369] [Structure of peptide structure 75]

[0370]

[0371] [Structure of peptide structure 76]

[0372]

[0373] [Structure of peptide structure 77]

[0374]

[0375] [Structure of peptide structure 78]

[0376]

[0377] [Structure of peptide structure 79]

[0378]

[0379] [Structure of peptide structure 80]

[0380]

[0381] [Structure of peptide structure 81]

[0382]

[0383] [Structure of peptide structure 82]

[0384]

[0385] [Structure of peptide structure 83]

[0386]

[0387] [Structure of peptide structure 84]

[0388]

[0389] [Structure of peptide structure 85]

[0390]

[0391] [Structure of peptide structure 86]

[0392]

[0393] [Structure of peptide structure 87]

[0394]

[0395] [Structure of peptide structure 88]

[0396]

[0397] [Structure of peptide structure 89]

[0398]

[0399] [Structure of peptide structure 90]

[0400]

[0401] [Structure of peptide structure 91]

[0402]

[0403] [Structure of peptide structure 92]

[0404]

[0405] [Structure of peptide structure 93]

[0406]

[0407] [Structure of peptide structure 94]

[0408]

[0409] [Structure of peptide structure 95]

[0410]

[0411] [Structure of peptide structure 96]

[0412]

[0413] [Structure of peptide structure 97]

[0414]

[0415] [Structure of peptide structure 98]

[0416]

[0417] [Structure of peptide structure 99]

[0418]

[0419] [Structure of Peptide Structure 100]

[0420]

[0421] [Structure of Peptide Structure 101]

[0422]

[0423] [Structure of peptide structure 102]

[0424]

[0425] [Structure of peptide structure 103]

[0426]

[0427] [Structure of peptide structure 104]

[0428]

[0429] [Structure of peptide structure 105]

[0430]

[0431] [Structure of peptide structure 106]

[0432]

[0433] [Structure of peptide structure 107]

[0434]

[0435] 1-7. Labeling of lutetium-175 (Lu-175)

[0436] For binding affinity experiments to the hCAIX protein, some peptide constructs containing the DOTA linker were labeled with the isotope lutetium-175 (Lu-175) instead of lutetium-177 (Lu-177).

[0437] The synthesized peptide structure was dissolved in DMSO at a concentration of 2 mM, and lutetium-175 (LuCl3, Cat#450960, Sigma-Aldrich) was prepared by dissolving it in sodium acetate buffer (pH 5.5) at a concentration of 10 mM. After adding 3 equivalents of lutetium-175 to the peptide structure (1 equivalent) dissolved in DMSO, 500 μL of sodium acetate buffer (pH 5.5) was added, and the mixture was reacted at 90 °C for 30 min in a thermoblock (thermo block, Cat#B10-48, BLE). After the reaction was completed, the mixture was cooled to room temperature and purified using a 1260 Infinity II LC system (Agilent) under the following conditions: (1) the stationary phase was a Kromasil 100-5-C18 column (10 X 250 mm, 5 μm), (2) the mobile phase solvent was a mixture of 3-distilled water and ACN with 0.1% acetic acid added, (3) the flow rate was 8 mL / min, and (4) the detection wavelengths were 214 and 254 nm. The purified peptide solution labeled with lutetium-175 was freeze-dried to obtain a powder form.

[0438]

[0439] Example 2. Confirmation of biological properties of peptide structures

[0440] 2-1. Analysis of binding affinity and binding kinetics for the extracellular domain (ECD) of human carbonic anhydrase IX (hCAIX)

[0441] The binding affinity and binding kinetic properties of the peptide constructs to hCAIX ECD were analyzed. To confirm the specific binding selectivity to hCAIX ECD, the binding kinetic properties of the peptide constructs were also analyzed to the extracellular domain of human carbonic anhydrogenase XII (hCAXII), another isoform of carbonic anhydrogenase.

[0442]

[0443] 2-1-1. Analysis using BLITZ equipment

[0444] Binding affinity was determined using the BLItz® system (Cat# 45-5000, Fort Bio) and the advanced kinetics module of BLItz Pro ver1.3 software were used. A streptavidin biosensor (Cat# 18-5019, Sartorius) was used for all measurements. In addition, a protein concentration of 0 nM was used as a reference for nonspecific binding and background correction. First, 10X Octet kinetics buffer (Cat# 18-1105, Sartorius) was diluted to 1X using DPBS (Cat# L0615, Biowest) and used as the assay buffer solution. Then, 200 μl / well of the assay buffer solution was dispensed into 96-well, black, flat-bottom polypropylene (Cat# 655209, Greiner Bio-One), and the streptavidin biosensor was immersed and hydrated for 10 minutes. After mounting the biosensor on the device, the peptide was loaded at a concentration of 1 μM for 120 seconds at 2200 rpm to attach it. After that, the protein was subjected to association and dissociation at each concentration for 120 seconds at 2200 rpm, and the kinetics data were analyzed using the global fitting function. D The values ​​were calculated as the ratio of kd to ka. Kinetic parameters were also obtained by fitting with a 1:1 binding model with baseline correction.

[0445] The results are shown in Fig. 1a. The lines in each graph shown in Fig. 1a represent the results of experiments with different protein concentrations. The protein concentrations corresponding to each line are indicated below the lines. For example, in Graph 2 shown in Fig. 1a, the line indicated at the top represents the results when the concentration of hCAIX (nM) is 125 nM, and the line indicated at the bottom represents the results when the concentration of hCAIX (nM) is 50 nM. The peptide structures showed significantly higher selectivity for hCAIX ECD than for hCAXII ECD.

[0446]

[0447] 2-1-2. Analysis using Octet equipment

[0448] The Octet®R8 system (Octet BLI Discovery 13 software) is an instrument based on the same principle as the BLItz® system, and proteins (Cat# CA9-H82E3, ACRO Biosystems) were attached to streptavidin biosensors (Cat# 18-5019, Sartorius) for analysis. The proteins were diluted to 300 nM in DPBS (Cat# L001-02, WELGENE) and loaded at 1000 rpm for 600 s. Afterwards, peptides (0.5% DMSO) diluted in DPBS were added at each concentration for association at 1000 rpm for 300 s and dissociation for 600 s. Nonspecific binding of peptides to the biosensor was confirmed to be absent at 100 nM of each peptide, and 0 nM of peptide concentration was used as a reference for background correction. K D The values ​​were obtained in the same manner as the BLItz® system, and the results are shown in Figs. 1b and 1c.

[0449]

[0450] 2-2. Fluorescence-activated cell sorting (FACS) analysis

[0451] To determine whether the peptide construct specifically binds to target cells expressing CAIX, in vitro cell binding properties were analyzed. The human renal cell carcinoma cell line SK-RC-52, which expresses CAIX, was purchased from Memorial Sloan Kettering Cancer Center (MSK, USA).

[0452] The peptide structure with fluorescent dye AF488 and Cy5 attached was analyzed using NanoPhotometer for accurate quantification. ® The absorbance was measured using (N60, IMPLEN) and the concentration was calculated according to Lambert-Beer's law. The freeze-dried peptide structure was dissolved in dimethyl sulfoxide (DMSO) to make a millimolar (mM) solution, and then measured using a NanoPhotometer. ® The absorbance of 1.5 μL of 100-fold diluted in PBS was measured at 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 absorption coefficient of Cy5 fluorescent dye is 271,000 L·mol -1 cm -1 am. The quantified peptide constructs were 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).

[0453] 2-3 days before FACS analysis, the SK-RC-52 cell line (T75 flask; 1.0-1.5 X 10 6 Dog, T175 flask; 5.0 ~ 8.0 X 10 6 (dog) were seeded in T75 or T175 flasks. On the day of the experiment, the culture medium was removed from the flask where the SK-RC-52 cell line was cultured at 80% confluency and washed twice with 10 mL of PBS buffer. 1 mL of Trypsin-EDTA was treated for 5 minutes in an incubator (37°C, 5% CO2, humidified conditions, Galaxy 170 S), and then 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 spun at 1000 rpm for 3 minutes using a VARISPIN 15R centrifuge. The supernatant of the centrifuged cell mixture was removed, and fresh culture medium was added to obtain 5 X 10 per 200 μL. 5After diluting to the cell number of dogs, 200 μL was dispensed into a 96-well round plate (Cat# 34096, SPL LIFE SCIENCE,). The cells were spun at 1000 RPM for 3 minutes using a VARISPIN 15R centrifuge, the supernatant was removed from each well, and 200 μL of the previously prepared 5 or 10 nM peptide construct solution was added and incubated in an incubator. After 1 hour, the cells were spun 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, mixed with the cells, and transferred to a 5 mL round-bottom test tube with a cell strainer (Cat# 352235, CORNING). 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).

[0454] The results of FACS analysis using the SK-RC-52 cell line are shown in Figures 2a to 2d. From the results of the FACS analysis, it was confirmed that the peptide construct of the present invention has high affinity for target cells expressing CAIX.

[0455]

[0456] 2-3. Biodistribution analysis using experimental animal models

[0457] 2 X 10 6-week-old female BALB / c nude mice (OrientBio or HanaBio, Korea) were used. 6 or 5 X 10 6SK-RC-52 cells were injected subcutaneously into the upper right forelimb of nude mice. Tumor size was measured 2-3 times a week starting 5 days after subcutaneous injection. Tumor size was measured using a caliper [volume = (width)] 2 It was measured as [X (length) / 2].

[0458] After xenografting with the SK-RC-52 cell line, the tumor size was 90 to 120 mm. 3 When the level was reached, 200 μL (1% DMSO in PBS) of 10 μM peptide constructs were injected into the tail vein, and ex vivo images were measured 24 hours later (Figs. 3a to 3c). The number of mice injected with each peptide construct was two, and the peptide constructs injected into more than two mice are shown in Table 1.

[0459]

[0460] Peptide number 33353637404243 Number of mice used 6343644

[0461]

[0462] For repeated experiments, seven peptide constructs (31, 33, 39, 40, 43, 45, 46) that showed high tumor signals in the results of Figures 3a to 3c were injected into three mice each, and 24-hour ex vivo images were taken (Figure 3d), and three peptide constructs (33, 39, 40) were injected into four mice each, and 72-hour ex vivo images were taken (Figure 3e).

[0463] Six peptide structures (55, 56, 57, 58, 59, and 60) were injected into two mice per type via tail vein at 5 μM and 200 μL (1% DMSO in PBS), and ex vivo images were taken 72 hours later to confirm the biodistribution (Fig. 3f). Among the six structures, 58 and 60, which had high fluorescence intensities, were injected into three mice each via tail vein at 10 μM and 200 μL (1% DMSO in PBS), and ex vivo images were taken on the fifth day to confirm the biodistribution (Fig. 3g).

[0464] Four peptide structures (61, 62, 63, 64) were injected into each group of five mice at 5 μM, 200 μL (5% DMSO in PBS) via tail vein injection, and the biodistribution was confirmed by measuring whole-body fluorescence images after 24 hours and ex vivo images after sacrificing the mice (Figs. 3h, 3i).

[0465] Fourteen peptide structures (65, 75, 80, 83, 84, 85, 87, 89, 90, 91, 92, 97, 98, 99) that had high fluorescence intensity in the tumor and low fluorescence intensity in the kidney were tail vein injected into 5 mice in each group at 5 μM, 200 μL (5% DMSO in PBS), and after 24 or 72 hours, whole-body fluorescence images were taken and ex vivo images were measured after sacrificing the mice to confirm the biodistribution (Figs. 3j to 3m).

[0466] To confirm the biodistribution, mice were sacrificed by injecting carbon dioxide, and then laparotomy was performed to remove tumors and organs (spleen, lung, heart, liver, kidney, stomach, pancreas, small intestine, and large intestine). Ex vivo images were taken using an in vivo imaging device, AMI HTX (Spectral Instruments Imaging), and the obtained fluorescence intensities were expressed as a bar graph including the deviation of each individual. The results were expressed as the area (0.08 cm) corresponding to the smallest tumor size. 2 ) based on the area with the highest fluorescence intensity in tumors, kidneys, liver, spleen, lungs, and heart (0.08 cm). 2 ) was applied. This work was performed using Aura imaging software (Spectral imaging, USA), and the unit of fluorescence intensity was photons / s / cm. 2 / sr. We confirmed that several substances that specifically bind to CAIX-overexpressing tumors compared to other organs persisted in the tumors even after 72 hours.

[0467]

[0468] 2-4. In vivo SPECT / CT imaging experiment of peptide structures labeled with isotope (177Lu)

[0469] To confirm the imaging diagnostic and therapeutic functionality of peptide structures 67, 68, 69, 70, 103, 104, and 105, each peptide structure was prepared by labeling with isotopes as follows. Each peptide structure was dissolved in DMSO to prepare a 1 mM stock solution, and 30 mCi isotopes ( diluted in 0.04 M hydrochloric acid solution) were prepared. 177LuCl3, ITM Medical Isotopes GmbH) was completely evaporated through a nitrogen purge. 200 μL of 0.2 M sodium acetate (pH 5.5) buffer solution was added to dissolve the isotope, and 7 mCi (approximately 50 μL) of the isotope was added and mixed with 7 μL of the peptide stock solution, and isotopic labeling was performed at 90°C for 30 min. After the labeling of each substance was completed, it was 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 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.

[0470] Obtained by refining 177 Lu-labeled peptide structures were purified using a Sep-Pak C18 column (Cat# 186005125, Waters) for organic solvent removal. The purified peptide structures were diluted 5-fold with distilled water, loaded onto an activated Sep-Pak column (5 mL of ethanol, 5 mL of distilled water), washed with 5 mL of distilled water to remove the organic solvent, eluted with 300 μL of 60% ethanol solution, and diluted with saline before use.

[0471] The average size of tumors generated by transplanting SK-RC-52, a human renal cancer cell line, was 120 mm. 3 200 μL of isotope equivalent to 500 μCi of radiation dose when reached inside and outside 177Lu-labeled peptide constructs were administered once via tail vein injection into mice, and SPECT / CT (Simens Inveon, software: Inveon Acqusition Workplace) was measured at 1.5 hours, 1 day, 2 days, 3 days, 7 days or 8 days, 10 days for constructs 67, 68, 69, and 70, and at 1.5 hours, 1 day, 2 days, and 3 days for constructs 103, 104, and 105, respectively. For imaging, mice were anesthetized with 0.2% isoflurane in oxygen for 3 to 5 minutes, and then the anesthetized mice were placed in the equipment and the anesthetic was continuously infused at 1.5 L / min. SPECT was measured for 40 minutes and CT for 5 minutes. Images were acquired using Inveon Research Workplace 4.2.

[0472] The SPECT / CT images obtained above are shown in Fig. 4a and Fig. 5a. Isotope 177 The Lu-labeled peptide construct was confirmed to bind to cancer cells SK-RC-52.

[0473]

[0474] 2-5. Analysis of the anticancer effect of CAIX target peptide labeled with isotope (177Lu)

[0475] Isotopes( 177 To confirm the anticancer effect of peptide structures 67, 68, 69, 70, 103, 104, and 105 labeled with Lu, 6-week-old female BALB / c nude mice (Orient Bio or Hana Bio, Korea) were used, and 5 X 10 6 SK-RC-52 cells were injected subcutaneously into the upper right forelimb of nude mice. After cell injection, the tumor size was 120 mm on average. 3 Grouping was performed in groups of 4 or more to ensure proper placement. Tumor size was measured using a caliper [volume = (width)]2 It was measured as [X (length) / 2].

[0476] After xenografting the SK-RC-52 cell line and grouping, the control group was injected with saline solution. 177 Approximately 500 μCi of each Lu-labeled peptide construct (67, 68, 69, 70, 103, 104, 105) was injected via tail vein, and body weight and tumor size were measured 2 to 3 times a week.

[0477] The results are shown in Figures 4b, 4c, 5b, and 5c, respectively. As seen in these figures, 177 Lu-labeled peptide constructs (67, 68, 69, 70, 103, 104, 105) exhibited anticancer effects that significantly reduced the size of CAIX-overexpressing tumors without causing significant changes in mouse body weight.

[0478] The description of the present invention as described above is for illustrative purposes, and a person having ordinary skill in the art to which the present invention pertains will understand that the scope of the present invention is not limited by the disclosed embodiments and the attached drawings, and that various modifications can be made in other specific forms within a scope that does not depart from the technical spirit of the present invention.

[0479]

[0480] Sequence list

[0481] Seq ID No. Amino Acid Sequences 1 YRFRR 2 RFRR 3 RARR 4 YRARR 5 ARFRR 6 ARARR 7 RXRR (wherein, X=DAP(Gal)) 8 RXRR (wherein, X=Uk(IB)) 9 RNRR 10 AAAA 11 RERR 12 YRXRR (wherein, X=DAP(Gal)) 13 RX1 RX2 (wherein, X1=DAP(Gal), X2=r(dimet)) 14 RX1 X2R (wherein, X1=DAP(Gal), X2=r(dimet)) 15 YSXRR (wherein, X=DAP(Gal)) 16 YRXSR (wherein, X=DAP(Gal)) 17 YRXRS (wherein, X=DAP(Gal)) 18 SRXRR (wherein, X=DAP(Gal)) 19 XRXRR (wherein, X=DAP(Gal))20XSXRR (wherein, X=DAP(Gal))21RXRS (wherein, X=DAP(Gal))22RXSR (wherein, X=DAP(Gal))23SXRR (wherein, X=DAP(Gal))24X1X2RR (wherein, X1=R(dimet), X2=DAP(Gal))25X1RX2RR (wherein, X1=DAP(Gal), X2=Uk(sIB))26RXRR (wherein, X=Uk(sIB))27ERXRR (wherein, X=DAP(Gal))28SSSS29SXRS (wherein, X=DAP(Gal))30SEX1EEX2 (wherein, X1=Uk(DOTA), X2=K(Biotin))31SEX1EEX2 (wherein, X1=Uk(DOTA), X2=Pra(AF488))32SX1X2EEX3 (where, X1=Gla, X2=DAP(DOTA), X3=Pra(AF488))33SEX1EEX2 (where, X1=DAP(DOTA), X2=Pra(AF488))34SX1X2EX3X4 (where, X1=Gla, X2=DAP(DOTA), X3=DAP(SZW), X4=Pra(AF488))35SEX1EEX2X3 (where, X1=DAP(DOTA), X2=DAP(SZW), X3=Pra(AF488))36EX1X2GVPTLQ (where, X1=Gla, X2=Pra(AF488))37EX1X2EGRGDK (where, X1=Gla,X2=Pra(AF488))38EX1X2KLPVM (wherein, X1=Gla, X2=Pra(AF488))39EX1X2EGKLPVM (wherein, X1=Gla, X2=Pra(AF488))40SX1X2EX3X4 (wherein, X1=Gla, X2=DAP(DOTA), X3=DAP(SZW), X4=Pra(Cy5))41SX1X2EX3 (wherein, X1=Gla, X2=DAP(DOTA), X3=Pra(Cy5))42SX1X2EEX3 (wherein, X1=Gla, X2=DAP(DOTA), X3=Pra(Cy5))43EX1X2EEX3 (wherein, X1=Gla, X2=DAP(DOTA), X3=Pra(Cy5))44SX1X2EX3X4 (wherein, X1=Gla, X2=DAP(DOTA), X3=Uk(IB), X4=Pra(Cy5))45EX1X2EX3X4 (wherein, X1=Gla, X2=DAP(DOTA), X3=Uk(IB), X4=Pra(Cy5))46SX1X2X1X3X4 (wherein, X1=Gla, X2=DAP(DOTA), X3=Uk(IB), X4=Pra(Cy5))47EX1X2EX3X4 (wherein, X1=Gla, X2=DAP(DOTA), X3=DAP(6OH), X4=Pra(Cy5))48SX1X2EVPTLQ (wherein, X1=Gla, X2=Pra(Cy5))49SX1X2EERGDK (wherein, X1=Gla, X2=Pra(Cy5))50EX1X2GVPTLQ (wherein, X1=Gla, X2=Pra(Cy5))51EX1X2EGRGDK (wherein, X1=Gla, X2=Pra(Cy5))52EX1X2KLPVM (wherein, X1=Gla, X2=Pra(Cy5))53EX1X2EGKLPVM (wherein, X1=Gla, X2=Pra(Cy5))54SEEX1EX2 (wherein, X1=Pra(Cy5), X2=DAP(SZW))55SEEX1EX2X3 (wherein, X1=Pra(Cy5), X2=DAP(SZW), X3=Uk(IB))56SEEXE (where, X=Pra(Cy5))57SEEX1EX2 (where, X1=DAP(DOTA),X2=DAP(SZW))58SEEX1EX2X3 (wherein, X1=DAP(DOTA), X2=DAP(SZW), X3=Uk(IB))59SEEXE (wherein, X1=DAP(DOTA))60EX1EX2 (wherein, X1=Pra(Cy5), X2=DAP(SZW))61SEEX1EX2 (wherein, X1=Pra(Cy5), X2=Uk(sIB))62X1EEX2 (wherein, X1=Pra(Cy5), X2=Uk(sIB))63X1EEX2 (wherein, X1=Pra(Cy5), X2=DAP(SZW))64X1EEE (wherein, X1=Pra(Cy5))65EEX1EX2* (wherein, X1=Pra(Cy5), X2=Uk(sIB))66EEX1X2* (wherein, X1=Pra(Cy5), X2=Uk(sIB))67SEEX1EX2X3 (wherein, X1=Pra(Cy5), X2=DAP(SZW), X3=Uk(sIB))68EEX1EX2X3 (wherein, X1=Pra(Cy5), X2=DAP(SZW), X3=Uk(sIB))69SEEX1EX2* (wherein, X1=Pra(Cy5), X2=Uk(sIB))70SSEXE (wherein, X=Pra(Cy5))71SEEXE (wherein, X=DAP(PSC))72SEEXE (wherein, X=DAP(TCMC))73X1YRFRRX2EX2 (wherein, X1=Uk(SFA), X2=Aha(SFA))74X1X2RFRRX2E (where, X1=Uk(SFA), X2=Aha(SFA))75X1X2RARRX2E (where, X1=Uk(SFA), X2=Aha(SFA))76XXRARRXE (where, X=Aha(SFA))77XYRFRRXEX (where, X=Aha(SFA))78X1YRARRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA))79X1ARFRRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA))80X1RFRRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA))81X1ARARRX2EX2 (where, X1=Uk(SFA), X2=Aha(SFA))82X1RARRX2EX2 (where, X1=Uk(SFA),X2=Aha(SFA))83X1X2RX3RRX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3=DAP(Gal))84X1X2RX3RRX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3=Uk(IB))85X1X2RNRRX2E (where, X1=Uk(SFA), X2=Aha(SFA))86X1X2AAAAX2E (where, X1=Uk(SFA), X2=Aha(SFA))87X1X2RERRX2E (where, X1=Uk(SFA), X2=Aha(SFA))88X1YRFRRX2E (where, X1=Uk(SFA), X2=Aha(SFA))89XYRFRRXE (where, X=Uk(SFA))90X1YRX2RRX3EX3 (where, X1=Uk(SFA), X2=DAP(Gal), X3=Aha(SFA))91X1X2RX3RX4X2E (where, X1=Uk(SFA), X2=Aha(SFA), X3= DAP(Gal), X4=r(dimet))92X1X2RX3X4RX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3= DAP(Gal), X4=r(dimet))93X1YSX2RRX3EX3 (where, X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA))94X1YRX2SRX3EX3 (where, X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA))95X1YRX2RSX3EX3 (where X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA))96X1SRX2RRX3EX3 (where X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA))97X1X2RX2RRX3EX3 (where, X1=Uk(SFA), X2= DAP(Gal), X3=Aha(SFA))98X1X2SX2RRX3EX3 (where, X1=Uk(SFA), X2=DAP(Gal), X3=Aha(SFA))99X1X2RX3RSX2E (where, X1=Uk(SFA), X2=Aha(SFA), X3= DAP(Gal))100X1X2RX3SRX2E (where, X1=Uk(SFA), X2=Aha(SFA),X3= DAP(Gal))101X1X2SX3RRX2E (wherein, X1=Uk(SFA), X2=Aha(SFA), X3= DAP(Gal))102X1X2X3X4RRX2E (wherein, X1=Uk(SFA), X2=Aha(SFA), X3=R(dimet), X4=DAP(Gal))103X1X2RX3RRX1E (wherein, X1=Uk(SFA), X2= DAP(Gal), X3=Uk(sIB))104X1X2RX3RRX2E (wherein, X1=Uk(SFA), X2=Aha(SFA), X3=Uk(sIB))105X1ERX2RRX3EX3 (wherein, X1=Uk(SFA), X2=DAP(Gal), X3=Aha(SFA))106X1X2SSSSX2E (wherein, X1=Uk(SFA), X2=Aha(SFA))107X1X2SX3RSX2E (wherein, X1=Uk(SFA), X2=Aha(SFA), X3=DAP(Gal))* trityl resin synthetic material having a C-terminal of -COOH. In the above sequence, the amino acid can be a D-amino acid or an L-amino acid. In addition, the chemical structures of the abbreviations used in each sequence are as follows. Abbreviation Chemical Structure DAP(Gal)2-amino-3-((2S,3R,4S,5R,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanoic acidUk(IB)N, 6-(4-(4-iodophenyl)butanoyl)-lysineUk(DOTA)2,2',2''-(10-(2-((5,6-diamino-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acidPra(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 acidDAP(DOTA)2,2',2''-(10-(2-((2-amino-2-carboxyethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acidDAP(SZW)3-((2-((2-amino-2-carboxyethyl)amino)-2-oxoethyl)(metheyliumyl)(methyl)-λ 4 -azaneyl)propane-1-sulfonateGlaγ-carboxyglutamic acidPra(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-sulfonateUk(sIB)N 6-(2-(4-iodophenyl)acetyl)-D-lysineDAP(6OH)(R)-2-amino-3-((2R,3R,4S,5R,6R)-2,3,4,5,6,7-hexahydroxyheptanamido)propanoic acidDAP(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 acidDAP(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 acidr(dimet)(R)-2-amino-5-((amino(dimethyl-l4-azaneylidene)methyl)amino)pentanoic acidR(dimet)(S)-2-amino-5-((amino(dimethyl-l4-azaneylidene)methyl)amino)pentanoic acidPEG12-(2-(2-aminoethoxy)ethoxy)acetic acidPEG(6atom)2-(2-aminoethoxy)acetic acidK(biotin)N6-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)-L-lysineUk(SFA)(R)-2-amino-6-(4-(4-sulfamoylphenyl)-1H-1,2,3-triazol-1-yl)hexanoic acidAha(SFA)(R)-2-amino-4-(4-(4-sulfamoylphenyl)-1H-1,2,3-triazol-1-yl)butanoic acid

[0482]

[0483] [서열번호 7의 구조]

[0484]

[0485] [Structure of sequence number 8]

[0486]

[0487] [Structure of sequence number 12]

[0488]

[0489] [Structure of sequence number 13]

[0490]

[0491] [Structure of sequence number 14]

[0492]

[0493] [Structure of sequence number 15]

[0494]

[0495] [Structure of sequence number 16]

[0496]

[0497] [Structure of sequence number 17]

[0498]

[0499] [Structure of sequence number 18]

[0500]

[0501] [Structure of sequence number 19]

[0502]

[0503] [Structure of sequence number 20]

[0504]

[0505] [Structure of sequence number 21]

[0506]

[0507] [Structure of sequence number 22]

[0508]

[0509] [Structure of sequence number 23]

[0510]

[0511] [Structure of sequence number 24]

[0512]

[0513] [Structure of sequence number 25]

[0514]

[0515] [Structure of sequence number 26]

[0516]

[0517] [Structure of sequence number 27]

[0518]

[0519] [Structure of sequence number 29]

[0520]

[0521] [Structure of sequence number 30]

[0522]

[0523] [Structure of sequence number 31]

[0524]

[0525] [Structure of sequence number 32]

[0526]

[0527] [Structure of sequence number 33]

[0528]

[0529] [Structure of sequence number 34]

[0530]

[0531] [Structure of sequence number 35]

[0532]

[0533] [Structure of sequence number 36]

[0534]

[0535] [Structure of sequence number 37]

[0536]

[0537] [Structure of sequence number 38]

[0538]

[0539] [Structure of sequence number 39]

[0540]

[0541] [Structure of sequence number 40]

[0542]

[0543] [Structure of sequence number 41]

[0544]

[0545] [Structure of sequence number 42]

[0546]

[0547] [Structure of sequence number 43]

[0548]

[0549] [Structure of sequence number 44]

[0550]

[0551] [Structure of sequence number 45]

[0552]

[0553] [Structure of sequence number 46]

[0554]

[0555] [Structure of sequence number 47]

[0556]

[0557]

[0558] [Structure of sequence number 48]

[0559]

[0560] [Structure of sequence number 49]

[0561]

[0562] [Structure of sequence number 50]

[0563]

[0564] [Structure of sequence number 51]

[0565]

[0566] [Structure of sequence number 52]

[0567]

[0568] [Structure of sequence number 53]

[0569]

[0570] [Structure of sequence number 54]

[0571]

[0572] [Structure of sequence number 55]

[0573]

[0574] [Structure of sequence number 56]

[0575]

[0576] [Structure of sequence number 57]

[0577]

[0578] [Structure of sequence number 58]

[0579]

[0580] [Structure of sequence number 59]

[0581]

[0582] [Structure of sequence number 60]

[0583]

[0584] [Structure of sequence number 61]

[0585]

[0586] [Structure of sequence number 62]

[0587]

[0588] [Structure of sequence number 63]

[0589]

[0590] [Structure of sequence number 64]

[0591]

[0592] [Structure of sequence number 65]

[0593]

[0594] [Structure of sequence number 66]

[0595]

[0596] [Structure of sequence number 67]

[0597]

[0598] [Structure of sequence number 68]

[0599]

[0600] [Structure of sequence number 69]

[0601]

[0602] [Structure of sequence number 70]

[0603]

[0604] [Structure of sequence number 71]

[0605]

[0606] [Structure of sequence number 72]

[0607]

[0608] [Structure of sequence number 73]

[0609]

[0610] [Structure of sequence number 74]

[0611]

[0612] [Structure of sequence number 75]

[0613]

[0614] [Structure of sequence number 76]

[0615]

[0616] [Structure of sequence number 77]

[0617]

[0618] [Structure of sequence number 78]

[0619]

[0620] [Structure of sequence number 79]

[0621]

[0622] [Structure of sequence number 80]

[0623]

[0624] [Structure of sequence number 81]

[0625]

[0626] [Structure of sequence number 82]

[0627]

[0628] [Structure of sequence number 83]

[0629]

[0630] [Structure of sequence number 84]

[0631]

[0632] [Structure of sequence number 85]

[0633]

[0634] [Structure of sequence number 86]

[0635]

[0636] [Structure of sequence number 87]

[0637]

[0638] [Structure of sequence number 88]

[0639]

[0640] [Structure of sequence number 89]

[0641]

[0642] [Structure of sequence number 90]

[0643]

[0644] [Structure of sequence number 91]

[0645]

[0646] [Structure of sequence number 92]

[0647]

[0648] [Structure of sequence number 93]

[0649]

[0650] [Structure of sequence number 94]

[0651]

[0652] [Structure of sequence number 95]

[0653]

[0654] [Structure of sequence number 96]

[0655]

[0656] [Structure of sequence number 97]

[0657]

[0658] [Structure of sequence number 98]

[0659]

[0660] [Structure of sequence number 99]

[0661]

[0662] [Structure of sequence number 100]

[0663]

[0664] [Structure of sequence number 101]

[0665]

[0666] [Structure of sequence number 102]

[0667]

[0668] [Structure of sequence number 103]

[0669]

[0670] [Structure of sequence number 104]

[0671]

[0672] [Structure of sequence number 105]

[0673]

[0674] [Structure of sequence number 106]

[0675]

[0676] [Structure of sequence number 107]

[0677]

Claims

1. Containing any one of the amino acid sequences of sequence numbers 1 to 29, At least one of the constituent amino acids is a D-amino acid, Among the constituent amino acids, the lysine (Lys) residue can be substituted with a chemical functional group in the side chain ε-amino group, or A peptide ligand wherein the 2,3-diaminopropionic acid (DAP) residue among the constituent amino acids can 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 linked directly or through a spacer to the peptide ligand, and characterized by having a structure represented by the following chemical formula 1. [Chemical Formula 1] In the above formula, P is the above peptide ligand, F1, F2 and F3 are each independently a sulfonamide functional group-containing amino acid residue, m is 0 or 1, F4 is a general formula -(F5) o -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is the base, where F5 is a sulfonamide functional group-containing amino acid residue, S1 and S2 are each independently spacers, F6 and F7 are each independently an amino acid residue containing a functional group other than sulfonamide, S3 is -NH2 or -OH, o is an integer of 0 or 1, p, q, r and s each independently represent an integer from 0 to 6.

2. A peptide structure characterized in that, in the first paragraph, the chemical functional group that can be substituted in 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).

3. A peptide structure according to claim 1 or 2, characterized in that the chemical functional group that can be substituted in 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).

4. A peptide structure according to any one of claims 1 to 3, wherein the sulfonamide functional group-containing amino acid residue has the following structure. or 5. A peptide structure according to any one of claims 1 to 4, characterized in that the peptide structure has a structure represented by the following chemical formula 2. [Chemical Formula 2] In the above formula, R5 is a general formula -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is the flag, Here, 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, respectively. Other substituents abbreviated as R1 to R4 and AA1 to AA5 are defined as follows.

6. A peptide structure according to any one of claims 1 to 5, characterized in that the functional group other than the sulfonamide is introduced through the side chain ε-amino group of a lysine residue or the side chain β-amino group of a 2,3-diaminopropionic acid (DAP) residue.

7. A peptide structure according to any one of claims 1 to 6, 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), a zwitteric ion (SZW), α-D-galacturonic acid (Gal), a cycloalkane having 5 to 15 carbon atoms, a fluorescent dye, or a cytotoxic agent.

8. In the 7th paragraph, the chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid-2,2',2'',2'''-(ethane-1,2-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-pentaazacyclopentadecane-N,N',N'',N''',N'''-pentaacetic acid (PEPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), (1R, 4R, 7R, 10R)-α, α', α'', α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)tetrasodium salt (DOTMA), 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA), triethylenetetramine (TETA), A peptide structure characterized by at least one selected from 1,4,7,10-tetraazacyclododecan-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).

9. A peptide structure according to any one of claims 1 to 8, characterized in that the spacer is at least one 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.

10. A peptide structure according to any one of claims 1 to 9, characterized in that the peptide structure has a structure represented by 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. Other substituents abbreviated as R1 to R5 and AA1 to AA5 are defined as follows.

11. A conjugate comprising the peptide structure of any one of claims 1 to 10, directly or via a linker, linked to a fluorescent dye, cytotoxic agent or radioisotope.

12. A conjugate according to claim 11, characterized in that the linker is at least one 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).

13. A conjugate according to claim 11 or 12, characterized in that the fluorescent dye is at least one selected from a near-infrared fluorescent dye, a fluorescein type, a rhodamine type, Alexa Fluor, 4,4-difluoro-4-boro-3a, 4a-diaza-s-indacene (BODIPY), Texas Red, dansyl, Lissamine, cyanine (Cy), and phycoerythrin.

14. In any one of paragraphs 11 to 13, the radioactive isotope is 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), A conjugate characterized by at least one selected from chromium-51 (Cr-51), krypton-81 (Kr-81), potassium-42 (K-42), holmium-166 (Ho-166), gallium-67 (Ga-67), gallium-68 (Ga-68), actinium-225 (Ac-225), zirconium-89 (Zr-89), 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).

15. A conjugate comprising a peptide structure selected from the group consisting of peptide structures 67 to 70 and 103 to 105, linked directly or via a linker to a radioisotope. [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] 16. A conjugate characterized in that in paragraph 15, the radioactive isotope is at least one 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).

17. A pharmaceutical composition for diagnosing, preventing or treating cancer, comprising the peptide structure of any one of claims 1 to 10.

18. A pharmaceutical composition for diagnosing, preventing or treating cancer, comprising a conjugate of any one of claims 11 to 16.

19. A pharmaceutical composition for diagnosing, preventing or treating cancer, wherein the cancer expresses carbonic anhydrase IX in the 17th paragraph.

20. A pharmaceutical composition for diagnosis, prevention or treatment of cancer, wherein the cancer in paragraph 17 is liver cancer, lung cancer, colon 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, anal 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.

21. A pharmaceutical composition for diagnosing, preventing or treating cancer, wherein the cancer expresses carbonic anhydrous enzyme IX in claim 18.

22. A pharmaceutical composition for diagnosing, preventing or treating cancer, wherein the cancer in paragraph 18 is liver cancer, lung cancer, colon 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, anal 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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