Peptide constructs targeting carbonic anhydrase ix and use thereof

Abstract

The present invention relates to a peptide ligand specifically binding to carbonic anhydrase IX (CAIX), to peptide constructs comprising same, and to a use thereof. The CAIX-binding peptide ligand of the present invention comprises a D-amino acid, and thus is stable in vivo and has high binding specificity to CAIX, and the cyclic CAIX-binding peptide constructs comprising same are capable of binding to CAIX in vivo with high affinity while minimizing side effects on the stomach, thereby being useful for diagnosis, prevention, inhibition or treatment of CAIX-mediated diseases.

Description

Peptide structures targeting carbonic anhydrous enzyme IX and their uses

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

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

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

[0004] Carbonic anhydrase (CA) is a compound found in zinc (Zn), which is commonly present in higher vertebrates, including humans. 2+ It is a metalloenzyme that catalyzes the reversible hydration reaction converting carbon dioxide into hydrogen ions and bicarbonate ions (CO2 + H2O ↔ HCO3 + H +Sixteen isozyme forms of these CAs have been identified, and in humans, they exist in various tissues including the gastrointestinal tract, reproductive tract, nervous system, kidneys, lungs, skin, and eyes. Most CA isozymes are known to be involved in important physiological processes such as respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid (Thiry et al., TRENDSin Pharmacological Sciences, 27(11): 566-573, 2006).

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

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

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

[0008] Meanwhile, CAIX is expressed in normal cells, such as the gastric mucosa and small intestine mucosa, rather than in tumors, and drug compounds labeled with radioisotopes that have a high binding affinity to CAIX protein can bind to normal cells of the stomach or small intestine and cause side effects.

[0009] Therefore, there is a need to develop novel, highly affinity, stable, and stable CAIX-specific binders and inhibitors that are suitable for the pharmaceutical use of cancer treatment, including treatment, prevention, diagnosis, prognosis prediction, and imaging of cancer, while minimizing side effects of radiation toxicity to the stomach.

[0010] Prior art literature

[0011] (Non-patent literature 1) Thiry et al., TRENDS in Pharmacological Sciences, 27(11): 566-573, 2006

[0012] (Non-patent Document 2) De Simone et al., Biochimica et Biophysica Acta, 1804:404-409, 2010

[0013] One objective of the present invention is to provide stable CAIX-specific binders and inhibitors suitable for use in the treatment, prevention, diagnosis, or prognosis prediction of cancer diseases.

[0014] One object of the present invention is to provide a high-affinity CAIX-specific peptide structure comprising one or more effectors or functional groups, including a sulfonamide functional group, together with a CAIX-specific binding peptide ligand.

[0015] One objective of the present invention is to provide a CAIX-specific peptide structure suitable for pharmaceutical use in cancer treatment, while minimizing side effects of radiation toxicity to the stomach.

[0016] An additional objective of the present invention is to provide a conjugate comprising the peptide structure.

[0017] An additional objective of the present invention is to provide a composition for the diagnosis, prevention, or treatment of cancer comprising the peptide structure or conjugate.

[0018] An additional objective of the present invention is to provide a method for diagnosing cancer using the peptide structure or conjugate.

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

[0020] An additional objective of the present invention is to provide a method for predicting the prognosis after cancer treatment using the peptide structure or conjugate.

[0021] As a result of diligent research to achieve the above objective, the inventors of the present application have developed novel CAIX-specific binders and inhibitors containing D-amino acids that are stable, highly affinity for CAIX, and capable of specifically binding to CAIX.

[0022] In one aspect of the present invention, the present invention

[0023] Includes the amino acid sequence of SEQ ID NO. 1 or 2, but,

[0024] At least one of the constituent amino acids is composed of a D-amino acid, and

[0025] A peptide ligand in which a 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

[0026] The present invention provides a peptide structure characterized by comprising a sulfonamide functional group-containing amino acid residue connected to the above-mentioned peptide ligand directly or through a spacer, and having the structure of the following chemical formula 1.

[0027] [Chemical Formula 1]

[0028]

[0029] In the above chemical formula,

[0030] P is the above-mentioned peptide ligand, and

[0031] F1, F2, and F3 are each independently sulfonamide functional group-containing amino acid residues, and

[0032] m is 0 or 1, and

[0033] F4 is the general formula -(F5) o -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is the basis, and here,

[0034] F5 is a sulfonamide functional group-containing amino acid residue, and

[0035] S1 is a spacer, and

[0036] S2 is a spacer or a residue having the amino acid sequence of SEQ ID NO. 3, and

[0037] F6 and F7 are each independently amino acid residues containing functional groups other than sulfonamide, and

[0038] S3 is -NH2, and

[0039] o is an integer of 0 or 1, and

[0040] p, q, r, and s each independently represent integers from 0 to 6, but,

[0041] When the above P is a peptide ligand containing the amino acid sequence of SEQ ID NO. 1, r is an integer from 1 to 6, and one or more of S2 are residues having the amino acid sequence of SEQ ID NO. 3.

[0042] Non-limiting examples of chemical functional groups that can be substituted at the side chain β-amino group of the 2,3-diaminopropionic acid (DAP) residue among the above constituent amino acids include α-D-galacturonic acid (α-D-galacturonic acid, Gal).

[0043] In the above CAIX-specific peptide structure, the sulfonamide functional group-containing amino acid residue may have the following structures, but is not limited to them.

[0044] or

[0045] In the above CAIX-specific peptide structure, functional groups other than sulfonamide include, for example, chelators, biotin, polyethylene glycol 12-biotin (PEG12-biotin), glucoheptonic acid, 4-(p-iodophenyl)butyric acid (IB), 2-(4-iodophenyl)acetic acid (sIB), zwitteric ion (SZW), α-D-galacturonic acid (Gal), cycloalkane having 5 to 15 carbon atoms, fluorescent dyes, or cytotoxic agents, and may be introduced through the side chain β-amino group of a 2,3-diaminopropionic acid (DAP) residue.

[0046] In one aspect, the present invention provides a conjugate comprising a peptide structure that is directly or through a linker coupled to a fluorescent dye, a cytotoxic agent, or a radioisotope to the CAIX-specific peptide structure.

[0047] In one aspect, the present invention provides a pharmaceutical composition for the diagnosis, prevention, or treatment of cancer comprising the peptide structure or conjugate. The cancer may be a cancer expressing carbonic anhydrous enzyme IX. The cancer may be selected from the group consisting of liver cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, prostatic cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, melanoma, thyroid cancer, astrocytoma, or glioblastoma, but is not limited thereto.

[0048] In one aspect, the present invention provides a method for diagnosing cancer comprising administering the CAIX-specific peptide structure or the conjugate to an individual.

[0049] In one aspect, the present invention provides a method for treating cancer comprising administering the CAIX-specific peptide structure or the conjugate to an individual.

[0050] In one aspect, the present invention provides a method for predicting the prognosis after cancer treatment, comprising administering the CAIX-specific peptide structure or the conjugate to an individual.

[0051] The present invention provides a stable, novel, high-affinity CAIX binder and inhibitor that specifically bind to CAIX while minimizing the side effects of radiation toxicity to the stomach. The CAIX binder of the present invention exhibits excellent stability in vivo and is useful for CAIX targeting by including a CAIX-specific binding peptide ligand comprising one or more D-amino acids. Furthermore, the present invention provides a peptide structure in which one or more effectors or functional groups, including a sulfonamide functional group, are introduced through the side chain of an amino acid residue to the CAIX-specific peptide ligand, thereby providing a CAIX binder that is particularly useful for cancer imaging or diagnosis due to its high binding affinity for CAIX. Additionally, the CAIX binder of the present invention can minimize the side effects of radiation toxicity to the stomach by introducing a linker containing an amino acid sequence that can be degraded by pepsin, a proteolytic enzyme that is specifically active only in the stomach. The CAIX-specific peptide structure of the present invention can also be used as an effective drug for the diagnosis, prevention, or treatment of cancer by conjugating it with fluorescent dyes, cytotoxic agents, or radioisotopes.

[0052] Figure 1a is a graph of the stability test results and HPLC results for human serum and plasma of peptide structure 1 according to one embodiment of the present invention.

[0053] FIG. 1b is a graph of the stability test results and HPLC results for human serum and plasma of peptide structure 2 according to one embodiment of the present invention.

[0054] FIG. 1c is a graph of the stability test results and HPLC results for human serum and plasma of peptide structure 7 according to one embodiment of the present invention.

[0055] FIG. 2 shows the binding affinity and binding kinetics characteristics of the hCAIX extracellular domain (ECD) of peptide structures 8 and 10 according to one embodiment of the present invention and the human carbonic anhydrous enzyme XII (hCAXII) ECD, BLItz ® This is a graph showing the results of the system analysis.

[0056] Figure 3a is a conceptual diagram showing the structure and molecular weight of peptides according to the cleavage site after reacting lutetium-175 (Lu-175) labeled peptide structures 1, 2, and 3 with human pepsin protease.

[0057] Figure 3b shows the HPLC and MS results for 0, 5, and 9 hours after reacting lutetium-175 (Lu-175) labeled peptide construct 1 with human pepsin protease.

[0058] Figure 3c shows the HPLC and MS results for 0, 5, and 9 hours after reacting lutetium-175 (Lu-175) labeled peptide construct 2 with human pepsin protease.

[0059] Figure 3d shows the HPLC and MS results for 0, 5, and 9 hours after reacting lutetium-175 (Lu-175) labeled peptide construct 3 with human pepsin protease.

[0060] Figure 4 is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using SK-RC-52 cell lines of peptide structures 4, 5, 6, and 9 according to one embodiment of the present invention.

[0061] Figure 5a shows the results of measuring mouse SPECT / CT images at 1 hour, 4 hours, 8 hours, 1 day, and 2 days in mice injected with lutetium-177 (Lu-177)-labeled peptide construct 1. Arrows indicate the location of the tumor.

[0062] Figure 5b shows the results of measuring mouse SPECT / CT images at 1.5 hours, 1 day, 2 days, 3 days, and 8 days in mice injected with lutetium-177 (Lu-177)-labeled peptide construct 7. Arrows indicate the location of the tumor.

[0063] Figure 6 is a graph showing the changes in tumor size (anticancer effect) and body weight measured in mice injected with lutetium-177 (Lu-177) labeled peptide structures 1 and 7, respectively.

[0064] Figure 7a shows the solvent conditions of the HPLC instrument used for the qualitative analysis of the synthesized material.

[0065] Figure 7b shows the HPLC chromatograms of synthesized peptide structures 1 and 2 and the molecular weights observed using electrospray ionization mass spectrometry (MS).

[0066] Figure 7c shows the HPLC chromatograms of synthesized peptide structures 3 and 4 and the molecular weights observed using electrospray ionization mass spectrometry (MS).

[0067] Figure 7d shows the HPLC chromatograms of synthesized peptide structures 5 and 6 and the molecular weights observed using electrospray ionization mass spectrometry (MS).

[0068] Figure 7e shows the HPLC chromatograms of synthesized peptide structures 7 and 8 and the molecular weights observed using electrospray ionization mass spectrometry (MS).

[0069] Figure 7f shows the HPLC chromatograms of synthesized peptide structures 9 and 10 and the molecular weights observed using electrospray ionization mass spectrometry (MS).

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Any method or material similar or equivalent to those described herein may be used in the practice or testing of the present invention.

[0071] The present invention allows for various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0072]

[0073] CAIX-specific peptide structure of the present invention

[0074] The present invention provides a peptide structure comprising a sulfonamide functional group-containing amino acid residue connected to a CAIX-specific peptide ligand directly or through a spacer.

[0075] The above CAIX-specific peptide ligand may include the amino acid sequence of SEQ ID NO. 1 or 2. The above peptide ligand may include D-amino acids or consist only of D-amino acids. Additionally, among the constituent amino acid residues of the peptide ligand, the 2,3-diaminopropionic acid (DAP) residue may include, but is not limited to, a chemical functional group, such as α-D-galacturonic acid (Gal), at the side chain β-amino group, and may be substituted with one or more chemical functional groups.

[0076] The above peptide ligand may be prepared such that amino acid residues having a specific sequence are combined with each other to form part of a peptide structure. The above peptide ligand may be prepared by a known peptide synthesis method and is not particularly limited. In one embodiment, the peptide ligand may be prepared by repeating the peptide synthesis process on a solid-phase single bead until a peptide of a desired length and sequence is completed.

[0077] The above CAIX-specific peptide ligand may also include its salt form.

[0078] The introduction of a sulfonamide functional group into the sulfonamide functional group-containing amino acid residue of the above-described peptide structure can be achieved through known synthetic reactions and is not particularly limited. In one embodiment of the present invention, the sulfonamide functional group can be introduced into the peptide structure of the present invention through a click chemistry reaction. Examples of preferred sulfonamide functional group-containing amino acid residues that can be introduced into the peptide structure of the present invention include, but are not limited to, the following structures.

[0079] or

[0080] The peptide structure may additionally include one or more other functional group-containing amino acid residues other than the sulfonamide functional group. The functional group other than the sulfonamide may be introduced through the side chain of the amino acid residue. The amino acid residue into which the functional group is introduced may be 2,3-diaminopropionic acid (DAP).

[0081] In one embodiment, non-limiting examples of functional groups other than sulfonamide that can be introduced into the peptide structure of the present invention may include a chelator, biotin, polyethylene glycol 12-biotin (PEG12-biotin), glucoheptonic acid, 4-(p-iodophenyl)butyric acid (IB), 2-(4-iodophenyl)acetic acid (sIB), zwitteric ion (SZW), α-D-galacturonic acid (Gal), cycloalkane having 5 to 15 carbon atoms, fluorescent dye, or cytotoxic agent.

[0082] The above chelator is, for example, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazcyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid-2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N'''',N''''-hexaacetic acid (HEHA), 2-[4-nitrobenzyl]-1,4,7,10,13-pentazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid (PEPA), 1,4,7,10-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), It may be one or more selected from 1,4,7,10-tetraazacyclododecane-7-acetamide-1,4,10-triacetic acid (PSC) and 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide (DOTAM or TCMC), but is not limited thereto.

[0083] The above cycloalkanes having 5 to 15 carbon atoms may be one or more selected from, for example, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, adamantan, norbonane, isobonane, and tricyclodecane, but are not limited thereto.

[0084] The peptide structure of the present invention may also connect a chemical functional group-containing amino acid residue, such as a sulfonamide functional group, to a CAIX-specific binding peptide ligand through a spacer. The spacer may be, for example, one or more selected from polyethylene glycol (PEG) linkers, glycine, sarcosine, γ-carboxyglutamic acid, and peptide linkers composed of 1 to 8 D-amino acids or L-amino acids, but is not limited thereto.

[0085] The peptide structure of the present invention has the structure of Chemical Formula 1 below.

[0086] [Chemical Formula 1]

[0087]

[0088] In the above chemical formula,

[0089] P, F1, F2, F3, F4 and m are each as defined above.

[0090] In one embodiment of the present invention, the peptide structure has the structure of the following chemical formula 2.

[0091] [Chemical Formula 2]

[0092]

[0093]

[0094]

[0095] In the above chemical formula,

[0096] R1 to R4 may include sulfonamides. AA1 to AA4 represent amino acid residues containing conventional amino acid residues or functional groups (Gal, IB, sIB, dimethyl), where lowercase letters indicate D-amino acids and uppercase letters indicate L-amino acids. U indicates a modified or unnatural D-amino acid. Substituents indicated by abbreviations in R1 to R4 and AA1 to AA4 are each defined as follows.

[0097]

[0098]

[0099]

[0100]

[0101] Although not bound by any specific theory, it is believed that cyclic structures, such as the cyclic peptide structure of the present invention, have less flexibility compared to linear peptides, resulting in less entropy loss upon target binding, higher binding affinity, and increased binding specificity to the target.

[0102] The peptide structure of the present invention exhibits high selectivity, specifically binding to CAIX but not to other isoenzymes of CAIX (e.g., carbonic anhydrous enzyme XII).

[0103]

[0104] Conjugate

[0105] The CAIX-specific peptide structure of the present invention can form a conjugate by being directly or through a linker bound to a fluorescent dye, a cytotoxic agent, or a radioisotope. The conjugate can target CAIX and label cancers expressing CAIX with a fluorescent dye or a radioisotope, or effectively deliver drugs such as radioisotopes or cytotoxic agents to said cancers, making it useful for the diagnosis, prevention, or treatment of cancer.

[0106] In one embodiment, the linker may be one or more selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 4-(2-pyridyldithio)butyric acid-N-hydroxysuccinimide ester (SPDB), and N-succinimidyl(4-iodo-acetyl)aminobenzoate (SIAB), but is not limited thereto.

[0107] In one embodiment, non-limiting examples of the fluorescent dye may include one or more selected from near-infrared fluorescent dyes, fluorescein type, rhodamine type, Alexa Fluor, 4,4-difluoro-4-boro-3a, 4a-diaza-s-indacen (BODIPY), Texas Red, dansyl, lissamine, cyanine (Cy), and phycoerythrin.

[0108] In one embodiment, the cytotoxic agent may be one or more selected from toxins, chemotherapy agents, drug moiety, antibiotics, and nucleases, but is not limited thereto.

[0109] In one embodiment, the radioisotopes are Fluorine-18 (F-18), Carbon-11 (C-11), Carbon-14 (C-14), Techtenium-99m (Tc-99m), Copper-64 (Cu-64), Copper-67 (Cu-67), Dysprosium-168 (Dy-168), Bismuth-213 (Bi-213), Samarium-153 (Sm-153), Strontium-89 (St-89), Strontium-90 (St-90), Erbium-169 (Er-169), Phosphorus-32 (P-32), Palladium-103 (Pd-103), Rhenium-186 (Re-186), Rhenium-188 (Re-188), Oxygen-15 (O-15), Selenium-75 (Se-75), Sodium-24 (Na-24), Strontium-85 (Sr-85), Lutetium-177 (Lu-177), Yttrium-90 (Y-90), Iodine-123 (I-123), Iodine-125 (I-125), Iodine-131 (I-131), Iridium-192 (Ir-192), Iridium-196 (Ir-196), Ytterbium-166 (Yb-166), Indium-111 (In-111), Xenon-133 (Xe-133), Nitrogen-13 (N-13), Calcium-47 (Ca-47), Cobalt-57 (Co-57), Cobalt-60 (Co-60), Chromium-51 (Cr-51), It may be one or more selected from krypton-81 (Kr-81), potassium-42 (K-42), holmium-166 (Ho-166), gallium-67 (Ga-67), gallium-68 (Ga-68), actinium-225 (Ac-225), zirconium-89 (Zr-89), thorium-227 (Th-227), radium-223 (Ra-223), tin-117m (Sn-117m), lead-103 (Pb-103), terbium-161 (Tb-161), lead-212 (Pb-212), and astatine-211 (At-211).

[0110]

[0111] Therapeutic administration and formulation

[0112] The present invention provides a pharmaceutical composition for the diagnosis, prevention, or treatment of cancer comprising the CAIX-specific peptide structure or conjugate of the present invention. Preferably, the cancer of the present invention is a solid tumor. More preferably, the cancer of the present invention is a cancer expressing CAIX. For example, the cancer of the present invention may be a solid tumor such as liver cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, prostatic cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renopelvic carcinoma, melanoma, thyroid cancer, astrocytoma, or glioblastoma, but is not limited thereto.

[0113] The subject to whom the pharmaceutical composition for the diagnosis, prevention, or treatment of cancer of the present invention is administered may be a mammal that is at risk of developing cancer, has been diagnosed with cancer, or has received cancer treatment. The mammal may be a human or a mammal other than a human.

[0114] The pharmaceutical composition for the diagnosis, prevention, or treatment of cancer according to the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external formulations, suppositories, and sterile injectable solutions according to conventional methods, and may include a suitable carrier, excipient, or diluent that is conventionally used in the manufacture of pharmaceutical compositions for formulation.

[0115] Various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used as the carrier, excipient, or diluent.

[0116] When formulating, it can be manufactured using diluents or excipients such as fillers, weights, binders, wetting agents, disintegrants, and surfactants commonly used in the pharmaceutical industry.

[0117] The preferred dosage of the pharmaceutical composition for the prevention or treatment of cancer according to the present invention may vary depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at a dose of 0.0001 to 2,000 mg / kg per day, preferably 0.001 to 2,000 mg / kg. The administration may be performed once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosage.

[0118] The pharmaceutical composition for the prevention or treatment of cancer according to the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes. The method of administration may be, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater, or intracerebroventricular injection.

[0119] The present invention also provides a method for treating cancer comprising administering the CAIX-specific peptide structure or conjugate of the present invention to an individual requiring treatment for cancer.

[0120] The CAIX-specific peptide structure and conjugate of the present invention may also be used to diagnose cancer by targeting and imaging cancer, or to predict or observe the treatment prognosis of an individual after cancer treatment by administering it to the individual who has received cancer treatment.

[0121] The present invention will be described in more detail below through examples. These examples are provided solely to illustrate the invention more specifically and are not intended to limit the legitimate scope of the invention; it will be obvious to those skilled in the art that various modifications are possible within the scope of the invention.

[0122]

[0123] Examples

[0124]

[0125] Example 1. Synthesis and purification of a peptide structure containing selected peptides

[0126] 1-1. Synthesis of reagents inserted into peptide structures

[0127] Fmoc-DAP(DOTA(Protected))-OH (S1) and Fmoc-DAP(Gal(Protected))-OH (S2) inserted into the peptide structure were synthesized directly and used. First, dissolve unprotected Fmoc-DAP-OH (1 equivalent) in N,N-dimethylformamide (DMF), and add Tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, DOTA-tris(tert-butyl ester), or 1,2:3,4-di-O-isopropylidene-aD-galacturonide (1.5 equivalents). A solution of 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (1.2 equivalents), N,N-Diisopropylethylamine (DIPEA) (3 equivalents), and activated solution dissolved in DMF was mixed and reacted for 2 hours. Subsequently, the progress of the reaction was monitored using LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent), and the solution was purified using a 1260 Infinity II LC system (Agilent) for use. The analytical conditions for each instrument are as follows: LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent): (1) The stationary phase was Agilent Poroshell 120 EC-C18 column (4.6 X 150 mm, 2.7 μm), (2) mobile phase solvent is 0.A mixture of triple-distilled water with 1% trifluoroacetic acid (TFA) and acetonitrile (ACN) was used, (3) the flow rate was 1 mL / min, (4) the detection wavelengths were 214 and 254 nm, and (5) the column temperature was maintained at 40 ℃. 1260 Infinity II LC system (Agilent): (1) the stationary phase was a Kromasil 100-5-C18 column (21.2 X 250 mm, 5 μm), (2) the mobile phase solvent was a mixture of triple-distilled water with 0.1% TFA and ACN, (3) the flow rate was 15 mL / min, and (4) the detection wavelengths were 214 and 254 nm. The synthesis process of S1 and S2 is as follows.

[0128] [Reaction Equation 1]

[0129]

[0130]

[0131] 1-2. Synthesis of Solid-Phase Peptides

[0132] Solid-phase peptide chains were synthesized using an automated synthesizer (Apex 396, AAPPTEC). The solid-phase resin used for the synthesis was Rink Amide-ChemMatrix ®Resin (0.41 mmole / g, Cat# 7-600-1310, Biotage) was used to have a C-terminus of -CONH2. For synthesis using an automated synthesizer (Apex 396, AAPPTEC), the resin swollen in NMP was coupled with Fmoc-amino acid (5 equivalents), TBTU (5 equivalents), and DIPEA (10 equivalents) in NMP solvent for 45 minutes at room temperature. Subsequently, the resin was washed with NMP and DCM, and the Fmoc protecting group was removed by reacting in a piperidine / NMP (v / v = 1:4) mixed solution for 10 minutes at room temperature. After repeating the Fmoc-amino acid synthesis process to achieve coupling, the beads were collected from the synthesizer. In addition, when the substances (S1 and S2) synthesized in Example 1-1 were used for solid-phase peptide synthesis, they were not synthesized by instrument but were directly subjected to manual reactions, and the number of equivalents of the reagents used in the synthesis was reduced to 2 equivalents. Fmoc-E(OAll)-OH (Cat#36613, GL Biochem) was used for the synthesis of cyclic peptides.

[0133]

[0134] 1-3. Synthesis of Structures and Functional Groups of Solid-Phase Peptides

[0135] 1-3-1. Cyclic reaction

[0136] The reaction to remove the allyl protecting group from glutamic acid of solid beads synthesized using an automated synthesizer was carried out. Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4, 1 equivalent, Cat# 216666, Sigma-Aldrich) and PhSiH3 (30 equivalents, Cat# 335150, Sigma-Aldrich) were dissolved in DCM solvent under an argon (Ar) atmosphere, placed in a filter-equipped cylinder containing solid beads, sealed with a lid, and reacted for 1 hour while suspended from a 180° shaker at room temperature. Afterward, the beads were washed 3 times with NMP, and then Pd impurities were removed by washing at least 5 times with an NMP solution containing sodium diethyldithiocarbamate (Cat# D3506, Sigma-Aldrich) dissolved at a concentration of 0.1 M. Afterward, the beads were washed three times with NMP, and 3 mL of piperidine / NMP (v / v = 1:4) solution was added to remove the N-terminal Fmoc protecting group. The beads were then hung on a 180° shaker at room temperature for 5 minutes, after which the solution was emptied and refilled, and the mixture was reacted for 10 minutes. After the reaction was complete, the beads were washed three times each with NMP, DCM, and NMP in that order. The cyclization reaction of beads from which both Fmoc and OAll had been removed was carried out by adding an NMP solution mixed with (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (5 equivalents, Cat# 36813, GL Biochem) and DIPEA (10 equivalents), sealing the container with a lid, and then hanging it on a 180° shaker at room temperature for 12 hours. The solid beads from the reaction were washed three times with NMP and DCM and used for the next reaction.An example of a cyclization reaction scheme for the synthesis of cyclic peptides is as follows.

[0137] [Reaction Equation 2]

[0138]

[0139]

[0140] 1-3-2. Synthesis of Sulfonamide Functional Groups

[0141] After the cyclization reaction, the solid-phase peptide synthesizes a sulfonamide functional group on the beads. The sulfonamide (SFA) functional group was synthesized by performing a click reaction between a peptide containing an azido functional group on the solid-phase beads and 4-ethynylbenzenesulfonamide (Cat# E1130, TCI). Under an Ar atmosphere, a peptide containing an azido functional group (1 equivalent), 4-ethynylbenzenesulfonamide (2 equivalents per azido functional group), CuI (1 equivalent per azido functional group), tris(benzyltriazolylmethyl)amine (TBTA, 2 equivalents per azido functional group, Cat# T2993, TCI), and DIPEA (10 equivalents) were mixed in NMP solvent on solid-phase beads and reacted at room temperature for 12 hours. After the reaction was complete, the beads were washed three times with NMP and at least five times with an NMP solution containing sodium diethyldithiocarbamate dissolved at a concentration of 0.1 M to remove copper impurities. An example of introducing a sulfonamide functional group into a peptide is as follows.

[0142] [Reaction Equation 3]

[0143]

[0144]

[0145] 1-4. Peptide cleavage, separation, and purification from solid-phase beads

[0146] Beads loaded with dried peptides were treated with a mixed solution of 95% TFA / 2.5% TIPS / 2.5% triple-distilled water at room temperature for 6 hours. The beads were removed using a filter, and the TFA mixed solution containing the peptides was collected in a conical tube and most of the mixed solution was removed by blowing nitrogen gas into it. Subsequently, diethyl ether was added to precipitate the peptides, and the mixture was placed in a centrifuge and spun at 3000 rpm for 5 minutes. After removing the supernatant diethyl ether and the small amount of remaining TFA mixed solution, the remaining solid peptides were vacuum dried.

[0147] The peptides cleaved from solid phase beads were dissolved in a mixed solution of ACN / triple distilled water (1:1), and insoluble impurities were removed using a 45 μm syringe filter. The solution was then purified and analyzed using the system mentioned in Example 1-1. The purified peptide solution was desolvated using a freeze-dryer and used in powder form.

[0148]

[0149] 1-5. Mounting Biotin Functional Groups on Purified Peptides

[0150] A purified peptide in powder form equipped with a lysine residue (1 equivalent), Biotin-PEG12-NHS (Cat# 271618, Chempep, 1 equivalent), and DIPEA (5 equivalents) were dissolved in DMSO solvent and reacted for 1 hour. After the reaction, the progress of the reaction was confirmed by LC-MS as mentioned in Example 1-1, and the confirmed peptide was purified using the same system as mentioned in Example 1-1 to obtain peptide structure 8. The reaction scheme for equipping the peptide with a Biotin functional group is as follows.

[0151] [Reaction Equation 4]

[0152]

[0153]

[0154] 1-6. Mounting fluorescent dye (Cy5) on purified peptide

[0155] For peptides requiring the introduction of a fluorescent dye, the fluorescent dye was loaded onto the peptide through click chemistry between the alkyne functional group contained in the peptide linker (introduced using Fmoc-propargyl-Gly-OH (Fmoc-Pra-OH, Cat# 21528, GL Biochem)) and the azido functional group contained in the fluorescent dye. Purified powdered peptide (1 equivalent), fluorescent dye Sulfo-Cyanine5 azide (Cy5 azide, cat# E3330, Lumiprobe) (1 equivalent), 1 M CuSO4 aqueous solution (3 equivalents), and 1 M ascorbic acid (6 equivalents) were dissolved in DMSO solvent and reacted for 1 hour under an Ar atmosphere. After the reaction, the progress of the reaction was confirmed by LC-MS as mentioned in Example 1-1, and the confirmed peptide was purified using the same system as mentioned in Example 1-1. The reaction equation for attaching a fluorescent dye to a peptide is as follows.

[0156] [Reaction Equation 5]

[0157]

[0158]

[0159] Peptide structures 1 to 10 having sulfonamide functional groups that specifically bind to human CAIX were obtained through the synthesis process of Examples 1-1 to 1-6 described above, and analyzed using LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent). An Agilent Poroshell 120 EC-C18 column (4.6 X 150 mm, 2.7 μm) was used as the stationary phase, and the column temperature was maintained at 40 ℃. In addition, a mixture of ACN containing 0.1% TFA and triple-distilled water containing 0.1% TFA was used as the mobile phase solvent, and the wavelengths were maintained at 214 and 254 nm, and the flow rate at 1 mL / min. The solvent conditions of the HPLC equipment are shown in Fig. 7a, and the chromatogram of the HPLC and the molecular weights observed using electrospray ionization mass spectrometry (MS) are shown in Figs. 7b to 7f.

[0160]

[0161] Material List

[0162] Peptide structureAA3R1DAP(Gal)se-EFLG-e-DAP(DOTA)-e-NH22DAP(Gal)PEG(6atom)-se-EFLG-e-DAP(DOTA)-e-NH23sse-EFLG-e-DAP(DOTA)-e-NH24DAP(Gal)se-EFLG-e-Pra(Cy5)-e-NH25DAP(G al) PEG(6atom)-se-EFLG-e-Pra(Cy5)-e-NH26sse-EFLG-e-Pra(Cy5)-e-NH27ssee-DAP(DOTA )-e-NH28sUk(PEG12-biotin)-NH29ssee-Pra(Cy5)-e-NH210DAP(Gal)Uk(PEG12-biotin)-NH2

[0163] In the above chemical formulas, lowercase letters represent D-amino acids and uppercase letters represent L-amino acids, respectively. U signifies a modified or unnatural D-amino acid. The chemical formulas excluding the usual amino acid residues have the following structures as defined herein. AA signifies an amino acid residue containing D-serine or a functional group (Gal), and R is an atomic group having various amino acid linkages.

[0164]

[0165]

[0166]

[0167]

[0168] [Structure of Peptide Structure 1]

[0169]

[0170] [Structure of Peptide Structure 2]

[0171]

[0172] [Structure of Peptide Structure 3]

[0173]

[0174] [Structure of Peptide Structure 4]

[0175]

[0176] [Structure of Peptide Structure 5]

[0177]

[0178] [Structure of Peptide Structure 6]

[0179]

[0180] [Structure of Peptide Structure 7]

[0181]

[0182] [Structure of Peptide Structure 8]

[0183]

[0184] [Structure of Peptide Structure 9]

[0185]

[0186] [Structure of Peptide Structure 10]

[0187]

[0188] Sequence list

[0189] Seq ID No. Amino Acid Sequences 1SXRR (where X=DAP(Gal)) 2SSRR3EFLG4SEEFLGEXE (where X=DAP(DOTA)) 5SEEFLGEXE (where X=Pra(Cy5)) 6SEEXE (where X=DAP(DOTA)) 7SEEXE (where X=Pra(Cy5)) In the above sequences, the amino acids may be D-amino acids or L-amino acids. Additionally, the chemical structural formulas of the abbreviations used in each sequence are as follows. Abbreviation Chemical Structural Formula DAP(Gal) 2-amino-3-((2S,3R,4S,5R,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanoic acidDAP(DOTA)2,2',2''-(10-(2-((2-amino-2-carboxyethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acidPra(Cy5)1-(6-((3-(4-(2-amino-2-carboxyethyl)-1H-1,2,3-triazol-1-yl)propyl)amino)-6-oxohexyl)-3,3-dimethy l-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium-5-sulfonate

[0190]

[0191] [Structure of Sequence No. 1]

[0192]

[0193] [Structure of Sequence No. 2]

[0194]

[0195] [Structure of Sequence No. 3]

[0196]

[0197] [Structure of Sequence No. 4]

[0198]

[0199] [Structure of Sequence No. 5]

[0200]

[0201] [Structure of Sequence No. 6]

[0202]

[0203] [Structure of Sequence No. 7]

[0204]

[0205]

[0206] Example 2. Evaluation of the stability of the peptide structure

[0207] Stability tests in serum and plasma were performed on peptide constructs 1, 2, and 7. Solutions of the peptide constructs, diluted to a final concentration of 300 μM, were aliquoted into 500 μL aliquots in 500 μL of 100% pure human serum (Cat# S1, Merk) and plasma (Cat# 70039.1, STEMCELL Technologies). The samples were stored at 37 ℃ and analyzed once a day for 7 days using an LC-MS (1260 infinity II, Infinity Lab LC / MSD, Agilent) equipped with an Agilent Poroshell 120 EC-C18 column (4.6 X 150 mm, 2.7 μm). Before injecting the sample into the LC, 50 μL of ACN (containing 0.1% TFA) was added to the serum or plasma solution containing the peptide, then placed in a centrifuge and spun to settle the precipitate, and only 10 μL of the supernatant was used for analysis.

[0208] The results of stability tests for each peptide structure in human serum and plasma are shown in Figures 1a to 1c.

[0209]

[0210] Example 3. Confirmation of biological properties of the peptide structure

[0211] 3-1. Analysis of Binding Affinity and Binding Kinetics for the Human Carbonic Anhydrous Seizure-IX (hCAIX) Extracellular Domain (ECD)

[0212] The binding affinity and binding kinetics of peptide constructs 8 and 10 for hCAIX ECD were analyzed. To confirm specific binding selectivity for hCAIX ECD (Cat# H5226, ACROBiosystems), the binding kinetics of the peptide constructs were also analyzed for the extracellular domain of human carbonic anhydrous enzyme XII (hCAXII, Cat# 10617-H08H, Sino Biological), another isoform of carbonic anhydrous enzyme.

[0213] Binding affinity is BLItz, based on Bio-Layer Interferometry (BLI) technology. ®The system (Cat# 45-5000, ForteBio) 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. To correct for non-specific binding and background of the biosensor, the results of associating and dissociating at the lowest protein concentration without peptide attachment were used as the reference. First, 10X Octet kinetics buffer (Cat# 18-1105, Sartorius) was prepared by diluting it to 5X using DPBS (Cat# L0615, Biowest) to serve as the analytical buffer solution, and 1X was prepared to serve as the peptide dilution solution. Analytical buffer was dispensed at a rate of 200 μl / well into 96-well black, flat-bottom polypropylene (Cat# 655209, Greiner Bio-One), and the streptavidin biosensor was immersed in it for 10 minutes for hydration. Subsequently, the biosensor was mounted on the instrument, and peptides were loaded at a concentration of 1 μM at 2200 rpm for 120 seconds to attach. Afterward, protein association and dissociation were performed at each concentration at 2200 rpm for 120 seconds, and kinetics data were analyzed using the global fitting function for K D The value is k d vs k a It was calculated at the ratio. In addition, the kinetic parameters were obtained by fitting a baseline correction and a 1:1 binding model.

[0214] The results are shown in Fig. 2. The curves shown in each graph in Fig. 2 represent the experimental results obtained by varying the protein concentration. The protein concentration corresponding to each curve is indicated below the curve. For example, in Graph 1 shown in Fig. 2, the curve indicated at the top represents the result when the concentration of hCAIX (nM) is 80 nM, and the curve indicated at the bottom represents the result when the concentration of hCAIX (nM) is 20 nM. The peptide structure exhibited significantly higher selectivity for hCAIX ECD compared to hCAXII ECD.

[0215]

[0216] 3-2. Experiment on the Cleavage of Linker Regions of Peptide Structures Using Human Pepsin Protease Activity

[0217] To minimize the side effects of radiation toxicity to the stomach, a linker containing an amino acid sequence that can be degraded by pepsin, a proteolytic enzyme that is specifically active only in the stomach, was introduced between the cyclic peptide region interacting with the CAIX protein and the DOTA chelator for radioisotope labeling. It is expected that the side effects of radiation toxicity to the stomach can be prevented because when pepsin present in the stomach cleaves the linker, the radioisotope-labeled DOTA chelator is separated from the CAIX target cyclic peptide and excreted from the body.

[0218] Pepsin is known to preferentially cleave aromatic amino acids such as phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). A four-amino acid combination of Glu-Phe-Leu-Gly containing aromatic amino acids was randomly selected and inserted into the linker region of the peptide construct, and peptide constructs 1, 2, and 3 were used by labeling the isotope Lu-175 (lutetium-175) with DOTA instead of Lu-177 (lutetium-177). The synthesized peptide constructs were dissolved in DMSO at a concentration of 2 mM, and Lu-175 (LuCl3, Cat#450960, Sigma-Aldrich) was prepared by dissolving it in sodium acetate buffer solution (pH 5.5) at a concentration of 10 mM. After adding 3 equivalents of Lu-175 to 1 equivalent of the peptide construct dissolved in DMSO, 500 μL of sodium acetate buffer (pH 5.5) was added, and the mixture was reacted in a thermoblock (Cat#B10-48, BLE) at 90 °C for 30 minutes. After the reaction, the mixture was cooled to room temperature and purified using a 1260 Infinity II LC system (Agilent) under the following conditions: (1) stationary phase was a Kromasil 100-5-C18 column (10 x 250 mm, 5 μm), (2) mobile phase solvent was a mixture of triple-distilled water with 0.1% acetic acid and ACN, (3) flow rate was 8 mL / min, and (4) detection wavelengths were 214 and 254 nm. The Lu-175-labeled peptide purification solution was freeze-dried to obtain a powder.

[0219] To confirm the possibility of cleavage of the linker region by human pepsin protein, the following experiment was conducted. Human pepsinogen (Cat# 13082-H08H, Sino Biological) protein was used, and this protein becomes pepsin when the pH is between 1 and 3. To activate the protein, a hydrochloric acid (HCl) solution at pH 1.3 was prepared, and the human pepsinogen protein was diluted to 200 μg / mL. The Lu-175-labeled peptide construct was dissolved in DMSO to a concentration of 10 mM and then diluted to 20 μM with the pH 1.3 hydrochloric acid solution.

[0220] 20 μL each of the prepared protein and peptide constructs were dispensed into 1.5 mL tubes, and then a thermomixer (Eppendorf ThermoMixer ® The reaction was carried out at 37°C / 800 rpm. At this time, the final concentrations of protein and peptide were 100 μg / mL and 10 μM, respectively. Three tubes were prepared for each peptide structure, and at 0, 5, and 9 hours, one tube was removed and 35 μL was injected into an LC-MS for analysis at a wavelength of 254 nm.

[0221] It was confirmed that the peptide structure was cleaved by the protein over time, and the results are shown in Figures 3a to 3d.

[0222]

[0223] 3-3. Fluorescence-activated Cell Sorting (FACS) Analysis

[0224] In vitro cell binding characteristics were analyzed to determine whether the peptide construct specifically binds to target cells expressing CAIX. The human renal cancer cell line SK-RC-52 expressing CAIX was purchased from Memorial Sloan Kettering Cancer Center (MSK, USA).

[0225] Peptide structures attached with the fluorescent dye Cy5 are measured using a NanoPhotometer for accurate quantification. ® Absorbance was measured using an N60 (IMPLEN) and concentration was calculated according to Lambert-Beer's law. The freeze-dried peptide constructs were dissolved in dimethyl sulfoxide (DMSO) to prepare a millimola (mM) solution, and then a NanoPhotometer ® The absorbance of 1.5 μL of Cy5 diluted 100-fold in PBS was measured using equipment at 650 nM. The molar extinction coefficient of the Cy5 fluorescent dye used in the calculation was 271,000 L·mol -1 ·cm -1 am. The quantified peptide construct was dissolved in DMSO to make a 1 mM solution, and then diluted to 1 nM using a mixture of Dulbecco's Modified Eagle Medium (DMEM) containing 1% fetal bovine serum (FBS) (buffer solution for FACS).

[0226] SK-RC-52 cell line (T75 flask; 1.0 ~ 1.5 X 10⁶ 2–3 days before FACS analysis 6 Pieces, T175 flasks; 5.0 ~ 8.0 X 10⁻⁶ 6Cells were seeded into T75 or T175 flasks. On the day of the experiment, the culture medium was removed from flasks containing SK-RC-52 cells cultured at 80% confluency and washed twice with 10 mL of PBS buffer. 1 mL of Trypsin-EDTA was added in an incubator (37 °C, 5% CO2, humid conditions, Galaxy 170 S) for 5 minutes, after which the Trypsin-EDTA was neutralized with 9 mL of culture medium. The neutralized cell mixture and an additional 10 mL of culture medium were added to the flask, and the collected cell mixture was placed in a 50 mL tube and centrifuged using a VARISPIN 15R centrifuge at 1000 rpm for 3 minutes. The supernatant of the centrifuged cell mixture was removed, and fresh culture medium was added at 5 x 10⁶ per 200 μL. 5 The solution was diluted to a cell count of 100 cells and dispensed in 200 μL aliquots into 96-well round plates (Cat# 34096, SPL LIFE SCIENCE). After centrifuging at 1000 RPM for 3 minutes using a VARISPIN 15R centrifuge, the supernatant was removed from each well, and 200 μL of the pre-prepared 1 nM peptide construct solution was added, followed by incubation in an incubator. After 1 hour, the solution was centrifuged at 1000 RPM for 3 minutes using a VARISPIN 15R centrifuge, the supernatant was removed, and the cells were washed with 200 μL of FACS buffer solution. After washing, 200 μL of FACS buffer was added to each well and mixed with the cells. The cells were then transferred to a 5 mL round-bottom tube equipped with a cell strainer (Cat# 352235, CORNING), and the fluorescence intensity of each cell was measured using a BD Accuri C6 Plus (BD Biosciences). The measured data were analyzed using FlowJo 10.7.1 (BD Biosciences).

[0227] The results of FACS analysis using the SK-RC-52 cell line are shown in Figure 4. From the FACS analysis results, it was confirmed that the peptide structure of the present invention has high affinity for target cells expressing CAIX.

[0228]

[0229] 3-4. Isotopes ( 177 In vivo SPECT / CT imaging experiment of Lu-labeled peptide constructs

[0230] To confirm the imaging diagnostic and therapeutic capabilities of peptide constructs 1 and 7, each peptide construct was prepared by labeling it with an isotope as follows. A 1 mM stock solution was prepared by dissolving each peptide construct in DMSO, and a 10 mCi isotope diluted in 0.04 M hydrochloric acid solution ( 177 LuCl3 (ITM Medical Isotopes GmbH, Lu-177, Lutetium-177) was completely evaporated from the solution by nitrogen purging. 50 μL of 0.2 M sodium acetate (pH 5.5) buffer solution was added to dissolve the isotope, and then 10 μL of peptide stock solution was added to perform isotope labeling at 90 °C for 30 minutes. Once labeling of each substance was complete, 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 250 mm, 2.7 μm), (2) the mobile phase solvent was a mixture of triple-distilled water and ACN (0.1% acetic acid), (3) the flow rate was 1 mL / min, (4) the detection wavelengths were 254 nm and 214 nm, and (5) the column temperature was 40℃.

[0231] The Lu-177-labeled peptide construct obtained by purification was processed using a Sep-Pak C18 column (Cat# 186005125, Waters) to remove organic solvents. The purified peptide construct was diluted 5-fold with distilled water, loaded onto an activated Sep-Pak column (5 mL ethanol, 5 mL distilled water), washed with 5 mL distilled water to remove organic solvents, eluted with 300 μL of 60% ethanol solution (180 μL ethanol, 120 μL distilled water), and diluted with saline solution for use.

[0232] The average size of tumors generated by transplanting the human kidney cancer cell line SK-RC-52 is 120 mm 3 When the mice reached the internal and external stages, 200 μL of a peptide construct labeled with the isotope Lu-177, corresponding to a radiation dose of 500 μCi, was administered once via tail vein injection to mice. SPECT / CT (Siemens Inveon, software: Inveon Acquisition Workplace) measurements were taken for Lu-177-labeled peptide construct 1 at 1 hour, 4 hours, 8 hours, 1 day, and 2 days, and for Lu-177-labeled peptide construct 7 at 1.5 hours, 1 day, 2 days, 3 days, and 8 days, respectively. For imaging, mice were respiratory anesthetized with 0.2% isoflurane in oxygen for 3–5 minutes. The anesthetized mice were then positioned in the equipment, and the anesthetic was controlled to be continuously infused at 1.5 L / min. SPECT measurements were taken at 40 minutes and CT at 7 minutes, and images were acquired using Inveon Research Workplace 4.2.

[0233] The SPECT / CT images obtained above are shown in Figures 5a and 5b. It was confirmed that the peptide construct labeled with the isotope Lu-177 binds to the cancer cell SK-RC-52. The location of the tumor is indicated by an arrow.

[0234]

[0235] 3-5. Analysis of the Anticancer Effects of Isotope (Lu-177) Labeled CAIX Targeted Peptide

[0236] To confirm the anticancer effect of peptide construct 1 labeled with the isotope (Lu-177), 6-week-old female BALB / c nude mice (Hana Bio, Korea) were used to 5 x 10 6 SK-RC-52 cells were injected subcutaneously into the upper right forelimb of nude mice. After cell injection, the average tumor size was 120 mm 3 Grouping was performed into groups of 5 animals to correspond to internal and external conditions. Tumor size was measured using a caliper [Volume = (Width)] 2 It was measured as [X (length) / 2].

[0237] After xenografting SK-RC-52 cell lines and performing grouping, physiological saline was injected into the control group, and 200 μL of the radioisotope Lu-177, corresponding to a radiation dose of 500 μCi, was injected into the tail veins of mice to inject peptide constructs 1 and 7, respectively, and body weight and tumor size were measured twice a week.

[0238] The results are shown in Figure 6. Lu-177-labeled peptide constructs 1 and 7 exhibited an anticancer effect that significantly reduced the size of tumors overexpressing CAIX without causing severe changes in mouse body weight.

[0239] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the scope of the present invention is not limited by the disclosed embodiments and attached drawings, and that various modifications can be made to other specific forms within the scope of the technical concept of the present invention.

Claims

1. Includes the amino acid sequence of SEQ ID NO. 1 or 2, but, At least one of the constituent amino acids is composed of a D-amino acid, and A peptide ligand in which a 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 characterized by comprising a sulfonamide functional group-containing amino acid residue connected to the peptide ligand directly or through a spacer, and having the structure of Chemical Formula 1 below. [Chemical Formula 1] In the above chemical formula, P is the above-mentioned peptide ligand, and F1, F2, and F3 are each independently sulfonamide functional group-containing amino acid residues, and m is 0 or 1, and F4 is the general formula -(F5) o -(S1) p -(F6) q -(S2) r -(F7) s -(S3) is the basis, and here, F5 is a sulfonamide functional group-containing amino acid residue, and S1 is a spacer, and S2 is a spacer or a residue having the amino acid sequence of SEQ ID NO. 3, and F6 and F7 are each independently amino acid residues containing functional groups other than sulfonamide, and S3 is -NH2, and o is an integer of 0 or 1, and p, q, r, and s each independently represent integers from 0 to 6, but, When P is a peptide ligand containing the amino acid sequence of SEQ ID NO. 1, r is an integer from 1 to 6, and at least one of S2 is a residue having the amino acid sequence of SEQ ID NO.

3.

2. A peptide structure according to claim 1, characterized in that the chemical functional group that can be substituted at the side chain β-amino group of the 2,3-diaminopropionic acid (DAP) residue among the constituent amino acids is α-D-galacturonic acid (α-D-galacturonic acid, Gal).

3. A peptide structure according to claim 1 or 2, characterized in that the sulfonamide functional group-containing amino acid residue has the following structure. or 4. A peptide structure according to any one of claims 1 to 3, wherein the functional group other than the sulfonamide is a chelator, biotin, polyethylene glycol 12-biotin (PEG12-biotin), glucoheptonic acid, 4-(p-iodophenyl)butyric acid (IB), 2-(4-iodophenyl)acetic acid (sIB), zwitteric ion (SZW), α-D-galacturonic acid (Gal), a cycloalkane having 5 to 15 carbon atoms, a fluorescent dye, or a cytotoxic agent.

5. In paragraph 4, the chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazcyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid-2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N'''',N''''-hexaacetic acid (HEHA), 2-[4-nitrobenzyl]-1,4,7,10,13-pentazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid (PEPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), (1R, 4R, 7R, 10R)-α, α', α'', α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)tetrasodium salt (DOTMA), 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA), triethylenetetramine (TETA), A peptide structure characterized by being one or more selected from 1,4,7,10-tetraazacyclododecane-7-acetamide-1,4,10-triacetic acid (PSC) and 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide (DOTAM or TCMC).

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 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 spacer is 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.

8. A peptide structure characterized by being selected from the group consisting of peptide structures having the structure of Chemical Formula 2 below. [Chemical Formula 2] In the above, Lowercase letters represent D-amino acids, and uppercase letters represent L-amino acids, respectively. The other substituents indicated by abbreviations in R1 to R4 and AA1 to AA4 are each defined as follows.

9. A conjugate comprising a peptide structure of any one of claims 1 to 8, coupled directly or through a linker to a fluorescent dye, cytotoxic agent, or radioisotope.

10. A conjugate according to claim 9, wherein the linker is one or more selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-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).

11. A conjugate according to claim 9 or 10, wherein the fluorescent dye is one or more selected from near-infrared fluorescent dyes, fluorescein type, rhodamine type, Alexa Fluor, 4,4-difluoro-4-boro-3a, 4a-diaza-s-indacen (BODIPY), Texas Red, dansyl, lissamine, cyanine (Cy), and phycoerythrin.

12. In any one of claims 9 to 11, the radioisotope 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), Chromium-51 (Cr-51), A conjugate characterized by being one or more selected from krypton-81 (Kr-81), potassium-42 (K-42), holmium-166 (Ho-166), gallium-67 (Ga-67), gallium-68 (Ga-68), actinium-225 (Ac-225), zirconium-89 (Zr-89), thorium-227 (Th-227), radium-223 (Ra-223), tin-117m (Sn-117m), lead-103 (Pb-103), terbium-161 (Tb-161), lead-212 (Pb-212), and astatine-211 (At-211).

13. A conjugate comprising a peptide structure selected from the group consisting of peptide structures 1, 2, 3 and 7, which is coupled to a radioisotope directly or through a linker. [Structure of Peptide Structure 1] [Structure of Peptide Structure 2] [Structure of Peptide Structure 3] [Structure of Peptide Structure 7] 14. A conjugate according to claim 13, characterized in that the radioisotope is one or more 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).

15. A pharmaceutical composition for the diagnosis, prevention, or treatment of cancer comprising a peptide structure according to any one of claims 1 to 8.

16. A pharmaceutical composition for the diagnosis, prevention, or treatment of cancer comprising a conjugate of any one of claims 9 to 14.

17. A pharmaceutical composition for the diagnosis, prevention, or treatment of cancer according to claim 15, wherein the cancer expresses carbonic anhydrous enzyme IX.

18. A pharmaceutical composition for the diagnosis, prevention, or treatment of cancer according to claim 15 or 17, wherein the cancer is liver cancer, lung cancer, colorectal cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, prostatic cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, melanoma, thyroid cancer, astrocytoma, or glioblastoma.

19. A pharmaceutical composition for the diagnosis, prevention, or treatment of cancer according to claim 16, wherein the cancer expresses carbonic anhydrous enzyme IX.

20. A pharmaceutical composition for the diagnosis, prevention, or treatment of cancer according to claim 16 or 19, wherein the cancer is liver cancer, lung cancer, colorectal cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, prostatic cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, prostate cancer, biliary tract cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, melanoma, thyroid cancer, astrocytoma, or glioblastoma.

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