Peptide ligands targeting carbonic anhydrase IX, peptide structures containing the same and their uses

Abstract

The present invention relates to a peptide ligand that specifically binds to carbonic anhydrase IX (CAIX), a peptide structure containing the same, and uses thereof. The CAIX-binding peptide ligand of the present invention includes D-amino acids, thereby being stable in vivo and having high binding specificity to CAIX, and a linear or annular CAIX-binding peptide structure containing the same can bind to CAIX with high affinity in vivo, thereby being effective in the diagnosis, prevention, inhibition or treatment of diseases mediated by CAIX.

Description

Technical Field

[0001] The present invention relates to a peptide ligand that specifically binds to carbonic anhydrase IX (CAIX) and a peptide structure comprising the same. Specifically, the present invention relates to a CAIX-binding peptide ligand that is specific to CAIX and contains D-amino acids to improve stability; a linear or cyclic high-affinity CAIX-binding peptide structure containing the above-mentioned peptide ligand; and their use in diagnosing, preventing, inhibiting or treating diseases mediated by CAIX. Background Art

[0002] Carbonic anhydrase (CA) is a zinc (Zn) enzyme that is common in higher vertebrates including humans. 2+ ) metalloenzyme, an enzyme that catalyzes the reversible hydration reaction that converts carbon dioxide into hydrogen ions and bicarbonate ions This CA has been found to have 16 isoenzyme forms, and is present in multiple tissues in the human body, including the gastrointestinal tract, reproductive tract, nervous system, kidney, lung, skin, eyeball, etc. Most of the known CA isoenzymes are involved in important physiological processes, such as respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid (Thiry et al., TRENDS in Pharmacological Sciences, 27(11):566-573, 2006).

[0003] It has been reported that among the CA family, carbonic anhydrase IX (CAIX) in particular is expressed very limitedly in normal tissues, but is abnormally overexpressed in most solid tumors. This is mainly due to the induction of hypoxia-inducible factor (HIF-1), a transcription factor, caused by hypoxia due to excessive proliferation of solid tumors, resulting in strong transcriptional activation of HIF-1 (De Simone et al., Biochimica et Biophysica Acta, 1804:404-409, 2010; Thiry et al., ditto).

[0004] Tumor hypoxia is caused by solid tumors growing at a rate that exceeds the blood supply provided by the host's vascular system, creating an oxygen-poor environment. Even in a hypoxic microenvironment, solid tumors maintain continued growth and proliferation through various genetic changes. Anticancer chemotherapeutics are difficult to deliver to such hypoxic tumor cells through the blood, and during radiotherapy, resistance to chemotherapy and radiotherapy increases due to insufficient oxygen required for the cytotoxic effects of free radicals derived from radiation. In addition, hypoxic tumor cells induce overexpression of CAIX on the cell surface, and CO is regulated by the extracellular catalytic domain of CAIX. 2 Hydration reaction can reduce the pH of the extracellular environment of tumor cells. The acidic tumor microenvironment thus formed can promote the invasion and migration of tumor cells and can render pH-sensitive drugs ineffective (Thiry et al., ditto). Therefore, tumor hypoxia is generally considered a poor prognostic factor for cancer patients.

[0005] Recently, research is being actively conducted on inhibiting the growth and proliferation of CAIX-related tumors by targeting CAIX overexpressed in tumor cells or disrupting the pH regulation caused by tumor cells by interfering with the catalytic activity of CAIX. The focus of this research is mainly on the development of monoclonal antibodies or sulfonamide-based small molecule inhibitors that bind to CAIX. However, in terms of solid tumor targeting, monoclonal antibodies with large molecular weight have the problem of being difficult to deliver effectively, and sulfonamide-based small molecule inhibitors are relatively unstable in solution, which limits their practicality as drug compounds.

[0006] Therefore, there is an urgent need to develop a novel CAIX-specific binder and inhibitor that is suitable for medical use in cancer treatment, including cancer treatment, prevention, diagnosis, prognosis prediction, and imaging, and that has high affinity and stability.

[0007] Prior art literature

[0008] Non-patent literature

[0009] Non-patent document 1: Thiry et al., TRENDS in Pharmacological Sciences, 27(11):566-573, 2006

[0010] Non-patent document 2: De Simone et al., Biochimica et Biophysica Acta, 1804: 404-409, 2010 Summary of the invention

[0011] Problems to be solved by the invention

[0012] One object of the present invention is to provide a stable CAIX-specific binding agent and inhibitor suitable for the treatment, prevention, diagnosis or prognosis of cancer.

[0013] One object of the present invention is to provide a stable CAIX-specific binding peptide ligand.

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

[0015] An additional object of the present invention is to provide a conjugate comprising the above peptide structure.

[0016] An additional object of the present invention is to provide a composition for diagnosing, preventing or treating cancer comprising the above-mentioned peptide ligand, peptide structure or conjugate.

[0017] An additional object of the present invention is to provide a method for diagnosing cancer using the above-mentioned peptide ligand, peptide structure or conjugate.

[0018] An additional object of the present invention is to provide a method for treating cancer using the above-mentioned peptide ligand, peptide structure or conjugate.

[0019] An additional object of the present invention is to provide a method for predicting prognosis after cancer treatment using the above-mentioned peptide ligand, peptide structure or conjugate.

[0020] Solutions for solving problems

[0021] As a result of intensive studies to achieve the above-mentioned objectives, the inventors of the present application have developed a novel CAIX-specific binder and inhibitor, which includes D-amino acids, thereby having stability and being able to specifically bind to CAIX with high affinity.

[0022] In one aspect of the present invention, a CAIX-specific peptide ligand comprising any one of the amino acid sequences of SEQ ID NO. 1 to 44 is provided. At least one of the constituent amino acids of the CAIX-specific peptide ligand is composed of D-amino acids, so that the CAIX-specific peptide ligand can be stably maintained in vivo. The side chain ε-amino group of the lysine (Lys) residue in the constituent amino acids of the CAIX-specific peptide ligand can be replaced by a chemical functional group. Non-limiting examples of the chemical functional group include pentafluorobenzoic acid or diphenolic acid.

[0023] One aspect of the present invention provides a peptide structure, which includes: an amino acid residue containing a sulfonamide functional group connected to the above-mentioned CAIX-specific peptide ligand directly or through a spacer. The above-mentioned peptide structure may further include amino acid residues containing one or more other functional groups other than the sulfonamide functional group, and may be a ring-shaped or linear structure.

[0024] In one aspect of the present invention, a CAIX-specific peptide structure having a ring structure as shown in the following Chemical Formula 1 is provided.

[0025]

Chemical formula 1

[0026]

[0027] In the above formula,

[0028] P is a CAIX-specific binding peptide of the present invention,

[0029] F 1 is glycine (Gly) or an amino acid residue containing a sulfonamide functional group,

[0030] F 2 is an amino acid residue containing a sulfonamide functional group,

[0031] F 3 is glycine (Gly) or an amino acid residue containing a functional group other than sulfonamide,

[0032] n and m are independently 0 or 1,

[0033] F 4 The general formula is -(S 1 ) o -(F 5 ) p -(S 2 ) q -(F 6 ) r -NH 2 A group, in which

[0034] S 1 and S 2 are independently a spacer,

[0035] F 5 and F 6 are each independently an amino acid residue containing a functional group other than sulfonamide,

[0036] o, p, q and r each independently represent an integer of 0 to 6.

[0037] In one aspect of the present invention, a CAIX-specific peptide structure having a linear structure as shown in Chemical Formula 2 is provided.

[0038]

Chemical formula 2

[0039] CH 3 C(=O)-Gly-(F 7 ) s -F 8 -(S 3 ) t -P-(F 9 ) u -(S 4 ) v -(F 10 ) w -NH 2

[0040] In the above formula,

[0041] P is a CAIX-specific binding peptide of the present invention,

[0042] F 8 is an amino acid residue containing a sulfonamide functional group,

[0043] F 7 、F 9 and F 10 are each independently an amino acid residue containing a functional group other than sulfonamide, or

[0044] In F 9 In case of multiple cases, at least one F 9 is an amino acid residue containing a sulfonamide functional group, and the remaining F 9 and F 7 、F 10 are each independently an amino acid residue containing a functional group other than sulfonamide,

[0045] S 3 and S 4 are independently a spacer,

[0046] s, t, u, v and w each independently represent an integer of 0 to 3.

[0047] In the above CAIX-specific peptide structure, the amino acid residue containing a sulfonamide functional group may have the following structure, but is not limited thereto.

[0048]

[0049] In the above-mentioned CAIX-specific peptide structure, amino acid residues containing functional groups other than sulfonamide can be introduced through the side chain ε-amino group of the lysine residue, for example, including chelators, cycloalkane, biotin, glucoheptonic acid, 4-(p-iodophenyl)butyric acid (IB), fluorescent dyes or cytotoxic agents.

[0050] In one aspect of the present invention, a conjugate is provided in which a fluorescent dye, a cytotoxic agent or a radioactive isotope is conjugated to the above CAIX-specific peptide structure.

[0051] In one aspect of the present invention, a pharmaceutical composition for diagnosing, preventing or treating cancer comprising the above CAIX-specific peptide structure or the above conjugate is provided. The above cancer may be a cancer expressing CAIX. The above 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, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, prostate cancer, bile duct cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic cancer, melanoma, thyroid cancer, astrocytoma and glioblastoma, but is not limited thereto.

[0052] In one aspect of the present invention, a method for diagnosing cancer is provided, which comprises administering the above-mentioned CAIX-specific peptide structure or the above-mentioned conjugate to an individual.

[0053] In one aspect of the present invention, a method for treating cancer is provided, which comprises administering the above-mentioned CAIX-specific peptide structure or the above-mentioned conjugate to an individual.

[0054] In one aspect of the present invention, a method for predicting prognosis after cancer treatment is provided, which comprises administering the above-mentioned CAIX-specific peptide structure or the above-mentioned conjugate to an individual.

[0055] Effects of the Invention

[0056] According to the present invention, novel high-affinity CAIX binders and inhibitors that specifically bind to CAIX and are stable are provided. The CAIX binders of the present invention include CAIX-specific binding peptide ligands containing one or more D-amino acids, thereby showing excellent stability in vivo and being effective in targeting CAIX. In addition, the present invention provides a peptide structure in which one or more effectors or functional groups including a sulfonamide functional group are introduced into the above-mentioned CAIX-specific peptide ligand through the side chain of an amino acid residue, thereby providing a CAIX binder that is particularly practical for imaging or diagnosing cancer due to its high binding affinity to CAIX. The CAIX-specific peptide structure of the present invention can also be used as an effective drug for diagnosing, preventing or treating cancer by combining with a fluorescent dye, a cytotoxic agent or a radioactive isotope, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 is a flow chart showing a peptide screening process for human carbonic anhydrase IX (hCAIX) protein according to one embodiment of the present invention.

[0058] Figure 2 Shown is a click reaction for introducing a sulfonamide functional group into a peptide library according to an embodiment of the present invention and a library structure for hCAIX screening.

[0059] Figure 3 The results of screening positive beads binding to hCAIX using COPAS according to an embodiment of the present invention are shown.

[0060] Figure 4a The mass spectrometry analysis results of hCAIX extracellular domain (ECD) binding peptides according to one embodiment of the present invention are shown.

[0061] Figure 4b The MS / MS sequence analysis results of the hCAIX ECD binding peptide according to one embodiment of the present invention are shown.

[0062] Figure 5a and Figure 5b : is a graph showing the results of a stability test of a peptide structure (No. 35) in serum and plasma according to one embodiment of the present invention.

[0063] Figure 6a and Figure 6b This is a graph showing the results of a stability test of a peptide structure (No. 48) according to one embodiment of the present invention in serum and plasma.

[0064] Figures 7a to 7dThis is a graph showing the results of analyzing the binding affinity and binding kinetics of hCAIX ECD and human carbonic anhydrase XII (hCAXII) ECD of a peptide structure according to an embodiment of the present invention.

[0065] Figures 8a to 8d FIG. 1 is a graph showing the results of fluorescence activated cell sorting (FACS) analysis of SK-RC-52 cell line using a peptide structure according to an embodiment of the present invention.

[0066] Figure 8e to Figure 8h This is a graph showing the results of FACS analysis of the A549 cell line using the peptide structure according to one embodiment of the present invention.

[0067] Fig. 9 This is a diagram showing the results of immunofluorescence analysis of SK-RC-52 cell line using the peptide structure (No. 22) according to one example of the present invention.

[0068] Fig.10 This is a Z-stack confocal microscope analysis image of a peptide structure (No. 94) according to one embodiment of the present invention.

[0069] Figures 11a to 11c The results of biodistribution analysis of the peptide structure according to one embodiment of the present invention in mice xenotransplanted with SK-RC-52 cell lines are shown. Fig.11a shows the experimental plan using mice, Fig.11b These are ex vivo images of tumors and organs removed from mice. Fig.11c The graph shows the results of homogenizing each tumor and organ and measuring the fluorescence intensity.

[0070] Fig.12a and Figure 12b Figure 2 shows the weight change of mice in which SK-RC-52 cell lines were xenotransplanted, according to the peptide structure-drug conjugate (No. 86) of one embodiment of the present invention. Fig.12a ) and changes in tumor size ( Figure 12b ) in the table below.

[0071] Fig.13 The isotope according to one embodiment of the present invention ( 177 In vivo SPECT / CT imaging results of the peptide structure (No. 95) labeled with Lu.

[0072] Fig.14 Is the isotope ( 177 95) was purified by LC.

[0073] Figures 15a to 15c Figure 2 shows the effect of isotope ( 177 Figure 2. Changes in tumor size in mice caused by Lu-labeled peptide structure (No. 95) ( Fig.15a ) and charts ( Fig.15c ), and a graph of weight change ( Fig.15b ). DETAILED DESCRIPTION

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the 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.

[0075] The present invention can be variously modified and applied within the scope of the following claims and the equivalent scope interpreted therefrom.

[0076] CAIX-specific peptide ligands of the present invention

[0077] The present invention provides a peptide ligand that specifically binds to CAIX. The CAIX-specific peptide ligand of the present invention may include any one of the amino acid sequences of SEQ ID NO.1 to 44. The above-mentioned peptide ligand may include D-amino acids or be composed only of D-amino acids. In addition, in the above-mentioned peptide ligand, the side chain ε-amino group of the lysine (Lys) residue in the constituent amino acid residues may be substituted by one or more chemical functional groups, for example, the above-mentioned functional groups include pentafluorobenzoic acid or diphenolic acid, but are not limited thereto. Moreover, the above-mentioned peptide ligand may also include one or more chemical modifications of the side chain of the phenylalanine (Phe) residue in the constituent amino acid residues. Such modifications include: the phenylalanine residue is modified to a homophenylalanine residue; the phenyl group is modified to a naphthyl group; or the substitution of a halo, amino or phenyl substituent in the phenyl group, etc., but are not limited thereto.

[0078] The peptide ligand of the present invention can be prepared in a manner that amino acid residues having a specific sequence are combined with each other to form a linear or annular molecule. The peptide ligand of the present invention can be prepared by a known peptide synthesis method, and is not particularly limited. In one embodiment, the peptide ligand of the present invention can be prepared by repeating the peptide synthesis process on a solid phase single bead until a peptide of the desired length and sequence is completed.

[0079] The CAIX-specific peptide ligand of the present invention also includes a salt form thereof.

[0080] CAIX-specific peptide structures of the present invention

[0081] The present invention provides a peptide structure, which includes: an amino acid residue containing a sulfonamide functional group connected to a CAIX-specific peptide ligand directly or through a spacer. The above-mentioned peptide structure may further include amino acid residues containing one or more other functional groups other than the sulfonamide functional group. The chemical functional groups including the sulfonamide functional group in the peptide structure of the present invention can be introduced through the side chain of the amino acid residue. The amino acid residue introduced with the above-mentioned functional group is preferably lysine.

[0082] The peptide structure of the present invention may include one or two sulfonamide functional groups. The introduction of the above-mentioned sulfonamide functional group can be achieved by a known synthesis reaction, which 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 by a click chemistry reaction. Examples of preferred amino acid residues containing sulfonamide functional groups that can be introduced into the peptide structure of the present invention include the following structures, but are not limited thereto.

[0083]

[0084] In one embodiment, non-limiting examples of functional groups other than sulfonamide that can be introduced into the peptide structure of the present invention include chelators, cycloalkane with a carbon number of 5 to 15, biotin, glucoheptonic acid, 4-(p-iodophenyl)butyric acid (IB), fluorescent dyes or cytotoxic agents.

[0085] The chelate can be selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid 2,2',2",2"'-(ethane-1,2-diyldinitro)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-pentaazacyclopentadienyl At least one of pentadecane-N,N',N",N"',N""-pentaacetic acid (PEPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), (1R,4R,7R,10R)-α,α',α",α"'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) tetrasodium salt (DOTMA), 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA) and triethylenetetramine (TETA), but not limited thereto.

[0086] For example, the cycloalkane having 5 to 15 carbon atoms may be one or more selected from cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, adamantane, norbornane, isobornane and tricyclodecane, but is not limited thereto.

[0087] The peptide structure of the present invention can also connect an amino acid residue containing a chemical functional group such as a sulfonamide functional group to a CAIX-specific binding peptide ligand via a spacer. For example, the spacer can be one or more selected from polyethylene glycol (PEG) linkers, glycine, sarcosine, and peptide linkers composed of 1 to 5 D-amino acids or L-amino acids, but is not limited thereto.

[0088] The peptide structure of the present invention may have a ring-shaped or linear structure. Preferably, the peptide structure of the present invention has a ring-shaped structure as shown in the following Chemical Formula 1.

[0089]

Chemical formula 1

[0090]

[0091] In the above formula,

[0092] P.F. 1 、F 2 、F 3 、F 4 , n and m are as defined above respectively.

[0093] Although not bound by a particular theory, the annular structure of the cyclic peptide structure of the present invention has poor flexibility compared to linear peptides. Therefore, it is believed that when targeted binding occurs, there is less entropy loss, resulting in higher binding affinity and increased binding specificity to the target.

[0094] The peptide structure of the present invention shows high selectivity, that is, the peptide structure specifically binds to CAIX but does not bind to other isozymes of CAIX (eg, carbonic anhydrase XII).

[0095] Conjugate

[0096] The CAIX-specific peptide structure of the present invention can be directly or through a linker to a fluorescent dye, a cytotoxic agent, a radioactive isotope, etc. to form a conjugate. The conjugate can target CAIX and can label cancers expressing CAIX with fluorescent dyes or radioactive isotopes or effectively deliver drugs such as radioactive isotopes or cytotoxic agents to the cancer, thereby being effective in the diagnosis, prevention or treatment of cancer.

[0097] In one embodiment, the linker can be selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (N-Succinimidyl The invention can be selected from the group consisting of, but not limited to, one or more of, 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).

[0098] In one embodiment, non-limiting examples of the fluorescent dye may include one or more selected from near-infrared fluorescent dyes, fluoresceins, rhodamines, Alexa Fluor, 4,4-difluoro-4-boron-3a,4a-diaza-s-indene (BODIPY), Texas Red, dansyl, Lissamine, Cy and phycoerythrin.

[0099] In one embodiment, the cytotoxic agent may be one or more selected from the group consisting of toxins, chemotherapeutic agents, drug moieties, antibiotics, and nucleases, but is not limited thereto.

[0100] In one embodiment, the radioactive isotope may be selected from fluorine-18 (F-18), carbon-11 (C-11), carbon-14 (C-14), technetium-99m (Tc-99m), copper-64 (Cu-64), copper-67 (Cu-67), dysprosium-168 (Dy-168), bismuth-213 (Bi-213), samarium-153 (Sm-153), strontium-89 (St-89), strontium-123 (Sm-123), strontium-124 (Sm-124), strontium-125 (Sm-125), strontium-126 (Sm-126), strontium-127 (Sm-127), strontium-128 (Sm-128), strontium-129 (Sm-129), strontium-130 (Sm-130), strontium-131 ​​(Sm-131), strontium-132 (Sm-132), strontium-133 (Sm-133), strontium-134 (Sm-134), strontium-135 (Sm-135), strontium-136 (Sm-136), strontium-137 (Sm-137), strontium-138 (Sm-138), strontium-139 (Sm-139), strontium-139 (Sm-139), strontium-131 ​​(Sm-139), strontium-139 (Sm-139), strontium-138 (Sm-139), strontium-139 (Sm-139), strontium-139 -90 (St-90), erbium-169 (Er-169), phosphorus-32 (P-32), palladium-103 (Pd-103), rhenium-186 (Re-186), rhenium-188 (Re-188), oxygen-15 (O-15), selenium-75 (Se-75), sodium-24 (Na-24), strontium-85 (Sr-85), lutetium-177 (Lu-177), yttrium-90 (Y- 90), iodine-123 (I-123), iodine-125 (I-125), iodine-131 (I-131), iridium-192 (Ir-192), iridium-196 (Ir-196), ytterbium-166 (Yb-166), indium-111 (In-111), xenon-133 (Xe-133), nitrogen-13 (N-13), calcium-47 (Ca-47), cobalt-57 (Co-57 ), cobalt-60 (Co-60), chromium-51 (Cr-51), krypton-81 (Kr-81), potassium-42 (K-42), holmium-166 (Ho-166), gallium-67 (Ga-67), gallium-68 (Ga-68), actinium-225 (Ac-225), zirconium-89 (Zr-89), lead-212 (Pb-212) and astatine-211 (At-211).

[0101] Therapeutic administration and dosage forms

[0102] The present invention provides a pharmaceutical composition for diagnosis, prevention or treatment of cancer, which comprises a CAIX-specific peptide ligand, peptide structure or conjugate of the present invention. The cancer of the present invention is preferably a solid cancer. More preferably, the cancer of the present invention is a cancer expressing CAIX. For example, the cancer of the present invention can be a solid cancer such as liver cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, prostate cancer, bile duct cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic cancer, melanoma, thyroid cancer, astrocytoma or glioblastoma, but is not limited thereto.

[0103] The subject of administration of the pharmaceutical composition for diagnosis, prevention or treatment of cancer of the present invention may be a mammal at risk of developing cancer, diagnosed with cancer or receiving cancer treatment. The mammal may be a human or a mammal other than a human.

[0104] The pharmaceutical composition for diagnosis, prevention or treatment of cancer according to the present invention can be used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc.; external medicines; suppositories; and sterile injection solutions according to conventional methods. For the purpose of dosage form, appropriate carriers, excipients or diluents conventionally used in the preparation of pharmaceutical compositions can be included.

[0105] As the above-mentioned carrier, excipient or diluent, various compounds or mixtures including lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil can be used.

[0106] When the formulation is carried out, it can be prepared by using diluents or excipients such as fillers, weight agents, binders, wetting agents, disintegrants, surfactants, etc. which are conventionally used in the pharmaceutical industry.

[0107] The preferred dosage of the pharmaceutical composition for the prevention or treatment of cancer according to the present invention varies according to the patient's state, body weight, degree of disease, drug form, route of administration and period, but can be appropriately selected by those skilled in the art. However, for the desired effect, it can be administered in an amount of 0.0001 mg / kg to 2000 mg / kg per day, preferably in an amount of 0.001 mg / kg to 2000 mg / kg. In terms of administration, it can be administered once a day or in multiple doses. However, the scope of the present invention is not limited to the above-mentioned dosage.

[0108] The pharmaceutical composition for the prevention or treatment of cancer according to the present invention can be administered to mammals such as mice, nude mice, livestock, humans, etc. through various routes. For example, the administration can be performed by oral, rectal, intravenous, intramuscular, subcutaneous, endometrial, or intracerebral (intracerebroventricular) injection.

[0109] The present invention also provides a method for treating cancer, which comprises administering the CAIX-specific peptide structure or conjugate of the present invention to an individual in need of cancer treatment.

[0110] The CAIX-specific peptide structures and conjugates of the present invention can also be used for diagnosing cancer by targeting and imaging cancer, or can be administered to individuals receiving cancer treatment for predicting treatment prognosis or for observation after cancer treatment.

[0111] Below, the present invention is described in more detail by examples. These examples are only used to illustrate the present invention more specifically, rather than to limit the legitimate scope of the present invention, and can be changed in many ways within the scope of the claims of the present invention, which is obvious to those skilled in the art.

[0112] [Example]

[0113] Example 1. Screening for peptides that bind to human carbonic anhydrase IX

[0114] A peptide library was synthesized to obtain peptides that specifically bind to the extracellular domain (ECD) of human carbonic anhydrase IX (hCAIX). The peptides selected by screening the synthesized peptide library were evaluated for binding and specificity to hCAIX ECD.

[0115] Figure 1 A flow chart of the peptide screening process against hCAIX ECD is shown in FIG.

[0116] 1-1. Synthesis of peptide library introduced into beads

[0117] To synthesize the peptide library, a random OBOC (combinatorial one-bead-one-compound) library was synthesized by using TentaGel beads. S-NH 2 The resin (Cat# NSD30902) was purchased from Rapp Polymere GmbH (Germany).

[0118] exist S-NH 2For each residue position on the beads (polyethylene glycol-grafted polystyrene beads), 18 kinds of D-amino acids other than cysteine ​​and methionine were used to synthesize by repeating the split and mix process using an automatic synthesizer (Apex 396, AAPPTEC). In particular, for Fmoc (fluorenylmethoxycarbonyl) -D-isoleucine (Fmoc (fluorenylmethoxycarbonyl) -D-isoleucine-OH) and Fmoc-D-glutamine (Fmoc-D-glutamine-OH), 10 mol% of Fmoc-glycine (Fmoc-glycine-OH) was added to distinguish isotopic residues when performing amino acid sequence analysis of the peptide. TentaGel was swollen in NMP (N-Methylpyrrolidone) solvent and loaded with a linker capable of photocleavage, and then the peptide library was synthesized by an automatic synthesizer. When the Fmoc-ANP linker (3-(Fmoc-amino)-3-(2-nitrophenyl) propionic acid, Cat#LSP308, Aipeptide) was introduced, the loading ratio was adjusted to 1 / 4 by adding N-acetylglycine (3 equivalents, Cat#A16300, Sigma-Aldrich), and reacted with TBTU (3 equivalents, Cat#12806, Sigma-Aldrich) and DIPEA (7.5 equivalents, Cat#8.00894, Sigma-Aldrich). The solid phase beads were washed with NMP, reacted with piperidine / NMP (1:4) solution to remove the Fmoc protecting group, and then washed with NMP, DCM (dichloromethane) and NMP in sequence. Next, Fmoc-Arg(pbf)-OH (3 equivalents, Cat#36404, GL Biochem) and Fmoc-PEG1-OH (3 equivalents, Cat#246201, ChemPep) were synthesized by the same method to prepare the linker. In order to synthesize the peptide library, the beads equipped with the linker were divided into the same amount and loaded into 18 wells of the automatic synthesizer RV (reaction vessel). When one cycle of coupling and removal of the Fmoc protecting group was completed, the solid phase beads were gathered in CV (collector vessel) for mixing, and then the same amount was divided into 18 RV wells and coupling and removal of the Fmoc protecting group were performed, and the separation and mixing process was repeated until the desired peptide length was reached.

[0119] 1-2. Synthesis of peptide library with sulfonamide functional group

[0120] After the azido substituent was introduced into the peptide synthesized in the above Example 1-1, the beads of 18 RVs were recovered into one tube and then subjected to a click reaction with 4-ethynylbenzenesulfonamide ( Figure 2 Specifically, under an argon (Ar) atmosphere, 1 equivalent of beads loaded with a peptide library, 4-ethynylbenzenesulfonamide (3 equivalents), CuI (1 equivalent), tris(benzyltriazolylmethyl)amine (TBTA, 3 equivalents, Cat#T2993, TCI), and DIPEA (10 equivalents) were mixed in an NMP solution and reacted at room temperature for 12 hours. After the reaction was completed, the beads were washed with NMP and 0.1M sodium diethyldithiocarbamate (Sigma-Aldrich, Cat#D3506) / NMP solution to remove copper impurities. In order to remove the protecting groups of amino acid residues, the mixture was reacted in trifluoroacetic acid (95%, TFA, Cat#299537, Sigma-Aldrich), triple distilled water (2.5%), and triisopropylsilane (2.5%, TIS, Cat#233781, Sigma-Aldrich) for 2 hours, then washed with DCM and dried under vacuum, and stored at 4°C in the dark.

[0121] Figure 2 An example of a click reaction for introducing a sulfonamide functional group into a peptide library and a library structure for hCAIX screening is shown in FIG.

[0122] Example 2. Screening of peptides binding to human CAIX

[0123] 2-1. Initial screening of peptide library

[0124] 50 mg of beads introduced with the peptide library synthesized in Example 1 were transferred to an Extract-Clean filter column (Cat#211104, S*PURE, Singapore), and then 2 mL of a phosphate buffer solution (PBS) at pH 7.4 was added and swollen by sonication using an ultrasonic cleaner (Cat#5210R-DTH, BRANSON, USA). After replacing the PBS solution with 2 mL of a blocking solution [10% FBS, 0.1% Tween 20 (Cat#69295-1601, Junsei Chemical, Japan) dissolved in pH 7.4 PBS], the beads were incubated at room temperature in a 360° Shaker (Cat#M04-238-157, Celocyk, USA). Then, a 60 nM solution of hCAIX ECD (Biopsy, Cat#CA9-H5226) loaded with fluorescent dye was added and the solution was removed after further isothermal treatment. The beads were transferred to a conical tube, and then diluted with 45 mL of PBST buffer solution containing 0.1% Tween 20 and divided into small portions. PBST buffer solution containing 0.1% Tween 20 was added to each of the solutions divided into small portions for further dilution, and the solution was placed in the COPAS sample vessel for screening. The screening was performed with an excitation wavelength of 640 nm and an emission wavelength of 680 / 30 BP, and about 5,000 beads with high fluorescence intensity were screened under the conditions of enrichment mode, PMT690, and gain 3.0. A total of 25,000 positive beads (i.e., beads loaded with peptides binding to hCAIX ECD) were screened for secondary screening by repeating the above process 5 times ( Figure 3 ).

[0125] 2-2. Secondary screening of peptide library

[0126] hCAIX ECD loaded with fluorescent dye was removed from the approximately 25,000 positive beads obtained by the first screening in Example 2-1 above. After that, 1 mL of blocking solution was added to the above beads and the solution was removed after isothermal treatment using a 360° oscillator. Then, 250 nM hCAIX ECD solution loaded with fluorescent dye was added and the solution was removed after further isothermal treatment. The beads were transferred to a conical tube, and then diluted with 45 mL of PBST buffer solution containing 0.1% Tween 20 and divided into small portions. PBST buffer solution containing 0.1% Tween 20 was added to each of the small portions for further dilution, and the solution was placed in the sample container of COPAS, and the solution was analyzed in two steps under the conditions of excitation wavelength (Excitation) 640 nm and emission wavelength (Emission680 / 30BP). In the first step, about 1,000 beads with high fluorescence intensity were screened under the conditions of enrichment mode (Enrichment mode), PMT630, and gain 3.0. The 1000 beads obtained were diluted with triple distilled water, and then a second COPAS screening was performed using a 96-well plate in Pure mode, PMT620, and gain 3.0.

[0127] 2-3. Isolation and analysis of peptides obtained through secondary screening

[0128] The peptides were separated from the solid phase single beads obtained by the secondary screening of Example 2-2 by photolysis reaction. Specifically, after sealing the 96-well plate containing the beads under argon atmosphere, UVP crosslinker (Cat#849-30101-2, Jena, Germany) was used at 365 nm wavelength and 9000 μJ / cm 2 After opening the 96-well plate, the plate was concentrated at room temperature using an acid benchtop concentrator (Cat#7310042, LABCONCO, USA).

[0129] Afterwards, the molecular weight and amino acid sequence of the peptides were analyzed based on MS and MS / MS obtained using an enhanced ultrafleXtreme MALDI-TOF / TOF mass spectrometer (Bruker, USA). Figure 4a (mass spectrometry results) and Figure 4b An example of each analysis result is shown in (MS / MS sequence analysis results).

[0130] 2-4. Three-step screening of peptide library for selecting candidate peptides

[0131] About 50 peptides were screened out from 250-300 peptides having the amino acid sequences obtained in Examples 2-3 above, and synthesized using an Apex396 automatic synthesizer on TentaGel beads (0.08 mmole / g, 10 mg for each peptide) equipped with a photodegradable linker by the same method as in Example 1 above, and then the protecting groups of the amino acid residues were removed using a mixed solution of TFA (95%) / tri-distilled water (2.5%) / TIS (2.5%).

[0132] Afterwards, in order to conduct three COPAS screenings for deriving the final peptide candidate substances, 1 mg (50 mg in total) of TentaGel beads containing more than 50 different peptides were taken, aggregated and mixed on a 4 mL Extract-Clean filter column, and then 2 mL of PBS was added for ultrasonic treatment to swell the beads. After that, the COPAS screening operation was performed by the same method as the secondary peptide library screening, while keeping the final concentration of hCAIX ECD containing fluorescent dye at 250 nM. In order to ensure the reproducibility of the results, this process was repeated three times. After the MS and MS / MS data analysis results, the peptides were sequenced in the order of positive hit numbers, and the candidate substances were selected based on the top peptides.

[0133] Example 3. Synthesis and purification of peptide structures containing the screened peptides

[0134] 3-1. Synthesis of biotin linker

[0135] An automatic microwave peptide synthesizer (Liberty Blue TM The solid phase biotin linker was synthesized by automated microwave peptide synthesizer (CEM). Specifically, Rink amide- The resin (0.45mmole / g) was treated with a piperidine / NMP (v / v=1:4, 0.1M Oxyma Pure) mixed solution to remove the Fmoc protecting group, and then washed with NMP. Thereafter, Fmoc-Lys(mtt)-OH (5 equivalents), Oxyma Pure (5 equivalents) and diisopropylcarbodiimide (DIC, Cat#D0254, TCI) (10 equivalents) were treated in NMP solvent and coupled to remove the Fmoc protecting group. Fmoc-PEG1-OH (5eq) was used and the same process was repeated twice for connection. The resin recovered from the microwave peptide synthesizer was treated with a 1.5% TFA / 2.5% TIS / 96% DCM mixed solution to remove the 4-methyltrityl protecting group (Mtt). To introduce biotin, biotin-NHS (3 equivalents) and DIPEA (10 equivalents) were reacted in an NMP solution at room temperature for 1 hour, and then washed with NMP and DCM in sequence and dried in vacuum.

[0136] Below, an exemplary reaction formula for synthesizing a biotin linker is shown.

[0137] [Reaction 1]

[0138]

[0139] 3-2. Synthesis of Screened Peptides

[0140] The peptide of the screened amino acid sequence is synthesized by using the biotin linker synthesized in the above Example 3-1. First, the Fmoc protecting group is removed from the beads introduced with the biotin linker using a mixed solution of piperidine / NMP (v / v=1:4), and then NMP, DCM, and NMP are used to wash in sequence. After reacting with an amino acid (3 equivalents), TBTU (3 equivalents), and DIPEA (7 equivalents) with an Fmoc protecting group at room temperature, it is washed with NMP. Repeat the above process until the peptide of the desired sequence is completed. In particular, in order to synthesize a peptide with a ring structure, Fmoc-E(OAll)-OH is first introduced into the biotin linker, and a peptide connection (cyclization) reaction is performed.

[0141] 3-3. Synthesis and purification of linear peptide structures

[0142] A linear peptide structure is prepared by introducing sulfonamide, adamantane, cyclooctane, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or biotin functional groups into the peptide synthesized in the above Example 3-2.

[0143] The sulfonamide functional group was introduced by subjecting the peptide containing an azido substituent to a click reaction as described in Examples 1-2 above.

[0144] The introduction of adamantane, cyclooctane or DOTA functional groups was performed by removing the Mtt or Dde protecting groups from beads loaded with peptides including lysine protected by Mtt or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) substituents. The Mtt protecting group was removed by treatment with a 1.5% TFA / 2.5% TIS / 96% DCM mixed solution, and Pd(PPh 3 ) 4 (1 equivalent) and 1,3-dimethylbarbituric acid (30 equivalents) were mixed in DCM solution and reacted to remove the Dde protecting group. Then, 1-adamantanecarboxylic acid (1-adamantanecarboxylic acid, Cat#106399-25G, Sigma-Aldrich), cyclooctanecarboxylic acid (Cat#EN300-85433, Enamine) or DOTA-tri(tert-butyl ester) (2 equivalents), TBTU (2 equivalents), and DIPEA (5 equivalents) were reacted in NMP solvent at room temperature to introduce Dde.

[0145] The biotin functional group was introduced by reacting biotin-NHS (3 equivalents) and DIPEA (10 equivalents) in NMP solvent at room temperature.

[0146] After the reaction was completed, the beads were washed with NMP and DCM in sequence and dried in vacuo. The following is an example of a reaction formula for introducing an adamantane functional group into the screened peptide.

[0147] [Reaction 2]

[0148]

[0149] 3-4. Synthesis and purification of ring-shaped peptide structures

[0150] The ring-shaped peptide structure is formed by a cyclization reaction in which a covalent bond is formed between the carboxyl group of glutamic acid bonded to the C-terminus of the amino acid and the amide group of the N-terminus of the amino acid in the peptide into which glutamic acid is introduced synthesized in the above Example 3-2.

[0151] First, the allyl protecting group of glutamic acid in the peptide on the solid phase beads was removed. Pd(PPh 3 ) 4 (0.5 equivalent), PhSiH 3 (20 equivalents), and then reacted at room temperature. The beads were washed with 0.1M sodium diethyldithiocarbamate / NMP solution to remove Pd impurities. The Fmoc protecting group was removed with a piperidine / NMP (v / v=1:4) mixed solution, and then washed with NMP, DCM, and NMP in sequence. The cyclization reaction was carried out at room temperature using PyAOP (3 equivalents, Cat#36813, Gill Biochemical) and DIPEA (10 equivalents). After the reaction was completed, the beads were washed with NMP and DCM in sequence and then vacuum dried.

[0152] The following is an example of a cyclization reaction formula for synthesizing a cyclic peptide.

[0153] [Reaction 3]

[0154]

[0155] 3-5. Separation and purification of the obtained linear or cyclic peptide structure

[0156] The beads containing the linear peptide or cyclic peptide obtained in the above Examples 3-3 and 3-4 in a dry state were treated with a 95% TFA / 2.5% TIS / 2.5% H2O mixed solution. The beads were removed using a filter, and after the TFA mixed solution containing the peptide was concentrated in a conical tube, most of the mixed solution was removed by purging with nitrogen. Next, the peptide was precipitated by adding diethyl ether, and then placed in a centrifuge for rotation, and then the upper ether and a small amount of residual TFA mixed solution were removed, and then the remaining solid phase peptide was vacuum dried.

[0157] The peptide cleaved from the solid phase beads was dissolved in ACN / H 2O (1:1) mixed solution, then use filter (45μm syringe filter, Cat#DISMIC-3HP, Advantec, Japan) to remove undissolved impurities, and 1260Infinity II LC system (Agilent) was used for purification under the following conditions: (1) stationary phase is Kromasil 100-5-C18 column (21.2×250mm, 5μm); mobile phase solvent is a mixture of triple distilled water and ACN with 0.1% TFA added; (3) flow rate is 15mL / min; (4) detection wavelength is 214nm and 254nm. For the determination of purity and molecular weight of the purified peptide, LC-MS (1260infinity II, Infinity Lab LC / MSD, Agilent) and MNOVA (v.14.2.0, Mestrelab research, Spain) were used to analyze the data.

[0158] For the stationary phase, an Agilent poroshell 120ec-c18 column (4.6×50 mm, 2.7 μm) was used, and the column temperature was maintained at 40°C. In addition, a mixture of triple distilled water and ACN with 0.1% TFA added was used as a mobile phase solvent, with wavelengths of 214 nm and 254 nm, and a flow rate of 1 mL / min. The purified peptide solution was used in a powdered form after removing the solvent using a freeze dryer.

[0159] As a result, unique linear or cyclic peptide structures of the following Chemical Formulae 3 to 8, including a peptide that specifically binds to human CAIX and has one or two sulfonamide functional groups, were obtained.

[0160] [Linear peptide structure of Chemical Formula 3]

[0161]

[0162] serial number α β γ <![CDATA[AA 1 ]]> <![CDATA[AA 2 ]]> <![CDATA[AA 3 ]]> <![CDATA[AA 4 ]]> <![CDATA[AA 5 ]]> <![CDATA[AA 6 ]]> <![CDATA[AA 7 ]]> θ δ 1 0 1 1 r a h k h y h 0 0 2 0 1 1 h r k h d d n 0 0 3 0 1 1 a y y r k k w 0 0 4 0 1 1 r r l l f s G 0 0 5 0 0 1 r r f h f t h 0 0 6 0 0 1 a y y r k k w 0 0 7 0 0 1 r r l l f d G 0 0 8 0 0 1 r r f h f t h 0 0 9 0 0 1 f h d r r d v 0 0 10 0 0 1 v l r n k s d 0 0 11 0 0 1 r s r h f h y 0 0 12 0 0 1 f h r i k h l 0 0 13 0 0 1 r i t a i n y 0 0 14 0 0 2 y h k h i r q 0 0 15 0 0 3 h f v k k f r 0 0 16 0 0 3 f f k r k h e 0 0 17 0 0 3 h G h l f k r 0 0 18 0 0 3 r f h h f r n 0 0 19 0 0 3 r l s k f h s 0 0 20 0 0 3 y e t f r t r 0 0 21 0 0 0 r r f r h y h 0 0 22 0 0 0 r r f r h y h 1 0 23 1 0 0 r r f r h y h 0 0 24 0 0 0 r r f r h y h 0 1

[0163] [Linear peptide structure of Chemical Formula 4]

[0164]

[0165] serial number <![CDATA[AA 1 ]]> <![CDATA[AA 2 ]]> <![CDATA[AA 3 ]]> <![CDATA[AA 4 ]]> <![CDATA[AA 5 ]]> <![CDATA[AA 6 ]]> <![CDATA[AA 7 ]]> <![CDATA[R 1 ]]> 25 r r f r h y h <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 26 r r f r h y h <![CDATA[Uk(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 27 r t U(1-Nal) r h <![CDATA[Uk(PhF 5 )]]> h <![CDATA[Uk(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]>

[0166] [Linear peptide structure of Chemical Formula 5]

[0167]

[0168] serial number <![CDATA[AA 1 ]]> <![CDATA[AA 2 ]]> <![CDATA[AA 3 ]]> <![CDATA[AA 4 ]]> <![CDATA[AA 5 ]]> <![CDATA[R 2 ]]> 28 r r f r y <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 29 r r f r y <![CDATA[Uk(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 30 r r U(1-Nal) r y <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 31 r r f r Uk(Biphenol) <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 32 r r U(1-Nal) r <![CDATA[Uk(PhF 5 )]]> <![CDATA[Uk(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 33 e r U(1-Nal) r <![CDATA[Uk(PhF 5 )]]> <![CDATA[Uk(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 34 r r U(1-Nal) e <![CDATA[Uk(PhF 5 )]]> <![CDATA[Uk(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]>

[0169] [Cyclic peptide structure of Chemical Formula 6]

[0170]

[0171] serial number <![CDATA[R 4 ]]> <![CDATA[R 3 ]]> α <![CDATA[AA 7 ]]> <![CDATA[AA 6 ]]> <![CDATA[AA 5 ]]> <![CDATA[AA 4 ]]> <![CDATA[AA 3 ]]> <![CDATA[AA 2 ]]> <![CDATA[AA 1 ]]> <![CDATA[R 5 ]]> 35 K(SFA) 1 r r f h f t h <![CDATA[NH 2 ]]> 36 K(SFA) 1 r r f h f t h <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 37 K(cyclooctane) K(SFA) 1 r r f h f t h <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 38 K(SFA) 0 r r f r h y h <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 39 K(Adamantane) K(SFA) 0 r r f r h y h <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 40 K(cyclooctane) K(SFA) 0 r r f r h y h <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 41 Uk(SFA) 0 h y h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 42 Uk(cyclooctane) Uk(SFA) 0 h y h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 43 uk(SFA) 0 h y h r f r r <![CDATA[(Sar) 6 -K(AF488)-NH 2 ]]> 44 ukl cyclooctane) uk(SFA) 0 h y h r f r r <![CDATA[(Sar) 6 -K(AF488)-NH 2 ]]> 45 Uk(Adamantane) Uk(SFA) 0 h y h r f r r <![CDATA[(PEG1) 2 -K(AF488)-NH 2 ]]> 46 Uk(Adamantane) Uk(SFA) 0 h y h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 47 Uk(Adamantane) Uk(SFA) O h y h r f r r <![CDATA[K(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 48 Uk(Adamantane) Uk(SFA) 0 h y h r f r r <![CDATA[(PEG1) 2 -K(DOTA)-NH 2 ]]> 49 Uk(Adamantane) Uk(SFA) 0 h y h r f r r <![CDATA[(Sar) 6 -K(AF488)-NH 2 ]]> 50 Uk(Adamantane) Uk(SFA) 0 h y h r f r r <![CDATA[K(AF488)-NH 2 ]]> 51 Uk(Adamantane) UAha(AZA) 0 h y h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 52 G UAha(AZA) 0 h y h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 53 Uk(Adamantane) Uk(thiophene) 0 h y h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 54 G Uk(thiophene) 0 h y h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 55 K(Adamantane) K(SFA) 0 h <![CDATA[u(Phe(F 5 ))]]> h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 56 K(Adamantane) K(SFA) 0 h <![CDATA[U(Phe(4-NH 2 ))]]> h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 57 K(Adamantane) K(SFA) 0 h U(1-Nal) h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 58 K(Adamantane) K(SFA) 0 h U(BiP(4,4-)) h r f r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 59 Uk(Adamantane) Uk(SFA) 0 h y h r <![CDATA[U(Phe(F 5 ))]]> r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 60 Uk(Adamantane) Uk(SFA) 0 h y h r <![CDATA[U(Phe(4-NH 2 ))]]> r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 61 Uk(Adamantane) Uk(SFA) 0 h y h r U(1-Nal) r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 62 Uk(Adamantane) Uk(SFA) 0 h y h r U(BiP(4,4-)) r r <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]>

[0172] [Cyclic peptide structure of Chemical Formula 7]

[0173]

[0174] serial number <![CDATA[R 7 ]]> <![CDATA[AA 7 ]]> <![CDATA[AA 6 ]]> <![CDATA[AA 5 ]]> <![CDATA[AA 4 ]]> <![CDATA[AA 3 ]]> <![CDATA[AA 2 ]]> <![CDATA[AA 1 ]]> <![CDATA[R 6 ]]> <![CDATA[R 8 ]]> 63 K(SFA) r r f r h y h KlSFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 64 Uk(SFA) h y h r f r r Uk(SFA) <![CDATA[(PEG1) 2 -K(biotin)-NH 2 ]]> 65 Uk(SFA) h y h r f r r Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]>

[0175] [Cyclic peptide structure of chemical formula 8]

[0176]

[0177] serial number <![CDATA[R 10 ]]> <![CDATA[AA 5 ]]> <![CDATA[AA 4 ]]> <![CDATA[AA 3 ]]> <![CDATA[AA 2 ]]> <![CDATA[AA 1 ]]> <![CDATA[R 9 ]]> <![CDATA[R 11 ]]> 66 K(SFA) r r f r y K(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 67 K(SFA) r r U(homo-f) r y K(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 68 K(SFA) r r U(1-Nal) r <![CDATA[uk(PhF 5 )]]> K(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 69 K(SFA) r r μ(1-Nal) e <![CDATA[Uk(PhF 5 )]]> K(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 70 K(SFA) e r U(1-Nal) e <![CDATA[Uk(PhF 5 )]]> K(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 71 Uk(SFA) y r f r r Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 72 Uk(SFA) y r f r r Uk(SFA) <![CDATA[Uk(6OH)-Uk(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 73 UAha(AZA) y r f r r UAha(AZA) <![CDATA[K(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 74 Uk(SFA) y R f r r Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 75 UOrn(SFA) y R f r r UOrn(SFA <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 76 UAha(SFA) y R f r r UAha(SFA <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 77 UAza(SFA) y R f r r UAza(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 <!-- 18 --> ]]> 78 Uk(SFA) y R f r r UAha(SFA <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 79 UAha(SFA) y R f r r Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 80 Uk(SFA) y R f r r Uk(SFA) se-k(DoTA)-ee-Pra(AF488)-NH2 81 UAha(SFA) y R f r r UAha(SFA) se-k(DOTA)-ee-Pra(AF488)-NH2 82 Uk(SFA) y R f r r UAha(SFA se-k(DOTA)-ee-pra(AF488)-NH2 83 Uk(SFA) y R f r r Uk(SFA) <![CDATA[sdrds-Uk(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 84 Uk(SFA) y R f r r Uk(SFA) <![CDATA[sdrd-Uk(6OH)-Uk(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 85 Uk(SFA) y R f r r Uk(SFA) <![CDATA[sd-Uk(6OH)-d-NH 2 ]]> 86 Uk(SFA) y R f r r Uk(SFA) sd-Uk(6OH)-d-valine-citrullinated acid-PABC-MMAE 87 Uk(SFA) y e f r r Uk(SFA) <![CDATA[UKfDOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 88 Uk(SFA) y r f e r Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 89 Uk(SFA) y r f r e Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 90 Uk(SFA) <![CDATA[Uk(PhF 5 )]]> r U(1-Nal) r r Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 91 Uk(SFA) y r U(1-Nal) r r Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 92 Uk(SFA) <![CDATA[uk(PhF 5 )]]> r f r r Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 93 Uk(SFA) <![CDATA[U(Phe(4-NH 2 ))]]> r U(1-Nal) r r Uk(SFA) <![CDATA[UK(DOTA)-(PEG1) 2 -K(biotin)-NH 2 ]]> 94 Uk(SFA) y R f r r Uk(SFA) <![CDATA[VPTLQ-Uk(AF488)-NH 2 ]]> 95 Uk(SFA) y R f r r Uk(SFA) <![CDATA[sd-Uk(DOTA)-dds-Uk(IB)-Uk(6OH)-NH 2 ]]>

[0178] In the above chemical formulae 3 to 8, AA1 to AA7 represent amino acid residues, lowercase letters represent D-amino acids, uppercase letters represent L-amino acids, and U refers to modified or unnatural D-amino acids, having the following structures defined in the present specification.

[0179]

[0180]

[0181]

[0182] Example 4. Evaluation of the stability of peptide structures

[0183] The stability test of the peptide structure obtained in Example 3 was conducted in serum and plasma. The peptide structure solution was diluted to a final concentration of 50 μM in 1 mL of 100% pure human serum (Cat#S1, Merck) or plasma (Cat#70039.1, STEMCELL Technologies) [or 100% pure mouse serum (Sigma, Cat#S7273) or plasma (Rockland, Cat#D508-06-0050)], aliquoted in 100 μL portions, and then stored at 37°C and analyzed once a day for 7 days using LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent) equipped with an Agilent poroshell 120ec-c18 column (4.6×50 mm, 2.7 μm). Before injecting the sample into the LC, 100 μL of ACN was added to the serum or plasma solution containing the peptide, which was then spun in a centrifuge, and after the precipitate was settled, only 10 μL of the upper layer was used for analysis.

[0184] Figure 5a and Figure 5b ,and Figure 6a and Figure 6b The results of the stability test of peptide structures 35 and 48 in serum and plasma are shown in Figure 1 and Figure 2, respectively. It was confirmed that peptide structure 35 containing D-amino acids was not completely degraded even after 6 days and remained intact in serum and plasma. However, when all the amino acid residues of peptide structure 35 were changed to L-form, it was completely degraded in serum within 1 day and in plasma within 3 days. Figure 5a and Figure 5b ).

[0185] Similarly, the following results were obtained: the peptide structure No. 48 containing D-amino acids was not completely degraded and remained intact in human or mouse serum and plasma after 7 days. However, when all the amino acid residues of No. 48 were changed to L-form, it was completely degraded in serum within 1 day and was mostly degraded in plasma within 7 days ( Figure 6a and Figure 6b ).

[0186] Example 5. Confirmation of biological properties of peptide structures

[0187] 5-1. Analysis of binding affinity and binding kinetics to hCAIX ECD

[0188] The binding affinity and binding kinetics of the peptide structures to hCAIX ECD were analyzed. In order to confirm the specific binding selectivity to hCAIX ECD, the binding kinetics of the peptide structures were also analyzed for the extracellular domain of human carbonic anhydrase XII (hCAXII), another isoform of carbonic anhydrase.

[0189] For binding affinity, we used the Bio-Layer Interferometry (BLI) technique. System (Cat#45-5000, Forte Bio, the United States) and the advanced kinetics module of BLItz Pro ver1.3 software. And in all determinations, streptavidin biosensor (Cat#18-5019, Sartorius, France) was used. In addition, for non-specific binding and background modification, protein concentration 0nM was used as reference. First, 10X Octet kinetics buffer (10X Octet kinetics buffer) (Cat#18-1105, Sartorius) was prepared as 1X using DPBS (Biowest, Cat#L0615) and used as analytical buffer solution. Then, the analytical buffer solution was seeded into a 96-well, black, flat-bottom polypylene plate (Cat#655209, Greiner Bio-One, Austria) at 200 μl / well, and then hydration was performed for 10 minutes while the streptavidin biosensor was immersed. After the biosensor was installed in the device, in order to attach the peptide, it was loaded at 2200 rpm for 120 seconds at a concentration of 1 μM. After that, the protein of each concentration was subjected to association and dissociation at 2200 rpm, and the kinetics data was calculated using the global fitting function by the ratio of kd to ka. D In addition, the kinetic parameters were obtained by baseline correction and fitting a 1:1 binding model.

[0190] The results are shown in Figures 7a to 7dThe peptide constructs showed significantly higher selectivity for hCAIX ECD compared to hCAXII ECD.

[0191] 5-2. Analysis of binding specificity to target cells

[0192] To confirm whether the peptide constructs specifically bind to target cells expressing CAIX, in vitro cell binding properties were analyzed. Human renal cancer cell line SK-RC-52 expressing CAIX was purchased from Memorial Sloan Kettering Cancer Center (MSK, USA), and human lung cancer cell line A549 expressing CAXII was purchased from the Korean Cell Line Bank (Cat#10185, KCLB, Korea).

[0193] Fluorescence activated cell sorting (FACS) analysis

[0194] The peptide structure used in the analysis was dissolved in DMSO to prepare a 1 mM solution, and then diluted to the desired concentration (10000 nM, 100 nM, 30 nM, 10 nM, 5 nM) using a DMEM mixed solution (FACS buffer solution) containing 1% FBS.

[0195] 2 to 3 days before FACS analysis, 5×10 5 For SK-RC-52 cell line, 1-1.5×10 6 On the day of the experiment, remove the culture medium from the T75 flasks cultured with A549 cells or SK-RC-52 cells at 80% confluence and wash once with 15 mL of PBS. 2 Constant temperature processor (37°C, 5% CO 2 After treatment with 9 mL of culture medium, trypsin-EDTA was neutralized. The neutralized cell mixture was placed in a 15 mL test tube and spun at 1000 RPM for 4 minutes using a VARISPIN 15R centrifuge. The supernatant of the centrifuged cell mixture was removed, and new culture medium was added to a concentration of 5 × 10 cells per 100 μL. 5After diluting in a certain manner, 100 μL was injected into each well of a 96-well round plate (Cat#34096, SPL Life Sciences). After spinning at 1000 RPM for 4 minutes using a VARISPIN 15R centrifuge, the supernatant was removed from each well, and 200 μL of the pre-prepared peptide structure solution was added, and then the mixture was centrifuged in a Galaxy 170S CO 2 The cells were incubated at 37°C and 5% CO 2 , and cultured under humid conditions. After 1 hour, the supernatant was removed and washed with 200 μL of FACS buffer solution after spinning at 1000 RPM for 4 minutes in a VARISPIN 15R centrifuge. After centrifugation (spinning at 1000 RPM for 4 minutes), the washing solution was removed and then treated with 200 μL of streptavidin-Alexa fluor488 (Cat#S11223, Invitrogen, USA) diluted to a concentration of 1 μg / mL in FACS buffer solution and incubated in a Galaxy 170S CO 2 The cells were incubated at 37°C and 5% CO 2 , cultured for 1 hour under humid conditions (streptavidin-Alexa fluor488 treatment was omitted for peptide structures 80, 81, and 82). After centrifugation through VARISPIN 15R (rotated for 4 minutes at 1000RPM), the supernatant was removed and washed with 200μL of FACS buffer solution. After further washing once by the same method, 200μL of FACS buffer solution was added to each well and mixed with the cells, and then transferred to a 5ml round bottom test tube (Cat#352235, Corning, the United States) with a cell strainer, and the fluorescence intensity of each cell was measured using BD AccuriC6 Plus (BD Biosciences, Singapore). The measured data were analyzed by FlowJo 10.7.1 (BD Biosciences, the United States).

[0196] The cell mixture after FACS analysis was centrifuged at 1000RPM for 4 minutes using a VARISPIN 15R centrifuge, and the supernatant was removed. Then, 200μL of Hoechst 33342 diluted to a concentration of 1μg / mL in a FACS buffer solution was treated and nuclear staining was performed at 4°C for 30 minutes. After centrifugation at 1000RPM for 4 minutes using a VARISPIN 15R centrifuge, the supernatant was removed and washed with 200μL of PBS (repeated twice). The cell mixture with 200μL of PBS added was transferred to a confocal dish (Cat#100350, SPL Life Sciences), and fluorescence was observed by Axio Observer 3 (Zeiss, Germany).

[0197] Figures 8a to 8d The results of FACS analysis using the SK-RC-52 cell line are shown in FIG. Figure 8e to Figure 8h The results of FACS analysis using the A549 cell line are shown in . From the results of FACS analysis, it was confirmed that the peptide structure of the present invention binds to target cells expressing CAIX with high affinity and selectivity.

[0198] Immunofluorescence analysis (colocalization)

[0199] For A549 cell line, 5×10 4 For SK-RC-52 cell line, 1×10 5 cells, and in the Galaxy 170S CO 2 Constant temperature processor (37°C, 5% CO 2 Next, each cell line was treated with M75-FITC antibody or peptide construct diluted in 2 mL of FACS buffer to a final concentration of 1:200 (dilution ratio to the stock solution) and 1 μM, respectively, and then incubated in a Galaxy 170S CO 2 The cells were incubated at 37°C and 5% CO 2 , and cultured for 1 hour under humid conditions. Then, after washing twice with 3 mL of FACS buffer solution, the cells were treated with streptavidin-Texas red and Hoechst 33342 diluted in 2 mL of FACS buffer solution to a final concentration of 1:50 (dilution ratio to the original solution) and 0.5 μg / mL, respectively, and incubated in a Galaxy 170S CO2 The cells were incubated at 37°C and 5% CO 2 The cells were incubated for 1 hour under humid conditions. Then, the cells were washed twice with 3 mL of FACS buffer and once with PBS, and fluorescence was observed using Axio Observer 3.

[0200] Fig. 9 ] The results of immunofluorescence analysis of peptide structure No. 22 are shown in . It was confirmed that peptide structure No. 22 specifically binds to the SK-RC-52 cell line expressing CAIX, similar to the M75 antibody.

[0201] Confocal microscopy imaging

[0202] For SK-RC-52 cell line, 1×10 5 Cells were seeded in confocal culture dishes and imaged on Galaxy170SCO 2 Constant temperature processor (37°C, 5% CO 2 Next, the peptide construct diluted in 2 mL of FACS buffer was treated in the cell line to a final concentration of 100 nM and then incubated in a Galaxy 170S CO 2 The cells were incubated at 37°C and 5% CO 2 Then, the cells were washed three times with 3 mL of FACS buffer solution, treated with Hoechst fluorescent dye 33342 diluted in 2 mL of FACS buffer solution to a final concentration of 0.5 μg / mL, and incubated under a Galaxy 170S CO 2 The cells were incubated at 37°C and 5% CO 2 After reacting for 15 min under humid conditions, the cells were washed three times with 3 mL of PBS, and then fluorescence was observed using a Leica SP5 confocal microscope.

[0203] Fig.10 Z-stack confocal microscopic images of peptide structure No. 94 using SK-RC-52 cell line are shown in FIG. It was confirmed that peptide structure No. 94 exhibited good binding to CAIX ECD expressed in cells.

[0204] 5-3. Biodistribution analysis using experimental animal models

[0205] Six-week-old female BALB / c nude mice (orientbio, Korea) were used to culture 2×10 6SK-RC-52 was injected subcutaneously into the upper side of the left forelimb of nude mice. The weight and tumor size were measured on days 13, 20, 27, and 35 after subcutaneous injection. Fig.11a The size of the tumor was measured using a caliper [volume = (width) 2 × (length) / 2] for measurement.

[0206] On day 36 after SK-RC-52 cell line xenografting, 5 μM of the peptide construct was injected via tail vein in 200 μL portions (5% DMSO in saline solution). For the mice imaged 24 hours after the injection, they were sacrificed by injecting an overdose of carbon dioxide, and the laparotomy and removal of tumors and organs (liver, kidney, intestine, lung, spleen, stomach, pancreas, heart) were performed for ex vivo imaging ( Fig.11b ).

[0207] For each group of tumors, liver, kidney, intestine, lung, spleen, stomach, and heart obtained from animal experiments, they were frozen with dry ice after ex vivo imaging and stored at -80°C before the experiment. After measuring the weight of each tumor before the experiment, ice-cold homogenization buffer (PBS with pH 7.4 containing a trace amount of deoxyribonuclease 1 (DNase 1) in 40mM EDTA, 6mg / mL trypsin, and 1.6% Triton X-100) was added to 100μL of buffer solution per 100mg of the organ weight, and homogenized for 5 minutes using FastPrep-245G (MP Biomedical, USA). For the homogenized organs, 100μL of the homogenate was injected into each well of a black 96-well plate, and the fluorescence ( Fig.11c ). It was confirmed that the peptide constructs specifically bind to tumors that overexpress CAIX compared to other organs.

[0208] 5-4. Analysis of the anticancer effect of peptide structure-drug conjugates

[0209] Animal experiments were conducted to confirm whether the drug-peptide conjugate exhibited anticancer effects in vivo. SK-RC-52 cells, which are renal cancer cells that overexpress CAIX, were incubated in PBS at 2 × 10 6The cells were diluted and injected subcutaneously into the right anterior flank of 6-week-old female BLAB / c nude mice (orientbio, Korea). The body weight of the mice transplanted with tumor cells was measured for 2-3 weeks to measure the health changes of the mice, and the size of the tumor (width × width × length / 2) was measured using a digital caliper (Digimatic Caliper) (Cat#500-151-30, Mitutoyo, Japan). When the tumor size reached about 100 mm 3 The mice were divided into the following groups: a control group in which saline containing 1% DMSO and 2% EtOH was injected into the tail vein; a peptide-treated group in which only the peptide structure was injected into the tail vein (i.e., peptide structure No. 85); and a PDC1-treated group in which the peptide structure and the drug (MMAE) were combined with a linker (i.e., peptide structure No. 86 in the form of a drug conjugate) in the tail vein. In the above PDC1, there were three mice in each group, and two mice were used as subjects for the peptide-treated group. The drug concentration was 250 nmole / kg, and 200 μl of saline containing 1% DMSO and 2% EtOH was administered as a carrier. Regarding the frequency of administration, the drug was administered through the tail vein three times a week at intervals of two days for a total of 7 times. The body weight and tumor size were measured every 1 to 3 days.

[0210] The results are shown in Fig.12a and Figure 12b .like Fig.12a and Figure 12b As shown, peptide construct 86 did not cause significant changes in mouse body weight ( Fig.12a ), and showed an anticancer effect that significantly reduced the size of tumors overexpressing CAIX ( Figure 12b ), the above-mentioned peptide structure No. 86 is a drug conjugate form in which the MMAE drug is conjugated to the peptide structure No. 85 via a linker.

[0211] 5-5. Isotopes ( 177 In vivo SPECT / CT imaging experiments of Lu-labeled peptide structures

[0212] The diagnostic imaging and therapeutic functionality of the peptide structure is confirmed by isotope labeling. Peptide structure No. 95 is dissolved in DMSO to prepare a 10mM stock solution, and then diluted with ammonium acetate buffer at pH 5.5 to prepare a 1mM peptide structure solution. 7nmole of the 1mM solution is taken and mixed with 1mCi of isotope ( 177 LuCl 3, Eckert&Ziegler Radiopharma GmbH) was mixed and the pH was adjusted to 5.5 by adding an ammonium acetate buffer solution of pH 7.0. Isotope labeling was performed at 90°C for 1 hour, and after the labeling was completed, it was cooled to room temperature and then purified using a Sep-Pak column (Cat#186005125, Waters). The sample was loaded into an activated Sep-Pak column (5 mL ethanol, 5 mL distilled water) and washed with 5 mL distilled water, and then eluted with 1.5 mL ethanol to obtain a solution, which was purged with nitrogen and concentrated. The concentrated ethanol solution was diluted with saline for use.

[0213] SK-RC-52 cells (Sloan-Kettering Cancer Institute (MSK), New York, USA), a human-derived renal cell carcinoma (RCC) cell line, were cultured at a rate of 2 × 10 cells / mouse. 6 The cells / 100 μL were administered subcutaneously in the right forelimb axilla of 6-week-old male BALB / c nude mice (orientbio, Seoul, Korea). The size of the tumors formed when the transplanted cancer cells SK-RC-52 were implanted was an average of 100 mm 3 (80~100mm 3 ), the groups were separated based on the size of the tumor. Body weight and tumor size were measured twice a week, and the tumor size was calculated by substituting the following formula into the width and length of the tumor measured with a Vernier Calipers (Mitutoyo, Kawasaki, Japan, tumor volume).

[0214] Tumor volume (mm 3 )=Width 2 × Length / 2

[0215] When the tumors generated by transplanting SK-RC-52, a renal cell carcinoma cell line, reached an average size of 100 mm 3 (80~100mm 3 ) isotope 100-150 μL equivalent to a radiation dose of 600-700 μCi 177Lu-labeled peptide structure No. 95 was administered once by tail vein injection at 100-150 μL, and SPECT / CT (Siemens Inveon, software: Inveon acquisition work platform) was measured at 6 hours, the second day, and the eighth day. For imaging, the mice were anesthetized by breathing with 0.2% isoflurane in oxygen for about 3-5 minutes, and then placed behind the device to continue to inject anesthetic at 1.5 L / min, and measured by SPECT for 50 minutes and CT for 7 minutes, and images were obtained by Inveon Research Workplace 4.2.

[0216] Fig.13 The SPECT / CT images obtained from the above are shown in FIG. 177 Lu-labeled peptide structure No. 95 also still exists in a form bound to the cancer cell SK-RC-52.

[0217] After that, for the mice that had completed the SPECT / CT measurement, the mice were given carbon dioxide gas (CO 2 ) to euthanize the animals and perform laparotomy to remove the tumor and organs (liver, kidney, spleen), and measure their weights. Then, the radiation dose of each organ was measured using a portable radioactivity meter (Inspector survey meter, INSPECTER (078-510)). 177 The remaining amount of Lu-labeled peptide structure No. 95 was converted into radiation intensity per unit weight, and then the relative radiation intensity of other organs was calculated based on the tumor value and is shown in Table 1 below.

[0218]

Table 1

[0219] Organ / Tissue 8 days after injection Tumor 1 liver 0.08 kidney 0.33 spleen 0.07

[0220] 5-6. Isotopes ( 177 Analysis of the anticancer effect of Lu-labeled CAIX targeting peptide (peptide structure 95)

[0221] Peptide structure No. 95 was dissolved in DMSO to prepare a 10 mM stock solution, and then diluted with pH 5.5 ammonium acetate buffer to prepare a 1 mM peptide structure solution. 40 nmole of the 1 mM solution was taken and mixed with 14 mCi isotope ( 177 LuCl 3, Eckert & Ziegler Radiopharma GmbH) and then adjusted to pH 5.5 by adding ammonium acetate buffer solution of pH 7.0. Isotope labeling was performed at 90°C for 1 hour, and after completion of labeling, the product was cooled to room temperature and then purified by 1200 Infinity LC system (Agilent) under the following conditions: (1) the stationary phase was Agilent Poroshell 120 EC-C18 column (4.6×50 mm, 2.7 μm); (2) the mobile phase solvent was a mixture of triple distilled water and ACN with 0.1% TFA added; (3) the flow rate was 1 mL / min; (4) the detection wavelengths were 254 nm and 214 nm. Fig.14 It is shown in 177 Chromatogram of Lu-labeled targeting peptide 95 construct.

[0222] The purified 177 The Lu-labeled targeting peptide No. 95 was loaded onto an activated Sep-Pak column (5 mL ethanol, 5 mL distilled water), washed with 5 mL distilled water to remove the organic solvent, and then washed with 500 μL of CAN:H 2 The obtained solution was eluted with 700 μL of ethanol and purged with nitrogen gas to concentrate the solution. The concentrated ethanol solution was diluted with saline (EtOH 20%, DMSO 1%) before use.

[0223] SK-RC-52 cells, which are renal cancer cells overexpressing CAIX, were incubated in PBS at 2 × 10 6 The cells were diluted and injected subcutaneously into the right anterior flank of 6-week-old female BLAB / c nude mice (orientbio, Korea). The body weight of the mice transplanted with tumor cells was measured for 2 to 3 weeks to measure the health changes of the mice, and the size of the tumor (width) was measured using a digital caliper (Digimatic Caliper) (Cat#500-151-30, Mitutoyo, Japan). 2 × length / 2). When the tumor size reaches about 100 mm 3 The animals were divided into the following groups: a control group of 6 mice injected with saline as a vehicle; and a control group of 6 mice injected with approximately 500 μCi of 177 Lu-labeled targeting peptide 95 constructs were injected into the tail vein of 6 experimental groups. Body weight and tumor size were measured three times a week.

[0224] The results are shown in Figures 15a to 15c .like Figures 15a to 15c As shown, 177The Lu-labeled targeting peptide construct 95 did not cause significant changes in the body weight of mice ( Fig.15b ), while also showing an anticancer effect that significantly reduced the size of tumors overexpressing CAIX ( Fig.15a and Fig.15c ).

[0225] The description of the present invention as described above is exemplary, and a person skilled in the art in the art to which the present invention belongs can understand that the protection scope of the present invention is not limited to the disclosed embodiments and drawings, and various changes can be made in other specific forms without departing from the technical idea of ​​the present invention.

[0226] Sequence Listing

[0227]

Table 2

[0228]

Claims

1. A carbonic anhydrase IX-specific peptide structure, comprising: A peptide ligand, which is composed of any one of the amino acid sequences selected from SEQ ID NO. 1 to 44, at least one of the constituent amino acids is composed of a D-amino acid, and the side chain ε-amino group of the lysine residue in the constituent amino acids can be replaced by a chemical functional group; and An amino acid residue containing a sulfonamide functional group connected to the peptide ligand directly or through a spacer, the carbonic anhydrase IX-specific peptide structure is represented by a ring structure of the following chemical formula 6, 7 or 8, 【Chemical formula 6】 【Chemical formula 7】 【Chemical formula 8】 In the chemical formulas 6 to 8, R5, R8 and R 11 They are respectively the general formula -(S1) o -(F5) p -(S2) q -(F6) r -NH2 group; wherein, S1 and S2 are independently spacers, F5 and F6 are each independently an amino acid residue containing a functional group other than sulfonamide, The functional group other than sulfonamide is a chelate, a cycloalkane having a carbon number of 5 to 15, biotin, glucoheptonic acid, 4-(p-iodophenyl)butyric acid, a fluorescent dye or a cytotoxic agent, o, p, q and r each independently represent an integer from 0 to 6, Lowercase letters represent D-amino acids, uppercase letters represent L-amino acids, The substituents represented by U are respectively defined as follows, 2. The carbonic anhydrase IX-specific peptide structure according to claim 1, characterized in that The functional groups other than sulfonamide are introduced via the side chain ε-amino group of the lysine residue.

3. The carbonic anhydrase IX-specific peptide structure according to claim 1, characterized in that The chelate is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, ethylenediaminetetraacetic acid 2,2',2",2"'-(ethane-1,2-diyldinitro)tetraacetic acid, 1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N",N"',N"",N""'-hexaacetic acid, 2-[4-nitrobenzyl]-1,4,7,10,13 -pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid), (1R,4R,7R,10R)-α,α',α",α"'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) tetrasodium salt, 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid and triethylenetetramine.

4. The carbonic anhydrase IX-specific peptide structure according to claim 1, characterized in that The cycloalkane having 5 to 15 carbon atoms is selected from at least one of cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, adamantane, norbornane, isobornane and tricyclodecane.

5. The carbonic anhydrase IX-specific peptide structure according to claim 1, characterized in that: The spacer is selected from at least one of a polyethylene glycol linker, glycine, sarcosine, and a peptide linker consisting of 1 to 5 D-amino acids or L-amino acids.

6. The carbonic anhydrase IX-specific peptide structure according to claim 1, characterized in that Having the following chemical formula 6, 7 or 8, 【Chemical formula 6】 【Chemical formula 7】 【Chemical formula 8】 In the chemical formula, lowercase letters represent D-amino acids, uppercase letters represent L-amino acids, and the substituents represented by U are defined as follows:

7. A conjugate comprising the peptide structure according to claim 1 or claim 6 bound to a fluorescent dye, a cytotoxic agent or a radioactive isotope directly or through a linker.

8. The conjugate according to claim 7, characterized in that The linker is selected from one or more of 6-maleimidocaproyl, maleimidopropionyl, valine-citrulline, alanine-phenylalanine, p-aminobenzyloxycarbonyl, N-succinimidyl 4-(2-pyridylthio)pentanoate, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 4-(2-pyridyldithio)butyric acid-N-hydroxysuccinimide ester and N-succinimidyl (4-iodo-acetyl)aminobenzoate.

9. The conjugate according to claim 7, characterized in that The fluorescent dye is selected from one or more of near-infrared fluorescent dyes, fluorescein, rhodamine, Alexa Fluor, 4,4-difluoro-4-boron-3a,4a-diaza-s-indene, Texas Red, dansyl, lissamine, cynerine and phycoerythrin.

10. The conjugate according to claim 7, characterized in that The cytotoxic agent is selected from at least one of a toxin, a chemotherapeutic agent, a drug moiety and a nuclease.

11. The conjugate according to claim 7, characterized in that The radioactive isotope is selected from fluorine-18, carbon-11, carbon-14, technetium-99m, copper-64, copper-67, dysprosium-168, bismuth-213, samarium-153, strontium-89, strontium-90, erbium-169, phosphorus-32, palladium-103, rhenium-186, rhenium-188, oxygen-15, selenium-75, sodium-24, strontium-85, lutetium-177, yttrium-90 , iodine-123, iodine-125, iodine-131, iridium-192, iridium-196, ytterbium-166, indium-111, xenon-133, nitrogen-13, calcium-47, cobalt-57, cobalt-60, chromium-51, krypton-81, potassium-42, holmium-166, gallium-67, gallium-68, actinium-225, zirconium-89, lead-212 and astatine-211.

12. A pharmaceutical composition for diagnosis or treatment of cancer expressing carbonic anhydrase IX, comprising the peptide structure according to claim 1 or claim 6.

13. A pharmaceutical composition for diagnosis or treatment of cancer expressing carbonic anhydrase IX, comprising the conjugate according to claim 7.

14. The pharmaceutical composition according to claim 12, 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, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, prostate cancer, bile duct cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic cancer, melanoma, thyroid cancer, astrocytoma or glioblastoma.

15. The pharmaceutical composition according to claim 13, 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, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, prostate cancer, bile duct cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic cancer, melanoma, thyroid cancer, astrocytoma or glioblastoma.

Images