Peptide construct targeting fibroblast activation protein-α (FAP-α) and use thereof

Abstract

The present invention relates to a peptide construct that specifically binds to fibroblast activation protein-α (FAP-α) and use thereof. The FAP-α peptide construct of the present invention comprises a peptide ligand that includes one or more D-amino acids and specifically binds to FAP-α, thereby exhibiting excellent stability in vivo and being effective in targeting FAP-α, and has a structure of chemical formula 1 including a substituted or unsubstituted pyrrolidinyl group linked to the peptide ligand, thereby exhibiting high binding affinity for FAP-α. Therefore, the FAP-α peptide construct is useful for diagnosing, preventing, suppressing or treating FAP-α-overexpressing diseases.

Description

Peptide constructs targeting fibroblast active protein alpha (FAP-α) and their uses

[0001] The present invention relates to peptide structures that specifically bind to Fibroblast Activation Protein-α (FAP-α) and their uses. Specifically, the present invention relates to a FAP-α binding peptide ligand that specifically binds to FAP-α and includes a D-amino acid to enhance stability, a high-affinity FAP-α binding peptide structure comprising a substituted or unsubstituted pyrrolidinyl group connected to the peptide ligand, and their uses for the diagnosis, prevention, inhibition, or treatment of FAP-α overexpression diseases.

[0002] FAP-α is a type II membrane peptidase belonging to the peptidyl peptidase (DPP) family that is selectively expressed in the stroma of a certain range of epithelial malignancies regardless of location and histological type. Accordingly, the concept of FAP-targeting has been developed for the diagnosis, prevention, and treatment of epithelial cancers or certain other diseases. Subsequently, the monoclonal antibody (mAb) F19, which specifically binds to FAP-α, was developed, revealing that FAP-α is a cell surface molecule with a molecular weight of 95,000 [Rettig et al. (1988) Proc. Natl. Acad. Sci. USA 85, 3110-3114; Rettig et al. (1993) Cancer Res 53, 3327-3335].

[0003] According to previous studies, FAP-α has been shown to be selectively highly expressed on the surfaces of reactive stromal fibroblasts of numerous histological types, including human epithelial carcinomas, granulation tissues, and malignant cells of certain bone and soft tissue sarcomas. Although it is generally not expressed in normal human tissues, most common types of epithelial carcinomas, including breast cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, and gastric cancer, contain FAP-α-responsive stromal fibroblasts (Scanlanet et al. (1994) Proc. Natl. Acad. Sci. USA 91, 5657-5661). Among these, a subgroup of fibroblasts called cancer-associated fibroblasts (CAFs) is known to be involved in tumor metastasis and growth, and additionally, to cause resistance to chemotherapy and immunosuppression. Therefore, FAP-α is considered an important target for tumor imaging and treatment.

[0004] Recently, in the field of tumor imaging, much research has been conducted on fibroblast activation protein inhibitors (FAPIs) in which quinoline derivatives are labeled with radionuclides. For example, PET / CT imaging agents such as FAPI-02 have enabled tumor-specific imaging (Lindner et al., Journal of Nuclear Medicine., 2018, 59 (9) 1415-1422). However, currently reported FAPIs are rapidly eliminated from the blood and simultaneously rapidly eluted from the tumor site. Due to their rapid elution and metabolism, FAPIs have a short residence time at the tumor site and a low effective dose, presenting a problem where high-dose administration or increased administration frequency is required to meet tumor treatment requirements. Consequently, the likelihood of side effects also increases significantly.

[0005] Therefore, there is a need to develop novel, high-affinity, stable, and novel FAP-α-specific binders and inhibitors suitable for the pharmaceutical use of treating FAP-α overexpression diseases, including treatment, prevention, diagnosis, prognosis prediction, and imaging.

[0006]

[0007] One objective of the present invention is to provide high-affinity, stable FAP-α specific binders and inhibitors suitable for use in the treatment, prevention, diagnosis, or prognosis prediction of FAP-α overexpression diseases.

[0008] One object of the present invention is to provide a high-affinity FAP-α specific peptide structure comprising an FAP-α specific binding peptide ligand and a substituted or unsubstituted pyrrolidinyl group connected to the peptide ligand directly or through a spacer.

[0009] An additional objective of the present invention is to provide a conjugate comprising the peptide structure.

[0010] An additional object of the present invention is to provide a composition for the diagnosis, prevention, or treatment of fibroblast-activated protein-alpha (FAP-α) overexpression diseases comprising the peptide structure or conjugate.

[0011] An additional object of the present invention is to provide a method for diagnosing a fibroblast-activated protein-alpha (FAP-α) overexpression disease using the peptide structure, conjugate, or a therapeutic composition containing the same.

[0012] An additional object of the present invention is to provide a method for treating fibroblast-activated protein-alpha (FAP-α) overexpression diseases using the peptide structure, conjugate, or a therapeutic composition containing the same.

[0013] An additional objective of the present invention is to provide a method for predicting the prognosis after treatment of a fibroblast-activated protein-alpha (FAP-α) overexpression disease using the peptide structure, conjugate, or a therapeutic composition containing the same.

[0014]

[0015] As a result of diligent research to achieve the above objective, the inventors of the present invention have developed novel fibroblast-activating protein-alpha (FAP-α)-specific binders and inhibitors that include at least one D-amino acid and are stable and capable of specifically binding to fibroblast-activating protein-alpha (FAP-α) with high affinity.

[0016] In one aspect of the present invention, the present invention provides a peptide structure comprising an amino acid sequence of any one of SEQ ID NOs 1 to 88, wherein at least one of the constituent amino acids is a D-amino acid, and the β-phenyl group of the side chain of the phenylalanine (Phe) residue among the constituent amino acids can be substituted with a halogen; and a substituted or unsubstituted pyrrolidinyl group connected to the peptide ligand directly or through a spacer, and having a structure of the following chemical formula 1.

[0017] [Chemical Formula 1]

[0018]

[0019] In the above formula,

[0020] P is the above-mentioned peptide ligand, and

[0021] F1 is halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 1-10 Alkanoylamin, C(O)-C 1-6 Alkyl, C(O)-C 1-6 Haloalkyl, C(O)OC 1-6 Alkyl, C(O)OC 1-6It is any one selected from the group consisting of haloalkyl, NH2, OH, CN, COOH, and NO2, and

[0022] m is an integer from 0 to 8, and

[0023] F2 is glycine (Gly), C(O)-C 1-6 Alkylene, C(O)-C 1-6 Any one selected from the group consisting of alkylene-(O)C and pyrrolidinyl group-containing amino acid residues, and

[0024] n is an integer from 0 to 3, and

[0025] F3 is -(S1) o -(F4) p -(S2) q -(F5) r -(S3) s -(F6) t -(S4) is the basis, and here

[0026] S1, S2, and S3 are each independently spacers, and

[0027] F4, F5, and F6 are each independently substituted or unsubstituted amino acid residues, and

[0028] S4 is -NH2 or -OH, and

[0029] o, p, q, r, s, and t each independently represent integers from 0 to 6.

[0030] In one embodiment, the pyrrolidinyl group in the pyrrolidinyl group-containing amino acid residue may be substituted with one or more X1s, wherein X1 is a halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 1-10 Alkanoylamin, C(O)-C 1-6 Alkyl, C(O)-C 1-6 Haloalkyl, C(O)OC 1-6 Alkyl, C(O)OC 1-6It may be any one selected from the group consisting of haloalkyl, NH2, OH, CN, COOH, and NO2.

[0031] In one embodiment, the pyrrolidinyl group-containing amino acid residue may have the following structure.

[0032] , or

[0033]

[0034] In one aspect of the present invention, one or more of F4, F5 and F6 are substituted amino acid residues, wherein the substituent of the amino acid residue may be introduced through any one selected from the group consisting of 1) a side chain ε-amino group of a lysine residue; 2) a side chain β-amino group of a 2,3-diaminopropionic acid (DAP) residue; 3) a side chain β-ethynyl group of a propargylglycine residue; 4) a side chain β-phenyl group of a phenylalanine (Phe) residue; 5) a side chain amino group of an arginine residue; and 6) a side chain γ-amino group of an azidohomoalanine residue.

[0035] In one embodiment, one or more of F4, F5, and F6 are substituted amino acid residues, wherein the substituents of the amino acid residues are hydroxyl groups (OH), chelators, indole, C 1-6 Alkyl, 4,4-(bis(4-hydroxyphenyl)valeric acid (BHPV), PEG24-biotin, hexanoylamino, 4-(p-iodophenyl)butyric acid (IB), pentafluorophenyl (PhF5), halogen, zwitteric ion (SZW), 1,2:3,4-di-O-isopropylidene-aD-galacturonide (1,2:3,4-Di-O-isopropylidene-aD-galacturonide, Gal), N 6 -Hexanoylglycyl-L-prolyl-L-prolyl-D-lysine(N 6It may include one or more selected from the group consisting of -hexanoylglycyl-L-prolyl-L-prolyl-D-lysine), fluorescent dyes, and cytotoxic agents.

[0036] In one aspect, the present invention provides a conjugate comprising the peptide structure directly or through a linker to one or more selected from the group consisting of fluorescent dyes, cytotoxic agents, and radioactive isotopes.

[0037] In one embodiment, the peptide structure may be combined with a fluorescent dye and a cytotoxic agent directly or through a linker.

[0038] In one embodiment, the peptide structure may be coupled to a fluorescent dye and a radioisotope directly or through a linker.

[0039] In one aspect, the present invention provides a pharmaceutical composition for the diagnosis, prevention, or treatment of FAP-α overexpression diseases comprising the peptide structure.

[0040] In one aspect, the present invention provides a pharmaceutical composition for the diagnosis, prevention, or treatment of FAP-α overexpression diseases comprising the conjugate.

[0041] In one embodiment, the FAP-α overexpression disease may be any one selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid.

[0042] In one aspect, the present invention provides a method for diagnosing cancer comprising administering the FAP-α specific peptide structure or the conjugate to an individual.

[0043] In one aspect, the present invention provides a method for treating cancer comprising administering the FAP-α specific peptide structure or the conjugate to an individual.

[0044] In one aspect, the present invention provides a method for predicting the prognosis after cancer treatment, comprising administering the FAP-α specific peptide structure or the conjugate to an individual.

[0045] The present invention provides novel high-affinity FAP-α binders and inhibitors that specifically bind to FAP-α while being stable.

[0046] The FAP-α peptide structure of the present invention includes a peptide ligand that specifically binds to FAP-α containing one or more D-amino acids, thereby exhibiting excellent stability in vivo and being useful for FAP-α targeting. By having the structure of Formula 1, which includes a substituted or unsubstituted pyrrolidinyl group connected to the peptide ligand, it can provide a FAP-α binder that is particularly useful for imaging or diagnosing FAP-α overexpression diseases due to its high binding affinity for FAP-α.

[0047] In addition, the FAP-α specific peptide structure of the present invention can be used as an effective agent for the diagnosis, prevention, or treatment of FAP-α overexpression diseases by conjugating it with a fluorescent dye, a cytotoxic agent, or a radioisotope.

[0048]

[0049] Figure 1 is a schematic diagram showing a peptide screening process for hFAP-α protein according to one embodiment of the present invention.

[0050] FIG. 2 illustrates the structure of the library used for hFAP-α screening and the binding reaction for introducing an HO-Su-GP-CN functional group into a peptide library according to one embodiment of the present invention.

[0051] Figure 3 shows the results of screening positive beads that bind to hFAP-α using COPAS according to one embodiment of the present invention.

[0052] Figure 4 shows the mass spectrometry results of an hFAP-α extracellular domain (ECD) binding peptide according to one embodiment of the present invention.

[0053] Figure 5 shows the results of a stability test in serum and plasma for a peptide structure 83 according to one embodiment of the present invention.

[0054] Figure 6 is a graph showing the results of analyzing the binding affinity and binding kinetics characteristics of the hFAP-α extracellular domain (ECD), PREP extracellular domain (ECD), and DPP4 extracellular domain (ECD) of peptide structure 84 according to one embodiment of the present invention using the BLItz® system.

[0055] Figure 7 is a graph showing the binding affinity to the hFAP-α extracellular domain (ECD) of peptide structure 82 according to one embodiment of the present invention as a result of fluorescence polarization analysis.

[0056] FIG. 8a is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 1 to 12 according to one embodiment of the present invention.

[0057] FIG. 8b is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 13 to 24 according to one embodiment of the present invention.

[0058] FIG. 8c is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 25 to 36 according to one embodiment of the present invention.

[0059] FIG. 8d is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 37 to 48 according to one embodiment of the present invention.

[0060] FIG. 8e is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 49 to 60 according to one embodiment of the present invention.

[0061] FIG. 8f is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 61 to 72 according to one embodiment of the present invention.

[0062] FIG. 8g is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 73 to 80, 82, and 84 to 86 according to one embodiment of the present invention.

[0063] FIG. 8h is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 87 to 98 according to one embodiment of the present invention.

[0064] FIG. 8i is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 99 to 109 and 111 according to one embodiment of the present invention.

[0065] FIG. 8j is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 112 to 123 according to one embodiment of the present invention.

[0066] FIG. 8k is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 124 to 135 according to one embodiment of the present invention.

[0067] FIG. 81 is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 136 to 147 according to one embodiment of the present invention.

[0068] FIG. 8m is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 148 to 159 according to one embodiment of the present invention.

[0069] FIG. 8n is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 163 to 173 and 179 according to one embodiment of the present invention.

[0070] FIG. 80 is a graph showing the activity of the hFAP-α extracellular domain (ECD) of peptide structures 181 to 182 according to one embodiment of the present invention.

[0071] FIG. 8p is a graph showing the activity of the PREP extracellular domain (ECD) of peptide structures 8, 87, 101 to 104, 119 to 122, 128 and 129 according to one embodiment of the present invention.

[0072] FIG. 8q is a graph showing the activity of the PREP extracellular domain (ECD) of peptide structures 130 to 133, 138 to 142, 147, 148 and 152 according to one embodiment of the present invention.

[0073] FIG. 8r is a graph showing the activity of the PREP extracellular domain (ECD) of 182 in peptide structures 173, 179, and 181 according to one embodiment of the present invention.

[0074] FIG. 8s is a graph showing the activity of the DPP4 extracellular domain (ECD) of peptide structures 8, 87, 99 to 102, 117 to 120, and 139 to 140 according to one embodiment of the present invention.

[0075] FIG. 8t is a graph showing the activity of the DPP4 extracellular domain (ECD) of peptide structures 145, 146, 150, 173, 179, 181 and 182 according to one embodiment of the present invention.

[0076] FIG. 9a is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-huFAP cell lines of peptide structures 1 to 20 according to one embodiment of the present invention.

[0077] FIG. 9b is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-huFAP cell lines of peptide structures 21 to 40 according to one embodiment of the present invention.

[0078] FIG. 9c is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-huFAP cell lines of peptide structures 41 to 60 according to one embodiment of the present invention.

[0079] FIG. 9d is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-huFAP cell lines of peptide structures 61 to 80 according to one embodiment of the present invention.

[0080] FIG. 9e is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-huFAP cell lines of peptide structures 84 to 88, 97, 98, 103 to 109 and 111 to 116 according to one embodiment of the present invention.

[0081] FIG. 9f is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-huFAP cell lines of peptide structures 121 to 125 and 132 to 135 according to one embodiment of the present invention.

[0082] FIG. 9g is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-NCV cell lines of peptide structures 1 to 20 according to one embodiment of the present invention.

[0083] FIG. 9h is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-NCV cell lines of peptide structures 21 to 40 according to one embodiment of the present invention.

[0084] FIG. 9i is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-NCV cell lines of peptide structures 41 to 60 according to one embodiment of the present invention.

[0085] FIG. 9j is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-NCV cell lines of peptide structures 61 to 80 according to one embodiment of the present invention.

[0086] FIG. 9k is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-NCV cell lines of peptide structures 84 to 88, 97, 98, 103 to 109 and 111 to 116 according to one embodiment of the present invention.

[0087] FIG. 91 is a graph of the results of fluorescence-activated cell sorting (FACS) analysis using HEK293-NCV cell lines of peptide structures 121 to 125 and 132 to 135 according to one embodiment of the present invention.

[0088] Figure 10 shows fluorescence images observed 1, 24, and 72 hours after treating HEK293-huFAP cell lines with peptide construct 8, respectively.

[0089] Figure 11a is a graph showing the results of measuring the total fluorescence of the cells after 1, 24, 48, and 72 hours, respectively, after treating the HEK293-huFAP cell line with peptide construct 8.

[0090] Figure 11b is a graph showing the results of measuring the total fluorescence of cells after 1 and 24 hours, respectively, after treating cell lines differentiated into myofibroblasts with peptide construct 8.

[0091] Figure 12a is a graph showing the results of ex vivo imaging and fluorescence intensity measurements of tumors and organs excised after 24 and 72 hours in mice injected with peptide structures 1, 6, and 8, respectively.

[0092] Figure 12b is a graph showing the results of ex vivo imaging and fluorescence intensity measurements of tumors and organs excised after 24 hours in mice injected with peptide structures 8, 85, 86, 87, 88, and 27, respectively.

[0093] Figure 12c is a graph showing the results of ex vivo imaging and fluorescence intensity measurements of tumors and organs excised after 24 hours in mice injected with peptide structures 131, 162, 169, 170, 171, and 172, respectively.

[0094] Figure 12d is a graph showing the results of ex vivo imaging and fluorescence intensity measurements of tumors and organs excised 48 hours after mice injected with peptide structures 176 and 177, respectively.

[0095] Figure 12e is a graph showing the results of ex vivo imaging and fluorescence intensity measurements of tumors and organs excised 48 hours after mice injected with peptide structures 173, 174, 175, 178, 179, and 180, respectively.

[0096] Figure 13 is a graph showing the results of ex vivo imaging and fluorescence intensity measurements of tumors and organs excised after 24 and 72 hours in mice injected with peptide structure 87, respectively.

[0097] Fig. 14a is 177 Lu-labeled peptide construct 145 ( 177 In mice injected with Lu-145, at 1.5 hours, 1 day, 177 Lu-labeled peptide construct 160 ( 177 This figure shows the results of measuring mouse SPECT / CT images at 1.5 hours, 1 day, and 2 days after injection of Lu-160. Arrows indicate tumors.

[0098] Fig. 14b is 177 Lu-labeled peptide construct 145 ( 177 Lu-145) and 160 ( 177 This figure shows a graph of the results of measuring changes in body weight in mice injected with Lu-160.

[0099] Fig. 14c is 177 Lu-labeled peptide construct 145 (177 Lu-145) and 160 ( 177 This figure shows a graph of the results of measuring changes in tumor size (anticancer effect) in mice injected with Lu-160.

[0100] Fig. 15a is 177 Lu-labeled peptide construct 128 ( 177 Lu-128), 129 ( 177 Lu-129), 161 ( 177 Lu-161) and 162 ( 177 This figure shows the results of measuring mouse SPECT / CT images at 1.5 hours, 1 day, and 2 days after injecting each of Lu-162. Arrows indicate tumors.

[0101] Fig. 15b is 177 Lu-labeled peptide construct 128 ( 177 Lu-128), 129( 177 Lu-129), 161 ( 177 Lu-161) and 162 ( 177 This is a graph showing the results of measuring changes in body weight in mice injected with Lu-162.

[0102] Fig. 15c is 177 Lu-labeled peptide construct 128 ( 177 Lu-128), 129( 177 Lu-129), 161 ( 177 Lu-161) and 162 ( 177 This figure shows a graph of the results of measuring changes in tumor size (anticancer effect) in mice injected with Lu-162.

[0103] Fig. 16a is 177 Lu-labeled peptide construct 181 ( 177 Lu-181) and 182 ( 177 This figure shows the results of measuring mouse SPECT / CT images at 1.5 hours, 1 day, and 2 days after injecting each of Lu-182. Arrows indicate tumors.

[0104] Fig. 16b is 177 Lu-labeled peptide construct 181 ( 177 Lu-181) and 182 ( 177 This figure shows a graph of the results of measuring changes in body weight in mice injected with Lu-182.

[0105] Fig. 16c is 177 Lu-labeled peptide construct 181 ( 177 Lu-181) and 182 ( 177 This figure shows a graph of the results of measuring changes in tumor size (anticancer effect) in mice injected with Lu-182.

[0106] Figure 17a shows the HPLC analysis conditions for the synthesized material.

[0107] Figure 17b shows the HPLC retention times and molecular weights of synthesized substances 1 to 28 observed using electrospray ionization mass spectrometry (MS).

[0108] Figure 17c shows the HPLC retention times and molecular weights of synthesized substances 29 to 68 observed using electrospray ionization mass spectrometry (MS).

[0109] Figure 17d shows the HPLC retention times and molecular weights of the synthesized substances 69 to 108 observed using electrospray ionization mass spectrometry (MS).

[0110] Figure 17e shows the HPLC retention times and molecular weights of synthesized substances 109 to 148 observed using electrospray ionization mass spectrometry (MS).

[0111] Figure 17f shows the HPLC retention times and molecular weights of synthesized substances 149 to 182 observed using electrospray ionization mass spectrometry (MS).

[0112]

[0113] 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.

[0114] The present invention allows for various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0115]

[0116] FAP-α specific peptide structure of the present invention

[0117] The present invention may provide a peptide structure comprising a peptide ligand that specifically binds to fibroblast-activated protein-alpha (FAP-α), and a substituted or unsubstituted pyrrolidinyl group connected to the peptide ligand directly or through a spacer.

[0118] The peptide ligand that specifically binds to FAP-α according to the present invention may include any one of the amino acid sequences of SEQ ID NOs 1 to 88. The amino acids constituting the peptide ligand may include D-amino acids or consist solely of D-amino acids. That is, at least one of the amino acids constituting the peptide ligand may consist of a D-amino acid. Additionally, the β-phenyl group of the side chain of the phenylalanine (Phe) residue among the constituent amino acid residues of the peptide ligand may be substituted with one or more chemical functional groups. Preferably, the chemical functional group may be a halogen, but is not limited thereto.

[0119] The peptide ligand of the present invention may be prepared such that amino acid residues having a specific sequence are combined with each other to form a linear molecule. The peptide ligand of the present invention may be prepared by known peptide synthesis methods and is not particularly limited. In one embodiment, the peptide ligand of the present invention 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. The peptide ligand of the present invention also includes its salt form.

[0120] The above peptide structure may have the structure of Chemical Formula 1 below.

[0121] [Chemical Formula 1]

[0122]

[0123] In the above formula,

[0124] P, F1, F2, F3, F4 and n are each as defined above.

[0125] In one embodiment, for example, P, m, n, F3, S1, S2, S3, S4, F4, F5, F6, o, p, q, r, s, and t are each as defined above, and F1 is a halogen, C 1-10 It is any one selected from the group consisting of alkanoylamino and CN, and F2 is glycine (Gly), C(O)-C 1-6 It may be any one selected from the group consisting of alkylene-(O)C and pyrrolidinyl group-containing amino acid residues.

[0126] In one embodiment, the pyrrolidinyl group within the pyrrolidinyl group-containing amino acid residue may be substituted with one or more X1s, wherein X1 is a halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 1-10 Alkanoylamin, C(O)-C 1-6Alkyl, C(O)-C 1-6 Haloalkyl, C(O)OC 1-6 Alkyl, C(O)OC 1-6 It may be any one selected from the group consisting of haloalkyl, NH2, OH, CN, COOH, and NO2.

[0127] In one embodiment, the pyrrolidinyl group-containing amino acid residue may have the following structure, but is not limited thereto.

[0128] , or

[0129]

[0130] In one embodiment, the peptide structure of the present invention may include the structure of the following chemical formula 2, 3, 4, 5, 6, or 7.

[0131]

[0132] [Chemical Formula 2]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] [Chemical Formula 3]

[0140]

[0141]

[0142]

[0143] [Chemical Formula 4]

[0144]

[0145]

[0146]

[0147] [Chemical Formula 5]

[0148]

[0149]

[0150]

[0151] [Chemical Formula 6]

[0152]

[0153]

[0154]

[0155] [Chemical Formula 7]

[0156]

[0157]

[0158]

[0159] In the above chemical formulas 2 to 7, F3 is as defined above.

[0160] In one embodiment, one or more of F4, F5 and F6 are substituted amino acid residues, wherein the substituent of the amino acid residue may be introduced through any one selected from the group consisting of 1) a side chain ε-amino group of a lysine residue; 2) a side chain β-amino group of a 2,3-diaminopropionic acid (DAP) residue; 3) a side chain β-ethynyl group of a propagylglycine residue; 4) a side chain β-phenyl group of a phenylalanine (Phe) residue; 5) a side chain amino group of an arginine residue; and 6) a side chain γ-amino group of an azidohomoalanine residue.

[0161] In one embodiment, in the peptide structure of the present invention, the F 4, One or more of F5 and F6 may include a substituted amino acid residue, wherein the substituent of the substituted amino acid residue is a hydroxyl group (OH), a chelator, an indole, or C 1-6Alkyl, 4,4-(bis(4-hydroxyphenyl)valeric acid (BHPV), PEG24-biotin, hexanoylamino, 4-(p-iodophenyl)butyric acid (IB), pentafluorophenyl (PhF5), halogen, zwitteric ion (SZW), 1,2:3,4-di-O-isopropylidene-aD-galacturonide (1,2:3,4-Di-O-isopropylidene-aD-galacturonide, Gal), N 6 -Hexanoylglycyl-L-prolyl-L-prolyl-D-lysine(N 6 It may include one or more selected from the group consisting of -hexanoylglycyl-L-prolyl-L-prolyl-D-lysine), fluorescent dyes, and cytotoxic agents, but is not limited thereto.

[0162] In one embodiment, the chelator is preferably 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.

[0163] In one embodiment, a spacer included in the peptide structure of the present invention may connect a substituted or unsubstituted pyrrolidinyl group to a FAP-α specific binding peptide ligand. Additionally, the spacer may independently connect F4, F5, and F6, which are substituted or unsubstituted amino acid residues, to a FAP-α specific binding peptide ligand. The spacer may be, for example, one or more selected from a polyethylene glycol (PEG) linker, glycine, sarcosine, and a peptide linker consisting of 1 to 8 D-amino acids or L-amino acids, but is not limited thereto. Preferably, the spacer may be a polyethylene glycol (PEG) linker or 1 to 8 D-amino acids or L-amino acids.

[0164] In one embodiment, the peptide structure of the present invention may be any one of compounds 1 to 182 having the structure of the following chemical formula 2, 3, 4, 5, 6 or 7.

[0165]

[0166] [Chemical Formula 2]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173] [Chemical Formula 3]

[0174]

[0175]

[0176]

[0177]

[0178] [Chemical Formula 4]

[0179]

[0180]

[0181]

[0182] [Chemical Formula 5]

[0183]

[0184]

[0185]

[0186] [Chemical Formula 6]

[0187]

[0188]

[0189]

[0190] [Chemical Formula 7]

[0191]

[0192]

[0193]

[0194] In the above, * or lowercase letters represent L-amino acids and uppercase letters represent D-amino acids, respectively, and the substituents indicated by abbreviations in AA1 to AA5 and F3 are as defined below.

[0195]

[0196]

[0197]

[0198]

[0199]

[0200] The peptide structure of the present invention exhibits high selectivity, specifically binding to FAP-α but not to other enzymes (e.g., DDP4 or PREP).

[0201]

[0202] Conjugate

[0203] The FAP-α-specific peptide structure of the present invention can form a conjugate by being directly or through a linker attached to one or more selected from the group consisting of fluorescent dyes, cytotoxic agents, and radioactive isotopes.

[0204] In one embodiment, the peptide structure may form a conjugate that is directly or through a linker connected to a fluorescent dye and a cytotoxic agent, or directly or through a linker connected to a fluorescent dye and a radioisotope.

[0205] The above conjugate can target FAP-α and label diseases expressing FAP-α with fluorescent dyes or radioisotopes, or effectively deliver drugs such as radioisotopes or cytotoxic agents to diseases expressing FAP-α, making it useful for the diagnosis, prevention, or treatment of FAP-α overexpression diseases.

[0206] In one embodiment, the linker may be one or more selected from, for example, 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyl oxycarbonyl (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.

[0207] In one embodiment, the fluorescent dye may be one or more selected from, for example, 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, but is not limited thereto.

[0208] 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.

[0209] In one embodiment, the radioisotope is, for example, fluorine-18 (F-18), carbon-11 (C-11), carbon-14 (C-14), tectonium-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), lead-212 (Pb-212), thorium-227 (Th-227), radium-223 (Ra-223), tin-117m (Sn-117m), lead-103 (Pb-103), lead-212 (Pb-212), and astatine-211 (At-211), but is not limited thereto.

[0210]

[0211] Therapeutic administration and formulation

[0212] The present invention provides a pharmaceutical composition for the diagnosis, prevention, or treatment of FAP-α overexpression diseases comprising the FAP-α-specific peptide structure or conjugate of the present invention. The FAP-α overexpression disease may be any one selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid. The cancer may preferably be a solid tumor, and more preferably, a cancer expressing FAP-α.

[0213] In one embodiment, the cancer may be a solid tumor such as, for example, 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, anal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, prostate cancer, bile duct cancer, bladder cancer, kidney cancer, heart cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, splenic cancer, melanoma, thyroid cancer, nasopharyngeal cancer, laryngeal cancer, myeloma, cholangiocarcinoma, clear cell carcinoma, neuroendocrine tumor, carcinogenic osteomalacia, sarcoma, carcinoma of unknown primary, thymic carcinoma, desmoid tumor, glioma, astrocytoma or glioblastoma, but is not limited thereto.

[0214] In one embodiment, the fibrosis may be pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, renal fibrosis, pancreatic fibrosis, cutaneous fibrosis, ocular fibrosis, skeletal muscle fibrosis, intestinal fibrosis, or pericardial fibrosis, but is not limited thereto.

[0215] A subject to whom the pharmaceutical composition for the diagnosis, prevention, or treatment of FAP-α of the present invention is administered may be a mammal that is at risk of developing a disease of FAP-α overexpression, has been diagnosed with a disease of FAP-α overexpression, or has received treatment for a disease of FAP-α overexpression. The mammal may be a human or a mammal other than a human.

[0216] The pharmaceutical composition for the diagnosis, prevention, or treatment of FAP-α overexpression disease 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, as well as topical 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.

[0217] 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.

[0218] When formulating, it can be manufactured using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants commonly used in the pharmaceutical industry.

[0219] The preferred dosage of the pharmaceutical composition for the prevention or treatment of FAP-α overexpression diseases according to the present invention may vary depending on the patient's condition, body weight, severity of the 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.

[0220] The pharmaceutical composition for the prevention or treatment of FAP-α overexpression diseases 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.

[0221] The present invention also provides a method for treating FAP-α overexpression diseases, comprising administering the FAP-α-specific peptide structure or conjugate of the present invention to an individual requiring treatment for cancer.

[0222] The FAP-α-specific peptide structure and conjugate of the present invention may also be used to diagnose FAP-α overexpression diseases by targeting and imaging FAP-α overexpression diseases, or to predict or observe the treatment prognosis of individuals after treatment for FAP-α overexpression diseases by administering them to individuals who have received treatment for FAP-α overexpression diseases.

[0223]

[0224] 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.

[0225]

[0226] Examples

[0227]

[0228] Example 1. Peptide screening binding to human fibroblast active protein alpha

[0229] A peptide library was synthesized to obtain peptides that specifically bind to human fibroblast activation protein-α (hFAP-α). The synthesized peptide library was screened, and the binding affinity and specificity for hFAP-α of the selected peptides were evaluated.

[0230] A schematic diagram illustrating the peptide screening process for hFAP-α is shown in Figure 1.

[0231]

[0232] 1-1. Synthesis of Bead-Introduced Peptide Libraries

[0233] For the synthesis of the peptide library, a random OBOC (combinatorial one-bead-one-compound) peptide library was synthesized using TentaGel® S-NH2 beads (TentaGel, Cat# NSD30902, Rapp Polymere GmbH).

[0234] Synthesized on TentaGel® S-NH2 beads (polyethylene glycol-grafted polystyrene beads) through a repetitive split-and-mix synthesis process using an automated synthesizer (Apex 396, AAPPTEC) with 17 D-type amino acids derivatized by Fmoc (fluorenylmethyloxycarbonyl) functional groups and pentafluoro-D-phenylalanine (f(F5), Cat# 23708, GL Biochem) derivatized by Fmoc functional groups, excluding cysteine, methionine, and histidine at each amino acid residue position. In particular, for Fmoc-D-isoleucine-OH and Fmoc-D-glutamine-OH, 10 mol% of Fmoc-glycine-OH was added to each to distinguish isotopic residues during amino acid sequence analysis of the peptides. TentaGel was swollen in NMP (N-Methylpyrrolidone) solvent, and after mounting a photocleavage-capable linker, a peptide library was synthesized using an automated synthesizer. When introducing the Fmoc-ANP linker (3-(Fmoc-amino)-3-(2-nitrophenyl)propanoic acid, Cat# LSP308, AAPPTEC), N-acetylglycine (3 equivalents, Cat# A16300, Sigma-Aldrich) was added to adjust the loading ratio to 1 / 4, and reacted with 3 equivalents of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU), Cat# 12806, Sigma-Aldrich) and DIPEA (7.5 equivalents, Cat# 8.00894, Sigma-Aldrich).The solid beads were washed with NMP, reacted with a piperidine / NMP (1:4) solution to remove the Fmoc protecting group, and then washed sequentially with NMP, dichloromethane (DCM), and NMP. Next, Fmoc-Arg(pbf)-OH (3 equivalents, Cat# 36404, GL Biochem) and Fmoc-PEG1-OH (3 equivalents, Cat# 246201, ChemPep) were synthesized in the same manner to create linkers. For the synthesis of the peptide library, the beads equipped with linkers were divided equally among 18 wells of an automated synthesizer RV (reaction vessel). Once one cycle of coupling and Fmoc protecting group removal was completed, the solid beads were collected and mixed in a CV (collector vessel), then divided into equal amounts into 18 RV wells for coupling and Fmoc protecting group removal, and the separation-mixing synthesis process was repeated until the desired peptide length was achieved.

[0235]

[0236] 1-2. Synthesis of Functional Groups HO-Su-GP-CN (S1), HO-Su-GP(F2)-CN (S2), and HO-Su-GP(Hex)-CN (S3)

[0237] First, for the synthesis of HO-Su-GP-CN (S1), (S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile (1 equivalent, Cat# BD44668, BLD Pharmatech Ltd) and ammonium hydroxide (NH4OH, 250 equivalents, Cat# 000A3781, Samjeon Soonyak Industrial Co., Ltd., wt. 25~30%) were placed in a round-bottom flask and reacted for 4 hours, after which the organic layer was extracted with DCM and the moisture was removed with MgSO4 (Cat# M1807, Samjeon Soonyak Industrial Co., Ltd.). (S)-1-glycylpyrrolidine-2-carbonitrile synthesized by the above method was reacted for 1 hour with succinic anhydride (0.8 equivalents, Cat# S0107, TCI, in ACN) and Me3N (2 equivalents, Cat# T2704, TCI, 1M in THF) in a round-bottom flask. Afterward, the solution was filtered using a 45 μm syringe filter (Cat# DISMIC-3HP, Advantec, Japan) to evaporate the solvent and remove insoluble impurities. The reaction progress was checked using LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent), and the solution was purified using a 1260 Infinity II LC system (Agilent) before use. The analytical instrument used was LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent), and data were analyzed using MNOVA (v. 14.2.0, Mestrelab research, Spain). An Agilent Poroshell 120 EC-C18 column (4.6 X 50 mm, 2.7 μm) was used as the stationary phase, and the column temperature was maintained at 40 °C.In addition, a mixture of acetonitrile (ACN) containing 0.1% trifluoroacetic acid (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. Purification was performed using a 1260 Infinity II LC system (Agilent) under the following conditions: (1) the stationary phase was a Kromasil 100-5-C18 column (21.2 X 250 mm, 5 μm), (2) the mobile phase solvent was a mixture of triple-distilled water and ACN with 0.1% TFA added, (3) the flow rate was 15 mL / min, and (4) the detection wavelengths were 214 and 254 nm.

[0238] The substance obtained by purification in this way was subjected to solvent removal using a rotary evaporator to obtain (S)-4-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)-4-oxobutanoic acid ((S)-4-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)-4-oxobutanoic acid (HO-Su-GP-CN). The synthesis process of S1 is as follows.

[0239]

[0240] [Reaction Equation 1]

[0241]

[0242]

[0243] The synthesis of the functional group HO-Su-GP(F2)-CN (S2) is as follows. N-(tert-butoxycarbonyl)glycine (1.1 equivalents, Cat# 15420, Sigma-Aldrich), 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU, 1.1 equivalents, Cat# 12806, Sigma-Aldrich), and DIPEA (N,N-Diisopropylethylamine, 5 equivalents, Cat# 8.00894, Sigma-Aldrich) were dissolved in ACN (acetonitrile) and reacted at room temperature for 20 minutes. This reaction solution was placed in a 50 mL round-bottom flask containing (S)-4,4-difluoropyrrolidine-2-carboxamide hydrochloride (1 equivalent, Cat# BD169957, BLD Pharmatech Ltd) and reacted for 2 hours, after which the solvent was removed using a rotary evaporator. Diethyl ether was added to the gel-like reaction product, centrifuged, and the precipitate was dried to obtain tert-butyl (S)-(2-(2-carbamoyl-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl) carbamate.

[0244] tert-butyl (S)-(2-(2-carbamoyl-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl) carbamate (1 equivalent, mixture; containing trace amounts of 1-Hydroxybenzotriazole), trifluoroacetic anhydride (2.2 equivalents, Cat# T0433, TCI), anhydrous pyridine (8 equivalents, Cat# 270970, Sigma-Aldrich), and anhydrous tetrahydrofuran (Cat# 401757, Sigma-Aldrich) were placed in a round-bottom flask and reacted at room temperature for 2 hours. The progress of the reaction was monitored by LC / MS, and if the reaction was not completed, additional trifluoroacetic anhydride (0.5 equivalents) and pyridine anhydride (2 equivalents) were added to complete the reaction. The solvent of the reaction mixture was removed using a rotary evaporator, and the gel-like reaction product was dissolved in ethyl acetate and transferred to a separatory funnel. It was then washed twice with 1 M HCl solution and Sat. NaHCO3, brine, discarded the aqueous layer, and only the organic layer was collected. The organic layer was dehydrated with sodium sulfate, and the filtered solution was desolvated using a rotary evaporator to obtain tert-butyl (S)-(2-(2-carbamoyl-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamate.

[0245] tert-butyl (S)-(2-(2-carbamoyl-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamate (1 equivalent, mixture; containing trace amounts of 1-Hydroxybenzotriazole), p-toluenesulfonic acid monohydrate (2 equivalents, Cat# T0267, TCI), and ACN obtained above were added to a round-bottom flask and reacted at room temperature for 24 hours. The solvent was removed using a rotary evaporator, and diethyl ether was added to obtain the precipitate (S)-4,4-difluoro-1-glycylpyrrolidine-2-carbonitrile.

[0246] The (S)-4,4-difluoro-1-glycylpyrrolidine-2-carbonitrile (1 equivalent, mixture; containing trace amounts of p-toluenesulfonic acid), succinic anhydride (1.2 equivalents, Cat# S0107, TCI), and trimethylamine (ca. 13% in tetrahydrofuran, ca. 2 mol / L) (3.2 equivalents, Cat# S0107, TCI) obtained above were placed in a round-bottom flask and reacted at room temperature for 1 hour. After removing the solvent, ACN was added to dissolve the mixture, and acetic acid was added to acidify it. And, HO-Su-GP(F2)-CN ((S)-4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)amino)-4-oxobutanoic acid) was obtained by purifying using HPLC under the same conditions as in the case of S1 and then freeze-drying. The synthesis process of S2 is as follows.

[0247]

[0248] [Reaction Equation 2]

[0249]

[0250] For the synthesis of the functional group HO-Su-GP(Hex)-CN (S3), (2S,4R)-4-Alloc-amino-1-Fmoc-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4R-NHAlloc)-OH, 2.5 equivalents, Cat# AF28682, A2B chem), TBTU (2.5 equivalents), and DIPEA (9 equivalents) were dissolved in a cylinder containing Rink Amide-ChemMatrix® resin (0.41 mmole / g, Cat# 7-600-1310, Biotage) swollen in NMP and reacted at room temperature for 2 hours. After the reaction, the beads were washed three times with NMP and DCM to remove the solvent and prepare for the next reaction.

[0251] Subsequently, to remove the Alloc protecting group, tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4, 1 equivalent, Cat# 216666, Sigma-Aldrich) and 1,3-dimethylbarbituric acid (10 equivalents, Cat# 39565, Sigma-Aldrich) were dissolved in anhydrous N,N-dimethylformamide (DMF) solvent under an argon (Ar) atmosphere and reacted at room temperature for 1 hour. After the reaction, the mixture was washed three times each with NMP and DCM, and washed at least five times with a 0.1M sodium diethyldithiocarbamate (Cat# D3506, Sigma-Aldrich) solution in NMP to remove the Pd impurities. The solution was removed. Then, the beads were washed with NMP and DCM, and hexanoic acid (2.5 equivalents, Cat# 153745, Sigma-Aldrich), TBTU (2.5 equivalents), and DIPEA (10 equivalents) were dissolved in NMP and reacted for 2 hours. After the reaction, the beads were washed three times each with NMP and DCM to remove the solvent and prepare for the next reaction.

[0252] To remove the Fmoc protecting group, a 20% (v / v) piperidine in NMP solution was added to the beads and treated twice for 10 minutes at room temperature, followed by washing five times each with NMP and DCM. Subsequently, Boc-Gly-OH (2.5 equivalents, Cat# 35301, GL Biochem), TBTU (2.5 equivalents), and DIPEA (10 equivalents) were dissolved in NMP and reacted for 2 hours. After the reaction, the beads were washed three times each with NMP and DCM to remove the solvent and prepare for the next reaction.

[0253] To cleave the peptides on the synthesized beads, a mixed solution of trifluoroacetic acid (TFA, Cat# T6508, Sigma-Aldrich) (95%) / triisopropylsilane (TIS, Cat# 233781, Sigma-Aldrich) (2.5%) / triple distilled water (2.5%) was used and treated at room temperature for 2 hours. The beads were removed using a filter, and the TFA mixed solution containing the peptides was collected in a conical tube and blew with nitrogen gas to remove most volatile substances. 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 discarding the supernatant diethyl ether, the remaining solid peptides were dried at room temperature.

[0254] Subsequently, succinic anhydride (4 equivalents, Cat# S0107, TCI) and Me3N (5 equivalents, Cat# T2704, TCI, 1M in THF) were added to the dried peptide and reacted at room temperature for 1 hour. After the reaction, the solvent was removed from the reaction solution using an evaporator, and the solution was dissolved in a mixed solvent of ACN / triple distilled water (1:1). To remove insoluble foreign substances, the solution was filtered using a 45 μm syringe filter, and after HPLC purification as described above, it was freeze-dried to obtain 4-((2-((2S,4R)-2-carbamoyl-4-hexanamidopyrrolidin-1-yl)-2-oxoethyl)amino)-4-oxobutanoic acid.

[0255] The above compounds 4-((2-((2S,4R)-2-carbamoyl-4-hexanamidopyrrolidin-1-yl)-2-oxoethyl)amino)-4-oxobutanoic acid (1 equivalent), trifluoroacetic anhydride (2.2 equivalents, Cat# T0433, TCI), and anhydrous pyridine (8 equivalents, Cat# 270970, Sigma-Aldrich) were dissolved in anhydrous THF (Cat# 401757, Sigma-Aldrich) and reacted at room temperature for 2 hours. The reaction extent was confirmed by LC / MS, and the solvent of the reaction mixture was removed using nitrogen. The gel-state reaction product was dissolved in a mixed solution of ACN and triple-distilled water (1:1) and purified using HPLC under the same conditions as in the case of S1. The purified substance was freeze-dried to obtain 4-((2-((2S,4R)-2-cyano-4-hexanamidopyrrolidin-1-yl)-2-oxoethyl)amino)-4-oxobutanoic acid (HO-Su-GP(Hex)-CN,S3). The synthesis process of S3 is as follows.

[0256]

[0257] [Reaction Equation 3]

[0258]

[0259] 1-3. Synthesis of a peptide library with an introduced functional group (HO-Su-GP-CN)

[0260] Eighteen RV beads equipped with the peptide synthesized in Example 1-1 were recovered into a single tube and mixed, and then HO-Su-GP-CN was introduced to the N-terminus of the bead-shaped peptide via an amide coupling reaction (Fig. 2). Specifically, HO-Su-GP-CN (2 equivalents), TBTU (2 equivalents), and DIPEA (10 equivalents) were mixed in NMP solvent and reacted at room temperature for 1 hour. After the reaction, the mixture was washed 3 times with NMP and 3 times with DCM. To remove the protecting group from the amino acid residue, the mixture was reacted for 2 hours in a mixed solution of TFA (95%) / TIS (2.5%) / triple distilled water (2.5%), washed 5 times with DCM, dried under vacuum, and then 4 in a light-blocked state o It was stored in C.

[0261] An example of a peptide library structure with the functional group HO-Su-GP-CN (S1) introduced for hFAP-α screening is shown in Figure 2.

[0262]

[0263] Example 2. Selection of peptides binding to human FAPα

[0264] 2-1. Peptide Library Primary Screening

[0265] 50 mg of beads containing the peptide library synthesized in Example 1 above were transferred to 4 mL of Extract-Clean Filter Columns (Cat# 211104, S*PURE), 2 mL of pH 7.4 phosphate buffer solution (PBS) was added, and the beads were swollen by sonication using an ultrasonic cleaner (Cat# 5210R-DTH, BRANSON). After removing the PBS, 2 mL of blocking solution (10% FBS, 0.1% Tween20 (Cat# 69295-1601, Junsei) in pH 7.4 PBS) was added, and the mixture was incubated at room temperature in a 360° shaker (Cat# M04-238-157, SCILOGEX). 60 nM of hFAP-α (Cat# FAP-H5244, ACROBiosystems) protein loaded with a fluorescent dye was added, and the solution was removed after the reaction. The beads were transferred to a conical tube, diluted with 45 mL of PBST buffer solution containing 0.1% Tween20, and divided into three conical tubes. PBST buffer solution containing 0.1% Tween20 was added to each aliquot for further dilution, and the tubes were mounted in the sample vessels of a COPAS for screening. Screening was performed with Excitation 640 nm and Emission 680 / 30 BP, under Enrichment mode, PMT 600, and Gain 3.0 conditions, and approximately 5,000 beads with high fluorescence intensity were selected. The above process was repeated 5 times to select a total of 25,000 positive beads (i.e., beads equipped with peptides binding to hFAP-α) to be used for secondary screening. (Fig. 3)

[0266]

[0267] 2-2. Peptide Library Secondary Screening

[0268] The fluorescent dye-equipped hFAP-α protein was removed from approximately 25,000 positive beads obtained from the primary screening in Example 2-1 above. Subsequently, 1 mL of blocking solution was added to the beads and reacted in a 360° shaker, and the fluorescent dye-equipped hFAP-α protein was added to a concentration of 250 nM and reacted, after which the solution was removed. The beads were transferred to a conical tube, diluted with 45 mL of PBST buffer solution containing 0.1% Tween20, and then aliquoted. Each aliquoted solution was further diluted by adding PBST solution containing 0.1% Tween20, mounted in a sample container of a COPAS, and processed in two steps under conditions of Excitation 640 nm and Emission 680 / 30 BP. In the first step, approximately 1,000 beads with high fluorescence intensity were selected under conditions of Enrichment mode, PMT 630, and Gain 3.0. The 1,000 obtained beads were diluted with triple-distilled water, and then a second COPAS screening was performed using a 96-well plate under conditions of Pure mode, PMT 540, and Gain 3.0.

[0269]

[0270] 2-3. Isolation and Analysis of Peptides Obtained from Secondary Screening

[0271] Peptides were isolated from solid-phase single beads obtained through the secondary screening of Example 2-2 above via a photolysis reaction. Specifically, a 96-well plate containing the beads was sealed under an argon (Ar) atmosphere, and then a UVP crosslinking agent (Cat# 849-30101-2, Analytikjena) was used at a wavelength of 365 nm and an irradiation dose of 9,000 μJ / cm². 2The photolysis reaction was carried out for 10 minutes under these conditions. After opening the 96-well plate, the solution was concentrated at room temperature using an acid benchtop concentrator (Cat# 7310042, LABCONCO).

[0272] Subsequently, the molecular weight and amino acid sequence of the peptide were analyzed based on MS and MS / MS obtained using an Enhanced ultrafleXtreme MALDI-TOF / TOF mass spectrometer (mass spectrometer, Bruker). An example is shown in Figure 4.

[0273]

[0274] 2-4. Tertiary Screening of Peptide Library for Candidate Peptide Selection

[0275] About 50 types of peptides were selected from 250 to 300 types of peptides having amino acid sequences obtained in Examples 2-3 above, synthesized on TentaGel beads (0.08 mmole / g, 10 mg per peptide) equipped with a photodegradation linker using an Apex 396 autosynthesizer in the same manner as in Example 1, and then the protecting groups of the amino acid residues were removed using a mixed solution of TFA (95%) / TIS (2.5%) / triple distilled water (2.5%).

[0276] Subsequently, for the third COPAS screening to derive final peptide candidates, 1 mg of each of approximately 50 different TentaGel beads loaded with various peptides (total 50 mg) were collected and mixed in 4 mL Extract-Clean Filter Columns, 2 mL of PBS was added, and the beads were swollen by sonication. Afterward, COPAS screening was performed using the same method as the second peptide library screening while maintaining the final concentration of hFAP-α loaded with a fluorescent dye at 250 nM. This process was repeated three times to ensure the reproducibility of the results. Based on the analysis of MS and MS / MS data, peptides were sequenced in order of positive hit numbers, and candidate substances were selected based on the top-ranked peptides.

[0277]

[0278] Example 3. Synthesis and purification of a peptide structure containing selected peptides

[0279] 3-1. Synthesis of reagents inserted into peptide structures

[0280] Fmoc-DAP(DOTA(Protected))-OH (S4), 3-(dimethylamino)propane-1-sulfonic acid (SZW) (S5), N-succinimidyl 2,3:4,5:6,7-tri-O-isopropylidene-D-glycero-D-gulo-heptonate (6OH(Protected)-NHS) (S6), and Fmoc-D-ADMA(pbf)-OH (r(dimet)) (S7), which are inserted into the peptide structure, were synthesized directly and used. First, for the synthesis of S4, Fmoc-DAP-OH (N αFirst, dissolve tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, DOTA-tris(tert-butyl ester), 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (1.2 equivalents), N,N-diisopropylethylamine, A solution of DIPEA (3 equivalents) and activated solution dissolved in DMF was mixed and reacted for 2 hours. Afterward, the progress of the reaction was checked using the same LC-MS (1260 Infinity II, Infinity Lab LC / MSD, Agilent) as used in Examples 1-2, and the solution was purified using the 1260 Infinity II LC system (Agilent) for use. The synthesis process of S4 is as follows.

[0281]

[0282] [Reaction Equation 4]

[0283]

[0284] For the synthesis of S5, 1 g of 1,3-propanesultone (1,3-Propanesultone (Cat# P0324, TCI)) and 16 mL of 2 M dimethylamine in THF solution (Cat# D3948, TCI) were added to a 100 mL round-bottom flask and stirred at room temperature for 24 hours. The reaction mixture was concentrated using an evaporator, and the resulting gel-like substance was first precipitated using ethyl acetate. The precipitate was then separated using a nylon membrane filter. The separated precipitate was dissolved in a minimal amount of water, DMF was added to recrystallize the substance, and after separation using a nylon membrane filter, it was washed with ethyl acetate and dried. An example of the synthesis of S5 is as follows.

[0285]

[0286] [Reaction Equation 5]

[0287]

[0288] For the synthesis of S6, methyl 2,3:4,5:6,7-tri-O-isopropylidene-D-glycero-D-gulo-heptonate (1 equivalent) and lithium hydroxide monohydrate (2.5 equivalents) were first added to a 50 mL round-bottom flask equipped with a magnetic stirrer bar, and then MeOH was added and the reaction was carried out at room temperature for 24 hours. After the reaction was complete, volatile substances and the solvent were removed from the reaction mixture using a rotary evaporator. The reaction mixture was dissolved in 50 mL of a 10% aqueous citric acid solution and transferred to a separatory funnel, after which the organic matter was extracted using ethyl acetate. The extracted ethyl acetate solution was washed with a saturated aqueous solution of NaCl in a separatory funnel and then dried with sodium sulfate in an Erlenmeyer flask. Subsequently, the sodium sulfate was removed from the ethyl acetate solution using filter paper, and the volatile solvent was removed using an evaporator. The reaction product was dissolved once again in DCM, and the volatile solvent was removed using a rotary evaporator.

[0289] The 2,3:4,5:6,7-tri-O-isopropylidene-D-glycero-D-gulo-heptonic acid (1 equivalent) synthesized in this way was added to a 50 mL round-bottom flask equipped with a magnetic stir bar for the following reaction with N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC hydrochloride, 1.6 equivalents), and then anhydrous DCM was added and the mixture was reacted at room temperature for 24 hours. After the reaction was complete, the reaction mixture was dissolved in ethyl acetate, transferred to a separatory funnel, and washed with a saturated aqueous solution of NaCl. The organic layer was further washed in a separatory funnel with a saturated aqueous solution of sodium bicarbonate and then washed with a saturated aqueous solution of NaCl. The organic layer was collected in a 300 mL Erlenmeyer flask and dried with anhydrous sodium sulfate. Subsequently, the sodium sulfate was removed from the organic solution using filter paper, and the volatile solvent was removed using an evaporator. The reaction product was dissolved once again in DCM, and the volatile solvent was completely removed using a rotary evaporator before use. An example of the synthesis of S6 is as follows.

[0290]

[0291] [Reaction Equation 6]

[0292]

[0293] The synthesis process of S7 is as follows.

[0294] Fmoc-D-Orn(boc)-OH (1 equivalent, Cat# BD131117, BLD Pharmatech), N, N-Dicyclohexylcarbodiimide (N, N-Dicyclohexylcarbodiimide, DCC, 1.5 equivalents, Cat# 36650, Sigma-Aldrich), 1-hydroxybenzotriazole (1-hydroxy benzotriazole, HOBt, 1.5 equivalents, Cat# CXZ010, Aapptec), 4-(Dimethylamino)pyridine (4-(Dimethylamino)pyridine, DMAP, 0.05 equivalents, Cat# 107700, Sigma-Aldrich) and anhydrous DCM were added to a round-bottom flask and stirred well, and reacted at room temperature for 3 hours. After confirming that sufficient precipitation had occurred, allyl alcohol (2 equivalents, Cat# 240532, Sigma-Aldrich) was added and the reaction was carried out at room temperature for 16 hours. After the reaction, most of the precipitate was filtered through a paper filter, and the resulting solution was concentrated using an evaporator. Then, the gel-like substance was completely dissolved in a small amount of ethyl acetate, and the solution was stored at 20°C for 16 hours to induce precipitation. The precipitate of the ethyl acetate solution containing the dissolved reactant was filtered through a paper filter, transferred to a separatory funnel, and 100 mL of ethyl acetate was added. The organic layer was washed once with a saturated sodium bicarbonate aqueous solution (Cat# S0341, Samjeon Soonyak Industrial Co., Ltd.), twice with triple distilled water, and once with a saturated sodium chloride aqueous solution (Cat# S0476, Samjeon Soonyak Industrial Co., Ltd.) using a separatory funnel. Anhydrous magnesium sulfate (Cat# M1806, Samjeon Soonyak Industrial Co., Ltd.) was added to the washed organic layer and dried, and then the solution obtained by filtering the precipitate through a paper filter was concentrated using an evaporator.The concentrated gel-form material was completely dissolved in a minimal amount of DCM, and half of the solution was purified using a column (40 x 210 mm) containing 200 g of silica gel (0.063-0.200 mm, Cat# 1.07734.1000, Sigma-Aldrich). An ethyl acetate : hexane (Cat# 1.07734.1000, Sigma-Aldrich) = 1 : 1 solution was used as the developing solvent. The remaining DCM solution was also purified in the same way using a silica gel column. The reaction product Fmoc-D-Orn(boc)-OAll had an Rf of 0.59 (ethyl acetate : hexane = 1 : 1), and the purified solution of the corresponding Rf was collected and concentrated using an evaporator to obtain Fmoc-D-Orn(boc)-OAll.

[0295] For the next reaction, to remove the Boc protecting group, Fmoc-D-Orn(boc)-OAll (1 equivalent) was placed in a round-bottom flask, and a TFA : DCM = 1 : 1 mixed solution was slowly poured in at room temperature, mixed, and stirred well for 1 hour. After the reaction, the solution was concentrated using an evaporator. The process of dissolving the concentrated material in DCM and concentrating it again using an evaporator was repeated 3 times. Then, the concentrated material was dissolved in anhydrous DCM (Cat# 270997, Sigma Aldrich), sealed, and stored in a freezer. The molecular weight and purity of the concentrated material were confirmed using LC-MS as in Examples 1-2 above, and then used in the following reaction without any special purification process.

[0296] 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl isothiocyanate (2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl isothiocyanate, pbf-NCS), which is required for the next reaction, was first synthesized as follows.

[0297] A solution of tetrabutylammonium thiocyanate (Bu4NNCS, 5 equivalents, Cat# T1278, TCI) and 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl chloride (pbf-Cl, 1 equivalent, Cat# BD41320, BLD Pharmatech) dissolved in anhydrous DCM was added to a round-bottom flask, and after adding a reflux condenser, the reaction solution was refluxed (75°C) and reacted for 1 hour. The reaction product pbf-NCS [Rf=0.49 (hexane : DCM = 1 : 1)] was confirmed by TLC. After the reaction was complete, the concentrated gel-like substance was completely dissolved in a minimal amount of DCM and purified using a column (40 x 210 mm) containing 200 g (0.063–0.200 mm) of silica gel. The developing solvent used was ethyl acetate : hexane = 1 : 1. The purified solution with an Rf of 0.49 was collected and concentrated using an evaporator to obtain a clear liquid pbf-NCS. The procedure of this reaction is as follows.

[0298]

[0299] [Reaction Equation 7]

[0300]

[0301]

[0302] For the next reaction, the Fmoc-D-Orn-OAll (1 equivalent), pbf-NCS (1.2 equivalents), and DIPEA (10 equivalents) synthesized above were dissolved in DCM in a round-bottom flask and reacted at room temperature for 1 hour. After the reaction, the mixture was concentrated using an evaporator, and the concentrated material was completely dissolved in a small amount of DCM and then purified using a column (40 x 210 mm) containing 200 g (0.063-0.200 mm) of silica gel. Ethyl acetate : hexane = 1 : 1 was used as the developing solvent. The reaction product Fmoc-D-Orn(SCN(pbf))-OAll had an Rf of 0.62 (ethyl acetate : hexane = 1 : 1), and the purified solution of the corresponding Rf was collected, concentrated using an evaporator, and used in the next reaction.

[0303] In a round-bottom flask, synthesized Fmoc-D-Orn(SCN(pbf))-OAll (1 equivalent) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, EDC hydrochloride, 2 equivalents, Cat# E7750, Sigma-Aldrich) were dissolved in DCM, and 2 M dimethylamine in THF (2 equivalents, Cat# 391956, Sigma-Aldrich) was added and reacted at room temperature for 10 minutes. After confirming the molecular weight and purity of the reaction mixture using LC-MS as in Example 1-1, the reaction mixture was concentrated using a rotary evaporator once the reaction was complete. The concentrated substance was dissolved in an ACN / triple distilled water (1:1) mixed solution and filtered through a 45 μm syringe filter (Cat# DISMIC-3HP, Advantec), and purified using HPLC in the same manner as in Examples 1-2 above. The purified substance, Fmoc-D-ADMA(pbf)-OAll, was obtained in powder form by freeze-drying.

[0304] Finally, under an argon (Ar) atmosphere, the synthesized Fmoc-D-ADMA(pbf)-OAll (1 equivalent), Pd(PPh3)4 (0.05 equivalents), and N-methyl aniline (3 equivalents, Cat# M29304, Sigma-Aldrich) were dissolved in anhydrous THF in a glass vial and reacted at room temperature for 1 hour. After confirming the molecular weight and purity of the reactants by LC-MS, the reactants were concentrated using a rotary evaporator. The concentrated material was dissolved in a mixed solution of ACN / triple distilled water (1:1), filtered through a 45 μm syringe filter, and purified using HPLC in the same manner as in Examples 1-2 above. The purified material was freeze-dried to obtain Fmoc-D-ADMA(pbf)-OH (N-α-Fmoc-N,N-ω-dimethyl-N-ωÆ-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-D-arginine, Fmoc-r(dimet)-OH) (S7) in powder form. The synthesis process of S7 is as follows.

[0305]

[0306] [Reaction Equation 8]

[0307]

[0308]

[0309] 3-2. Synthesis of Selected Peptides

[0310] Solid-phase peptide chains were synthesized using an automated ultrasonic peptide synthesizer (Liberty Blue™ automated microwave peptide synthesizer, CEM Corporation) or an automated synthesizer (Apex 396, AAPPTEC). Two types of solid-phase resins were used for the synthesis, differing in the C-terminus of the resulting peptides. Rink Amide-ChemMatrix® Resin (0.41 mmole / g, Cat# 7-600-1310, Biotage), used for most materials, has a -CONH2 C-terminus, while 2-Chlorotrityl Chloride Resin has a -COOH C-terminus. Rink Amide-ChemMatrix® Resin can be used directly for synthesis, but 2-chlorotritryl chloride resin (2-Chlorotrityl Chloride Resin, 1.07 mmole / g, Cat# GLS210819-48101, BL BioChem) was used after mounting by dissolving the first amino acid (5 equivalents) and DIPEA (10 equivalents) in anhydrous DCM solvent under an Ar atmosphere and reacting for 12 hours. In an automated ultrasonic peptide synthesizer, a resin swollen in N-methyl-2-pyrrolidone (NMP) was treated with an amino acid (5 equivalents) equipped with a Fmoc protecting group, Oxyma Pure (5 equivalents, Cat# cxz021, AApptec), and N,N'-diisopropylcarbodiimide (DIC, Cat# D0254, TCI) (10 equivalents) in NMP solvent, coupled for 5 minutes at 75°C, washed 3 times with NMP, and then reacted with a piperidine / NMP (v / v = 1:4, 0.1 M OxymaPure) mixed solution for 1 minute at 90°C to remove the Fmoc protecting group.After completing a peptide with the desired amino acid sequence by repeating the same process, the beads loaded with the peptide were collected from the synthesizer. Similarly, in an automated synthesizer (Apex 396, AAPPTEC), resin swollen in NMP was treated with an amino acid (5 equivalents) loaded with a Fmoc protecting group, TBTU (5 equivalents), and DIPEA (10 equivalents) in NMP solvent and coupled at room temperature for 45 minutes. Afterward, the resin was washed with NMP and DCM, and the Fmoc protecting group was removed by reacting with a piperidine / NMP (v / v = 1:4) mixed solution at room temperature for 10 minutes. After completing a peptide with the desired amino acid sequence by repeating the same process, the beads loaded with the peptide were collected from the synthesizer. Peptides requiring additional functional group attachment were collected without removing the Fmoc protecting group, and the following operations were performed.

[0311] The introduction of DOTA, IB, PhF5, Hex, PEG24-biotin, BHPV, and Gal functional groups was carried out by removing the Mtt or Dde protecting group from beads equipped with peptides containing lysine protected by 4-methyltrityl (Mtt) or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) substituents. The Mtt protecting group was removed by treating with a mixed solution of 1.5% TFA / 2.5% TIS / 96% DCM at room temperature for 5 minutes each, for a total of 6 times, and the Dde protecting group was removed by mixing hydroxylamine hydrochloride / imidazole (1.3:1 w / w) in NMP solvent and reacting at room temperature for 2 hours twice or 12 hours once.Subsequently, on beads having amine functional groups from which the protecting group has been removed, DOTA-tris(tert-butyl ester) (DOTA(protected)-OH, 2 equivalents) or 4-(4-iodophenyl)butanoic acid (4-(4-Iodophenyl)butanoic acid, IB-OH, 2 equivalents, Cat# BD11901, BLD Pharmatech) or pentafluorobenzoic acid (Pentafluorobenzoic acid, PhF5-OH, Cat# P0806, Tokyo Chemical Industry) or hexanoic acid (Hexanoic acid, Hex-OH, 2 equivalents, Cat# 153745, Sigma-Aldrich) or biotin-PEG24-NHS ester (PEG24-biotin-NHS, Cat# AP10528, AxisPharm) or 4,4-(bis(4-hydroxyphenyl)valeric acid, BHPV-OH, Cat# B47707, Sigma-Aldrich) or 1,2:3,4-di-O-isopropylidene-aD-galacturonide, Cat# BD52588, BLD Pharmatech, 1.5 equivalents) were reacted with TBTU (2 equivalents) and DIPEA (10 equivalents) in NMP solvent at room temperature for 30 minutes. After the reaction was complete, the beads were washed sequentially three times each with NMP and DCM, and the Fmoc protecting group was removed as a final step.

[0312] The introduction of an amphoteric ionic functional group (SZW) is performed using a peptide containing a lysine residue or a DAP (Diaminopimelic acid) residue protected by Mtt or Dde. The N-terminus of the peptide synthesized on the bead was formed into a protected amine form by removing the Mtt or Dde protecting group as described above. Subsequently, 2-bromoacetic acid (30 equivalents, Cat#17000, Sigma-Aldrich) and DIC (30 equivalents) were dissolved in DMF, added to the beads containing the protected amine, and reacted for 3 hours. After the reaction was complete, the beads were washed sequentially three times each with NMP and DCM. Then, directly synthesized S5 (30 equivalents) was dissolved in triple distilled water and added to the beads submerged in DMF along with DIPEA (30 equivalents), and reacted for 12 hours. After the reaction was complete, the beads were washed three times each with NMP and DCM in sequence and vacuum dried.

[0313] The introduction of the SZW-DAP-indole functional group was performed using a peptide containing lysine residues or DAP residues protected by Mtt or Dde. First, the Fmoc protecting group at the N-terminus of the peptide was removed, and the group was converted to Boc protecting group by reacting di-tert-butyl dicarbonate (5 equivalents, Cat# 34660, Sigma-Aldrich) and DIPEA (10 equivalents) dissolved in NMP for 1 hour, followed by washing with NMP and DCM. Then, the Mtt or Dde protecting groups on the lysine residues or DAP residues were removed according to the method described above, thereby converting the N-terminus of the peptide synthesized on the beads into a amine form with the protecting group removed. Subsequently, Fmoc-DAP(mtt)-OH (2 equivalents), TBTU (2 equivalents), and DIPEA (10 equivalents) were dissolved in NMP and reacted for 1 hour, followed by washing with NMP and DCM. Next, the Fmoc protecting group was removed, and azido groups were introduced by reacting 4-azidobutyric acid (3 equivalents, Cat# 180201, ChemPep, Inc.), TBTU (3 equivalents), and DIPEA (10 equivalents) dissolved in NMP for 1 hour. Then, the Mtt protecting group on the DAP residue was removed, and an amphoteric functional group (SZW) was mounted as described above. Subsequently, the introduction of an indole functional group to the azido group was carried out after peptide purification.

[0314] The introduction of the PPG(hex) functional group is performed using a peptide containing a lysine residue protected by Mtt or Dde. The N-terminus of the peptide synthesized on the bead was formed into an amine form with the protecting group removed by removing the Mtt or Dde protecting group as described above. Subsequently, amino acids of Fmoc-L-Pro-OH twice and Fmoc-Gly-OH were sequentially synthesized using an autosynthesizer, and finally, hexanoic acid was synthesized in the same manner to complete the process.

[0315] To cleave the peptide from the beads, a mixed solution of TFA (95%) / TIS (2.5%) / triple distilled water (2.5%) was used and reacted at room temperature for 2 hours. Afterward, the beads were removed using a filter, and the TFA mixed solution containing the peptide was collected in a conical tube and most of the mixed solution was removed by blowing with nitrogen gas. Subsequently, diethyl ether was added and the mixture was centrifuged to precipitate the peptide; after removing the supernatant diethyl ether and a small amount of the remaining TFA mixed solution, the remaining solid peptide was vacuum dried.

[0316]

[0317] 3-3. Mounting of Functional Groups (HO-Su-GP-CN, HO-Su-GP(F2)-CN, or HO-Su-GP(Hex)-CN) on Peptide N-Terminus and Purification

[0318] Based on a dry peptide (1 equivalent), HO-Su-GP-CN (S1) or HO-Su-GP(F2)-CN (S2) (2 equivalents), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 1.5 equivalents, Cat# A1797, TCI), and DIPEA (10 equivalents) were dissolved in DMF solvent and pre-activated for about 10 minutes, then added to the dry peptide and reacted for 30 minutes, and the reaction was confirmed by LC / MS. In the case of Su-GP(F2)-CN, which is also mounted to DAP or Uk residues, since there are two reaction sites (peptide N-terminus and DAP or Uk residue), it can be mounted simultaneously by reacting twice the equivalent amount of the above reaction.

[0319] HO-Su-GP(Hex)-CN (S3) (2 equivalents) was reacted after pre-activating N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium Tetrafluoroborate (TSTU, 1.5 equivalents, Cat# T2224, TCI) and DIPEA (10 equivalents) in DMF solvent for about 10 minutes. The reaction solution was dissolved in a 1:1 mixture of ACN and triple-distilled water, and insoluble impurities were removed using a filter (45 μm syringe filter, Cat# DISMIC-3HP, Advantec). The solution was then purified and analyzed using HPLC as described in Examples 1-2. The purified peptide solution was freeze-dried to obtain a powder form.

[0320]

[0321] 3-4. Mounting 6OH functional groups on purified peptides

[0322] Purified peptide in powder form, 6OH(Protected)-NHS (S6) (6 equivalents), and DIPEA (15 equivalents) were dissolved in DMF in a glass vial and reacted for 2 hours under magnetic stirring. After the reaction, impurities were removed using a 45 μm syringe filter, and the solution was purified using HPLC as described in Examples 1-2. The purified peptide solution was lyophilized to remove the solvent and then converted into a powder. To remove the acetal protecting group of 6OH(Protected), a mixed solution of 95% (v / v) TFA, 2.5% (v / v) TIS, and 2.5% (v / v) water was added to the purified peptide and reacted for about 2 minutes. Care must be taken at this time, as exceeding 2 minutes causes the 6OH group to detach from the lysine (Lys) residue. Immediately after the reaction, a mixed solution of ACN and triple-distilled water (1:1) was added. To neutralize the solution, trimethylamine (Cat# T0424, TCI) was slowly added under 0 °C ice water in an amount equal to that of the TFA solution. This solution was purified once again under the same conditions as above, and the resulting peptide solution was obtained in powder form using a freeze-dryer.

[0323]

[0324] 3-5. Mounting of indole, AZA functional groups and fluorescent dye (Cy5, AF488) on purified peptides

[0325] Indole, AZA functional groups and fluorescent dyes were mounted on purified peptides using click chemistry. First, purified powdered peptide (1 equivalent) having an indole, AZA functional group and an Azido functional group, 3-(Prop-2-yn-1-yl)-1H-indole (3 equivalents, Cat# BD01165374, BLD Pharmatech Ltd.) or N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)hex-5-ynamide (3 equivalents, Cat# CSSB00102954486, ChemSpace), 1 M CuSO4 (Cat#C1297, Sigma-Aldrich) aqueous solution (3 equivalents), and 1 M ascorbic acid (Cat#A0278, Sigma-Aldrich) (6 equivalents) were reacted under an Ar atmosphere for 1 hour, the extent of the reaction was checked by LC-MS, and the identified peptide was purified using a 1260 Infinity II LC system. The purified peptide solution was freeze-dried to obtain a powder form. A fluorescent dye was mounted to the alkyl (alkyne) functional group site of Fmoc-L-propargyl-Gly-OH (Fmoc-L-propargyl-Gly-OH, Fmoc-Pra-OH, Cat# 21528, GL Biochem). The purified powdered peptide (1 equivalent), Sulfo-Cyanine5 azide (Cy5 azide, Cat#E3330, Lumiprobe) or Alexa Fluor 488 azide (AF488 azide, Cat#1275, Click Chemistry Tools,) (1 equivalent), 1 M CuSO4 aqueous solution (3 equivalents), and 1 M ascorbic acid (6 equivalents) were reacted under an Ar atmosphere for 1 hour.After the reaction, the progress of the reaction was checked by LC-MS as described in Examples 1-2. For the peptide containing the DOTA functional group, sodium diethyldithiocarbamate (5 equivalents) was added to remove the copper coordinated to DOTA, and after a reaction of 20 minutes, the removal of copper was confirmed by LC-MS. The identified peptide was purified using HPLC. The purified peptide solution was obtained in powder form using a freeze-dryer.

[0326]

[0327] 3-6. Labeling of Lutetium-175 (Lu-175)

[0328] Peptide constructs containing a DOTA linker were labeled with lutetium-175 (Lu-175) instead of the isotope lutetium-177 (Lu-177) to conduct binding affinity and activity experiments for hFAP-α protein.

[0329] The synthesized peptide construct was dissolved in DMSO at a concentration of 2 mM, and lutetium-175 (LuCl3, Cat#450960, Sigma-Aldrich) was dissolved in sodium acetate buffer solution (pH 5.5) at a concentration of 10 mM. After adding 3 equivalents of lutetium-175 to the peptide construct (1 equivalent) dissolved in DMSO, 500 μL of sodium acetate buffer solution (pH 5.5) was added, and the mixture was reacted in a thermoblock (Cat#B10-48, BLE) at 90 °C for 30 minutes. After the reaction was complete, the solution was cooled to room temperature and purified using a 1260 Infinity II LC system (Agilent) under the following conditions: (1) stationary phase was a Kromasil 100-5-C18 column (10 x 250 mm, 5 μm), (2) mobile phase solvent was a mixture of triple-distilled water with 0.1% TFA 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.

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337] In the above chemical formulas, AA1 to AA5 represent a conventional amino acid residue or an amino acid residue containing a functional group (dimethyl), lowercase letters represent D-amino acids, and uppercase letters or * symbols represent L-amino acids. U represents a modified or unnatural D-amino acid. Chemical formulas excluding conventional amino acid residues have the following structures defined in this specification.

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344] [Structure of Peptide Structure 1]

[0345]

[0346] [Structure of Peptide Structure 2]

[0347]

[0348] [Structure of Peptide Structure 3]

[0349]

[0350] [Structure of Peptide Structure 4]

[0351]

[0352] [Structure of Peptide Structure 5]

[0353]

[0354] [Structure of Peptide Structure 6]

[0355]

[0356] [Structure of Peptide Structure 7]

[0357]

[0358] [Structure of Peptide Structure 8]

[0359]

[0360] [Structure of Peptide Structure 9]

[0361]

[0362] [Structure of Peptide Structure 10]

[0363]

[0364] [Structure of Peptide Structure 11]

[0365]

[0366] [Structure of Peptide Structure 12]

[0367]

[0368] [Structure of Peptide Structure 13]

[0369]

[0370] [Structure of Peptide Structure 14]

[0371]

[0372] [Structure of Peptide Structure 15]

[0373]

[0374] [Structure of Peptide Structure 16]

[0375]

[0376] [Structure of Peptide Structure 17]

[0377]

[0378] [Structure of Peptide Structure 18]

[0379]

[0380] [Structure of Peptide Structure 19]

[0381]

[0382] [Structure of Peptide Structure 20]

[0383]

[0384] [Structure of Peptide Structure 21]

[0385]

[0386] [Structure of Peptide Structure 22]

[0387]

[0388] [Structure of Peptide Structure 23]

[0389]

[0390] [Structure of Peptide Structure 24]

[0391]

[0392] [Structure of Peptide Structure 25]

[0393]

[0394] [Structure of Peptide Structure 26]

[0395]

[0396] [Structure of Peptide Structure 27]

[0397]

[0398] [Structure of Peptide Structure 28]

[0399]

[0400] [Structure of Peptide Structure 29]

[0401]

[0402] [Structure of Peptide Structure 30]

[0403]

[0404] [Structure of Peptide Structure 31]

[0405]

[0406] [Structure of Peptide Structure 32]

[0407]

[0408] [Structure of Peptide Structure 33]

[0409]

[0410] [Structure of Peptide Structure 34]

[0411]

[0412] [Structure of Peptide Structure 35]

[0413]

[0414] [Structure of Peptide Structure 36]

[0415]

[0416] [Structure of Peptide Structure 37]

[0417]

[0418] [Structure of Peptide Structure 38]

[0419]

[0420] [Structure of Peptide Structure 39]

[0421]

[0422] [Structure of Peptide Structure 40]

[0423]

[0424] [Structure of Peptide Structure 41]

[0425]

[0426] [Structure of Peptide Structure 42]

[0427]

[0428] [Structure of Peptide Structure 43]

[0429]

[0430] [Structure of Peptide Structure 44]

[0431]

[0432] [Structure of Peptide Structure 45]

[0433]

[0434] [Structure of Peptide Structure 46]

[0435]

[0436] [Structure of Peptide Structure 47]

[0437]

[0438] [Structure of Peptide Structure 48]

[0439]

[0440] [Structure of Peptide Structure 49]

[0441]

[0442] [Structure of Peptide Structure 50]

[0443]

[0444] [Structure of Peptide Structure 51]

[0445]

[0446] [Structure of Peptide Structure 52]

[0447]

[0448] [Structure of Peptide Structure 53]

[0449]

[0450] [Structure of Peptide Structure 54]

[0451]

[0452] [Structure of Peptide Structure 55]

[0453]

[0454] [Structure of Peptide Structure 56]

[0455]

[0456] [Structure of Peptide Structure 57]

[0457]

[0458] [Structure of Peptide Structure 58]

[0459]

[0460] [Structure of Peptide Structure 59]

[0461]

[0462] [Structure of Peptide Structure 60]

[0463]

[0464] [Structure of Peptide Structure 61]

[0465]

[0466] [Structure of Peptide Structure 62]

[0467]

[0468] [Structure of Peptide Structure 63]

[0469]

[0470] [Structure of Peptide Structure 64]

[0471]

[0472] [Structure of Peptide Structure 65]

[0473]

[0474] [Structure of Peptide Structure 66]

[0475]

[0476] [Structure of Peptide Structure 67]

[0477]

[0478] [Structure of Peptide Structure 68]

[0479]

[0480] [Structure of Peptide Structure 69]

[0481]

[0482] [Structure of Peptide Structure 70]

[0483]

[0484] [Structure of Peptide Structure 71]

[0485]

[0486] [Structure of Peptide Structure 72]

[0487]

[0488] [Structure of Peptide Structure 73]

[0489]

[0490] [Structure of Peptide Structure 74]

[0491]

[0492] [Structure of Peptide Structure 75]

[0493]

[0494] [Structure of Peptide Structure 76]

[0495]

[0496] [Structure of Peptide Structure 77]

[0497]

[0498] [Structure of Peptide Structure 78]

[0499]

[0500] [Structure of Peptide Structure 79]

[0501]

[0502] [Structure of Peptide Structure 80]

[0503]

[0504] [Structure of Peptide Structure 81]

[0505]

[0506] [Structure of Peptide Structure 82]

[0507]

[0508] [Structure of Peptide Structure 83]

[0509]

[0510] [Structure of Peptide Structure 84]

[0511]

[0512] [Structure of Peptide Structure 85]

[0513]

[0514] [Structure of Peptide Structure 86]

[0515]

[0516] [Structure of Peptide Structure 87]

[0517]

[0518] [Structure of Peptide Structure 88]

[0519]

[0520] [Structure of Peptide Structure 89]

[0521]

[0522] [Structure of Peptide Structure 90]

[0523]

[0524] [Structure of Peptide Structure 91]

[0525]

[0526] [Structure of Peptide Structure 92]

[0527]

[0528] [Structure of Peptide Structure 93]

[0529]

[0530] [Structure of Peptide Structure 94]

[0531]

[0532] [Structure of Peptide Structure 95]

[0533]

[0534] [Structure of Peptide Structure 96]

[0535]

[0536] [Structure of Peptide Structure 97]

[0537]

[0538] [Structure of Peptide Structure 98]

[0539]

[0540] [Structure of Peptide Structure 99]

[0541]

[0542] [Structure of Peptide Structure 100]

[0543]

[0544] [Structure of Peptide Structure 101]

[0545]

[0546] [Structure of peptide structure 102]

[0547]

[0548] [Structure of peptide structure 103]

[0549]

[0550] [Structure of peptide structure 104]

[0551]

[0552] [Structure of Peptide Structure 105]

[0553]

[0554] [Structure of peptide structure 106]

[0555]

[0556] [Structure of peptide structure 107]

[0557]

[0558] [Structure of peptide structure 108]

[0559]

[0560] [Structure of Peptide Structure 109]

[0561]

[0562] [Structure of Peptide Structure 110]

[0563]

[0564] [Structure of Peptide Structure 111]

[0565]

[0566] [Structure of Peptide Structure 112]

[0567]

[0568] [Structure of Peptide Structure 113]

[0569]

[0570] [Structure of Peptide Structure 114]

[0571]

[0572] [Structure of Peptide Structure 115]

[0573]

[0574] [Structure of Peptide Structure 116]

[0575]

[0576] [Structure of Peptide Structure 117]

[0577]

[0578] [Structure of Peptide Structure 118]

[0579]

[0580] [Structure of Peptide Structure 119]

[0581]

[0582] [Structure of Peptide Structure 120]

[0583]

[0584] [Structure of Peptide Structure 121]

[0585]

[0586] [Structure of Peptide Structure 122]

[0587]

[0588] [Structure of Peptide Structure 123]

[0589]

[0590] [Structure of Peptide Structure 124]

[0591]

[0592] [Structure of Peptide Structure 125]

[0593]

[0594] [Structure of Peptide Structure 126]

[0595]

[0596] [Structure of Peptide Structure 127]

[0597]

[0598] [Structure of Peptide Structure 128]

[0599]

[0600] [Structure of Peptide Structure 129]

[0601]

[0602] [Structure of Peptide Structure 130]

[0603]

[0604] [Structure of Peptide Structure 131]

[0605]

[0606] [Structure of Peptide Structure 132]

[0607]

[0608] [Structure of Peptide Structure 133]

[0609]

[0610] [Structure of Peptide Structure 134]

[0611]

[0612] [Structure of Peptide Structure 135]

[0613]

[0614] [Structure of Peptide Structure 136]

[0615]

[0616] [Structure of Peptide Structure 137]

[0617]

[0618] [Structure of Peptide Structure 138]

[0619]

[0620] [Structure of Peptide Structure 139]

[0621]

[0622] [Structure of Peptide Structure 140]

[0623]

[0624] [Structure of Peptide Structure 141]

[0625]

[0626] [Structure of Peptide Structure 142]

[0627]

[0628] [Structure of Peptide Structure 143]

[0629]

[0630] [Structure of Peptide Structure 144]

[0631]

[0632] [Structure of Peptide Structure 145]

[0633]

[0634] [Structure of Peptide Structure 146]

[0635]

[0636] [Structure of Peptide Structure 147]

[0637]

[0638] [Structure of Peptide Structure 148]

[0639]

[0640] [Structure of Peptide Structure 149]

[0641]

[0642] [Structure of Peptide Structure 150]

[0643]

[0644] [Structure of Peptide Structure 151]

[0645]

[0646] [Structure of Peptide Structure 152]

[0647]

[0648] [Structure of Peptide Structure 153]

[0649]

[0650] [Structure of Peptide Structure 154]

[0651]

[0652] [Structure of Peptide Structure 155]

[0653]

[0654] [Structure of Peptide Structure 156]

[0655]

[0656] [Structure of Peptide Structure 157]

[0657]

[0658] [Structure of Peptide Structure 158]

[0659]

[0660] [Structure of Peptide Structure 159]

[0661]

[0662] [Structure of Peptide Structure 160]

[0663]

[0664] [Structure of Peptide Structure 161]

[0665]

[0666] [Structure of Peptide Structure 162]

[0667]

[0668] [Structure of Peptide Structure 163]

[0669]

[0670] [Structure of Peptide Structure 164]

[0671]

[0672] [Structure of Peptide Structure 165]

[0673]

[0674] [Structure of Peptide Structure 166]

[0675]

[0676] [Structure of Peptide Structure 167]

[0677]

[0678] [Structure of Peptide Structure 168]

[0679]

[0680] [Structure of Peptide Structure 169]

[0681]

[0682] [Structure of Peptide Structure 170]

[0683]

[0684] [Structure of Peptide Structure 171]

[0685]

[0686] [Structure of Peptide Structure 172]

[0687]

[0688] [Structure of Peptide Structure 173]

[0689]

[0690] [Structure of Peptide Structure 174]

[0691]

[0692] [Structure of Peptide Structure 175]

[0693]

[0694] [Structure of Peptide Structure 176]

[0695]

[0696] [Structure of Peptide Structure 177]

[0697]

[0698] [Structure of Peptide Structure 178]

[0699]

[0700] [Structure of Peptide Structure 179]

[0701]

[0702] [Structure of Peptide Structure 180]

[0703]

[0704] [Structure of Peptide Structure 181]

[0705]

[0706] [Structure of Peptide Structure 182]

[0707]

[0708]

[0709] Example 4. Evaluation of the stability of the peptide structure

[0710] Stability tests in serum and plasma were performed on 83 of the peptide structures obtained in Example 3 above. A peptide structure solution diluted to a concentration of 400 μM was aliquoted into 1 mL of 100% pure human serum (Cat# S1, Merk) or plasma (Cat# 70039.1, STEMCELL Technologies) in 50 μL aliquots and then analyzed for 9 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 50 mm, 2.7 μm) while stored at 37 ℃. Before injecting the sample into the LC, 100 μL of ACN was added to a serum or plasma solution containing peptides, and after centrifugation to settle the precipitate, only 20 μL of the supernatant was used for analysis.

[0711] Figure 5 shows the results of the stability test of peptide structure No. 83 in serum and plasma, and it was completely maintained without any degradation in serum and plasma even after 9 days.

[0712]

[0713] Example 5. Confirmation of biological properties of the peptide structure

[0714] 5-1. Analysis of binding affinity and binding kinetics for hFAP-α

[0715] The binding affinity and binding kinetics of peptide construct 84 for hFAP-α were analyzed. To confirm specific binding selectivity for hFAP-α, the binding kinetics of the peptide construct were also analyzed for dipeptidylpeptidase 4 (DPP4, Cat# DP4-H5221, ACROBiosystems) and prolyl oligopeptidase (PREP, Cat# 4308-SE-010, R&D systems) proteins.

[0716] Binding affinity was analyzed using a streptavidin biosensor (Cat# 18-5019, Sartorius) with the advanced kinetics module of BLItz Pro ver1.3 software and a BLItz® system (Cat# 45-5000, Fortibio, USA) based on Bio-Layer Interferometry (BLI) technology. In addition, to correct for background of the streptavidin biosensor protein, the lowest concentration among the protein concentrations used was used as the reference value. 10X Octet kinetics buffer (Cat# 18-1105, Sartorius) was dispensed at a rate of 200 μL / well into 96-well black, flat-bottom polypylene (Cat# 655209, Greiner Bio-One), and the streptavidin biosensor was immersed and hydrated for 10 minutes. The peptide was diluted to a concentration of 1 μM by preparing 1X peptide buffer with DPBS (Cat# L0615, Biowest) using 10X Octet Kinetics Buffer. After attaching the biosensor to the instrument, the peptide was loaded at 2200 rpm for 120 seconds to induce peptide attachment. Subsequently, protein association and dissociation were performed at each concentration at 2200 rpm for 120 seconds, and the kinetics data were analyzed using the global fitting function. D The value is k d vs k a It was calculated at the ratio. In addition, kinetic parameters were obtained through baseline correction and fitting a 1:1 binding model.

[0717] The results are shown in Figure 6. Peptide structure 84 showed high selectivity for hFAP-α compared to DPP4 and PREP.

[0718]

[0719] 5-2. Analysis of Binding Affinity Using Fluorescence Polarization

[0720] K for hFAP-α through fluorescence polarization analysis capable of confirming binding / interaction with proteins D The values ​​were calculated. The fluorescence polarization experiment was performed on 96-well, black, flat-bottom polystyrene (Cat# 655209, Costar ® It was performed in a ) and a microplate reader (TECAN SPARK ® Fluorescence polarization was measured in ). hFAP-α protein was diluted with 50 mM Tris (Cat# TR2073-050-75, Biosesang), 100 mM NaCl (Cat# SR1009-500-00, Biosesang), and 1 mM EDTA (ML005-01, welgene) buffer solutions and dispensed 50 μL into each well of a 96-well plate, and the Alexa488-bound peptide (No. 82) was also diluted with the same buffer solutions and injected into each well at a concentration of 1 nM. The volume per well of the 96-well, black, flat-bottom polystyrene plate was 100 μL, and the experiment was repeated 3 times. The microplate reader was set to 25°C in kinetic mode to take measurements every 30 minutes for 2 hours, and K for hFAP-α was measured using Prism 10 with the 2-hour measurement values ​​at which equilibrium is reached for each protein concentration. D The value was derived (Fig. 7).

[0721]

[0722] 5-3. Analysis of hFAP-α activity

[0723] Recombinant hFAP-α protein was diluted to 3.6 nM and the peptide to a concentration range of 0.03–3 μM using pH 7.5 buffer solution (50 mM Tris, 1 M NaCl, 1 mg / mL bovine serum albumin (BSA, Cat# P6154-100GR, Biowest)), and Z-Gly-Pro-7-amino-4-methylcoumarin (Z-GP-AMC, Cat# HY-D1670, MedChemExpress), used as the reaction substrate, was diluted to 150 μM. The final DMSO concentration was maintained constant during the experiment to exclude solvent effects, and all buffer solutions and samples used were stored at 37 °C before use. 30 μL of the hFAP-α protein dilution and 30 μL of the peptide dilution were added to a 96-well plate and incubated at 37 °C for 5 minutes; afterward, 30 μL of the reaction substrate (Z-GP-AMC) Microplate reader set to 37 °C by adding (TECAN SPARK ® The AMC emission rate (Excitation 380 nm, Emission 465 nm) was measured using Prism 10 and IC 50 Values ​​were derived (Figs. 8a to 8o).

[0724] To confirm the selectivity of the peptide construct for hFAP-α binding, the activities of DPP4 and PREP were analyzed, performed in the same manner as the hFAP-α activity analysis, except for the conditions below. The DPP4 activity analysis was performed with recombinant human DPP4 protein in DPP4 analysis buffer (25 mM Tris, pH 8.0), using Gly-Pro-7-amido-4-methylcoumarin (Gly-Pro-AMC,Cat# HY-137834A, MedChemExpress) as the substrate (Figs. 8p to 8t).

[0725]

[0726] 5-4. Analysis of Binding Specificity to Target Cells

[0727] In vitro cell binding characteristics were analyzed to determine whether the peptide construct specifically binds to target cells expressing hFAP-α. HEK293-huFAP cells (positive control), in which high levels of hFAP-α expression were artificially induced in HEK293 MZ cells that do not naturally express hFAP-α, and HEK293-NCV (non-coding-vector) cells (negative control), which do not express hFAP-α, were purchased from INSCREENEX (ID #T33J-7 / 14, #T25J-2 / 18 K10, Germany).

[0728]

[0729] 5-4-1. Fluorescence-activated Cell Sorting (FACS) Analysis

[0730] Peptide structures attached with the fluorescent dye Cy5 are measured using a NanoPhotometer for accurate quantification. ® Absorbance was measured using (N60, IMPLEN), and concentration was calculated according to Lambert-Beer's law. The freeze-dried peptide constructs were dissolved in dimethyl sulfoxide (DMSO) to prepare a millimola (mM) solution, and then, using a NanoPhotometer ® The absorbance of 1.5 μL diluted 100-fold in PBS was measured at 650 nm using equipment. 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 20 μM solution, and then diluted to 5 nM using a mixture of Dulbecco's Modified Eagle Medium (DMEM) containing 1% fetal bovine serum (FBS) (buffer solution for FACS).

[0731] 2–3 days before performing FACS analysis, the HEK293-NCV cell line and HEK293-huFAP cell line were 5 X 10 6 Canine cells were seeded. On the day of the experiment, the culture medium was removed from T175 flasks containing HEK293-NCV or HEK293-huFAP cell lines cultured at 80% confluency and washed twice with 10 mL of PBS buffer. 2 mL of trypsin-EDTA was added in an incubator (37°C, 5% CO2, humid conditions, Galaxy 170 S) for 2 minutes, after which the trypsin-EDTA was neutralized with 8 mL of culture medium. The neutralized cell mixture and 10 mL of culture medium were added to the flask, and the excess cell mixture was transferred to 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. 5The cells were diluted to a cell count and dispensed in 200 μL aliquots into 96-well round plates (Cat# 34096, SPL LIFE SCIENCE). After centrifuging at 1000 rpm for 3 minutes using a VARISPIN 15R centrifuge, the supernatant was removed from each well, 200 μL of the pre-prepared peptide construct solution was added, and the cells were incubated in an incubator. After 1 hour, the cells were centrifuged at 1000 rpm for 3 minutes using a VARISPIN 15R centrifuge, the supernatant was removed, and the cells were washed with 200 μL of FACS buffer. After washing, the cells were centrifuged at 1000 rpm for 3 minutes, and the supernatant was removed. After washing once more using the same method, 200 μL of FACS buffer was added to each well and mixed with the cells. Next, the cells were transferred to a 5 mL round-bottom tube (Cat# 352235, CORNING) equipped with a cell strainer, and the fluorescence intensity of each cell was measured using a BD Accuri C6 Plus (BD Biosciences, Singapore). The measured data were analyzed using FlowJo 10.7.1 (BD Biosciences, USA).

[0732] The results of FACS analysis using the HEK293-huFAP cell line are shown in FIGS. 9a to 9f, and the results of FACS analysis using the HEK293-NCV cell line are shown in FIGS. 9g to 9l. From the FACS analysis results, it was confirmed that the peptide structure of the present invention binds to target cells expressing hFAP-α with high affinity and selectivity.

[0733]

[0734] 5-4-2. Fluorescence Image Analysis (Cellular Internalization)

[0735] HEK293-huFAP cell line 1 X 10 5Canine cells were seeded into an imaging dish (Cat#81156, Ibidi GmbH) and cultured in an incubator for one day. Subsequently, each cell line was treated with peptide construct 8 diluted in 1 mL of FACS buffer to a final concentration of 5 nM, and cultured for 1 hour under the same conditions as above. Then, the dish was washed once with 1 mL of FACS buffer. For the dishes to be analyzed at 24 and 72 hours, 2 mL of culture medium was added, and fluorescence images were observed by performing the following procedures at each time. For the 1-hour fluorescence image analysis dish, the dish was washed with FACS buffer, then treated with Hoechst 33342 diluted in 1 mL of FACS buffer to a final concentration of 0.5 μg / mL, and cultured in an incubator for 10 minutes. Afterward, the 1 mL of FACS buffer was removed, the dish was washed once with PBS, and fluorescence was observed using an Axio Observer 3 (ZEISS, Germany).

[0736] Figure 10 shows the results of the fluorescence analysis of peptide construct 8. It was confirmed that peptide construct 8 specifically binds to the HEK293-huFAP cell line expressing hFAP-α.

[0737]

[0738] 5-4-3. Total Fluorescence Analysis

[0739] The peptide construct to be used for analysis was dissolved in DMSO to make a 20 μM solution, and then diluted to a desired concentration of 5 nM using FACS buffer.

[0740] HEK293-huFAP cell line 1 X 10 5Canine cells were seeded into 35 mm dishes (Cat# 20035, SPL LIFE SCIENCE) and cultured in an incubator for one day. Subsequently, each cell line was treated with peptide construct 8 diluted in 1 mL of FACS buffer to a final concentration of 5 nM, and then cultured in an incubator for 1 hour. Afterward, the cells were washed once with 1 mL of PBS solution. For the dishes to be analyzed for total fluorescence at 24, 48, and 72 hours, 2 mL of culture medium was added, and fluorescence was observed by performing the following procedures at each time. For the 1-hour total fluorescence analysis, after washing with PBS, 200 μL of trypsin-EDTA was treated in an incubator for 2 minutes, and then the trypsin-EDTA was neutralized with 800 μL of culture medium. The neutralized cell mixture was placed in a 1.5 mL e-tube and centrifuged at 3000 rpm for 3 minutes using a Labogene 1736R centrifuge, after which the supernatant was removed. Next, 1 mL of PBS was added and mixed with the cells, followed by centrifugation to remove the supernatant. Then, 200 μL of PBS was added and mixed with the cells, transferred to a 96-well plate (Cat# 3916, CORNING), and placed on a microplate reader (TECAN SPARK ® The total fluorescence intensity was measured using ) (Fig. 11a).

[0741] Primary lung fibroblast (Cat# PCS-201-013, ATCC) cell line 2 5Canine cells were seeded into a 35 mm dish (Cat# 20035, SPL LIFE SCIENCE) and cultured in an incubator for one day. Afterward, the existing culture medium was removed, 2 ml of FBS-free starvation medium was added, and the cells were cultured in the incubator for one day. Subsequently, the starvation medium was removed, and 2 ml of myofibroblast differentiation medium was added and cultured for 3 days. The culture medium of the differentiated cells was removed, and the cell line was treated with peptide construct 8 diluted in 1 ml of starvation medium to a final concentration of 10 nM. The cells were then incubated at 4 ℃ for 1 hour and in an incubator for 5 minutes. Afterward, the cells were washed once with 1 ml of PBS solution. For the dish to be analyzed for total fluorescence at 24 hours, 2 ml of culture medium was added, and after 24 hours in the incubator, fluorescence was observed by performing the procedure as follows. For the analysis of 1-hour total fluorescence, the dish was washed with PBS solution, treated with 500 μL of accutase cell dissociation reagent (Cat# A1110501, Gibco) in an incubator for 3 minutes, then transferred to a 1.5 mL e-tube and centrifuged using a Labogene 1736R centrifuge at 3000 rpm for 3 minutes to remove the supernatant. Next, 200 μL of PBS was added and mixed with the cells, then transferred to a 96-well plate (Cat# 3916, CORNING) and placed on a microplate reader (TECAN SPARK ® The total fluorescence intensity was measured using ) (Fig. 11b).

[0742] In Figures 11a and 11b, it was confirmed that peptide construct 8 specifically binds to the HEK293-huFAP cell line and myofibroblast expressing hFAP-α and remains in the cells for 72 hours.

[0743]

[0744] 5-5. Biodistribution Analysis Using Experimental Animal Models

[0745] 5-5-1. HEK293-huFAP xenograft model

[0746] Using 6-week-old female BALB / c nude mice (Hana Biotech, Korea) 5 X 10 6 HEK293-huFAP cells were injected subcutaneously into the upper right forelimb of nude mice. After cell injection, the average tumor size was 110–120 mm 3 Grouping was performed into groups of 3 animals to correspond to [Volume = (Width)]. Tumor size was measured using a caliper [Volume = (Width)] 2 It was measured as [X (length) / 2].

[0747] After xenografting and grouping the HEK293-huFAP cell line, three peptide constructs (1, 6, 8) were injected intravenously at 200 μL (1% DMSO in PBS) at 10 μM each. Bioavailability was confirmed by whole-body fluorescence imaging 24 or 72 hours after injection and ex vivo imaging after sacrificing the mice (Fig. 12a).

[0748] Six peptide constructs (8, 85, 86, 87, 88, 27) were injected via tail IV at 200 μL (0.5% DMSO in PBS) at 5 μM. Bioavailability was confirmed by whole-body fluorescence imaging 24 hours after injection and ex vivo imaging after sacrificing the mice (Fig. 12b).

[0749] Six peptide constructs (131, 162, 169, 170, 171, 172) were injected via tail IV at 200 μL (0.5% DMSO in PBS) at 5 μM. Bioavailability was confirmed by whole-body fluorescence imaging 24 hours after injection and ex vivo imaging after sacrificing the mice (Fig. 12c).

[0750] Two types of peptide constructs (176, 177) were injected via tail IV at 200 μL (0.5% DMSO in PBS) at 5 μM. Bioavailability was confirmed by whole-body fluorescence imaging 48 hours after injection and ex vivo imaging after sacrificing the mice (Fig. 12d).

[0751] Six types of peptide constructs (173, 174, 175, 178, 179, 180) were injected via tail IV at 200 μL (0.5% DMSO in PBS) at 5 μM. Bioavailability was confirmed by whole-body fluorescence imaging 48 hours after injection and ex vivo imaging after sacrificing the mice (Fig. 12e).

[0752] To verify biodistribution, mice were anesthetized by inhalation using the in vivo imaging device AMI HTX (Spectral Instruments Imaging) to capture whole-body fluorescence images. Afterward, the mice were sacrificed by carbon dioxide injection, laparotomy was performed, and tumors and organs (spleen, lungs, heart, liver, kidneys, stomach, pancreas, small intestine, and large intestine) were ex vivo images were taken. The fluorescence intensities obtained were presented as bar graphs including individual variations. The results were based on the area adjusted for the smallest tumor size (0.09 cm²). 2 Based on ), the same area (0.09 cm²) in the part with the strongest fluorescence intensity in the tumor, kidney, liver, spleen, lung, and heart 2It was derived by applying ). This work was performed using Aura imaging software (Spectral imaging, USA), and the unit of fluorescence intensity is photons / s / cm. 2 / sr. It was confirmed that peptide constructs that specifically bind to tumors overexpressing hFAP-α compared to other organs remain in the tumor after 24, 48, or 72 hours.

[0753]

[0754] 5-5-2. HT-29 xenograft model

[0755] Using 6-week-old female BALB / c nude mice (Hana Biotech, Korea) 5 X 10 6 The HT-29 colorectal cancer cell line was injected subcutaneously into the upper right forelimb of nude mice. After cell injection, the average tumor size was 80–120 mm 3 Grouping was performed into groups of 6 animals to correspond to [Volume = (Width)]. Tumor size was measured using a caliper [Volume = (Width)] 2 It was measured as [X (length) / 2].

[0756] After xenografting and grouping HT-29 colorectal cancer cell lines, peptide construct 87 was injected via tail IV at 200 μL (0.5% DMSO in PBS) at a concentration of 5 μM. Bioavailability was confirmed by whole-body fluorescence imaging 24 or 72 hours after injection and ex vivo imaging after sacrificing the mice (Fig. 13).

[0757] To verify biodistribution, mice were anesthetized by inhalation using the in vivo imaging device AMI HTX (Spectral Instruments Imaging) to capture whole-body fluorescence images. Afterward, the mice were sacrificed by carbon dioxide injection, laparotomy was performed, and tumors and organs (spleen, lungs, heart, liver, kidneys, stomach, pancreas, small intestine, and large intestine) were ex vivo images were taken. The fluorescence intensities obtained were presented as bar graphs including individual variations. The results were based on the area adjusted for the smallest tumor size (0.09 cm²). 2 Based on ), the same area (0.09 cm²) in the part with the strongest fluorescence intensity in the tumor, kidney, liver, spleen, lung, and heart 2 It was derived by applying ). This work was performed using Aura imaging software (Spectral imaging, USA), and the unit of fluorescence intensity is photons / s / cm. 2 / sr. It was confirmed that peptide construct 87 specifically binds to cancer-associated fibroblasts expressing FAP-α compared to other organs, and remained in the cancer-associated fibroblasts even after 72 hours.

[0758]

[0759] 5-6. Isotopes ( 177 In vivo SPECT / CT imaging experiment of Lu-labeled peptide constructs

[0760] To confirm the imaging diagnostic and therapeutic capabilities of peptide constructs 128, 129, 145, 160, 161, 162, 181, and 182, 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 the isotope diluted in 0.04 M hydrochloric acid solution ( 17710 mCi of LuCl3 (ITM Medical Isotopes GmbH) was dispensed and the solution was completely evaporated by nitrogen purging. To this, 90 μL of 0.2 M sodium acetate (pH 5.5) buffer solution was added, mixed with 10 μL of peptide stock solution, and isotope labeling was performed at 90°C for 30 minutes. Once the labeling of each substance was complete, the solution was cooled to room temperature, and the remaining 177 A Sep-Pak C18 column (Cat# 186005125, Waters) was used for Lu removal. The peptide construct was diluted with 5 mL of distilled water, loaded onto an activated Sep-Pak column (5 mL of ethanol, 5 mL of distilled water), washed with 5 mL of distilled water, eluted with 300 μL of 60% ethanol solution, and diluted with saline solution for use.

[0761] The average size of tumors generated by transplanting the HEK293-huFAP cell line was 120 or 200 mm 3 When reaching the inner and outer, 177 Lu-labeled peptide constructs 128, 129, 145, 160, 161, and 162 had a radiation dose of 600 μCi, 177 Lu-labeled peptide constructs 181 and 182 were administered once via tail vein injection of 200 μL of the isotope, corresponding to a radiation dose of 500 μCi, to mice, and SPECT / CT (Siemens Inveon, software: Inveon Acquisition Workplace) measurements were taken at 90 minutes, 1 day, and 2 days. For imaging, mice were respiratory anesthetized with 0.2% isoflurane in oxygen for 3–5 minutes, then the anesthetized mice were placed in the equipment and the anesthetic was controlled to be continuously infused at 1.5 L / min. SPECT measurements were taken at 35 minutes and CT at 7 minutes, and images were acquired using Inveon Research Workplace 4.2.

[0762] The SPECT / CT images obtained above are shown in Figures 14a, 15a, and 16a.

[0763]

[0764] 5-7. Isotopes ( 177 Analysis of the anticancer effects of CAIX-targeted peptides labeled with Lu)

[0765] Isotope 177 To confirm the anticancer effects of Lu-labeled peptide constructs 145, 160, 128, 129, 161, 162, 181, and 182, 6-week-old female BALB / c nude mice (Orient Bio or Hana Bio, Korea) were used to 5 x 10 6 HEK293-huFAP cells were injected subcutaneously into the upper right forelimb of nude mice. After cell injection, the average tumor size was 120 or 200 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].

[0766] A control group that was xenografted with HEK293-huFAP cell lines, grouped, and then injected with saline, and 177 Approximately 500 μCi of each Lu-labeled peptide construct (145, 160, 128, 129, 161, 162, 181, and 182) was injected via tail vein, and body weight and tumor size were measured twice a week. To comply with the humane termination criteria for the tumor experiment, the tumor size was 2000 mm². 3 Euthanasia was performed upon exceeding the limit.

[0767] The results are shown in FIGS. 14b, 14c, 15b, 15c, 16b, and 16c, respectively. As can be seen in these figures, 177Lu-labeled peptide constructs (145, 160, 128, 129, 161, 162, 181, 182) exhibited an anticancer effect that significantly reduced the size of tumors overexpressing hFAP-α without causing serious changes in mouse body weight.

[0768] Ranking list

[0769] Seq ID No.Amino Acid Sequences1GPFXQ (here, X=f(F5))2PVSLR3EXRGL (here, X=f(F5))4GDXQR (here, X=f(F5))5SDXRR (here, X=f(F5))6GEXLR (here, X=f(F5))7SXRYL (here, X=f(F5))8RPPXR (here, X=f(F5))9PDXIR (here, X=f(F5))10PSQXR (here, X=f(F5))11PVEFR12ENIXR (here, X=f(F5))13RAYXR (here, X=f(F5))14AXRYT (here, X=f(F5))15PXVNR (here, X=f(F5))16PSXNR (Here, X=f(F5))17RLDYR18RAWXN (Here, X=f(F5))19RSWXA (Here, X=f(F5))20RNWXN (Here, X=f(F5))21DFNXR (Here, X=f(F5))22PQDXR (Here, X=f(F5))23PXTPR (Here, X=f(F5))24PFTFR25RSWXR (Here, X=f(F5))26RSWXK (Here, X=f(F5))27RLWXR (Here, X=f(F5))28RAWXK (Here, X=f(F5))29RWGXK (Here, X=f(F5))30RLAXK (Here, X=f(F5))31PVDXR (Here, X=f(F5))32DVWXR (where, X=f(F5))33NVWFR34RAWFD35DNRFV36IDVXR (where, X=f(F5))37FFEFR38GPEXR (where, X=f(F5))39XPDTR (where, X=f(F5))40TYRYL41APELR42RAWXR (where, X=f(F5))43RVWXR (where, X=f(F5))44RIWXR (where, X=f(F5))45RWDXR (where, X=f(F5))46PEIXR (where, X=f(F5))47RSDFR48RADYR49DYTXR (where, X=f(F5))50RLAYR51EIRNF52PEILR53EXRVT (Here, X=f(F5))54PIDFR55RNWXA (Here,X=f(F5))56PGDFR57DYRPY58YYEVR59AVEXR (here, X=f(F5))60DFYLR61EGXXR (here, X=f(F5))62FVETR63PIEPR64DSTFR65DQXYR (here, X=f(F5))66DQVFR67SESXR (here, X=f(F5))68RWDXK (here, X=f(F5))69FPEYR70DNRWR71ADTXR (here, X=f(F5))72DXPXR (here, X=f(F5))73EXVNR (here, X=f(F5))74DYPIR75FVDYR76RAWYR77DNRYX (here, X=f(F5))78DWQFR79PXDSR (Here, X=f(F5))80XFESR (Here, X=f(F5))81APPXR (Here, X=f(F5))82RAPXR (Here, X=f(F5))83RPAXR (Here, X=f(F5))84RPPAR85RPPXA (Here, X=f(F5))86X1PPX2R (Here, X1=R(dimet), X2=f(F5))87RPPX1X2 (Here, X1=f(F5), X2=R(dimet))88X1PPX2X1 (Here, X1=R(dimet), X2=f(F5))89EREEX (Here, X=Pra(Cy5))90EREEX (Here, X=Pra(AF488))91EREEX (Here, X=Uk(DOTA))92EX1EX2 (where, X1=Pra(Cy5), X2=Uk(IB))93EX1EX2X3 (where, X1=f(F5), X2=Uk(IB), X3=Uk(DOTA))94EX1EEX2X3 (where, X1=f(F5), X2=Uk(IB), X3=Uk(DOTA))95EX1EX2X3 (where, X1=Uk(PhF5), X2=Uk(IB), X3=Uk(DOTA))96EX1EEX2X3 (where, X1=Uk(PhF5), X2=Uk(IB), X3=Uk(DOTA))97EWEX1X2 (where, X1=Uk(IB), X2=Uk(DOTA))98EREEX1X2X3 (where, X1=Pra(Cy5), X2=Uk(IB),X1=Pra(Cy5), X2=Uk(DOTA))100EX1EX2 (here, X1=Pra(Cy5), X2=Uk(Hex))101EREEWX (here, X=Uk(DOTA))102EREEX1X2 (here, X1=K(BHPV), X2=Uk(DOTA))103EREEX1X2 (here, X1=K(indole), X2=Uk(DOTA))104EREEX1X2 (here, X1=*Aha(indole), X2=Uk(DOTA))105EREEX1X2 (here, X1=Pra(Cy5), X2=DAP(DOTA))106EREEX1X2 (here, X1=Pra(Cy5), X2=DAP(DOTA))107AAAEREEX (here, X=Pra(Cy5))108AAGEREEX (here, X=Pra(Cy5))109AGGEREEX (here, X=Pra(Cy5))110REEEX (here, X=Pra(Cy5))111EEREX (here, X=Pra(Cy5))112EEERX (here, X=Pra(Cy5))113EREEX1X2 (here, X1=K(SZW-DAP-indole), X2=Uk(DOTA))114EX1EEX2X3 (here, X1=Uk(SZW), X2=K(indole), X3=Uk(DOTA))115EREX1EX2 (here, X1=K(indole), X2=DAP(DOTA))116EREEX1X2 (here, X1=K(indole), X2=DAP(DOTA))117EX1EEX2 (here, X1=R(dimet), X2=Pra(Cy5))118EREEX1X2 (here, X1=K(SZW-DAP-indole), X2=DAP(DOTA))119EREEX1X2 (here, X1=K(SZW-DAP-indole), X2=DAP(DOTA))120EREX1EX2 (here, X1=K(indole), X2=DAP(DOTA))121EREEX1X2 (here, X1=*DAP(SZW-DAP-indole), X2=DAP(DOTA))122EREEX1X2X3 (here, X1=K(indole), X2=DAP(SZW),X3=DAP(DOTA))123EREEX1X2X3 (where, X1=K(indole), X2=DAP(SZW), X3=DAP(DOTA))124EREX1X1X2 (where, X1=Pra(Cy5), X2=DAP(DOTA))125X1EEX2 (where, X1=DAP(DOTA), X2=K(indole))126EREEX1X2 (where, X1=Pra(Cy5), X2=DAP(DOTA))127X1EREEX2 (where, X1=DAP(DOTA), X2=Pra(Cy5))128X1EREEX2 (where, X1=Pra(Cy5), X2=DAP(DOTA))129X1EREEX2 (where, X1=Uk(PPG(hex)), X2=Pra(Cy5))130EREX1X2X3 (where X1=K(indole), X2=Pra(Cy5), X3=DAP(DOTA))131X1REEX2 (where X1=Uk(Gal), X2=Pra(Cy5))132EX1EEX2 (where X1=Uk(Gal), X2=Pra(Cy5))133X1X2REEX3 (where X1=DAP(DOTA), X2=Uk(Gal), X3=Pra(Cy5))134X1X2REEX3 (where X1=DAP(DOTA), X2=Uk(Gal), X3=Pra(Cy5)) In the above sequence, the amino acids may be D-amino acids or L-amino acids. In addition, the chemical structural formulas of the abbreviations used in each sequence are as follows: Same. Abbreviation Chemical structural formulaUk(DOTA)2,2',2''-(10-(2-((5,6-diamino-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acidDAP(DOTA)2,2',2''-(10-(2-((2-amino-2-carboxyethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acidUk(6OH)N, 6-((2R,3R,4S,5R,6R)-2,3,4,5,6,7-hexahydroxyheptanoyl)-D-lysineUk(PhF5)N 6 -(perfluorobenzoyl)-D-lysineUk(IB)N 6 -(4-(4-iodophenyl)butanoyl)-lysineUk(Hex)N 6 -hexanoyl-D-lysineUk(SZW)(R)-3-((2-((5-amino-5-carboxypentyl)amino)-2-oxoethyl)dimethylammonio)propane-1-sulfonateDAP(SZW)3-((2-((2-amino-2-carboxyethyl)amino)-2-oxoethyl)(metheyliumyl)(methyl)-λ 4 -azaneyl)propane-1-sulfonateK(BHPV)N 6-(4,4-bis(4-hydroxyphenyl)pentanoyl)-L-lysineK(indole)(S)-6-(4-((1H-indol-3-yl)methyl)-1H-1,2,3-triazol-1-yl)-2-aminohexanoic acid*Aha(indole)(S)-4-(4-((1H-indol-3-yl)methyl)-1H-1,2,3-triazol-1-yl)-2-aminobutanoic acidf(F5)(R)-2-amino-3-(perfluorophenyl)propanoic acidr(dimet)(R)-2-amino-5-((amino(dimethyl-l4-azaneylidene)methyl)amino)pentanoic acidR(dimet)(S)-2-amino-5-((amino(dimethyl-l4-azaneylidene)methyl)amino)pentanoic acidPEG12-(2-(2-aminoethoxy)ethoxy)acetic acidPEG(6atom)2-(2-aminoethoxy)acetic acidK(SZW-DAP-indole)3-((2-(((R)-2-(4-(4-((1H-indol-3-yl)methyl)-1H-1,2,3-triazol-1-yl)butanamido)-3-(((S)-5-amino-5-carboxypentyl)amino)-3-oxopropyl)amino)-2-oxoethyl)dimethylammonio)propane-1-sulfonate*DAP(SZW-DAP-indole)3-((2-(((R)-2-(4-(4-((1H-indol-3-yl)methyl)-1H-1,2,3-triazol-1-yl)butanamido)-3-(((S)-2-amino-2-carboxyethyl)amino)-3-oxopropyl)amino)-2-oxoethyl)dimethylammonio)propane-1-sulfonatePra(Cy5)1-(6-((3-(4-(2-amino-2-carboxyethyl)-1H-1,2,3-triazol-1-yl)propyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium -5-sulfonatePra(AF488)5-((6-(4-(2-amino-2-carboxyethyl)-1H-1,2,3-triazol-1-yl)hexyl)carbamoyl)-2-(6-amino-3-imino-4,5-disulfo-3H-xanthen-9-yl)benzoic acidUk(PPG(hex))N, 6 -hexanoylglycyl-L-prolyl-L-prolyl-D-lysineUk(Gal)N 6 -((2S,3R,4S,5R,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carbonyl)-D-lysine

[0770]

[0771] [Structure of Sequence No. 1]

[0772]

[0773] [Structure of Sequence No. 2]

[0774]

[0775] [Structure of Sequence No. 3]

[0776]

[0777] [Structure of Sequence No. 4]

[0778]

[0779] [Structure of Sequence No. 5]

[0780]

[0781] [Structure of Sequence No. 6]

[0782]

[0783] [Structure of Sequence No. 7]

[0784]

[0785] [Structure of Sequence No. 8]

[0786]

[0787] [Structure of Sequence No. 9]

[0788]

[0789] [Structure of Sequence No. 10]

[0790]

[0791] [Structure of Sequence No. 11]

[0792]

[0793] [Structure of Sequence No. 12]

[0794]

[0795] [Structure of Sequence No. 13]

[0796]

[0797] [Structure of Sequence No. 14]

[0798]

[0799] [Structure of Sequence No. 15]

[0800]

[0801] [Structure of Sequence No. 16]

[0802]

[0803] [Structure of Sequence No. 17]

[0804]

[0805] [Structure of Sequence No. 18]

[0806]

[0807] [Structure of Sequence No. 19]

[0808]

[0809] [Structure of Sequence No. 20]

[0810]

[0811] [Structure of Sequence No. 21]

[0812]

[0813] [Structure of Sequence No. 22]

[0814]

[0815] [Structure of Sequence No. 23]

[0816]

[0817] [Structure of Sequence No. 24]

[0818]

[0819] [Structure of Sequence No. 25]

[0820]

[0821] [Structure of Sequence No. 26]

[0822]

[0823] [Structure of Sequence No. 27]

[0824]

[0825] [Structure of Sequence No. 28]

[0826]

[0827] [Structure of Sequence No. 29]

[0828]

[0829] [Structure of Sequence No. 30]

[0830]

[0831] [Structure of Sequence No. 31]

[0832]

[0833] [Structure of Sequence No. 32]

[0834]

[0835] [Structure of Sequence No. 33]

[0836]

[0837] [Structure of Sequence No. 34]

[0838]

[0839] [Structure of Sequence No. 35]

[0840]

[0841] [Structure of Sequence No. 36]

[0842]

[0843] [Structure of Sequence No. 37]

[0844]

[0845] [Structure of Sequence No. 38]

[0846]

[0847] [Structure of Sequence No. 39]

[0848]

[0849] [Structure of Sequence No. 40]

[0850]

[0851] [Structure of Sequence No. 41]

[0852]

[0853] [Structure of Sequence No. 42]

[0854]

[0855] [Structure of Sequence No. 43]

[0856]

[0857] [Structure of Sequence No. 44]

[0858]

[0859] [Structure of Sequence No. 45]

[0860]

[0861] [Structure of Sequence No. 46]

[0862]

[0863] [Structure of Sequence No. 47]

[0864]

[0865] [Structure of Sequence No. 48]

[0866]

[0867] [Structure of Sequence No. 49]

[0868]

[0869] [Structure of Sequence No. 50]

[0870]

[0871] [Structure of Sequence No. 51]

[0872]

[0873] [Structure of Sequence No. 52]

[0874]

[0875] [Structure of Sequence No. 53]

[0876]

[0877] [Structure of Sequence No. 54]

[0878]

[0879] [Structure of Sequence No. 55]

[0880]

[0881] [Structure of Sequence No. 56]

[0882]

[0883] [Structure of Sequence No. 57]

[0884]

[0885] [Structure of Sequence No. 58]

[0886]

[0887] [Structure of Sequence No. 59]

[0888]

[0889] [Structure of Sequence No. 60]

[0890]

[0891] [Structure of Sequence No. 61]

[0892]

[0893] [Structure of Sequence No. 62]

[0894]

[0895] [Structure of Sequence No. 63]

[0896]

[0897] [Structure of Sequence No. 64]

[0898]

[0899] [Structure of Sequence No. 65]

[0900]

[0901] [Structure of Sequence No. 66]

[0902]

[0903] [Structure of Sequence No. 67]

[0904]

[0905] [Structure of Sequence No. 68]

[0906]

[0907] [Structure of Sequence No. 69]

[0908]

[0909] [Structure of Sequence No. 70]

[0910]

[0911] [Structure of Sequence No. 71]

[0912]

[0913] [Structure of Sequence No. 72]

[0914]

[0915] [Structure of Sequence No. 73]

[0916]

[0917] [Structure of Sequence No. 74]

[0918]

[0919] [Structure of Sequence No. 75]

[0920]

[0921] [Structure of Sequence No. 76]

[0922]

[0923] [Structure of Sequence No. 77]

[0924]

[0925] [Structure of Sequence No. 78]

[0926]

[0927] [Structure of Sequence No. 79]

[0928]

[0929] [Structure of Sequence No. 80]

[0930]

[0931] [Structure of Sequence No. 81]

[0932]

[0933] [Structure of Sequence No. 82]

[0934]

[0935] [Structure of Sequence No. 83]

[0936]

[0937] [Structure of Sequence No. 84]

[0938]

[0939] [Structure of Sequence No. 85]

[0940]

[0941] [Structure of Sequence No. 86]

[0942]

[0943] [Structure of Sequence No. 87]

[0944]

[0945] [Structure of Sequence No. 88]

[0946]

[0947] [Structure of Sequence No. 89]

[0948]

[0949] [Structure of Sequence No. 90]

[0950]

[0951] [Structure of Sequence No. 91]

[0952]

[0953] [Structure of Sequence No. 92]

[0954]

[0955] [Structure of Sequence No. 93]

[0956]

[0957] [Structure of Sequence No. 94]

[0958]

[0959] [Structure of Sequence No. 95]

[0960]

[0961] [Structure of Sequence No. 96]

[0962]

[0963] [Structure of Sequence No. 97]

[0964]

[0965] [Structure of Sequence No. 98]

[0966]

[0967] [Structure of Sequence No. 99]

[0968]

[0969] [Structure of Sequence No. 100]

[0970]

[0971] [Structure of Sequence No. 101]

[0972]

[0973] [Structure of Sequence No. 102]

[0974]

[0975] [Structure of Sequence No. 103]

[0976]

[0977] [Structure of Sequence No. 104]

[0978]

[0979] [Structure of Sequence No. 105]

[0980]

[0981] [Structure of Sequence No. 106]

[0982]

[0983] [Structure of Sequence No. 107]

[0984]

[0985] [Structure of Sequence No. 108]

[0986]

[0987] [Structure of Sequence No. 109]

[0988]

[0989] [Structure of Sequence No. 110]

[0990]

[0991] [Structure of Sequence No. 111]

[0992]

[0993] [Structure of Sequence No. 112]

[0994]

[0995] [Structure of Sequence No. 113]

[0996]

[0997] [Structure of Sequence No. 114]

[0998]

[0999] [Structure of Sequence No. 115]

[1000]

[1001] [Structure of Sequence No. 116]

[1002]

[1003] [Structure of Sequence No. 117]

[1004]

[1005] [Structure of Sequence No. 118]

[1006]

[1007] [Structure of Sequence No. 119]

[1008]

[1009] [Structure of Sequence No. 120]

[1010]

[1011] [Structure of Sequence No. 121]

[1012]

[1013] [Structure of Sequence No. 122]

[1014]

[1015] [Structure of Sequence No. 123]

[1016]

[1017] [Structure of Sequence No. 124]

[1018]

[1019] [Structure of Sequence No. 125]

[1020]

[1021] [Structure of Sequence No. 126]

[1022]

[1023] [Structure of Sequence No. 127]

[1024]

[1025] [Structure of Sequence No. 128]

[1026]

[1027] [Structure of Sequence No. 129]

[1028]

[1029] [Structure of Sequence No. 130]

[1030]

[1031] [Structure of Sequence No. 131]

[1032]

[1033] [Structure of Sequence No. 132]

[1034]

[1035] [Structure of Sequence No. 133]

[1036]

[1037] [Structure of Sequence No. 134]

[1038]

Claims

1. Includes any one of the amino acid sequences of SEQ ID NOs 1 to 88, wherein At least one of the constituent amino acids is composed of a D-amino acid, and A peptide ligand in which the β-phenyl group of the side chain of a phenylalanine (Phe) residue among the constituent amino acids can be substituted with a halogen; and A peptide structure characterized by comprising a substituted or unsubstituted pyrrolidinyl group connected directly or through a spacer to the above-mentioned peptide ligand, and having the structure of the following chemical formula 1. [Chemical Formula 1] In the above formula, P is the above-mentioned peptide ligand, and F1 is halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 1-10 Alkanoylamin, C(O)-C 1-6 Alkyl, C(O)-C 1-6 Haloalkyl, C(O)OC 1-6 Alkyl, C(O)OC 1-6 It is any one selected from the group consisting of haloalkyl, NH2, OH, CN, COOH, and NO2, and m is an integer from 0 to 8, and F2 is glycine (Gly), C(O)-C 1-6 Alkylene, C(O)-C 1-6 Any one selected from the group consisting of alkylene-(O)C and pyrrolidinyl group-containing amino acid residues, and n is an integer from 0 to 3, and F3 is -(S1) o -(F4) p -(S2) q -(F5) r -(S3) s -(F6) t -(S4) is the basis, and here S1, S2, and S3 are each independently spacers, and F4, F5, and F6 are each independently substituted or unsubstituted amino acid residues, and S4 is -NH2 or -OH, and o, p, q, r, s, and t each independently represent integers from 0 to 6.

2. In Paragraph 1, P, m, n, F3, S1, S2, S3, S4, F4, F5, F6, o, p, q, r, s and t are as defined in paragraph 1, and F1 is halogen, C 1-10 It is any one selected from the group consisting of alkanoylamino and CN, and F2 is glycine (Gly), C(O)-C 1-6 A peptide structure characterized by being selected from the group consisting of alkylene-(O)C and pyrrolidinyl group-containing amino acid residues.

3. In Paragraph 1 or 2, The pyrrolidinyl group within the above pyrrolidinyl group-containing amino acid residue may be substituted with one or more X1s, wherein X1 is a halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 1-10 Alkanoylamin, C(O)-C 1-6 Alkyl, C(O)-C 1-6 Haloalkyl, C(O)OC 1-6 Alkyl, C(O)OC 1-6 A peptide structure characterized by being any one selected from the group consisting of haloalkyl, NH2, OH, CN, COOH, and NO2.

4. In any one of paragraphs 1 through 3, A peptide structure characterized in that the above-mentioned pyrrolidinyl group-containing amino acid residue has the following structure. , or 5. In any one of paragraphs 1 through 4, A peptide structure characterized by comprising the structure of the following chemical formula 2, 3, 4, 5, 6, or 7. [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] In the above chemical formulas 2 to 7, F3 is as defined in paragraph 1.

6. In any one of paragraphs 1 through 5, One or more of the above F4, F5, and F6 are substituted amino acid residues, where The substituent of the above amino acid residue is 1) The ε-amino group of the side chain of a lysine residue; 2) Side chain β-amino group of 2,3-diaminopropionic acid (DAP) residue; 3) β-ethynyl group of the side chain of the propargylglycine residue; 4) β-phenyl group of the side chain of the phenylalanine (Phe) residue; 5) the side-chain amino group of the arginine residue; and 6) γ-amino group of the side chain of the azidohomoalanine residue A peptide structure characterized by being introduced through any one selected from the group consisting of 7. In any one of paragraphs 1 through 6, One or more of the above F4, F5, and F6 are substituted amino acid residues, where The substituents of the above amino acid residues are OH, chelator, indole, C 1-6 Alkyl, 4,4-(bis(4-hydroxyphenyl)valeric acid (BHPV), PEG24-biotin, hexanoylamino, 4-(p-iodophenyl)butyric acid (IB), pentafluorophenyl (PhF5), halogen, zwitteric ion (SZW), 1,2:3,4-di-O-isopropylidene-aD-galacturonide (1,2:3,4-Di-O-isopropylidene-aD-galacturonide, Gal), N 6 -Hexanoylglycyl-L-prolyl-L-prolyl-D-lysine(N 6 A peptide structure comprising one or more selected from the group consisting of -hexanoylglycyl-L-prolyl-L-prolyl-D-lysine), fluorescent dyes, and cytotoxic agents.

8. In Paragraph 7, The above 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), 1,4,7,10-tetraazacyclododecane-7-acetamide-1,4,10-triacetic acid (PSC) and A peptide structure characterized by being one or more selected from 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazcyclododec-1-yl]acetamide (DOTAM or TCMC).

9. In any one of paragraphs 1 through 8, A peptide structure characterized in that the above spacer is one or more selected from a polyethylene glycol (PEG) linker, glycine, sarcosine, and a peptide linker composed of 1 to 8 D-amino acids or L-amino acids.

10. In any one of paragraphs 1 through 9, A peptide structure characterized in that the above peptide structure is any one of compounds 1 to 182 having the structure of the following chemical formula 2, 3, 4, 5, 6 or 7. [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] As mentioned above, * or lowercase letters represent L-amino acids and uppercase letters represent D-amino acids, respectively, and other substituents indicated by abbreviations in AA1 to AA5 and F3 are as defined below.

11. A conjugate comprising a peptide structure according to any one of claims 1 to 10, which is directly or through a linker linked to one or more selected from the group consisting of fluorescent dyes, cytotoxic agents, and radioactive isotopes.

12. In claim 11, the peptide structure comprises a structure that is directly or through a linker coupled to a fluorescent dye and a cytotoxic agent, forming a conjugate.

13. The conjugate according to claim 11 or 12, wherein the peptide structure comprises a structure that is directly or through a linker coupled to a fluorescent dye and a radioisotope.

14. A conjugate according to any one of claims 11 to 13, 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).

15. A conjugate according to any one of claims 11 to 14, 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.

16. In any one of claims 11 to 15, 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), A conjugate characterized by being one or more selected from chromium-51 (Cr-51), krypton-81 (Kr-81), potassium-42 (K-42), holmium-166 (Ho-166), gallium-67 (Ga-67), gallium-68 (Ga-68), actinium-225 (Ac-225), zirconium-89 (Zr-89), thorium-227 (Th-227), radium-223 (Ra-223), tin-117m (Sn-117m), lead-103 (Pb-103), lead-212 (Pb-212), and astatine-211 (At-211).

17. A pharmaceutical composition for the diagnosis, prevention, or treatment of fibroblast-activated protein-alpha (FAP-α) overexpression diseases comprising a peptide structure according to any one of claims 1 to 10.

18. A pharmaceutical composition for the diagnosis, prevention, or treatment of fibroblast-activated protein-alpha (FAP-α) overexpression diseases comprising a conjugate according to any one of claims 11 to 16.

19. A pharmaceutical composition according to claim 17 or 18, wherein the fibroblast-activated protein-alpha (FAP-α) overexpression disease is any one selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid.

20. A pharmaceutical composition according to claim 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, heart cancer, ureteral cancer, renal cell carcinoma, renal-pelvic carcinoma, splenic cancer, melanoma, thyroid cancer, nasopharyngeal cancer, laryngeal cancer, myeloma, cholangiocarcinoma, clear cell carcinoma, neuroendocrine tumor, carcinogenic osteomalacia, sarcoma, carcinoma of unknown primary, thymic carcinoma, desmoid tumor, glioma, astrocytoma, or glioblastoma.

21. A pharmaceutical composition according to claim 19, wherein the fibrosis is pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, renal fibrosis, pancreatic fibrosis, cutaneous fibrosis, ocular fibrosis, skeletal muscle fibrosis, intestinal fibrosis, or pericardial fibrosis.

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