EpCAM-targeting cyclic peptide and radionuclide small molecule probe thereof and application

Abstract

The application belongs to the field of biological medicine, and relates to an EpCAM-targeting cyclic peptide and a nuclide small-molecule probe and application thereof. The sequence of the cyclic peptide is shown as formula I. Through systematic screening and optimization, the application obtains a candidate peptide probe suitable for EpCAM-targeting nuclear medical imaging or treatment application, and provides a feasible new-type molecular tool for early diagnosis and treatment of EpCAM-related tumors with high expression. Formula I.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an EpCAM-targeting cyclic peptide, a further labeled EpCAM-targeting nuclide small molecule probe, and its application. Background Technology

[0002] Epithelial cell adhesion molecule (EpCAM, also known as CD326) is a type I transmembrane glycoprotein with a molecular weight of approximately 40 kDa, first identified in colorectal cancer tissue in 1979. Encoded by the TACSTD1 gene, EpCAM is located on the short arm of chromosome 2 (2p21). Its molecular structure consists of an extracellular domain (approximately 80% of the total sequence length), a single transmembrane region, and a relatively short intracellular domain. Although it mediates cell adhesion, EpCAM does not belong to the four traditional families of cell adhesion molecules in terms of sequence and structure, and its adhesive activity is characterized by Ca²⁺. + It is independent of other processes and mainly participates in the regulation of epithelial cell adhesion, polarity maintenance, and migration by modulating the strength of intercellular adhesion. It plays an important role in cell signal transduction, interepithelial interactions, and tissue homeostasis.

[0003] In normal tissues, EpCAM is mainly expressed in the basolateral membrane of embryonic epithelial cells and most mature epithelial cells, while its expression is low or absent in connective tissue, hematopoietic cells, brain tissue, and vascular endothelium. Pathologically, EpCAM is widely upregulated in various epithelial tumors and is often enriched at tight junctions. Numerous studies have reported high expression of EpCAM in solid tumors such as breast cancer, liver cancer, gastrointestinal tumors, ovarian cancer, prostate cancer, and lung cancer, as well as in tumor stem cells and undifferentiated embryonic stem cells. In some tumors (e.g., pancreatic cancer, urothelial carcinoma, and gallbladder cancer), high EpCAM expression is associated with increased invasiveness and poor prognosis. Based on its broad tumor-specific expression and functional role in tumorigenesis, progression, and metastasis, EpCAM is considered a diagnostic and therapeutic target of significant clinical value.

[0004] Molecular imaging and targeted therapy research on EpCAM has been conducted for many years, and research on antibody- and its derivative-based radioactive probes is currently the most mature. Although antibody probes have high affinity and good specificity, their large molecular weight leads to long circulating half-life and slow pharmacokinetic characteristics. At the same time, their synthesis and modification are costly, and repeated dosing may induce immunogenic reactions. These characteristics significantly limit their application in routine clinical molecular imaging and radiotherapy.

[0005] In contrast, peptide molecular probes have become an important direction in nuclear medicine research and clinical translation due to their small molecular weight, convenient synthesis and structural modification, good tissue penetration, high targeting specificity, and low immunogenicity. Peptide probes are easy to label and functionalize with isotopes, offering adjustable advantages in imaging quality, renal clearance, and tissue background signal. However, current systematic research and preclinical validation of peptide-based EpCAM-targeting radionuclide probes are still limited. Further efforts are needed to screen superior ligands, optimize structures, and conduct comprehensive in vitro and in vivo characterization to meet imaging and therapeutic needs. Summary of the Invention

[0006] This invention uses mRNA display library technology to screen and obtain cyclic peptide sequences that specifically target EpCAM. Then, the target sequence is rationally structured, functionalized, chemically labeled, and radiolabeled. Finally, a series of in vitro binding properties, in vivo imaging, and pharmacokinetic evaluations are completed.

[0007] A first aspect of the present invention provides an EpCAM-targeting cyclic peptide, the sequence of which is shown in Formula I:

[0008] Formula I

[0009] Where C represents cysteine, X1 represents threonine, proline, valine, arginine, phenylalanine, or histidine, X2 represents tryptophan or threonine, X3 represents tyrosine, serine, histidine, or valine, X4 represents isoleucine, tryptophan, lysine, valine, or glutamine, X5 represents isoleucine or arginine, X6 represents serine, valine, glycine, or aspartic acid, X7 represents arginine, threonine, or serine, X8 represents asparagine, lysine, tyrosine, or histidine, X9 represents glutamic acid or histidine, X... 10 Indicates arginine, glutamic acid, tyrosine, or phenylalanine, X 11 Indicates leucine, phenylalanine, or isoleucine, X 12 The presence of valine is optional, with R1 being H or a carboxyl group and R2 being H or an amino group.

[0010] According to a preferred embodiment of the present invention, X1 represents threonine or valine, X2 represents tryptophan, X3 represents tyrosine, X4 represents isoleucine or lysine, X5 represents isoleucine, X6 represents serine or glycine, X7 represents arginine or threonine, X8 represents asparagine or tyrosine, X9 represents glutamic acid, and X... 10 X represents arginine or tyrosine. 11 X represents leucine or isoleucine. 12 It does not exist.

[0011] According to a more preferred embodiment of the present invention, X1 represents threonine, X2 represents tryptophan, X3 represents tyrosine, X4 represents isoleucine, X5 represents isoleucine, X6 represents serine, X7 represents arginine, X8 represents asparagine, X9 represents glutamic acid, and X... 10 Represents arginine, X 11 This represents leucine, X 12 It does not exist.

[0012] More preferably, the peptide sequence of the linear portion of the cyclic peptide (i.e., from the N-terminus to the C-terminus is A) C -CX1X2X3X4X5X6X7X8X9X 10 X 11 X 12 C-NH2) can be any one of the following sequences:

[0013] R1-CTWYIISRNERLC-R2;

[0014] R1-CPTSWRVTKHEFVC-R2;

[0015] R1-CVWYKIGTYEYIC-R2;

[0016] R1-CRWYIIDRNHYLC-R2;

[0017] R1-CFWHVIDSNHFLC-R2;

[0018] R1-CHWVQISSHEYLC-R2.

[0019] R1 can be H or carboxyl, and the N-terminus of the synthesized polypeptide is usually H. After further modification, a carboxyl N-terminus can be obtained. R2 can also be H or amino, and the C-terminus of the synthesized polypeptide is usually amino. After further treatment, a carboxyl C-terminus can be obtained.

[0020] According to some specific embodiments of the present invention, the peptide sequence of the linear portion of the cyclic peptide is any one of the following sequences:

[0021] A C -CTWYIISRNERLC-NH2 (SEQ ID NO: 1); The overall cyclic peptide structure is PYT1-1 in Table 1;

[0022] A C -CPTSWRVTKHEFVC-NH2 (SEQ ID NO: 2); The overall cyclic peptide structure is PYT1-2 as shown in Table 1;

[0023] A C-CVWYKIGTYEYIC-NH2 (SEQ ID NO: 3); The overall cyclic peptide structure is PYT1-3 in Table 1;

[0024] A C -CRWYIIDRNHYLC-NH2 (SEQ ID NO: 4); The overall cyclic peptide structure is PYT1-4 in Table 1;

[0025] A C -CFWHVIDSNHFLC-NH2 (SEQ ID NO: 5); The overall cyclic peptide structure is 2CT1-1 in Table 1;

[0026] A C -CHWVQISSHEYLC-NH2 (SEQ ID NO: 6); the overall cyclic peptide structure is 2CT1-3 in Table 1.

[0027] As is known in the art, the above letters are all abbreviations for amino acids, specifically: glycine (Gly / G), alanine (Ala / A), valine (Val / V), leucine (Leu / L), isoleucine (Ile / I), proline (Pro / P), phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W), serine (Ser / S), threonine (Thr / T), cysteine ​​(Cys / C), methionine (Met / M), asparagine (Asn / N), glutamine (Gln / Q), aspartic acid (Asp / D), glutamic acid (Glu / E), lysine (Lys / K), arginine (Arg / R), and histidine (His / H).

[0028] A second aspect of the present invention provides an EpCAM-targeted small molecule probe, wherein the probe is a cyclic peptide labeled with the aforementioned radionuclide.

[0029] The method of labeling peptides with radionuclides is well known in the art. The most common method is to achieve this by coupling with a bifunctional chelating agent. Specifically, the probe includes a cyclic peptide, a bifunctional chelating agent and a radionuclide. The bifunctional chelating agent is covalently linked to the cyclic peptide and the radionuclide is chelated with the bifunctional chelating agent.

[0030] According to a preferred embodiment of the present invention, the bifunctional chelating agent is DOTA, NOTA, NODA, NODAGA, DOTP, TETA, RESCA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A, NODAM, CB-DO2A, Cyclen, SHNH, NODA-AA, NETA, HETA, TRITA, DAR, DO3A, DO3AP, HYNIC, MAG2, MAS3, MAG3, TPEN, m-MeATE, AAZTA, DOTAGA, NODAGA, DOTMA, DOTAM, BAPTA-AM, TRAP, CB-TE2A, Cyclam, DADT, CE-DTS, NS3, CP256, PCTA, NODA-MPAA, HBED, THP, Macropa, isonitriles, porphyrins, polyamines, crown ethers, dithiocarbamates, or polyoximes and their derivatives.

[0031] The radionuclide can be a diagnostic radionuclide or a therapeutic radionuclide. The diagnostic radionuclide is preferably... 11 C 13 N、 14 C 15 O、 18 F, 24 Na、 32 P, 33 P, 35 S, 42 K, 43 Sc、 44 Sc、 45 Ti、 47 Sc、 51 Cr 51 Mn, 52 Mn, 52 Fe、 55 Co、 57 Co、 58m Co、 59 Fe、 60 Cu、 61 Cu、 62 Cu、 63 Zn, 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Sc、 75 Br、 76 Br、 77 Br、 77 As、 81m Kr、82 Rb、 86 Y、 87 Y、 90 Y、 89 Sr、 89 Zr、 94 Tc, 97 Ru、 99 Tc, 99m Tc, 99 Mo、 101m Rh、 103m Rh、 105 Pd, 105 Rh、 111 Ag、 111 ln、 117m Sn、 119 Sb、 123 I, 124 I, 125 I, 131 I, 133 Xe, 137 Cs、 142 Pr, 143 Pr, 149 Pm, 149 Tb, 152 Tb, 153 Sm、 154- 159 Gd, 161 Tb, 165 Dy、 166 Dy、 166 Ho、 169 Er、 169 Yb、 175 Yb、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 191m Pt, 193m Pt, 195m Pt, 194 lr、 197 Pt, 198 Au、 199 Au、 201 Tl、 203 Pb, 211 At、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra、 224 Ra and 225 At least one of Ac. The therapeutic radionuclide is preferably...32 P, 47 Sc、 51 Cr 57 Co、 58m Co、 60 Co、 61 Cu、 67 As、 89 Sr、 90 Y、 103 Pd, 103m Rh、 105 Rh、 106 Ru、 117m Sn、 119 Sb、 124 I, 125 I, 131 I, 131 Cs、 137 Cs、 149 Tb, 149 Pm, 161 Ho、 165 Dy、 177 Lu、 177 Yb、 186 Re、 188 Re、 192 Ir、 193m Pt, 195m Pt, 197 Pt, 199 Au、 201 Tl、 203 Pb, 211 As、 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 224 Ra、 225 Ac、 226 Th、 227 Th and 229 At least one of Th.

[0032] According to the present invention, the bifunctional chelating agent can be directly covalently linked to the cyclic peptide, for example, by linking it to the Ac at the N-terminus of the cyclic peptide, or it can be covalently linked via a linker, for example, by linking it to the NH2 at the C-terminus of the cyclic peptide via a linker selected from -(CH2CH2O). m -(CH2) n -(Lys) p -、-NH-(CH2) q -CO-、-(Gly) x -or-(GS) y -

[0033] Linker -(CH2CH2O) m -(CH2) n -(Lys) p - is a PEG-type connector, where m is an integer from 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, n is an integer from 1 to 6, for example, 1, 2, 3, 4, 5, 6, and p is 0 or 1, preferably 1.

[0034] Linker -NH-(CH2) q -CO- is a carbon chain linker, where q is an integer from 4 to 8, for example, 4, 5, 6, 7, 8, preferably 4, 5, 6, and the corresponding compounds are 4-aminobutyric acid group, 5-aminovaleric acid group, and 6-aminohexanoic acid group.

[0035] Connector - (Gly) x - is a polyglycine-type linker, where x is an integer from 3 to 8, for example, 3, 4, 5, 6, 7, 8.

[0036] Connector-(GS) y - is a flexible chain connector, where y is an integer from 1 to 6, for example, 1, 2, 3, 4, 5, 6.

[0037] The third aspect of this invention provides the following applications of the above-described EpCAM-targeted small molecule probe for targeting radionuclides:

[0038] (i) Prepare reagents for detecting EpCAM-related diseases; preferably, the reagents are kits for detecting EpCAM-related diseases;

[0039] (ii) To prepare a drug for the treatment of EpCAM-related diseases;

[0040] (iii) Detection of EpCAM expression level in test samples for non-diagnostic purposes.

[0041] In this invention, the EpCAM-related diseases mainly refer to epithelial tissue diseases, including but not limited to pneumonia, enteritis, lung cancer, gastric cancer, liver cancer, breast cancer, colorectal cancer, bladder cancer, testicular cancer, prostate cancer, pancreatic cancer, esophageal cancer, bile duct cancer, ovarian cancer, nasopharyngeal carcinoma, kidney cancer, spleen cancer, brain cancer, head and neck cancer, squamous cell carcinoma of the skin, urothelial carcinoma, endometrial cancer, oral squamous cell carcinoma, cervical squamous cell carcinoma, gallbladder cancer, hematologic malignancies, gastric ulcer, duodenal ulcer, or reflux esophagitis.

[0042] Through systematic screening and optimization, this invention has obtained candidate peptide probes suitable for EpCAM-targeted nuclear medicine imaging or therapeutic applications, providing a feasible new molecular tool for the early diagnosis and treatment of EpCAM-related tumors.

[0043] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0044] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0045] Figure 1-4 The images show the LC-MS spectra of the cyclic peptides byPYT1-1, byPYT1-3, byPYT1-4, and by2CT1-3, respectively.

[0046] Figure 5 The cyclic peptide probe in Example 4 [ 68 High performance liquid chromatogram of Ga]Ga-byPYT1-1.

[0047] Figure 6 This illustrates the different injection methods used in PC3 subcutaneous tumor mice in Example 4. 68 Comparison of PET / CT imaging results after 1 hour of Ga-labeled cyclic peptide probe.

[0048] Figure 7 The image shows the LC-MS spectrum of the probe byPYT1-1-FAM in Example 5.

[0049] Figure 8 The results of fluorescence confocal imaging of cells stained with the fluorescent probe by PYT1-1-FAM in Example 5 are shown.

[0050] Figure 9 The labeled probe in Example 6 [ 68 Imaging of Ga]Ga-byPYT1-1 in a subcutaneous tumor model of breast cancer.

[0051] Figure 10 The labeled probe in Example 7 [ 68 In vivo biodistribution map of Ga]Ga-byPYT1-1 at 30 min and 60 min after injection.

[0052] Figure 11 The labeled probe in Example 8 [ 68 Toxicological experimental results of Ga]Ga-byPYT1-1. Detailed Implementation

[0053] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0054] Unless otherwise specified in the examples, all procedures were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0055] Example 1: Screening of EpCAM-targeted cyclic peptides

[0056] The mRNA display technology was used to screen for cyclic peptides that bind to EpCAM: First, a random DNA library was constructed (encoding a peptide segment with cysteine ​​residues at both ends and 8-12 random amino acids in the middle). This was transcribed to obtain a random mRNA library, which was then ligated to DNA-PEG-CC-Pu to obtain an mRNA-Pu library. The mRNA-Pu library and 20 natural amino acids were then added to a cell-free protein expression (PURE) system for in vitro translation. The resulting peptide library was reverse transcribed, and dibromomethylpyridine was added to obtain a cyclic peptide library for mRNA display. This library was then affinity-eluted with magnetic beads immobilized with EpCAM target proteins. The sequences binding to EpCAM target proteins were then amplified by PCR, transcribed in vitro, and then screened again. After 3-5 rounds of "adsorption-elution-amplification," cyclic peptides specifically binding to the EpCAM target were highly enriched. Since one gene sequence corresponds to one cyclic peptide sequence, the specific sequence of the cyclic peptide binding to the target protein could be determined by gene sequencing. This invention successfully enriched cyclic peptides that bind to EpCAM through four rounds of screening. The sequences and structures of the cyclic peptides were obtained by next-generation sequencing using Novaseq 6000 and are shown in Table 1.

[0057] Table 1

[0058]

[0059]

[0060] Example 2 Synthesis of EpCAM-targeted cyclic peptide

[0061] Peptide synthesis was performed on Rink amide resin using standard Fmoc-SPPS. The resin was first swelled in DCM (dichloromethane) for 30 min, followed by deprotection of the Fmoc protecting group for 20 min under nitrogen bubbling with 20% piperidine / DMF (N,N-dimethylformamide) (3 times the resin volume), and then washed sequentially with DMF (×3) and DCM (×1). Fmoc-Cys(Trt)-OH and Oxyma (ethyl 2-oxime cyanoacetate) were dissolved in DMF, and DIC (diisopropylcarbodiimide) was added before being poured into the resin. The reaction was carried out under nitrogen bubbling for 1.5 h. After the reaction, the Fmoc deprotection was repeated and the resin was thoroughly washed. Subsequent amino acids were sequentially coupled to the target sequence according to the same procedure, and after removing the N-terminal Fmoc protecting group, acetylation was carried out in the presence of acetic anhydride and triethylamine. After sequence assembly, the resin was placed in a lysis buffer of TFA (trifluoroacetic acid) / H2O / EDT (1,2-ethylenedithiol) / TIS (triisopropylsilane) = 95 / 1 / 2 / 2 and reacted at room temperature for 2 h to achieve peptide cleavage and side chain deprotection. The lysis buffer was poured into pre-cooled anhydrous diethyl ether to precipitate the peptide, centrifuged and dried to obtain the crude peptide. The crude peptide was dissolved in DMSO (dimethyl sulfoxide) (1:1), and the pH was adjusted to >8 with triethylamine. Free thiols were reduced by rotation for 30-60 min in the presence of TCEP (tris(2-carboxyethyl)phosphine). Subsequently, the peptide solution was diluted to 0.1-0.5 mM, and 1.1 eq of 2,6-dibromomethylpyridine was slowly added at 0-4°C. The reaction was carried out for 2-3 h to achieve disulfide alkylation and cyclization. The reaction progress was monitored by liquid chromatography-mass spectrometry (LC-MS). After the reaction was completed, the reaction was quenched by acidification with 1% TFA, and purified by C18 RP-HPLC using a water / acetonitrile gradient containing 0.1% TFA. The target peak was collected and lyophilized to obtain a cyclic peptide with a purity >95%. The molecular weight was finally confirmed by ESI-MS.

[0062] PYT1-1, PYT1-2, PYT1-3, PYT1-4, 2CT1-1, and 2CT1-3 were all prepared using the above method, and the sequences and structures of each cyclic peptide are shown in Table 1.

[0063] Example 3: Determination of EpCAM-targeted ligand affinity

[0064] The affinity of the cyclic peptide for EpCAM was determined using surface plasmon resonance (SPR) with a Biacore T200 and CAP chip. The running buffer was 1×HBS-EP+ containing 0.1% DMSO. EpCAM protein was diluted to 250 nM, and cyclic peptides were prepared at concentration gradients of 1000 nM, 200 nM, 40 nM, 8 nM, and 1.6 nM, with a blank control under the same conditions. The appropriate conjugation amount was determined manually, and the minimum and maximum conjugation RU of the protein were calculated using formulas. The conjugation time was ultimately set to 1500 s, achieving a conjugation level of approximately 3200 RU. A single-cycle kinetic mode was then used for the assay, with a protein conjugation flow rate of 2 μL / min and a cyclic peptide injection time of 120 s at a flow rate of 30 μL / min. Finally, the equilibrium dissociation constants (KD) of the cyclic peptides and EpCAM were calculated using Biacore T200 software, as shown in Table 1. All cyclic peptides exhibited high affinity for EpCAM, with PYT1-1, PYT1-3, PYT1-4, 2CT1-1, and 2CT1-3 having KD values ​​below 4 × 10⁻⁶. -9 M.

[0065] Example 4: EpCAM-targeted high-affinity ligand modification and radionuclide imaging screening

[0066] Based on the results of in vitro affinity screening, four candidate cyclic peptide molecules with high affinity—2CT1-3, PYT1-1, PYT1-3, and PYT1-4—were selected for PEG4 modification and then coupled with the chelating agent DOTA to prepare... 68 Ga-labeled imaging probes were used. The tumor uptake capacity and tumor / background signal-to-noise ratio of each probe were compared through preliminary in vivo imaging to determine the candidate probe with the best imaging performance.

[0067] 1. EpCAM-targeted high-affinity ligand modification

[0068] Peptide synthesis was performed on Rink amide resin using standard Fmoc-SPPS. The resin was first expanded in DCM (dichloromethane) for 30 min, followed by deprotection of the Fmoc protecting group for 20 min under nitrogen bubbling with 20% piperidine / DMF (N,N-dimethylformamide) (3 times the resin volume), and then washed sequentially with DMF (×3) and DCM (×1). Fmoc-Lys(Mtt)-OH and Oxyma (ethyl 2-oxime cyanoacetate) were dissolved in DMF, and DIC (diisopropylcarbodiimide) was added before being poured into the resin. The reaction was carried out under nitrogen bubbling for 1.5 h. After the reaction, the Fmoc deprotection was repeated and the resin was thoroughly washed. Fmoc-NH-PEG4-CH2CH2-COOH and subsequent amino acids were sequentially coupled to the target sequence according to the same procedure. After deprotection of the N-terminal Fmoc protecting group, acetylation was carried out in the presence of acetic anhydride and triethylamine. After sequence assembly, the Mtt protecting group of Lys ε-NH2 was removed by multiple short-term treatments with 1% TFA (trifluoroacetic acid) / DCM + 2% TIS (triisopropylsilane). Subsequently, DOTA-tris(t-Bu)-COOH was activated with HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate) / DIPEA (N,N-diisopropylethylamine) and coupled at room temperature for 2-3 h. After coupling, the resin was thoroughly washed with DMF and DCM. The resin was placed in a TFA / H2O / EDT / TIS = 95 / 1 / 2 / 2 lysis buffer and reacted at room temperature for 2 h to achieve peptide cleavage and side chain deprotection. The lysis buffer was poured into pre-cooled anhydrous diethyl ether to precipitate the peptide, centrifuged, and dried to obtain the crude peptide. The crude peptide was dissolved in DMSO (dimethyl sulfoxide) (1:1), and the pH was adjusted to >8 with triethylamine. Free thiols were reduced by rotation for 30-60 min in the presence of TCEP (tris(2-carboxyethyl)phosphine). The peptide solution was then diluted to 0.1-0.5 mM, and 1.1 eq of 2,6-dibromomethylpyridine was slowly added at 0-4°C. The reaction was allowed to proceed for 2-3 h to achieve disulfide alkylation and cyclization, with the reaction progress monitored by LC-MS. After the reaction was complete, the solution was quenched with 1% TFA and purified by a water / acetonitrile gradient semi-preparative C18 RP-HPLC containing 0.1% TFA. The target peak was collected and lyophilized to obtain the cyclic peptide-PEG4-Lys-DOTA with a purity >95%.

[0069] PYT1-1-PEG4-Lys-DOTA (by PYT1-1), PYT1-3-PEG4-Lys-DOTA (by PYT1-3), PYT1-4-PEG4-Lys-DOTA (by PYT1-4), and 2CT1-3-PEG4-Lys-DOTA (by 2CT1-3) were all prepared using the above method. The molecular weights were finally confirmed by ESI-MS, and the spectra are shown below. Figure 1-4 As shown, the specific sequence and structure are shown in Table 2.

[0070] Table 2

[0071]

[0072] 2. Nuclide labeling and quality control

[0073] (1) 68 Ga labeling: Accurately weigh a certain mass of byPYT1-1 ligand and dissolve it in DMSO to a concentration of 25 μg / μL. Pipette 4 μL of the ligand solution and 130 μL of NaOAc solution (1 mol / L) into a vial, and add 2 mL of freshly rinsed solution. 68 Ga 3+ An ionic solution (0.05 mol / L hydrochloric acid solution with a radioactivity of 10⁻¹⁵ mCi) was prepared, shaken well, sealed, and reacted at 95 °C for 10 minutes. After adding 5 mL of water and rapidly cooling to room temperature, the reaction solution was passed through an activated reverse-phase solid-phase extraction column (Sep-Pak™ Light C18), followed by rinsing with 5 mL of H₂O. 18 Column. Finally, rinse C with 1 mL of 80% EtOH physiological saline solution. 18 The column was used, and the final eluent was passed through a 0.22 μm sterile filter membrane and transferred to the final product vial. Finally, the quality control was performed by radio-HPLC (radio-high performance liquid chromatography).

[0074] (2) Quality control: 68 The radiochemical purity of Ga-labeled byPYT1-1 was determined by radio-HPLC. The eluent was 20 μL, the flow rate was 1.0 mL / min, and the mobile phase consisted of 0.1% (v / v) TFA / water as phase A and 0.1% TFA / acetonitrile as phase B. A C18 column was used, and the elution gradient was identified using the analytical parameters shown in Table 3. 68 Radiochemical purity of Ga]Ga-byPYT1-1.

[0075] Table 3. Parameters for setting the mobile phase gradient in radio-HPLC analysis

[0076]

[0077] like Figure 5 As shown, radio-HPLC detection [ 68 The radiochemical purity of Ga]Ga-byPYT1-1 is greater than 98%.

[0078] Prepare according to the above method [ 68 Ga]Ga-byPYT1-3、[68 Ga]Ga-byPYT1-4、[ 68 Ga]Ga-by2CT1-3, and confirmed that the radiochemical purity was greater than 98%.

[0079] 3. EpCAM-targeted high-affinity ligand radionuclide screening

[0080] A subcutaneous tumor model in male Balb / c nude mice was established using the PC3 prostate cancer cell line expressing EpCAM. 0.1 mL of freshly prepared […] 68 Ga]Ga-by2CT1-3 (5.55 MBq), [ 68 Ga]Ga-byPYT1-1 (5.55 MBq), [ 68 Ga]Ga-byPYT1-3 (5.55 MBq) with [ 68 Ga]Ga-byPYT1-4 (5.55 MBq) was injected into mice via the tail vein (tumor diameter approximately 0.5 cm). One hour later, the mice were anesthetized with isoflurane and subjected to small animal PET / CT (uMicroEXPLORER Pro, United Imaging Healthcare, China) imaging. The regions of interest were delineated using standard uptake values ​​(SUVs).

[0081] The results are as follows Figure 6 As shown in Table 4, it can be seen that, 68 Ga]Ga-byPYT1-1、[ 68 Ga]Ga-byPYT1-3 showed good uptake in the tumor region, while [ 68 Ga]Ga-by2CT1-3、[ 68 Ga-byPYT1-4 showed relatively low accumulation at tumor sites. All four radioactive probes are primarily metabolized by the kidneys. [Among them...] 68 Ga-byPYT1-1 showed the highest uptake ratio between tumor and muscle, indicating a better imaging signal-to-noise ratio. Therefore, [ 68 Ga]Ga-byPYT1-1 can be used as the optimal cyclic peptide probe for subsequent tumor imaging studies targeting EpCAM.

[0082] Table 4. SUVmax image acquisition 1 hour after injection of different probes in PC3 subcutaneous tumor mice (n=3)

[0083]

[0084] Example 5: Cell fluorescence experiment

[0085] Preparation of probe PYT1-1-PEG4-Lys-FAM (by PYT1-1-FAM): Peptide synthesis was performed on Rink amide resin using standard Fmoc-SPPS. The resin was first swelled in DCM (dichloromethane) for 30 min, followed by deprotection of the Fmoc protecting group for 20 min under nitrogen bubbling with 20% piperidine / DMF (N,N-dimethylformamide) (3 times the resin volume), and then washed sequentially with DMF (×3) and DCM (×1). Fmoc-Lys(Mtt)-OH and Oxyma (ethyl 2-oxime cyanoacetate) were dissolved in DMF, and DIC (diisopropylcarbodiimide) was added before being poured into the resin. The reaction was carried out under nitrogen bubbling for 1.5 h. After the reaction, the Fmoc deprotection was repeated and the resin was thoroughly washed. Fmoc-NH-PEG4-COOH and subsequent amino acids were sequentially coupled to the target sequence according to the same procedure. After sequence assembly, the Mtt protecting group of Lys ε-NH2 was removed by multiple short-term treatments with 1% TFA (trifluoroacetic acid) / DCM + 2% TIS (triisopropylsilane). Then, a DMF solution containing 1.5 eq FAM-NHS and 3 eq DIPEA (N,N-diisopropylethylamine) was added, and coupling was performed at room temperature for 2-3 h. After coupling, the resin was thoroughly washed with DMF and DCM. The resin was placed in a lysis buffer of TFA / H2O / EDT / TIS = 95 / 1 / 2 / 2 and reacted at room temperature for 2 h to achieve peptide cleavage and side chain deprotection. The lysis buffer was poured into pre-cooled anhydrous diethyl ether to precipitate the peptide, centrifuged, and dried to obtain the crude peptide. The crude peptide was dissolved in DMSO (dimethyl sulfoxide) (1:1), and the pH was adjusted to > 8 with triethylamine. Free thiols were reduced by rotation for 30-60 min in the presence of TCEP (tris(2-carboxyethyl)phosphine). The peptide solution was then diluted to 0.1–0.5 mM, and 1.1 eq of 2,6-dibromomethylpyridine was slowly added at 0–4°C. The reaction was allowed to proceed for 2–3 h to achieve disulfide alkylation and cyclization, with the reaction progress monitored by LC-MS. After the reaction was complete, the solution was quenched with 1% TFA acid, and purified by a water / acetonitrile gradient semi-preparative C18 RP-HPLC method containing 0.1% TFA. The target peak was collected and lyophilized to obtain a cyclic peptide, PEG4-Lys-FAM, with a purity >95%. The molecular weight was finally confirmed by ESI-MS. Results are as follows: Figure 7 As shown.

[0086] Two types of breast cancer cells, HCC1806 and MDA-MB-231, expressing high and low levels of EpCAM respectively, were seeded in 35-mm confocal microplates (200,000 cells / plate) and cultured in corresponding mediums for 24 h in an incubator. Experiments were conducted after complete cell adhesion. In the experimental group, HCC1806 and MDA-MB-231 cells were incubated for 1 h at 37 ℃ in corresponding medium containing 10 μM byPYT1-1-FAM. After washing twice with PBS, the cell nuclei were stained with Hoechst for 10 min, and images were captured using a confocal microscope. In the control group (blocked), HCC1806 cells were first incubated for 30 min at 37 ℃ in medium containing 100 μM byPYT1-1, followed by co-incubation at 37 ℃ for 1 h in medium containing both 100 μM byPYT1-1 and 10 μM byPYT1-1-FAM. Subsequent procedures were the same as in the experimental group. The excitation wavelength was 488 nm, and the emission wavelength was 520-530 nm. Image quantization was performed using ImageJ software.

[0087] The results are as follows Figure 8 As shown, Figure 8 a. Fluorescence microscopy imaging of HCC1806 cells, MDA-MB-231 cells, and HCC1806 cells after pre-incubation with byPYT1-1 to block the receptor (green represents byPYT1-1-FAM; blue represents hoechst 33342). Scale bar: 20 μm. b. Quantitative analysis of fluorescence intensity of cells in image a (n = 15 cells). P < 0.0001, two-tailed unpaired t-test. au is an abbreviation for arbitrary units.

[0088] The results showed that byPYT1-1-FAM could effectively bind to EpCAM, and significant fluorescence signals were observed on the cell membrane. Quantitative analysis of the fluorescence signal showed that the fluorescence intensity of HCC1806 cells expressing high levels of EpCAM was 15 times higher than that of MDA-MB-231 cells, and the fluorescence signal could be competitively inhibited by the uncoupled FAM cyclic peptide, verifying the specificity of byPYT1-1-FAM binding to EpCAM.

[0089] Example 6 [ 68 Ga]Ga-byPYT1-1 live imaging

[0090] Take the freshly prepared [ 68Ga]Ga-byPYT1-1 (0.2 mL, 7.4 MBq) was injected via the tail vein into female Balb / c nude mice with HCC1806 tumors that highly expressed EpCAM and MDA-MB-231 tumors that lowly expressed EpCAM (tumor diameter approximately 0.5 cm). After 30 min and 1 h, the mice were anesthetized with isoflurane and subjected to small animal PET / CT (uMicroEXPLORER Pro, United Imaging Healthcare, China) static imaging. The regions of interest were delineated using standard uptake values ​​(SUVs).

[0091] like Figure 9 As shown, [ 68 After injection of Ga]Ga-byPYT1-1 for 30 min and 1 h, significant uptake was observed in the tumor region of the HCC1806 breast cancer model with high EpCAM expression, while no significant uptake was observed in the tumor model with low expression. At 30 min, the uptake values ​​at the two tumor sites were 0.64 ± 0.18 vs 0.31 ± 0.06, and at 60 min, they were 0.48 ± 0.07 vs 0.18 ± 0.04, respectively. Tumor uptake of the probe showed a slight decreasing trend over time. Furthermore, high physiological uptake of the probe was observed in tissues such as the thyroid and gastrointestinal tract in both tumor models.

[0092] Example 7 [ 68 In vivo biodistribution of Ga]Ga-byPYT1-1

[0093] Female BALB / c nude mouse models bearing HCC-1806 and MDA-MB-231 breast cancer were established, respectively. 68 The Ga-byPYT1-1 labeled solution was diluted to an isotonic solution and injected into mice via the tail vein (0.1 mL, 0.74 MBq). Mice in each group were anesthetized and sacrificed at 30 min and 60 min, respectively. Blood was collected from each group of mice, and the heart, liver, spleen, lungs, kidneys, flesh, bones, stomach, large intestine, small intestine, brain, and tumors were dissected, weighed, and their radioactivity counts were performed. The radioactivity counts were measured using a gamma counter, and the %ID / g of each organ was calculated. All data were attenuated and corrected.

[0094] like Figure 10 As shown in Table 5, at the 30 min and 60 min time points, [ 68The uptake of HCC1806 tumors by Ga-byPYT1-1 was 2.12 and 2.07 times that of MDA-MB-231 tumors, respectively (1.08±0.23 %ID / g vs 0.51±0.04 %ID / g; 0.81±0.19 %ID / g vs 0.39±0.16 %ID / g). Simultaneously, the tumor / muscle ratio in the HCC1806 tumor-bearing model was 3.23 and 3.17 times that in the MDA-MB-231 tumor-bearing model, respectively. This indicates that the probe can be effectively taken up by tumors highly expressing EpCAM, consistent with imaging results. Furthermore, the physiological uptake of this probe was mainly concentrated in the kidney and gastrointestinal tract tissues, with lower uptake in the heart, liver, and brain tissues, which may be related to the physiological expression distribution of EpCAM in normal tissues.

[0095] Table 5 [ 68 In vivo biodistribution of Ga-byPYT1-1 in a mouse model of breast cancer (mean ± SD, n = 4, %ID / g)

[0096]

[0097] Example 8 [ 68 Toxicological and safety verification of Ga-byPYT1-1

[0098] Twelve 6-week-old KM mice, weighing approximately 25-35 g, were selected. Six mice were assigned to the experimental group, and six to the control group. All mice were fed under normal conditions before the experiment and throughout the observation period. Each mouse in the experimental group received a tail vein injection of […]. 68 Ga-byPYT1-1 labeled solution (0.5 mL, 18.5 MBq) was injected uniformly over 5 seconds; 6 mice were injected with the same volume of physiological saline as a blank control. Mice were observed for 21 consecutive days, including monitoring their diet and activity levels, and their weight was recorded every 3 days. At the end of the observation period, mice were sacrificed and dissected, and the color and morphology of various organs in both groups were observed. Tissue samples were collected, including sections of major organs (heart, liver, lungs, kidneys, and spleen) for HE staining, and blood was collected for serum AST / ALT / BUN and other biochemical tests.

[0099] like Figure 11 As shown, injection [ 68 There was no significant difference in body weight between the Ga-byPYT1-1 probe group and the saline-injected control group (Figure a). Blood biochemical indicators, including ALT, AST, and BUN, also showed no statistically significant differences between the two groups (Figure b). HE staining results showed that major organs, including the heart, liver, spleen, lungs, and kidneys, showed significant differences after injection. 68 No significant pathological changes were observed after using the Ga]Ga-byPYT1-1 probe group (Figure c). These results indicate that using this probe for in vivo specific EpCAM imaging diagnosis has good safety.

[0100] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An EpCAM-targeting cyclic peptide, characterized in that, The sequence of the cyclic peptide is shown in Formula I: Equation I Wherein, the linear portion of the cyclic peptide is R1-CX1X2X3X4X5X6X7X8X9X 10 X 11 (X 12 The peptide sequence of C-R2 is R1-CTWYIISRNERLC-R2, where C represents cysteine, R1 is H or -Ac, and R2 is -NH2.

2. The EpCAM-targeting cyclic peptide according to claim 1, characterized in that, The structure of the cyclic peptide is shown below: 。 3. An EpCAM-targeted small molecule probe, characterized in that, The probe is a cyclic peptide labeled with a radionuclide as described in any one of claims 1-2; the probe comprises a cyclic peptide, a bifunctional chelating agent and a radionuclide, wherein the radionuclide is chelated with the bifunctional chelating agent and the bifunctional chelating agent is covalently linked to the R2 group of the cyclic peptide via a linker.

4. The EpCAM-targeted small molecule probe according to claim 3, characterized in that, The bifunctional chelating agents are DOTA, NOTA, NODAGA, RESCA, HBEDCC, DTPA, SBAD, DFO, NOTAM, CB-DO2A, NOTA-AA, NETA, HETA, TRITA, DAR, D O3A, HYNIC, MAG2, MAS3, MAG3, AAZTA, DOTAGA, NOTAGA, DOTMA, DOTAM, TRAP, CB-TE2A, PCTA, NODA-MPAA, THP, Macropa; The radionuclide is a diagnostic radionuclide; the diagnostic radionuclide is 18 F, 43 Sc、 44 Sc、 45 Ti、 47 Sc、 51 Cr 51 Mn, 52 Mn, 52 Fe、 55 Co、 57 Co、 58m Co、 59 Fe、 60 Cu、 61 Cu、 62 Cu、 63 Zn, 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Sc、 86 Y、 87 Y、 90 Y、 89 Sr、 89 Zr、 94 Tc, 97 Ru、 99 Tc, 99m Tc, 101m Rh、 103m Rh、 105 Pd, 105 Rh、 111 Ag、 111 ln、 117m Sn、 119 Sb, 142 Pr, 143 Pr, 149 Pm, 149 Tb, 152 Tb, 153 Sm、 154-159 Gd, 161 Tb, 165 Dy、 166 Dy、 166 Ho、 169 Er、 169 Yb、 175 Yb、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 191m Pt, 193m Pt, 195m Pt, 194 lr、 197 Pt, 198 Au、 199 Au、 201 Tl、 203 Pb, 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra、 224 Ra and 225 At least one of Ac.

5. The EpCAM-targeted small molecule probe according to claim 3, characterized in that, The linker is selected from -(CH2CH2O). m -(CH2) n -(Lys) p -、-NH-(CH2) q -CO-、-(Gly) x - or - (GS) y - where m is an integer from 1 to 10, n is an integer from 1 to 6, p is 0 or 1, q is an integer from 4 to 8, x is an integer from 3 to 8, and y is an integer from 1 to 6.

6. The following applications of the EpCAM-targeted small molecule probe according to any one of claims 3-5: (i) Preparation of reagents for detecting EpCAM-related diseases; (ii) To detect the expression level of EpCAM in the test sample for non-diagnostic purposes; The EpCAM-related diseases are breast cancer or prostate cancer.

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