KR20240066484A - Compound for targeting cancer comprising EGFR L858R mutation and uses thereof - Google Patents

Abstract

The present invention relates to a compound targeting cancer containing the EGFR L858R mutation, a dual-modality contrast agent containing a compound targeting cancer containing the EGFR L858R mutation, a composition for diagnosing cancer, and a method of providing information for cancer monitoring. will be. When the cancer-targeting compound containing the EGFR L858R mutation according to the present invention is treated with an organ or tissue in vivo, it is ingested into the cancer tissue and radiological images and fluorescence images can be acquired simultaneously, and each of them can diagnose EGFR-positive cancer. And it can be provided as an image for staging and surgical guidance, so it can be used for cancer diagnosis and treatment using molecular imaging techniques.

Description

Compound for targeting cancer comprising EGFR L858R mutation and uses thereof}

The present invention relates to a compound targeting cancer containing the EGFR L858R mutation, a dual-modality contrast agent containing a compound targeting cancer containing the EGFR L858R mutation, a composition for diagnosing cancer, and a method of providing information for cancer monitoring. will be.

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein composed of 1,186 amino acids and is involved in various cellular processes such as cell growth, differentiation, proliferation, and apoptosis. EGFR is overexpressed in malignant tumors and is associated with angiogenesis, progression, and metastasis. The mutation status of EGFR in non-small cell lung carcinoma (NSCLC) tumors is correlated with prognosis and treatment response. A kinase domain mutation in exon 21 resulting in a substitution of leucine for arginine at position 858 (L858R) is associated with responsiveness to EGFR tyrosine kinase inhibitors (TKIs). Patients with L858R mutant EGFR show improved overall survival and progression-free survival after TKI treatment compared to patients with wild-type EGFR. Therefore, activating mutations in the EGFR kinase domain are useful biomarkers for selecting patients who will benefit from TKI treatment.

In clinical practice, EGFR mutation testing is performed for non-small cell lung cancer through tumor tissue biopsies. However, tumor biopsy is an invasive procedure that provides limited information due to the heterogeneity of tumor tissue. Small portions of tumor obtained from biopsy may not represent the mutational status of the entire tumor and metastatic disease. In contrast, molecular imaging techniques, including positron emission tomography (PET) and single-photon emission computed tomography (SPECT), enable noninvasive detection and determination of the mutational status of EGFR. Provides information. In this regard, the development of molecular imaging agents targeting EGFR with the L858R mutation may be helpful in the selection and monitoring of patients treated with TKIs.

Accordingly, the present inventors completed the present invention by developing a cancer-targeting compound labeled with a radioactive isotope and a fluorescent substance.

Therefore, the purpose of the present invention is to provide a cancer-targeting compound that can specifically detect cancer.

Another object of the present invention is to provide a dual-modality contrast agent containing a cancer-targeting compound, a cancer diagnostic composition, and a cancer monitoring method using the cancer-targeting compound.

In order to achieve the above object, the present invention provides a cancer targeting peptide that binds to a fluorescent substance and a radioactive isotope and includes a chelating ligand represented by the amino acid sequence of SEQ ID NO: 3 and a cancer targeting peptide represented by the amino acid sequence of SEQ ID NO: 1. Provides a compound.

Additionally, the present invention provides a dual-modality contrast agent containing the cancer-targeting compound.

Additionally, the present invention provides a composition for diagnosing cancer containing the cancer targeting compound.

Additionally, the present invention provides a method of providing information for cancer monitoring, including the step of treating a biological sample with the cancer-targeting compound.

When the cancer-targeting compound containing the EGFR L858R mutation according to the present invention is treated with an organ or tissue in vivo, it is ingested into the cancer tissue and radiological images and fluorescence images can be acquired simultaneously, and each of them can diagnose EGFR-positive cancer. And it can be provided as an image for staging and surgical guidance, so it can be used for cancer diagnosis and treatment using molecular imaging techniques.

1 is a diagram showing the chemical structure of a cancer targeting compound according to the present invention.
Figure 2 is a diagram showing the results of analyzing the in vitro receptor binding affinity of the cancer targeting compound according to the present invention.
Figure 3 is a diagram showing the results of cell uptake analysis of a compound for targeting cancer according to the present invention using a confocal microscope.
Figure 4 is a diagram showing the results of in vivo gamma camera imaging of the cancer targeting compound according to the present invention in a tumor mouse model.
Figure 5 is a diagram showing the in vitro fluorescence imaging results of the cancer-targeting compound according to the present invention in a sacrificed tumor mouse model (Lu; lung, Hr; heart, Lv; liver, St; stomach, Sp; spleen, Co; Colon, Kd; Muscle, NCI-H1975 tumor; Tu2;
Figure 6 is a diagram showing the results of immunohistochemical staining of a cancer-targeting compound according to the present invention in tumor tissue derived from a sacrificed tumor mouse model.

Hereinafter, the present invention will be described in detail.

The present invention provides a compound for targeting cancer, which binds to a fluorescent substance and a radioactive isotope and includes a chelating ligand represented by the amino acid sequence of SEQ ID NO: 3 and a cancer targeting peptide represented by the amino acid sequence of SEQ ID NO: 1.

In the present invention, cancer refers to a malignant tumor that grows abnormally due to excessive growth. Includes. The cancer is selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, glioma, ovarian cancer, pancreatic cancer, stomach cancer, thyroid cancer, and uterine cancer. It may be possible.

Additionally, the cancer preferably contains the epidermal growth factor receptor (EGFR) L858R mutation.

In the present invention, the fluorescent material is a material that develops color in response to light of a specific wavelength, and its type is not limited. Fluorescent substances include light-emitting molecules that emit light in an excited state, fluorescent proteins, metal ions, complex compounds, organic dyes, conductors, semiconductors, insulators, quantum dots, or quantum wires.

Examples of the above fluorescent substances include fluorescent proteins such as EGFP (enhanced green fluorescent protein), ECFP (enhanced cyan fluorescent protein), EBFP (enhanced blue fluorescent protein), EYFP (enhanced yellow fluorescent protein), and RFP (red fluorescent protein). there is.

Additionally, examples of the fluorescent substances include Pyrene or its derivatives, Cyanine (Cy) series, Alexa Fluor series, BODIPY series, DY series, rhodamine or Its derivatives, Fluorescein or its derivatives, coumarin or its derivatives, Acridine homodimer or its derivatives, Acridine Orange or its derivatives, 7-amino solution 7-aminoactinomycin D (7-AAD) or a derivative thereof, Actinomycin D (Actinomycin D) or a derivative thereof, 9-amino-6-chloro-2-methoxyacridine (ACMA) or a derivative thereof , DAPI or a derivative thereof, Dihydroethidium or a derivative thereof, Ethidium bromide or a derivative thereof, Ethidium homodimer-1 (EthD-1) or a derivative thereof, ethidium homo Dimer-2 (EthD-2) or a derivative thereof, Ethidium monoazide or a derivative thereof, Hexidium iodide or a derivative thereof, bisbenzimide (Hoechst 33258) or a derivative thereof , Hoechst 33342 or a derivative thereof, Hoechst 34580 or a derivative thereof, hydroxystilbamidine or a derivative thereof, LDS 751 or a derivative thereof, propidium Propidium Iodide (PI) or a derivative thereof, Calcein or a derivative thereof, Oregon Green or a derivative thereof, Magnesium Green or a derivative thereof, Calcium Green or a derivative thereof Derivative, JOE or a derivative thereof, Tetramethylrhodamine or a derivative thereof, TRITC or a derivative thereof, Tamra (N,N,N',N'-tetrametyl-6-carboxyrhodamine, TAMRA) or a derivative thereof, Pyronin Y (Pyronin Y) or a derivative thereof, Lissamine or a derivative thereof, ROX or a derivative thereof, Calcium Crimson or a derivative thereof, Texas Red or a derivative thereof, Nile Red or It may be a derivative thereof, thiadicarboxyanine or a derivative thereof, dansylamide or a derivative thereof, cascade blue, or DAPI (4',6-diamidino-2-phenylindole).

Quantum dots can be used as the fluorescent material. Quantum dots are particles centered on nano-sized II-IV or III-V semiconductor particles, and have a core of about 2 to 10 nm in size and a shell (mainly made of ZnS, etc.) shell), and even if it is the same material, the fluorescence wavelength varies depending on the size of the particle, allowing fluorescence in various wavelength ranges to be obtained. Group II-VI or III-V compounds constituting the quantum dots include CdSe, CdSe/ZnS, CdTe/CdS, CdTe/CdTe, ZnSe/ZnS, ZnTe/ZnSe, PbSe, PbS InAs, InP, InGaP, InGaP/ZnS and HgTe. It may be selected from the group consisting of, and may be in the form of a single core or core/shell.

In one embodiment of the present invention, TAMRA was used as the fluorescent substance.

In the present invention, the radioactive isotope is an isotope whose atomic nucleus is unstable, emits radiation, and converts into a stable atomic nucleus, and its type is not limited. Examples of the radioactive isotopes include Technetium-99m (Technetium-99m, Tc-99m), Gallium-67, Gallium-68, Copper-64, Copper-67, Gold-198, Lead-210, Nickel-63, Dysprosium-165, Rhenium-186, Rhenium-188, Rubidium-82, Ruthenium-177, Manganese-177, Molybdenum-99, Fluorine-18, Bismuth-213, Samarium-153, Oxygen-15, Cesium- 137, Selenium-75, Sodium-24, Strontium-85, Strontium-89, Strontium-90, Americium-241, Zinc-65, Erbium-169, Chlorine-36, Iodine-123, Iodine-124, Iodine -125, Iodine-129, Iodine-131, Uranium-234, Uranium-235, Silver-110m, Iridium-192, Ytterbium-169, Ytterbium-177, Yttrium-90, Phosphorus-32, Phosphorus-33 , Indium-111, Germanium-68, Xenon-133, Nitrogen-13, Iron-55, Cadmium-109, Calcium-47, California-252, Cobalt-57, Cobalt-60, Quarium-244, Chromium-51 , Krypton-81, Krypton-85, Carbon-11, Carbon-14, Thallium-201, Thallium-204, Thorium-229, Thorium-230, Tritium, Palladium-103, Potassium-42, Polonium-210, There are therapeutic radioactive isotopes such as promethium-147, plutonium-238, holmium-166, and sulfur-35.

In one embodiment of the present invention, Technetium-99m (Tc-99m) was used as the radioactive isotope.

In the present invention, the chelating ligand is preferably represented by the amino acid sequence of SEQ ID NO: 3. The chelating ligand shows particularly strong and stable chelation with the radioactive isotope Tc-99m.

In the present invention, a peptide refers to a linear molecule formed by linking amino acid residues to each other through peptide bonds. The peptide may be manufactured according to chemical synthesis methods known in the art, and preferably, may be manufactured according to solid phase synthesis technology, but is not limited thereto.

The cancer targeting peptide is preferably represented by the amino acid sequence of SEQ ID NO: 1. It was confirmed through examples that the cancer targeting peptide targets EGFR of cancer cells, especially EGFR containing the L858R mutation, and thus accumulates in cancer tissues.

The cancer targeting compound may further include a spacer to reduce fluorescence disturbance of the cancer targeting peptide. In detail, the spacer is preferably represented by the amino acid sequence of SEQ ID NO: 2. Additionally, as a spacer, a histidine inserted into the sequence of a conjugate of peptidehydrazinonicotinamide (HYNIC) can be used. By inserting the above-described spacer into a cancer targeting compound, tumor-targeting performance can be improved in vitro and in vivo. In addition, it can reduce the disturbance of cancer-targeting peptides and fluorescent substances of cancer-targeting compounds.

The cancer targeting compound is preferably arranged in the form of structural formula 1 below.

[Structural Formula 1]

Cancer targeting peptide-spacer-chelating ligand-fluorescent substance

The cancer targeting compound is preferably represented by the following formula (1).

[Formula 1]

In another aspect, the present invention provides a dual-modality contrast agent containing a cancer-targeting compound.

In the present invention, the contrast medium is a substance injected into tissue to increase the contrast of the image during radiological examination, but is not limited thereto. When using a conventional contrast agent, only radiological images such as magnetic resonance imaging (MRI) or computed tomography (CT) could be acquired. However, when using a dual-modality contrast agent containing the cancer-targeting compound according to the present invention, radiological images and fluorescence images can be acquired simultaneously.

The dual-modality contrast agent containing the cancer-targeting compound may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those commonly used in preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly Includes, but is not limited to, vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The dual mode contrast agent is preferably administered to the subject parenterally. When administered parenterally, intravenous injection, intramuscular injection, intra-articular injection, intra-synovial injection, intrathecal injection, intrahepatic injection, intralesional injection, or It can be administered by intracranial injection. The appropriate dosage of the dual-modality contrast agent can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. there is.

The method of obtaining a radiological image and a fluorescence image using the dual contrast agent can be performed according to a conventional method.

In another aspect, the present invention provides a composition for diagnosing cancer containing a cancer targeting compound.

In the present invention, diagnosis refers to medically determining the name, etiology, type, severity, condition, and prognosis of the disease, and is not limited thereto.

For administration, the composition for diagnosing cancer may further include a pharmaceutically acceptable carrier in addition to the active ingredients described above. Pharmaceutically acceptable carriers are those commonly used in preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly Includes, but is not limited to, vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

A cancer diagnostic composition containing the cancer targeting compound can be used as a cancer diagnostic kit.

In another aspect, the present invention provides a method of providing information for cancer monitoring, including the step of treating a biological sample with a cancer-targeting compound.

The biological sample is preferably a variety of living organs or tissues.

The method of providing information for the above cancer monitoring is preferably one that provides information such as the location, prognosis, and treatment process of the cancer in vivo or in vitro.

A sample treated with a cancer-targeting compound can be subjected to gamma camera imaging and optical imaging according to a conventional method, thereby simultaneously acquiring a radiological image and a fluorescence image. The acquired radiological images and fluorescence images can provide objective basic information necessary for monitoring the location, prognosis, and treatment process of cancer, and the doctor's clinical judgment or opinion is excluded.

Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention pertains.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Experimental Materials

Acetone, 1N-HCl, 1M NaOH, SnCl ₂ and sodium tartrate were purchased from Sigma Aldrich Korea (Seoul, Korea). Tc-99m pertechnetate was eluted from our institution's commercial technetium generator (EnviroKorea, Daejeon, Korea). A Radio-Cap ^® capillary column (cat. FC-D1012, silica gel size = 38-75 μm; Futurechem, Seoul, Korea) filled with silica material was used for radio-chromatography. NCI-H1975 (human NSCLC cells expressing EGFR with the L858R mutation) and NCI-H1650 (human NSCLC cells expressing EGFR without the L858R mutation) cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea).

Example 2. Synthesis of cancer targeting compound (STHHYYP-GHEG-ECG-TAMRA)

2-1. Synthesis of compounds targeting cancer

The cancer targeting compound (STHHYYP-GHEG-ECG-K(TAMRA)) was synthesized by Peptron, Inc. (Daejeon, Korea). The cancer targeting compound is an EGFR L858R targeting peptide (SEQ ID NO: 1), a spacer (SEQ ID NO: 2), a chelating ligand (SEQ ID NO: 3), and a fluorescent substance sequentially and operably linked. The chemical structure of the cancer targeting compound is shown in Figure 1.

Specifically, the EGFR L858R target peptide (SEQ ID NO: 1) was synthesized using Fmoc-based solid-phase peptide synthesis (SPPS). The peptide-bound resin was then treated with TAMRA-succinimidyl ester in N-methyl-2-pyrrolidone and diisopropylethylamine. The resulting compounds were analyzed using reverse phase high-performance liquid chromatography (RP-HPLC) with a C18 analytical column (C18, 5 μm, 100 Å column, 4.6 x 250 mm; Shimadzu, Kyoto, Japan). refined. For elution, a linear gradient from 0 to 70% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA) was used. The mass of the synthesized peptide was analyzed by mass spectrometry (AXIMA-CFR, MALDI-TOF Mass Spectrometer, Shimadzu).

In the examples described later, the cancer targeting compound was described as ‘STHHYYP-GHEG-ECG-TAMRA’.

2-2. Radiolabeling of cancer targeting compounds using Tc-99m

The cancer targeting compound synthesized in Example 2-1 was radioactively labeled with Tc-99m. Specifically, cancer targeting compounds (0.005 mg/ml in 300 μl of nitrogen-purged water) and sodium tartrate (100 mg/ml in 300 μl of nitrogen-purged water) were mixed in a microcentrifuge tube. Next, Tc-99m pertechnetate (1.0 ml, approximately 1,110 MBq) and SnCl ₂ (1 mg/ml in 30 μl nitrogen-purged 0.01 M HCl) were added. The solution was heated at 95°C for 15 minutes and cooled to room temperature. Gilson 321 HPLC pump (Gilson, Inc., Middleton, WI, USA), Bioscan FC-1000 radiation detector (Bioscan, Inc., Washington DC, USA), Trilution LC software (Gilson, Inc.) and YMC-Triart C18 column. (4.6 × 100 mm, YMC, Kyoto, Japan) was used to analyze a radiolabeled cancer targeting compound (Tc-99m STHHYYP-GHEG-ECG-TAMRA). Solvents A and B were 0.1% TFA in water and acetonitrile, respectively. The analysis used a linear gradient from 3% to 70% solvent B over 17 minutes at a flow rate of 1.0 ml/min. An ultraviolet detector (230 nm) and a gamma radiation detector were used for monitoring.

To evaluate the radiochemical stability of the radiolabeled cancer targeting compound (Tc-99m STHHYYP-GHEG-ECG-TAMRA), the labeled complex (0.1 ml) was incubated with 0.9 ml of saline at room temperature and incubated at 37°C. Freshly collected human serum was cultured. Samples were incubated at 30 minutes and 1, 3, and 24 hours in two mobile phases, saline (Rf of Tc-99m STHHYYP-GHEG-ECG-TAMRA and free pertechnetate = 0.9-1.0, Rf of colloid = 0.0-0.1) and acetone (Rf of Analysis was performed using a capillary column with free pertechnetate = 0.9-1.0, Rf of Tc-99m STHHYYP-GHEG-ECG-TAMRA and colloid = 0.0-0.1). All experiments were repeated three times (n=3).

In the examples described later, the radioactively labeled cancer targeting compound was described as ‘Tc-99m STHHYYP-GHEG-ECG-TAMRA’.

2-3. Characterization of cancer-targeting compounds

The cancer targeting compound synthesized in Example 2-1 (STHHYYP-GHEG-ECG-TAMRA)); and the radioactively labeled cancer-targeting compound (Tc-99m STHHYYP-GHEG-ECG-TAMRA) of Example 2-2; were analyzed.

As a result of the characterization, it was confirmed that the synthesized cancer targeting compound (STHHYYP-GHEG-ECG-TAMRA) had a chemical formula of C ₉₈ H ₁₂₀ N ₂₄ O ₂₈ S ₁ and a molecular weight of 2113.13.

After radiolabeling with Tc-99m, RP-HPLC detected a single radioactive compound (retention time = 6.6 min). As a result, it was confirmed that the radiochemical purity of Tc-99m STHHYYP-GHEG-ECG-TAMRA was over 93% after labeling. Additionally, Tc-99m STHHYYP-GHEG-ECG-TAMRA showed high stability in saline and serum for 24 hours. The intact percentage of Tc-99m STHHYYP-GHEG-ECG-TAMRA measured with a capillary column filled with silica material in saline solution was 93.1 ± 0.4, 92.7 ± 0.5, 91.9 ± 1.1 and 90.8 ± 1.9% at 30 min 1 h 3 . hour and 24 hours, respectively. In serum, it was 92.6±0.7, 92.4±0.8, 91.6±1.0, and 90.3±1.6% at 30 minutes, 1 hour, 3 hours, and 24 hours, respectively.

Example 3. In vitro receptor binding affinity

The cell affinity of Tc-99m STHHYYP-GHEG-ECG-TAMRA was determined using the NCI-H1975 human NSCLC cell line. Specifically, NCI-H1975 cells were cultured at 37°C in 5% CO ₂ and humidified conditions. Cells were incubated with 10% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), D-glucose (2.5 mg/mL), and gentamicin ( were cultured in RPMI-1640 medium containing 50 mg/mL). Afterwards, cells were inoculated into a 96-well plate at a density of 1 x 10 ⁴ cells/well and cultured at 37°C overnight. On the day of the experiment, cells were washed twice (5 minutes each) using ice-cold binding buffer (25 mM HEPES and 1% bovine serum albumin).

Labeled peptides were incubated at different concentrations (3.6, 10, 20, 36, 50, 80, 130, 200, and 360 nM) in the presence (non-specific binding) or absence (total binding) of a 500-fold excess of unlabeled peptide. was added to the well. The total volume of each well was 200 μl, and cells were maintained at 37°C for 1 hour. Afterwards, the culture medium was removed and the cells were washed twice using cold binding buffer. Finally, the cells were lysed with 1M NaOH, and the solution in each well was counted using a gamma counter (1480 Wizard 3 gamma counter; PerkinElmer Life and Analytical Sciences, Wallingford, CT, USA). Equilibrium dissociation constant (K _d ) was determined by nonlinear regression of the data according to a single-site binding model using GraphPad Prism software version 5.03 (GraphPad Software, La Jolla, CA, USA). Each data point is the average of four values. The results of in vitro receptor binding affinity analysis are shown in Figure 2.

As shown in Figure 2, the Kd value of Tc-99m STHHYYP-GHEG-ECG-TAMRA for NCI-H1975 cells was estimated to be 130.6 ± 29.2 nM, and B _max was confirmed to be 6353 ± 647 fmol/mg.

Example 4. Cellular uptake analysis using confocal microscopy

NCI-H1975 and NCI-H1650 cells (1 x ¹⁰⁵ cells) were seeded on the top of a cover slip slide and incubated at 37°C for 24 hours. Cells were cultured in fresh serum-free medium (500 μL) containing Tc-99m STHHYYP-GHEG-ECG-TAMRA (200 nM). The cultured cells were incubated at 37°C for 1 hour. These cells were washed three times with phosphate-buffered saline. Cells were incubated with anti-human L858R mutant-specific EGFR antibody (cat. 3197S; Cell Signaling Technology, Danvers, MA, USA) and Alexa Fluor® 488-conjugated goat anti-rabbit secondary antibody (cat. 111-545-003, 1: 100 dilution; Jackson Immuno Research Inc., West Grove, PA, USA). Afterwards, fluorescent mounting medium (Dako, Glostrup, Denmark) was added to the cover slip slide and the slide was closed with a cover glass. Microscopic imaging studies were performed with an FV1200 confocal microscope (Olympus, Pittsburgh, PA, USA) with a 40x oil immersion lens. The results of cell uptake analysis using a confocal microscope are shown in Figure 3.

As shown in Figure 3, the fluorescence activity of STHHYYP-GHEG-ECG-TAMRA was detected in NCI-H1975 cells. In the merged image, the activity of STHHYYP-GHEG-ECG-TAMRA (red) matched well with the anti-L858R mutant EGFR signal (green, Figure 3A). In contrast, the fluorescence activity of STHHYYP-GHEG-ECG-TAMRA and anti-L858R mutant EGFR was barely detectable in NCI-H1650 cells (Figure 3B).

These results indicate that Tc-99m STHHYYP-GHEG-ECG-TAMRA is useful for imaging EGFR with the L858R mutation.

Example 5. Tumor mouse model

All animal studies were conducted strictly in accordance with the Korean Animal Protection Act. Guidelines for the care and use of laboratory animals (Law No. 14651, 2017). The protocol was approved by the Animal Care Committee of Wonkwang University Animal Research Institute (No. 2019-07-004). All animal experiments were performed under ketamine/xylazine anesthesia. Animals were sacrificed by cervical dislocation under deep anesthesia, and every effort was made to minimize animal suffering and sacrifice.

Six-week-old female homozygous athymic BALB/c nu/nu mice (weight 16-18 g) were purchased from Damul Science (Daejeon, Korea) and kept in cages. They had access to alfalfa-free (non-fluorescent) food and water ad libitum. After 1 week of adaptation to laboratory conditions, mice were inoculated subcutaneously with 1 × 10 ⁷ NCI-H1975 (L858R mutation positive) and NCI-H1650 (L858R mutation negative) cells (0.1 ml PBS) on the left and right sides of the prothoracic region, respectively. did. They were raised to grow to a volume of about 350 to 450 mm ³ . Tumor volume was calculated using equation 1.

[Calculation Formula 1]

V(mm ³ ) = 0.5 × a × b ² ,

where a and b represent the long axis and vertical short axis of the tumor by caliper measurement, respectively.

Gamma camera imaging and biodistribution studies were performed approximately 28 days after inoculation.

Example 6. In vivo gamma camera imaging

Tumor-bearing mice were anesthetized by intraperitoneal injection of ketamine (60 mg/kg) and xylazine (5 mg/kg). Anesthetized mice were injected intravenously with 55.5 MBq (200 nM in 150 μL) of Tc-99m STHHYYP-GHEG-ECG-TAMRA. In vivo imaging was performed 1, 2, and 3 hours after injection. A gamma camera (Vertex; ADAC Laboratories, Milpitas, CA, USA) equipped with a 3 mm pinhole collimator (window setting 140 keV, width 20%) was used. The acquisition time was 150 seconds, and the matrix size of the image was 512 × 512. Regions of interest (ROI) (15 × 15 pixels) were drawn on the tumor of the chest wall. Additional ROIs were drawn on the right arm muscles to assess normal muscle uptake (n = 5). The average number per pixel in the ROI was measured and the target-to-normal muscle uptake ratio was calculated. The in vivo gamma camera imaging results are shown in Figure 4.

As shown in Figure 4, diffuse renal uptake was observed 1 h after injection of Tc-99m STHHYYP-GHEG-ECG-TAMRA, indicating that unbound Tc-99m STHHYYP-GHEG-ECG-TAMRA is mainly excreted through the renal system. It means that it becomes. Additionally, radioactivity was detected in the liver and colon at 2 and 3 hours, suggesting that part of Tc-99m STHHYYP-GHEG-ECG-TAMRA was eliminated from the body through the hepatobiliary system.

Tc-99m STHHYYP-GHEG-ECG-TAMRA accumulated substantially in NCI-H1975 tumors (Figure 4A, arrow). The tumor to normal muscle uptake ratio of Tc-99m STHHYYP-GHEG-ECG-TAMRA increased over time (3.1 ± 0.4, 4.7 ± 0.7, and 5.9 ± 0.9 at 1, 2, and 3 h, respectively).

In contrast, Tc-99m STHHYYP-GHEG-ECG-TAMRA did not significantly accumulate in NCI-H1650 tumors (Figure 4A, arrowhead). The NCI-H1650 tumor-to-normal muscle uptake ratio (2.0 ± 0.2, 2.2 ± 0.4, and 2.7 ± 0.5 at 1, 2, and 3 h, respectively) was significantly lower than that of NCI-H1975 tumors (p < 0.05, *, Fig. 4b). .

Example 7. In vitro fluorescence imaging and immunohistochemical staining

7-1. In vitro fluorescence imaging

After in vivo gamma camera imaging studies, mice (n = 5) were sacrificed by cervical dislocation. Several different organs and tumors were resected and ex vivo fluorescence imaging studies were performed using a fluorescence imaging system (VISQUE™ InVivo Smart-LF, Vieworks, Anyang, Korea). The emission band from 520 to 675 nm was used for TAMRA (peak absorbance and peak emission wavelength = 565 and 580 nm, respectively). Exposure time was 5.0 seconds per image. Images were analyzed using dedicated software (CluVue™, Vieworks, Anyang, Korea). The in vitro fluorescence imaging results are shown in Figure 5.

As shown in Figure 5, in the in vitro fluorescence images of mouse organs, among healthy organs, high fluorescence activity of Tc-99m STHHYYP-GHEG-ECG-TAMRA was observed in the kidney, and muscle showed faint TAMRA activity. NCI-H1975 tumors (arrows) showed significantly higher fluorescence activity than NCI-H1650 tumors (arrowheads). The results of in vitro fluorescence imaging were confirmed to show similar trends to in vivo gamma camera imaging.

7-2. Immunohistochemical staining

After ex vivo fluorescence imaging studies, tumors were snap frozen using liquid nitrogen. Frozen tumors were cut into slices (10 μm thick). After drying at room temperature, the slices were fixed in ice-cold acetone for 10 min. Afterwards, the slices were air-dried at room temperature for 20 min and maintained at room temperature for 30 min with 5% goat serum to block nonspecific binding. Afterwards, tumor sections were stained with anti-human L858R mutant specific EGFR antibody for 1 hour at room temperature. After washing with PBS, tumor slides were incubated with Alexa Fluor ^® 488-conjugated goat anti-rabbit secondary antibody. Tumor slides were washed three times with PBS. Washed tumor slides were incubated in Prolong ^® Gold Antifade Reagent containing 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA). Soaked and closed the cover glass. Imaging studies were performed using an FV1200 confocal microscope (Olympus) with a 20x lens. The results of immunohistochemical staining are shown in Figure 6.

As shown in Figure 6, significant fluorescence of Tc-99m STHHYYP-GHEG-ECG-TAMRA was detected in NCI-H1975 tumor tissue. The fluorescence correlated with the fluorescence activity of the anti-L858R mutant EGFR antibody (Figure 6A). However, the fluorescence activity of TAMRA and anti-L858R mutant EGFR antibodies was barely detectable in NCI-H1650 tumor tissue (Figure 6b).

Example 8. Biodistribution study

Tumors and small portions of selected organs were excised 1 and 3 hours after injection of 18.5 MBq of Tc-99m STHHYYP-GHEG-ECG-TAMRA. Tissue was weighed and placed into pre-weighed gamma counter tubes. Tissue radioactivity was counted using a gamma counter (1480 Wizard 3; PerkinElmer Life and Analytical Sciences), and the resulting minute counts were decay corrected. The total injected radioactivity per animal was calculated by measuring the radioactivity of the syringe before and after injection. Results were expressed as percentage of dose injected per gram of tissue (%ID/g). The results of the biodistribution study are shown in Table 1.

Organ Mean %ID/g (SD) 1h 3h Lungs 1.37 (0.72) 0.85 (0.33) Heart 1.04 (0.13) 0.73 (0.28) Blood 2.59 (0.95) 1.64 (0.45) Liver 1.93 (0.43) 1.94 (0.58) Stomach 1.79 (0.77) 0.96 (0.24) Colon 0.64 (0.24) 0.55 (0.18) Kidneys 19.76 (3.00) 9.18 (2.18) Muscles 0.83 (0.24) 0.61 (0.34) NCI-H1975 tumor
(EGFR with L858R mutation) 2.77 (0.70) 3.48 (1.01) NCI-H1650 tumor
(EGFR without L858R mutation) 1.84 (0.55) 1.47 (0.64)

As shown in Table 1, the kidney showed the highest activity at 1 hour, followed by the blood, stomach, lung, and liver. At 3 hours, the activity of normal organs except the kidneys decreased significantly, resulting in a Tc-99m STHHYYP-GHEG-ECG-TAMRA target-to-non-target ratio. ) has been improved. The %ID/g values for NCI-H1975 tumors were significantly higher than those for NCI-H1650 tumors. The NCI-H1975 tumor to normal muscle uptake ratio of %ID/g values was 3.35 ± 0.12 and 6.20 ± 1.83 at 1 and 3 hours, respectively.

Example 9. Statistical analysis

SPSS version 18.0 (IBM Corp., Armonk, NY, USA) was used for data analysis. The experimental results were expressed as mean ± standard deviation (SD). Target-to-normal muscle uptake ratio of gamma camera images was compared using one-way analysis of variance (ANOVA) with appropriate post hoc tests. In all tests, a p-value <0.05 indicated statistical significance.

Overall, the present inventors confirmed that radioisotope-labeled cancer-targeting compounds had high absorption rates and biodistribution values when administered to cancer tissues with the EGFR L858R mutation. This means that radiographic images and fluorescence images can be obtained together when administering a radioactively labeled cancer-targeting compound, and the cancer-targeting compound of the present invention can be used in a variety of ways in the field of cancer diagnosis.

As above, specific parts of the present invention have been described in detail, and it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Sequence list electronic file attached

Claims (11)

fluorescent substance,
A chelating ligand that binds to a radioactive isotope and is represented by the amino acid sequence of SEQ ID NO: 3, and
A cancer-targeting compound comprising a cancer-targeting peptide represented by the amino acid sequence of SEQ ID NO: 1.
According to paragraph 1,
The fluorescent substance is a light-emitting molecule, fluorescent protein, metal ion, complex compound, organic dye, conductor, semiconductor, insulator, quantum dot or quantum wire, and a cancer targeting compound.
According to paragraph 1,
The cancer is selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, glioma, ovarian cancer, pancreatic cancer, stomach cancer, thyroid cancer, and uterine cancer. A compound targeting cancer.
According to paragraph 1,
A compound for targeting cancer, wherein the cancer contains the epidermal growth factor receptor (EGFR) L858R mutation.
According to paragraph 1,
The cancer targeting compound further includes a spacer to reduce fluorescence disturbance of the targeting peptide.
According to clause 5,
A compound for targeting cancer, wherein the spacer is represented by the amino acid sequence of SEQ ID NO: 2.
According to clause 5,
The cancer targeting compound is arranged in the form of structural formula 1.
[Structural Formula 1]
Cancer targeting peptide-spacer-chelating ligand-fluorescent substance
According to clause 5,
The cancer-targeting compound is a cancer-targeting compound, characterized in that represented by Formula 1
[Formula 1]

A dual modality contrast agent comprising the cancer-targeting compound according to any one of claims 1 to 8.
A composition for diagnosing cancer comprising the cancer targeting compound according to any one of claims 1 to 8.
A method of providing information for cancer monitoring, comprising the step of treating a biological sample with the cancer-targeting compound according to any one of claims 1 to 8.