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WO2025063823A1 - Composition pour prédire la sensibilité au cétuximab dans le cancer - Google Patents

Composition pour prédire la sensibilité au cétuximab dans le cancer Download PDF

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WO2025063823A1
WO2025063823A1 PCT/KR2024/096185 KR2024096185W WO2025063823A1 WO 2025063823 A1 WO2025063823 A1 WO 2025063823A1 KR 2024096185 W KR2024096185 W KR 2024096185W WO 2025063823 A1 WO2025063823 A1 WO 2025063823A1
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cetuximab
probe
cetux
egfr
fluorescence
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Korean (ko)
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류주희
권익찬
홍승택
성예진
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Korea Institute of Science and Technology KIST
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Korea Institute of Science and Technology KIST
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Priority claimed from KR1020240126408A external-priority patent/KR20250042670A/ko
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a composition for predicting the responsiveness of cetuximab in cancer, and more specifically, to a composition for predicting the responsiveness of cetuximab, which comprises a complex (Cetux-probe) in which a peptide cleaved by a lysosomal enzyme is centered, a fluorescent substance and a quencher are conjugated to both sides thereof, and cetuximab is bound to the C-terminus of the peptide.
  • a complex Cetux-probe
  • EGFR epidermal growth factor receptor
  • Activation of the epidermal growth factor receptor (EGFR) plays a critical role in the growth and aggressiveness of various cancers, including colorectal, head and neck, lung, and pancreatic cancers, making EGFR an important target in cancer therapy.
  • Adding the EGFR-targeting antibody cetuximab to standard chemotherapy significantly improves the treatment efficacy in patients with metastatic colorectal cancer, but the response to cetuximab varies considerably among individuals, and the clinical benefit appears to be limited to certain subgroups of colorectal cancer patients.
  • EGFR monoclonal antibodies cetuximab, panitumumab, necitumumab, and nimotuzumab
  • cetuximab panitumumab
  • necitumumab nimotuzumab
  • nimotuzumab nimotuzumab
  • KRAS mutation status is the only biomarker that can be used to predict response to anti-EGFR therapy in combination with chemotherapy in colorectal cancer.
  • KRAS mutant colorectal cancers are considered unresponsive to cetuximab because KRAS mutations lead to constitutive activation of downstream EGFR signaling.
  • Anti-EGFR antibodies exert their antitumor activity through several mechanisms. They can bind to the EGFR extracellular domain, blocking ligand binding and downstream signaling, and can bind to Fc ⁇ receptors, inducing antibody-dependent cytotoxicity. Another important mechanism of action of anti-EGFR antibodies is to induce EGFR internalization and degradation. Internalization and degradation of activated receptors are essential mechanisms by which growth-promoting signals are downregulated within cells (H. Sunada, et al. , Proc. Natl. Acad. Sci. USA , 83:3825-3829, 1986).
  • Cetux-probe a novel fluorescent probe, Cetux-probe, was developed to evaluate EGFR degradation after anti-EGFR treatment.
  • the method by which anti-EGFR antibody (Cetuximab) induces EGFR degradation was carefully investigated, and then Cetux-probe was manufactured to operate by the same mechanism as this therapeutic mechanism.
  • Cetuximab binds to EGFR and is internalized into cells in the form of a Cetuximab:EGFR complex. This complex is transported to lysosomes via early endosomes, leading to proteolysis of EGFR.
  • Cetux-probe was rationally designed to share this mechanism of action, binding to EGFR, being taken up into cells, and transported to lysosomes for degradation. Just as degradation of cetuximab:EGFR complex in lysosomes promotes the anticancer effect of cetuximab, degradation of cetux-probe:EGFR complex in lysosomes can activate the fluorescent signal.
  • the purpose of the present invention is to provide a composition for predicting the responsiveness of cetuximab in cancer or for confirming the prognosis of cetuximab treatment effect.
  • Another object of the present invention is to provide a method for providing information for predicting responsiveness to cetuximab using the composition.
  • Another object of the present invention is to provide a composition for diagnosing EGFR positive cancer.
  • the present invention provides a composition for predicting the reactivity of cetuximab or confirming the prognosis of cetuximab treatment effect, comprising a complex in which a peptide represented by the amino acid sequence of SEQ ID NO: 1, which is cleaved by a lysosomal enzyme present in a cancer cell, is centered on a peptide, a fluorescent substance and a quencher are conjugated to both sides, and cetuximab is bound to the C-terminus of the peptide.
  • the fluorescent substance may be bound to the N-terminus of the peptide, and a quencher may be bound to the epsilon amine group of a lysine or arginine amino acid residue of the peptide.
  • the fluorescent substance is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), Oregon green, Texas red, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, indocarbocyanine, rhodamine, oxacarbocyanine, thiacarbocyanine, merocyanine, pyrodyloxazole, nitrobenzoxadiazole, benzoxadiazol, Nile red, Nile orange, acridine yellow, aumarine, crystal violet, and It may be any one selected from the group consisting of malachite green.
  • FITC fluorescein isothiocyanate
  • Texas red Texas red
  • Cy2, Cy3, Cy3B Cy3.5
  • Cy5 Cy5.5
  • Cy7 indocarbocyanine
  • rhodamine oxacarbocyanine
  • thiacarbocyanine merocyanine
  • pyrodyloxazole nitrobenzoxadiazol
  • the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher, and Iowa black.
  • TAMRA 6-carboxytetramethyl-rhodamine
  • BHQ1 black hole quencher 1
  • BHQ2 black hole quencher 2
  • BHQ3 black hole quencher 3
  • NFQ nonfluorescent quencher
  • dabcyl Eclipse
  • DDQ Deep Dark Quencher
  • Blackberry Quencher and Iowa black.
  • the cancer may be an epidermal growth factor receptor (EGFR) positive cancer, and preferably an EGFR positive colon cancer.
  • EGFR epidermal growth factor receptor
  • the complex can penetrate into cancer cells and be cleaved by an enzyme present in the lysosome of the cancer cells, thereby eliminating the quenching effect of the fluorophore by the quencher, thereby generating fluorescence.
  • an enzyme present in the lysosome of the cancer cells thereby eliminating the quenching effect of the fluorophore by the quencher, thereby generating fluorescence.
  • the present invention provides a method for providing information for predicting responsiveness to cetuximab or therapeutic effect to cetuximab, comprising the step of treating a sample isolated from a cancer patient with the composition and measuring the fluorescence intensity.
  • the subject when the fluorescence intensity is detected, can be determined to have a reaction to cetuximab or to have a therapeutic effect on cetuximab.
  • the present invention provides a composition for diagnosing EGFR-positive cancer, which comprises a complex in which a peptide represented by the amino acid sequence of sequence number 1, which is cleaved by a lysosomal enzyme present in a cancer cell, is centered on a peptide, and a fluorescent substance and a quencher are conjugated to both sides thereof, and cetuximab is bound to the C-terminus of the peptide.
  • the present invention provides a method for screening for a cancer treatment drug having EGFR-degrading ability, the method comprising the step of treating a candidate substance with a complex comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1, which is cleaved by a lysosomal enzyme present in cancer cells, a fluorescent substance and a quencher conjugated to both sides of the peptide, and cetuximab bound to the C-terminus of the peptide, and then measuring the fluorescence intensity by the complex.
  • cetuximab-conjugated probe (Cetux-probe) of the present invention operates in the same manner as the cetuximab therapeutic mechanism, it can rapidly and accurately predict the therapeutic effect and responsiveness of cetuximab.
  • the predictive ability of Cetux-probe activated fluorescence was confirmed to be much higher than that of EGFR expression or KRAS mutation status, the Cetux-probe of the present invention can be usefully utilized to predict the response to cetuximab therapy by assessing EGFR degradation.
  • Figure 1 is a schematic representation of the mechanism of action of Cetux-probe, a novel fluorescent probe for visualizing target protein (EGFR) degradation, and the binding of Cetux-probe and its potential applications in precision medicine.
  • Cetux-probe was synthesized by conjugating C-probe to cetuximab via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) reaction, and C-probe is an activatable peptide-based fluorescent probe activated by lysosomal enzymes.
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • sulfo-NHS 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • sulfo-NHS 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • sulfo-NHS 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • sulfo-NHS 1-ethy
  • Figure 2 is a schematic diagram showing the synthetic process of Cetux-probe, where the conjugate is synthesized by amide coupling between the carboxylic acid group of C-probe and the free amine group of cetuximab.
  • Figure 3 is data confirming the characterization of Cetux-probe.
  • Figure 3a is the chromatogram data obtained by SEC-FPLC for the crude reaction mixture, cetuximab, and C-probe at 280 nm (blue line) and 610 nm (red line), respectively.
  • Figure 3b shows non-reducing SDS-PAGE analysis of cetuximab, Cetux-probe, Cetux-FPR675, C-probe, and FPR675 obtained in a gel stained with InstantBlueTM Coomassie stain (left) and in fluorescence mode (right).
  • M represents a molecular weight marker.
  • Figure 3c shows the absorbance spectra of Cetux-probe (7.5 ⁇ M; top), cetuximab (0.6 mg/mL; light blue line, bottom), and C-probe (10.0 ⁇ g/mL; orange line, bottom) in PBS, respectively.
  • Figure 3d is data showing UV-Vis absorption spectra of 1) cetuximab and 2) C-probe in PBS, 3) absorbance plot at 280 nm versus cetuximab concentration in PBS, and absorbance plots at 4) 280 nm and 5) 610 nm versus C-probe concentration in PBS, respectively.
  • Figure 3e shows the observed and quantified fluorescence activation of C-probe, Cetux-probe, FPR675, and Cetux-FPR675 when treated with lysosomal enzymes, papain (top), and cathepsin B (bottom).
  • Figure 3f shows data confirming the serum stability of C-probe and Cetux-probe.
  • C-probe and Cetux-probe were each cultured with 10% FBS at 37°C for 24 hours.
  • Figure 3g is the normalized fluorescence spectra of Cetux-probe (1.5 ⁇ M, red curve) and Cetux-FPR675 (1.5 ⁇ M, black curve) activated by papain (5 unit/ml) treatment; Cetux-probe did not emit fluorescence without papain treatment (blue curve).
  • Figure 3h shows the microplate fluorescence image (top) of Cetux-probe and its quantitative analysis data (bottom) in the presence of 0 to 2.0 unit/ml of papain and 0 to 10.0 ⁇ g/ml of cathepsin B.
  • Figure 3i shows fluorescence images (top) of microplates containing lysates of CCK-81 cells treated with various concentrations of Cetux-probe (0 to 100 ⁇ g/mL) and data from quantitative analysis of Cetux-probe concentration and fluorescence intensity (bottom).
  • Figure 3j is data confirming the binding affinity of Cetux-probe, cetuximab, and PD-L1 antibodies to EGFR analyzed by ELISA.
  • Figure 4 shows data confirming (a) the fluorescence activity for lysosomal protein hydrolase (cathepsin B) and (b) the sensitivity of the fluorescence inhibition effect for a protein degradation inhibitor (chloroquine) in cellular lysosomes using a Cetux-probe manufactured based on two peptide sequences cleaved by lysosomal enzymes.
  • Figure 5 shows data confirming fluorescence activation of Cetux-probe in CCK-81 cells.
  • Figure 5b is a fluorescence confocal microscopy image of CCK-81 cells 10 min after probe treatment.
  • Cetux-probe red
  • Cetux-FPR675 red
  • surface EGFR sky blue
  • the white dotted rectangle represents an enlarged area with a scale bar of 160 ⁇ m).
  • Figure 5c shows fluorescence confocal microscopy images (top) of CCK-81 cells treated with Cetux-probe (red) and LysoTracker (green) 4 h and 24 h after probe treatment, and fluorescence intensity profile data of Cetux-probe and LysoTracker along the white lines in the merged confocal images (bottom) (scale bar: 20 ⁇ m.
  • White dotted rectangle indicates an enlarged area with a scale bar of 160 ⁇ m).
  • Figure 5d shows the fluorescence intensity profile data of Cetux-probe and Cetux-FPR675 over time, respectively.
  • the fluorescence intensities were obtained through simultaneous confocal imaging of the probe (red) and markers such as surface EGFR (blue) or lysosome (green) markers, respectively, in CCK-81 cells.
  • Figure 5e shows data confirming the intracellular location of Cetux-probe (red) over time.
  • Figure 5f shows data confirming the subcellular localization of Cetux-FPR675 (red) over time.
  • the fluorescence localization of the probe (red) in Figures 5e and 5f was analyzed by simultaneous confocal imaging using surface EGFR (sky blue) and lysosome (green) markers in KRAS mutant colon cancer cells, respectively, and the degree of colocalization was quantified by the Pearson correlation coefficient (r) (the white dotted rectangle indicates the enlarged area).
  • Figure 6 shows data confirming Cetux-probe activation according to inhibitor treatment.
  • Figures 6a and 6b show confocal microscopy images (top) and quantitative analysis data (bottom) of CCK-81 cells treated with Cetux-probe (1 ⁇ M, 4 h) and Cetux-FPR675 (1 ⁇ M, 4 h).
  • FI means fluorescence intensity (scale bar: 40 ⁇ m).
  • Figures 6c and 6d are confocal microscopy images (top) and quantitative analysis data (bottom) of HCT-8 cells treated with Cetux-probe (1 ⁇ M, 4 h) and Cetux-FPR675 (1 ⁇ M, 4 h).
  • FI means fluorescence intensity (scale bar: 20 ⁇ m).
  • Figure 7 shows data monitoring EGFR degradation using Cetux-probe in colon cancer cells.
  • Figure 7a shows Western blot data confirming total EGFR levels and quantifying relative EGFR levels according to cetuximab treatment (0 - 100 ⁇ g/mL) in CCK-81, HCT-8, LoVo, and COLO 320DM cells.
  • Figure 7b is a fluorescence confocal microscopy image of total EGFR expression (green) and Cetux-probe (red, 1 ⁇ M, 24 h) in CCK-81, HCT-8, LoVo, and COLO 320DM cells treated with cetuximab (100 ⁇ g/ml, 24 h) (scale bar: 20 ⁇ m).
  • Figure 7d shows the EGFR levels in the presence of inhibitor CQ (100 ⁇ M) in HCT-8 cells treated with cetuximab (100 ⁇ g/ml) for 24 hours, as confirmed by Western blot (top), and the quantification data (bottom).
  • Figure 7e shows the EGFR levels in HCT-8 cells in the presence of inhibitors BafA1 (100 nM) and Z-FA-FMK (100 ⁇ M) when treated with cetuximab (100 ⁇ g/ml) for 24 hours, as determined by Western blot (top), and the quantification data (bottom).
  • Figure 7f shows the data confirming the fluorescence intensity of Cetux-probe (1 ⁇ M) in HCT-8 cells when pretreated with various concentrations (0 to 100 ⁇ M) of CQ.
  • the fluorescence intensity was measured as the mean fluorescence intensity (MFI) using a flow cytometer.
  • Figure 8 shows data confirming the cytotoxicity of cetuximab in eight colon cancer cells.
  • KRAS wild-type (CCK-81, HT-29, LIM-1215, COLO 320DM) and KRAS mutant (HCT-8, HCT-116, HCT-15, LoVo) cells were treated with various concentrations of cetuximab (0 to 100 ⁇ g/ml), and cell viability was measured.
  • Figure 9 shows data analyzing the correlation between the cytotoxicity of cetuximab and the fluorescence intensity of Cetux-probe in various colon cancer cell lines.
  • Figure 9a shows flow cytometry histograms (left) and quantitative analysis data of mean fluorescence intensity (MFI) (right) of eight colon cancer cell lines treated with Cetux-probe at a concentration of 10 nM for 24 hours.
  • Figure 9b shows data analyzing the correlation between the cytotoxicity of cetuximab and the average fluorescence intensity of the Cetux-probe when eight colon cancer cell lines were treated with various cetuximab treatment concentrations (100 ⁇ g/mL, 50 ⁇ g/mL, 10 ⁇ g/mL, 5 ⁇ g/mL, 1 ⁇ g/mL, 0.5 ⁇ g/mL) for 48 hours.
  • Figure 9c shows data analyzing the correlation between the cytotoxicity of cetuximab and the average fluorescence intensity of the Cetux-probe when cetuximab (0.5 ⁇ g/ml) was treated in 12 colon cancer cell lines.
  • Figure 9e shows data confirming the cytotoxicity of cetuximab (0.5 ⁇ g/ml) in KRAS wild-type and KRAS mutant cells. Red dots in the plot represent KRAS mutant cells, and blue dots represent KRAS wild-type cells.
  • Figure 10 shows data observing the biodistribution and tumor-focused fluorescence of Cetux-probe in a xenograft mouse model derived from colon cancer cells.
  • Figure 10a is an IVIS fluorescence imaging photograph of Cetux-probe and Cetux-FPR675 in HT-29 tumor-bearing mice 7 days after probe injection.
  • Figure 10b is data analyzing the average radiant efficiency in the tumor area of mice injected with Cetux-probe by separately normalizing the fluorescence images at 2, 4, and 8 hours.
  • Figure 11 is data predicting therapeutic response to cetuximab treatment by measuring in vivo EGFR degradation using Cetux-probe.
  • Figure 11a is a schematic diagram showing the in vivo experimental timeline of Cetux-probe imaging and cetuximab treatment in animal experiments.
  • Figure 11b is IVIS fluorescence imaging of mice bearing HCT-8 tumors, HT-29 tumors, and LoVo tumors 32 hours after intravenous administration of Cetuximab probe (10 nmol). Tumor sizes in HCT-8, HT-29, and LoVo xenograft mice were nearly identical when imaged before starting cetuximab treatment.
  • Figure 11c shows data quantifying the average radioactivity of Cetux-probe in the tumor area in vivo at 8, 16, 24, and 32 hours after Cetux-probe administration.
  • Figure 11d is a fluorescence image of tumor tissue sections from HCT-8 and LoVo mice treated with Cetux-probe (red) (scale bar: 50 ⁇ m).
  • Figure 11e shows the ex vivo fluorescence imaging data of major organs and tumors resected from HCT-8 and LoVo tumor-bearing mice 32 hours after intravenous administration of Cetux-probe, and the quantitative analysis of the average radiative efficiency of Cetux-probe in the indicated tumors (H: heart, Lu: lung, Li: liver, Sp: spleen, Ki: kidney, Tu: tumor).
  • FIG. 11g Western blot images of EGFR and GAPDH expression in tumor lysates (top) and data showing quantitative EGFR/GAPDH levels (bottom).
  • Figure 11h shows immunohistochemical staining of EGFR (left) and quantitative analysis data of EGFR levels (right) for tumor tissues collected from mice bearing HCT-8 tumors and LoVo tumors treated with cetuximab.
  • the relative fluorescence intensity of EGFR in the control tumor tissues was set to 100%.
  • the present invention relates to a composition for predicting the responsiveness of cetuximab or confirming the prognosis of cetuximab therapeutic effect, comprising a complex in which a peptide represented by the amino acid sequence of SEQ ID NO: 1, which is cleaved by a lysosomal enzyme present in a cancer cell, is centered on the peptide, a fluorescent substance and a quencher are conjugated to both sides, and cetuximab is bound to the C-terminus of the peptide.
  • the fluorescent substance may be bound to the N-terminus of the peptide, and a quencher may be bound to the epsilon amine group of a lysine or arginine amino acid residue of the peptide.
  • the fluorescent substance is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), Oregon green, Texas red, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, indocarbocyanine, rhodamine, oxacarbocyanine, thiacarbocyanine, merocyanine, pyrodyloxazole, nitrobenzoxadiazole, benzoxadiazol, Nile red, Nile orange, acridine yellow, aumarine, crystal violet, and malachite green. It can be any one of the selected ones.
  • the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher, and Iowa black.
  • TAMRA 6-carboxytetramethyl-rhodamine
  • BHQ1 black hole quencher 1
  • BHQ2 black hole quencher 2
  • BHQ3 black hole quencher 3
  • NFQ nonfluorescent quencher
  • dabcyl Eclipse
  • DDQ Deep Dark Quencher
  • Blackberry Quencher and Iowa black.
  • the cancer is an epidermal growth factor receptor (EGFR) positive cancer, which may be colon cancer, head and neck cancer, or lung cancer known to be treated with cetuximab, and preferably may be EGFR positive colon cancer.
  • EGFR epidermal growth factor receptor
  • the complex can penetrate into cancer cells and be cleaved by an enzyme present in the lysosome of the cancer cells, thereby relieving the quenching effect of the fluorescent substance by the quencher, thereby generating fluorescence.
  • EGF-Probe The inventors of the present invention have developed EGF-Probe in a previous study (Korean Patent Publication No. 10-2022-0021509). Although EGF-Probe and the Cetux-probe of the present invention are identical in that they bind to EGFR with high specificity, EGF is known to promote the generation and growth of cancer as a natural ligand of EGFR. Therefore, the present invention sought to develop a substance that is safer than EGF-Probe.
  • Cetuximab is also a targeted therapy that targets EGFR to treat cancer, and is expected to be suitable for use as a tool for predicting the responsiveness of cancer treatment. Therefore, in the present invention, a predictive probe that operates with the same mechanism as the therapeutic agent was developed with the purpose of predicting the responsiveness to treatment with an EGFR targeted therapy such as cetuximab.
  • the mechanism by which cetuximab binds to EGFR and is internalized and degraded by lysosomal enzymes has the advantage that the Cetux-probe, a probe introduced into cetuximab, can properly mimic the mechanism compared to the EGF-probe.
  • cetuximab-conjugated probe capable of predicting the drug reactivity of cetuximab was prepared using the schematic diagram of FIG. 1 and the method of FIG. 2. It was confirmed that the Cetux-probe proportionally activated fluorescence depending on the lysosomal enzyme concentration and the amount of probe used, and its binding affinity for human recombinant EGFR protein was found to be almost the same as that of cetuximab.
  • cetuximab was confirmed using Cetux-probe in an animal model, and the fluorescence intensity of Cetux-probe was confirmed to be correlated with the degree of EGFR degradation and the therapeutic efficacy of cetuximab in mice with colon cancer.
  • the present invention relates to a method for providing information for predicting a therapeutic effect on cetuximab response to cetuximab, comprising the steps of treating a sample isolated from a cancer patient with the composition and measuring the fluorescence intensity.
  • the subject when the fluorescence intensity is detected, the subject can be determined to have a reaction to cetuximab or to have a therapeutic effect on cetuximab.
  • the present invention relates to a composition for diagnosing EGFR-positive cancer, which comprises a complex in which a peptide represented by the amino acid sequence of SEQ ID NO: 1, which is cleaved by a lysosomal enzyme present in a cancer cell, is centered on the peptide, a fluorescent substance and a quencher are conjugated to both sides, and cetuximab is bound to the C-terminus of the peptide.
  • the Cetux-probe internalized into cells by EGFR is activated due to lysosomal degradation and the fluorescence intensity increases, and therefore, it can be utilized as a diagnostic composition for EGFR-positive cancer, preferably EGFR-positive colon cancer.
  • the present invention relates to a method for screening for a cancer treatment drug having EGFR-degrading ability, comprising the steps of treating a candidate substance with a complex comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1, which is cleaved by a lysosomal enzyme present in cancer cells, a fluorescent substance and a quencher conjugated to both sides of the peptide, and cetuximab bound to the C-terminus of the peptide, and then measuring the fluorescence intensity caused by the complex.
  • the candidate substance when the fluorescence intensity is detected, can be selected as having EGFR decomposition ability.
  • cetuximab-induced EGFR degradation and fluorescence activation of Cetux-probe, it can be utilized for screening candidate substances having EGFR degradation ability equal to or superior to that of cetuximab.
  • cetuximab-conjugated probe capable of predicting the drug reactivity of cetuximab was prepared.
  • a Cetux-probe was developed by conjugating a lysosomal enzyme-activating peptide-based probe (C-probe) with cetuximab.
  • C-probe was synthesized by a previously reported method (25) and consists of a GFLG substrate peptide reported to exhibit lysosomal enzyme-specific cleavage, conjugated to a near-infrared fluorophore, FlammaTM Fluors 675 (FPR675), and a dark quencher (BHQ-3).
  • the peptide (GFLGGKGG; SEQ ID NO: 1) and FPR675 NHS ester were reacted with dimethylaminopyridine and N-methylmorpholine, and the peptide-FPR675 conjugate was reacted with BHQ-3 NHS ester to generate C-probe.
  • the product was purified by RP-HPLC on a C18 column.
  • Cetux-probe was prepared by conjugating C-probe to Cetuximab via EDC/N-hydroxysulfosuccinimide (sulfo-NHS) reaction.
  • C-probe (5.12 mg, 2.57 ⁇ mol), sulfo-NHS (6.98 mg, 32.16 ⁇ mol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 2.47 mg, 12.86 ⁇ mol) were shaken in phosphate-buffered saline (PBS; pH 8.0) for 15 min, and then 1.8 mL of Cetuximab (21 mg/mL) was added.
  • PBS phosphate-buffered saline
  • the mixture was reacted at 25°C for 2 h and purified using an AKTA Purifier 100 FPLC system (Cytiva, USA) equipped with a Superdex 200 Increase 10/300 GL column (#28-9909-44, Cytiva).
  • the purified Cetux-probe was stored in PBS (pH 7.4) at 4°C for further use.
  • Cetux-FPR675 was synthesized by conjugating only a fluorophore (FPR675) to cetuximab. Cetux-FPR675 was synthesized by conjugating FPR675 NHS ester (1.07 mg, 1.03 ⁇ mol) and 1.8 mL of cetuximab (21 mg/mL) in the same manner as the Cetux-probe conjugate.
  • SEC-FPLC fast protein size-exclusion liquid chromatography
  • cetuximab was eluted earlier from the crude reaction mixture at 9 to 11 mL of elution volume. Simultaneous absorbance signals were observed at 280 nm and 610 nm, indicating that the C-probe was conjugated to cetuximab.
  • Cetux-probe was purified by SEC-FPLC and then identified using non-reducing SDS-PAGE. First, cetuximab, Cetux-probe, and Cetux-FPR675 were quantified at the same concentration, and then the samples mixed with 4X non-reducing loading buffer were loaded onto 8% (w/v) SDS-PAGE. FPR675 fluorescence of the gel was obtained using the FPR675 fluorescence mode (excitation: 608–632 nm, emission 675–720 nm) using an iBrightTMFL1000 imaging system (Invitrogen, USA). The gel was then stained with InstantBlueTMCoomassie dye, and the proteins in each sample were identified using the protein visible light mode of the same instrument.
  • Cetux-probe did not emit fluorescence in SDS-PAGE gel, whereas Cetux-FPR675, used here as a comparative formulation for Cetux-probe, showed strong fluorescence (Fig. 3b, right lanes 2 and 3), which is consistent with the activatable property of Cetux-probe.
  • the average number of C-probes conjugated to cetuximab in Cetux-probe was evaluated by measuring the absorbance of cetuximab and C-probe at various concentrations using UV/Vis spectroscopy. As shown in Fig. 3c and Fig. 3d, the absorbance of Cetux-probe at 280 nm for cetuximab and at 610 nm for C-probe were compared with the standard curves generated for cetuximab and C-probe. As a result, 1.30 ⁇ 0.11 C-probes were conjugated to each cetuximab. This means that the C-probe was successfully attached to cetuximab in Cetux-probe.
  • a new activation probe (C2 probe) was developed based on a peptide of a different sequence.
  • the present invention is composed of a lysosomal enzyme-activating peptide, a near-infrared fluorescent agent, FlammaTMFluors 675 (FPR675), and a dark quencher (BHQ-3).
  • the C2 probe differs from the C-probe manufactured in ⁇ Example 1-1> in that it is composed of a peptide whose core cleavage sequence is GGFG-based (GGFGGKGG; SEQ ID NO: 2).
  • the peptide-FPR675 conjugate 1 equivalent of peptide and 1.2 equivalents of FPR675 NHS ester are used, and the reaction is carried out for 2 hours. After the reaction, the product is purified by RP-HPLC using a C18 column and then lyophilized to obtain it. Thereafter, for the conjugation of BHQ-3, the peptide-FPR675 conjugate is treated with 50% trifluoroacetic acid/dichloromethane for 30 minutes to expose the amine group, and 1 equivalent of the reactant, 1.2 equivalents of BHQ-3 NHS ester, and 10 equivalents of N,N-Diisopropylethylamine are added thereto and reacted for 1 hour. The final product is purified by RP-HPLC using a C18 column and then lyophilized to obtain it.
  • C1 probe refers to a Cetux-probe manufactured using C-probe
  • C2 probe refers to a Cetux-probe manufactured using C2 probe (Cetux-C2 probe).
  • both probes were in a quenched state in the absence of cathepsin B, and a fluorescence signal appeared as the concentration of cathepsin B increased.
  • the C1 probe reacted more sensitively to cathepsin B than the C2 probe, and the fluorescence signal was activated more strongly.
  • both probes detected activated fluorescence intensity in EGFR-expressing colon cancer cells, and in cells pretreated with chloroquine, the fluorescence intensity of each probe decreased as the concentration of chloroquine increased.
  • the C1 probe showed stronger intracellular fluorescence signal activity and reacted more sensitively to chloroquine than the C2 probe, resulting in stronger inhibition of fluorescence intensity.
  • the C1 probe and C2 probe have very similar fluorescence restoration characteristics for basic lysosomal enzymes, and when the C1 probe and C2 probe were introduced into the EGF-probe previously produced by the inventors of the present invention, the fluorescence restoration characteristics were also very similar.
  • Cetux-probe was designed to be activated by lysosomal enzymes, activation of Cetux-probe by lysosomal enzymes was confirmed.
  • Cetux-probe, C-probe, Cetux-FPR675, and FPR675 were adjusted to the same concentration (1 ⁇ M of FPR675 fraction) and dispensed into a 96-well microplate. Each sample was incubated with 2-(N-morpholino)ethanesulfonic acid (MES) containing papain (0 to 2.0 unit/ml) in phosphate-buffered saline (PBS) buffer (pH 7.4) and cathepsin B (0 to 10.0 ⁇ g/ml) at 37°C for 1 h, and then the fluorescence intensity of each well was measured in the FPR675 fluorescence mode of iBrightTM. Fluorescence intensity was quantified using Image J software (National Institutes of Health, USA), and the coefficient of determination (R 2 ) was determined using Prism GraphPad software (MA, USA).
  • MES 2-(N-morpholino)ethanesulfonic acid
  • PBS phosphate-buffered saline
  • FRET fluorescence resonance energy transfer
  • the fluorescence emission spectrum of the activated Cetux-probe was identical to that of Cetux-FPR675, indicating that FPR675 was not altered by activation (Fig. 3g).
  • ELISA Enzyme-linked immunosorbent assay
  • the binding affinity for human recombinant EGFR was assessed using a cetuximab (human) ELISA kit according to the manufacturer's protocol. Briefly, the assay buffer was added to each well, followed by the addition of standard reagent (cetuximab), cetuximab, Cetux-probe, or PD-L1 antibody. The samples were then incubated for 30 min at room temperature (RT), the reaction solution was removed, the wells were washed three times, and then the HRP-conjugated probe solution was added and incubated for another 30 min at RT. After repeated washing, the TMB (3, 3′, 5, 5-tetramethylbenzidine) chromogen substrate solution was added and incubated for 10 min at RT under light-protected conditions.
  • cetuximab human ELISA kit according to the manufacturer's protocol. Briefly, the assay buffer was added to each well, followed by the addition of standard reagent (cetuximab), cetuximab, Cetux-probe, or
  • the CCK-81 cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank) and cultured in a humidified environment with 5% CO2 at 37°C in the recommended medium containing 10% FBS and 1% antibiotics (streptomycin and 100 units/mL penicillin).
  • Cells were seeded at a density of 3.0 ⁇ 10 5 cells/dish in confocal dishes and treated with Cetux-probe and Cetux-FPR675 at a concentration of 1 ⁇ M, respectively.
  • To co-label lysosomes cells were treated with 2 ⁇ M LysoTracker Green for 2 h, and fluorescence images of the cells were acquired with a TCS SP8 confocal laser microscope (Leica, Wetzlar, Germany). Pearson's correlation coefficient (r) for co-localization was measured to quantify the degree of overlap between the two fluorescence signals.
  • the fluorescence intensity profile showed the distribution of each fluorescence along a line, and the fluorescence intensity was calculated as the fluorescence intensity per cell nucleus in each image using Image J software and expressed as the relative fluorescence intensity.
  • the treated probes were washed and the cells were incubated with rabbit anti-human EGFR antibody for 10 min at 4°C. After washing twice with PBS (pH 7.4), the cells were treated with goat anti-rabbit antibody labeled with Alexa Fluor 488 as a secondary antibody for 30 min at 4°C.
  • the cells were fixed and permeabilized with Cytofix/CytopermTM kit and blocked with 1% (w/v) BSA for 30 min at room temperature. The cells were then incubated with the same anti-EGFR antibody for 30 min at room temperature, washed with PBS, and stained with the same secondary antibody for 30 min at room temperature. The fluorescence images of the stained cells were obtained through a confocal microscope and quantified using Image J software.
  • CCK-81 cells which are KRAS wild-type colon cancer cells, showed rare fluorescence after treatment with Cetux-probe for 10 minutes and strong fluorescence after 24 hours, and cells treated with Cetux-FPR675 showed strong fluorescence both at 10 minutes and 24 hours (Fig. 5a).
  • confocal microscopy images were obtained over time after treatment with Cetux-probe for 10 min, 4 h, and 24 h (Fig. 5b, Fig. 5c, Fig. 5d).
  • a very weak signal was detected from the surface EGFR of the cell membrane (Fig. 5b, sky blue), and at 4 h and 24 h, Cetux-probe showed strong activation fluorescence (Fig. 5c, red), most of which was combined with the fluorescence of LysoTracker in lysosomes (Fig. 5c, green), showing a rich yellow signal.
  • Cetux-FPR675 which has no quenching effect, showed strong fluorescence 10 min after treatment. At this time point, the signal was merged with that of EGFR, which was mainly distributed in the cell membrane (Fig. 5b).
  • Cetux-FPR675 was shown to emit fluorescence throughout the entire process from membrane entry to lysosomal processing. In contrast, Cetux-probe fluoresced primarily in lysosomes, which is thought to be due to protein degradation by lysosomal enzymes.
  • Cetux-probe in order to investigate the mechanism of action of Cetux-probe in more detail, the activity of Cetux-probe was confirmed when treated with various inhibitors. Cetuximab, BafA1, and Z-FA-FMK were treated as inhibitors, and it was expected that cetuximab would inhibit the EGFR-mediated uptake of Cetux-probe or Cetux-FPR675 by competing for binding to EGFR.
  • CCK-81 cells were cultured in the same manner as in ⁇ Example 3>, and then treated with Cetux-probe or Cetux-FPR675 and cetuximab (1 mg/ml, co-treated for 4 hours), BafA1 (20 nM, co-treated for 4 hours), or Z-FA-FMK (100 ⁇ M, pre-treated for 1 hour), and then cell nuclei were stained with Hoechst solution.
  • Chloroquine (CQ, 0–100 ⁇ M) was pretreated for 3 h before Cetux-probe treatment to inhibit EGFR degradation. Changes in EGFR expression were monitored by Western blot, and the fluorescence intensity of Cetux-probe in cells was evaluated by flow cytometry.
  • the Western blot method for EGFR quantification was performed as follows. Before protein extraction from each cell, cells were washed twice with ice-cold PBS, and then cell lysates were collected using RIPA buffer containing 100X protease inhibitor cocktail. The samples were centrifuged at 16,000 g for 20 min at 4°C, the supernatant was collected, and the protein concentration was measured by BCA assay, and the cell lysates containing the same amount of protein were loaded onto 8% SDS-PAGE. After electrophoresis at 90 V for 90 min, the separated proteins were transferred to a poly(vinylidene fluoride) membrane. The membrane was blocked with 5% (w/v) skim milk for 2 h at room temperature, and then incubated overnight at 4°C with rabbit anti-human EGFR antibody and mouse anti-human GAPDH antibody as primary antibodies.
  • the membrane was reacted with HRP-linked goat anti-rabbit secondary antibody or HRP-linked goat anti-mouse secondary antibody at room temperature for 1 hour, then the membrane was washed and treated with enhanced chemiluminescence solution to observe the expression levels of EGFR and GAPDH using iBrightTM.
  • the EGFR expression level was quantitatively expressed as the relative EGFR level by calculating the intensity of EGFR/GAPDH using Image J software.
  • Flow cytometry was performed as follows. Cells were dissociated into single cells using trypsin-EDTA, and then the fluorescence intensity of Cetux-probe was analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, USA). The fluorescence data of each sample was quantitatively measured as the mean fluorescence intensity (MFI) value in an equal number of cells by gating on the live cell population using FlowJo software (BD bioscience, USA). The MFI of Cetux-probe was calculated by subtracting the MFI value of the sample that was not treated with Cetux-probe from the MFI value of each sample.
  • MFI mean fluorescence intensity
  • Bafilomycin A1 (BafA1), a vacuolar H+-ATPase inhibitor, is known to inhibit lysosomal degradation by increasing the pH within the lysosome.
  • Cells treated with the probe together with BafA1 showed 97.4 ⁇ 0.7% inhibition of fluorescence activation of Cetux-probe, confirming that the fluorescence of Cetux-probe was activated by lysosomal degradation.
  • cetuximab (10, 25, 50, and 100 ⁇ g/ml) was treated to four colon cancer cell lines, CCK-81, HCT-8, LoVo, and COLO 320DM cells, respectively, and then Western blot and EGFR immunocytochemical experiments were performed using the same methods as in ⁇ Example 3> and ⁇ Example 4>.
  • cetuximab-induced EGFR degradation was inhibited by treatment with chloroquine (CQ), which inhibits lysosomal degradation.
  • CQ chloroquine
  • HCT-8 cells were treated with CQ (100 ⁇ M), BafA1 (100 nM), and Z-FA-FMK (100 ⁇ M), and then treated with cetuximab (100 ⁇ g/ml) for 24 hours.
  • CQ 100 ⁇ M
  • BafA1 100 nM
  • Z-FA-FMK 100 ⁇ M
  • cetuximab 100 ⁇ g/ml
  • HCT-8 cells were pretreated with various concentrations of CQ (0 to 100 ⁇ M) for 3 h and then treated with Cetux-probe (1 ⁇ M) for 24 h, and the degree of EGFR degradation was confirmed by Western blot.
  • cytotoxicity following cetuximab treatment was evaluated using the Cell Counting Kit-8 (CCK-8) assay.
  • the growth of CCK-81 cells was inhibited by more than 30% at the lowest concentration (0.5 ⁇ g/mL) of cetuximab, indicating that CCK-81 cells are highly sensitive to cetuximab.
  • the growth of HCT-8 cells was inhibited by more than 20% at 0.5 ⁇ g/mL to 100 ⁇ g/mL of cetuximab, indicating that HCT-8 cells are moderately sensitive to cetuximab.
  • HCT 116, LIM1215, and HT-29 cells were inhibited by 10–20% at all tested cetuximab concentrations, indicating that these cell lines were moderately responsive to cetuximab.
  • HCT-15 and LoVo cells were assessed as having low sensitivity to cetuximab, whereas COLO 320DM cells were determined to be unresponsive to cetuximab, showing less than 10% growth inhibition at all tested concentrations.
  • the eight cell lines were cultured for 24 hours by treating them with 10 nM Cetux-probe, and then flow cytometry analysis was performed, and the degree of EGFR degradation was confirmed by Western blotting.
  • COLO 320DM cells showed a tendency to deviate significantly from the correlation line, which may be due to the extremely low expression of EGFR in COLO 320DM cells, which may limit the ability of the Cetux-probe to enter these cells.
  • KRAS mutations have been established as a biomarker predicting response to cetuximab, we evaluated the correlation between cetuximab sensitivity and KRAS mutation status.
  • Cetux-probe can be usefully utilized as a powerful tool for predicting response to cetuximab.
  • mice BALB/c nu/nu mice were purchased from Orient Bio (Korea), and the mice were raised in a pathogen-free environment at the Korea Institute of Science and Technology (KIST). All live animal experiments were performed in compliance with the relevant laws and institutional guidelines of the Institutional Animal Care and Use Committee (IACUC) of KIST, and the experiments were approved by the IACUC (Approval No. 2022-04-5052).
  • IACUC Institutional Animal Care and Use Committee
  • HT-29, HCT-8, and LoVo cells were subcutaneously inoculated into the right flank of BALB/c nu/nu mice at a density of 4 ⁇ 106 cells/mouse, respectively, and then in vivo imaging was performed when the tumor size reached approximately 160 mm3 .
  • Cetux-FPR675 (15 nmol) and Cetux-probe (15 nmol) were intravenously injected into tumor-bearing mice, respectively, and in vivo images were taken for 7 days using IVIS (PerkinElmer, USA) at 660 nm excitation and 710 nm emission. After that, three mice from each group were sacrificed, and the heart, lungs, liver, spleen, kidney, and tumor were extracted and imaged ex vivo.
  • Fluorescence intensity was measured as the average radiant efficiency using Living Image software (PerkinElmer, USA), and the fluorescence focus efficiency for the tumor was calculated as a percentage by dividing the fluorescence intensity of the isolated tumor by the total fluorescence intensity combined from other organs and the tumor.
  • Cetux-probe was intravenously injected into mice bearing HT-29 colon cancer, and the fluorescence signal was observed over time.
  • the fluorescence signal appeared predominantly in the tumor from 16 h after injection (Fig. 10a and Fig. 10b).
  • the fluorescence signal rapidly increased to the peak on day 1 and then slowly decreased until day 7 after injection, whereas the Cetux-FPR675 signal developed very rapidly until 2 h after injection.
  • the activatable Cetux-probe showed more concentrated fluorescence in the tumor area compared to the "always-on" Cetux-FPR675 in vivo.
  • the extracted tumors were fixed with 4% paraformaldehyde, and tissue section slides were obtained from paraffin blocks. The slides were incubated at 60°C for 1 h and then immersed in xylene for paraffin removal. The slides were then immersed in 100% to 70% ethanol, rinsed with distilled water, rehydrated, and treated with antigen retrieval buffer for 20 min and blocked with 1% (w/v) BSA/PBS buffer for 30 min. Primary staining was then performed with anti-EGFR antibody at 4°C overnight. The following day, secondary staining was performed with goat anti-rabbit antibody labeled with Alexa Fluor 488 for 1 h at room temperature, and then the nuclei were stained. EGFR levels were expressed as the fluorescence intensity of Alexa Fluor 488 using a confocal microscope.
  • the tumor sizes were found to be almost the same in HCT-8, HT-29, and LoVo xenograft mice (HCT-8; 166.46 ⁇ 15.42 mm 3 , HT-29; 166.79 ⁇ 9.74 mm 3 , and LoVo; 165.66 ⁇ 17.46 mm 3 ) when imaged after Cetux-probe administration.
  • cetuximab significantly inhibited tumor growth in mice bearing HCT-8 tumors and mice bearing HT-29 tumors compared to the untreated control mice. In contrast, in mice bearing LoVo tumors, cetuximab only slightly inhibited tumor growth, and there was no significant difference in tumor volume between cetuximab-treated and untreated control mice.
  • the cetuximab-conjugated probe (Cetux-probe) of the present invention can rapidly and accurately predict the therapeutic effect and responsiveness of cetuximab because it operates in the same manner as the therapeutic mechanism of cetuximab.
  • the activated fluorescence of Cetux-probe can be used to measure EGFR degradation, and a strong linear correlation was observed with the cytotoxicity of cetuximab in colon cancer cells and tumor-bearing mice.
  • the Cetux-probe of the present invention can be usefully utilized to predict the response to cetuximab therapy by assessing EGFR degradation.

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Abstract

La présente invention concerne une composition pour prédire la sensibilité au cétuximab dans le cancer, et plus spécifiquement une composition pour prédire la sensibilité au cétuximab, comprenant un complexe (Cetux-sonde) ayant un fluorophore et un extincteur de luminescence liés aux deux côtés d'un peptide clivé par une enzyme lysosomale, et ayant du cétuximab conjugué à l'extrémité C-terminale du peptide. Le complexe Cetux-sonde de la présente invention fonctionne de la même manière que le mécanisme thérapeutique du cétuximab, et ainsi l'effet thérapeutique et la sensibilité au cétuximab peuvent être prédits rapidement et avec précision. La fluorescence activée par Cetux-sonde peut être utilisée pour mesurer la dégradation de l'EGFR, et une forte corrélation linéaire entre la fluorescence activée par Cetux-sonde et la cytotoxicité du cétuximab a été observée dans des cellules de cancer colorectal et des souris porteuses de tumeurs. De plus, la capacité prédictive de la fluorescence activée par Cetux-sonde a été confirmée comme étant beaucoup plus élevée que celle de l'expression d'EGFR ou de l'état de mutation de KRAS, et ainsi le complexe Cetux-sonde de la présente invention peut être utilement employé pour prédire la sensibilité à une thérapie au cétuximab en évaluant la dégradation d'EGFR.
PCT/KR2024/096185 2023-09-19 2024-09-19 Composition pour prédire la sensibilité au cétuximab dans le cancer Pending WO2025063823A1 (fr)

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