Composition for predicting cetuximab responsiveness in cancer

Abstract

The present invention relates to a composition for predicting cetuximab responsiveness in cancer, and more specifically to a composition for predicting cetuximab responsiveness, comprising a complex (Cetux-probe) having a fluorophore and a quencher bound to both sides of a peptide cleaved by a lysosomal enzyme, and having cetuximab conjugated to the C-terminus of the peptide. The Cetux-probe of the present invention operates in the same manner as the therapeutic mechanism of cetuximab, and thus the therapeutic effect and responsiveness of cetuximab may be quickly and accurately predicted. Cetux-probe activated fluorescence may be used to measure EGFR degradation, and a strong linear correlation between the Cetux-probe activated fluorescence and the cytotoxicity of cetuximab was observed in colorectal cancer cells and tumor-bearing mice. In addition, the predictive ability of the Cetux-probe activated fluorescence was confirmed to be much higher than that of EGFR expression or KRAS mutation status, and thus the Cetux-probe of the present invention may be usefully employed for predicting a response to cetuximab therapy by assessing EGFR degradation.

Description

Composition for predicting responsiveness to cetuximab in cancer

[0001] 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, and cetuximab is bound to the C-terminus of the peptide.

[0002]

[0003] Activation of the epidermal growth factor receptor (EGFR) plays a crucial 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 efficacy of patients with metastatic colorectal cancer. However, responses to cetuximab vary significantly among individuals, and clinical benefits appear to be limited to specific subgroups of colorectal cancer patients.

[0004] Early differentiation between responders and non-responders can minimize unnecessary prolonged treatment, avoiding significant limitations such as serious toxicity and high costs. Therefore, developing methods to predict response to anti-EGFR therapy is essential to maximize treatment outcomes. Accordingly, recent research has primarily focused on predicting response to anti-EGFR therapy.

[0005] Currently, four EGFR monoclonal antibodies (cetuximab, panitumumab, necitumumab, and nimotuzumab) are on the market, and several other EGFR monoclonal antibodies are in clinical trials. In early clinical trials, only patients with EGFR positivity confirmed by immunohistochemical staining were considered for anti-EGFR therapy. However, several studies have shown that initial EGFR expression levels are not associated with response to anti-EGFR therapy.

[0006] To date, 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 cancer is considered refractory to cetuximab because KRAS mutations lead to constitutive activation of downstream EGFR signaling.

[0007] However, even in patients with KRAS wild-type colorectal cancer, the response rate to anti-EGFR therapy is low at 40%, so a new and reliable approach to predict response to anti-EGFR therapy is needed (W. De Roock,et al.,Ann. Oncol., 19:508-515, 2008).

[0008] 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 Fcγ 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 for downregulating growth-promoting signals within cells (H. Sunada, et al., Proc. Natl. Acad. Sci. USA, 83:3825-3829, 1986).

[0009] As the importance of effective degradation of specific disease-associated proteins, such as EGFR, for disease treatment has been emphasized, various targeted protein degradation strategies, such as proteolysis-targeting chimeras (PROTACs) and lysosome-targeting chimeras (LYTACs), have recently attracted considerable attention. Accumulating evidence suggests that the degree of EGFR degradation, rather than the degree of EGFR expression following EGFR-mediated internalization, significantly correlates with the anticancer efficacy of anti-EGFR antibodies (Y. Okada, et al., Mol. Cancer Res., 15:1445-1454, 2017). Previous studies suggest that monitoring EGFR degradation following EGFR-mediated internalization may serve as a means of predicting response to anti-EGFR therapy.

[0010]

[0011] Accordingly, the present invention developed a novel fluorescent probe, Cetuximab, for assessing EGFR degradation following anti-EGFR treatment. Specifically, the present invention carefully investigated how the anti-EGFR antibody (cetuximab) induces EGFR degradation, and then developed Cetuximab to operate by the same mechanism as this therapeutic mechanism. As shown in Figure 1, 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 EGFR proteolysis.

[0012] Cetuximab probes are rationally designed to share this mechanism of action: they bind to EGFR, are internalized into cells, and are transported to lysosomes for degradation. Just as degradation of the cetuximab:EGFR complex in lysosomes promotes cetuximab's anticancer effects, degradation of the cetuximab:EGFR complex in lysosomes can activate a fluorescent signal.

[0013]

[0014] Accordingly, 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 the cetuximab treatment effect.

[0015] Another object of the present invention is to provide a method for providing information for predicting responsiveness to cetuximab using the composition.

[0016] Another object of the present invention is to provide a composition for diagnosing EGFR-positive cancer.

[0017]

[0018] To achieve the above-mentioned purpose,

[0019] The present invention provides a composition for predicting the responsiveness 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, and a fluorescent substance and a quencher are conjugated to both sides of the peptide, and cetuximab is bound to the C-terminus of the peptide.

[0020] In a preferred embodiment of the present invention, 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.

[0021] In another preferred embodiment of the present invention, the fluorescent substance is 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.

[0022] In another preferred embodiment of the present invention, 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.

[0023] In another preferred embodiment of the present invention, the cancer may be an epidermal growth factor receptor (EGFR) positive cancer, and preferably an EGFR positive colon cancer.

[0024] In another preferred embodiment of the present invention, 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 fluorescent substance by the quencher and generating fluorescence. The higher the fluorescence, the higher the reactivity to cetuximab.

[0025]

[0026] To achieve other purposes,

[0027] 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 fluorescence intensity.

[0028] In a preferred embodiment of the present invention, 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.

[0029]

[0030] To achieve another purpose,

[0031] The present invention provides a composition for diagnosing EGFR-positive cancer, 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, and a fluorescent substance and a quencher are conjugated to both sides thereof, and cetuximab is bound to the C-terminus of the peptide.

[0032] In addition, the present invention provides a method for screening a cancer treatment drug having EGFR degradation capability, 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 of the complex.

[0033]

[0034] The cetuximab-conjugated probe (Cetux-probe) of the present invention operates in the same manner as the cetuximab therapeutic mechanism, enabling rapid and accurate prediction of cetuximab's therapeutic efficacy and responsiveness. Furthermore, the predictive power of Cetux-probe-activated fluorescence was confirmed to be significantly higher than that of EGFR expression or KRAS mutation status. Therefore, the Cetux-probe of the present invention can be usefully utilized to predict response to cetuximab therapy by assessing EGFR degradation.

[0035]

[0036] Figure 1 is a schematic diagram depicting the mechanism of action of Cetux-probe, a novel fluorescent probe for visualizing target protein (EGFR) degradation, and its binding and potential applications in precision medicine. (a) Schematic representation of (a) cetuximab and (b) Cetux-probe, which share the same mechanism of action - EGFR-mediated internalization and lysosomal degradation. (c) Cetux-probe was synthesized by conjugating C-probe to cetuximab via the 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. In addition, (d) Cetux-probe can be utilized to predict patient-specific responses to anti-EGFR therapy and to visualize target protein degradation.

[0037]

[0038] Figure 2 is a schematic diagram showing the synthesis process of Cetux-probe, and the conjugate is synthesized by amide coupling between the carboxylic acid group of C-probe and the free amine group of cetuximab.

[0039]

[0040] Figure 3 is data confirming the characterization of Cetux-probe.

[0041] Figure 3a shows 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.

[0042] Figure 3b shows the results of non-reducing SDS-PAGE analysis of cetuximab, Cetux-probe, Cetux-FPR675, C-probe, and FPR675 obtained in a gel stained with InstantBlue™ Coomassie stain (left) and in fluorescence mode (right). In the figure, M represents a molecular weight marker.

[0043] Figure 3c shows the absorbance spectra of Cetux-probe (7.5 μM; top), cetuximab (0.6 mg / mL; light blue line, bottom), and C-probe (10.0 μg / mL; orange line, bottom) in PBS, respectively.

[0044] Figure 3d shows 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 4) absorbance plots at 280 nm and 5) 610 nm versus C-probe concentration in PBS, respectively.

[0045] Figure 3e shows data showing the fluorescence activation of C-probe, Cetux-probe, FPR675, and Cetux-FPR675 observed and quantified when treated with lysosomal enzymes, papain (top), and cathepsin B (bottom).

[0046] 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.

[0047] Figure 3g shows the normalized fluorescence spectra of Cetux-probe (1.5 μM, red curve) and Cetux-FPR675 (1.5 μM, black curve) activated by papain (5 unit / ml) treatment, while Cetux-probe did not emit fluorescence without papain treatment (blue curve).

[0048] 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 μg / ml of cathepsin B.

[0049] Figure 3i shows a fluorescence image (top) of a microplate containing lysates of CCK-81 cells treated with various concentrations of Cetux-probe (0 to 100 μg / ml) and data from a quantitative analysis of Cetux-probe concentration and fluorescence intensity (bottom).

[0050] Figure 3j is data confirming the binding affinity of Cetux-probe, cetuximab, and PD-L1 antibody to EGFR analyzed by ELISA.

[0051]

[0052] Figure 4 shows data confirming (a) the fluorescence activity against a lysosomal protein hydrolase (cathepsin B) and (b) the sensitivity of the fluorescence inhibition effect against a cellular lysosomal protein degradation inhibitor (chloroquine) using a Cetux-probe manufactured based on two peptide sequences that are cleaved by lysosomal enzymes.

[0053]

[0054] Figure 5 is data confirming fluorescence activation of Cetux-probe in CCK-81 cells.

[0055] Figure 5a shows confocal microscopy images of CCK-81 cells treated with activatable Cetux-probe (1 μM, red) and 'always-on' Cetux-FPR675 (1 μM, red) at 10 min and 24 h after probe treatment. Cetux-probe showed barely detectable fluorescence at 10 min, but strong fluorescence was observed at 24 h. Cetux-FPR675 showed fluorescence at both 10 min and 24 h (scale bar: 20 μm).

[0056] Figure 5b is a fluorescence confocal microscopy image of CCK-81 cells 10 min after probe treatment. Cetux-probe (red) emitted a weak signal that was barely traceable on the cell membrane of CCK-81 cells, whereas Cetux-FPR675 (red) showed strong fluorescence mainly combined with surface EGFR (light blue) distributed on the cell membrane under a confocal microscope (scale bar: 20 μm. The white dotted rectangle represents an enlarged area with a scale bar of 160 μm).

[0057] Figure 5c shows fluorescence confocal microscopy images (top) of CCK-81 cells treated with Cetux-probe (red) and LysoTracker (green) 4 and 24 hours after probe treatment, and data profiling the fluorescence intensity of Cetux-probe and LysoTracker along the white lines in the merged confocal images (bottom) (scale bar: 20 μm. White dotted rectangle indicates an enlarged area with a scale bar of 160 μm).

[0058] 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 (light blue) or lysosomal (green) markers, respectively, in CCK-81 cells.

[0059] Figure 5e shows data confirming the intracellular location of Cetux-probe (red) over time.

[0060] Figure 5f shows data confirming the intracellular localization of Cetux-FPR675 (red) over time. The fluorescent localization of the probe (red) in Figures 5e and 5f was analyzed via simultaneous confocal imaging using surface EGFR (light blue) and lysosomal (green) markers in KRAS mutant colon cancer cells, respectively, and the degree of colocalization was quantified by the Pearson correlation coefficient (r) (white dotted rectangles indicate enlarged areas).

[0061]

[0062] Figure 6 is data confirming Cetux-probe activation according to inhibitor treatment.

[0063] Figures 6a and 6b show confocal microscopy images (top) and quantitative analysis data (bottom) of CCK-81 cells treated with Cetux-probe (1 μM, 4 h) and Cetux-FPR675 (1 μM, 4 h). In the figures, FI represents fluorescence intensity (scale bar: 40 μm).

[0064] Figures 6c and 6d show confocal microscopy images (top) and quantitative analysis data (bottom) of HCT-8 cells treated with Cetux-probe (1 μM, 4 h) and Cetux-FPR675 (1 μM, 4 h). In the figures, FI represents fluorescence intensity (scale bar: 20 μm).

[0065]

[0066] Figure 7 shows data monitoring EGFR degradation using Cetux-probe in colon cancer cells.

[0067] Figure 7a shows Western blot data confirming total EGFR levels and quantifying relative EGFR levels according to cetuximab treatment (0 - 100 μg / mL) in CCK-81, HCT-8, LoVo, and COLO 320DM cells. Relative EGFR levels were expressed as relative levels, with the EGFR / GAPDH value in the cetuximab 0 μg / mL group set at 100% (n = 3).

[0068] Figure 7b is a fluorescence confocal microscopy image of total EGFR expression (green) and Cetux-probe (red, 1 μM, 24 hours) in CCK-81, HCT-8, LoVo, and COLO 320DM cells treated with cetuximab (100 μg / ml, 24 hours) (scale bar: 20 μm).

[0069] Figure 7c shows data confirming the correlation between the degraded EGFR (%) and the relative fluorescence intensity of Cetux-probe, and the plot was evaluated by the Pearson correlation coefficient (r = 0.95, P = 0.0498).

[0070] Figure 7d shows the EGFR levels in HCT-8 cells in the presence of inhibitor CQ (100 μM) when treated with cetuximab (100 μg / ml) for 24 hours, as confirmed by Western blot (top), and the quantified data (bottom).

[0071] Figure 7e shows the EGFR levels in HCT-8 cells in the presence of inhibitors BafA1 (100 nM) and Z-FA-FMK (100 μM) when treated with cetuximab (100 μg / ml) for 24 hours, as determined by Western blot (top), and the quantified data (bottom).

[0072] Figure 7f shows data confirming the fluorescence intensity of Cetux-probe (1 μM) in HCT-8 cells when pretreated with various concentrations (0 to 100 μM) of CQ. The fluorescence intensity was measured as mean fluorescence intensity (MFI) using a flow cytometer.

[0073]

[0074] 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 μg / ml), and cell viability was measured.

[0075]

[0076] 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.

[0077] Figure 9a shows the flow cytometry histogram (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.

[0078] Figure 9b shows data analyzing the correlation between the cytotoxicity of cetuximab and the average fluorescence intensity of Cetux-probe when eight colon cancer cell lines were treated with various cetuximab treatment concentrations (100 μg / ml, 50 μg / ml, 10 μg / ml, 5 μg / ml, 1 μg / ml, 0.5 μg / ml) for 48 hours.

[0079] Figure 9c shows data analyzing the correlation between the cytotoxicity of cetuximab and the average fluorescence intensity of Cetux-probe when cetuximab (0.5 μg / ml) was treated in 12 colon cancer cell lines.

[0080] Figure 9d shows Western blot analysis of EGFR levels in eight colon cancer cell lines (top) and a correlation plot between relative EGFR levels and cytotoxicity of cetuximab (0.5 μg / mL) (bottom). The correlation was assessed using the Pearson correlation coefficient (r = 0.24).

[0081] Figure 9e shows data confirming the cytotoxicity of cetuximab (0.5 μg / 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.

[0082]

[0083] Figure 10 shows data observing the biodistribution and tumor-focused fluorescence of Cetux-probe in a xenograft mouse model derived from colon cancer cells.

[0084] 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.

[0085] Figure 10b is data analyzing the average radiant efficiency in the tumor area of ​​mice injected with Cetux-probe by separately normalizing the fluorescence imaging at 2, 4, and 8 hours.

[0086] Figure 10c shows ex vivo fluorescence imaging images of major organs and tumors resected from mice bearing HT-29 tumors (top) and quantified data of fluorescence focus efficiency for tumors as a ratio of tumor radiance efficiency to total radiance efficiency in ex vivo imaging (bottom) using Living Image software (n = 3) (H: heart, Lu: lung, Li: liver, Sp: spleen, Ki: kidney, Tu: tumor).

[0087]

[0088] Figure 11 is data predicting therapeutic response to cetuximab treatment by measuring in vivo EGFR degradation using Cetux-probe.

[0089] Figure 11a is a schematic diagram showing the in vivo experimental timeline of Cetux-probe imaging and cetuximab treatment in animal experiments.

[0090] Figure 11b shows 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 initiating cetuximab treatment.

[0091] Figure 11c shows data quantifying the average radiation efficiency of Cetux-probe in the tumor area in vivo at 8, 16, 24, and 32 hours after Cetux-probe administration.

[0092] Figure 11d is a fluorescence image of tumor tissue sections from HCT-8 and LoVo mice treated with Cetux-probe (red) (scale bar: 50 μm).

[0093] Figure 11e shows the ex vivo fluorescence imaging data of major organs and tumors resected from mice bearing HCT-8 and LoVo tumors 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).

[0094] Figure 11f shows data observing the extent of tumor growth in mice bearing HCT-8 tumors, HT-29 tumors, and LoVo tumors. Control mice were injected with PBS (control group n = 3, cetuximab treatment group n = 5).

[0095] Figure 11g Western blot images of EGFR and GAPDH expression in tumor lysates (top) and data showing quantitative EGFR / GAPDH levels (bottom).

[0096] Figure 11h shows immunohistochemical staining of EGFR (left) and quantitative analysis data of EGFR levels in tumor tissues collected from mice bearing HCT-8 and LoVo tumors treated with cetuximab (right). The relative fluorescence intensity of EGFR in control tumor tissues was set to 100%.

[0097]

[0098] Hereinafter, the present invention will be described in detail.

[0099]

[0100] The present invention relates to a composition for predicting the responsiveness 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 the peptide, a fluorescent substance and a quencher are conjugated to both sides, and cetuximab is bound to the C-terminus of the peptide.

[0101] In the present invention, 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.

[0102] In the present invention, 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 choices.

[0103] In the present invention, 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.

[0104] In the present invention, the cancer is an epidermal growth factor receptor (EGFR) positive cancer, and 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.

[0105] In the present invention, 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 fluorescent substance by the quencher, thereby generating fluorescence. The higher the fluorescence, the higher the reactivity to cetuximab.

[0106]

[0107] The inventors of the present invention previously developed an EGF-Probe in a previous study (Korean Patent Publication No. 10-2022-0021509). While both the EGF-Probe and the Cetuximide-probe of the present invention bind to EGFR with high specificity, EGF is known to be a natural ligand for EGFR and promote cancer formation and growth. Therefore, the present invention sought to develop a substance safer than the EGF-Probe.

[0108] Cetuximab, a targeted therapy that targets EGFR for cancer treatment, is expected to be suitable as a tool for predicting cancer treatment responsiveness. Therefore, in the present invention, a predictive probe that operates through the same mechanism as the therapeutic agent was developed with the goal of predicting treatment responsiveness to EGFR-targeted therapeutics such as cetuximab. The mechanism by which cetuximab binds to EGFR, is internalized, and degraded by lysosomal enzymes, and the Cetux-probe, a probe incorporated into cetuximab, has the advantage of being able to more accurately mimic the mechanism than the EGF-probe.

[0109] In a specific embodiment of the present invention, a cetuximab-conjugated probe (Cetux-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 identical to that of cetuximab.

[0110] In another specific embodiment of the present invention, when colon cancer cell lines were treated with Cetux-probe, it was confirmed that the Cetux-probe internalized into the cells was activated through lysosomal degradation. Furthermore, when various colon cancer cell lines were treated with Cetux-probe, it was confirmed that there was a strong correlation between cetuximab-induced toxicity and Cetux-probe fluorescence intensity.

[0111] In addition, the drug responsiveness of 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.

[0112]

[0113] From another aspect, the present invention relates to a method for providing information for predicting the therapeutic effect of cetuximab on a sample isolated from a cancer patient, comprising the step of treating the composition with the composition and measuring the fluorescence intensity.

[0114] In the present invention, 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.

[0115]

[0116] From another perspective, the present invention relates to a composition for diagnosing EGFR-positive cancer, 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.

[0117] In the present invention, it was confirmed that the Cetux-probe internalized into cells by EGFR is activated by lysosomal degradation and the fluorescence intensity increases, and therefore, it can be used as a diagnostic composition for EGFR-positive cancer, preferably EGFR-positive colon cancer.

[0118]

[0119] From another aspect, the present invention relates to a method for screening 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 of the complex.

[0120] In the present invention, when the fluorescence intensity is detected, the candidate substance can be selected as having EGFR decomposition ability.

[0121] In the present invention, it was confirmed that there is a significant and strong correlation between cetuximab-induced EGFR degradation and fluorescence activation of Cetux-probe, and therefore, it can be used for screening candidate substances having EGFR degradation ability equal to or superior to that of cetuximab.

[0122]

[0123] Hereinafter, the present invention will be described in more detail through examples.

[0124] These examples are intended solely to illustrate 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 being limited by these examples.

[0125]

[0126] Example 1: Preparation of cetuximab-conjugated probe (Cetux-probe)

[0127] 1-1: Cetux-probe manufacturing

[0128] In the present invention, a cetuximab-conjugated probe (Cetux-probe) capable of predicting the drug response of cetuximab was prepared. As shown in Figure 1, the Cetux-probe was developed by conjugating a lysosomal enzyme-activating peptide-based probe (C-probe) to cetuximab.

[0129] 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, Flamma™ Fluors 675 (FPR675), and a dark quencher (BHQ-3).

[0130] Specifically, as shown in Fig. 2, 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.

[0131] Next, Cetux-probe was prepared by conjugating C-probe to cetuximab via EDC / N-hydroxysulfosuccinimide (sulfo-NHS) reaction. C-probe (5.12 mg, 2.57 μmol), sulfo-NHS (6.98 mg, 32.16 μmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 2.47 mg, 12.86 μmol) 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. 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.

[0132] As a control, 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 μmol) and 1.8 ml of cetuximab (21 mg / ml) in the same manner as the Cetux-probe conjugate.

[0133]

[0134] 1-2: Checking Cetux-probe characteristics

[0135] To confirm the characteristics of the manufactured Cetux-probe, SEC-FPLC (fast protein size-exclusion liquid chromatography) was used and monitored by detecting specific absorbance wavelengths for Cetux-probe and Cetux-FPR675 (λmax 280 nm for cetuximab and λmax 610 nm for C-probe).

[0136] Absorption and fluorescence spectra were recorded with a Cary 300 spectrophotometer (Agilent Technologies, USA) and a fluorescence spectrophotometer F-7000 (Hitachi-High-Tech, Japan), respectively, and the fluorescence spectra were obtained by exciting at 672 nm and scanning the emission fluorescence from 680 nm to 800 nm, respectively.

[0137] As a result, as shown in Fig. 3a, compared to free cetuximab, cetuximab was eluted earlier from the crude reaction mixture at 9 to 11 ml elution volume. Simultaneous absorbance signals were observed at 280 nm and 610 nm, indicating that the C-probe was conjugated to cetuximab.

[0138] Cetuximab 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 in the gel was acquired using the iBright™ FL1000 imaging system (Invitrogen, USA) in the FPR675 fluorescence mode (excitation: 608–632 nm, emission: 675–720 nm). The gel was then stained with InstantBlue™ Coomassie dye, and the proteins in each sample were identified using the protein visible light mode of the same instrument.

[0139] As a result, as shown in Fig. 3b, when the protein was stained with InstantBlue™ Coomassie dye, Cetux-probe showed a strong band only at the cetuximab position in SDS-PAGE, and there was no band at the C-probe position, indicating that unbound C-probe was successfully removed (Fig. 3b, left lanes 2 and 4).

[0140] Furthermore, in SDS-PAGE gels, Cetux-probe did not emit fluorescence, whereas Cetux-FPR675, used here as a comparative formulation for Cetux-probe, exhibited strong fluorescence (Fig. 3b, right lanes 2 and 3). This is consistent with the activatable property of Cetux-probe.

[0141]

[0142] The average number of C-probes conjugated to cetuximab in the Cetux-probe was assessed by measuring the absorbance of cetuximab and C-probe at various concentrations using UV / Vis spectroscopy. As shown in Figures 3c and 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 indicates that C-probes were successfully attached to cetuximab in the Cetux-probe.

[0143]

[0144] 1-3: Fluorescence intensity according to C-probe sequence

[0145] In the present invention, 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 fluorophore, Flamma™Fluors 675 (FPR675), and a dark quencher (BHQ-3). The C2 probe differs from the C-probe prepared in <Example 1-1> in that it is composed of a peptide whose core cleavage sequence is GGFG-based (GGFGGKGG; SEQ ID NO: 2).

[0146] Specifically, to synthesize the peptide-FPR675 conjugate, 1 equivalent of the peptide and 1.2 equivalents of FPR675 NHS ester were used and reacted for 2 hours. After the reaction, the product was purified by RP-HPLC using a C18 column and then lyophilized. Then, for the conjugation of BHQ-3, the peptide-FPR675 conjugate was 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 were added and reacted for 1 hour. The final product was purified by RP-HPLC using a C18 column and then lyophilized.

[0147] Next, a Cetux-probe was manufactured using a C2 probe as in <Example 1-1> above.

[0148]

[0149] Using Cetux-probes manufactured based on two peptide sequences cleaved by lysosomal enzymes, the fluorescence activity against lysosomal proteolytic enzymes (cathepsin B) and the sensitivity of the fluorescence inhibition effect to a lysosomal protein degradation inhibitor (chloroquine) were confirmed. In Fig. 5, C1 probe refers to a Cetux-probe manufactured using C-probe, and C2 probe refers to a Cetux-probe manufactured using C2 probe (Cetux-C2 probe).

[0150] As a result, as shown in Fig. 5a, 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. At this time, the C1 probe reacted more sensitively to cathepsin B than the C2 probe, and the fluorescence signal was activated more strongly.

[0151] Furthermore, as shown in Fig. 5b, 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. At this time, 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.

[0152]

[0153] The C1 probe and C2 probe have very similar fluorescence restoration properties for basic lysosomal enzymes, and when the C1 probe and C2 probe were introduced into the EGF-probe previously produced by the inventor of the present invention, the fluorescence restoration properties were also very similar.

[0154] On the other hand, when the C1 probe and C2 probe were introduced into Cetuximab, the fluorescence restoration ability of the Cetux-probe was confirmed to be much superior to that of the Cetux-C2 probe. Accordingly, in the present invention, subsequent experiments were performed using the Cetux-probe, which has excellent fluorescence restoration ability.

[0155]

[0156] Example 2: Verifying Cetux-probe Activation

[0157] 2-1: Confirmation of Cetux-probe activation by lysosomal enzymes

[0158] Since Cetux-probe was designed to be activated by lysosomal enzymes, activation of Cetux-probe by lysosomal enzymes was confirmed.

[0159] Cetux-probe, C-probe, Cetux-FPR675, and FPR675 were adjusted to the same concentration (1 μM 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 PBS buffer (pH 7.4) and cathepsin B (0 to 10.0 μg / mL) at 37°C for 1 h, and then the fluorescence intensity of each well was measured in the FPR675 fluorescence mode of iBright™. The 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).

[0160]

[0161] As a result, in the absence of enzyme, FPR675 fluorescence was significantly suppressed by BHQ-3, and C-probe and Cetux-probe did not show fluorescence, while application of papain and cathepsin B dramatically increased the fluorescence of Cetux-probe by 13.5±0.2-fold and 24.0±4.8-fold, respectively. In contrast, unlike the activatable Cetux-probe, strong fluorescence was observed with Cetux-FPR675, an “always-on” probe, regardless of the presence of enzyme (Fig. 3e).

[0162] In the uncleaved probe, fluorescence is quenched by fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher. Consistent with this, both C-probe and Cetux-probe remained quenched after incubation with 10% FBS at 37°C for 24 h (Fig. 3f).

[0163] 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).

[0164] Additionally, Cetux-probe was used to treat papain (R 2 = 0.99) or cathepsin B (R 2 = 0.97) when exposed to different concentrations or treated with different concentrations of Cetux-probe (R 2 = 0.97) fluorescence intensity increased dose-dependently (Fig. 3h to 3i). These results indicate that Cetux-probe proportionally activates fluorescence depending on the lysosomal enzyme concentration and the amount of probe used.

[0165]

[0166] 2-2: Affinity evaluation of Cetux-probe for EGFR

[0167] To determine whether conjugation of cetuximab to C-probe could decrease cetuximab's binding ability to EGFR, the affinity of cetuximab for EGFR was assessed. Enzyme-linked immunosorbent assay (ELISA) for human recombinant EGFR protein was performed with various concentrations (100–3000 ng / mL) of cetuximab, cetuximab, and anti-PD-L1 antibody.

[0168]

[0169] Binding affinity to human recombinant EGFR was assessed using a cetuximab (human) ELISA kit according to the manufacturer's protocol. Briefly, assay buffer was added to each well, followed by the addition of standard reagents (cetuximab), cetuximab, Cetux-probe, or PD-L1 antibody. The samples were then incubated at room temperature (RT) for 30 minutes. The reaction solution was removed, and the wells were washed three times. HRP-conjugated probe solution was added and incubated for an additional 30 minutes at RT. After repeated washing, TMB (3,3′,5,5-tetramethylbenzidine) chromogen substrate solution was added and incubated for 10 minutes at RT under light-protected conditions. Finally, stop solution was added, and the absorbance of each well was detected at 450 nm using a VersaMax™ microplate reader (Molecular Devices Corp., USA). The concentrations of the standard reagents used were 0, 100, 300, 1000, and 3000 ng / mL of cetuximab moiety.

[0170]

[0171] As a result, as shown in Fig. 3j, the binding affinity of Cetux-probe for human recombinant EGFR protein was confirmed to be almost the same as that of cetuximab, while anti-PD-L1 used as a control showed almost no binding affinity for human recombinant EGFR protein. These results indicate that conjugation of cetuximab to C-probe does not reduce the affinity of cetuximab for EGFR.

[0172]

[0173] Example 3: Confirmation of Cetux-probe activation in KRAS wild-type colon cancer cells.

[0174] Cetux-probe activation following lysosomal degradation was confirmed in CCK-81 cells, a KRAS wild-type colon cancer cell line.

[0175] The CCK-81 cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank) and cultured in a high-humidity environment with 5% CO2 at 37°C in the recommended medium containing 10% FBS and 1% antibiotics (streptomycin and 100 units / mL penicillin).

[0176] Cells were plated at 3.0 × 10 in a confocal dish. 5 Cells were seeded at a density of 10 cells / dish and treated with Cetux-probe and Cetux-FPR675 at a concentration of 1 μM each. To co-label lysosomes, cells were treated with 2 μM LysoTracker Green for 2 h, and fluorescence images of the cells were acquired using a TCS SP8 confocal laser microscope (Leica, Wetzlar, Germany). The Pearson 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.

[0177]

[0178] To co-label EGFR on the cell surface, the treated probes were washed, and the cells were incubated with rabbit anti-human EGFR antibodies for 10 minutes at 4°C. After washing twice with PBS (pH 7.4), the cells were treated with Alexa Fluor 488-labeled goat anti-rabbit antibodies as a secondary antibody for 30 minutes at 4°C. To stain total EGFR in cells, the cells were fixed and permeabilized with the Cytofix / Cytoperm™ kit, and then blocked with 1% (w / v) BSA for 30 minutes at room temperature. The cells were then incubated with the same anti-EGFR antibodies for 30 minutes at room temperature, washed with PBS, and stained with the same secondary antibodies for 30 minutes at room temperature. Fluorescence images of the stained cells were acquired using a confocal microscope and quantified using Image J software.

[0179]

[0180] As a result, 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).

[0181]

[0182] To more specifically monitor the intracellular activity of Cetux-probe, confocal microscopy images were obtained over time after treatment with Cetux-probe for 10 min, 4 h, and 24 h (Fig. 5b, Fig. 5c, and Fig. 5d). After 10 min of treatment with Cetux-probe, a very weak signal was detected from the surface EGFR of the cell membrane (Fig. 5b, light blue). At 4 and 24 h, Cetux-probe exhibited strong activated fluorescence (Fig. 5c, red), most of which was combined with the fluorescence of LysoTracker in lysosomes (Fig. 5c, green), resulting in a rich yellow signal.

[0183] The colocalization levels of Cetux-probe and LysoTracker were calculated by analyzing the Pearson correlation coefficient (r), and r increased from r = 0.82 at 4 hours to r = 0.92 at 24 hours.

[0184] In contrast to the results obtained with Cetux-probe, Cetux-FPR675, which has no quenching effect, showed strong fluorescence 10 min after treatment. At this point, the signal was merged with that of EGFR, which was mainly distributed on the cell membrane (Fig. 5b).

[0185] Cetux-FPR675 was found to emit fluorescence throughout the entire process, from plasma 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.

[0186]

[0187] To measure the cellular internalization and fluorescence activation of the Cetuximab in other colon cancer cells, the same assay was performed using KRAS mutant HCT-8 cells (purchased from the Korean Cell Line Bank). Similar results were observed as in KRAS wild-type CCK-81 cells (Figs. 5e and 5f). These results indicate that the internalized Cetuximab is activated in lysosomes.

[0188]

[0189] Example 4: Confirmation of Cetux-probe activation following inhibitor treatment

[0190] In order to further investigate the mechanism of action of Cetux-probe, the activity of Cetux-probe was confirmed when treated with various inhibitors. The inhibitors used were cetuximab, BafA1, and Z-FA-FMK. It was anticipated that cetuximab would inhibit EGFR-mediated uptake of Cetux-probe or Cetux-FPR675 by competing for binding to EGFR.

[0191]

[0192] CCK-81 cells were cultured in the same manner as in <Example 3> above, 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 μM, pre-treated for 1 hour), and then cell nuclei were stained with Hoechst solution.

[0193] Chloroquine (CQ, 0–100 μM) was pretreated for 3 h before Cetuximab treatment to inhibit EGFR degradation. Changes in EGFR expression were monitored by Western blot, and the fluorescence intensity of Cetuximab in cells was assessed by flow cytometry.

[0194]

[0195] Western blot analysis for EGFR quantification was performed as follows. Before protein extraction from individual cells, cells were washed twice with ice-cold PBS, and cell lysates were collected using RIPA buffer containing a 100X protease inhibitor cocktail. Samples were centrifuged at 16,000 g for 20 minutes at 4°C, the supernatant was collected, and the protein concentration was determined by BCA assay. Cell lysates containing equal amounts of protein were loaded onto 8% SDS-PAGE. After electrophoresis at 90 V for 90 minutes, the separated proteins were transferred to a poly(vinylidene fluoride) membrane. The membrane was blocked with 5% (w / v) skim milk for 2 hours at room temperature, and then incubated overnight at 4°C with rabbit anti-human EGFR and mouse anti-human GAPDH antibodies as primary antibodies.

[0196] After washing three times with 1X Tris-buffered saline, 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 iBright™. The EGFR expression level was quantitatively expressed as the relative EGFR level by calculating the intensity of EGFR / GAPDH using Image J software.

[0197]

[0198] Flow cytometry was performed as follows. Cells were dissociated into single cells using trypsin-EDTA, and the fluorescence intensity of the Cetux-probe was analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, USA). The fluorescence data of each sample were 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 the Cetux-probe was calculated by subtracting the MFI value of the sample not treated with Cetux-probe from the MFI value of each sample.

[0199]

[0200] As a result, as shown in Figures 6a and 6b, when cells were co-treated with the probe and cetuximab, the fluorescence of Cetux-probe was inhibited by 85.1±9.1%, and the fluorescence of Cetux-FPR675 was inhibited by 87.2±5.2%. This indicates that both probes are capable of EGFR-mediated internalization.

[0201] Bafilomycin A1 (BafA1), a vacuolar H+-ATPase inhibitor, is known to inhibit lysosomal degradation by increasing intralysosome pH. Cells co-treated with the probe and BafA1 showed a 97.4±0.7% inhibition of fluorescence activation of Cetux-probe, confirming that fluorescence of Cetux-probe is activated by lysosomal degradation.

[0202] In addition, pretreatment of CCK-81 cells with Z-FA-FMK (Z-Phe-Ala fluoromethyl ketone), a well-known inhibitor of the lysosomal enzyme cathepsin B, resulted in a 94.1±2.0% inhibition of fluorescence activation of Cetux-probe. On the other hand, co-treatment with BafA1 or pretreatment with Z-FA-FMK had little effect on fluorescence of Cetux-FPR675.

[0203]

[0204] The experiment was performed using the same method as above using HCT-8 cells, which are KRAS mutant cells, and as a result, similar results to those of KRAS wild-type CCK-81 cells were observed, as shown in Figures 6c and 6d. That is, it was confirmed that the Cetux-probe was internalized through EGFR binding and activated through lysosomal degradation, resulting in fluorescence activation.

[0205]

[0206] Example 5: Monitoring EGFR degradation using Cetux-probe

[0207] 5-1: Monitoring EGFR degradation by Cetuximab in different colon cancer cell lines

[0208] We investigated whether Cetux-probe could monitor cetuximab-induced EGFR degradation.

[0209] First, cetuximab (10, 25, 50, and 100 μg / 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>.

[0210]

[0211] As a result, as shown in Figures 7a and 7b, 100 μg / mL of cetuximab promoted significant EGFR degradation in CCK-81 and HCT-8 cells, but no EGFR degradation was observed in LoVo or COLO 320DM cells at any of the tested cetuximab concentrations. These results are consistent with the fluorescence activation of the Cetux-probe in the four cell lines. CCK-81 and HCT-8 cells showed strong fluorescence intensity, whereas LoVo and COLO 320DM cells showed low fluorescence intensity.

[0212] Indeed, a significant and strong correlation was observed between cetuximab-induced EGFR degradation and fluorescence activation of Cetux-probe in four cell lines (r = 0.95, P = 0.0498; Fig. 7c).

[0213]

[0214] 5-2: Monitoring EGFR degradation by Cetuximide probe following treatment with lysosomal degradation inhibitors

[0215] To further investigate whether the Cetux-probe can monitor EGFR degradation, in the present invention, cetuximab-induced EGFR degradation was inhibited by treatment with chloroquine (CQ), which inhibits lysosomal degradation.

[0216] In the same manner as in <Example 4> above, HCT-8 cells were treated with CQ (100 μM), BafA1 (100 nM), and Z-FA-FMK (100 μM), respectively, and then treated with cetuximab (100 μg / ml) for 24 hours. The degree of EGFR degradation was confirmed by Western blot.

[0217]

[0218] As a result, as shown in Fig. 7d, we confirmed that CQ inhibited cetuximab-induced EGFR degradation in HCT-8 cells, whereas it had no effect on EGFR expression in cetuximab-untreated cells. Other lysosomal degradation inhibitors, such as BafA1 and Z-FA-FMK, were also found to similarly inhibit cetuximab-induced EGFR degradation in HCT-8 cells (Fig. 7e).

[0219]

[0220] Additionally, HCT-8 cells were pretreated with various concentrations of CQ (0 to 100 μM) for 3 h and then treated with Cetux-probe (1 μM) for 24 h, and the degree of EGFR degradation was confirmed by Western blot.

[0221] As a result, as shown in Fig. 7e, the reduced EGFR degradation by CQ was monitored by changes in the fluorescence intensity of the Cetux-probe. 100 μM CQ reduced EGFR degradation by 29.1 ± 4.8% and the fluorescence intensity of the Cetux-probe by 86.1 ± 1.1% in Western blot analysis.

[0222] This high sensitivity of Cetux-probe to EGFR degradation was also observed in the fluorescence signals of cells co-treated with 1, 20, and 50 μM CQ, indicating that Cetux-probe can be used to measure EGFR degradation in cells.

[0223]

[0224] Example 6: Confirmation of drug responsiveness of cetuximab using Cetux-probe in colon cancer cells.

[0225] To investigate the relationship between the fluorescence intensity of Cetux-probe and the anticancer activity of cetuximab, we evaluated the cytotoxicity by cetuximab and the fluorescence activation by Cetux-probe in eight colon cancer cell lines, including four KRAS wild-type (CCK-81, HT-29, COLO 320DM and LIM1215) cell lines and four KRAS mutant (HCT-8, HCT-15, HCT 116 and LoVo) cell lines.

[0226]

[0227] 6-1: Confirmation of cytotoxicity by cetuximab

[0228] First, cytotoxicity following cetuximab treatment was evaluated using the Cell Counting Kit-8 (CCK-8) assay.

[0229] Each cell line (5000 cells / well) was seeded in a 96-well microplate, and the cells were treated with various concentrations of cetuximab ranging from 0 to 100 μg / ml, and then cultured at 37°C for 48 h. Afterwards, the absorbance was measured at 450 nm using a microplate reader, and the cell viability of each group (n = 6) was determined by taking the absorbance of untreated cells as 100%, and the cytotoxicity was calculated as (100 - cell viability)%.

[0230]

[0231] As a result, as shown in Fig. 8, the growth of CCK-81 cells was inhibited by more than 30% at the lowest concentration (0.5 μg / 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 μg / ml to 100 μg / ml of cetuximab, indicating that HCT-8 cells are moderately sensitive to cetuximab.

[0232] Growth of HCT 116, LIM1215, and HT-29 cells was inhibited by 10–20% at all tested cetuximab concentrations, indicating that these cell lines showed a moderately poor response to cetuximab.

[0233] HCT-15 and LoVo cells were assessed as having low sensitivity to cetuximab, whereas COLO 320DM cells were determined to be nonresponsive to cetuximab, showing less than 10% growth inhibition at all tested concentrations.

[0234]

[0235] 6-2: Fluorescence intensity confirmation using Cetux-probe

[0236] To analyze the fluorescence intensity of Cetux-probe, the above eight cell lines were cultured for 24 hours by treating them with Cetux-probe at a concentration of 10 nM, and then flow cytometry analysis was performed, and the degree of EGFR degradation was confirmed by Western blot.

[0237]

[0238] As a result, as shown in Fig. 9a, it was confirmed that the fluorescence intensity by Cetux-probe was high in cell lines showing high cytotoxicity to cetuximab.

[0239] To confirm their correlation, the fluorescence intensity of Cetux-probe was compared with the degree of cytotoxicity induced by various concentrations of cetuximab (1, 5, 10, 50, and 100 μg / ml). As shown in Fig. 9b, a strong correlation was confirmed between the toxicity caused by cetuximab and the fluorescence intensity of Cetux-probe.

[0240] Among the tested cell lines, only 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.

[0241]

[0242] By adding 4 KRAS mutant cell lines (SW480, LS513, LS 174T DLD-1) reported to have low cetuximab responsiveness to the above 8 cell lines, the correlation between the fluorescence activity of Cetux-probe and the cytotoxicity of cetuximab (0.5 ㎍ / ㎖) was analyzed in a total of 12 cell lines. As shown in Fig. 9c, even when KRAS mutant cell lines reported to have low cetuximab responsiveness were added, the Pearson correlation index was 0.92, confirming that Cetux-probe still showed high accuracy for cetuximab treatment.

[0243]

[0244] 6-3: Comparison of Cetux-probe with other biomarkers

[0245] To confirm the superiority of Cetux-probe in assessing cetuximab drug responsiveness, we analyzed it using other biomarkers used to predict cetuximab responders and non-responders in colorectal cancer.

[0246] First, we evaluated the level of EGFR expression in cells known as a predictive biomarker for response to anti-EGFR antibody treatment. Western blot of EGFR expression in eight colon cancer cell lines showed that HCT 116 and LIM1215 cells showed high levels of EGFR expression, followed by HT-29, HCT-8, HCT-15, LoVo, and CCK-81 cells, while COLO 320DM cells showed the lowest level of EGFR expression (Fig. 9d, top). There was no clear linear correlation between the level of EGFR expression and the degree of cetuximab-induced cytotoxicity (r = 0.24, Fig. 9d, bottom).

[0247]

[0248] Additionally, because KRAS mutations have been established as a biomarker predicting response to cetuximab, we evaluated the correlation between cetuximab sensitivity and KRAS mutation status.

[0249] As a result, as shown in Figure 9e, KRAS mutations showed low predictive power (P = 0.7549). This discrepancy may be due to the fact that KRAS mutations have been identified as a predictive biomarker for anti-EGFR therapy combined with chemotherapy in metastatic colorectal cancer, whereas in the present invention, KRAS mutations were evaluated to predict response to anti-EGFR monotherapy. Taken together, these results confirmed that the Cetux-probe can be usefully utilized as a powerful tool for predicting response to cetuximab.

[0250]

[0251] Example 7: Confirmation of drug responsiveness of cetuximab using Cetux-probe in an animal model

[0252] To investigate the in vivo relevance of the strong correlation observed between fluorescence activation of Cetux-probe and in vitro cetuximab cytotoxicity, we generated xenograft mouse models using HCT-8, HT-29 and LoVo cell lines, which represent high-, medium- and low-responsive cell lines, respectively, using Cetux-probe, and then predicted their responsiveness to cetuximab.

[0253]

[0254] 7-1: Animal model production and probe administration

[0255] BALB / c nu / nu mice were purchased from Orient Bio (Korea) and maintained in a pathogen-free environment at the Korea Institute of Science and Technology (KIST). All live animal experiments were performed in compliance with relevant laws and institutional guidelines of the KIST Institutional Animal Care and Use Committee (IACUC) and were approved by the IACUC (Approval No. 2022-04-5052).

[0256] To prepare colon cancer xenograft models, HT-29, HCT-8, and LoVo cells were cultured at 4 × 10 6 Subcutaneously inoculated into the right flank of BALB / c nu / nu mice at a density of 10 cells / mouse, and tumor size was approximately 160 mm 3 In vivo imaging was performed when reached.

[0257] Cetux-FPR675 (15 nmol) and Cetux-probe (15 nmol) were intravenously injected into tumor-bearing mice, respectively, and in vivo imaging was performed for 7 days using IVIS (PerkinElmer, USA) at 660 nm excitation and 710 nm emission. Afterwards, three mice from each group were sacrificed, and the heart, lungs, liver, spleen, kidney, and tumor were extracted and imaged ex vivo.

[0258] 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.

[0259]

[0260] To determine the optimal time to visualize fluorescence after Cetux-probe administration, Cetux-probe was injected intravenously into HT-29 colon cancer-bearing mice, and observations were made over time. The fluorescence signal appeared predominantly in the tumor starting 16 hours after injection (Fig. 10a and Fig. 10b). After Cetux-probe injection, the fluorescence signal rapidly increased to a peak on day 1 and then slowly decreased until day 7 after injection, whereas the Cetux-FPR675 signal developed very rapidly until 2 hours after injection. In addition, the activatable Cetux-probe showed more concentrated fluorescence in the tumor area compared to the "always-on" Cetux-FPR675 in vivo.

[0261] In vitro analysis further confirmed that the fluorescence focus efficiency for tumors in mice treated with Cetux-probe (37.2 ± 2.5%) was higher than that of Cetux-FPR675 (26.1 ± 3.1%, P < 0.01; Fig. 10c).

[0262]

[0263] 7-2: Cetuximab treatment of tumor-bearing mice

[0264] Mice administered with Cetuximab probe in the same manner as in <Example 7-1> above were monitored using an IVIS imaging system (Fig. 11a). After imaging, the mice were injected intraperitoneally with cetuximab at a dose of 25 mg / kg every 3 days for a total of 8 administrations. Tumor size was measured every 2 days using calipers, and the tumor volume was calculated as length × (width) 2 × was calculated as 0.53 (control group n=3, cetuximab treatment group n=5).

[0265] The day after all treatments were completed, tumors were extracted, and a portion of each tumor was used to prepare lysates for Western blot and paraffin blocks for immunohistochemistry.

[0266] Specifically, to perform immunohistochemical staining for EGFR, 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, and rehydrated. The slides were then 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 overnight at 4°C with an anti-EGFR antibody. The following day, secondary staining was performed with an Alexa Fluor 488-labeled goat anti-rabbit antibody for 1 h at room temperature, followed by nuclei staining. EGFR levels were expressed as the fluorescence intensity of Alexa Fluor 488 using a confocal microscope.

[0267]

[0268] As a result, as shown in Fig. 11b, the tumor size was 166.46 ± 15.42 mm in HCT-8, HT-29, and LoVo xenograft mice (HCT-8; 166.46 ± 15.42 mm) when imaged after Cetux-probe administration. 3 , HT-29; 166.79 ± 9.74 mm 3 and LoVo; 165.66 ± 17.46 mm 3 ) were found to be almost identical.

[0269] Quantification of the average radiative efficiency of Cetux-probe in the tumor area in vivo at 8, 16, 24, and 32 hours after Cetux-probe administration, as shown in Figures 11c, 11d, and 11e, showed that the fluorescence intensity of Cetux-probe in the tumor was 1.34±0.07×10 at 32 hours in mice with HCT-8 tumors. 10 ) in mice bearing HT-29 tumors (1.13±0.07×10 at 32 h 10 ) and mice with LoVo tumors (0.95±0.06×10 at 32 h10 ) was found to be significantly higher.

[0270]

[0271] After treating mice with cetuximab, the degree of tumor suppression was observed. As shown in Fig. 11f, cetuximab significantly inhibited tumor growth in mice bearing HCT-8 tumors and HT-29 tumors compared to 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.

[0272]

[0273] Next, to determine whether the growth inhibitory effect of cetuximab was related to EGFR degradation, tumors were resected the day after all treatments were completed, and EGFR levels were determined by Western blot and immunohistochemistry.

[0274] As a result, as shown in Figures 11g and 11h, it was confirmed that EGFR levels induced by cetuximab were significantly reduced in HCT-8 tumors compared to LoVo tumors. This was further supported by the finding that the fluorescence intensity of Cetuximab was higher in HCT-8 tumor tissues compared to LoVo tumor tissues. Overall, the results of the present invention imply that the fluorescence intensity of Cetuximab in tumors correlates with the degree of EGFR degradation and the therapeutic efficacy of cetuximab in mice with colon cancer.

[0275]

[0276] The cetuximab-conjugated probe (Cetux-probe) of the present invention operates in the same manner as the cetuximab therapeutic mechanism, allowing rapid and accurate prediction of cetuximab's therapeutic efficacy and responsiveness. The activated fluorescence of the Cetux-probe can be used to measure EGFR degradation, and a strong linear correlation was observed with cetuximab's cytotoxicity in colon cancer cells and tumor-bearing mice. Furthermore, the predictive power of the Cetux-probe's activated fluorescence was confirmed to be much higher than that of EGFR expression or KRAS mutation status. Therefore, the Cetux-probe of the present invention can be usefully utilized to predict responses to cetuximab therapy by assessing EGFR degradation.

Claims

1. A composition for predicting the responsiveness of cetuximab or confirming the prognosis of cetuximab treatment effect, comprising 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 the peptide, a fluorescent substance and a quencher are conjugated to both sides, and cetuximab is bound to the C-terminus of the peptide.

2. In paragraph 1, A composition for predicting the responsiveness of cetuximab or confirming the prognosis of cetuximab treatment effect, characterized in that the fluorescent substance is bound to the N-terminus of the peptide and the quencher is bound to the epsilon amine group of the lysine or arginine amino acid residue of the peptide.

3. In paragraph 1, The above fluorescent substance is any one 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. A composition for predicting the responsiveness of cetuximab or confirming the prognosis of cetuximab treatment effect, characterized by:

4. In paragraph 1, A composition for predicting the responsiveness of cetuximab or confirming the prognosis of the cetuximab treatment effect, characterized in that the quencher is any 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.

5. In paragraph 1, A composition for predicting the responsiveness to cetuximab or confirming the prognosis of cetuximab treatment effect, characterized in that the cancer is epidermal growth factor receptor (EGFR) positive colon cancer.

6. In paragraph 1, The above complex can penetrate into cancer cells and be cleaved by an enzyme present in the lysosome of colon cancer cells, and when the quenching effect of the fluorescent substance by the quencher is relieved, fluorescence can be generated, and the higher the fluorescence, the higher the responsiveness to cetuximab or the higher the therapeutic effect to cetuximab, characterized in that a composition for predicting the responsiveness to cetuximab or confirming the prognosis of the therapeutic effect of cetuximab.

7. 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 a composition according to any one of claims 1 to 6 and measuring fluorescence intensity.

8. In paragraph 7, A method for providing information for predicting responsiveness to cetuximab or therapeutic effect to cetuximab, characterized in that when fluorescence intensity is detected in the above method, the cancer patient is judged to have responsiveness to cetuximab or is judged to have a therapeutic effect to cetuximab.

9. A method for screening a drug for cancer treatment having EGFR degradation ability, comprising the step of treating a candidate substance with an EGFR-positive cancer cell line treated with the composition of any one of claims 1 to 6, and then measuring the fluorescence intensity by the composition.

10. A composition for diagnosing EGFR-positive cancer, comprising 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 the peptide, a fluorescent substance and a quencher are conjugated to both sides, and cetuximab is bound to the C-terminus of the peptide.

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