Construction of CD24 / TROP2 dual antibody magnetic beads and their application in the detection of circulating tumor cells in breast cancer.

Abstract

This invention relates to the field of biotechnology, specifically to a method for constructing CD24 / TROP2 dual-antibody magnetic beads and their application in the detection of circulating tumor cells (CTCs) in breast cancer. The method involves chemically conjugating anti-CD24 monoclonal antibodies and anti-TROP2 monoclonal antibodies onto the surface of magnetic beads, thereby constructing dual-antibody functionalized magnetic beads. The invention also provides a method for capturing CTCs using the aforementioned dual-antibody magnetic beads, comprising mixing and incubating the magnetic beads with a blood sample to be tested, allowing tumor cells expressing CD24 and / or TROP2 to bind to the magnetic beads, obtaining enriched CTCs after magnetic separation, and identifying and analyzing them by immunofluorescence staining. Compared with existing technologies, this invention, by introducing a synergistic recognition mechanism of CD24 and TROP2 dual markers, can effectively improve the capture efficiency of circulating tumor cells, especially exhibiting higher sensitivity and accuracy in the detection of CTCs with low EpCAM expression during epithelial-mesenchymal transition (EMT), thus overcoming the limitations of traditional single-marker detection methods.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the construction of a CD24 / TROP2 dual antibody magnetic bead and its application in the detection of circulating tumor cells in breast cancer. Background Technology

[0002] Circulating tumor cells (CTCs) are tumor cells that detach from the primary tumor or metastases and enter the peripheral blood circulation. Although few in number, they have significant clinical importance. CTC detection has been widely used in early tumor screening, treatment monitoring, and recurrence risk assessment, and is particularly valuable in personalized treatment of breast cancer.

[0003] Currently, CTC enrichment technologies mainly include capture methods based on physical properties (such as particle size and charge) and biological markers. Among them, antibody magnetic bead capture methods based on immunomagnetic separation have become the mainstream technical route due to their simplicity and high specificity. Existing commercial systems mostly rely on epithelial cell adhesion molecules (EpCAM) for CTC capture.

[0004] However, with a deeper understanding of tumor biology, it has been found that CTCs undergo epithelial-mesenchymal transition (EMT) during blood circulation, which leads to a significant decrease or even absence of expression of some CTC cell surface epithelial markers (such as EpCAM), resulting in a high false negative rate for capture methods based on a single epithelial marker.

[0005] To improve the sensitivity of CTC detection, existing technologies have attempted to employ multi-antibody combination strategies, such as combining EpCAM with markers like HER2 and EGFR, aiming to cover more CTC subpopulations. However, these approaches primarily focus on epithelial-derived markers, offering insufficient coverage for CTCs with stem cell or mesenchymal characteristics, and thus failing to fully reflect CTC heterogeneity. Studies have shown that CD24, a molecule associated with tumor stemness and metastatic potential, is persistently expressed in some breast cancer CTCs. TROP2, a transmembrane glycoprotein, is highly expressed in various epithelial-derived tumor cells, particularly showing high detection value in breast cancer. Therefore, theoretically, the combined use of CD24 and TROP2 holds promise for covering different phenotypes of CTCs, including epithelial and some stem cell-like CTCs.

[0006] However, current technologies lack a systematic approach for the synergistic use of CD24 and TROP2 dual biomarkers in CTC capture, particularly regarding antibody magnetic bead construction, antibody distribution structure, and capture performance optimization, which require further investigation. Therefore, developing a dual antibody magnetic bead system capable of simultaneously recognizing multiple CTC phenotypes, improving capture efficiency, and reducing false negative rates is of great significance for enhancing the accuracy of CTC detection. Summary of the Invention The purpose of this invention is to address the problems in existing CTC capture technologies, such as reliance on a single epithelial marker leading to missed detections, the difficulty in covering CTC heterogeneity with multiple antibody combinations, and insufficient recognition ability for CTCs in low EpCAM or EMT states. This invention provides a structurally controllable and functionally synergistic CD24 / TROP2 dual-antibody magnetic bead and its application method to achieve efficient capture of CTCs with different phenotypes, thereby improving detection sensitivity and accuracy.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: As a first aspect of the present invention, the present invention provides a dual-antibody functionalized magnetic bead, characterized by the synergistic introduction of anti-CD24 antibody and anti-TROP2 antibody onto the surface of the magnetic microspheres to construct a CTC capture system with multi-target recognition capability. CD24 is used to recognize CTCs with stem cell characteristics or partial mesenchymal phenotypes, while TROP2 is used to recognize epithelial-derived tumor cells. The synergistic distribution of both on the same magnetic bead surface enables the magnetic bead to simultaneously recognize CTC cells of different phenotypes in a single capture process.

[0008] As a second aspect of the invention, the invention optimizes the spatial distribution and synergistic mechanism of antibodies. In a preferred embodiment, anti-CD24 antibody and anti-TROP2 antibody are immobilized on the surface of magnetic beads in a mixed or partitioned distribution. By regulating the antibody ratio and coupling density, selective enhanced capture of different CTC subsets is achieved. This structure is beneficial for increasing the probability of cell binding to magnetic beads, enhancing multi-site binding stability, and reducing capture failure caused by insufficient expression of a single antigen.

[0009] As a third invention, a method for preparing CD24 / TROP2 dual antibody magnetic beads is provided. The magnetic beads are prepared in the following manner: ① Activate carboxyl magnetic beads using EDC / NHS; ② Control the reaction pH, time, and antibody concentration to couple the magnetic beads to the antibody; ③ Different antibody distribution structures can be constructed using synchronous coupling or stepwise coupling methods; This results in the production of dual-antibody magnetic beads with stable antibody immobilization and high biological activity.

[0010] As the fourth invention of this invention, a method for detecting CTCs is provided. When using the aforementioned dual-antibody magnetic beads for CTC detection, the magnetic beads are first added to a blood sample for incubation. Then, CTCs are enriched through specific binding of the antigen and antibody. Finally, target cells are separated using magnetic separation. Compared to traditional monoclonal antibody magnetic beads, this invention can simultaneously capture epithelial and some mesenchymal CTCs, increasing the detection probability of cells with low marker expression.

[0011] Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly improves CTC capture efficiency. Due to the synergistic effect of the two antibodies, the probability of CTC binding to magnetic beads is increased, resulting in an overall capture efficiency increase of approximately 20%–50%. (2) Enhances the coverage of CTC subpopulations and can identify different phenotypes of CTC, including epithelial CTC, stem cell-like CTC, and some EMT-state CTC; (3) Reduce the false negative rate by reducing reliance on a single biomarker, thereby effectively reducing missed detections caused by the lack of biomarker expression; (4) It has good scalability. The dual antibody magnetic bead construction strategy provided by the present invention can be extended to other tumor marker combinations. Attached Figure Description

[0012] Figure 1 The effect of different antibody ratios on magnetic bead performance; Figure 2 Figure showing the CTC capture rate results of dual-antibody magnetic beads; Figure 3 Performance comparison test between dual antibody magnetic beads and traditional EpCAM magnetic beads; Figure 4 Comparison of positive rate detection results of clinical samples between dual antibody magnetic beads and traditional EpCAM magnetic beads. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0014] Example 1: Preparation of CD24 / TROP2 dual antibody magnetic beads (1) Antibody preparation In the early stages, anti-CD24 antibody and anti-TROP2 antibody sequences were obtained through phage display technology screening. The amino acid sequence of the heavy chain variable region of the anti-CD24 antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2. The amino acid sequence of the heavy chain variable region of the anti-TROP2 antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4. The expression and preparation of the two antibodies were outsourced to GenScript to obtain anti-CD24 antibody and anti-TROP2 antibody.

[0015]

[0016] (2) Magnetic bead antibody conjugation material ① Magnetic microspheres: Carboxylated magnetic beads, 0.5~3μm (Carboxylated Magnetic Beads, catalog number M2010002); ② Anti-CD24 monoclonal antibody ③ Anti-TROP2 monoclonal antibody ④EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (Sangon Biotech, Product No. C600433-0025) ⑤NHS (N-hydroxysuccinimide) (Sigma, catalog number E7750-5G) ⑥PBS buffer (pH 7.4) (Sangon Biotech, catalog number E607008-0500) (3) Preparation steps ① Vortex and sonicate the carboxyl magnetic beads for 20 seconds, then transfer 1 mg of the magnetic beads into a centrifuge tube.

[0017] ② Separate the magnetic beads magnetically, discard the supernatant, and wash the magnetic beads twice with 200 μL of PBS buffer (pH 7.4).

[0018] ③ Separate the magnetic beads magnetically, discard the supernatant, add 180 μL of PBS buffer (pH 7.4) to resuspend the magnetic beads, add 10 μL of LEDC (10-100 mg / mL, dissolved in PBS buffer (pH 7.4)); add 10 μL of Lulfo-NHS (10-100 mg / mL, dissolved in PBS buffer (pH 7.4)).

[0019] ④ Incubate at room temperature by flipping for 25 minutes.

[0020] ⑤ Separate the magnetic beads magnetically, discard the supernatant, add 200 μL of coupling buffer to resuspend the magnetic beads, and immediately add 50 μg of anti-CD24 antibody and 100 μg of anti-TROP2 antibody.

[0021] ⑥ Incubate at room temperature by flipping for 2.5 hours.

[0022] ⑦ Separate the magnetic beads magnetically, discard the supernatant, and resuspend the magnetic beads in 500 μL of blocking solution (1% BSA solution).

[0023] ⑧ Incubate at room temperature by flipping for 30 minutes.

[0024] ⑨ Separate the magnetic beads magnetically, discard the supernatant, and wash the magnetic beads once with 500 μL of blocking solution (1% BSA solution).

[0025] ⑩ Separate the magnetic beads magnetically, discard the supernatant, and wash the magnetic beads twice with 500 μL of magnetic bead preservation solution (PBST solution of 1 mg / mL BSA).

[0026] 11. Separate the magnetic beads magnetically, discard the supernatant, resuspend the magnetic beads in an appropriate volume of preservation solution (PBST solution of 1 mg / mL BSA), and store at 4°C.

[0027] (4) Results Protein quantification showed that the antibody conjugation efficiency was approximately 70%–85%, and the antibodies were evenly distributed on the surface of the magnetic beads.

[0028] Example 2: Effect of different antibody ratios on magnetic bead performance (1) Set up groups with different antibody ratios:

[0029] (2) Experimental methods ① Blood sample preparation: 1×10⁻⁵ MCF-7 (Wuxi Xinrun Biotechnology Co., Ltd., catalog number CL1123) was added to 5 mL of peripheral blood from healthy volunteers to simulate the addition of breast cancer cell line MCF-7. 4 One PBMC was separated by density gradient centrifugation after red blood cells were removed using red blood cell lysis buffer. ②Capture Incubation: Add the corresponding magnetic beads (magnetic beads: cell mass ratio of 10:1) to the isolated PBMC cells and incubate at a constant temperature of 37℃ for 25 minutes, maintaining gentle shaking during incubation; ③ Magnetic separation: An external magnetic field is applied for sedimentation, the supernatant is discarded, and then PBS is used. Wash five times with Tween elution buffer to remove non-specifically bound components; ④ Dissociation: Add dissociation buffer to the complex, gently mix and incubate at 4°C for 15 minutes, remove the microspheres magnetically, and recover the released CTCs; ⑤ Physiological activity detection and analysis: Flow cytometry was used to statistically analyze capture and recovery efficiency; AO / PI double staining was used to assess cell viability.

[0030] (3) Experimental results

[0031] result Figure 1 As shown, the capture rate of the monoclonal antibody group (A, B) was 40%–60%, while the capture rate of the bispecific antibody group (C–E) was significantly improved to 65%–85%. Among them, the 1:2 ratio (group D) had the highest capture efficiency. The antibody ratio has a significant impact on the capture performance, and there is an optimal synergistic range.

[0032] Implementation Case 3: CTC Simulation Capture Experiment (1) Sample preparation: 5000 MCF-7 breast cancer cell lines (epithelial type, Wuxi Xinrun Biotechnology Co., Ltd., catalog number CL1123) and 5000 MDA-MB-231 cells (mesenchymal type, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., catalog number ZQ0118) were added to 5 mL of peripheral blood from healthy volunteers. Red blood cells were removed using red blood cell lysis buffer, and then PBMCs were separated by density gradient centrifugation. (2) CTC cell capture and incubation: Add the coupled magnets (magnetic beads: cell mass ratio of 10:1) of Example 1 to the isolated PBMC cells, and incubate at a constant temperature of 37°C for 25 minutes, with gentle shaking during incubation; (3) Immunofluorescence staining: Add the three-color labeled immunocapture solution to the sample and incubate in a constant temperature water bath at 37°C for 30 minutes while slowly rotating to mix and promote the binding of magnetic microspheres to CTC; then place it in a magnetic separator to separate, discard the unbound components, and wash twice with elution buffer. (4) Observation of capture status: Take about 20µL of the captured complex and drop it into the center of a pre-cooled glass slide. After covering with a coverslip, fix with methanol and stain the nucleus with DAPI. Under a laser confocal microscope, acquire three-channel images: green (anti-CD24 antibody labeling), blue (cell nucleus), and red (anti-TROP2 antibody labeling). Perform colocalization analysis and count the capture rate.

[0033] (5) Experimental results, such as Figure 2 As shown, the dual-antibody magnetic beads not only improved the capture efficiency of epithelial CTCs, but also significantly enhanced the capture ability of mesenchymal CTCs.

[0034] Implementation Case 4: Performance Comparison Test with Traditional EpCAM Magnetite Beads (1) Set up different experimental groups

[0035] (2) Experimental method: ① Blood sample preparation: 5000 MCF-7 breast cancer cell lines (epithelial type, Wuxi Xinrun Biotechnology Co., Ltd., catalog number CL1123) and 5000 MDA-MB-231 cells (mesenchymal type, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., catalog number ZQ0118) were added to 5 mL of peripheral blood from healthy volunteers. Red blood cells were removed using red blood cell lysis buffer, and then PBMCs were separated by density gradient centrifugation. ②Capture Incubation: Add the corresponding magnetic beads (magnetic beads: cell mass ratio of 10:1) to the isolated PBMC cells and incubate at a constant temperature of 37℃ for 25 minutes, maintaining gentle shaking during incubation; ③ Magnetic separation: An external magnetic field is applied for sedimentation, the supernatant is discarded, and then PBS is used. Wash five times with Tween elution buffer to remove non-specifically bound components; ④ Dissociation: Add dissociation buffer to the complex, gently mix and incubate at 4°C for 15 minutes, remove the microspheres magnetically, and recover the released CTCs; ⑤ Immunofluorescence staining: Add tricolor-labeled immunocapture solution to the sample and incubate in a 37°C constant temperature water bath for 30 minutes while slowly rotating to mix and promote the binding of magnetic microspheres to CTC; then place it in a magnetic separator to separate, discard unbound components, and wash twice with elution buffer; ⑥ Observation of capture status: Take about 20µL of the captured complex and drop it into the center of a pre-cooled glass slide. After covering with a coverslip, fix with methanol and stain the nucleus with DAPI. Under a laser confocal microscope, acquire three-channel images of green (EpCAM label), blue (cell nucleus), and red (dual antibody label), and perform colocalization analysis to calculate the capture rate.

[0036] (3) Experimental results: The results are as follows Figure 3 As shown, EpCAM magnetic beads exhibit a high capture rate for MCF-7 cells (approximately 80%), but only about 25% for MDA-MB-231 cells; dual-antibody magnetic beads demonstrate a high capture rate (over 70%) for both cell types, and the combined approach further enhances the overall capture rate. This invention offers significant advantages in the detection of CTCs with low EpCAM expression.

[0037] Implementation Case 5: Preliminary Validation of Clinical Samples (1) Sample preparation: Peripheral blood samples (n=10) from breast cancer patients were selected for testing. Red blood cells were removed using red blood cell lysis buffer and then separated by density gradient centrifugation. (2) Capture and incubation: Add the corresponding magnetic beads (magnetic beads: cell mass ratio of 10:1) to the isolated PBMC cells and incubate at a constant temperature of 37°C for 25 minutes, while gently shaking during incubation; (3) Magnetic separation: An external magnetic field is applied for sedimentation, the supernatant is discarded and then PBS is used. Wash five times with Tween elution buffer to remove non-specifically bound components; (4) Add dissociation buffer to the complex, gently mix and incubate at 4°C for 15 minutes, remove the microspheres magnetically, and recover the released CTCs; (5) Immunofluorescence staining: Add tricolor-labeled immunocapture solution to the sample and incubate in a constant temperature water bath at 37°C for 30 minutes while slowly rotating to mix and promote the binding of magnetic microspheres to CTC; then place it in a magnetic separator to separate, discard unbound components, and wash twice with elution buffer. (6) Observation of the captured state: Take about 20µL of the captured complex and drop it into the center of the pre-cooled glass slide. After covering with a coverslip, fix it with methanol and stain the nucleus with DAPI. Under a laser confocal microscope, collect three-channel images of green (anti-CD24 antibody labeling), blue (cell nucleus), and red (anti-TROP2 antibody labeling) and perform colocalization analysis to calculate the positive rate.

[0038] (7) Experimental results: The results are as follows Figure 4 As shown, the positive rate of CTC detection by the dual-antibody magnetic beads was higher than that of the monoclonal antibody group. CTC was still detected in some EpCAM-negative samples, indicating that the present invention has good application prospects in clinical samples.

[0039] The above description is a preferred embodiment of the invention. Those skilled in the art will understand that, without departing from the spirit of the invention, several improvements and modifications can be made, and these improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A dual-antibody magnetic bead for capturing circulating tumor cells, characterized in that... The surface of the magnetic microspheres was coated with anti-CD24 antibodies and anti-TROP2 antibodies obtained by phage display technology.

2. The magnetic bead as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-CD24 antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2; the amino acid sequence of the heavy chain variable region of the anti-TROP2 antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

3. The magnetic bead as described in claim 1, characterized in that, The magnetic microspheres have a particle size of 0.5~3μm, preferably 1μm; the microspheres and anti-CD24 antibody and anti-TROP2 antibody are fixed by covalent coupling, and the mass ratio of the two antibodies on the surface of the magnetic beads is 1:(0.5~5).

4. The magnetic beads of claim 1, wherein the anti-CD24 antibody and the anti-TROP2 antibody present any of the following structures: (1) Uniform random distribution; (2) Local enrichment distribution; (3) Core-shell distribution structure.

5. The magnetic bead as described in claim 1, characterized in that, The antibody conjugation density is 10~500μg antibody / mg magnetic beads.

6. The method for preparing magnetic beads as described in claim 1, characterized in that, Covalent coupling was performed using an EDC / NHS system activation reaction at pH 5.0–7.

5. The antibody was added in the order of simultaneous addition of anti-CD24 antibody and anti-TROP2 antibody.

7. The magnetic bead as described in claim 1, characterized in that, The magnetic beads can simultaneously capture epithelial CTCs and mesenchymal CTCs.

8. A method for detecting circulating tumor cells, comprising the following steps: (1) Contacting a blood sample with the magnetic beads described in any one of claims 1 to 7; (2) Incubate for 15-60 minutes; (3) Magnetic separation and washing; (4) Detect target cells; The capture efficiency is ≥20% higher than that of the monoclonal antibody system.

9. The magnetic bead according to any one of claims 1 to 7, characterized in that, Magnetic beads are used for the detection of circulating tumor cells in breast cancer or for the detection of CTCs with low EpCAM expression.

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