An EGFR fluorescent antibody, its preparation method and application

Abstract

To overcome the problems of non-targeted uptake, high false positive rate, short half-life, and instability of existing fluorescent dyes, this invention provides an EGFR fluorescent antibody, comprising a fluorescent dye, a linker, an EGFR antibody, and maleimide. The EGFR antibody covalently binds a maleimide-modified fluorescent dye molecule carrying the linker. This invention also discloses a method for preparing the above-mentioned EGFR fluorescent antibody and its applications. The EGFR fluorescent antibody provided by this invention can directly target EGFR in tumor cells, exhibits a low false positive rate, and can be efficiently and specifically taken up by tumor tissue through endocytosis and remain in tumor tissue for a long time, providing stable fluorescence imaging and navigation characteristics.

Description

Technical Field

[0001] This invention belongs to the field of biological antibody-fluorescent dye technology, specifically relating to an EGFR fluorescent antibody, its preparation method, and its application. Background Technology

[0002] Radical surgical resection is the foundation of solid tumor treatment, but surgical techniques have not fundamentally changed in the past few decades. Traditional oncology surgery relies primarily on the surgeon's subjective assessment of tissue structure, color, and texture to distinguish the tumor from surrounding normal tissue and to remove the tumor as completely as possible. However, this approach inevitably carries the risk of residual tumor or excessive removal of normal tissue. Current medical imaging techniques cannot detect micro-tumor lesions at the millimeter level or smaller. If these micro-lesions are not completely removed, they may cause tumor recurrence and metastasis, endangering the patient's life. Therefore, achieving radical tumor resection requires more precise navigation and intraoperative imaging techniques for micro-lesions. Fluorescence-guided surgery, especially immunofluorescence-guided surgery, involves labeling antibodies with fluorescent groups. These antibodies target tumor surface antigens, and real-time fluorescence imaging is achieved during surgery using a near-infrared excitation light source, a highly sensitive near-infrared fluorescence camera, and a computer image processing system. This allows for real-time visualization of the tumor tissue during surgery, helping surgeons more accurately determine tumor boundaries and metastases, thereby determining the extent of surgical resection. Fluorescent navigation surgical systems were first applied in the field of neurosurgery. In recent years, their clinical application has gradually expanded to spinal surgery, otolaryngology, hepatobiliary surgery, and gastrointestinal surgery, and has been promoted with the advent of the concept of precision surgery.

[0003] Traditional real-time navigation technology for tumor surgery based on indocyanine green fluorescence imaging involves the following basic steps: intravenous or local injection of indocyanine green preoperatively or intraoperatively; acquisition and preservation of indocyanine green fluorescence images during surgery; and removal of fluorescent tumors and suspicious lymph nodes for pathological examination. Traditional real-time navigation technology for tumor surgery based on fluorescently labeled antibodies first requires the preparation of fluorescently labeled antibodies. The basic steps involve using an antibody labeling kit to allow a fluorescent dye carrying an active ester to react with the free amino group of lysine on the antibody via an NHS ester reaction, followed by purification to obtain the desired fluorescently labeled antibody. For example, Ito et al. (DOI:10.1007 / s10120-013-0316-0) conducted a mouse fluorescence imaging study based on indocyanine green-labeled EGFR monoclonal antibodies. The research team used an indocyanine green antibody labeling kit (Dojindo, Kumamoto, Japan) to prepare indocyanine green-labeled cetuximab, allowing the indocyanine green carrying an active ester to react with the free amino group of lysine on the cetuximab via an NHS ester reaction. The research team also constructed a gastric cancer cell line stably expressing luciferase and established a mouse peritoneal metastatic tumor CDX model. They then performed three-dimensional (3D) dual bioluminescence and fluorescence imaging of peritoneal metastases by intravenously injecting indocyanine green-labeled cetuximab. While such traditional techniques can achieve fluorescence imaging of tumors, their limitations make them difficult to apply universally.

[0004] In real-time tumor surgery navigation technology based on indocyanine green fluorescence imaging, indocyanine green has a certain false positive rate due to its non-targeting nature, which may lead to excessive removal of normal tissue. After intravenous injection, indocyanine green immediately binds to plasma albumin and α- and β-lipoproteins, and is rapidly and efficiently taken up by hepatocytes via blood circulation. It is then secreted into bile in a free form by hepatocytes, without enterohepatic circulation. Therefore, indocyanine green is cleared by hepatocytes using first-order kinetics, resulting in a short residence time in the body (retention rate <10% after 15 minutes). Real-time tumor surgery navigation technology based on fluorescently labeled antibodies... The first-generation antibody labeling technology used in this study resulted in heterogeneous fluorescently labeled antibody products (inconsistent numbers of fluorescent groups in the antibody), inconsistent fluorescence signals, unclear clinical indications, and the fact that it has not yet been commercialized and is still in the clinical trial stage. The reported mouse fluorescence imaging based on indocyanine green-labeled cetuximab also used first-generation antibody labeling technology to prepare the fluorescent antibody. The synthesis principle involves a random NHS ester reaction between a fluorescent dye carrying an active ester and the free amino group of lysine on the antibody. The stability and biosafety of this method are unpredictable, and there is a lack of pharmacokinetic data of the labeled antibody in mice. Summary of the Invention

[0005] To address the problems of non-targeted uptake, high false positives, short half-life, and instability of fluorescent dyes in existing immunofluorescence-guided surgery techniques, this paper provides an EGFR fluorescent antibody, its preparation method, and its application.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0007] On one hand, the present invention provides an EGFR fluorescent antibody, wherein the EGFR antibody covalently binds a maleimide-modified fluorescent dye molecule having the linker.

[0008] Optionally, the fluorescent dye includes one or more of indocyanine green, anthocyanin, and rhodamine.

[0009] Optionally, the connector substructure is as follows:

[0010]

[0011] The value of a ranges from 1 to 5, and the value of b ranges from 0 to 5.

[0012] Optionally, the fluorescent dye is linked to the maleimide in the following structural formula:

[0013]

[0014] On the other hand, the present invention provides a method for preparing an EGFR fluorescent antibody, comprising the following steps:

[0015] Maleimide-modified fluorescent dyes were synthesized and prepared by linker connection between maleimide and fluorescent dye molecules;

[0016] After mixing and reacting EGFR antibody with a protein reducing agent, the reduced antibody is obtained.

[0017] The reduced antibody was mixed with a modified fluorescent dye and then purified to obtain the EGFR fluorescent antibody.

[0018] Optionally, the protein reducing agent includes one or more of tris(2-carboxyethyl)phosphine, DTT, and GSH.

[0019] Optionally, the concentration of the protein reducing agent is 5–10 mM.

[0020] Optionally, the purification process also includes high-speed centrifugation, with a centrifugation time of 3-5 minutes and a centrifugation speed of 15,000-20,000 g.

[0021] On the other hand, the EGFR fluorescent antibody provided by the present invention is used in tumor diagnostic imaging agents.

[0022] Optionally, the tumor diagnosis includes colon cancer surgery.

[0023] In this invention, a fluorescent dye and maleimide are linked by a linker to obtain a modified fluorescent dye. The modified fluorescent dye then binds to the reduced EGFR antibody. This invention differs from traditional fluorescent dye-labeled antibody technology and indocyanine green-labeled cetuximab technology. The EGFR antibody provided by this invention can directly target EGFR in tumor cells with a low false positive rate. Furthermore, the EGFR fluorescent antibody can be efficiently taken up by tumor tissues with high EGFR expression, can remain in tumor tissues for a long time, and has a long in vivo half-life. In addition, the preparation method of the EGFR fluorescent antibody provided in this invention has the advantages of high product stability and ease of production. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the EGFR fluorescent antibody synthesis process provided by the present invention;

[0025] Figure 2 These are the mouse fluorescence in vivo imaging images provided by this invention; (a) Serial fluorescence in vivo imaging of ICG in a mouse subcutaneous colon cancer tumor model; b) Serial fluorescence in vivo imaging of EGFR fluorescent antibody in a mouse subcutaneous colon cancer tumor model; c) Metabolic curve of EGFR fluorescent antibody in mice.

[0026] Figure 3 The images provided by this invention show the fluorescence intensity detection of EGFR fluorescent antibody versus ICG in a mouse subcutaneous colon cancer tumor model under fluorescence laparoscopy (a, ICG imaging in the control group of the mouse subcutaneous colon cancer tumor model; b, EGFR fluorescent antibody imaging in the experimental group of the mouse subcutaneous colon cancer tumor model).

[0027] Figure 4 This is a fluorescence intensity detection diagram of the EGFR fluorescent antibody under fluorescence laparoscopy in a mouse orthotopic colon tumor model provided by the present invention. Detailed Implementation

[0028] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0029] The present invention provides an EGFR fluorescent antibody, wherein the EGFR antibody covalently binds a maleimide-modified fluorescent dye molecule having the linker.

[0030] In the technical solution of the present invention, the fluorescent dye and the maleimide are connected by the linker to obtain the modified fluorescent dye. The modified fluorescent dye is then bound to the EGFR antibody label, which can directly target the EGFR of tumor cells with a low false positive rate. Moreover, the EGFR antibody can be efficiently taken up by tumor tissues with high EGFR expression, can remain in tumor tissues for a long time, and has a long half-life in vivo.

[0031] In some embodiments, the fluorescent dye includes one or more of indocyanine green, anthocyanin, and rhodamine.

[0032] It should be noted that indocyanine green, as a tricyanine dye, exhibits near-infrared absorption characteristics (peak absorption at approximately 800 nm) and a maximum emission wavelength of 807 nm, but shows almost no absorption in the visible light range. This explains its low autofluorescence, tissue absorption, and scattering at near-infrared wavelengths (700–900 nm). The maleimide active group can selectively attach indocyanine green dye to thiol groups (free thiol, R-SH) on neutral pH and various substrates (antibodies, peptides, proteins, oligonucleotides, small molecule drugs, etc.) without any activation. Because maleimide has very low reactivity with amines, alcohols, and phenols (including tyrosine and histidine), and does not react with histidine and methionine, it exhibits very high labeling selectivity. Furthermore, indocyanine green has good human safety and low toxicity, and has been a clinically used fluorescent contrast agent for many years.

[0033] Specifically, in a preferred embodiment of this application, the fluorescent dye includes indocyanine green and sulfonate-substituted indocyanine green.

[0034] The linker, also called a chemical linker, is generally a small molecular weight group or fragment. Its main function is to connect two chemical structures or molecules that are difficult to react with, so that chemical structures or intermolecular reactions can proceed smoothly.

[0035] The linker described in this application is used to connect the maleimide and the fluorescent dye; specifically, the linker structure in the embodiments of this application is as follows:

[0036]

[0037] In some embodiments, the fluorescent dye is linked to the maleimide in the following structural formula:

[0038]

[0039] In a preferred embodiment, the fluorescent dye and the maleimide are linked by the following structural formula:

[0040]

[0041] It should be noted that the maleimide is a structural element in the reaction of maleic anhydride with amine derivatives, and can easily form a stable target product through a nucleophilic Michael addition reaction;

[0042] The disulfonic acid-ICG-maleimide is a sulfonated derivative of ICG-maleimide. Sulfonation enhances its water solubility, enabling it to be stably dissolved in aqueous buffers, cell culture media, and other aqueous solutions. Furthermore, disulfonic acid-ICG-maleimide retains the fluorescent properties of the original fluorescent dye ICG, exhibiting fluorescence emission in the near-infrared wavelength range, allowing it to form a stable covalent bond during the labeling reaction and provide a durable fluorescent signal.

[0043] Specifically, the synthesis method refers to the synthesis method reported in existing literature (Targeting Fluorescence Imaging of RGD-Modified Indocyanine Green Micelles on Gastric Cancer. FrontBioeng Biotechnol.doi:10.3389 / fbioe.2020.575365).

[0044] An embodiment of the present invention provides a method for preparing an EGFR fluorescent antibody, comprising the following steps:

[0045] A fluorescent dye modified with maleimide was synthesized, and the fluorescent dye was linked to maleimide via a linker;

[0046] After mixing and reacting EGFR antibody with a protein reducing agent, the reduced antibody is obtained.

[0047] The reduced antibody was mixed with a modified fluorescent dye and then purified to obtain the EGFR fluorescent antibody.

[0048] In some embodiments, the protein reducing agent includes one or more of tris(2-carboxyethyl)phosphine, DTT, and GSH.

[0049] In some embodiments, the concentration of the protein reducing agent is 5–10 mM.

[0050] In a preferred embodiment, the concentration of the protein reducing agent is 10 mM.

[0051] In some embodiments, the amount of protein reducing agent added is 0.5 to 2 equivalents.

[0052] In some embodiments, the amount of EGFR antibody added is 0.5 to 2 equivalents.

[0053] In some embodiments, the purification further includes high-speed centrifugation, wherein the centrifugation time is 3 to 5 minutes and the centrifugation speed is 15,000 to 20,000 g.

[0054] Specifically, in the preparation method of EGFR fluorescent antibody, the EGFR antibody solution is mixed with the protein reducing agent and then centrifuged to remove residual protein reducing agent; specifically, the centrifugation time is 5 min and the centrifugation speed is 20000 g.

[0055] The EGFR fluorescent antibody provided in another embodiment of the present invention is used in tumor diagnostic imaging agents.

[0056] Specifically, the fluorescent material used in this technology targets the tumor cell target protein (EGFR), which has high targeting and low false positive rate. The fluorescent dye can be efficiently taken up by tumor tissues with high EGFR expression, has a long half-life in vivo, and can remain in tumor tissues for a long time, improving the resolution and imaging efficiency of lesions. It can accurately locate tumor tissues with high target protein expression in real time during surgery.

[0057] In some embodiments, the tumor diagnosis includes colon cancer surgery.

[0058] Specifically, the EGFR fluorescent antibody targets the target protein of tumor cells, binding to the target protein in vivo to enable fluorescence imaging of the corresponding lesions. EGFR is highly expressed in colorectal cancer; therefore, the EGFR fluorescent antibody described in this application is beneficial for the efficient and convenient tracing of colorectal cancer lesions during surgery. The following examples further illustrate the invention.

[0059] Example 1

[0060] This embodiment is used to illustrate the EGFR fluorescent antibody and its preparation method disclosed in this invention. Figure 1 The diagram illustrates the following steps:

[0061] Maleimide and fluorescent dye were linked separately using linkers to obtain modified fluorescent dyes;

[0062] EGFR antibody was mixed with 2.0 equivalents of tri-(2-carboxyethyl)phosphine reducing agent and reacted at 37°C for 2 hours to obtain the reduced antibody;

[0063] The reduced antibody was mixed with 4.0 equivalents of the modified fluorescent dye and reacted at 25°C for 4 hours. The mixture was then centrifuged at 20000g for 5 minutes. The supernatant was collected and desalted. PBS was added as an equilibration solution to adjust the pH to 7.4, thus obtaining the EGFR fluorescent antibody.

[0064] animal experiments

[0065] The detection of the pharmacokinetic curve of the EGFR fluorescent antibody prepared in Example 1 in mice includes the following steps:

[0066] Establishing a colon cancer cell line with stable luciferase expression:

[0067] Mouse strain: NCG; Tumor cell line: Human colon cancer cell line HT29 (luciferase stable cell line);

[0068] Fluorescent materials are administered via intravenous injection.

[0069] Amount of fluorescent material used:

[0070] TBSA = kW 2 / 3 / 10000

[0071] TBSA=total body surface area(m 3 )

[0072] W = weight(g)

[0073] K mouse =9.1

[0074] TBSA mouse =7.57E-3m 3

[0075] Initial dose: 250 mg / m³ 2

[0076] W mouse =24g

[0077] Standard treatment dose per mouse: 250 mg / m² 2 Based on previous literature reports, at 1 / 10 of the therapeutic dose (25 mg / m²) 2 As a diagnostic dosage;

[0078] Since the (drug-antibody ratio) DAR of the EGFR fluorescent antibody is 8, meaning that each EGFR antibody molecule covalently binds 8 indocyanine green molecules, the diagnostic dose of indocyanine green for each mouse was calculated to be 5.74 μg as a control group.

[0079] EGFR fluorescent antibody (25 mg / m² per mouse) was injected via the tail vein into mice. 2 Using IVIS Spectrum in vivo imaging, with excitation light at 780 nm and emission light at 830 nm, observation was conducted for 2 weeks (once daily in the first week and once every other day in the second week).

[0080] The fluorescence intensity of the EGFR fluorescent antibody prepared in Example 1 was detected in mice, including the following steps:

[0081] Establish mouse xenograft models of human colon cancer cell lines, including subcutaneous tumor models and orthotopic colon models;

[0082] The experimental group was injected with EGFR fluorescent antibody via the tail vein of mice, while the control group was injected with indocyanine green. Three days later, the fluorescence intensity of the experimental and control groups was observed using a fluorescence endoscopy imaging system (808nm laser).

[0083] Figure 2 Figure a shows that after intravenous administration of ICG, no specific fluorescence was observed in subcutaneous tumors in mice, while non-tumor sites (especially the liver) showed non-specific fluorescence (as shown in D0). The fluorescence signal disappeared after 24 hours (the circle represents the actual size of the tumor). Figure b shows that after intravenous administration of EGFR fluorescent antibody, specific fluorescence was observed in subcutaneous tumors 1–4 days later (the circle represents the actual size of the tumor). The fluorescence signal gradually decreased 5–7 days after administration. Figure c shows the metabolic curve of EGFR fluorescent antibody in mouse serum, with a half-life of approximately 4.5 days.

[0084] Figure 3 This is a mouse subcutaneous tumor model. Figure a shows that the fluorescence signal in the control group, which was injected with ICG intravenously, was diffusely distributed throughout the mouse body. Figure b shows that the fluorescence signal in the experimental group, which was injected with EGFR fluorescent antibody intravenously, was enriched in the subcutaneous tumor, indicating that the EGFR fluorescent antibody specifically targets the tumor tissue.

[0085] Figure 4 To establish a mouse colonic orthotopic tumor model, EGFR fluorescent antibody was administered intravenously 3 days prior, and imaging was performed using a fluorescence endoscopy system. Preoperatively (grossly), colonic tumors invisible under ordinary light sources were visualized under near-infrared (808nm laser) and fluorescence modes (arrows indicate locations). Intraoperatively (post-laparotomy), colonic tumors were visible under ordinary light sources, and specific fluorescence imaging of colonic tumors was visible under both near-infrared (808nm laser) and fluorescence modes (arrows indicate locations), while non-tumor tissues showed no fluorescence signal.

[0086] The EGFR fluorescent antibody provided in this application can be efficiently taken up by tumor tissues with high EGFR expression and can remain in the tumor tissue for a long time, prolonging the half-life of the EGFR fluorescent antibody in vivo, thereby providing a good navigation and tracking effect for immunofluorescence labeling surgery of related tumors and improving the accuracy of tumor resection.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An EGFR fluorescent antibody, characterized in that, It includes a fluorescent dye, a linker, an EGFR antibody, and maleimide, wherein the EGFR antibody is covalently bound to a maleimide-modified fluorescent dye molecule with the linker. The fluorescent dye and the maleimide are linked in the following structural formula: ； The method for preparing the EGFR fluorescent antibody includes the following steps: Maleimide-modified fluorescent dyes were synthesized and prepared by linker connection between maleimide and fluorescent dye molecules; After mixing and reacting EGFR antibody with a protein reducing agent, the reduced antibody is obtained; The reduced antibody was mixed with a modified fluorescent dye and then purified to obtain the EGFR fluorescent antibody.

2. The EGFR fluorescent antibody according to claim 1, characterized in that, The protein reducing agent includes one or more of tris(2-carboxyethyl)phosphine, DTT, and GSH.

3. An EGFR fluorescent antibody according to claim 1, characterized in that, The concentration of the protein reducing agent is 5-10 mM.

4. An EGFR fluorescent antibody according to claim 1, characterized in that, The purification process includes high-speed centrifugation, with a centrifugation time of 3-5 minutes and a centrifugation speed of 15,000-20,000 g.

5. The use of the EGFR fluorescent antibody as described in any one of claims 1 to 4 in the preparation of a colorectal cancer diagnostic imaging agent.

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