Bispecific antibody-photosensitizer conjugate targeting EGFR and MET as well as preparation method and application of bispecific antibody-photosensitizer conjugate

Abstract

The invention relates to the technical field of biological medicines, and particularly relates to a bispecific antibody-photosensitizer conjugate targeting EGFR and MET as well as a preparation method and application of the bispecific antibody-photosensitizer conjugate. The invention provides a bispecific antibody-photosensitizer conjugate targeting EGFR (Epidermal Growth Factor Receptor) and MET (Methyl Epidermal Growth Factor Receptor). The bispecific antibody-photosensitizer conjugate targeting the EGFR and the MET is formed by connecting a bispecific antibody, namely an Ertentoin monoclonal antibody and a photosensitizer through a covalent bond. According to the photosensitizer conjugate provided by the invention, the synergistic problem of double-target blocking, BBB penetration, long circulation and efficient photo-thermal / ROS output is solved at one time, so that glioma cell immunogenicity death is efficiently induced under 690-800nm near-infrared irradiation, and NIR-PIT of intracranial in-situ tumors is realized; and an accurate, minimally invasive and repeatable brand-new treatment strategy is provided for the recurrent / refractory EGFR / MET expression glioma.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to a bispecific antibody-photosensitizer conjugate targeting EGFR and MET, its preparation method and application. Background Technology

[0002] Gliomas are the most common primary malignant tumors of the central nervous system, and their treatment resistance and high recurrence rate are leading causes of death. According to the World Health Organization (WHO) classification of tumors of the central nervous system, gliomas are graded from 1 to 4; data indicate that nucleated astrocytomas are the most common type of glioma in children. Diffuse gliomas in children are classified into clinically indolent low-grade tumors and high-grade tumors with aggressive behavior. Diffuse midline gliomas with histone 3K27 alterations are a leading cause of glioma-related death in children.

[0003] The current standard of care for glioblastoma is surgical resection, radiotherapy, and chemotherapy combined with temozolomide (TMZ). TMZ is an alkylating agent that induces tumor cell death by alkylating DNA at multiple sites. However, TMZ often leads to resistance. Various proto-oncogenes in gliomas may be amplified and overexpressed, particularly epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor receptor (MET). However, clinical translation of single-target therapies against EGFR and MET has repeatedly failed. Small molecule inhibitors such as gefitinib and cabozantinib have failed to improve prognosis due to insufficient blood-brain barrier penetration or limited efficacy; monoclonal antibodies (such as depatuxizumab) and targeted vaccines (such as rindopepimut) have also not shown survival benefits in late-stage clinical trials.

[0004] Therefore, based on the pathological characteristics of gliomas driven by EGFR / MET, and the urgent clinical need to overcome the shortcomings of existing therapies such as poor penetration, single targeting, easy drug resistance, and high systemic toxicity, there is an urgent need to develop a molecule that can efficiently target and penetrate brain tissue, and has both dual pathway blocking function and high phototherapy activity. Summary of the Invention The purpose of this application is to provide a bispecific antibody-photosensitizer conjugate targeting EGFR and MET, its preparation method and application, aiming to solve the problems of poor penetration, single targeting, easy drug resistance and systemic toxicity in existing treatment regimens.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides a bispecific antibody-photosensitizer conjugate targeting EGFR and MET, wherein the bispecific antibody-photosensitizer conjugate targeting EGFR and MET is formed by covalently linking a bispecific antibody, ervastatin monoclonal antibody, and a photosensitizer.

[0006] In some embodiments, the photosensitizer is selected from ICG-COOH.

[0007] In some embodiments, the carboxyl group of ICG-COOH is activated by an activator to form an active ester intermediate; the active ester intermediate undergoes an amidation reaction with the amino group on the bispecific antibody ervantuo monoclonal antibody molecule to form a covalent amide bond.

[0008] Secondly, this application provides a method for preparing a bispecific antibody-photosensitizer conjugate targeting EGFR and MET as described above, comprising the following steps: The photosensitizer is dissolved in an organic solvent to obtain a photosensitizer solution; A solution containing a catalyst and a photosensitizer solution are mixed, and the pH is adjusted to 8-8.5; the reaction is carried out at room temperature in the dark for 15-30 minutes to obtain an activated photosensitizer. The photosensitizer and the bispecific antibody ervastatin monoclonal antibody solution were mixed, the pH was adjusted to 8-8.5, and the reaction was carried out at room temperature in the dark for 2-2.5 hours. The reaction product was then purified to obtain the photoimmunoconjugate.

[0009] In some embodiments, the catalyst-containing solution comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of (1-1.2):(1-1.2).

[0010] In some embodiments, the molar ratio of photosensitizer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 1:(10-11):(10-11).

[0011] In some embodiments, the photosensitizer in the photosensitizer solution is selected from ICG-COOH, and the concentration of the photosensitizer is 10-100 nM.

[0012] In some embodiments, purification is performed using a BeyoDesalt™ G-25 Mini desalting column.

[0013] Thirdly, this application provides the use of the above-mentioned bispecific antibody-photosensitizer conjugate targeting EGFR and MET in the preparation of a drug for near-infrared photoimmunotherapy of glioma.

[0014] In some embodiments, the glioma is an EGFR and / or c-MET-expressing glioma, including glioblastoma, diffuse midline glioma, or recurrent glioma.

[0015] The first aspect of this application provides a bispecific antibody-photosensitizer conjugate targeting EGFR and MET. By using the bispecific antibody Amivantamab as the target head, the conjugate can simultaneously bind with high affinity to the overexpressed EGFR and MET on tumor cells, achieving synergistic blocking of both pathways. This significantly inhibits tumor migration, invasion, and drug resistance, overcoming the limitations of single-target therapy. Secondly, the covalently conjugated photosensitizer, under near-infrared light excitation, simultaneously generates efficient photothermal and photodynamic effects. This photothermal / photodynamic synergistic effect ensures powerful killing of glioma cells in deep tissues. The conjugate exhibits long-circulating characteristics; its induced immunogenic death combines local phototherapy with a systemic anti-tumor immune response, precisely eliminating the tumor in situ while stimulating a durable immune memory effect, providing a possibility for controlling tumor recurrence and metastasis. It has high safety and few toxic side effects, providing a precise, minimally invasive, and repeatable new treatment strategy for relapsed / refractory EGFR / MET-expressing gliomas.

[0016] The second aspect of this application provides a method for preparing a bispecific antibody-photosensitizer conjugate targeting EGFR and MET. This method is simple, easy to operate, has mild reaction conditions, and is easy to apply.

[0017] The application of the aforementioned bispecific antibody-photosensitizer conjugate targeting EGFR and MET, provided in the third aspect of this application, in the preparation of a drug for near-infrared photoimmunotherapy of gliomas, enables the bispecific antibody-photosensitizer conjugate targeting EGFR and MET to achieve precise enrichment and efficient penetration of intracranial tumors with the help of dual-targeting antibodies, overcoming the blood-brain barrier limitation; by activating the photosensitizer through near-infrared light irradiation, a synergistic photothermal / photodynamic effect is generated, which induces immunogenic death of tumor cells while stimulating systemic anti-tumor immunity; this treatment strategy has the comprehensive advantages of precise targeting, minimally invasive and controllable, and repeatable implementation, effectively overcoming the drug resistance and systemic toxicity problems of traditional therapies, and providing a new treatment paradigm for relapsed / refractory EGFR / MET-positive gliomas. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This describes the proliferation status of murine glioma cells after NIR-PIT, a bispecific antibody-photosensitizer conjugate targeting EGFR and MET provided in this application embodiment. Figure 2 This application provides examples of the inhibitory effects of different concentrations of ICG solutions on the proliferation of human GL261 glioma cells. Figure 3 This application demonstrates the inhibitory effect of the Amivantamab monoclonal antibody on the proliferation of human GL261 glioma cells. Figure 4 The results are the tumor volume and tumor inhibition rate of the combined application of immune checkpoint blockade and photodynamic therapy (PDT) provided in the embodiments of this application. Detailed Implementation

[0020] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0021] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0022] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0023] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0024] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0025] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0026] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0027] The first aspect of this application provides a bispecific antibody-photosensitizer conjugate targeting EGFR and MET, wherein the bispecific antibody-photosensitizer conjugate targeting EGFR and MET is formed by covalently linking a bispecific antibody, ervastatin monoclonal antibody, and a photosensitizer.

[0028] The first aspect of this application provides a bispecific antibody-photosensitizer conjugate targeting EGFR and MET. By using the bispecific antibody Amivantamab as the target head, the conjugate can simultaneously bind with high affinity to the overexpressed EGFR and MET on tumor cells, achieving synergistic blocking of both pathways. This significantly inhibits tumor migration, invasion, and drug resistance, overcoming the limitations of single-target therapy. Secondly, the covalently conjugated photosensitizer, under near-infrared light excitation, simultaneously generates efficient photothermal and photodynamic effects. This photothermal / photodynamic synergistic effect ensures powerful killing of glioma cells in deep tissues. The conjugate exhibits long-circulating characteristics; its induced immunogenic death combines local phototherapy with a systemic anti-tumor immune response, precisely eliminating the tumor in situ while stimulating a durable immune memory effect, providing a possibility for controlling tumor recurrence and metastasis. It has high safety and few toxic side effects, providing a precise, minimally invasive, and repeatable new treatment strategy for relapsed / refractory EGFR / MET-expressing gliomas.

[0029] In some embodiments, the bispecific antibody Evantu monoclonal antibody (JNJ-61186372) can specifically target the extracellular domains of EGFR and MET; CAS number 2171511-58-1; molecular weight 145.88kD.

[0030] In some embodiments, the photosensitizer is selected from ICG-COOH. ICG-COOH has the CAS number 181934-09-8 and the molecular formula C2. 45 H 50 N₂O₅S, molecular weight 730.95; λ ex It is 785nm, λ em The wavelength is 810 nm; the molar extinction coefficient is 2.3 × 10⁻⁶. 5 M -1 cm -1 .

[0031] ICG-COOH is achieved by attaching an alkyl chain (such as -(CH2) to the polycyclic skeleton of ICG. n The carboxyl group is introduced by introducing the -COOH group; the carboxyl group is a very versatile active functional group in chemistry, which can readily undergo an efficient amidation reaction with the primary amino group on the antibody molecule in the presence of a catalyst to form a stable, irreversible amide bond.

[0032] Using ICG-COOH as a photosensitizer, ICG-COOH still exhibits strong absorption in the near-infrared region and can generate photothermal effects and reactive oxygen species. Through the reaction of carboxyl and amino groups, ICG can be linked to antibodies in a specific stoichiometry and with a defined orientation. This ensures that the covalent bond does not prematurely dissociate during in vivo circulation, and also avoids the coupling process occurring in the antigen-binding region of the antibody, thereby maximizing its ability to target EGFR / MET.

[0033] In some embodiments, the carboxyl group of ICG-COOH is activated by an activator to form an active ester intermediate; the active ester intermediate undergoes an amidation reaction with the amino group on the bispecific antibody ervantuo monoclonal antibody molecule to form a covalent amide bond.

[0034] The second aspect of this application provides a method for preparing a bispecific antibody-photosensitizer conjugate targeting EGFR and MET as described above, comprising the following steps: S01. Dissolve the photosensitizer in an organic solvent to obtain a photosensitizer solution; S02. Mix the catalyst-containing solution and the photosensitizer solution, adjust the pH to 8-8.5; react at room temperature in the dark for 15-30 minutes to obtain the activated photosensitizer; S03. Mix the photosensitizer and the bispecific antibody ervastatin monoclonal antibody solution, adjust the pH to 8-8.5, and react at room temperature in the dark for 2-2.5 hours; purify the reaction product to obtain the photoimmunoconjugate.

[0035] The second aspect of this application provides a method for preparing a bispecific antibody-photosensitizer conjugate targeting EGFR and MET. This method is simple, easy to operate, has mild reaction conditions, and is easy to apply.

[0036] In step S01, the photosensitizer is dissolved in an organic solvent to obtain a photosensitizer solution.

[0037] In some embodiments, the photosensitizer in the photosensitizer solution is selected from ICG-COOH, and the concentration of the photosensitizer is 10-100 nM.

[0038] In some embodiments, the organic solvent is selected from DMSO solvent.

[0039] In step S02, a solution containing a catalyst and a photosensitizer solution are mixed and the pH is adjusted to 8-8.5; the mixture is reacted at room temperature in the dark for 15-30 minutes to obtain an activated photosensitizer.

[0040] In some embodiments, the catalyst-containing solution comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of (1-1.2):(1-1.2).

[0041] In some specific embodiments, the catalyst-containing solution includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of 1:1.

[0042] In some embodiments, the molar ratio of photosensitizer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 1:(10-11):(10-11).

[0043] In some embodiments, the molar ratio of photosensitizer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 1:10:10.

[0044] Among these factors, the pH must be strictly controlled between 8 and 8.5. If the pH is too high, antibody aggregation is likely to occur, and if the pH is too low, DAR will decrease.

[0045] In step S03, the photosensitizer and the bispecific antibody ervastatin monoclonal antibody solution are mixed, the pH is adjusted to 8-8.5, and the reaction is carried out at room temperature in the dark for 2-2.5 hours; the reaction product is purified to obtain the photoimmunoconjugate.

[0046] In some embodiments, purification is performed using a BeyoDesalt™ G-25 Mini desalting column.

[0047] In some embodiments, the quality of the prepared bispecific antibody-photosensitizer conjugate targeting EGFR and MET meets at least one of the following criteria: (1) The antibody-photosensitizer coupling ratio determined by ultraviolet absorption spectroscopy is calculated with the molar extinction coefficient of ICG at 780 nm being 2.0 × 10⁻⁶. 5 L·mol -1 ·cm -1 The molar extinction coefficient of the antibody at 280 nm is 1.4 × 10⁻⁶. 5 L·mol -1 ·cm -1 ; (2) The residual amount of free ICG is not higher than 1.0%; (3) The content of polymer aggregates in the solution is not higher than 5.0%; (4) The endotoxin content should be less than 0.1 EU / mg; (5) Sterility testing shall comply with the relevant provisions of the Chinese Pharmacopoeia; (6) The photothermal conversion efficiency should meet the following conditions: after irradiation with 808 nm laser (1 W / cm²) for 5 minutes, the temperature rise of 1 μM coupling aqueous solution should not be less than 25 degrees Celsius; (7) The plasma stability should meet the following requirements: after incubation in plasma at 37°C for 24 hours, the SEC-MALS recovery rate should not be less than 90%, and the amount of free ICG generated should not exceed 0.5%.

[0048] The third aspect of this application provides the use of the above-mentioned bispecific antibody-photosensitizer conjugate targeting EGFR and MET in the preparation of a medicament for near-infrared photoimmunotherapy of glioma.

[0049] The application of the above-mentioned EGFR and MET bispecific antibody-photosensitizer conjugate provided in the third aspect of this application in the preparation of a drug for near-infrared photoimmunotherapy of gliomas, allows the EGFR and MET bispecific antibody-photosensitizer conjugate to achieve precise enrichment and efficient penetration of intracranial tumors with the help of dual-targeting antibodies, overcoming the blood-brain barrier limitation; by activating the photosensitizer through near-infrared light irradiation, a synergistic photothermal / photodynamic effect is generated, which induces immunogenic death of tumor cells while stimulating systemic anti-tumor immunity; this treatment strategy has the comprehensive advantages of precise targeting, minimally invasive controllability and repeatability, effectively overcoming the drug resistance and systemic toxicity problems of traditional therapies, and providing a new treatment paradigm for relapsed / refractory EGFR / MET positive gliomas.

[0050] In some embodiments, the glioma is an EGFR and / or c-MET-expressing glioma, including glioblastoma, diffuse midline glioma, or recurrent glioma.

[0051] In some embodiments, bispecific antibody-photosensitizer conjugates targeting EGFR and MET can be used for near-infrared photoimmunotherapy (NIR-PIT) of intracranial malignant gliomas. Administration: Intravenous infusion of 3 mg / kg → 24 h later, irradiation with an 808 nm laser (100–300 mW / cm², 30–100 J / cm²) through a cranial optical window or via a naso-brain fiber optic transillumination; once every 4 weeks, up to a maximum of 6 cycles.

[0052] In some embodiments, a bispecific antibody-photosensitizer conjugate targeting EGFR and MET can be used for intraoperative real-time fluorescence-guided resection. Application: During surgery, a handheld 780 nm excitation light source is used, and ICG fluorescence (820 nm bandpass filtered) is used to delineate the tumor margins; the light dose is below the treatment threshold to avoid premature PIT triggering.

[0053] In some embodiments, bispecific antibody-photosensitizer conjugates targeting EGFR and MET can be used for photothermal-immunotherapy synergistic neoadjuvant therapy. Administration: 7 days preoperatively, a low dose of Ami-ICG (1 mg / kg) + a subablation dose of light (30 J / cm²) is administered to induce immunogenic cell death, followed by surgical resection and standard TMZ; 14 days postoperatively, a second PIT treatment is performed to clear micrometastases.

[0054] In some embodiments, bispecific antibody-photosensitizer conjugates targeting EGFR and MET can be applied to head and neck squamous cell carcinoma (HNSCC): external fiber optic irradiation is used in the maxillofacial region to avoid radiation necrosis of the jawbone.

[0055] In some embodiments, a bispecific antibody-photosensitizer conjugate targeting EGFR and MET can be applied to brain metastases of non-small cell lung cancer (NSCLC): Ami-ICG is administered before whole-brain irradiation, and local photoselective enhancement of the dose to metastatic lesions is added.

[0056] In some embodiments, bispecific antibody-photosensitizer conjugates targeting EGFR and MET can be applied to peritoneal metastases of gastric / esophageal adenocarcinoma: laparoscopic 808 nm fiber optic irradiation to achieve photoimmunoassay clearance of the peritoneal cavity after CRS.

[0057] In some embodiments, a bispecific antibody-photosensitizer conjugate targeting EGFR and MET can be applied to pediatric diffuse midline glioma (DMG, H3K27M). The application method is jugular vein-basilar artery perfusion + transnasal fiber optic irradiation; taking advantage of the thin skull and low light penetration loss (<15%) in children.

[0058] In some embodiments, bispecific antibody-photosensitizer conjugates targeting EGFR and MET can be applied to recurrent / dural metastases of spinal gliomas. Application involves inserting a cylindrical optical fiber through the interlaminar foramen of the frontal vertebra for 360° circumferential irradiation; it can also be co-doped with ICG-microspheres in vertebroplasty bone cement to achieve secondary photothermal effects.

[0059] In some embodiments, a bispecific antibody-photosensitizer conjugate targeting EGFR and MET can be combined with CAR-T cells to achieve "optical ignition". The specific application method is as follows: first, EGFR / MET-CAR-T is infused → 24 h later, a subtherapeutic dose of ervastatin monoclonal antibody-ICG is injected → local light irradiation to raise the temperature to 42 degrees Celsius to promote CAR-T migration across the vascular endothelium (mediated by heat shock protein 90).

[0060] The following description is based on specific embodiments.

[0061] Example 1 Bispecific antibody-photosensitizer conjugate targeting EGFR and MET and its preparation method I. Antibody and ICG Sources 1. Evantu monoclonal antibody (JNJ-61186372): can be purchased through TargetMol Chemicals Inc.

[0062] 2. ICG-COOH: Commercially available active pharmaceutical ingredient (Ruixi Biotechnology); or it can be prepared by one-step hydrolysis of ICG-sulfo-OSu according to the method in J Med Chem 2017, 60: 5420-5431, with HPLC purity ≥98% and carboxyl content ≥95%.

[0063] II. Coupling Methods Linkage is achieved through a non-specific acylation reaction between a highly active ester and lysine residues in the antibody; Step 1, Solution preparation: Dissolve ICG-COOH in DMSO at a concentration of 10-100 mM to prepare an EDC and NHS solution with a concentration 10 times that of the ICG solution (solvent is PBS). Step 2, Activation of carboxyl groups: Mix ICG, EDC and NHS solutions thoroughly to form a mixture with a final concentration ratio of 1:10:10. It can be further diluted to the target concentration with PBS. Adjust the pH of the mixture to 8-8.5 with NaOH solution. Shake gently at room temperature in the dark for 15-30 minutes to activate the carboxyl groups in ICG-COOH into active esters. Step 3, antibody ligation: Mix the activated ICG-COOH solution with the Evanto monoclonal antibody solution to obtain mixture 2. Adjust the pH of mixture 2 to 8-8.5 with NaOH solution. React at room temperature in the dark with gentle shaking for 2 hours.

[0064] Step 4, Purification: After the reaction is complete, use a desalting column (Beyotime, BeyoDesalt™ G-25 Mini desalting column, P2615) to purify according to the instructions.

[0065] III. Animal Models and Experimental Procedures 1. Model Establishment Cells: GL261-hEGFRvIII / hMET (C57BL / 6 background, transduced with lentivirus, flow cytometry confirmed double positivity rate >90%).

[0066] Stereoscopic localization: 5 × 10⁻⁶ dextrose was injected into the right caudate nucleus of the mouse (0.5 mm anterior to the anterior fontanelle, 2.0 mm to the right, and 3.0 mm deep). 5 Cells / 3 µL. Tumor volume was verified by MRI (T2WI) on day 7 post-inoculation, with tumors measuring 30–50 mm³ before enrollment.

[0067] 2. Grouping and drug administration a (n=15); a. Control (PBS): Equal volume of PBS was injected, without light exposure; b. Free ICG, 0.3 mg / kg, 808 nm, 200 mW / cm², 100 J / cm²; c. Evantu monoclonal antibody, 3 mg / kg, in the absence of light; d. Evantu monoclonal antibody-ICG, 3 mg / kg, in the absence of light; e. NIR-PIT (single dose), 3 mg / kg, 808 nm, 200 mW / cm², 100 J / cm²; f. NIR-PIT (repeated, q7d×3), 3 mg / kg, 808 nm, 200 mW / cm², 100 J / cm².

[0068] 3. Lighting method Transcranial optical window: After hair removal, the dental drill is thinned to approximately 100 µm, preserving the dura mater; an optical fiber (Ø 600 µm, NA 0.22) is placed perpendicularly to the skull at a distance of 2 mm, with the irradiation field covering the tumor area.

[0069] Temperature monitoring: MR-compatible infrared thermal imager, peak temperature controlled at ≤46℃; avoid thermal damage to normal tissues.

[0070] 4. Evaluation Indicators Fluorescence / photoacoustic imaging: IVIS Spectrum, 780 nm excitation, 820–840 nm emission, 24 h TBR (tumor / contralateral brain) ≥6. MRI: 9.4 T Bruker, volume change measured, inhibition rate calculated = [1-(V t / V0)]×100%. Survival period: Kaplan–Meier, Log-rank test, P<0.05 was considered significant. Immunology: Flow cytometry on day +7 (tumor-infiltrating CD8) + CD4 + Treg), ELISPOT (IFN-γ spot), and a second challenge experiment (mice surviving >60 days were re-inoculated with 2×10⁻⁶ cells in the contralateral brain). 5 (Cells, observe the tumor-free rate).

[0071] Safety: Record weight and behavioral data (Rotarod, Morris water maze); collect major organs for HE staining 24 hours after the last light exposure; blood biochemistry (ALT, CREA, CK-MB); skin phototoxicity test (after hair removal on the back, use a sun simulator at 200 J / cm² to observe the incidence of erythema / edema ≥ Grade 2).

[0072] IV. Key Points of Repeatability 1. Strictly control the pH to 8.2±0.05. Too high a pH can easily lead to antibody aggregation, while too low a pH will cause a decrease in DAR.

[0073] 2. Before irradiation, it must be confirmed that the thickness of the skull window is ≤100 µm. Otherwise, the irradiation time should be extended or the power increased to ensure that the tumor area reaches above 42 degrees Celsius.

[0074] 3. Model cells should be at passage number ≤ P6. Double positivity rate should be validated by flow cytometry for each batch. If the rate is below 85%, re-transfection or sorting is required.

[0075] Comparative Example 1 ICG, a photosensitizing agent.

[0076] Comparative Example 2 Antibody: Evantu monoclonal antibody.

[0077] Comparative Example 3 The provided mixture is a simple mixture of free ICG and Evantu monoclonal antibody.

[0078] Comparative Example 4 Cetuximab-IR700.

[0079] Property Testing and Result Analysis First, the bispecific antibody-photosensitizer conjugate targeting EGFR and MET prepared in Example 1 is provided for drug treatment of murine glioma cells CT-2A. After adding the drug for 2H, the cells are subjected to light treatment. Figure 1 Irradiation conditions: 808 nm, 1 w, 5 min; 24 h after illumination, cell proliferation status was detected using the CCK8 kit.

[0080] Meanwhile, the effective concentrations of ICG (Comparative Example 1) and ervastatin monoclonal antibody (Comparative Example 2) against human glioma cells GL261 were determined under the same light conditions, and the results are as follows. Figure 2 and Figure 3 As shown, preliminary in vitro results of glioma cells indicate that a single NIR-PIT treatment of the ervantuo monoclonal antibody-ICG formulation significantly inhibited the proliferation of murine glioma cells, and its effect was the additive effect of ICG and ervantuo monoclonal antibody used alone. Simultaneously, both ervantuo monoclonal antibody and ICG significantly inhibited the proliferation of human GL261 glioma cells, suggesting that this photoimmunotherapy agent can significantly suppress tumor growth in a mouse GL261 orthotopic tumor model via photoimmunotherapy, and also holds promise for clinical glioma patients expressing human EGFR / c-MET.

[0081] Second, the following experiments were conducted with either the mixture of free ICG and Evantu monoclonal antibody (Comparative Example 3) or Cetuximab-IR700 (Comparative Example 4): (I) Simultaneous Blocking Experiment at Two Targets Experimental steps: 1. Cell pretreatment EGFRvIII-GL261 (5×10 5 Cells / wells (6-well plate) were starved with serum-free DMEM for 4 h.

[0082] Add Amivantamab-ICG, free ICG, a simple mixture of ICG and amivantamab, and Cetuximab-IR700, and incubate at 37 °C for 30 min.

[0083] Adding EGF (50 ng / mL) + HGF (40 ng / mL) for 10 min induces dual receptor phosphorylation. 2. Pyrolysis and Quantification Wash twice with ice-cold PBS, lyse with RIPA and phosphatase inhibitor, and quantify with BCA.

[0084] Detection was performed using a Meso Scale Discovery (MSD) phosphorylated-EGFR (Y1068) / MET (Y1234 / 1235) dual-channel electrochemiluminescence plate, following the kit instructions.

[0085] 3. Data Analysis Inhibition rate (%) = (1 – sample p-receptor signal / stimulation control signal) × 100.

[0086] IC50 was calculated using GraphPad Prism 10 nonlinear regression (log[inhibitor] vs. response).

[0087] Ultimately, the experiment demonstrated that the ervatuo monoclonal antibody-ICG inhibited EGFR and MET phosphorylation in EGFRvIII-GL261 cells by ≥90%, which was significantly higher than that of the single-target antibody group (<50%). (II) Ultra-long circulation and brain enrichment Experimental steps: 1. Establishment of an intracranial in situ model Mice were anesthetized (ketamine / xylazine 100 / 10 mg / kg ip), and stereotactically injected with GL261-luc (5×10) 5 (cells / 5 μL, 2 mm to the right of the anterior fontanelle, 3 mm depth).

[0088] On day 7, bioluminescence assay was performed to verify the tumor burden (≥1×10⁻⁶). 6 photons / s).

[0089] 2. Drug administration and sampling A single intravenous injection of Amivantamab-ICG 3 mg / kg via tail vein.

[0090] Blood collection time points: 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h (orbital venous plexus, heparin lithium tube).

[0091] Brain tissue: euthanized at 24 h and 72 h, perfused with physiological saline, the tumor hemisphere was harvested, weighed, and ground with liquid nitrogen.

[0092] 3. Quantitative PCR using ICG fluorescence Plasma: 100 μL + 900 μL methanol to precipitate proteins, centrifuge and collect the supernatant, measure fluorescence at Ex / Em 780 / 820 nm. Brain homogenate: Add 1 mL of 0.5% Triton X-100 / PBS, sonicate to disrupt, and perform fluorescence assay as above.

[0093] Concentration was converted using the ICG standard curve (matrix-matched).

[0094] 4. PK Calculation Plasma data were analyzed using a Phoenix WinNonlin 8.3 non-compartmental model to calculate t1 / 2β; brain AUC from 0 to 24 h was analyzed using a trapezoidal method.

[0095] Brain / plasma ratio = Brain concentration / Concentration of plasma at the same time.

[0096] Ultimately, the experimental results showed that in the mouse intracranial orthotopic model, the concentration of ervastatin monoclonal antibody-ICG in brain tissue reached 6.8±0.7 μM after 24 h, which was 18 times that of free ICG, and the plasma half-life t1 / 2β=14.6 h.

[0097] (III) High photothermal conversion efficiency Experimental steps: 1. Sample Preparation Prepare 1 μM Amivantamab-ICG with PBS and take 1 mL into a quartz cuvette.

[0098] Control: ① Pure water ② Free ICG.

[0099] 2. Laser irradiation An 808 nm continuous wave laser with a power density of 1 W / cm² was used for irradiation for 5 minutes, and a thermal imager (FLIR A700) recorded the temperature every second.

[0100] Ambient temperature 25 °C, humidity 50% RH, protected from light.

[0101] 3. Calculate the photothermal conversion efficiency η According to the Roper method: η = (hSΔTmax – Qdis) / I(1 – 10^-A808), where hS is fitted by the cooling curve, Qdis is the solvent blank heat dissipation, I is the laser power, and A808 is the absorbance at 808 nm.

[0102] Judgment: η≥40% is acceptable; record 1 μM temperature rise ΔT≥28 °C.

[0103] Ultimately, the experimental results proved that when DAR=4, the photothermal conversion efficiency η=46%, and the 1 μM solution heated 28 degrees Celsius in 5 min under 808 nm (1 W / cm²) irradiation, meeting the ≥40% index.

[0104] (iv) Complete ablation with a single irradiation Experimental steps: 1. Model Establishment Same as method (ii).

[0105] 2. Drug administration and irradiation On day 7, Amivantamab-ICG 3 mg / kg was injected via the tail vein. 24 hours later, the head was shaved to expose the skull.

[0106] 808 nm laser (1 W / cm², 10 min, 8 mm diameter, covering the tumor area), thermal imager real-time monitoring temperature ≤46 °C.

[0107] 3. Evaluation of therapeutic effect Bioluminescence: Imaging on days 0, 1, 3, 7, 14, and 21, with ROI quantification.

[0108] Complete disappearance is defined as: bioluminescent signal ≤ background + 3σ.

[0109] Survival observation: Time of death was recorded, and median survival was analyzed using Kaplan-Meier analysis and Log-rank test.

[0110] Ultimately, the experimental results showed that in GL261-hEGFR / hMET double-positive mice, the tumor complete disappearance rate was 70% 21 days after 1×PIT, and the median survival was >90 days, compared to <30 days in the control group.

[0111] (v) Immunological memory effect: Re-vaccination Experimental steps: 1. Initial treatment Complete 1×PIT using the same method as in (iv), and collect mice that have completely regressed 21 days later (n=10).

[0112] 2. Re-vaccination GL261-hEGFR / hMET (1×10) was injected again into the contralateral hemisphere. 5 (cells / 2 μL), with untreated naive mice as a control.

[0113] 3. Monitoring Weekly bioluminescence imaging for 6 months; palpation and weight recording.

[0114] 4. ELISPOT Three animals were sacrificed on day 30, and their spleens were harvested to prepare a single-cell suspension (5 × 10⁻⁶ cells / mL). 5 (cells / pores).

[0115] Pre-coated with Mouse IFN-γ ELISPOT Plate (BD), with hEGFR added. 805-893 or hMET 995-1003 Peptide pool (10 μg / mL), incubated for 20 h.

[0116] The spots were counted using an AID ELISPOT Reader, and the result was expressed as "number of spots / 10". 6 "Spleen cells" is the term used to describe this cell type.

[0117] Ultimately, the experimental results showed that after re-inoculation with tumor cells, 100% of mice in the Evantu monoclonal antibody-ICG PIT group had tumor-free survival for ≥6 months, and ELISPOT showed a 4.5-fold increase in IFN-γ+ CD8+ T cells. (vi) Safety: 3 mg / kg × 3 times, no toxicity ≥ Grade 2. Experimental steps: 1. Animals and Grouping Healthy C57BL / 6J mice (tumor-free), half male and half female, were divided into: Solvent comparison Amivantamab-ICG 3 mg / kg × 3 (days 0, 7, and 14) 24 h after each administration, 808 nm laser treatment was performed (1 W / cm², 10 min, 2 cm of the shaved area on the back).

[0118] 2. Clinical observation Record weight, hair, and skin erythema / eschar daily (according to CTCAE v5.0 classification).

[0119] Neurological assessment: rotarod, gait, Morris water maze (day 28).

[0120] 3. Pathology and Blood On day 29, the animals were sacrificed, and their brains, skin, livers, and kidneys were harvested. The organs were fixed with 4% PFA, embedded in paraffin, and subjected to immunohistochemistry for H&E, GFAP, and Iba1.

[0121] Serum biochemistry (ALT, AST, BUN, Cr), complete blood count.

[0122] 4. Judgment Criteria No skin or central nervous system toxicity ≥ Grade 2; no neuronal degeneration or thermal damage vacuolation in brain tissue; biochemical indicators <20% different from control.

[0123] Ultimately, the experimental results proved that repeated toxicity tests (3 mg / kg × 3 times, 28 days) showed no phototoxic side effects in the central nervous system or skin at ≥ Grade 2, and pathological examination showed no thermal damage to normal brain tissue.

[0124] Fourth, to evaluate the potential of combining immune checkpoint blockade with photodynamic therapy (PDT), a murine orthotopic tumor model was constructed: tumor-bearing mice were randomly assigned to four intervention groups: (i) monospecific antibody (mAb) group, (ii) bispecific antibody (bsAb) group, (iii) mAb combined with photosensitizer-mediated PDT (mAb + PS + light) group, and (iv) bsAb combined with photosensitizer-mediated PDT (bsAb + PS + light) group. Treatment began on day 0, and tumor volume was dynamically monitored daily using digital calipers. Endpoint analysis showed a clear gradient of antitumor activity.

[0125] like Figure 4 As shown, the tumor growth inhibition rate (TGI) of mAb monotherapy was less than 2%, and the final tumor burden was not significantly different from that of the control group (P>0.99). After upgrading to bsAb, the TGI significantly increased to 25%, and the median tumor volume decreased to about 650 mm³, suggesting that simultaneous blocking of two immune checkpoints can produce measurable but still unsatisfactory anti-tumor immunity.

[0126] The combination of PDT significantly amplified the therapeutic index. In the mAb + PS + phototherapy group, tumor growth was suppressed to approximately 530 mm³, with a TGI of 36%. More remarkably, the bsAb + PS + phototherapy group achieved a TGI of approximately 50%, with a tumor volume of only approximately 450 mm³. No significant toxicity was observed in any group (weight loss <5%, normal serum biochemistry), confirming the safety and feasibility of the combined regimen.

[0127] In summary, photochemoimmunotherapy based on bispecific antibodies demonstrated superior tumor control compared to monotherapy or adjuvant PDT regimens based on monoclonal antibodies. This incremental benefit aligns with a synergistic mechanism: PDT-induced immunogenic cell death increases neoantigen release, thereby amplifying the efficacy of dual immune checkpoint blockade. This proof-of-concept study provides a solid basis for the clinical translation of bsAb-PDT combination therapy for solid tumors resistant to traditional immuno-oncology drugs.

[0128] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A bispecific antibody-photosensitizer conjugate targeting EGFR and MET, characterized in that, The bispecific antibody-photosensitizer conjugate targeting EGFR and MET is composed of a bispecific antibody, Evanto monoclonal antibody, and a photosensitizer linked by a covalent bond.

2. The bispecific antibody-photosensitizer conjugate targeting EGFR and MET according to claim 1, characterized in that, The photosensitizer is selected from ICG-COOH.

3. The bispecific antibody-photosensitizer conjugate targeting EGFR and MET according to claim 2, characterized in that, The carboxyl group of the ICG-COOH is activated by an activator to form an active ester intermediate; the active ester intermediate undergoes an amidation reaction with the amino group on the bispecific antibody ervantuo monoclonal antibody molecule to form a covalent amide bond.

4. A method for preparing a bispecific antibody-photosensitizer conjugate targeting EGFR and MET as described in any one of claims 1-3, characterized in that, Includes the following steps: The photosensitizer is dissolved in an organic solvent to obtain a photosensitizer solution; A solution containing a catalyst and the photosensitizer solution are mixed, and the pH is adjusted to 8-8.5; the reaction is carried out at room temperature in the dark for 15-30 minutes to obtain an activated photosensitizer. Mix the photosensitizer and the bispecific antibody ervastatin monoclonal antibody solution, adjust the pH to 8-8.5, and react at room temperature in the dark for 2-2.5 hours; The reaction product was purified to obtain the bispecific antibody-photosensitizer conjugate targeting EGFR and MET.

5. The method for preparing the bispecific antibody-photosensitizer conjugate targeting EGFR and MET according to claim 4, characterized in that, The catalyst-containing solution comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of (1-1.2):(1-1.2).

6. The method for preparing the bispecific antibody-photosensitizer conjugate targeting EGFR and MET according to claim 5, characterized in that, The molar ratio of the photosensitizer, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and the N-hydroxysuccinimide is 1:(10-11):(10-11).

7. The method for preparing the bispecific antibody-photosensitizer conjugate targeting EGFR and MET according to claim 4, characterized in that, The photosensitizer in the photosensitizer solution is selected from ICG-COOH, and the concentration of the photosensitizer is 10-100 nM.

8. The method for preparing the bispecific antibody-photosensitizer conjugate targeting EGFR and MET according to claim 4, characterized in that, The purification process was performed using a BeyoDesalt™ G-25 Mini desalting column.

9. The use of a bispecific antibody-photosensitizer conjugate targeting EGFR and MET as described in any one of claims 1-3 in the preparation of a medicament for near-infrared photoimmunotherapy of glioma.

10. The application according to claim 9, characterized in that, The glioma is an EGFR and / or c-MET expressing glioma, including glioblastoma, diffuse midline glioma, or recurrent glioma.

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