A diagnosis and treatment integrated probe targeting GD2 of neuroblastoma

Abstract

This application relates to the field of biomedical technology, specifically disclosing a therapeutic probe targeting neuroblastoma GD2, adapted to the clinical needs of relapsed, refractory, and high-risk neuroblastoma. This therapeutic probe comprises paired diagnostic and therapeutic components, both using the humanized anti-GD2 monoclonal antibody hu3F8 as the sole targeting carrier; the diagnostic component is a zirconium-89-labeled deferoxamine-hu3F8 conjugate, and the therapeutic component is an actinium-225-labeled DOTA-hu3F8 conjugate. This application also discloses its preparation method, obtaining a high-purity product through three steps: antibody-chelating agent conjugation, radionuclide labeling, and purification. This probe system can be used to prepare a therapeutic drug composition for neuroblastoma, exhibiting strong targeting specificity, enabling imaging-guided precise radioimmunotherapy, and demonstrating good synergistic diagnostic and therapeutic effects.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and more specifically, to a therapeutic probe targeting neuroblastoma GD2. Background Technology

[0002] Neuroblastoma is the most common extracranial solid malignant tumor in children, with a 5-year overall survival rate of less than 40% for high-risk patients with relapse and refractory disease, resulting in extremely poor clinical prognosis. The tumor cells universally express GD2 ganglioside antigen, which is limited in expression in normal tissues and is a recognized ideal target for targeted diagnosis and treatment. Currently, the diagnosis of GD2-positive neuroblastoma mainly employs positron emission tomography (PET) imaging technology, using targeted vectors to locate and assess the extent of tumor lesions; treatment methods include monoclonal antibody immunotherapy and radioimmunotherapy, utilizing radiolabeled targeted molecules to specifically kill tumor cells.

[0003] In existing technologies, studies have verified that the anti-GD2 humanized antibody hu3F8 can label diagnostic radionuclides. 89 Zr, therapeutic radionuclide 225 Ac's individual performance: 89 Zr-labeled probes can be used to achieve PET imaging of GD2-positive tumors. 225 Ac-labeled probes can inhibit tumor growth in tumor-bearing mouse models. However, existing technologies still have core shortcomings: the diagnostic and treatment processes are independent, failing to form a coordinated diagnostic and treatment system based on the same target carrier. The tumor lesion uptake information obtained from diagnosis cannot directly and accurately guide the individualized selection of treatment doses, leading to the blindness of clinical treatment where "low doses are ineffective and high doses have significant off-target toxicity." Furthermore, there is a widespread technical bias in the field that "the combined use of two radionuclide probes increases the radiation load on normal tissues," resulting in a lack of targeted integrated diagnostic and treatment solutions and failing to achieve synergistic therapeutic effects and reduced toxicity. In clinical practice, diagnostic and treatment procedures are often performed separately, which not only increases the complexity of the process but may also affect the targeting and overall efficacy of treatment due to differences in the targeting properties of the diagnostic and treatment carriers. Therefore, developing a GD2-targeted integrated diagnostic and treatment probe system that can achieve precise diagnostic and treatment coordination, improve the treatment index, and reduce toxicity has significant clinical value and practical implications. Summary of the Invention

[0004] To address the core issues in existing technologies regarding the independent diagnosis and treatment of GD2-positive neuroblastoma, the lack of a coordinated diagnostic and treatment system, the blind application of treatment dosage, and significant off-target toxicity, this application provides an integrated diagnostic and therapeutic probe targeting GD2 in neuroblastoma.

[0005] Firstly, this application provides a diagnostic and therapeutic probe targeting neuroblastoma GD2, employing the following technical solution:

[0006] A diagnostic and therapeutic probe targeting neuroblastoma GD2, the probe comprising a paired diagnostic component and a therapeutic component for synergistic use;

[0007] The diagnostic component is a zirconium-89 labeled deferoxamine-humanized anti-GD2 monoclonal antibody conjugate, namely [ 89 Zr]Zr-DFO-hu3F8;

[0008] The therapeutic component is an actinol-225-labeled DOTA-humanized anti-GD2 monoclonal antibody conjugate, namely [ 225 Ac]Ac-DOTA-hu3F8;

[0009] The ratio of the dosing activity of the diagnostic component to the therapeutic component is (200~300):1, and both are targeted by the humanized anti-GD2 monoclonal antibody hu3F8. The individualized dosing of the therapeutic component is guided by the quantitative results of PET imaging of the diagnostic component, thereby achieving an integrated synergistic effect in the diagnosis and treatment of GD2-positive neuroblastoma.

[0010] By employing the above technical solution, using the FDA-approved humanized anti-GD2 monoclonal antibody hu3F8 as the sole targeting vector, the diagnostic and therapeutic components are ensured to have consistent targeting distribution patterns in vivo, fundamentally resolving the diagnostic and therapeutic bias issues caused by different targeting vectors. The diagnostic component is selected with a half-life (78.4 h) that matches the antibody's in vivo distribution cycle. 89 Zr is an imaging nuclide that can be used to precisely quantify the GD2 expression level, uptake capacity, and systemic distribution of tumor lesions via PET imaging; the therapeutic component is an alpha nuclide with high linear energy transfer (LET). 225 Ac, requiring only 2-3 alpha particles within a cell to cause irreversible DNA double-strand breaks in tumor cells, exhibits a killing efficiency far exceeding that of beta nuclides, while causing minimal damage to surrounding normal tissues. More importantly, through precise dosage ratios and synergistic combination rules, an organic linkage system of "diagnosis-decision-treatment" is formed, overcoming the technical biases inherent in the use of dual nuclides in this field, achieving synergistic effects and reduced toxicity in diagnosis and treatment, and solving the core problem of separation between diagnosis and treatment in existing technologies.

[0011] Secondly, this application provides a diagnostic and therapeutic probe targeting neuroblastoma GD2, employing the following technical solution:

[0012] A therapeutic probe targeting neuroblastoma GD2 includes the following steps:

[0013] S1. Preparation of antibody-chelating agent conjugates: Humanized anti-GD2 monoclonal antibody hu3F8 is provided; the antibody hu3F8 is coupled with a first chelating agent and a second chelating agent dissolved in dimethyl sulfoxide, respectively, in a reaction system isolated from oxygen and exogenous metal ions, thereby obtaining the first conjugate hu3F8-DFO and the second conjugate hu3F8-DOTA;

[0014] S2. Labeling and purification of diagnostic probes: The first conjugate hu3F8-DFO obtained in step S1 is reacted with the radionuclide zirconium-89 in HEPES buffer pretreated with Chelex® 100 resin at a pH of 6.8 to 7.2 to obtain a crude labeled product; the crude labeled product is purified to obtain a diagnostic probe with a radiochemical purity of not less than 99%. 89 Zr]Zr-DFO-hu3F8;

[0015] S3. Labeling and purification of the therapeutic probe: The second conjugate hu3F8-DOTA obtained in step S1 is reacted with the radionuclide actinium-225 in sodium acetate buffer solution pretreated with Chelex® 100 resin at a pH of 5.3 to 5.7 to obtain a crude labeled product; the crude labeled product is purified to obtain a therapeutic probe with a radiochemical purity of not less than 99%. 225 Ac]Ac-DOTA-hu3F8.

[0016] By adopting the above technical solution, a stable, reproducible probe preparation process suitable for clinical translation is formed by first completing the directional coupling reaction between the antibody and the chelating agent, and then separately labeling and purifying the radionuclide. By controlling the reaction system to isolate oxygen and exogenous metal ions, the oxidative inactivation of the antibody and the non-specific binding of the chelating agent are avoided, ensuring the activity and purity of the conjugate. By removing metal impurities through pretreatment buffer and optimizing the core parameters of the labeling reaction, quantitative labeling of radionuclides is achieved, significantly improving the radiochemical purity and in vitro stability of the probe, providing a stable and qualified product foundation for in vivo diagnostic and therapeutic applications.

[0017] Preferably, in step S1, the molar ratio of the antibody hu3F8 to the first chelating agent and the second chelating agent is 1:10 to 1:20.

[0018] By adopting the above technical solution, this molar ratio is the optimal ratio for antibody-chelating agent conjugation. It can provide sufficient chelating agent binding sites for antibody molecules, promote the full conjugation reaction, and ensure a stable conjugation efficiency of ≥85%. At the same time, it can avoid changes in antibody spatial conformation and decreased antigen-binding activity caused by excessive chelating agent. It also reduces the difficulty of subsequent purification. It is the optimal parameter that balances conjugation efficiency and antibody activity. The significant effect of this specific ratio has not been disclosed in the prior art.

[0019] Preferably, in step S1, the first chelating agent is benzyl-deferroamine p-isothiocyanate, used to specifically chelate the diagnostic radionuclide zirconium-89; the second chelating agent is benzyl-DOTA p-isothiocyanate, used to specifically chelate the therapeutic radionuclide actinium-225; both chelating agents are directionally coupled to the lysine residues of the antibody Fc segment, without affecting the GD2 antigen binding activity of the antibody Fab segment.

[0020] By adopting the above technical solution, benzyl-deferroamine isothiocyanate (p-SCN-Bn-DFO) and 89 Zr exhibits extremely high complexation stability and a very low dissociation constant, effectively preventing free radionuclides from being deposited in bone due to in vivo delabeling; it also exhibits good complexation stability against benzyl-DOTA isothiocyanate (p-SCN-Bn-DOTA) and... 225 Ac has an extremely high complexation constant, which can stably complex α-nuclides in vivo, avoiding radiation damage to normal tissues caused by free nuclides. More importantly, through directional coupling design, the chelating agent binds only to lysine residues of the antibody's Fc segment, without affecting the antigen-binding activity of the Fab segment, thus ensuring the probe's targeting specificity. This is a key design not disclosed in existing technologies.

[0021] Preferably, in step S1, the coupling reaction is carried out at a temperature of 36-38°C for a reaction time of 1.5 to 2.5 hours.

[0022] By adopting the above technical solution, the temperature range is close to the physiological temperature of the human body, which can maintain the natural spatial conformation and immune activity of the antibody to the greatest extent and avoid antibody denaturation and inactivation caused by high temperature. The 2-hour reaction time can ensure that the coupling reaction reaches a full equilibrium. While maximizing the coupling efficiency, it avoids antibody aggregation and increased byproducts caused by excessive reaction time. It is the optimal condition that balances reaction efficiency and antibody activity.

[0023] Preferably, in step S2, the concentration of the HEPES buffer is 0.4-0.6M; the reaction is carried out at 36-38°C with continuous shaking for 0.5-1.5 hours; and the ratio of the hu3F8-DFO conjugate to zirconium-89 is 6 to 10 microcuries of radioactivity per microgram of protein.

[0024] By employing the above technical solution, this concentration of HEPES buffer provides a strong pH buffering capacity for the labeling reaction, maintaining the pH of the reaction system stable at around 7.0, ensuring... 89The efficient complexation of Zr with DFO; at the same time, isothermal oscillation at this temperature allows the conjugate to fully contact the nuclide, resulting in a labeling reaction with a stable labeling yield of ≥98%; this feed ratio ensures high probe specific activity while avoiding radioactive waste and non-specific labeling caused by excessive nuclide, making it the optimal parameter for achieving quantitative labeling.

[0025] Preferably, in step S3, the concentration of the sodium acetate buffer is 0.4-0.6M; the reaction is carried out with continuous shaking at 36-38°C for 0.5-1.5 hours; and the ratio of the hu3F8-DOTA conjugate to actinium-225 is such that each microgram of protein corresponds to 6 to 10 microcuries of radioactivity.

[0026] By adopting the above technical solution, this concentration of sodium acetate buffer solution can be... 225 The complexation reaction between Ac and DOTA provides an optimal pH 5.5 environment, promoting efficient complexation. Isothermal oscillation at this temperature enables the labeling reaction, with a stable labeling yield of ≥98%. This feed ratio ensures that the therapeutic probe has precise specific activity, meeting the cytotoxicity requirements for treatment while avoiding off-target toxicity caused by excessive radionuclide dosage, thus providing a stable dosage basis for subsequent in vivo treatment.

[0027] Preferably, the purification described in step S2 and the purification described in step S3 are both performed using an activated PD-10 size exclusion chromatography column. The size exclusion chromatography is performed using an activated chromatography column, the packing material of the chromatography column is cross-linked dextran, and the mobile phase is sterile pyrogen-free phosphate buffer with pH 7.2-7.4.

[0028] By adopting the above technical solution, the activated PD-10 size exclusion chromatography column has excellent molecular sieving effect, which can efficiently separate high molecular weight labeled antibodies from low molecular weight free nuclides, uncoupled chelating agents and other impurities based on molecular size. The purification process is mild and does not affect the biological activity of the antibody or the radiochemical purity of the probe. Using sterile and pyrogen-free phosphate buffer as the mobile phase, the purified probe can be directly used for in vivo administration without additional sterilization and pyrogen removal treatment, which meets the requirements of clinical translation.

[0029] Preferably, the HEPES buffer solution in step S2 and the sodium acetate buffer solution in step S3 are both treated with Chelex® 100 resin to remove metal impurities, and the concentration of free metal ions is ≤10 ppb.

[0030] By adopting the above technical solution, Chelex® 100 resin can specifically adsorb divalent and trivalent metal ion impurities in the buffer solution, control the concentration of free metal ions below 10 ppb, avoid the competition between exogenous metal impurities and chelating agents for binding to radionuclides, eliminate the generation of non-specific labeled products, significantly improve the specificity and success rate of the labeling reaction, and ensure the quality stability of different batches of probe products.

[0031] Thirdly, this application provides an application of a diagnostic and therapeutic probe targeting neuroblastoma GD2, employing the following technical solution:

[0032] The application of a therapeutic probe targeting neuroblastoma GD2, wherein the application is in the preparation of a pharmaceutical composition for personalized diagnosis and treatment of GD2-positive neuroblastoma;

[0033] The integrated diagnosis and treatment process is as follows:

[0034] A1. Diagnostic Imaging Stage: Administration of Diagnostic Components [ 89 Zr]Zr-DFO-hu3F8 was administered, and PET imaging was performed 120-168 hours after administration to quantitatively obtain the radioactive uptake %ID / g value of tumor tissue and the ratio of tumor / normal tissue (T / NT).

[0035] A2. Individualized Dosage Decision Stage: With a tumor %ID / g ≥ 40 and a T / NT ratio ≥ 10 as the threshold, subjects above the threshold are matched with a treatment dose of 11.1 kBq, and subjects below the threshold are matched with a treatment dose of 18.5 kBq.

[0036] A3. Precision Treatment Phase: Within 72 hours after diagnostic imaging is completed, administer the corresponding dose of the therapeutic component. 225 Ac]Ac-DOTA-hu3F8, complete a single targeted radioimmunotherapy session;

[0037] A4. Efficacy verification and follow-up stage: The diagnostic component was administered again 28 days after treatment, and the tumor remission was assessed by PET imaging. The imaging results guided the adjustment of subsequent treatment plans and follow-up.

[0038] The diagnosis involves precise localization and quantitative imaging of GD2-positive neuroblastomas using positron emission tomography (PET), and the treatment involves individualized targeted radioimmunotherapy for GD2-positive neuroblastomas.

[0039] By employing the above-mentioned technical solution, the imaging results of diagnostic probes are quantitatively linked to the dosage selection of therapeutic probes, forming a complete diagnostic-therapeutic closed loop. This solves the problem of dosage blindness in existing treatments: for subjects with high tumor uptake and abundant GD2 expression, a medium dose of 11.1 kBq can achieve 100% tumor remission, avoiding off-target toxicity caused by high doses; for subjects with low tumor uptake and heterogeneous GD2 expression, a high dose of 18.5 kBq ensures anti-tumor efficacy, avoiding treatment ineffectiveness. Simultaneously, strictly controlling the dosing interval between diagnosis and treatment within 72 hours ensures treatment is completed within the optimal time window for antibody distribution in vivo, maximizing targeted enrichment in tumor tissue and minimizing radiation exposure in normal tissues, achieving the core effect of synergistic therapeutic enhancement and toxicity reduction. Furthermore, post-treatment re-imaging allows for non-invasive and precise evaluation of treatment efficacy, providing objective evidence for adjusting subsequent treatment plans, significantly improving the scientific rigor and standardization of clinical diagnosis and treatment, and possessing extremely high clinical translational value.

[0040] In summary, this application has the following beneficial effects:

[0041] 1. This application constructs a diagnostic and therapeutic probe system with hu3F8 as the sole target carrier, and provides clear rules for synergistic use and dosage ratios, forming a complete closed loop of diagnosis, decision-making, treatment, and verification. It effectively solves the core pain point of the separation and lack of linkage between the diagnosis and treatment links in the existing technology, realizes the synergistic effect of diagnosis and treatment and reduces toxicity, and significantly improves the problem of blind selection of radioimmunotherapy dosage in clinical treatment.

[0042] 2. This application optimizes the core process parameters for probe preparation, clarifying key technical features such as the preferred antibody-chelating agent ratio of 1:15, Fc segment directional coupling design, and metal ion quality control standards for buffer pretreatment. This enables stable probe labeling yield ≥98%, radiochemical purity ≥99%, and the radiochemical purity can still be maintained at ≥90% after 7 days of incubation in serum, effectively improving probe preparation efficiency, product purity, storage stability, and batch-to-batch quality consistency.

[0043] 3. The integrated diagnosis and treatment application solution of this application effectively broadens the treatment window and improves the treatment index by using individualized dose matching guided by tumor uptake threshold, and reduces the risk of off-target toxicity caused by blindly using high doses. The synergistic effect of this technology is something that those skilled in the art cannot reasonably expect based on existing imaging and treatment solutions alone.

[0044] 4. The probe system and integrated diagnostic and therapeutic operation process of this application can be directly adapted to the PET imaging equipment and radioimmunotherapy clinical operation specifications used in clinical practice. The preparation process is stable and reproducible, and the purified formulation meets the basic quality control requirements for in vivo administration. It has good clinical operability and translational application potential. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the radiolabeling strategy for the integrated diagnostic and therapeutic probe targeting neuroblastoma GD2 provided in this application and its application in an animal model;

[0046] Figure 2 This is an image showing the results of immunoPET imaging and in vitro biodistribution of the diagnostic probe targeting neuroblastoma GD2 provided in this application;

[0047] Figure 3 This is a graph showing the efficacy evaluation results of IMR32 tumor-bearing mice treated with a therapeutic probe targeting neuroblastoma GD2, as provided in this application.

[0048] Figure 4 This is a diagram showing the H&E staining pathological evaluation results of tumors and major organs in different treatment groups of tumor-bearing mice provided in this application. Detailed Implementation

[0049] The present application will be further described in detail below with reference to embodiments and comparative examples. Unless otherwise specified, the experimental methods used below are conventional methods. Unless otherwise specified, the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels or prepared according to literature methods.

[0050] Technical Concept: In the clinical diagnosis and treatment of GD2-positive neuroblastoma, current technologies mostly employ independent diagnostic and therapeutic approaches. The diagnostic process relies on a targeted carrier for positron emission tomography (PET) imaging, while the therapeutic process utilizes a different type of targeted molecule for radioimmunotherapy. The targeted carriers used in these two approaches are often different. The core reason for this situation is the lack of a technical solution that simultaneously carries both diagnostic and therapeutic radionuclides on the same targeted carrier. This prevents the tumor lesion information obtained from diagnosis from directly and accurately guiding the formulation and implementation of treatment plans. This not only increases the complexity of clinical procedures but also reduces the specificity of treatment due to differences in targeted carriers, ultimately affecting the overall treatment outcome.

[0051] This technical solution uses the humanized anti-GD2 monoclonal antibody hu3F8, which specifically recognizes the GD2 antigen, as the core targeting carrier. It is conjugated with two chelating agents, deferoxamine and DOTA, and then chelated with zirconium-89 and actinium-225 radionuclides to construct a diagnostic component.89 Zr]Zr-DFO-hu3F8 and therapeutic components[ 225 The therapeutic probe is composed of Ac]Ac-DOTA-hu3F8. Simultaneously, through a series of technical means, including optimizing the molar ratio of antibody to chelating agent, controlling the temperature and time of the coupling and labeling reactions, using a size exclusion chromatography column filled with cross-linked dextran, and using buffer solution treated with Chelex® 100 resin, the probe's high radiochemical purity and stability are ensured. Ultimately, this achieves synergistic operation of tumor diagnostic imaging and radioimmunotherapy using the same targeting carrier, solving the core problem of the separation of diagnosis and treatment in existing technologies.

[0052] This implementation method details the preparation, detection, treatment, and efficacy verification process of a therapeutic probe targeting neuroblastoma GD2. The specific details are as follows:

[0053] I. Probe preparation and related detection methods

[0054] 1.1 Antibody-conjugated antibodies

[0055] Humanized anti-GD2 monoclonal antibody hu3F8 was purchased from Hangzhou Nester Biotechnology Co., Ltd., and isotope control IgG protein was purchased from MedChemExpress. The hu3F8 antibody was replaced with 0.1M sodium bicarbonate buffer (pH 8.5) to adjust the protein concentration to 5 mg / mL, and aliquoted into 1 mL tubes. The entire process was performed in an anaerobic clean bench to prevent the use of exogenous metal ions.

[0056] Dissolve p-SCN-Bn-deferoxamine (DFO, Macrocyclic, CAS No.: 1222468-90-7) and p-SCN-Bn-DOTA (DOTA, Macrocyclic, CAS No.: 127985-74-4) separately in anhydrous dimethyl sulfoxide, preparing each solution immediately before use. Add the chelating agent solution dropwise to the antibody solution at a molar ratio of antibody to chelating agent of 1:15, gently invert to mix, and incubate at 37°C for 2 hours.

[0057] After the reaction was completed, the reaction mixture was purified by passing it through an activated PD-10 column (GE Healthcare, catalog number 17-0851-01), eluted with sterile phosphate buffer at pH 7.4, and the protein elution peak was collected to obtain hu3F8-DFO and hu3F8-DOTA conjugates. The protein concentration was determined by the BCA method and the mixture was aseptically aliquoted and stored at -20°C.

[0058] 1.2, Zirconium-89 and Actinium-225 radiolabeling

[0059] Buffer pretreatment: Take 0.5M MEPES buffer (pH 7.0) and 0.5M sodium acetate buffer (pH 5.5), add Chelex® 100 resin respectively, shake at room temperature for 2 hours, filter to remove resin, and test to find that the concentration of free metal ions in the treated buffer is ≤10 ppb. Store aseptically at 4℃ for later use.

[0060] 89 Zr labeling: Following a feed ratio of 8 microcuries of radioactivity per microgram of protein, pretreated 0.5 M H EPES buffer, hu3F8-DFO conjugate, and other components were added sequentially. 89 Zr oxalic acid solution, gently mixed, and incubated at 37°C with shaking for 1 hour. After the reaction, the protein was purified using a PD-10 column equilibrated with sterile, pyrogen-free PBS at pH 7.4, and the protein elution peak was collected to obtain […]. 89 Zr]Zr-DFO-hu3F8 final product.

[0061] 225 Ac labeling: Following a feed ratio of 8 microcuries of radioactivity per microgram of protein, pretreated 0.5M sodium acetate buffer, hu3F8-DOTA conjugate, and other ingredients were added sequentially. 225 After gently mixing with Ac hydrochloric acid solution, the mixture was incubated at 37°C with shaking for 1 hour. Following the reaction, the protein was purified using a PD-10 column equilibrated with sterile, pyrogen-free PBS at pH 7.4. The protein elution peak was collected to obtain […]. 225 Ac]Ac-DOTA-hu3F8 final product.

[0062] Control probe [ 89 Zr]Zr-DFO-IgG and [ 225 Ac]Ac-DOTA-IgG was prepared using the exact same method.

[0063] 1.3 Quality Inspection and Stability Assessment

[0064] The labeling yield and radiochemical purity were determined using radio-TLC (BIOSCAN). 89 The mobile phase for the Zr-labeled product was 0.05 M citric acid / sodium citrate buffer (pH 5.0). 225 The mobile phase for the Ac-labeled product was a 50 mM EDTA-2Na solution (pH 4.5).

[0065] In vitro stability assessment: The prepared [ 89 Zr]Zr-DFO-hu3F8 and [ 225 Ac]Ac-DOTA-hu3F8 were placed in 10% fetal bovine serum at 37℃ and incubated for 7 days. Samples were taken on days 1, 2, 5, and 7 to determine their radiochemical purity.

[0066] 1.4 ImmunoPET Imaging and In Vitro Biodistribution

[0067] Female BALB / c nude mice aged 4-6 weeks were selected and inoculated with GD2-positive IMR32 neuroblastoma cells in the right axilla. Tumors were used for experiments when they reached a volume of 180-300 mm³. Tumor-bearing mice (n=3) were injected intravenously with 5.55 MBq of […]. 89 Zr]Zr-DFO-hu3F8 or [ 89 Zr-DFO-IgG was administered to animals anesthetized with 2% isoflurane. The animals were fixed in a prone position on the scanning bed and small animal PET (MOLECUBES, Belgium) imaging was performed at 2, 24, 48, 72, 120, and 168 hours after injection. All images were reconstructed and attenuated using a system-integrated workstation.

[0068] After the final PET scan, the mice were euthanized, and tumor tissue and major organs such as the heart, liver, spleen, lungs, kidneys, stomach, intestines, pancreas, bladder, bones, muscles, skin, and brain were collected. After weighing, the radioactivity was measured using an automatic gamma counter (PerkinElmer2470WIZARD²). The biodistribution data were expressed as percentage of the injected dose per gram of tissue (%ID / g).

[0069] 1.5 In vivo validation experiment of the integrated diagnosis and treatment regimen

[0070] 1.5.1 Experimental Animals and Grouping: IMR32 tumor-bearing nude mice with tumor volume of 180-300 mm³ were selected and randomly divided into 4 groups, with 5 mice in each group:

[0071] Experimental group (therapeutic group): The combined use procedure of this application was followed, starting with an injection of 5.55 MBq. 89 Zr-DFO-hu3F8 mice underwent PET imaging at 168 hours. All mouse tumors had a %ID / g ≥40 and a T / NT ratio ≥10. 11.1 kBq was injected 48 hours after imaging. 225 Ac]Ac-DOTA-hu3F8;

[0072] Control group 1 (single medium-dose treatment group): direct injection of 11.1 kBq [ 225 Ac]Ac-DOTA-hu3F8 (the medium-dose group disclosed in the paper);

[0073] Control group 2 (high-dose treatment group): direct injection of 18.5 kBq [ 225 Ac]Ac-DOTA-hu3F8 (the high-dose group published in the paper);

[0074] Blank control group: injected with an equal volume of sterile PBS.

[0075] 1.5.2 Observation indicators: Mouse body weight and tumor volume were monitored twice a week. The tumor volume was calculated using the formula V = length × width² × 0.5. Mice were sacrificed 28 days after treatment, and blood, serum, tumors and major organs were collected for hematological analysis, serum biochemical detection, H&E staining and immunohistochemical staining (GD2, γ-H2AX).

[0076] 1.6 Biotoxicity Assessment

[0077] At the end of treatment, venous blood was collected from mice into anticoagulant tubes for hematological analysis, including white blood cell (WBC), red blood cell (RBC), lymphocyte (LYM), and monocyte (MON) counts. Serum was separated, and the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine (Cr) were measured to assess liver and kidney function. Major organs such as the heart, liver, spleen, lungs, and kidneys were collected, fixed in 4% paraformaldehyde, and stained with H&E to assess histopathological changes.

[0078] 1.7 Histological staining

[0079] Tumor tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and subjected to GD2 and γ-H2AX immunohistochemical staining. Brown staining represents GD2 expression and DNA double-strand break damage, respectively. All images were taken and observed using a Leica microscope.

[0080] II. Experimental Results

[0081] 2.1 Radiosynthesis and Quality Detection of Probes

[0082] Please refer to Figure 1 The figure illustrates the radiolabeling strategy based on hu3F8 and the integrated diagnostic and therapeutic process, where (a) shows the detailed radiosynthesis steps of the two probes and (b) shows the complete integrated diagnostic and therapeutic process.

[0083] Radio-TLC analysis showed that the [[] prepared in this embodiment... 89 Zr]Zr-DFO-hu3F8 and [ 225 The labeling yield of Ac]Ac-DOTA-hu3F8 was ≥98%, and the radiochemical purity was ≥99%, which is significantly better than the level of "labeling yield >90% and radiochemical purity >95%" disclosed in the prior art.

[0084] In vitro stability results showed that after incubation in 10% fetal bovine serum at 37°C for 7 days, the radiochemical purity of the two probes remained above 90%, which is far superior to the stability of "purity above 85% after 2 days of incubation" disclosed in the existing technology, and has excellent potential for in vivo application.

[0085] 2.2 ImmunoPET Imaging and Biodistribution Results

[0086] ImmunoPET images show that, 89 Zr-DFO-hu3F8 was rapidly taken up by IMR32 tumors within 2 hours of injection, and this high uptake level was maintained for up to 168 hours post-injection, demonstrating excellent tumor-targeting specificity; while [ 89 Zr-DFO-IgG showed no significant specific uptake in tumors.

[0087] Please refer to Figure 2 (a) shows PET images at different time points, and (b) shows the in vitro biodistribution results 168 hours after injection. The in vitro biodistribution analysis showed that... 89 The tumor uptake in the Zr-DFO-hu3F8 group was 58.47 ± 3.64% ID / g, significantly higher than that in the [Zr-DFO-hu3F8 group]. 89 The Zr-DFO-IgG group showed 5.65±1.40%ID / g (P<0.001), a tumor / muscle ratio ≥25, and a tumor / blood ratio ≥15, demonstrating excellent tumor targeting and imaging contrast.

[0088] 2.3 Anti-tumor effect of integrated diagnosis and treatment regimen

[0089] Please refer to Figure 3 (a) shows tumor images of mice in each group 28 days after treatment, (b) shows the standardized tumor volume curve of mice in each group, and (c) shows the standardized body weight curve of mice in each group.

[0090] Tumor growth monitoring results showed that 28 days after treatment, all 5 mice in the integrated diagnosis and treatment experimental group achieved complete tumor remission, with a complete remission rate of 100%; 4 / 5 mice in the single medium-dose treatment group achieved complete remission, with a complete remission rate of 80%; only 3 / 5 mice in the single high-dose treatment group achieved complete remission, and 1 mouse reached the humanitarian endpoint due to a weight loss of more than 20%; the tumor in the blank control group continued to grow rapidly, and all mice reached the humanitarian endpoint within 21 days.

[0091] Immunohistochemical results showed that the tumor tissue in the integrated diagnosis and treatment experimental group was completely negative for GD2 staining and strongly positive for γ-H2AX staining, confirming that the tumor cells had undergone extensive irreversible DNA double-strand breaks, and the treatment effect was thorough; while in the single treatment group, GD2 positive expression was still visible in the residual tumor foci, and the intensity of γ-H2AX staining was lower than that in the experimental group.

[0092] 2.4 Safety Assessment Results

[0093] Weight monitoring results showed that 28 days after treatment, the final weight of mice in the integrated diagnosis and treatment experimental group was 97.6±3.2% of the initial weight, which was not significantly different from the blank control group (P>0.05), and there was no weight loss; the final weight of mice in the single high-dose treatment group was 84.8±7.6% of the initial weight, and there was a significant weight loss (P<0.001).

[0094] Hematological and serum biochemical results showed that there were no significant differences in blood routine and liver and kidney function indicators between the integrated diagnosis and treatment experimental group and the blank control group (P>0.05); the high-dose treatment group showed a decrease in lymphocyte count and a slight increase in ALT / AST levels, indicating potential blood and liver toxicity.

[0095] H&E staining results showed that no obvious pathological damage was observed in the major organs such as the heart, liver, spleen, lungs, and kidneys in the integrated diagnosis and treatment experimental group; mild hepatocellular enlargement and vacuolar degeneration of renal tubular epithelial cells were observed in the high-dose treatment group, which is consistent with the toxicity results disclosed in the existing technology.

[0096] Please refer to Figure 4 The results of H&E staining of tumors and major organs in mice of each group are shown. The tumor tissue of the integrated diagnosis and treatment experimental group showed extensive nuclear condensation, nuclear dissolution and cell necrosis, while normal organs showed no obvious pathological abnormalities.

[0097] III. Comparative Verification Experiment

[0098] Comparative Example 1: The preparation method disclosed in the paper was used, with an antibody to chelating agent ratio of 1:10, and the remaining steps were the same as in Example 1. After testing, [ 89 The labeling yield of Zr-DFO-hu3F8 was 91.2%, the radiochemical purity was 95.3%, and the radiochemical purity after incubation with serum at 37°C for 2 days was 86.1%, which was significantly lower than the optimized preparation method of this application.

[0099] Comparative Example 2: A simple combination of two probes without dose matching was used, with tumor-bearing mice simultaneously injected with 5.55 MBq. 89 Zr]Zr-DFO-hu3F8 and 11.1kBq[ 225 Ac]Ac-DOTA-hu3F8, with the remaining observations the same as in the previous example. Results showed that only 3 / 5 of the mice in this group achieved complete tumor remission, and the mice exhibited mild weight loss and decreased lymphocyte counts. This confirms that simple combinations without clear rules for combination therapy cannot achieve the synergistic therapeutic effect, but instead increase toxicity.

[0100] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A diagnostic and therapeutic probe targeting neuroblastoma GD2, characterized in that: The probe consists of a diagnostic component and a therapeutic component used in synergistic pairing. The diagnostic component is a zirconium-89 labeled deferoxamine-humanized anti-GD2 monoclonal antibody conjugate, namely [ 89 Zr]Zr-DFO-hu3F8; The therapeutic component is an actinol-225-labeled DOTA-humanized anti-GD2 monoclonal antibody conjugate, namely [ 225 Ac]Ac-DOTA-hu3F8; The ratio of the dosing activity of the diagnostic component to the therapeutic component is (200~300):1, and both are targeted by the humanized anti-GD2 monoclonal antibody hu3F8. The individualized dosing of the therapeutic component is guided by the quantitative results of PET imaging of the diagnostic component, thereby achieving an integrated synergistic effect in the diagnosis and treatment of GD2-positive neuroblastoma.

2. A method for preparing a diagnostic and therapeutic probe targeting neuroblastoma GD2, characterized in that, The preparation of the therapeutic probe targeting neuroblastoma GD2 as described in claim 1 comprises the following steps: S1. Preparation of antibody-chelating agent conjugates: Humanized anti-GD2 monoclonal antibody hu3F8 is provided; the antibody hu3F8 is coupled with a first chelating agent and a second chelating agent dissolved in dimethyl sulfoxide, respectively, in a reaction system isolated from oxygen and exogenous metal ions, thereby obtaining the first conjugate hu3F8-DFO and the second conjugate hu3F8-DOTA; S2. Labeling and purification of diagnostic probes: The first conjugate hu3F8-DFO obtained in step S1 is reacted with the radionuclide zirconium-89 in HEPES buffer pretreated with Chelex® 100 resin at a pH of 6.8 to 7.2 to obtain a crude labeled product; the crude labeled product is purified to obtain a diagnostic probe with a radiochemical purity of not less than 99%. 89 Zr]Zr-DFO-hu3F8; S3. Labeling and purification of the therapeutic probe: The second conjugate hu3F8-DOTA obtained in step S1 is reacted with the radionuclide actinium-225 in sodium acetate buffer solution pretreated with Chelex® 100 resin at a pH of 5.3 to 5.7 to obtain a crude labeled product; the crude labeled product is purified to obtain a therapeutic probe with a radiochemical purity of not less than 99%. 225 Ac]Ac-DOTA-hu3F8.

3. The method for preparing a diagnostic and therapeutic probe targeting neuroblastoma GD2 according to claim 2, characterized in that: In step S1, the molar ratio of antibody hu3F8 to the first chelating agent and the second chelating agent is 1:10 to 1:

20.

4. The method for preparing a diagnostic and therapeutic probe targeting neuroblastoma GD2 according to claim 2, characterized in that: In step S1, the first chelating agent is benzyl-deferroamine p-isothiocyanate, used to specifically chelate the diagnostic radionuclide zirconium-89; the second chelating agent is benzyl-DOTA p-isothiocyanate, used to specifically chelate the therapeutic radionuclide actinium-225; both chelating agents are directionally coupled to the lysine residues of the antibody Fc segment and do not affect the GD2 antigen binding activity of the antibody Fab segment.

5. The method for preparing a diagnostic and therapeutic probe targeting neuroblastoma GD2 according to claim 2, characterized in that: In step S1, the coupling reaction is carried out at a temperature of 36-38°C for a reaction time of 1.5 to 2.5 hours.

6. The method for preparing a diagnostic and therapeutic probe targeting neuroblastoma GD2 according to claim 2, characterized in that: In step S2, the concentration of the HEPES buffer is 0.4-0.6M; the reaction is carried out at 36-38°C with continuous shaking for 0.5-1.5 hours; the ratio of the hu3F8-DFO conjugate to zirconium-89 is 6 to 10 microcuries of radioactivity per microgram of protein.

7. The method for preparing a diagnostic and therapeutic probe targeting neuroblastoma GD2 according to claim 2, characterized in that: In step S3, the concentration of the sodium acetate buffer is 0.4-0.6M; the reaction is carried out at 36-38°C with continuous shaking for 0.5-1.5 hours; the ratio of the hu3F8-DOTA conjugate to actinium-225 is 6 to 10 microcuries of radioactivity per microgram of protein.

8. The method for preparing a diagnostic and therapeutic probe targeting neuroblastoma GD2 according to claim 2, characterized in that: The purification described in step S2 and step S3 are both performed using an activated PD-10 size exclusion chromatography column. The size exclusion chromatography is performed using an activated column, the column packing material is cross-linked dextran, and the mobile phase is sterile pyrogen-free phosphate buffer with pH 7.2-7.

4.

9. The method for preparing a diagnostic and therapeutic probe targeting neuroblastoma GD2 according to claim 2, characterized in that: The HEPES buffer solution mentioned in step S2 and the sodium acetate buffer solution mentioned in step S3 are both treated with Chelex® 100 resin to remove metal impurities, and the concentration of free metal ions is ≤10 ppb.

10. The application of a diagnostic and therapeutic probe targeting neuroblastoma GD2, characterized in that, The therapeutic probe for targeting neuroblastoma GD2 as described in claim 1 is used in the preparation of a pharmaceutical composition for personalized diagnosis and treatment of GD2-positive neuroblastoma. The integrated diagnosis and treatment process is as follows: A1. Diagnostic Imaging Stage: Administration of Diagnostic Components [ 89 Zr]Zr-DFO-hu3F8 was administered, and PET imaging was performed 120-168 hours after administration to quantitatively obtain the radioactive uptake %ID / g value of tumor tissue and the ratio of tumor / normal tissue (T / NT). A2. Individualized Dosage Decision Stage: With a tumor %ID / g ≥ 40 and a T / NT ratio ≥ 10 as the threshold, subjects above the threshold are matched with a treatment dose of 11.1 kBq, and subjects below the threshold are matched with a treatment dose of 18.5 kBq. A3. Precision Treatment Phase: Within 72 hours after diagnostic imaging is completed, administer the corresponding dose of the therapeutic component. 225 Ac]Ac-DOTA-hu3F8, complete a single targeted radioimmunotherapy session; A4. Efficacy verification and follow-up stage: The diagnostic component was administered again 28 days after treatment, and the tumor remission was assessed by PET imaging. The imaging results guided the adjustment of subsequent treatment plans and follow-up. The diagnosis involves precise localization and quantitative imaging of GD2-positive neuroblastomas using positron emission tomography (PET), and the treatment involves individualized targeted radioimmunotherapy for GD2-positive neuroblastomas.

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