A radiolabeled monoclonal antibody formulation, methods of making, kits and uses thereof

Abstract

This invention provides a radioactive monoclonal antibody formulation, its preparation method, kit, and application, belonging to the field of biomedical technology. It addresses the limitations of existing B7-H3-targeted radiopharmaceuticals in terms of administration methods and formulations, which are unsuitable for the minimally invasive local treatment needs of brainstem gliomas. The radioactive monoclonal antibody formulation includes: a radionuclide. 177 The radioactive monoclonal antibody formulation contains Lu, the chelating agent DOTA, and B7-H3 monoclonal antibody; the radioactive concentration of the formulation is ≥370 MBq / mL, the protein content is 5-10 μg / dose, the pH value is 6.8-7.4, the osmotic pressure is 270-320 mOsm / kg, and the viscosity at 20°C is 1.2-2.5 mPa·s. This radioactive monoclonal antibody formulation is suitable for "stereotactic micro-injection" into the brainstem parenchyma, achieving high-concentration deposition in small volumes and inhibiting backflow upon needle cessation, reducing long-term perfusion dependence and backflow risk, and reducing operation time and procedural complexity in high-risk areas of the brainstem.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a radioactive monoclonal antibody preparation, its preparation method, reagent kit, and application. Background Technology

[0002] Brainstem gliomas, especially diffuse endophytic pontine gliomas (DIPG) and diffuse midline gliomas (DMG), are highly malignant and aggressive tumors of the central nervous system in children and adults, with extremely poor prognosis. Because the brainstem is the center of life, with dense anatomical structures and extremely high surgical risks, radiotherapy and chemotherapy are the main treatment methods in clinical practice. However, the overall efficacy is limited, and patient survival is short. Therefore, there is an urgent need for safe, precise, and effective local targeted therapy.

[0003] In recent years, B7-H3 (CD276), as an immune checkpoint molecule, has been shown to be highly expressed in pediatric central nervous system tumors and lowly expressed in normal brain tissue, making it an ideal targeted therapeutic target for brainstem gliomas. Radiolabeled anti-B7-H3 monoclonal antibodies can achieve integrated diagnosis and treatment of tumors, combining lesion imaging and local radiotherapy functions, and have become an important direction in central nervous system tumor research.

[0004] In existing technologies, there are already... 124 I-omburtamab was clinically explored for intratumoral perfusion in DIPG / DMG patients via convection-enhanced delivery (CED). The drug was distributed within the tumor through slow perfusion via an intratumoral catheter, and dosimetry and safety assessments were conducted to verify the feasibility of local administration of B7-H3-targeting radioantibodies to brainstem tumors.

[0005] However, existing technologies still have the following drawbacks: (1) Path-dependent CED: CED can achieve a large volume distribution, but it is sensitive to catheter position, drug administration rate, etc. Moreover, the operation time is long, the implementation is difficult, and the risk of drug backflow and misdistribution is high. Especially in functionally dense areas such as the brainstem, long-term perfusion brings concerns about complex procedures and complications.

[0006] (2) Insufficient coupling between nuclides and processes: 124 I is primarily used in PET imaging and dosimetry research.

[0007] In summary, the existing administration methods, formulations, and dosimetric procedures of B7-H3 targeted radiopharmaceuticals are not adequate for the minimally invasive, safe, controllable, and highly effective local treatment needs of brainstem gliomas. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a radioactive monoclonal antibody preparation, its preparation method, kit and application. The radioactive monoclonal antibody preparation is applicable to "stereotactic micro-injection" in the brainstem parenchyma, with small volume high concentration deposition and needle cessation to suppress backflow, reducing long-term perfusion dependence and backflow risk, and reducing operation time and process complexity in high-risk areas of the brainstem.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: On the one hand, the present invention provides a radioactive monoclonal antibody formulation, wherein the radioactive monoclonal antibody formulation is... 177 Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation, including: nuclide 177 Lu, chelating agent DOTA and B7-H3 monoclonal antibody; The radioactive monoclonal antibody preparation has a radioactive concentration ≥370 MBq / mL, a protein content of 5-10 μg / dose, a pH value of 6.8-7.4, an osmotic pressure of 270-320 mOsm / kg, and a viscosity of 1.2-2.5 mPa·s at 20℃.

[0010] Optionally, the radiochemical purity is ≥90%, the immunoreactivity is ≥80%, and the radiochemical purity decreases by <5% after 7 days at 2-8℃.

[0011] Optionally, the B7-H3 monoclonal antibody is a full-length antibody or a fragment antibody that retains the B7-H3 targeting property.

[0012] Optionally, the B7-H3 monoclonal antibody is at least one of F(ab')2, scFv, and nanobody.

[0013] In a second aspect, the present invention provides a method for preparing a radioactive monoclonal antibody formulation, comprising: Step (1): Place the purified B7-H3 monoclonal antibody in a weakly alkaline buffer system, add the chelating agent DOTA, and carry out the coupling reaction at room temperature in the dark for 1-2 hours. Remove the unbound free DOTA through an ultrafiltration tube, and then replace it with the buffer system required for metallization labeling to obtain the DOTA-B7-H3 precursor solution. Step (2), containing 177 The solution of Lu ions is mixed with the DOTA-B7-H3 precursor solution to carry out a metallization reaction, yielding a crude product. Step (3) purify the crude product to remove unbound free radicals. 177 Lu ions were eluted, and the main peak component was collected. After concentration via ultrafiltration and adjustment of formulation parameters, it was then aseptically filtered to obtain... 177 Lu-DOTA-B7-H3 radioactive monoclonal antibody preparation.

[0014] Optionally, in step (1), the molar ratio of DOTA to B7-H3 monoclonal antibody is 2-4:1; Optionally, the pH of the weakly alkaline buffer system is 8.5-9.0.

[0015] Optionally, in step (2), the conditions for the metallization reaction include: pH value of 4.5-5.0, temperature of 37-42℃, and time of 30-45 min.

[0016] Optionally, in step (3), the purification is performed by purifying with a PD-10 desalting column.

[0017] Thirdly, the present invention also provides a kit for stereotactic micro-injection of brainstem gliomas, comprising: The aforementioned radioactive monoclonal antibody preparations; Glass microneedles with an outer diameter of 50-100 μm at their tips; A short-range injection device connected to the glass microneedles, in which the radioactive monoclonal antibody preparation is located.

[0018] Fourthly, the present invention also provides the use of the above-mentioned radioactive monoclonal antibody preparation in the preparation of a medicament for stereotactic micro-injection therapy for brainstem gliomas.

[0019] The above-described solution of the present invention has at least the following beneficial effects: (1) Controllable distribution and concentrated hot spots: small volume high concentration deposition and needle cessation to suppress backflow, reducing long-term perfusion dependence and backflow risk (drug backflow rate is less than 10%), reducing operation time and process complexity in high-risk areas of the brainstem.

[0020] (2) Visualized dosimetry integration: radionuclides 177 Lu has its own gamma rays, which facilitates multi-timepoint SPECT quantification. Using D_tumor (Gy) / MBq as the threshold improves the objectivity and repeatability of efficacy assessment.

[0021] (3) Path differentiation: Unlike existing technologies, human CED is combined with 124 The I-omburtamab scheme, the present invention 177 The Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation can be combined with a micro-injection kit to inhibit drug reflux, which is beneficial for achieving high concentrations of the drug in the brainstem and reducing the risk of neurotoxicity in non-target areas. Attached Figure Description

[0022] Figure 1 This is a Radio-TLC scan of the crude product prepared in Example 1 of this invention; Figure 2This is a Radio-TLC scan of the crude product prepared in Comparative Example 1 of this invention. Detailed Implementation

[0023] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0024] Explanation of technical terms: B7-H3: B7 homolog 3 (CD276), an immune checkpoint molecule, is highly expressed in pediatric central nervous system tumors and lowly expressed in normal brain tissue. It is the tumor target of the radioactive monoclonal antibody used in this invention.

[0025] 177 Lu: Radiouclide Lutetium-177, a therapeutic radionuclide that emits beta-emitting radiation. - The particles, accompanied by 113 / 208 keV gamma rays, can be used for SPECT imaging.

[0026] DOTA: 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid. A chelating agent used to... 177 Lu is conjugated with antibodies.

[0027] SPECT: Single Photon Emission Computed Tomography, this invention is used for quantitative assessment of tumors at multiple time points after drug administration.

[0028] CED: Convection-Enhanced Delivery (slow perfusion); compared with the "stereotactic micro-injection (non-CED)" of this invention.

[0029] D_tumor(Gy) / MBq: Tumor-absorbed dose per unit administered activity (Gray per Megabecquerel), the absorbed dose per unit of tumor; used as a threshold for efficacy and as a basis for enrollment and treatment response.

[0030] Gy (Gray): a unit of absorbed dose.

[0031] MBq (Megabecquerel): a unit of radioactivity.

[0032] μL (microliter): a unit of volume.

[0033] DIPG: Diffuse Intrinsic Pontine Glioma (the core population for brainstem gliomas).

[0034] DMG: Diffuse Midline Glioma; it belongs to the same high-risk midline glioma lineage as DIPG.

[0035] scFv / F(ab')2: Single-chain variable fragment / Fragment antigen-binding (dimer), which are structural variants of the B7-H3 antibody in this invention.

[0036] On the one hand, this invention proposes a radioactive monoclonal antibody formulation, wherein the radioactive monoclonal antibody formulation is... 177 Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation, including: nuclide 177 Lu, chelating agent DOTA and B7-H3 monoclonal antibody; The radioactive monoclonal antibody preparation has a radioactive concentration ≥370 MBq / mL, preferably 370-740 MBq / mL, a protein content of 5-10 μg / dose, a pH value of 6.8-7.4, an osmotic pressure of 270-320 mOsm / kg, and a viscosity of 1.2-2.5 mPa·s at 20°C.

[0037] For example, the radiochemical purity of the radioactive monoclonal antibody preparation is ≥90%, the immunoreactivity is ≥80%, and the radiochemical purity decreases by <5% after 7 days at 2-8°C.

[0038] The present invention 177 Lu-DOTA-B7-H3 radioactive monoclonal antibody preparations at extremely high radioactive concentrations 177The particles emitted by Lu cause a large number of water molecules to ionize, generating free radicals (ROS). This is the root cause of antibody peptide chain breakage, exposure of hydrophobic regions (leading to aggregation), and radionuclide shedding (reduced radiochemical purity). This application maintains the thickness of the "hydration layer" on the surface of B7-H3 monoclonal antibody to the greatest extent by strictly controlling the pH at 6.8-7.4 and using an isotonic environment (270–320 mOsm / kg). This prevents the cross-linking and aggregation between high concentration protein molecules from the perspective of steric hindrance, thereby achieving high concentration of radioactivity (≥370 MBq / mL) in a very small volume, while ensuring that the B7-H3 monoclonal antibody protein does not aggregate (precipitate) and that the radiochemical purity is maintained at ≥90%.

[0039] The present invention 177 The viscosity of the Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation is 1.2-2.5 mPa·s. This viscosity is designed to create specific hydrodynamic resistance with ultrafine glass microneedles (50-100 μm outer diameter), unlike existing brain CED (long-term convection perfusion) techniques which are prone to backflow and leakage. 177 The Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation can be used with a kit that allows for "short-course micro-injection + 3-5 min pause". By controlling the formulation viscosity, microneedle aperture, injection volume and pause time, it can reduce drug reflux along the needle path and spillage outside the target area (reflux <10%), thus preventing drug backflow.

[0040] For example, the B7-H3 monoclonal antibody is a full-length antibody or a fragment antibody that retains the targeting property of B7-H3.

[0041] For example, the B7-H3 monoclonal antibody is at least one of F(ab')2, scFv, and nanobody.

[0042] Secondly, the present invention also provides a method for preparing a radioactive monoclonal antibody formulation, comprising: Step (1): Place the purified B7-H3 monoclonal antibody in a weakly alkaline buffer system, add the chelating agent DOTA, and carry out the coupling reaction at room temperature in the dark for 1-2 hours. Remove the unbound free DOTA through an ultrafiltration tube, and then replace it with the buffer system required for metallization labeling to obtain the DOTA-B7-H3 precursor solution. Step (2), containing 177 The solution of Lu ions is mixed with the DOTA-B7-H3 precursor solution to carry out a metallization reaction, yielding a crude product. Step (3) purify the crude product to remove unbound free radicals. 177 Lu ions were eluted, and the main peak component was collected. After concentration via ultrafiltration and adjustment of formulation parameters, it was then aseptically filtered to obtain...177 Lu-DOTA-B7-H3 radioactive monoclonal antibody preparation.

[0043] For example, in step (1), the molar ratio of DOTA to B7-H3 monoclonal antibody is 2-4:1. This molar ratio can avoid altering the antibody's hydrophilicity due to excessive coupling. If this molar ratio is less than 2, it will result in insufficient chelating agent conjugated to the antibody, making it impossible to load sufficient tumor-killing radioactivity in a very small volume (≤5μL). 177 Lu); if the molar ratio is higher than 4, the excessive DOTA strong hydrophilic structure will seriously change the conformation of B7-H3 monoclonal antibody, reduce its immunoreactivity to brainstem tumor surface antigen, and cause non-specific protein aggregation and rapid clearance in local brain tissue.

[0044] For example, the weakly alkaline buffer system is a 0.1M carbonate buffer solution with a pH of 8.5-9.0.

[0045] For example, the buffer system required for metallization labeling is 0.2-0.5M sodium acetate buffer.

[0046] For example, in step (2), the pH value of the metallization reaction is 4.5-5.0. The optimal pH for lutetium ions to form thermodynamically stable complexes with DOTA is in the acidic range. However, excessive acidity can cause antibody peptide chain breakage and denaturation. The slightly acidic window of 4.5-5.0 can ensure that the reaction kinetics reach their maximum while protecting the integrity of the antibody.

[0047] For example, in step (2), the metallization reaction is carried out at a temperature of 37-42°C for 30-45 minutes. Traditional DOTA labeling often uses high temperatures (such as 90-95°C), but higher temperatures may cause thermal denaturation or aggregation of antibody proteins. This invention uses mild labeling conditions of 37-42°C, which avoids thermal denaturation and aggregation of B7-H3 antibodies and can achieve a crude radiochemical purity of ≥85% within 30-45 minutes. When the crude radiochemical purity reaches ≥85%, the reaction solution only needs to be purified by a conventional desalting column (such as PD-10) to remove free nuclides (i.e., Figure 1 (The small amount of impurity peaks on the right side of the middle section) can ensure that the radiochemical purity of the final formulation is stable at ≥90%, which can meet the quality control requirements for brainstem micro-injection.

[0048] For example, the containing 177 The solution of Lu ions is lutetium chloride ( 177 LuCl3 solution.

[0049] For example, in step (3), the purification is performed using a PD-10 desalting column to remove free nuclides, achieving a radiochemical purity ≥90%. The brainstem is the vital center, and free nuclides...177 Lu can cause severe radiation necrosis of non-targeted nerve cells. Purification was used to increase the purity to ≥90% to meet the requirements for safe administration to the brainstem.

[0050] For example, in step (3), the elution is performed using a final formulation buffer.

[0051] For example, in step (3), the aseptic filtration is terminal aseptic filtration through a 0.22 μm filter membrane.

[0052] Collect after elution 177 The main peak component of Lu-DOTA-B7-H3 was concentrated via ultrafiltration and replaced with buffer. The radioactivity concentration, protein content, pH, osmotic pressure, and viscosity were adjusted, and then aseptically filtered through a 0.22 μm filter to obtain the final radioactive monoclonal antibody formulation. The ultrafiltration concentration is preferably centrifugal ultrafiltration; for full-length B7-H3 monoclonal antibodies, an ultrafiltration membrane with a molecular weight cutoff of 30–50 kDa is preferred. The parameters of the radioactive monoclonal antibody formulation are limited to: radioactivity concentration ≥370 MBq / mL, single-dose protein content 5–10 μg, osmotic pressure 270–320 mOsm / kg, and viscosity (20℃) 1.2–2.5 mPa·s. Isotonicity (270-320 mOsm / kg) is used to prevent acute brain parenchymal edema (space-occupying effect) during injection; a specific viscosity (1.2-2.5 mPa·s) is used to accommodate micro-injection glass microneedles with extremely fine pores (tip outer diameter 50-100 μm). If the viscosity is too low, the drug will flow back and overflow rapidly along the needle insertion gap like water (high backflow rate, high neurotoxicity). If the viscosity is too high, the microneedle injection resistance will be too high, causing a sudden increase in local pressure, which may damage the brainstem nerve bundles. Moreover, this viscosity is used in conjunction with the "stop needle for 3-5 minutes" operation, which can effectively reduce the risk of drug backflow along the needle path and keep the backflow rate below 10%.

[0053] Thirdly, the present invention also provides a kit for stereotactic micro-injection of brainstem gliomas, comprising: The aforementioned radioactive monoclonal antibody preparations; Glass microneedles with an outer diameter of 50-100 μm at their tips; A short-range injection device connected to the glass microneedles, in which the radioactive monoclonal antibody preparation is located.

[0054] The needle insertion path of the kit is stereotactic via neuronavigation to the core area of ​​the tumor lesion.

[0055] For example, the injection time of the short-range injection device is 200-300s / point, the single-point injection volume is ≤5μL, and the total injection volume is ≤10μL.

[0056] For example, the reagent kit is also provided with a timing module.

[0057] For example, the kit is set to stop injection for 3-5 minutes after injection to suppress backflow.

[0058] The kit of this invention is a completely new delivery method for the extremely high-risk brainstem microenvironment, from the physical parameters of the formulation and the drug administration strategy to the hydrodynamic matching of backflow prevention. It is conducive to achieving high concentration enrichment of drugs in the brainstem and reducing the risk of neurotoxicity in non-target areas.

[0059] This invention combines a high-concentration formulation with a high-precision stereotactic micro-injection device and 50-100μm glass microneedles. At a specific viscosity (1.2–2.5 mPa·s), the short-range injection device delivers the formulation at a constant rate, short distance, and extremely low flow rate (200-300 s / s per point), maintaining a stable laminar flow within the needle. This laminar flow avoids the physical damage to antibodies caused by turbulence during conventional manual injection or high-flow-rate CED perfusion, ensuring that high-concentration radioactive proteins do not mechanically aggregate or precipitate upon injection into the brain parenchyma.

[0060] Fourthly, the present invention also provides the use of the above-mentioned radioactive monoclonal antibody preparation in the preparation of a medicament for stereotactic micro-injection therapy for brainstem gliomas.

[0061] Example 1 This embodiment provides a method for preparing a radioactive monoclonal antibody formulation, including: (1) Antibody conjugation: The purified B7-H3 monoclonal full-length antibody was placed in 0.1M carbonate buffer (pH 8.5), and the chelating agent DOTA was added. The reaction was carried out at room temperature in the dark for 2 hours. Then, the unbound free DOTA was removed by ultrafiltration and replaced with 0.4M sodium acetate buffer to obtain DOTA-B7-H3 precursor solution; the molar ratio of DOTA to B7-H3 monoclonal full-length antibody was 3. (2) Radionuclide labeling: A solution of lutetium chloride with extremely high radioactivity concentration was added to the above DOTA-B7-H3 precursor tube for metallization reaction. The pH value of the metallization reaction was 4.7, the temperature was 40℃, and the time was 40 min, to obtain a crude product. The radiochemical purity of the crude product was 88.68% (e.g., Figure 1 (as shown) (3) Purification, desalting and final formulation preparation: The crude product was purified by passing it through a PD-10 desalting column to remove unbound free radicals. 177 Lu ions, collection 177 The main peak component of Lu-DOTA-B7-H3 was concentrated using a 30-50 kDa ultrafiltration membrane and then replaced with buffer. The radioactivity concentration, protein content, pH, osmotic pressure, and viscosity were adjusted. Finally, it was aseptically filtered through a 0.22 μm membrane to obtain...177 Lu-DOTA-B7-H3 radioactive monoclonal antibody preparation.

[0062] The preparation of this example 177 The Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation has a radioactive concentration of 555 MBq / mL, a protein content of 8 μg / dose, a pH of 7.2, an osmotic pressure of 300 mOsm / kg, a viscosity of 1.8 mPa·s at 20°C, a radiochemical purity of 98.2%, an immunoreactivity of 86.5%, and a 2.3% decrease in radiochemical purity over 7 days at 2-8°C.

[0063] Example 2 This embodiment provides a kit for stereotactic micro-injection of brainstem gliomas, comprising: The preparation of Example 1 177 Lu-DOTA-B7-H3 radioactive monoclonal antibody preparation; Glass microneedles with an outer diameter of 80 μm at their tips; A short-range injection device connected to the glass microneedles, in which the radioactive monoclonal antibody preparation is located.

[0064] The short-range injection device has an injection time of 240 s / point, a single-point injection volume of 4 μL, and a total injection volume of 8 μL. The kit is set to stop injection for 4 min after injection.

[0065] Comparative Example 1 This comparative example provides a method for preparing a radioactive monoclonal antibody formulation, which is basically the same as that in Example 1, except that the radionuclide labeling step does not use the slightly acidic heating labeling conditions specified in this invention, but is carried out under near-neutral and room temperature conditions. Specifically, it includes: (1) Antibody conjugation: The purified B7-H3 monoclonal full-length antibody was placed in 0.1M carbonate buffer at pH 8.5, and the chelating agent DOTA was added. The reaction was carried out at room temperature in the dark for 2 hours. Then, the unbound free DOTA was removed by ultrafiltration and replaced with 0.4M sodium acetate buffer to obtain DOTA-B7-H3 precursor solution. The molar ratio of DOTA to B7-H3 monoclonal full-length antibody was 3.

[0066] (2) Radionuclide labeling: labeling with radionuclides 177 The lutetium chloride solution containing Lu ions was added to the above DOTA-B7-H3 precursor solution, the pH of the reaction system was adjusted to 7.2, and the reaction was carried out at 25°C in the dark for 40 min to obtain the crude product.

[0067] (3) Detection: The crude product was analyzed by radio-TLC, and the results are as follows. Figure 2As shown. Compared to Example 1, Comparative Example 1 did not use a slightly acidic reaction window of pH 4.5–5.0 and mild heating conditions of 37–42°C. 177 The complexation reaction of Lu with the DOTA-B7-H3 precursor was insufficient, and free Lu... 177 The Lu ratio increased significantly, and the radiochemical purity of the crude product was 56.42%.

[0068] The results indicate that the experiment was conducted solely under near-neutral and room temperature conditions. 177 Lu mark, 177 The complexation of Lu with the DOTA-B7-H3 precursor is insufficient, making it difficult to obtain a crude radioactive monoclonal antibody product that meets the requirements for subsequent local micro-injection into the brainstem. The metallization reaction conditions of pH 4.5-5.0, 37-42℃, and 30-45min used in this invention can significantly improve the purity of the crude standard radiochemical reagent and provide a basis for subsequent purification by a PD-10 desalting column to obtain a final formulation with high radiochemical purity.

[0069] Test Example 1: 177 Evaluation of local delivery, imaging distribution, and preliminary therapeutic effect of Lu-DOTA-B7-H3 radioactive monoclonal antibody in an orthotopic model of brainstem glioma.

[0070] To verify the preparation of Example 1 177 The study investigated the controllability, safety, and preliminary antitumor effects of Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation in local micro-injection of brainstem gliomas, established an orthotopic xenograft model of B7-H3 positive human brainstem gliomas, and conducted the following animal experiments.

[0071] Thirty 6-8 week old female immunodeficient mice, contract-purchased under the name NTG mice, were selected. Human brainstem glioma 190326 cell line expressing luciferase was used, with 5 μL of 1×10⁻⁶ cells... 5 Cells were inoculated into the brainstem region of mice using a stereotactic method at a density of [number] cells. After modeling, animals were selected for evaluation based on survival, general condition, and bioluminescence imaging results; a total of 29 animals were selected for subsequent treatment and follow-up analysis. Bioluminescence imaging was performed on day 7 post-inoculation to confirm tumor formation and record baseline fluorescence signals; stereotactic local drug administration was administered on day 13 post-inoculation.

[0072] The 29 animals included were divided into a blank control group (8 animals) and a low-activity group. 177 Seven animals in the Lu-DOTA-B7-H3 group, with high activity. 177 Seven animals in the Lu-DOTA-B7-H3 group, with high activity. 177Seven animals were in the Lu-DOTA-B7-H3 combined with traditional Chinese medicine intervention group. The blank control group received an equal volume of PBS and underwent sham surgery; the low-activity group, high-activity group, and high-activity combined with traditional Chinese medicine group all used glass microneedles and microinfusion pumps to deliver the drug. 177 Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation was delivered in situ to the brainstem tumor region.

[0073] The animals used in this experiment 177 Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation is made from 177 Lu and the DOTA-B7-H3 precursor were obtained through radiolabeling. The radiochemical purity of the officially administered batch was 98.2%, and the immunoreactivity was 86.5%, meeting the quality control requirements of this invention: radiochemical purity not less than 90% and immunoreactivity not less than 80%. Due to the high initial specific activity, the drug was diluted before animal administration to ensure the accuracy of micro-injection. The actual administration volume per animal in the high-activity group and the high-activity combined with traditional Chinese medicine group was 4.5 μL; the actual administration volume per animal in the low-activity group was 2.5 μL. After dose correction, the actual in vivo dose in the high-activity group was approximately 40.3-52.8 μCi / animal, equivalent to approximately 1.49-1.95 MBq / animal; the actual in vivo dose in the low-activity group was approximately 20-26 μCi / animal, equivalent to approximately 0.74-0.96 MBq / animal. In this test case, the animal dosage was obtained by appropriately diluting the final formulation obtained in Example 1 to verify the local delivery, imaging distribution, and preliminary therapeutic effect of the formulation; the radioactivity concentration, pH, osmotic pressure, and viscosity parameters of the final formulation in Example 1 were based on the formulation test results.

[0074] SPECT / CT imaging was performed at 1 hour, 24 hours, D7, and D19 after drug administration. Results showed that... 177 Lu-DOTA-B7-H3 formed clear radioactive concentration foci in the brainstem tumor region, with the local signal largely consistent with the location of the brainstem lesion. This suggests that after local delivery via glass microneedles and a microinfusion pump, the formulation can achieve visible deposition and local retention in the brainstem tumor region. During administration, except for a few animals that showed mild diffusion, most animals achieved accurate drug delivery to the tumor region.

[0075] To evaluate the treatment effect, bioluminescence imaging was performed again on day 17 after drug administration, i.e., day 30 after model establishment. The results showed that, compared with the PBS control group, 177 The growth of bioluminescent signals in brainstem tumors was significantly inhibited in the Lu-DOTA-B7-H3 treatment group, suggesting that radiopharmaceutical therapy can effectively control tumor progression in an orthotopic brainstem glioma model. The low-activity group, high-activity group, and high-activity group combined with traditional Chinese medicine all showed varying degrees of tumor progression control trends. The high-activity group combined with traditional Chinese medicine was used as an exploratory expansion group, and its results are not intended to limit the scope of protection of the radioactive monoclonal antibody preparation itself.

[0076] Subsequent efficacy evaluation was primarily based on overall survival and longitudinal changes in tumor bioluminescence signal. The self-administration diary was designated as D0, with bioluminescence imaging performed on D7, D14, D21, and D28. If animal condition permitted, follow-up continued every 7 days from D35 until D56 or the humanitarian endpoint. Survival analysis employed the Kaplan-Meier method, focusing on comparing whether the high-activity group prolonged overall survival compared to the control group, and whether the high-activity group combined with traditional Chinese medicine further improved survival compared to the high-activity group alone.

[0077] This round of experiments will no longer involve the mid-term voluntary sacrifice of animals for mechanism testing. All animals will be followed up until natural death or the humanitarian endpoint, at which point tissue will be collected uniformly. At the endpoint, the whole brain will be collected, with a focus on preserving the brainstem tumor area. Liver, spleen, kidney, lung, and other major organs will also be collected for safety evaluation. Brain tissue will be preferentially stained with hematoxylin and eosin (HE), Ki-67 immunohistochemistry, and TUNEL staining to evaluate terminal tumor burden, tumor cell proliferation activity, and apoptosis, respectively. If tissue conditions permit, γ-H2AX, CD68, and CD8 assays can be further performed to supplement the evaluation of DNA damage and treatment-related immune microenvironment changes.

[0078] The above results indicate that the material prepared in Example 1... 177 The Lu-DOTA-B7-H3 radioactive monoclonal antibody formulation can be accurately delivered to the brainstem glioma region via a stereotactic microinfusion pump and can be visualized in vivo using SPECT / CT. Simultaneously, preliminary bioluminescence imaging suggests that this formulation can inhibit tumor progression in an orthotopic brainstem glioma model. These results support the feasibility of using the formulation of this invention for stereotactic microinfusion therapy of brainstem gliomas.

[0079] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A radioactive monoclonal antibody formulation, characterized in that, The radiomab formulation is 177 A Lu-DOTA-B7-H3 radiomab formulation comprising: a radionuclide 177 Lu, a chelator DOTA, and a B7-H3 monoclonal antibody; The radioactive monoclonal antibody preparation has a radioactive concentration ≥370 MBq / mL, a protein content of 5-10 μg / dose, a pH value of 6.8-7.4, an osmotic pressure of 270-320 mOsm / kg, and a viscosity of 1.2-2.5 mPa·s at 20℃.

2. The radioactive monoclonal antibody formulation according to claim 1, characterized in that, Radiochemical purity ≥90%, immunoreactivity ≥80%, radiochemical purity decreases by <5% over 7 days at 2-8℃.

3. The radioactive monoclonal antibody formulation according to claim 1, characterized in that, The B7-H3 monoclonal antibody is a full-length antibody or a fragment antibody that retains the B7-H3 targeting property.

4. The radioactive monoclonal antibody formulation according to claim 3, characterized in that, The B7-H3 monoclonal antibody is at least one of F(ab')2, scFv, and nanobody.

5. A method for preparing a radioactive monoclonal antibody formulation, characterized in that, For preparing the radioactive monoclonal antibody formulation according to any one of claims 1-4, comprising: Step (1): Place the purified B7-H3 monoclonal antibody in a weakly alkaline buffer system, add the chelating agent DOTA, and carry out the coupling reaction at room temperature in the dark for 1-2 hours. Remove the unbound free DOTA through an ultrafiltration tube, and then replace it with the buffer system required for metallization labeling to obtain the DOTA-B7-H3 precursor solution. Step (2), the solution containing 177 The solution containing Lu ions is mixed with the DOTA-B7-H3 precursor solution to perform a metallization reaction to obtain a crude product; Step (3), the crude product is purified to remove unbound free 177 Lu ions, the main peak fraction is collected after elution, concentrated by ultrafiltration membrane and adjusted for formulation parameters, and then sterilized to obtain 177 Lu-DOTA-B7-H3 radiomab formulation.

6. The preparation method according to claim 5, characterized in that, In step (1), the molar ratio of DOTA to B7-H3 monoclonal antibody is 2-4:1; Optionally, the pH of the weakly alkaline buffer system is 8.5-9.

0.

7. The preparation method according to claim 5, characterized in that, In step (2), the conditions for the metallization reaction include: pH value of 4.5-5.0, temperature of 37-42℃, and time of 30-45 min.

8. The preparation method according to claim 5, characterized in that, In step (3), the purification is performed by passing the PD-10 desalting column.

9. A reagent kit for stereotactic micro-injection of brainstem gliomas, characterized in that, include: The radioactive monoclonal antibody formulation according to any one of claims 1-4; Glass microneedles with an outer diameter of 50-100 μm at their tips; A short-range injection device connected to the glass microneedles, in which the radioactive monoclonal antibody preparation is located.

10. Use of the radioactive monoclonal antibody formulation according to any one of claims 1-4 in the preparation of a medicament for stereotactic micro-injection therapy of brainstem gliomas.

Images