A molecular probe targeting Trop-2, its preparation method and application

Abstract

This invention belongs to the fields of biomedicine and molecular imaging, specifically relating to a molecular probe targeting Trop-2, its preparation method, and its application. The molecular probe is a conjugate formed by the following (a) and (b) through a bifunctional chelating agent: (a) an antibody fragment that can specifically bind to Trop-2, wherein the antibody fragment is scFv or Fab; and (b) a radionuclide. The Trop-2-targeting molecular probe of this invention has the following outstanding characteristics: (1) high targeting specificity, significant uptake in tumors with high Trop-2 expression, and rapid clearance in normal tissues, resulting in high target-background contrast; (2) excellent pharmacokinetics, rapid blood clearance, and high-contrast tumor PET / CT images can be obtained 1-2 hours after injection; (3) good tumor retention, meeting the time window requirements for diagnostic imaging; and (4) high sensitivity, capable of detecting small tumor lesions in animal models.

Description

Technical Field

[0001] This invention relates to the field of molecular imaging, specifically to a molecular probe targeting Trop-2, its preparation method, and its application. Background Technology

[0002] Radioimmunoimaging (RII) is a technique that combines the high specificity of radiolabeled monoclonal antibodies with the high sensitivity of radioactive detectors, and it is used for the early diagnosis of tumors. This technique provides a non-invasive method to study the distribution, quantity, and function of antigens in the body.

[0003] Radioimmunotherapy (RIT) is a treatment method that uses therapeutically labeled monoclonal antibodies or fragments thereof that emit alpha or beta particles to specifically irradiate tumor cells. Compared with the widespread damage and severe toxic side effects that chemotherapy drugs may cause to normal tissue cells, and the limitation of radiotherapy that only treats specific areas, RIT combines the systemic and targeted nature of treatment and has developed rapidly in recent years.

[0004] Currently, molecular probes used for RII and RIT are mainly murine antibodies or humanized human-mouse chimeric antibodies. However, in clinical applications, the heterologous nature of these antibodies may cause adverse immune responses in the body, which is an obstacle to clinical translation. Summary of the Invention

[0005] This invention provides a molecular probe with high specificity and superior pharmacokinetic performance in vivo for the diagnosis and treatment of tumor diseases.

[0006] The Trop-2-targeting molecular probe provided by this invention is a conjugate formed by the following (a) and (b) chelating agents: (a) An antibody fragment capable of specifically binding to Trop-2; said antibody fragment is scFv or Fab; (b) Radionuclides; in: The scFv is an scFv having the amino acid sequence shown in SEQ ID NO: 10; The Fab is a Fab composed of the light chain shown in SEQ ID NO: 8 and the heavy chain shown in SEQ ID NO: 9; The bifunctional chelating agent is p-SCN-Bn-NOTA; The radioactive nuclide is 68 Ga.

[0007] This invention also provides a method for preparing the above-mentioned Trop-2-targeting molecular probe, comprising the following steps: The above antibody fragments are coupled with a bifunctional chelating agent to generate a bifunctional chelating agent-coupled antibody fragment. The antibody fragment conjugated with the bifunctional chelating agent is reacted with a radionuclide to obtain the molecular probe targeting Trop-2.

[0008] In some embodiments of the present invention, the molar ratio of antibody fragment to bifunctional chelating agent is 1:30.

[0009] In some embodiments of the present invention, the pH conditions for the reaction of the antibody fragment coupled with the bifunctional chelating agent with the radionuclide are 4.0 to 4.5.

[0010] In some embodiments of the present invention, the coupling reaction is carried out in a Na2CO3 buffer solution with a pH of 8.5 to 9.0 and a concentration of 0.1 M.

[0011] The present invention also provides the application of the above-mentioned Trop-2-targeting molecular probe in the preparation of PET / CT imaging reagents for the diagnosis of non-small cell lung cancer.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The molecular probes provided by this invention are based on humanized antibody fragments (scFv or Fab), which have significantly reduced immunogenicity compared to traditional murine or chimeric antibodies. This effectively reduces the risk of immune side effects such as human anti-mouse antibody (HAMA) reactions in clinical applications, thereby improving the safety and feasibility of treatment.

[0013] 2. The molecular probe of this invention has a small molecular weight, exhibiting a faster blood clearance rate and a reduction in background levels in non-target organs in vivo. This allows for the achievement of extremely high tumor / blood (T / B) and tumor / liver (T / L) ratios early after injection, enabling high-contrast PET / CT imaging, which is beneficial for clear tumor localization and differential diagnosis.

[0014] 3. The molecular probe of this invention can specifically target and bind to tumor cells that highly express Trop-2, and effectively concentrate and remain at the tumor site. Experiments show that even 4 hours after injection, the tumor outline remains clear, while the control Trop-2 negative tumors show no significant uptake, demonstrating its high targeting specificity.

[0015] 4. In the NCI-H292 lung cancer orthotopic tumor model, the molecular probe of this invention can clearly display tiny tumor lesions with a diameter of only about 5 mm within 1 hour after injection, and its detection capability is superior to that with high background uptake. 18F-FDG demonstrates great potential for application in early tumor diagnosis and detection of small metastases.

[0016] 5. The molecular probe preparation method of the present invention has high labeling efficiency, and the product has radiochemical purity >95% and good stability in in vitro serum. Attached Figure Description

[0017] Figure 1 In the image, A: SEC-HPLC chromatogram of aTrop-2-scFv; B: SEC-HPLC chromatogram of NOA-aTrop-2-scFv.

[0018] Figure 2 Western blot analysis was performed on the expression of Trop-2 in BxPC-3, MDA-MB-468, MCF-7, NCI-H292, A549 and HCC827 cells (GAPDH was used as an internal control).

[0019] Figure 3 In the middle, A: 68 PET / CT images of NCI-H292 tumor-bearing mice at time points of 0.5 h, 1 h, 2 h, and 4 h after Ga-NOTA-aTrop-2-scFv injection (attenuation correction; fused images, coronal and axial views; arrows indicate NCI-H292 subcutaneous tumors); B: 68 Blood, liver, kidney, muscle, and tumor data of NCI-H292 tumor-bearing mice at time points of 0.5h, 1h, 2h, and 4h after Ga-NOTA-aTrop-2-scFv injection. 68 Quantitative analysis of the ROI of Ga-NOTA-aTrop-2-scFv uptake; C: 68 Biodistribution of molecular probe uptake in major organs such as blood, liver, kidney, muscle, and tumors 4 h after injection of Ga-NOTA-aTrop-2-scFv.

[0020] Figure 4 In the middle, A: 68 PET / CT images of A549 tumor-bearing mice at 1 h, 2 h, and 4 h post-Ga-NOTA-aTrop-2-scFv injection (attenuation corrected; fused images; white dashed circles indicate A549 subcutaneous tumors); B: Blood, liver, kidney, muscle, and tumor data of A549 tumor-bearing mice. 68 Quantitative analysis of the ROI of Ga-NOTA-aTrop-2-scFv uptake.

[0021] Figure 5 H&E staining and immunohistochemical staining analysis of NCI-H292 and A549 subcutaneous tumors, and Trop-2.

[0022] Figure 6 For NCI-H292 tumor-bearing mice 68 Ga-NOTA-aTrop-2-Fab PET / CT Images and Analysis: A: Representative PET / CT images of the NCI-H292 subcutaneous tumor model at different time points (attenuation correction; fused images, coronal and transverse images; arrows indicating NCI-H292 subcutaneous tumors); B: Semi-quantitative analysis of the mean PET signal intensity of blood, liver, kidney, muscle and tumor in PET images of the NCI-H292 subcutaneous tumor model (n=3); C: Analysis of tumor / blood (T / B), tumor / liver (T / L) and tumor / muscle (T / M) ratios of the NCI-H292 subcutaneous tumor model based on PET images (n=3).

[0023] Figure 7 For NCI-H292 tumor-bearing mice 68 Ga-NOTA-aTrop-2-scFv and 68 Ga-NOTA-aTrop-2-Fab PET / CT Imaging Comparison Analysis: A: NCI-H292 Subcutaneous Tumor Model Injection 68 Representative PET / CT images of Ga-NOTA-aTrop-2-scFv at 1 h, 2 h, and 4 h (attenuation correction; fused images; arrows indicate NCI-H292 subcutaneous tumors); B: NCI-H292 subcutaneous tumor model after injection. 68 Representative PET / CT images of Ga-NOTA-aTrop-2-Fab at 1 h, 2 h, and 4 h; C: Tumor / blood ratio analysis based on PET images of the NCI-H292 subcutaneous tumor model (n=3); D: Tumor / liver ratio analysis based on PET images of the NCI-H292 subcutaneous tumor model (n=3); E: Tumor / muscle ratio analysis based on PET images of the NCI-H292 subcutaneous tumor model (n=3). White arrows indicate tumors. : P <0.05.

[0024] Figure 8 In the image, A: SEC-HPLC chromatogram of aTrop-2-Fab; B: SEC-HPLC chromatogram of NOA-aTrop-2-Fab.

[0025] Figure 9 for 68 ITLC map of Ga-NOTA-aTrop-2-scFv.

[0026] Figure 10 for 68ITLC map of Ga-NOTA-aTrop-2-Fab. Detailed Implementation

[0027] The present invention will be described in detail below with reference to specific embodiments, thereby making its advantages and various effects more clearly apparent. Those skilled in the art should understand that these specific embodiments are for illustrative purposes only and not for limiting the invention.

[0028] To ensure clarity and rigor in this specification, the key technical terms and abbreviations used in this document are defined as follows: Trop-2: Trophoblastic cell surface antigen 2. A transmembrane glycoprotein highly expressed in various epithelial tumors (such as breast cancer, lung cancer, pancreatic cancer, etc.), and an important target for tumor diagnosis and treatment.

[0029] mAb: a complete, full-length monoclonal antibody.

[0030] scFv: Single-chain variable region fragment. A small molecule antibody fragment composed of the heavy chain variable region (VH) and light chain variable region (VL) of mAb linked by a short peptide linker. It retains the antigen-binding specificity of mAb but has a smaller molecular weight.

[0031] Fab: Antigen-binding fragment. It is composed of a light chain (VL+CL) and a portion of the heavy chain (VH+CH1) of the antibody linked by disulfide bonds. It is the part of the antibody responsible for specifically binding to the antigen, and its molecular weight is greater than scFv but less than mAb.

[0032] p-SCN-Bn-NOTA: A commonly used bifunctional chelating agent. Its structure consists of an isothiocyanate group (-SCN) at one end, which can couple with the amino group of proteins (such as antibody fragments); and a NOA macrocycle at the other end, which can efficiently and stably chelate various metal radionuclides.

[0033] aTrop-2-scFv: scFv targeting Trop-2.

[0034] aTrop-2-Fab: A Fab that targets Trop-2.

[0035] PET / CT: Positron Emission Tomography / Computed Tomography. An advanced medical imaging technology that combines functional metabolic imaging (PET) with anatomical imaging (CT).

[0036] ROI: Region of Interest. In image analysis, it refers to a specific area that is manually or automatically delineated for quantitative measurements (such as calculating radioactive uptake).

[0037] %ID / g: Percentage of injected dose taken up per gram of tissue. It is a standard unit used in nuclear medicine and pharmacokinetics studies to quantify the concentration of radioactive tracers taken up in tissues or organs.

[0038] SEC-HPLC: Size exclusion high-performance liquid chromatography. A chromatographic technique that separates and analyzes molecules based on their size, commonly used to detect protein purity, polymers, or fragments.

[0039] ITLC: Transient thin-layer chromatography. A rapid and simple chromatographic method commonly used to determine the radiochemical purity of radiolabeled compounds.

[0040] To address the issue of potential immune side effects such as human anti-mouse antibody (HAMA) reactions caused by commonly used murine or chimeric antibodies in clinical practice, this invention employs humanized antibody fragments (scFv or Fab) as targeting carriers. This significantly reduces the immunogenicity of molecular probes, improving their safety and feasibility for use in humans. Furthermore, the small molecular weight antibody fragments optimize the pharmacokinetic properties of the molecular probes, enabling rapid clearance from the blood and non-target organs in vivo. This allows for extremely high tumor / background contrast in the early post-injection period (e.g., 1-2 hours), achieving clear tumor localization and highly sensitive detection of small lesions. This solves the problems of slow blood clearance, high background signal, and long imaging waiting time associated with large molecular weight intact antibodies.

[0041] The Trop-2-targeting molecular probe provided by this invention is a conjugate formed by the following (a) and (b) chelating agents: (a) An antibody fragment capable of specifically binding to Trop-2; said antibody fragment is scFv or Fab; (b) Radionuclides; in: The scFv is an scFv having the amino acid sequence shown in SEQ ID NO: 10; The Fab is a Fab composed of the light chain shown in SEQ ID NO: 8 and the heavy chain shown in SEQ ID NO: 9; The bifunctional chelating agent is p-SCN-Bn-NOTA; The radioactive nuclide is 68 Ga.

[0042] The scFv and Fab used in this invention are derived from the variable regions of specific humanized antibodies, which can significantly reduce the immunogenicity of the molecular probe, thereby improving its safety and tolerability in human application and overcoming the clinical translation barriers of existing technologies. The smaller molecular weight of scFv and Fab compared to intact antibodies allows the molecular probe to be cleared from the bloodstream and non-target organs more quickly in vivo, thus reducing background signal. In particular, the scFv molecular probe, due to its smaller size and shorter blood half-life, can be cleared more quickly, resulting in significantly higher tumor / blood and tumor / liver ratios at various time points (1h, 2h, 4h). This molecular probe specifically accumulates significantly in tumors with high Trop-2 expression, while taking up very little in Trop-2-negative tumors. Simultaneously, the molecular probe exhibits a certain degree of retention at the tumor site, and the signal duration meets the requirements of the imaging time window.

[0043] In this invention, the bifunctional chelating agent is p-SCN-Bn-NOTA. One end of the p-SCN-Bn-NOTA structure (isothiocyanate group, -SCN) undergoes a covalent reaction with the amino group on the surface of the antibody fragment to form a stable thiourea bond; the other end, the NOA chelating group, is specifically designed to efficiently and specifically chelate radioactive metal cations, firmly immobilizing radionuclides and preventing them from detaching during in vivo imaging, thus ensuring the targeting specificity and accuracy of PET / CT imaging signals.

[0044] This invention also provides a method for preparing the above-mentioned Trop-2-targeting molecular probe, comprising the following steps: The above antibody fragments are coupled with a bifunctional chelating agent to generate a bifunctional chelating agent-coupled antibody fragment. The antibody fragment conjugated with the bifunctional chelating agent is reacted with a radionuclide to obtain the molecular probe targeting Trop-2.

[0045] Radionuclides are time-sensitive. This invention prepares stable, long-term-storeable bifunctional chelating agent-conjugated antibody fragments, which can be rapidly radiolabeled and purified when needed, minimizing radionuclide decay loss, improving labeling efficiency, and facilitating on-demand preparation.

[0046] In some embodiments of the present invention, the molar ratio of antibody fragment to bifunctional chelating agent is 1:30; and / or the bifunctional chelating agent is p-SCN-Bn-NOTA; and / or the pH conditions for the reaction between the antibody fragment conjugated with the bifunctional chelating agent and the radionuclide are 4.0-4.5. These conditions ensure the high efficiency and specificity of the ligation reaction, generating structurally uniform antibody fragments conjugated with the bifunctional chelating agent, avoiding random ligation or side reactions, and laying a reliable foundation for subsequent radiolabeling. The ligation reaction is carried out in a 0.1 M Na₂CO₃ buffer at pH 8.5-9.0. This condition achieves a high labeling rate, approximately 59%-90% in the examples, and after purification, a molecular probe targeting Trop-2 with high radiochemical purity (>95%) is obtained.

[0047] The present invention also provides the application of the above-mentioned Trop-2-targeting molecular probe in the preparation of PET / CT imaging reagents for the diagnosis of non-small cell lung cancer.

[0048] In some embodiments of the present invention, the imaging reagent is an imaging reagent for positron emission tomography / computed tomography imaging.

[0049] The technical solution of the present invention will be described in detail below through specific embodiments: Example 1: Preparation of Fab and scFv targeting Trop-2 In Example 1, the VH and VL of mAb, Fab, and scFv are derived from VH (SEQ ID NO: 1 in US 63 / 713,425; note: SEQ ID NO: 1 here is only for citation purposes and is not SEQ ID NO: 1 in Table 1) and VL (SEQ ID NO: 2 in US 63 / 713,425) in patent application US 63 / 713,425. The entire contents of that patent application are incorporated herein by reference. Note: In the prior patent application (US 63 / 713,425) cited here, "SEQ ID NO: 1" refers to the complete amino acid sequence of the antibody heavy chain variable region (VH) as described in its entirety, and "SEQ ID NO: 2" corresponds to the complete amino acid sequence of its antibody light chain variable region (VL). These two sequences serve as the source of the humanized variable region of the antibody fragment of this invention. Based on the VH and VL sequences, the present invention further defines and lists their complementarity determination regions (CDRs), wherein the sequence “GFTFSDY” of the heavy chain CDR1 (HCDR1) is independently defined as SEQ ID NO: 1 in this specification, and the sequence “ISSSDDFK” of the heavy chain CDR2 (HCDR2) is independently defined as SEQ ID NO: 2 in this specification, and is included in the sequence list (Table 1) at the end of this specification.

[0050] VH and VL were linked via linker (G4S)3, and six histidine residues were introduced at the C-terminus to obtain the complete sequence of scFv (SEQ ID NO: 10). SEQ ID NO: 10 was cloned into a mammalian expression vector. It was expressed in Expi293F cells using a standard transient transfection protocol and purified from cell culture supernatant using Ni Sepharose High Performance to obtain aTrop-2-scFv.

[0051] VH and VL were cloned into a mammalian Fab expression vector, which provided the constant regions of human Fab (including CL and CH1). The final sequence of the light chain of Fab is shown in SEQ ID NO:8, and the final sequence of the heavy chain is shown in SEQ ID NO:9. The Fab was expressed in ExpiCHO-S cells using a standard transient transfection protocol and purified from the cell culture supernatant using IgG Select Prism A to obtain aTrop-2-Fab.

[0052] Example 2: 68 Preparation of Ga-NOTA-aTrop-2-scFv 2.1 Preparation of NOTA-aTrop-2-scFv aTrop-2-scFv was purified using a PD-10 desalting chromatography column. The purity of aTrop-2-scFv was determined by size exclusion chromatography (SEC-HPLC). 20 nmol of purified aTrop-2-scFv was taken, and the pH was adjusted to 8.5–9.0 with Na₂CO₃ (0.1 M, pH 9.0). p-SCN-Bn-NOTA (dissolved in a small amount of DMSO) was added at a 30-fold molar ratio, and the mixture was reacted at 25°C in the dark for 8 h to obtain NOA-aTrop-2-scFv.

[0053] Excess unreacted NOA was purified using a PD-10 desalting chromatography column. The purified NOA-aTrop-2-scFv was concentrated using ultrafiltration centrifuge tubes (Millipore 0.5 ml / 3 kDa) at 4 °C, 12000 × g, for 10 min, and then stored at -20 °C. The concentrated solution was stable at -20 °C for one month.

[0054] 2.2 68 Preparation of Ga-NOTA-aTrop-2-scFv Rinse with 4 mL of 0.05 M HCl 68 Ge / 68 Ga generator, using sodium acetate (0.25 M, pH 6.8) to... 68The pH of GaCl3 was adjusted to 4.0–4.5. The purified NOA-aTrop-2-scFv (6 nmol) was then mixed with… 68 After thorough mixing of GaCl3 (111 MBq, 3 mCi), the mixture was reacted at 37°C for 20 min. The labeled mixture was then purified by PD-10 desalting chromatography to obtain... 68 Ga-NOTA-aTrop-2-scFv. 68 The radiochemical purity of Ga-NOTA-aTrop-2-scFv was determined by instant thin-layer chromatography (ITLC) using a citrate-sodium citrate buffer (pH 4.0) as the developing solvent.

[0055] 2.3 68 Radiochemical characterization of Ga-NOTA-aTrop-2-scFv Take 3.70 MBq respectively 68 Ga-NOTA-aTrop-2-scFv was added to 100 μL of phosphate-buffered saline (PBS) and 100 μL of fetal bovine serum (FBS), and incubated at room temperature for 1 h and 2 h, respectively. The PBS and FBS samples were then directly subjected to SEC-HPLC for radiochemical purity identification, using the same elution conditions as described in section 2.1, i.e., purification using a PD-10 desalting column.

[0056] The prepared 68 Ga-NOTA-aTrop-2-scFv was identified and characterized using analytical SEC-HPLC. SEC-HPLC results showed that the purity of Nota-aTrop-2-scFv was greater than 99%. Figure 1 B). Measured by ITLC 68 The radiochemical purity of Ga-NOTA-aTrop-2-scFv is greater than 95%. Figure 9 ).

[0057] In summary, a successful preparation has been achieved. 68 The Ga-NOTA-aTrop-2-scFv labeling rate was approximately 59.1%. After purification of the labeled mixture using a PD-10 desalting column, the radiochemical purity was greater than 95%.

[0058] Example 3: 68 Preparation of Ga-NOTA-aTrop-2-Fab 3.1 Preparation of NOTA-aTrop-2-Fab aTrop-2-Fab was purified using a PD-10 desalting chromatography column. The purity of aTrop-2-Fab was determined by size exclusion chromatography (SEC-HPLC). 20 nmol of purified aTrop-2-Fab was taken, and the pH was adjusted to 8.5–9.0 with Na₂CO₃ (0.1 M, pH 9.0). p-SCN-Bn-NOTA (dissolved in a small amount of DMSO) was added at a 30-fold molar ratio, and the mixture was reacted at 25°C in the dark for 8 h to obtain NOA-aTrop-2-Fab. Excess unreacted NOA was removed by purification using a PD-10 desalting chromatography column. The purified NOA-aTrop-2-Fab was concentrated using ultrafiltration centrifuge tubes (Millipore 0.5 ml / 3 kDa) at 4°C, 12000 × g, for 10 min, and then stored at -20°C. The concentrated solution was stable at -20°C for one month.

[0059] 3.2 68 Preparation of Ga-NOTA-aTrop-2-Fab Rinse with 4 mL of 0.05 M HCl 68 Ge / 68 Ga generator, using sodium acetate (0.25 M, pH 6.8) to... 68 The pH of GaCl3 was adjusted to 4.0–4.5. The purified NOA-aTrop-2-Fab (6 nmol) was then mixed with… 68 After thorough mixing of GaCl3 (111 MBq, 3 mCi), the mixture was reacted at 37°C for 20 min. The labeled mixture was then purified by PD-10 desalting chromatography to obtain... 68 Ga-NOTA-aTrop-2-Fab. The radiochemical purity of the purified product was determined by transient thin-layer chromatography (ITLC) using a citrate-sodium citrate buffer (pH 4.0) as the developing solvent.

[0060] 3.3 68 Radiochemical characterization of Ga-NOTA-aTrop-2-Fab Take 3.70 MBq respectively 68 Ga-NOTA-aTrop-2-Fab was added to 100 mL of phosphate-buffered saline (PBS) and 100 mL of fetal bovine serum (FBS), and incubated at room temperature for 1 h and 2 h, respectively. PBS and FBS samples were then directly subjected to SEC-HPLC for radiochemical purity identification, using the same elution conditions as described in section 3.1, i.e., purification using a PD-10 desalting column.

[0061] The prepared 68Ga-NOTA-aTrop-2-Fab was identified and characterized using analytical SEC-HPLC. SEC-HPLC results showed that the purity of NOTA-aTrop-2-Fab was greater than 99%. Figure 8 B). Measured by ITLC 68 The radiochemical purity of Ga-NOTA-aTrop-2-Fab is greater than 95%. Figure 10 ).

[0062] In summary, a successful preparation has been achieved. 68 The Ga-NOTA-aTrop-2-Fab labeling rate was approximately 89.1%. After purification of the labeled mixture using a PD-10 desalting column, the radiochemical purity was greater than 95%.

[0063] Example 4: Analysis of Trop-2 expression in cells Human non-small cell lung cancer cell lines NCI-H292 (Trop-2 positive) and A549 (Trop-2 negative) were cultured in complete DMEM medium (containing 10% FBS and 1% penicillin-streptomycin) and placed in a cell culture incubator at 37°C with 5% CO2.

[0064] NCI-H292 and A549 cells were cultured separately in culture dishes. When the cells reached 75%–80% confluence, total protein was extracted. Protein concentration was determined using a BCA kit, and then SDS-PAGE stacking and separating gels were prepared according to standard ratios. The prepared protein samples and markers were added to the wells, with a total protein loading of 40 μg per sample, and SDS-PAGE electrophoresis was performed. After electrophoresis, the membrane was transferred and blocked with PVDF. The primary antibody (rabbit anti-Trop-2 monoclonal antibody) was diluted 1:1000 and incubated overnight at 4°C with the PVDF membrane. After incubation with the secondary antibody on a shaker at room temperature for 1 h, development was performed. The grayscale values ​​of the bands were measured using ImageJ software. Trop-2 expression in human non-small cell lung cancer cell lines was assessed by Western blot. Figure 2 The results showed that the NCI-H292 cell line was positive for Trop-2 expression, while the A549 cell line was negative for it.

[0065] Example 5: 68 PET / CT imaging of a subcutaneous tumor model of Ga-NOTA-aTrop-2-scFv All animal studies were conducted in accordance with the guidelines of the Laboratory Animal Use and Management Committee of Tongji Medical College, Huazhong University of Science and Technology. Female Balb / c nude mice (4-5 weeks old) were purchased from Hubei Beiente Biotechnology Co., Ltd. and housed in an SPF-grade environment at the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology.

[0066] NCI-H292 and A549 cells in the logarithmic growth phase were collected, digested with routine trypsin, and the cell pellet was collected and counted. Subsequently, the cells were washed twice with pre-cooled PBS and centrifuged to collect the cell pellet. A cell resuspension was prepared at a PBS:Matrix gel ratio of 1:1 (v / v), and the cells were thoroughly pipetted. The cells were then subcutaneously inoculated into the right upper limb of mice near the axilla, with 5 × 10⁶ cells per mouse. 6 One NCI-H292 or A549 cell (125 μL / cell). Tumors were allowed to grow to 8-10 mm in diameter for subsequent experiments.

[0067] Nude mice bearing tumors (NCI-H292 in the experimental group and A549 in the control group) were injected via tail vein. 68 Following Ga-NOTA-aTrop-2-scFv (3.7-5.55 MBq, 100-150 μCi), PET / CT scans were performed under isoflurane inhalation anesthesia at 0.5, 1, 2, 3, and 4 hours. Each scan acquisition time was approximately 15 minutes. After the scan, PET and CT images were reconstructed after attenuation correction, and regions of interest (ROIs) were delineated using Amide software to calculate uptake values ​​(%ID / g).

[0068] In order to evaluate 68 In vivo targeting specificity of Ga-NOTA-aTrop-2-scFv, administered via tail vein injection in NCI-H292 tumor-bearing mice. 68 Static PET / CT scans were performed at 30 min, 1 h, 2 h, and 4 h after Ga-NOTA-aTrop-2-scFv administration. Figure 3 As shown, injection 68 Tumors became visible 30 minutes after Ga-NOTA-aTrop-2-scFv administration, with tumor uptake reaching 2.07% ID / g. Subsequently, tumor uptake remained stable, reaching 1.74% ID / g at 1 h and 1.62% ID / g at 2 h. At 4 h, the tumor outline remained very clear, with an uptake of 0.94% ID / g, indicating that the molecular probe has a certain affinity for the target and good retention within the tumor. The molecular probe was rapidly cleared from the bloodstream and liver. At 2 h, the images showed a high tumor / background ratio. ROI quantitative analysis showed that blood uptake decreased from 1.97% ID / g at 30 min to 0.90% ID / g, and liver uptake decreased from 1.33% ID / g to 0.84% ​​ID / g. At 4 h, the tumor / blood ratio was 1.94, the tumor / liver ratio was 2.32, and the tumor / muscle ratio was 8.24. Conversely, in the control group A549 tumor-bearing mice, no tumor contrast was observed at any time point. Figure 4 As shown, the tumor uptake values ​​at 1 h, 2 h, and 4 h were 0.81%ID / g, 0.79%ID / g, and 0.58%ID / g, respectively, all significantly lower than those in NCI-H292 tumor-bearing mice. In the imaging of both groups of tumor-bearing mice, significant radioactive concentrations were observed in the non-target organs, the kidneys and bladder, indicating that this molecular probe is primarily metabolized by the kidneys.

[0069] Example 6: Histological identification Tumor tissue was fixed with 4% paraformaldehyde, dehydrated with graded ethanol, cleared with xylene, infiltrated with paraffin and embedded to prepare paraffin tissue blocks, and then sectioned (5 μm) using a tissue sectioning machine.

[0070] H&E staining: Stain with hematoxylin (Harris) solution for 4 minutes, rinse with tap water for 2 minutes until no excess staining solution is removed from the slide. Differentiate the stained slide with 0.8% hydrochloric acid alcohol for 2 seconds, rinse with tap water, or use lithium carbonate solution to return to blue, then rinse with water for 2 minutes, stain with eosin solution (alcohol-soluble) for 20 seconds, no need to rinse with water, directly add 95% ethanol for 5 seconds to adjust the color, add anhydrous ethanol (1) and anhydrous ethanol (2) for 2 minutes to dehydrate, then use an environmentally friendly clearing agent to clear, seal, and examine under a microscope.

[0071] Trop-2 immunohistochemical staining: After antigen retrieval, endogenous peroxidase activity is blocked. The slides are then incubated with 10% serum (identical to the source of the secondary antibody) at 37°C for 30 minutes to reduce non-specific staining. The serum is discarded, and the antigen solution is diluted with 10% serum to prepare the primary antibody working solution. The diluted primary antibody working solution (anti-Trop-2 monoclonal antibody, 1:500 dilution) is then added, and the slides are incubated overnight at 4°C. The next day, the slides are placed at room temperature for 15 minutes (warming). They are then rinsed three times with TBST, followed by three immersion rinses, each for 3 minutes. The secondary antigen solution is diluted with TBST to prepare the secondary antibody working solution. The secondary antibody working solution is added to each slide, and the slides are incubated at 37°C for 45 minutes. The slides are rinsed three times with TBST, followed by three immersion rinses, each for 3 minutes. The TBST is discarded, and tyramine salt working solution is added to each slide, and the slides are incubated at room temperature in the dark for 10 minutes. The slides are rinsed three times with TBST, followed by three immersion rinses, each for 3 minutes. Then, freshly prepared tyramine working solution was added to each slide for staining. After counterstaining with hematoxylin, the slides were then blued back to their original color. The slides were then mounted and examined under a microscope.

[0072] Immunohistochemical analysis of tumor tissues was used to determine the expression of Trop-2 in tumor tissues of the NCI-H292 and A549 subcutaneous tumor models. Figure 5As shown, NCI-H292 tumor tissue exhibits extensive and significant Trop-2 expression, while A549 tumor tissue shows lower Trop-2 expression. This corresponds to the imaging results, suggesting that the molecular probe can effectively image tumors with different levels of Trop-2 expression.

[0073] Example 7: 68 PET / CT imaging of subcutaneous and orthotopic tumor models of Ga-NOTA-aTrop-2-Fab Human non-small cell lung cancer cell line NCI-H292 (Trop-2 positive) was cultured in complete DMEM medium (containing 10% FBS and 1% penicillin-streptomycin bispecific antibody) and placed in a cell culture incubator at 37°C with 5% CO2.

[0074] Obtaining a subcutaneous tumor model – NCI-H292 cells in the logarithmic growth phase were collected, digested with routine trypsin, and the cell pellet was collected and counted. Subsequently, the cells were washed twice with pre-cooled PBS and centrifuged to collect the cell pellet. A cell resuspension was prepared at a PBS:Matrix gel ratio of 1:1 (v / v), and the cells were thoroughly pipetted and then subcutaneously inoculated into the right upper limb of mice near the axilla, with 5 × 10⁶ cells per mouse. 6 Each tumor cell (100 μL / tumor) was collected. Tumors were allowed to grow to 8-10 mm in diameter for subsequent experiments.

[0075] Obtaining an orthotopic tumor model: NCI-H292 cells in the logarithmic growth phase were collected, digested with routine trypsin, and the cell pellet was collected and counted. Subsequently, the cells were washed twice with pre-cooled PBS and centrifuged to collect the cell pellet. A cell resuspension was prepared at a volume ratio of PBS:Matrix gel = 1:1. Healthy BALB / c-nu mice were continuously anesthetized using a small animal gas anesthesia system (2% isoflurane) in a laminar flow hood. The mice were fixed in a supine position, and the skin was routinely disinfected with povidone-iodine solution. A 1 cm incision was made approximately 1 cm above the lower edge of the left costal arch (between the fourth and fifth costal arches). The skin and muscle tissue were dissected layer by layer, and the cell suspension was slowly injected into the left lung along the incision to a depth of approximately 3 mm. The syringe plunger was gently pushed, and 0.05 mL (approximately 5 × 10⁶ cells) of the cell suspension was slowly injected. 5 (NCI-H292 cells). The cut mouse skin was sutured back together, and the mouse was placed in a right lateral decubitus position (with the wound facing upward) on a 37°C constant temperature heating plate until the mouse woke up. It was then returned to its original cage for continued feeding.

[0076] Tail vein injection of NCI-H292 tumor-bearing nude mice and NCI-H292 lung cancer orthotopic tumor models 68Following Ga-NOTA-aTrop-2-Fab (3.7-5.55 MBq, 100-150 μCi), PET / CT scans were performed under isoflurane inhalation anesthesia at 1, 2, and 4 hours. Each scan acquisition time was approximately 15 minutes. After the scan, PET and CT images were reconstructed after attenuation correction, and regions of interest (ROIs) were delineated using Amide software to calculate uptake values ​​(%ID / g).

[0077] NCI-H292 subcutaneously tumor-bearing mice were injected via tail vein. 68 Static PET / CT scans were performed at 1 h, 2 h, and 4 h after Ga-NOTA-aTrop-2-Fab implantation. Figure 6 As shown, NCI-H292 tumor-bearing mice were injected with... 68 One hour after exposure to Ga-NOTA-aTrop-2-Fab, the tumor became clearly visible, exhibiting a high tumor / background ratio. ROI delineation and semi-quantitative analysis of the PET images showed that tumor uptake continuously decreased over time, with uptake values ​​of 3.01±0.25 %ID / g, 2.90±0.16 %ID / g, and 2.39±0.61 %ID / g at 1 h, 2 h, and 4 h, respectively. The molecular probe was rapidly cleared from the bloodstream; at 1 h, the tumor / blood ratio was 0.53±0.01, the tumor / liver ratio was 0.67±0.01, and the tumor / muscle ratio was 5.48±1.29, decreasing over time, while the tumor / blood and tumor / liver ratios continuously increased. Significant radioactive concentrations were observed in the kidneys and bladder, suggesting that the molecular probe is primarily metabolized by the kidneys. The long retention time of this molecular probe at the tumor site and its rapid clearance from the background demonstrate its high affinity for the target and its promising potential for early post-injection imaging.

[0078] like Figure 7 As shown, in 68 Ga-NOTA-aTrop-2-scFv and 68 In a comparative analysis of PET imaging of the NCI-H292 subcutaneous tumor model using Ga-NOTA-aTrop-2-Fab, both molecular probes showed high uptake at the tumor site 1 hour after injection, and the tumor uptake gradually increased over time. 68 Ga-NOTA-aTrop-2-scFv has a shorter blood half-life, enabling faster clearance from the blood and non-target organs such as the liver, thus showing better tumor / background contrast at various time points. Semi-quantitative analysis of PET images showed that at various time points... 68The tumor / blood ratio and tumor / liver ratio of Ga-NOTA-aTrop-2-scFv were significantly higher than those of other strains. 68 Ga-NOTA-aTrop-2-Fab, tumor / muscle ratio slightly higher 68 Ga-NOTA-aTrop-2-Fab.

[0079] Example 8: Mice with NCI-H292 non-small cell lung cancer orthotopic tumor 68 Ga-NOTA-aTrop-2-scFv, 68 Comparison of Ga-NOTA-aTrop-2-Fab and 18F-FDG PET / CT Imaging Use respectively 68 Ga-NOTA-aTrop-2-scFv, 68 Ga-NOTA-aTrop-2-Fab and 18 F-FDG was used to image the NCI-H292 orthotopic tumor model in the same group to compare the detection capabilities of the three methods for lung cancer orthotopic tumors. 68 Ga-NOTA-aTrop-2-scFv and 68 Ga-NOTA-aTrop-2-Fab clearly visualized the location of the in situ lung cancer tumor 1 hour after injection, while 18 Due to high uptake in the circulating background, F-FDG is difficult to distinguish between tumors and normal organs. Autopsy of an in situ lung cancer tumor revealed that the tumor grew in the left lobe of the mouse lung, approximately 5 mm in diameter. The tumor location was similar to... 68 Ga-NOTA-aTrop-2-scFv and 68 The results are consistent with those shown in Ga-NOTA-aTrop-2-Fab PET imaging, indicating that... 68 Ga-NOTA-aTrop-2-scFv and 68 Both Ga-NOTA-aTrop-2-Fab can clearly detect lesions as small as 5 mm.

[0080] Table 1. Antibody information used in this invention.

[0081] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A molecular probe targeting Trop-2, characterized in that: It is a conjugate formed by the following (a) and (b) through a bifunctional chelating agent: (a) An antibody fragment capable of specifically binding to Trop-2; said antibody fragment is scFv or Fab; (b) Radionuclides; in: The scFv is an scFv having the amino acid sequence shown in SEQ ID NO: 10; The Fab is a Fab composed of the light chain shown in SEQ ID NO: 8 and the heavy chain shown in SEQ ID NO: 9; The bifunctional chelating agent is p-SCN-Bn-NOTA; The radioactive nuclide is 68 Ga.

2. A method for preparing a Trop-2-targeting molecular probe as described in claim 1, characterized in that, Includes the following steps: The antibody fragment is coupled with a bifunctional chelating agent to generate a bifunctional chelating agent-coupled antibody fragment. The antibody fragment conjugated with the bifunctional chelating agent is reacted with a radionuclide to obtain the molecular probe targeting Trop-2.

3. The method for preparing the Trop-2-targeting molecular probe according to claim 2, characterized in that: The molar ratio of antibody fragment to bifunctional chelator is 1:

30.

4. The method for preparing the Trop-2-targeting molecular probe according to claim 2, characterized in that: The pH conditions for the reaction of the antibody fragment conjugated with the bifunctional chelator with the radionuclide are 4.0–4.

5.

5. The method for preparing the Trop-2-targeting molecular probe according to claim 2, characterized in that: The coupling reaction was carried out in a 0.1 M Na2CO3 buffer solution with a pH of 8.5–9.

0.

6. The use of the Trop-2-targeting molecular probe of claim 1 in the preparation of PET / CT imaging reagents for the diagnosis of non-small cell lung cancer.

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