A theranostic polypeptide nuclide probe composition targeting TNFR2 and application thereof

Abstract

The application belongs to the field of nuclear medicine and molecular imaging technology, and discloses a kind of targeted TNFR2 diagnosis and treatment integrated polypeptide nuclide probe composition and its application.The composition takes TNFR2 targeting polypeptide P20 (amino acid sequence SHWGGNVRGTQT) and its full D type D-P20 as the skeleton, synthesizes [ 64 Cu]Cu-NOTA-P20、 68 Ga]Ga-DOTA-D-P20 and [¹ 77 Lu]Lu-DOTA-D-P20, constructs diagnostic and therapeutic probes.Diagnostic probes select patients, monitor and predict efficacy, and therapeutic probes target and kill TNFR2 positive tumor cells and eliminate Treg in TME.Animal experiments show that the probe has good radiochemical purity, stability, TNFR2 specificity;And in the colon cancer model, the imaging is clear, the tumor growth is significantly inhibited, and the precise diagnosis and treatment of "what you see is what you get" is realized.

Description

Technical Field

[0001] This invention relates to the field of nuclear medicine and molecular imaging, specifically to a method based on the TNFR2 target. 68 Ga / 177 Molecular probes for lu-labeled peptides, particularly a therapeutic probe for simultaneous use in tumor PET imaging and radiotherapy, and its preparation method and application. Background Technology

[0002] Tumor necrosis factor receptor 2 (TNFR2) is a member of the tumor necrosis factor receptor superfamily. Unlike TNFR1, TNFR2 does not contain a death domain, and its activation mainly mediates cell survival and immune regulation-related signaling pathways. TNFR2 is expressed at low levels in normal tissues, but is abnormally highly expressed on the surface of various tumor cells (colorectal cancer, non-small cell lung cancer, ovarian cancer, breast cancer, etc.), participating in tumor-associated inflammatory responses and tumor progression. Simultaneously, it is also highly expressed on regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment, participating in the maintenance of immunosuppressive cell function and thus promoting tumor immune escape. Therefore, this unique "dual-targeting" expression characteristic makes TNFR2 an ideal therapeutic target with both tumor-targeting and immune microenvironment-regulating functions. Molecular intervention strategies targeting TNFR2 hold promise for enhancing the body's anti-tumor immune response by regulating immunosuppression, thereby exerting anti-tumor effects.

[0003] Previous studies have reported on the antagonistic peptide P20 targeting TNFR2, which can specifically bind to TNFR2 and regulate related immune functions. However, existing P20 peptides are usually composed of natural L-amino acids, which are easily degraded by proteases in vivo, have poor stability, and short blood circulation time, limiting their in vivo application, especially in molecular imaging and radionuclide therapy. Furthermore, current research mainly focuses on its biological function, lacking structural optimization and therapeutic integration with radionuclide labeling. Therefore, it is necessary to optimize the structure of existing P20 peptides by introducing all-D amino acids to improve their resistance to enzymatic degradation and in vivo stability, and further introduce chelating groups to construct molecular probe systems that can be used for radionuclide labeling.

[0004] Based on the above modification strategy, a fully D-type DOTA-P20 radiolabeled probe was constructed. In diagnosis, it can achieve specific imaging of TNFR2-positive tumor tissues through in vivo tracing of TNFR2 expression, thereby improving the sensitivity and specificity of tumor detection. In treatment, it carries a therapeutic radionuclide. 177Lu's probes can not only achieve targeted radioactive killing of TNFR2-positive tumor cells, but also, by combining the functional characteristics of TNFR2 in the tumor immune microenvironment, regulate the local immunosuppressive state, thereby enhancing the overall anti-tumor effect.

[0005] With the continuous development of radionuclide labeling technology and peptide molecular design strategies, therapeutic molecular probes based on TNFR2 targets have promising applications in precision tumor diagnosis, treatment efficacy evaluation, and personalized treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a diagnostic and therapeutic polypeptide nuclide probe composition targeting TNFR2, which solves the technical problems of existing TNFR2-targeting drugs lacking supporting diagnostic tools and making it difficult to achieve patient screening and efficacy monitoring.

[0007] This invention is achieved through the following technical solution: 1. TNFR2-specific antagonistic peptide P20 (SEQ: SHWGGNVRGTQT) and D-P20 were selected as target molecules; 2. Transform the P20 sequence from L-type to D-type P20; 3. Connecting P20 via a Nota chelating agent; Connecting all-D type P20 via a DOTA chelating agent; 4. Use them separately 68 Ga and 177 Lu markers are used to build an integrated diagnosis and treatment system; The following probes are formed: [ 64 Cu-NOTA-P20 (for PET imaging) [ 68 Ga-DOTA-D-P20 (for PET imaging) [ 177 Lu-DOTA-D-P20 (for radiotherapy) Beneficial effects

[0008] Compared with the prior art, the present invention has the following beneficial effects: For the first time, integrated diagnosis and treatment of TNFR2 targeting has been achieved: "diagnosis-treatment" paired probes based on the same polypeptide backbone realize the precision medicine concept of "what is seen is what is treated".

[0009] Filling the technological gap in peptide-based TNFR2-targeted PET drugs: Excellent diagnostic performance: Small animal PET / CT imaging shows, [ 64Cu-NOTA-P20 specifically accumulated in TNFR2-positive tumors, with the tumor / muscle ratio peaking 4-7 hours post-injection, showing a significant difference compared to the TNFR2 knockout group (P < 0.01). 68 Ga-DOTA-D-P20 specifically aggregated in TNFR2-positive tumors and remained stable during uptake within 1 hour. The difference was significant compared to the TNFR2 knockout group (P < 0.01).

[0010] It exhibits good in vitro serum stability: In in vitro serum stability experiments, [ 68 When Ga]Ga-DOTA-D-P20 was incubated in serum at 37℃ with valence adjustment, the radiochemical purity of the probe remained above 70% after 1 h and 2 h, and no obvious degradation products were observed, indicating that it has excellent in vitro stability.

[0011] Significant therapeutic effects: [ 177 Lu-DOTA-D-P20 significantly inhibited tumor growth and reduced tumor tissue uptake in a colon cancer model. 18 F]FDG kills tumors and reduces tumor metabolism.

[0012] Good safety profile: No significant weight loss or immune-related adverse events were observed during treatment.

[0013] Figure 1 Differential analysis of TNFR2 expression between common tumors and normal tissues showed that the expression level in tumor tissues of colorectal cancer was significantly higher than that in normal tissues (P<0.01).

[0014] Figure 2 Probe quality control and synthesis: A. [ 64 Synthesis of Cu-NOTA-P20; B. [ 68 Synthesis of Ga-DOTA-D-P20; C. [ 177 Synthesis of Lu-DOTA-D-P20; D.[ 68 Ga]Ga-DOTA-D-P20, [ 177 Lu]Radio-TLC of Lu-DOTA-D-P20; E.[ 64 Cu]Cu-NOTA-P20, [ 68 In vitro serum stability of Ga]Ga-DOTA-D-P20, [177Lu]Lu-DOTA-D-P20.

[0015] Figure 3 . [ 64Cu]Cu-NOTA-P20 PET / CT imaging in MC38 tumor-bearing mice. A. [ 64 PET images of Cu-NOTA-P20 at different time points in the MC38-WT and MC38-KO groups; B. 64 Quantitative analysis of the Cu-NOTA-P20 tumor / muscle ratio. C. [ 64 Cu]Cu-NOTA-P20 PET / CT imaging in tumor-bearing mice in the CT26-WT and CT26-KO groups. D. [ 64 Quantitative analysis of the Cu-NOTA-P20 tumor / muscle ratio.

[0016] Figure 4 . [ 64 Biodistribution and specificity validation of Cu-NOTA-P20. A. MIP 3D images showing spleen and tumor uptake; BC. For injection [ 64 PET / CT images of CT26 and MC38 tumor-bearing mice after Cu-NOTA-P20; DE. represents the quantitative analysis results of tracer uptake in different tissues at different time points.

[0017] Figure 5 . [ 68 PET / CT imaging of Ga-DOTA-D-P20 in MC38 tumor-bearing mice. A. [ 68 Ga]Ga-DOTA-D-P20 dynamic PET images at 1 hour in the MC38-WT and MC38-KO groups; B. Quantitative SUV uptake by probe at different time points in the MC38-WT and MC38-KO groups. C. [ 68 Quantitative analysis of the tumor / muscle ratio using Ga]Ga-DOTA-D-P20.

[0018] Figure 6 . [ 177 The antitumor therapeutic effect of Lu-DOTA-P20. A. Schematic diagram of experimental protocol; B. Image of ex vivo tumor; C. Tumor weight curve; D. Tumor growth curve; E. Survival curve; F. Evaluation of 18F-FDG efficacy; G. Quantitative analysis of 18F-FDG. Detailed Implementation

[0019] Example 1: Diagnostic components [ 64 Preparation and characterization of Cu-NOTA-P20: The NOTA-P20 precursor was prepared using the Fmoc solid-phase synthesis method, and a DOTA chelating group was introduced at the N-terminus. After HPLC purification, the purity was >95%. 400 μg of NOTA-P20 was dissolved in 400 μL of sodium acetate buffer (0.5 M, pH 5.5), and […]. 64 Cu]Cu(OAc)2 (approximately 333 MBq) was reacted at room temperature for 15 minutes. The labeling rate was monitored by radioactive TLC, and the radiochemical purity was determined by HPLC. The results showed a labeling rate >99% and a radiochemical purity >99%. After incubation at 37°C for 12 hours in PBS and fetal bovine serum, the radiochemical purity remained >90%, indicating good in vitro stability.

[0020] Example 2: Diagnostic components [ 64 Cu]Cu-NOTA-P20 PET / CT Imaging: Constructing MC38-WT (TNFR2) + ) and MC38-KO (TNFR2) - C57BL / 6 tumor-bearing mouse model. When the tumor diameter reaches 5-6 mm, […]. 64 Cu-NOTA-P20 (12.70-20.77 MBq) was used for static PET / CT scans (Mediso, 10 minutes / scan) at 1, 4, 7, and 12 hours post-injection. Results showed that tumors in the MC38-WT group exhibited clear contrast enhancement 4-7 hours post-injection, with the tumor / muscle ratio (T / M) reaching its peak; no significant uptake was observed in the MC38-KO group. Splenic uptake was significantly positively correlated with tumor uptake (Spearman correlation coefficient r=0.82, P<0.01), confirming that the probe could simultaneously target TNFR2-positive Tregs in both tumor cells and the spleen.

[0021] Example 3: Diagnostic components [ 68 Preparation and characterization of Ga-DOTA-D-P20: The DOTA-D-P20 precursor was prepared using the Fmoc solid-phase synthesis method, with a DOTA chelating group introduced at the N-terminus. It was purified by HPLC to a purity >95%. 200 μg of DOTA-D-P20 was dissolved in sodium acetate buffer (0.5 M, pH 5.0), and […]. 68 [Ga]GaCl3 (370-555 MBq), reacted at 80°C for 10 minutes. Radio-TLC showed that the product had a radiochemical purity >95% and a specific activity >37 GBq / μmol.

[0022] Example 4: Diagnostic components [ 68 PET / CT imaging of Ga-DOTA-D-P20: Constructing MC38-WT (TNFR2) + ) and MC38-KO (TNFR2⁻) tumor-bearing C57BL / 6 mouse models. When the tumor diameter reached 5-6 mm, [tail vein injection] 68 Ga-DOTA-D-P20 (12.70-20.77 MBq) was administered via dynamic PET / CT scan (Mediso, 60 minutes / scan) post-injection. Results showed that tumors in the MC38-WT group exhibited clear contrast 1 hour post-injection, with the tumor / muscle ratio (T / M) reaching its peak. Tumor uptake was reduced in the MC38-KO group compared to the MC38-WT group. P <0.05), confirming that the probe can target TNFR2-positive colon cancer cells.

[0023] Example 5: Therapeutic components [ 177 Preparation of Lu]Lu-DOTA-D-P20: The DOTA-P20 precursor was prepared using the Fmoc solid-phase synthesis method, with a DOTA chelating group introduced at the N-terminus. After HPLC purification, the purity was >95%. 200 μg of DOTA-D-P20 was dissolved in sodium acetate buffer (0.5 M, pH 4.0), and […]. 177 Lu]LuCl3 (185-370 MBq), reacted at 95°C for 20 minutes. Radio-TLC showed that the product had a radiochemical purity >95% and a specific activity >37 GBq / μmol.

[0024] Example 6: [ 177 The anti-tumor therapeutic effects of Lu-DOTA-D-P20: MC38 tumor-bearing C57bl / 6 mouse models were established (n=5 / group). Drug administration began when the tumor volume reached 50 mm³, and was repeated seven days later: PBS control group (no treatment), […]. 177 Lu]Lu-DOTA-D-P20 low-dose treatment group (18.5 MBq / animal, administered in two divided doses), [ 177 Lu]Lu-DOTA-D-P20 medium-dose treatment group (37 MBq / animal) and [ 177 Lu]Lu-DOTA-D-P20 high-dose treatment group (55.5 MBq / animal). Tumor volume and body weight were continuously monitored. Results showed that the medium-dose and high-dose groups significantly inhibited tumor growth compared with the PBS group (P<0.001), and were superior to the low-dose group. No significant weight loss was observed during treatment.

Claims

1. A therapeutically integrated polypeptide radionuclide probe composition targeting TNFR2, characterized in that, The composition comprises: (a) a diagnostic component: a PET tracer consisting of a TNFR2-targeting peptide labeled with a diagnostic radionuclide via a bifunctional chelator. 64 The structure of Cu]Cu-NOTA-P20 is as follows: tracer 68 The structure of Ga-DOTA-D-P20 is as follows: (b) Therapeutic component: a radiopharmaceutical formed by labeling the TNFR2-targeting peptide with a therapeutic radionuclide via a bifunctional chelator. 177 The structure of Lu-DOTA-D-P20 is as follows: The amino acid sequence of the TNFR2-targeting peptide P20 is shown in SEQ ID NO.3: SHWGGNVRGTQT; the TNFR2-targeting peptide D-P20 is a peptide obtained by replacing all L-type amino acids in the sequence SHWGGNVRGTQT shown in SEQ ID NO.3 with their corresponding D-type enantiomers; the bifunctional chelating agent is selected from NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or its derivatives; the diagnostic radionuclide is selected from... 64 Cu or 68 Ga; the therapeutic radionuclide is selected from... 177 Lu.

2. The composition according to claim 1, characterized in that, The diagnostic components are [ 64 Cu]Cu-NOTA-P20 or [ 68 Ga]Ga-NOTA-P20, the therapeutic component is [ 177 Lu]Lu-DOTA-P20.

3. The composition according to claim 1 or 2, characterized in that, The diagnostic component has a radiochemical purity ≥95% and a specific activity ≥37 GBq / μmol; the therapeutic component has a radiochemical purity ≥95% and a specific activity ≥37 GBq / μmol.

4. The use of the composition according to any one of claims 1-3 in the preparation of TNFR2-positive tumor diagnostic and therapeutic drugs, characterized in that, The diagnostic component is used to: (i) screen patients with positive TNFR2 expression; (ii) assess the heterogeneity of tumor TNFR2 expression levels; (iii) predict patient responsiveness to the treatment component; and (iv) monitor the therapeutic response during treatment.

5. The application according to claim 4, characterized in that, The therapeutic component is used to: (i) target and kill TNFR2-positive tumor cells; (ii) eliminate TNFR2-positive regulatory T cells (Tregs) in the tumor microenvironment; and (iii) reverse the tumor immunosuppressive microenvironment.

6. The application according to claim 4, characterized in that, The diagnostic and therapeutic components share the same polypeptide backbone, enabling an integrated diagnostic and therapeutic strategy of "what is seen is what is treated".

7. A TNFR2-targeted integrated diagnostic and therapeutic method, characterized in that, Includes the following steps: (a) administering a diagnostically effective amount of the diagnostic component of claim 1 to a subject and performing PET / CT or PET / MRI imaging; (b) Assess the tumor TNFR2 expression level of the subjects based on the imaging results and determine the TNFR2 positive expression threshold; (c) Administering a therapeutically effective amount of the therapeutic component of claim 1 to a subject who is positive for TNFR2; (d) After treatment, the diagnostic component is administered again to assess the efficacy, and the subsequent treatment plan is adjusted based on the quantitative indicators of PET.

8. The method according to claim 7, characterized in that, The dosing interval between the diagnostic component and the therapeutic component is 1-7 days; the single dose range of the therapeutic component is 37-370 MBq, and it can be administered once or in multiple doses.

9. The method according to claim 7, characterized in that, The PET quantitative indicators include SUVmax, SUVmean, tumor / muscle ratio (T / M), and tumor / blood ratio (T / B); the TNFR2 positive expression threshold is tumor SUVmax ≥ 2.5 or T / M ratio ≥ 1.5.

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