A pet imaging agent precursor targeting pd-l1 of tumor, the imaging agent and the synthesis method and application thereof

Abstract

The application discloses a PET imaging agent labeling precursor targeting tumor PD-L1, an imaging agent and a synthesis method and application thereof, and a structure of the PET imaging agent labeling precursor is shown as formula I. The application is scientific in design, ingenious in conception, and creatively uses theoretical calculation to explore the action mode of the molecule and the target protein, and according to the nature of the molecule itself, a suitable linker and a chelating fragment are introduced to design a novel tumor PD-L1 targeting macrocyclic peptide radionuclide precursor. The binding mode of the precursor is explored through theoretical calculation, and the rationality of the design is verified. The precursor compound can be labeled with Ga to obtain a compound of formula II, which has good stability, excellent pharmacokinetics, good specificity in combination with tumor PD-L1, high tumor uptake and high tumor / muscle uptake ratio, and has a good clinical application prospect. 68 Ga labeling can obtain the compound of formula II, which has good stability, excellent pharmacokinetics, good specificity in combination with tumor PD-L1, high tumor uptake and high tumor / muscle uptake ratio, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of nuclear medicine and radiopharmaceuticals, specifically relating to a class of PET imaging agent precursors, imaging agents, their synthesis methods, and applications that target tumor PD-L1. Background Technology

[0002] In recent years, immunotherapy has become the fourth major cancer treatment technology after surgery, radiotherapy, and chemotherapy. Novel cancer immunotherapies, including tumor vaccines, CAR-T therapy, and immune checkpoint therapy, have achieved a series of major breakthroughs, bringing hope for a cure for cancer. It was for their outstanding contributions to cancer immunotherapy that American scholar James P. Alison and Japanese scholar Tasuku Honjo were awarded the 2018 Nobel Prize in Physiology or Medicine (Front Oncol, 2019, 9:248). Undoubtedly, cancer immunology has become one of the most noteworthy scientific fields globally.

[0003] Immune checkpoints consist of a series of co-stimulatory and co-inhibitory molecules, playing a crucial regulatory role in the body's immune system. As a core member of the immune checkpoint family, PD-1 is a member of the CD28 immunoglobulin (Ig) superfamily and contains two ligands, PD-L1 and PD-L2, with PD-L1 being the primary ligand. The PD-1 / PD-L1 signaling pathway not only maintains normal immune tolerance but also plays a vital role in tumor immune escape as an important immune checkpoint molecule. In the tumor microenvironment, tumor cells and related antigen-presenting cells overexpress PD-L1, continuously activating the PD-1 / PD-L1 signaling pathway, inhibiting tumor antigen-specific T cell activation, suppressing T cell receptor signal transduction, downregulating the expression of anti-apoptotic molecules and inflammatory factors, thereby mediating tumor immune escape (N Engl J Med. 2016, 375(18):1767-1778.).

[0004] Currently, several PD-1 / PD-L1 monoclonal antibody drugs, including Keytruda, Opdivo, Imfinzi, Tecentriq, and Bavencio, are available globally and are widely used in the clinical treatment of various solid tumors and hematological cancers, such as malignant melanoma, non-small cell lung cancer, gastric cancer, liver cancer, kidney cancer, and bladder cancer. Their clinical efficacy is significant, and their indications are continuously expanding. Meanwhile, clinical trials of PD-1 / PD-L1 monoclonal antibody drugs in combination with other drugs for the treatment of various malignant tumors are actively underway. However, these immune checkpoint monoclonal antibodies also face significant scientific bottlenecks, most notably low patient response rates (~20%) and the development of acquired resistance in some patients (Lancet Oncol. 2019, 20(10):1395-1408; Cancer Cell, 2020, 37(4):443-455).

[0005] Clinical studies have shown that the therapeutic efficacy and prognosis of PD-1 / PD-L1 monoclonal antibodies for various tumors are related to the patient's PD-L1 expression level (Int J Cancer. 2020, 147(1): 116-127). Therefore, assessing the PD-L1 expression level in patients is of great significance in determining whether they are suitable for immune checkpoint blockade therapy.

[0006] Immunohistochemistry (IHC) remains the "gold standard" for detecting PD-L1 levels in tumor tissue. However, IHC has several limitations in practical clinical applications: it is an invasive procedure, and tumor PD-L1 expression levels change dynamically during treatment, making it difficult to perform multiple samplings for dynamic monitoring. Tumor heterogeneity arises from different locations, metastases, and disease stages; biopsies only represent a portion of the tumor and cannot reflect the full extent of PD-L1 expression, especially in patients with multiple metastases. Furthermore, the release of immune antigens from tumor tissue after radiotherapy and chemotherapy can alter the immune status, affecting PD-L1 test results. The types of antibodies, staining methods, and lack of standardized PD-L1 cutoff values ​​used in different clinical laboratories also influence PD-L1 test results. In addition, systemic variability in immunohistochemical testing can lead to false negatives. Therefore, current PD-L1 testing in clinical evaluation still suffers from incompleteness and poor timeliness.

[0007] Positron emission tomography (PET) is one of the most advanced in vivo imaging techniques, enabling in vivo, non-invasive, real-time, and quantitative assessment of target expression levels. PET imaging can dynamically and quantitatively characterize PD-L1 expression in primary and metastatic tumors, allowing for early screening of patients who may benefit from immune checkpoint blockade therapy. Simultaneously, it can monitor PD-L1 expression and its dynamic changes before and after treatment, assessing treatment efficacy and potential toxic side effects. Molecular probes are crucial for PET molecular imaging; developing radioactive molecular probes targeting PD-L1 is a key focus of current research in nuclear medicine and radiopharmaceuticals, and a critical technical challenge that researchers in this field urgently need to address.

[0008] Currently, molecular probes targeting PD-L1 mainly utilize medium- to long-half-life radionuclide-labeled biomolecules, especially monoclonal antibodies. These imaging agents have large molecular weights, long in vivo circulation times, and high radiation doses; they also provide clear imaging of highly vascularized organs and have low target / background ratios. Conversely, research on small-molecule PD-L1 tracers is relatively limited. Compared to monoclonal antibodies, small molecules offer advantages such as flexible modification potential, controllable pharmacokinetic properties, non-immunogenicity, and low production costs. Similarly, radioactive small-molecule drugs offer advantages over monoclonal antibodies, including a reasonable half-life, stronger tumor penetration, and the ability to provide accurate assessment results in a shorter time.

[0009] In addition, in recent years, combining diagnostic imaging and therapeutic radionuclides with targeted molecules to achieve integrated diagnosis and treatment has become a future trend in radiopharmaceutical development. 68 Ga / 177 Lu-DOTATATE has been approved by the FDA for the diagnosis and treatment of neuroendocrine tumors, and for the diagnosis and treatment of prostate cancer. 68 Ga / 177 Lu-PSMA-617 is currently in clinical trials. At present, many common clinical tumors, including non-small cell lung cancer, breast cancer, and melanoma, show elevated PD-L1 expression levels. Developing radionuclide-labeled therapeutic drugs targeting tumor PD-L1 could enable radionuclide-targeted therapy for various tumors. Currently, there are no reported radionuclide therapeutic drugs targeting tumor PD-L1. Summary of the Invention

[0010] Purpose of the invention: To address the deficiencies of existing technologies, this invention provides a PET imaging agent precursor targeting tumor PD-L1, an imaging agent, its synthesis method, and its application. The resulting imaging agent can specifically bind to tumor PD-L1, exhibiting excellent imaging effects, and is worthy of further promotion and application.

[0011] Technical solution:

[0012] In a first aspect, the present invention provides a PET imaging agent labeling precursor targeting tumor PD-L1, the structure of which is shown in Formula I:

[0013]

[0014] Secondly, the present invention provides a PET imaging agent targeting tumor PD-L1, wherein the PET imaging agent is a radionuclide-labeled PET imaging agent precursor targeting tumor PD-L1.

[0015] Furthermore, the radionuclide is a diagnostic radionuclide or a therapeutic radionuclide; preferably, the diagnostic radionuclide is selected from... 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I and 124 At least one of I; preferably, the therapeutic radionuclide is selected from... 177 Lu、 125 I, 131 I, 211 At、 111 In、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

[0016] As one embodiment, the structure of the PET developer is shown in Formula II:

[0017]

[0018] Thirdly, the present invention provides a method for synthesizing the PET imaging agent labeling precursor targeting tumor PD-L1, comprising using the required amino acids of Cyclo[SAc-F-Phe(n-Me)-Nle(n-Me)-(D-trans-Pro-OH)-NV-Phe(n-Me)-Y-(D-pipec olicacid)-WYL-C]-Lys and DOTA as raw materials, and synthesizing it using the peptide Fmoc synthesis method.

[0019] Fourthly, the present invention provides a method for synthesizing the PET imaging agent targeting tumor PD-L1, comprising reacting the PET imaging agent labeled precursor for targeting tumor PD-L1 as described in claim 1 with a salt of a radionuclide to generate the PET imaging agent.

[0020] As one implementation scheme, the salt of the radionuclide is 68 The reaction system for Ga salts has a pH of 3-5, a reaction temperature of 80-120℃, and a reaction time of 5-15 min. The reaction formula is shown below:

[0021]

[0022] A buffer solution is added to the reaction system to adjust the pH value. The buffer solution is selected from the sodium acetate / acetic acid system, ammonium acetate / acetic acid system, sodium acetate / hydrochloric acid system, HEPES system, or Tris system.

[0023] Furthermore, the synthesis method also includes a purification step, in which the reaction solution after the reaction is completed is purified using a C18 column.

[0024] Fifthly, the present invention provides the aforementioned PET imaging agent precursor targeting tumor PD-L1 and the application of the aforementioned PET imaging agent targeting tumor PD-L1 in the preparation of immunotherapeutic kits.

[0025] In a sixth aspect, the present invention provides the use of the aforementioned PD-L1-targeting PET imaging agent precursor, and the aforementioned PD-L1-targeting PET imaging agent in the preparation of diagnostic reagents / drugs and / or therapeutic drugs for PD-L1-highly expressed tumors.

[0026] Furthermore, the PD-L1 highly expressed tumors include any one or more of non-small cell lung cancer, melanoma, and breast cancer.

[0027] The applicant, through computer-aided drug design and extensive calculations, designed a precursor for a PD-L1-targeting macrocyclic peptide nucleoside probe. First, CADD simulation technology was used to study the PD-L1-targeting cyclic peptide composed of 13 amino acid residues, where the glycine fragment faces the solvent region, making it a suitable linker binding site. Therefore, based on the aforementioned PD-L1-targeting cyclic peptide parent structure, the applicant introduced a butyramine fragment as a linker to the glycine fragment for connecting the metal chelate fragment. The applicant further investigated the binding mechanism of the newly designed molecule to the target protein (e.g., Figure 1 As shown in the figure, it was found that the added butylamine fragment was completely oriented towards the solvent region and did not affect the binding of the cyclic peptide to the protein. Therefore, the applicant designed and synthesized a novel labeled precursor for a PD-L1 macrocyclic peptide nuclide probe using computer-aided drug design technology.

[0028] Beneficial Effects: This invention is scientifically designed and ingeniously conceived. It creatively employs theoretical calculations to explore the interaction mode between the molecule and the target protein, and introduces suitable linkers and chelating fragments based on the molecule's inherent properties to design a novel tumor-targeting PD-L1 macrocyclic peptide precursor. The binding mode of the precursor is further explored through theoretical calculations, validating the rationality of the design. This precursor compound, used... 68 The Ga-labeled compound of formula II exhibits good stability, excellent pharmacokinetics, and high specificity for binding to tumor PD-L1. It also shows high tumor uptake and a high tumor-to-muscle uptake ratio, indicating promising clinical application prospects. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the interaction pattern between the macrocyclic peptide precursor and the target protein PD-L1.

[0030] Figure 2 The mass spectrum of the compound of formula I obtained in Example 2;

[0031] Figure 3 Affinity map of the binding between the PD-L1 macrocyclic peptide precursor and the target protein;

[0032] Figure 4 Figure A shows the Radio-iTLC detection results of Example 4. Figure B shows the labeling rate of the PD-L1 macrocyclic peptide nuclide probe and the radiochemical purity of the PD-L1 macrocyclic peptide nuclide probe after purification.

[0033] Figure 5 200 μCi was injected into Balb / c nude mice bearing the melanoma A375-PD-L1 model. 68 Whole-body Micro-PET / CT MIP images after Ga-DOTA-G1 at 30 min, 1 h, and 2 h respectively;

[0034] Figure 6 200 μCi was injected into Balb / c nude mice bearing the A375 melanoma model. 68 Whole-body Micro-PET / CT MPI images taken at 30 min, 1 h, and 2 h after Ga-DOTA-G1. Detailed Implementation

[0035] To further illustrate the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. These embodiments are purely illustrative and are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

[0036] This invention provides a PET molecular probe targeting PD-L1, its preparation method, and its application, aiming to solve the problem of providing detection probes for early non-invasive screening of PD-L1 patients.

[0037] Example 1

[0038] The binding mode of PD-L1 cyclic peptide 71 to the target protein was studied using computer simulation technology (CADD). The binding sites in 71 that can be linked to chelate fragments were revealed, providing a theoretical basis and technical guidance for the design and structural optimization of the small molecule precursor G.

[0039] Example 2

[0040] This embodiment discloses a method for synthesizing a macrocyclic peptide targeting PD-L1, specifically as follows:

[0041] Using Fmoc-amine resin and following the Fmoc solid-phase synthesis strategy, Fmco-lysine, S-triphenylmethyl-L-cysteine, leucine, tyrosine, tryptophan, D-piperidin-2-carboxylic acid, tyrosine, N-methylphenylalanine, valine, protected aspartic acid, trans-D-hydroxyproline, N-methyl-ortholeucine, N-methylphenylalanine, phenylalanine, and chloroacetic acid were coupled sequentially. After coupling, the Fmco protecting group was removed, followed by coupling with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester (DOTA). After completing the coupling of all amino acids and the DOTA fragment, the peptide was cleaved from the resin to obtain a crude linear peptide, which was then purified by preparative liquid chromatography to obtain a pure linear peptide. The linear peptide was cyclized under weakly basic conditions to obtain the macrocyclic peptide (the compound shown in Formula I), and its mass spectrum is shown below. Figure 2 As shown.

[0042] Example 3

[0043] Binding affinity assay for PD-L1 macrocyclic peptide precursor:

[0044] The binding affinity of the target PD-L1 macrocyclic peptide precursor was determined using the SPR assay, with concentration gradients of 3 nM, 6 nM, 12 nM, 24 nM, and 48 nM. The results are attached. Figure 3 As shown, its K D The value is 9.98 nM.

[0045] Example 4

[0046] This embodiment discloses the synthesis of PD-L1 macrocyclic peptide nuclide probe compounds, specifically as follows:

[0047] Prepare a distilled aqueous solution (1 mg / mL) of the PD-L1 macrocyclic peptide precursor. Place 37 μL of the precursor solution into a reaction flask, add 400 μL of 0.25 M sodium acetate and 1 mL of freshly rinsed solution. 68 Ga rinsing solution ( 68 Ga was eluted in 0.05 M hydrochloric acid solution. The pH of the reaction solution was approximately 4.0. After mixing, the reaction was carried out at 100°C for 10 min. After the reaction was completed, the product was purified using a C18 column. Radio-iTLC analysis showed that the radiochemical purity of the product was greater than 99%. The radiolabeling reaction equation is as follows:

[0048]

[0049] Example 5

[0050] This embodiment discloses the compound of formula II of the present invention. 68 In vivo imaging test of Ga-DOTA-G-1:

[0051] 1. Micro-PET / CT imaging of nude mice bearing melanoma A375-PD-L1 model

[0052] In nude mice with an A375-PD-L1 xenograft tumor model, tumor tissue was expanded to 100-300 mm. 3 Administer via tail vein injection at the appropriate time. 68 Ga-DOTA-G-1 (200 μCi / 1 μg antibody / animal) was used for static scanning at different time points (30, 60, and 120 min) after injection, with clear tumor visualization. After the scan, iterative reconstruction (OSEM 3D) scan data was used, and ASIProVM software was used to delineate two tumor sites and other tissues as regions of interest (ROIs) to calculate the radioactive uptake %ID / g of each ROI.

[0053] 2. Micro-PET / CT imaging of nude mice bearing melanoma A375 model

[0054] In nude mice with an A375 xenograft tumor model, tumor tissue was expanded to 100-300 mm. 3 Administer via tail vein injection at the appropriate time. 68 Ga-DOTA-G-1 (200 μCi / 1 μg antibody / animal) was used for static scanning at different time points (30, 60, and 120 min) after injection, with clear tumor visualization. After the scan, iterative reconstruction (OSEM 3D) scan data was used, and ASIProVM software was used to delineate two tumor sites and other tissues as regions of interest (ROIs) to calculate the radioactive uptake %ID / g of each ROI.

[0055] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention used to illustrate the technical solutions of the present invention, and are not intended to limit the invention, nor are they intended to limit the patent scope of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. That is to say, any changes or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but whose technical problems are still consistent with the present invention, should be included within the protection scope of the present invention. In addition, the direct or indirect application of the technical solutions of the present invention to other related technical fields are similarly included within the patent protection scope of the present invention.

Claims

1. A PET imaging agent labeling precursor targeting tumor PD-L1, the structure of which is shown in Formula I: 。 2. A PET imaging agent targeting tumor PD-L1, characterized in that, The PET imaging agent is a radionuclide-labeled PET imaging agent precursor for targeting tumor PD-L1 as described in claim 1; the radionuclide is selected from... 68 Ga; the structure of the PET developer is shown in Formula II: 。 3. The method for synthesizing the PET imaging agent labeling precursor targeting tumor PD-L1 as described in claim 1, characterized in that, It is synthesized using the peptide Fmoc synthesis method, which uses the amino acids required for Cyclo[SAc-F-Phe(n-Me)-Nle(n-Me)-(D-trans-Pro-OH)-NV-Phe(n-Me)-Y-(D-pipecolicacid)-WYL-C]-Lys and DOTA as raw materials.

4. The method for synthesizing the PET imaging agent targeting tumor PD-L1 according to claim 2, characterized in that, The PET imaging agent is generated by reacting the PET imaging agent labeled precursor of the tumor-targeting PD-L1 as described in claim 1 with a salt of a radionuclide.

5. The method for synthesizing a PET imaging agent targeting tumor PD-L1 according to claim 4, characterized in that, The reaction system has a pH of 3-5, a reaction temperature of 80-120℃, and a reaction time of 5-15 min.

6. The use of the PET imaging agent precursor targeting tumor PD-L1 as described in claim 1 and the PET imaging agent targeting tumor PD-L1 as described in claim 2 in the preparation of immunotherapeutic kits.

7. The use of the PET imaging agent precursor targeting tumor PD-L1 as described in claim 1 and the PET imaging agent targeting tumor PD-L1 as described in claim 2 in the preparation of diagnostic reagents / drugs and / or therapeutic drugs for tumors with high PD-L1 expression.

8. The application according to claim 7, characterized in that, The tumors that highly express PD-L1 are any one or more of non-small cell lung cancer, melanoma, and breast cancer.

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