Polypeptide targeting FGL1 and use thereof

Abstract

Provided are a polypeptide targeting FGL1 and a use thereof. The polypeptide targeting FGL1 can bind to / target an FGL1 protein. A probe targeting FGL1 is constructed by means of a chelating agent and a radionuclide. The probe can specifically target a tumor site, has good uptake and retention capabilities at the tumor site, and can be used for early diagnosis of tumors with high FGL1 expression and real-time non-destructive in-situ monitoring of early malignant tumors; and in addition, the concentration and retention of the probe targeting FGL1 at the tumor site can achieve a better tumor imaging effect.

Description

A polypeptide targeting FGL1 and its application Technical Field

[0001] The present application belongs to the field of biomedical engineering, and in particular relates to a polypeptide targeting FGL1 and its application. Background Art

[0002] Cancer is a major disease that seriously threatens human health. According to the World Health Organization, approximately 20 million new cancer cases and 9.7 million cancer deaths were reported worldwide in 2022. Traditional chemotherapy drugs act directly on tumor cells, resulting in severe adverse reactions and high rates of drug resistance. Activating the immune system to fight tumors has become a new strategy for cancer treatment.

[0003] Immune Checkpoint Blockade (ICB) is a revolutionary tumor immunotherapy that destroys tumor cells by activating the body's own immune system. Immune checkpoint blockade has shown significant efficacy in the treatment of diseases such as melanoma, non-small cell lung cancer, glioma, renal cancer, and colorectal cancer, bringing new hope for the cure of cancer. Programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) inhibitors are commonly used immune checkpoint blockade drugs in clinical practice. Currently, the overall response rate of this drug treatment is only about 30%. How to improve efficacy and expand the number of patients who benefit is a difficult problem in immunotherapy, and the discovery of new immune checkpoints is expected to break through this bottleneck.

[0004] Research has found that fibrinogen-like protein 1 (FGL1), expressed by tumor cells, can bind to the inhibitory receptor lymphocyte activation gene-3 (LAG3) expressed on T cells, forming a novel tumor immune escape pathway, FGL1 / LAG3, independent of PD-1 / PD-L1. Under normal physiological conditions, FGL1 is only expressed at low levels in liver and pancreatic tissues. However, it is highly expressed in solid tumors such as liver cancer, lung cancer, gastric cancer, breast cancer, kidney cancer, and hepatocellular carcinoma, with a positive rate reaching 50-60%. Clinical studies have confirmed that tumors with high FGL1 expression are more malignant, have strong drug resistance, and have a poor prognosis. FGL1 inhibitors can enhance immune responses and improve survival. New therapeutic strategies targeting FGL1, such as antibodies, vaccines, and nanoparticles, are under development.

[0005] Real-time, accurate, and comprehensive detection of FGL1 expression in tumors is essential for implementing novel immunotherapies. Biopsy is the primary method for clinically measuring FGL1 expression in tumors, but it is invasive, poorly tolerated by patients, and limited repeat testing. Furthermore, biopsy cannot monitor changes in the tumor microenvironment in real time. Due to tumor heterogeneity, serological testing cannot distinguish and identify FGL1 expression in every lesion throughout the body.

[0006] Positron emission tomography (PET) is an advanced clinical molecular imaging technology that focuses on the functional changes at the cellular and molecular levels during the pathophysiological process of an organism. It can provide a comprehensive, accurate, and visually objective evaluation of the functional status and biochemical changes of normal organs, soft tissues, bones, and lesions throughout the body. It has the advantages of being non-invasive, trace, quantitative, real-time, specific, and highly sensitive. 18 Although F-FDG can locate tumors and monitor tumor spread and invasion, it cannot provide accurate information on the expression of tumor surface receptors or ligands. 18 F-FDG PET has low specificity and is difficult to distinguish whether abnormal glucose metabolism is caused by tumor cell proliferation or immune cell infiltration, which brings confusion to tumor diagnosis and treatment, especially the judgment of efficacy.

[0007] FGL1 is an ideal marker for highly sensitive and specific cancer lesion localization imaging and novel immune checkpoint therapy, but there is currently a lack of reports on probes targeting FGL1. Summary of the Invention

[0008] The technical problem to be solved by the present invention is: to solve the problem that there is still a lack of probes targeting FGL1 in the prior art, thereby providing a polypeptide targeting FGL1 and its application.

[0009] The technical solution adopted by the present invention to solve its technical problem is:

[0010] A polypeptide targeting FGL1, selected from one or more of the following polypeptides or modified forms thereof:

[0011] FGLP1:

[0012] Pro-Cys-Ser-Pro-Thr-Ile-Pro-Leu-Gln-Asp-Leu-Ser-Leu-Leu-Arg-Arg-Ala-Gly-Val-Thr;

[0013] FGLP2:

[0014] Pro-Cys-Ser-Pro-Thr-Ile-Pro-Leu-Gln-Asp-Cys-Leu-Ser-Leu-Leu-Arg-Arg-Ala-Asp-Val-Cys-Thr;

[0015] FGLP3:

[0016] Pro-Cys-Ser-Pro-Thr-Hyp-Pro-Leu-Gln-Asp-Cys-Leu-Ser-Leu-Leu-Arg-Arg-Ala-Gly-Val-Cys-Thr;

[0017] FGLP4:

[0018] Pro-Cys-Ser-Pro-Thr-Ile-Pro-Leu-Gln-Asp-Leu-Ser-Leu-Leu-Arg-Arg-Ala-Gly-Val-Thr-Asn-Asp-Arg-Gly-Gly-Gly.

[0019] Preferably, the configuration of each amino acid in the polypeptide targeting FGL1 is independently selected from D-form or L-form.

[0020] Preferably, each amino acid in the targeting FGL1 polypeptide can be independently selected from substituted amino acids, and the substitution includes chloro, fluoro, bromo, or methyl, nitro, or methoxy substitution.

[0021] The present invention also provides use of the above-mentioned polypeptide targeting FGL1 in preparing tumor diagnosis or imaging tracers, preferably in preparing radionuclide imaging tracers.

[0022] Preferably, the tumor includes one or more of liver cancer, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, brain glioma and breast cancer.

[0023] A polypeptide ligand targeting FGL1 is prepared from the above-mentioned polypeptide targeting FGL1 and a chelating agent.

[0024] Preferably, the chelating agent is a macrocyclic ligand or an acyclic ligand, and the macrocyclic ligand is preferably 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonic acid) (NOTP) or a derivative thereof.

[0025] Preferably, the polypeptide ligand targeting FGL1 is selected from one of the following structures:

[0026] NOTA-FGLP1:

[0027] NODA-FGLP2:

[0028] NODA-FGLP3:

[0029] NOTA-FGLP4:

[0030] A probe targeting FGL1 comprises a polypeptide ligand targeting FGL1 and a radionuclide.

[0031] Preferably, the radionuclide is selected from 99m Tc, 68 Ga, 64 Cu, 67 Ga, 90 Y. 111 In, 177 Lu, 125 One of I.

[0032] The beneficial effects of the present invention are:

[0033] The FGL1-targeting polypeptide of the present invention can have affinity with / target the FGL1 protein. A probe targeting FGL1 is constructed by combining a chelating agent with a radionuclide. The probe can be specifically targeted to the tumor site and has good uptake and retention capabilities at the tumor site. It can be used for the early diagnosis of tumors with high FGL1 expression and can monitor early malignant tumors in real time and non-destructively. At the same time, the concentration and retention of the FGL1-targeting probe at the tumor site can achieve better tumor imaging effects, making it more conducive to clinical promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The technical solution of the present application is further described below with reference to the accompanying drawings and embodiments.

[0035] Figure 1 is a diagram of the embodiment of the present invention. 68 Ga-NOTA-FGLP1(A), 68 Ga-NODA-FGLP2(B), 68 Ga-NODA-FGLP3(C) and 68 HPLC profile of Ga-NOTA-FGLP4 (D);

[0036] Figure 2 shows the injection of tumor-bearing mice 68 Ga-NOTA-FGLP1 (A, B), 68 Ga-NODA-FGLP2(C), 68 Ga-NODA-FGLP3(D) and 68 Images at different time points after Ga-NOTA-FGLP4 (E), the circled area indicates the tumor. DETAILED DESCRIPTION

[0037] It should be noted that, unless there is any conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0038] The technical solution of the present application will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0039] Unless otherwise specified, the raw materials, reagents, and instruments used in the following examples or experimental examples can be obtained through commercial channels.

[0040] Example 1

[0041] This embodiment provides a 68 Ga-labeled probe targeting FGL1 ( 68 The preparation method of Ga-NOTA-FGLP1) is as follows:

[0042] The specific preparation method is as follows:

[0043] S1: Preparation of NOTA-FGLP1:

[0044] p-SCN-Bn-NOTA (0.67 mg, 1.2 μmol) and peptide FGLP1 (2.13 mg, 1 μmol) were dissolved in DMF and placed in an EP tube. After mixing, DIPEA (N,N-diisopropylethylamine) was added and the mixture was reacted at 40°C overnight. Preparative HPLC was used for purification (stationary phase: reverse-phase C18 column 10×250 mm; mobile phase: A: water + 1‰ TFA, B: acetonitrile + 1‰ TFA; flow rate: 5 ml / min). Fractions were collected and lyophilized to obtain 2.28 mg of a white powder; LC-MS: [MH] + = 2589.16 (m / z), which is consistent with the calculated value of the labeled precursor, i.e., NOTA-FGLP1 (2588.03 g / mol);

[0045] S2: 68 Preparation of Ga-NOTA-FGLP1:

[0046] To freshly rinsed 68 Add an appropriate volume of 1M sodium acetate aqueous solution to the Ga solution and adjust the solution pH to 4; accurately weigh 100 μg NOTA-FGLP1 in a centrifuge tube, dissolve it in water, and add 185 MBq 68The Ga solution was shaken and reacted at 60°C for 10 minutes. The reaction solution was cooled, diluted with water, and injected into an activated C18 solid-phase extraction column, followed by elution with 0.3 mL of 10 mM hydrochloric acid and ethanol. The labeled product was collected, diluted with physiological saline, and filtered through a sterile filter membrane into a sterile penicillin bottle. The sample was sampled and determined by HPLC (stationary phase: reverse-phase C18 column 4.6×250 mm; mobile phase: A: water + 1‰ TFA, B: acetonitrile + 1‰ TFA; flow rate: 5 ml / min). The radiochemical purity of the product was greater than 95%, and the yield was 73.2%. The HPLC spectrum is shown in Figure 1A.

[0047] Example 2

[0048] This embodiment provides a 68 Ga-labeled probe targeting FGL1 ( 68 The preparation method of Ga-NODA-FGLP2) is as follows:

[0049] The specific preparation method is as follows:

[0050] S1: Preparation of NODA-FGLP2:

[0051] p-NCS-benzyl-NODA-GA (0.63 mg, 1.2 μmol) and peptide FGLP2 (2.40 mg, 1 μmol) were dissolved in DMF and placed in an EP tube. After mixing, DIPEA was added and the mixture was reacted at 40°C overnight. Preparative HPLC was used for purification (stationary phase: reverse-phase C18 column 10×250 mm; mobile phase: A: water + 1‰ TFA, B: acetonitrile + 1‰ TFA; flow rate: 5 ml / min). Fractions were collected and lyophilized to obtain 2.15 mg of a white powder; LC-MS: [MH] + = 2924.37 (m / z), which is consistent with the calculated value of the labeled precursor, i.e., NOTA-FGLP2 (2923.41 g / mol);

[0052] S2: 68 Preparation of Ga-NODA-FGLP2:

[0053] To freshly rinsed 68 Add an appropriate volume of 1M sodium acetate aqueous solution to the Ga solution and adjust the solution pH to 4; accurately weigh 100 μg NODA-FGLP2 in a centrifuge tube, dissolve it in water, and add 185 MBq 68The Ga solution was shaken and reacted at 60°C for 10 minutes. The reaction solution was cooled, diluted with water, and injected into an activated C18 solid-phase extraction column, followed by elution with 0.3 mL of 10 mM hydrochloric acid and ethanol. The labeled product was collected, diluted with physiological saline, and filtered through a sterile filter membrane into a sterile penicillin bottle. The sample was sampled and determined by HPLC (stationary phase: reverse-phase C18 column 4.6×250 mm; mobile phase: A: water + 1‰ TFA, B: acetonitrile + 1‰ TFA; flow rate: 5 ml / min). The radiochemical purity of the product was greater than 95%, and the yield was 68.7%. The HPLC spectrum is shown in Figure 1B.

[0054] Example 3

[0055] This embodiment provides a 68 Ga-labeled probe targeting FGL1 ( 68 The preparation method of Ga-NODA-FGLP3) is as follows:

[0056] The specific preparation method is as follows:

[0057] S1: Preparation of NODA-FGLP3: p-NCS-benzyl-NODA-GA (0.63 mg, 1.2 μmol) and peptide FGLP3 (2.63 mg, 1 μmol) were dissolved in DMF and placed in an EP tube. After mixing, DIPEA was added and the mixture was reacted at 40°C overnight. The product was purified by preparative HPLC (stationary phase: reverse-phase C18 column 10 × 250 mm; mobile phase: A: water + 1‰ TFA, B: acetonitrile + 1‰ TFA; flow rate: 5 ml / min). Fractions were collected and lyophilized to obtain 2.53 mg of a white powder; LC-MS: [MH] + = (3150.62 m / z), which is consistent with the calculated value of the labeled precursor, namely NODA-FGLP3 (3149.64 g / mol);

[0058] S2: 68 Preparation of Ga-NODA-FGLP3:

[0059] To freshly rinsed 68 Add an appropriate volume of 1M sodium acetate aqueous solution to the Ga solution and adjust the solution pH to 4; accurately weigh 100 μg NODA-FGLP3 in a centrifuge tube, dissolve it in water, and add 185 MBq 68The Ga solution was shaken and reacted at 60°C for 10 minutes. The reaction solution was cooled, diluted with water, and injected into an activated C18 solid-phase extraction column, followed by elution with 0.3 mL of 10 mM hydrochloric acid and ethanol. The labeled product was collected, diluted with physiological saline, and filtered through a sterile filter membrane into a sterile penicillin bottle. The sample was sampled and determined by HPLC (stationary phase: reverse C18 column 4.6×250 mm; mobile phase: A: water + 1‰ TFA, B: acetonitrile + 1‰ TFA; flow rate: 5 ml / min). The radiochemical purity of the product was greater than 95%, and the yield was 65.5%. The HPLC spectrum is shown in Figure 1C.

[0060] Example 4

[0061] This embodiment provides a 68 Ga-labeled probe targeting FGL1 ( 68 The preparation method of Ga-NOTA-FGLP4) is as follows:

[0062] The specific preparation method is as follows:

[0063] S1: Preparation of NOTA-FGLP4: p-SCN-Bn-NOTA (0.67 mg, 1.2 μmol) and peptide FGLP4 (2.70 mg, 1 μmol) were dissolved in DMF and placed in an EP tube. After mixing, DIPEA was added and the mixture was reacted at 40°C overnight. The product was purified by preparative HPLC (stationary phase: reverse-phase C18 column 10 × 250 mm; mobile phase: A: water + 1‰ TFA, B: acetonitrile + 1‰ TFA; flow rate: 5 ml / min). Fractions were collected and lyophilized to obtain 2.71 mg of a white powder; LC-MS: [MH] + = (3145.60 m / z), which is consistent with the calculated value of the labeled precursor, namely NOTA-FGLP3 (3144.56 g / mol);

[0064] S2: 68 Preparation of Ga-NODA-FGLP3:

[0065] To freshly rinsed 68 Add an appropriate volume of 1M sodium acetate aqueous solution to the Ga solution and adjust the solution pH to 4; accurately weigh 100 μg NODA-FGLP3 in a centrifuge tube, dissolve it in water, and add 185 MBq 68The Ga solution was shaken and reacted at 60°C for 10 minutes. The reaction solution was cooled, diluted with water, and injected into an activated C18 solid-phase extraction column, followed by elution with 0.3 mL of 10 mM hydrochloric acid and ethanol. The labeled product was collected, diluted with physiological saline, and filtered through a sterile filter membrane into a sterile penicillin bottle. The sample was sampled and determined by HPLC (stationary phase: reverse-phase C18 column 4.6×250 mm; mobile phase: A: water + 1‰ TFA, B: acetonitrile + 1‰ TFA; flow rate: 5 ml / min). The radiochemical purity of the product was greater than 95%, and the yield was 67.2%. The HPLC spectrum is shown in Figure 1D.

[0066] Effect Examples

[0067] The FGL1-positive tumor cells (Huh7) and FGL1-negative tumor cells (U87MG) were inoculated into the axilla of nude mice and fed normally. The tumor mass at the inoculation site was 100-300 mm. 3 When the cells were stained with cytochrome P60, the transplanted tumor model mice were obtained.

[0068] The results of in vivo PET imaging experiments and cell uptake experiments are shown in Figures 2A, 2B and Tables 1 and 2. 68 Ga-NOTA-FGLP1 was concentrated in FGL1-positive Huh7 (human liver cancer cell) transplanted tumors compared with FGL1-negative U87MG (human brain astroglioma) transplanted tumors. At 60 minutes after injection, the Huh7 transplanted tumors showed a significant decrease in the expression of Ga-NOTA-FGLP1. 68 Ga-NOTA-FGLP1 uptake reached 1.71±0.10 ID% / g, 8.55 times the corresponding uptake value (0.20±0.04 ID% / g) in U87MG xenografts during the same period. The probe was primarily excreted via the kidneys, and the ratio of tumor to muscle uptake increased over time. At 60 and 120 minutes after injection, the tumor-to-muscle ratios were 4.27±0.40 and 5.28±0.39, respectively.

[0069] As shown in Figure 2 and Tables 1-5, 68 Ga-NODA-FGLP2, 68 Ga-NODA-FGLP3, 68 Compared with Ga-NOTA-FGLP4, 68 Ga-NOTA-FGLP1 is more likely to accumulate on FGL1-positive Huh7 transplanted tumors. 30 minutes after injection, 68 Ga-NOTA-FGLP1, 68 Ga-NODA-FGLP2, 68 Ga-NODA-FGLP3 and 68The uptake values ​​of Ga-NOTA-FGLP4 in FGL1-positive tumor Huh7 were 2.63±0.07ID% / g, 1.77±0.11ID% / g, 2.09±0.12ID% / g and 0.91±0.05ID% / g, respectively. 68 The uptake of Ga-NOTA-FGLP1 in FGL1-positive Huh7 xenografts was significantly higher than that in FGL1-negative U87MG xenografts (0.44±0.06 ID% / g). 68 At 120 minutes after the addition of Ga-NOTA-FGLP1, the ratio of tumor to muscle (5.28±0.39) was significantly higher than that of 68 Ga-NODA-FGLP2 and 68 The corresponding values ​​of Ga-NODA-FGLP3 were (4.39±0.27 and 4.50±0.33). 68 Ga-NODA-FGLP2, 68 Ga-NODA-FGLP3 and 68 Compared with Ga-NOTA-FGLP4, 68 Ga-NOTA-FGLP1 is more suitable for monitoring and judging the expression level of FGL1 in tumors and can play a unique role in tumor immunotherapy.

[0070] Table 1 68 Ga-NOTA-FGLP1 uptake values ​​(ID% / g) in tumors and major organs and tumor-to-muscle ratio in nude mice bearing Huh7 transplanted tumors

[0071] Table 2 68 Ga-NOTA-FGLP1 uptake values ​​(ID% / g) in tumors and major organs and tumor-to-muscle ratio in nude mice bearing U87MG transplanted tumors

[0072] Table 3 68 Ga-NODA-FGLP2 uptake values ​​(ID% / g) in tumors and major organs and tumor-to-muscle ratio in nude mice bearing Huh7 transplanted tumors

[0073] Table 4 68 Ga-NODA-FGLP3 uptake values ​​(ID% / g) in tumors and major organs and tumor-to-muscle ratio in nude mice bearing Huh7 transplanted tumors

[0074] Table 5 68Ga-NOTA-FGLP4 uptake values ​​(ID% / g) in tumors and major organs and tumor-to-muscle ratio in nude mice bearing Huh7 transplanted tumors

[0075] Based on the above-mentioned ideal embodiments of this application, and in accordance with the above description, relevant personnel can make various changes and modifications without departing from the scope of the technical concept of this application. The technical scope of this application is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A polypeptide targeting FGL1, characterized in that One or more selected from the following polypeptides or modified forms thereof: FGLP1: Pro-Cys-Ser-Pro-Thr-Ile-Pro-Leu-Gln-Asp-Leu-Ser-Leu-Leu-Arg-Arg-Ala-Gly-Val-Thr; FGLP2: Pro-Cys-Ser-Pro-Thr-Ile-Pro-Leu-Gln-Asp-Cys-Leu-Ser-Leu-Leu-Arg-Arg-Ala-Asp-Val-Cys-Thr; FGLP3: Pro-Cys-Ser-Pro-Thr-Hyp-Pro-Leu-Gln-Asp-Cys-Leu-Ser-Leu-Leu-Arg-Arg-Ala-Gly-Val-Cys-Thr; FGLP4: Pro-Cys-Ser-Pro-Thr-Ile-Pro-Leu-Gln-Asp-Leu-Ser-Leu-Leu-Arg-Arg-Ala-Gly-Val-Thr-Asn-Asp-Arg-Gly-Gly-Gly.

2. The polypeptide targeting FGL1 according to claim 1, characterized in that The conformation of each amino acid in the polypeptide targeting FGL1 is independently selected from D-form or L-form.

3. The polypeptide targeting FGL1 according to claim 1 or 2, characterized in that Each amino acid in the targeting FGL1 polypeptide can be independently selected from substituted amino acids, and the substitution includes chloro, fluoro, bromo, or methyl, nitro, or methoxy substitution.

4. Use of the polypeptide targeting FGL1 according to any one of claims 1 to 3 in the preparation of a tumor diagnosis or imaging tracer reagent, preferably in the preparation of a radionuclide imaging tracer.

5. Use of the polypeptide targeting FGL1 according to claim 4 in the preparation of a tumor diagnosis or tracing reagent, characterized in that: The tumor includes one or more of liver cancer, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, brain glioma and breast cancer.

6. A polypeptide ligand targeting FGL1, characterized in that The invention is prepared from the polypeptide targeting FGL1 according to any one of claims 1 to 3 and a chelating agent.

7. The polypeptide ligand targeting FGL1 according to claim 6, characterized in that The chelating agent is a macrocyclic ligand or an acyclic ligand, and the macrocyclic ligand is preferably 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonic acid) (NOTP) or a derivative thereof.

8. The polypeptide ligand targeting FGL1 according to claim 6 or 7, characterized in that The polypeptide ligand targeting FGL1 is selected from one of the following structures:

9. A probe targeting FGL1, characterized in that The method comprises the FGL1-targeting polypeptide ligand according to any one of claims 6 to 8 and a radionuclide.

10. The probe targeting FGL1 according to claim 9, characterized in that The radionuclide is selected from 99m Tc, 68 Ga, 64 Cu, 67 Ga, 90 Y. 111 In, 177 Lu, 125 One of I.

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