Sortilin-based lysosome targeting chimera and application thereof

Abstract

The invention discloses a lysosome targeted chimera based on Sortilin and application thereof. The lysosome targeted chimera comprises a ligand molecule of a lysosome targeted receptor and a ligand molecule of a targeted protein to be degraded, wherein the ligand molecule and the ligand molecule are connected through a linker; the lysosome targeting receptor is Sortilin, and the ligand molecule of the lysosome targeting receptor is a polypeptide targeting the Sortilin; the protein to be degraded is membrane protein or extracellular protein. Furthermore, the polypeptide of the targeted Sortilin is connected with an enzyme response sequence and an integrin receptor targeting sequence. According to the invention, the Sortilin-combined ligand neurotensin is functionally coupled with the neutralizing antibody or polypeptide of overexpression protein in targeted tumor or inflammatory diseases, and the Sortilin-combined ligand neurotensin has obvious treatment gain in malignant solid tumor and psoriasis models. The discovery promotes the transformation potential of the lysosome participating in the biological agent, and provides a dual strategy for realizing accurate degradation through receptor recruitment and microenvironment induction.

Description

Technical Field

[0001] The present invention belongs to the field of drug research and development, and specifically relates to a lysosome-targeted chimera based on Sortilin (neurotensin receptor 3) and applications thereof. Background Art

[0002] Cell surface proteins account for over 60% of existing therapeutic targets and play a central role in regulating disease progression and therapeutic intervention. Despite their significant biomedical significance, traditional regulatory strategies continue to face persistent technical bottlenecks, including inherent drawbacks such as insufficient ligand selectivity, incomplete functional inhibition, and difficulty in tissue-specific targeting.

[0003] Lysosome-mediated protein degradation is a novel therapeutic approach that can eliminate pathogenic targets by integrating with endogenous cellular waste clearance mechanisms. While promising degradation technologies such as antibody-based lysosome-targeting chimeras (LYTACs) have shown promise, their efficacy is limited by the lack of disease relevance of existing lysosome-targeting receptors (LTRs), such as the cation-independent mannose-6-phosphate receptor (CI-M6PR). Recent efforts to develop novel LTRs, such as integrins and transferrin receptors, have yet to effectively address key scientific challenges, including cancer-specific recognition and microenvironment-adaptive regulation.

[0004] Sortilin receptors are significantly overexpressed in malignancies and inflammatory diseases, including thyroid cancer, ovarian cancer, and colorectal cancer. They regulate neurotensin (NT)-mediated endocytosis and lysosomal trafficking, and are involved in the biological regulation of key inflammatory factors (such as TNF-α and IL-6) in pathological processes such as psoriasis. Based on this, we propose engineering NT-coupled antibodies / peptides / small molecules to create LYTAC molecules (NeuroTACs). These molecules can synergistically exploit the dual functional properties of sortilin as a disease-associated receptor and lysosomal trafficking factor, thereby achieving selective degradation of targeted membrane or extracellular proteins in tumor and inflammatory microenvironments. Summary of the Invention

[0005] In order to solve the problems in the prior art, the present invention proposes a lysosome-targeting chimera based on Sortilin and its application.

[0006] The technical solutions of the present invention are as follows:

[0007] The present invention first provides a lysosome-targeting chimera based on Sortilin, which comprises: a ligand molecule of a lysosome-targeting receptor and a ligand molecule targeting a protein to be degraded, connected by a linker; the lysosome-targeting receptor is Sortilin, and the ligand molecule of the lysosome-targeting receptor is a polypeptide targeting Sortilin;

[0008] The linker is a polypeptide chain composed of a PEG chain, a carbon chain, and amino acids.

[0009] The protein to be degraded is a membrane protein or an extracellular protein.

[0010] According to a preferred embodiment of the present invention, the polypeptide targeting Sortilin is neurotensin or an analogue thereof.

[0011] According to a preferred embodiment of the present invention, the amino acid sequence of the neurotensin is LYENKPRRPYIL, as shown in SEQ ID No. 1.

[0012] According to a preferred embodiment of the present invention, the linker is an SMCC linker, a polyethylene glycol linker, a C2-C100 carbon chain, or a polypeptide chain composed of different numbers of amino acids, and the number of ethylene glycol units in the polyethylene glycol linker is 2-100.

[0013] According to a preferred embodiment of the present invention, the polypeptide targeting Sortilin is a biologically responsive polypeptide targeting Sortilin, to which an enzyme-responsive sequence and an integrin receptor-targeting sequence are attached. The lysosome-targeting chimera is called BioresTAC.

[0014] According to a preferred embodiment of the present invention, the enzyme response sequence is a matrix metalloproteinase, and the integrin receptor targeting sequence is an RGD sequence.

[0015] According to a preferred embodiment of the present invention, the amino acid sequence of the polypeptide targeting Sortilin connected with the enzyme response sequence and the integrin receptor targeting sequence is LYENKPRRPYILPLGARGD, as shown in SEQ ID No. 2.

[0016] The present invention also provides the use of the aforementioned lysosome-targeting chimera in the preparation of anti-tumor drugs or drugs for treating autoimmune system diseases.

[0017] Preferably, the tumor is liver cancer, osteosarcoma, colon cancer, lung cancer, melanoma or breast cancer. Preferably, the autoimmune system disease is psoriasis.

[0018] Compared with the existing technology, the present invention first designed NeuroTAC, a lysosome-targeting chimera (LYTAC), which functionally couples the sortilin-binding ligand neuropeptide (neurotensin, NT) with therapeutic antibodies targeting overexpressed proteins in tumors or inflammatory diseases. In this study, the experimental results showed that NeuroTAC has the ability to efficiently degrade membrane proteins and extracellular proteins. Based on the pathological characteristics of abnormally elevated activity of matrix metalloproteinases (MMPs) in tumor and inflammatory microenvironments, the present invention further developed a bio-responsive LYTAC, BioresTAC. This intelligent molecular system integrates MMP-specific cleavage sites and RGD targeting peptides to achieve protease-activated spatially precise degradation mediated in the pathological microenvironment. Subsequent studies have shown that BioresTAC selectively mediates broad-spectrum protein degradation through the microenvironment, and can establish a selective broad-spectrum protein degradation platform, which has shown significant therapeutic benefits in both malignant solid tumors and psoriasis models. These findings advance the translational potential of lysosomal-engaged biologics, providing a dual strategy for precise degradation through receptor recruitment and microenvironment sensing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The following are the synthetic routes of NeuroTACs and the molecular structures of the three intermediates obtained. a is the synthetic route of the lysosome-targeting chimera NeuroTAC; b and d are the mass spectra and chemical structures of the intermediates NT-SMCC-MAL, NT-PEG4-MAL, and NT-PEG6-MAL, respectively.

[0020] Figure 2 The results of PD-L1 degradation in osteosarcoma K7M2 cells by NeuroTAC coupled with different linkers, where a is the Western Blot (WB) evaluation result; b is the flow cytometry evaluation result; c is the quantitative analysis of the result in b.

[0021] Figure 3 The molecular weight of the unmodified PD-L1 antibody was measured by mass spectrometry. PD-L1 The molecular weight was determined by mass spectrophotometer.

[0022] Figure 4 The experimental results of Example 3 of the present invention show that NeuroTACs can promote the uptake of extracellular proteins in vitro. a is the flow cytometry evaluation result showing that NeuroTAC IgGIt can promote the uptake of extracellular protein IgG by cells. b is the quantitative analysis of the results in a. c is the quantitative analysis of the results in a. TSLP Confocal images of Mgct-812 cells after 1 hour of treatment.

[0023] Figure 5 Figure 2 is the degradation effect of NeuroTAC in cell lines with different EGFR expression levels. EGFR Western blot analysis of EGFR levels in Mgct-812 cells after treatment. cd is WB analysis of NeuroTAC EGFR Degradation effect on EGFR in H1975 cells and HepG2 cells.

[0024] Figure 6 The figure shows the degradation effect of NeuroTAC in cell lines with different endogenous PD-L1 expression levels.

[0025] Wherein, ab is the expression of NeuroTAC at different time points (a) and concentrations (b). PD-L1 Western blot analysis of PD-L1 levels in K7M2 cells after treatment. c is the expression of PD-L1 on the surface of K7M2 cells detected by live cell flow cytometry, under treatment conditions of 40 nM anti-PD-L1 antibody or NeuroTAC PD-L1 48 hours. d is a quantitative analysis of the results in c. ef is a quantitative analysis of the results in c. PD-L1 Western blot analysis of total cellular PD-L1 levels after 48 h of treatment of LLC (e) and MC38 (f) cells.

[0026] Figure 7 The results of the in vivo anti-tumor efficacy evaluation of NeuroTAC targeting PD-L1 in Example 5 of the present invention are shown in Table 1. Wherein, a is the number of NeuroTAC cells in the BALB / c mouse K7M2-luciferin tumor model. PD-L1 Schematic diagram of treatment. b is a typical in vivo bioluminescence imaging of K7M2-luc tumors at different time points after treatment. c is the tumor growth curve after different treatment regimens (n=5). de is the image (d) and weight (e) of the resected tumor on day 14 after treatment (n=5). fh are the images of K7M2-luc tumors treated with saline (f), Ab-PD-L1 (g), or NeuroTAC PD-L1 (h) Volume changes of each tumor after treatment.

[0027] Figure 8 Figures 1 and 2 show the mass spectra of the raw materials and synthetic products of the RGD-MMP-SMCC-NT-MAL intermediate. Figure a shows the mass spectrometry results of the RGD-MMP-NT polypeptide. Figure b shows the mass spectrometry results of the intermediate RGD-MMP-NT-SMCC-MAL.

[0028] Figure 9 The synthetic route of BioresTACs and the characterization of purified products are shown in Figure 2. PD-L1 The molecular weight was determined by mass spectrophotometer.

[0029] Figure 10 The results of the MMP-responsive LYTAC (BioresTAC) in Example 7 on protein degradation in vivo and in vitro. Among them, a is the Western blot technique for detecting K7M2 cells treated with 40nM anti-PD-L1 antibody and NeuroTAC PD-L1 、BioresTAC PD-L1 or MMP9 combined with BioresTAC PD-L1 The expression level of total PD-L1 protein after 48 hours of treatment. b, c are live cell flow cytometry detection of K7M2 cells after 40nM antibody, NeuroTAC PD-L1 or BioresTAC PD-L1 d is the Western blot analysis of the total PD-L1 level in K7M2 cells: pretreated with 40 μM neurotensin or RGD peptide for 2 hours, and then treated with 40 nM NeuroTAC PD-L1 or BioresTAC PD-L1 Incubate for 48 hours. e is Western blot detection of total PD-L1 levels in K7M2 cells: 40nM anti-PD-L1 antibody, 40μM MMP9 inhibitor + BioresTAC PD-L1 、NeuroTAC PD-L1 or BioresTAC PD-L1 After 48 hours of treatment, the total PD-L1 level in HT22 cells was detected by Western blot: 40nM anti-PD-L1 antibody, NeuroTAC PD-L1 or BioresTAC PD-L1 After 48 hours of treatment.

[0030] Figure 11Figure 1 shows the results of PD-L1-targeted treatment in BALB / c mice. (a) Schematic diagram of the NeuroTAC and BioresTAC treatment regimens in the BALB / c mouse H22-luciferin tumor model. (b) Tumor growth curves (n = 6) following treatment according to the regimen shown in (a). (c) Representative in vivo bioluminescence images of H22-luc tumors at different time points after treatment.

[0031] Figure 12 The experimental results of LYTACs in Example 8 for scavenging TSLP and alleviating psoriasis symptoms in vivo are shown. a is the experimental timeline: BALB / c mice were topically applied with 5% IMQ cream and then received normal saline, MTX (1 mg / kg / day), Ab-TSLP (1 mg / kg / day), NeuroTAC for 7 consecutive days. TSLP (1 mg / kg / day) or BioresTAC TSLP (1 mg / kg / day) by intraperitoneal injection. b is a schematic diagram of LYTACs degrading TSLP protein in psoriasis and causing changes in downstream related cytokines. c is a representative clinical image and H&E staining of the back skin on the 7th day. Scale bar: 100 μm. d is the daily body weight change (n=5). e is the degree of splenomegaly. f is a quantitative analysis of epidermal hyperplasia on the 7th day. g is the Psoriasis Area and Severity Index (PASI) score, which reflects the erythema, scaling and skin thickening within 7 days. h is the TSLP protein level in the skin lesions on the 7th day. i is the IL-17A protein level in the skin lesions on the 7th day. j is the intraperitoneal injection of Cy7-Ab-TSLP and Cy7-NeuroTAC in psoriasis model mice and healthy mice TSLP or Cy7-BioresTAC TSLP Afterwards, representative fluorescence images of the living body and skin at different time points.

[0032] Figure 13 The results of in vivo tumor targeting and safety evaluation of Example 9 are shown. PD-L1 -Cy5 or BioresTAC PD-L1 Representative in vivo fluorescence images of mice at different time points after injection of Ab-PD-L1-Cy5. b is the quantitative analysis of the mean fluorescence intensity (MFI) of tumors at different time points after injection. c is the quantitative analysis of the mean fluorescence intensity (MFI) of tumors at different time points after injection of Ab-PD-L1-Cy5 and NeuroTAC. PD-L1 -Cy5 or BioresTAC PD-L1 Representative fluorescence images of tumors and major organs in vitro 72 hours after Cy5 injection. d is the quantitative analysis of MFI of tumors and major organs 72 hours after injection. e is the subcutaneous injection of saline, Ab-PD-L1, and NeuroTAC. PD-L1or BioresTAC PD-L1 Serum biochemical parameters were analyzed on the 7th day after treatment. The test parameters included blood urea nitrogen (BUN), creatinine (CRE), alanine aminotransferase (GPT), glucose (GLU), amylase (AMYL), alkaline phosphatase (ALP), albumin (ALB), total protein (TP), uric acid (UA), and lactate dehydrogenase (LDH). DETAILED DESCRIPTION

[0033] The present invention will be further described and illustrated below in conjunction with specific embodiments. The embodiments are merely illustrative of the present disclosure and do not limit its scope. The technical features of the various embodiments of the present invention may be combined accordingly, provided that there is no conflict between them.

[0034] Unless otherwise specified, all raw materials and reagents used were commercially available.

[0035] The K7M2 cells used in the examples were from ELGBIO (Guangzhou) Biotechnology Co., Ltd., K7M2-luc cells were from Warner Bio (Wuhan) Co., Ltd. (batch number WN-25497), LLC cells, MC38 cells, HT22 cells, MEF cells, Hepa1-6 cells, H1975 cells, HepG2 cells, and liver cancer H22 / Luc cells were from ORiCells Biotechnology Co., Ltd. (Shanghai). Primary human Mgct-812 cells were obtained from patients with malignant tendon sheath giant cell tumors. Informed consent was obtained from the patients, and the procedure was approved by the Ethics Committee of Hunan Cancer Hospital (KY2022233). Adherent cells were cultured at 37°C and 5% CO2, and all cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0036] Example 1 Synthesis of intermediate

[0037] Synthesis route: Intermediates with maleimide groups were prepared by coupling reaction of NT with a series of NHS esters.

[0038] a. Synthesis of NT-SMCC-MAL intermediate

[0039] NT (10 mg, 0.006 mmol) was added to a solution of SMCC (2.2 mg, 0.0066 mmol) in 10 ml of PBS / THF (1:1 volume ratio). The reaction mixture was stirred at room temperature for 8 hours. The reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the THF was removed under reduced pressure. The crude product was then lyophilized in a freeze dryer for 48 hours to obtain the NT-SMCC-MAL intermediate. MALDI-TOF MS (m / z) calculated for C90H135N22O23+: 1892.0065, found: 1892.236.

[0040] b. Synthesis of NT-PEG4-MAL intermediate

[0041] NT (10 mg, 0.006 mmol) was added to a 10 mL PBS / THF (1:1 volume ratio) solution containing Mal-PEG4-NHS ester (2.9 mg, 0.0066 mmol). The reaction mixture was stirred at room temperature for 8 hours. The reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the THF was removed under reduced pressure. The crude product was then lyophilized in a freeze dryer for 48 hours to obtain the NT-PEG4-MAL intermediate. MALDI-TOF MS (m / z) calculated for C93H143N22O27+: 2000.0488, found: 2000.408.

[0042] c. Synthesis of NT-PEG6-MAL intermediate

[0043] NT (10 mg, 0.006 mmol) was added to a 10 mL PBS / THF (1:1 volume ratio) solution containing Mal-PEG6-NHS ester (3.5 mg, 0.0066 mmol). The reaction mixture was stirred at room temperature for 8 hours. The reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the THF was removed under reduced pressure. The crude product was then lyophilized in a freeze dryer for 48 hours to obtain the NT-PEG6-MAL intermediate. MALDI-TOF MS (m / z) calculated for C97H151N22O29+ was 2088.1012, and the actual value was 2159.408.

[0044] Example 2 Synthesis and Optimization of NeuroTACs

[0045] Synthesis route: The target antibody was fully reduced using tris(2-carboxyethyl)phosphine (TCEP) at room temperature to ensure efficient exposure of the thiol group. Finally, the intermediate obtained in Example 1 was coupled with the reduced antibody, and NeuroTACs with high specificity and yield were obtained through a series of purification steps.

[0046] A 40-fold molar excess of TCEP (Tris(2-carboxyethyl)phosphine) reagent was added to 0.5 ml of PBS containing an appropriate amount of antibody (6.7 μmol) and slowly rotated at room temperature for 30 minutes. Three experimental groups were set up, and each group added one of the intermediates synthesized in Example 1 (253 μg, 267 μmol), mixed at room temperature for 2 hours, and then shaken at 4°C overnight. After the reaction was completed, the solution was purified by Sephadex G-25 column. Three NeuroTACs were obtained respectively. The synthetic route of NeuroTACs and the molecular structures of the three intermediates obtained are shown as follows. Figure 1 shown.

[0047] To identify the optimal linker system, we conducted degradation studies using programmed cell death ligand 1 (PD-L1) as a model target. As a key immune checkpoint molecule, PD-L1 inhibits T cell activity and mediates tumor immune escape by binding to programmed cell death protein 1 (PD-1) on the surface of immune cells. However, existing PD-L1 blockade therapies have clinical limitations such as low response rates and significant toxic side effects due to off-target inhibition in healthy tissues. We propose that cancer-specific PD-L1 degraders could provide a more selective, effective, and safer treatment strategy.

[0048] To verify this hypothesis, we first used anti-mouse PD-L1 antibody to construct a NeuroTAC molecule targeting mouse PD-L1, named SMCC-NeuroTAC PD-L1 PEG4-NeuroTAC PD-L1 PEG6-NeuroTAC PD-L1The subscript "PD-L1" in the name indicates the protein to be degraded (the subscripts in the corresponding names in the subsequent examples all indicate the corresponding proteins to be degraded and will not be repeated here). Subsequently, the degradation ability of NeuroTAC with different linkers on PD-L1 protein was evaluated by protein immunoblotting (Western blot, WB) and flow cytometry (Flowcytometry, FC). The results showed that NeuroTAC with SMCC linker (SMCC-NeuroTAC PD-L1 ) showed the best degradation effect: compared with antibodies and PEG4-NeuroTAC PD-L1 PEG6-NeuroTAC PD-L1 The degradation rate of PD-L1 can reach up to 70% ( Figure 2 We also further measured NeuroTAC using a mass spectrometer. PD-L1 Compared with the molecular weight of the antibody alone, the Figure 3 ), which was consistent with the expected results. Based on these results and considerations of linker stability, we selected the SMCC linker for all subsequent studies. NeuroTACs in subsequent examples refer to SMCC-NeuroTACs.

[0049] Example 3 NeuroTACs can effectively promote the uptake of extracellular proteins in vitro

[0050] We further evaluated the ability of NeuroTAC-mediated protein uptake in Mgct-812 cells. Mgct-812 cells are primary human cells derived from patients with malignant tenosynovial giant cell tumors. Their use has been approved by the Ethics Committee of Hunan Cancer Hospital (Approval No. KY2022233), and informed consent was obtained from the patients. In the experiment, IgG proteins were labeled with the fluorescent dye Cy3 and incubated with IgG-Cy3, anti-IgG, or NeuroTAC. IgG The cells were treated for 1 hour. Flow cytometry showed that NeuroTAC IgG The intracellular fluorescence intensity of the treated group increased fourfold. In addition, we further verified the potential of NeuroTAC to mediate the internalization of extracellular proteins by detecting the uptake of extracellular pathogenic proteins, thymic stromallymphopoietin (TSLP), by Mgct-812 cells. TSLP is a cytokine that is highly expressed in inflammatory sites, and its abnormal function is closely related to the occurrence and development of many diseases (especially autoimmune diseases such as psoriasis). We observed that TSLP, similar to IgG, can also effectively promote its internalization into Mgct-812 cells after treatment with NeuroTAC ( Figure 4), which further expands the therapeutic application range of NeuroTAC. This shows that NeuroTAC can efficiently mediate the internalization of extracellular proteins.

[0051] Example 4 NeuroTACs mediate membrane protein degradation in vitro

[0052] After confirming that NeuroTAC can induce the uptake of soluble proteins through the lysosomal pathway, we further explored its potential to selectively degrade membrane proteins in cancer cells. First, we evaluated the degradation effect of epidermal growth factor receptor (EGFR). EGFR is a key driver of cancer growth, and even if its receptor tyrosine kinase activity is inhibited, it can still exert its effects through a variety of structure-dependent mechanisms. In Mgct-812 cells overexpressing EGFR, NeuroTAC treatments of different concentrations and action times showed a concentration- and time-dependent EGFR degradation trend. Through system optimization, we determined that a concentration of 40nM and an incubation time of 48 hours were selected as the optimal conditions for EGFR degradation in subsequent experiments. To further verify the effectiveness, we evaluated the degradation efficiency of NeuroTAC (40nM, 48h) in cell lines (H1975 and HepG2) with different EGFR expression levels, and the results showed that EGFR was significantly degraded ( Figure 5 To expand the universality of NeuroTAC in membrane protein targeted degradation, we selected PD-L1 as another model protein for study. Similar to EGFR, NeuroTAC treatment can induce PD-L1 degradation in a time- and concentration-dependent manner, with the most significant degradation effect after 48 hours of treatment at a concentration of 40 nM ( Figure 6 NeuroTAC (40 nM, 48 h) effectively degraded PD-L1 in cell lines (LLC and MC38) with varying levels of endogenous PD-L1 expression, a finding further validated by flow cytometry. These results demonstrate that NeuroTAC is highly effective in degrading membrane proteins.

[0053] Example 5 In vivo evaluation of the anti-tumor efficacy of NeuroTAC targeting PD-L1

[0054] Establishment of tumor model: All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) and approved by the Animal Care and Use Committee of Zhejiang University (approval number ZJU20240840). 4-6 week-old female BALB / c mice were purchased from GemPharmatech Biotechnology Co., Ltd. (Nanjing, China). To establish tumor models in BALB / c mice, 5 × 10 6K7M2-luc cells or Hepatoma 22 / luc cells were resuspended in PBS and then injected into the right back of each 6-7 week old mouse. The tumor volume reached approximately 40-60 mm 3 The same dose of treatment factors were intraperitoneally injected twice a week. The treatment factors were: Ab-PD-L1 (20 μg / kg), NeuroTAC PD-L1 (20 μg / kg) or BioresTAC PD-L1 (20 μg / kg). A saline group served as a negative control. Tumor size was measured with a vernier caliper, and tumor volume was calculated using the formula: Tumor volume = tumor major diameter * tumor minor diameter * tumor minor diameter / 2. Tumor size and mouse body weight were measured every two days. On day 14, mice were sacrificed, and tumor and major normal tissues were collected for further analysis.

[0055] We further evaluated NeuroTAC in the K7M2-luc tumor model in BALB / c mice. PD-L1 When the average tumor volume reaches 100mm 3 At 4 hr, normal saline, Ab-PD-L1 and NeuroTAC were administered by intraperitoneal injection. PD-L1 (Dose 1 mg / kg, once every three days, for a total of four doses) Figure 7 (a) The results showed that NeuroTAC PD-L1 The tumor growth was significantly inhibited and the tumor weight was reduced, while the anti-tumor effect of Ab-PD-L1 was weak. By using in vivo bioluminescence imaging after the injection of luciferin substrate, we dynamically monitored the tumor volume on days 0, 7, and 14, confirming that only NeuroTAC PD-L1 It can effectively delay tumor progression, and its inhibition rate is significantly higher than that of the saline group or Ab-PD-L1 treatment group ( Figure 7 b). It is worth noting that NeuroTAC PD-L1 Almost completely eliminated the tumor, while Ab-PD-L1 only slightly inhibited tumor growth ( Figure 7 Taken together, these results highlight the importance of NeuroTAC PD-L1 Powerful anti-tumor effect, especially against solid tumors.

[0056] Example 6 Design of MMP-responsive LYTAC (BioresTAC)

[0057] a. Synthesis of RGD-MMP-SMCC-NT-MAL intermediate

[0058] RGD-MMP-NT (10 mg, 0.006 mmol, purchased from Hefei Guopeptide Biotechnology Co., Ltd.) was added to a solution of 10 ml PBS / THF (volume ratio 1:1) containing SMCC (2.2 mg, 0.0066 mmol). The reaction mixture was stirred at room temperature for 8 hours. The reaction was monitored by thin layer chromatography (TLC). After the reaction was completed, THF was removed under reduced pressure. The crude product was then placed in a freeze dryer and lyophilized for 48 hours to obtain the RGD-MMP-NT-SMCC-MAL intermediate, which was then used directly in the next step. The molecular weight was measured by MALDI-TOF MS (m / z) to be 2825.2920.257. The mass spectra of the raw materials and synthetic products are shown in Figure 8 .

[0059] b. Synthesis of BioresTACs

[0060] A 40-fold molar excess of TCEP reagent was added to 0.5 ml of PBS containing an appropriate amount of antibody (6.7 μmol) and slowly rotated at room temperature for 30 minutes. RGD-MMP-NT-SMCC-MAL (253 μg, 267 μmol) was then added and mixed at room temperature for 2 hours, followed by shaking at 4°C overnight. After the reaction was complete, purification was performed using a Sephadex G-25 column. The synthesis route and characterization of the purified product are shown in the figure. Figure 9 shown.

[0061] Example 7 Effects of MMP-responsive LYTAC (BioresTAC) on protein degradation in vivo and in vitro

[0062] We confirmed that NeuroTAC PD-L1 with BioresTAC PD-L1 Both mediate the degradation of the membrane protein PD-L1 in cancer cells. In K7M2 cells with high expression of PD-L1, WB analysis showed that both can achieve more than 60% PD-L1 protein degradation ( Figure 10 (a) Flow cytometry further verified that NeuroTAC PD-L1 with BioresTAC PD-L1 The degradation rate of cell surface PD-L1 was about 60% ( Figure 10 b,c in Figure 2). Subsequently, to verify the MMP enzyme responsiveness, we found that after adding MMP inhibitors, BioresTAC PD-L1 The PD-L1 degradation effect of the PD-L1 inhibitor was significantly weakened, confirming the success of its MMP responsiveness design ( Figure 10e in the figure). We then pre-incubated with excess free NT to competitively bind to sortilin and found that PD-L1 degradation in K7M2 cells was reduced, suggesting that sortilin is involved in the degradation process; however, pre-incubation with excess RGD did not affect the degradation effect of BioresTAC, indicating that its effect is not dependent on the RGD pathway ( Figure 10 NeuroTAC was further validated in normal cell lines (such as hippocampal neuron HT22 cells). PD-L1 with BioresTAC PD-L1 MMP9 dependence ( Figure 10 f) in NeuroTAC PD-L1 PD-L1 degradation mediated by BioresTAC is independent of MMP9, while PD-L1 The total degradation rate of PD-L1 was less than 40%, highlighting its specific targeting effect on MMP9-expressing cells. To evaluate the therapeutic potential of PD-L1, we established an H22-luc liver cancer ascites model in BALB / c mice. When the average tumor volume reached 100 mm 3 At the same time, normal saline, Ab-PD-L1 antibody, NeuroTAC PD-L1 or BioresTAC PD-L1 (Dose 1 mg / kg, once every three days, for a total of four doses) Figure 11 In vivo bioluminescence imaging and tumor volume measurement at multiple time points ( Figure 11 b,c) show that all treatment groups can inhibit tumor growth. PD-L1 with BioresTAC PD-L1 The inhibition rate of NeuroTAC was significantly higher than that of Ab-PD-L1 antibody alone. PD-L1 with BioresTAC PD-L1 It showed a strong anti-tumor effect and was also effective in the liver cancer ascites model that was sensitive to immunotherapy.

[0063] Example 8 LYTACs can clear TSLP and alleviate psoriasis symptoms in vivo

[0064] Given the therapeutic potential of TSLP in psoriasis and the characteristic macrophage infiltration at the epidermal-dermal junction in patients, we evaluated NeuroTAC in vivo. TSLP with BioresTAC TSLP Therapeutic effect on psoriasis. Using an Imiquimod (IMQ)-induced psoriasis mouse model, BALB / c mice were shaved and IMQ cream was applied to the back skin for 7 consecutive days. Then, NeuroTAC was intraperitoneally injected. TSLP 、BioresTAC TSLP, Ab-TSLP, methotrexate (MTX) or saline as control ( Figure 12 The results showed that compared with the positive control group, NeuroTAC TSLP or BioresTAC TSLP Treatment significantly reduced psoriasis severity, as demonstrated by improvements in clinical and pathological features, weight maintenance, decreased splenomegaly, reduced acanthosis, and significant improvement in overall disease severity, including skin thickness, erythema, and scaling. Figure 12 Of note, MTX alone did not significantly reduce weight loss, acanthosis, or PASI scores ( Figure 12 e), which highlights the superiority of LYTACs over traditional inhibitors. We further verified the ability of LYTACs to target TSLP in vivo ( Figure 12 j) and treatment effect ( Figure 12 In h,i), we detected the levels of TSLP and IL-17A in the back skin of mice. The results showed that NeuroTAC TSLP with BioresTAC TSLP Treatment significantly reduced TSLP and IL-17A levels in the lesions. In summary, our study demonstrated that LYTACs can efficiently degrade TSLP, exert potent anti-inflammatory effects, and successfully improve IMQ-induced psoriasis symptoms in mice.

[0065] Example 9 In vivo tumor targeting and safety assessment

[0066] Subsequently, we injected Cy5-labeled Ab-PD-L1 and NeuroTAC into the osteosarcoma mouse model intraperitoneally. PD-L1 and BioresTAC PD-L1 , and monitored its distribution in vivo to verify the targeting. The results showed that NeuroTAC PD-L1 and BioresTAC PD-L1 The highest accumulation in the tumor site was observed 12 hours after injection, and the signal lasted until 24 hours, which was significantly better than that of other groups. PD-L1 and BioresTAC PD-L1 The tumor fluorescence intensity of the Ab-PD-L1 group was significantly higher than that of the Ab-PD-L1 group. To exclude the passive targeting effect that may be caused by peptide functionalization, we quantitatively analyzed the fluorescence signal of the isolated organ and normalized it to the organ surface area (cm 2). The results showed that the accumulation of LYTACs in tumors and liver increased significantly, while no significant changes were observed in other organs. These results indicate that our LYTACs exhibited potent tumor targeting activity both in vitro and in vivo, and effectively inhibited tumor growth in an osteosarcoma mouse model. In addition, whole blood analysis of blood samples revealed no significant differences in the levels of multiple biochemical markers between the groups, indicating that LYTACs have good safety in cancer treatment. The overall results are shown in Figure 13 These data further confirm the safety and targeting advantages of LYTACs in tumor treatment.

[0067] The above-described embodiments merely illustrate several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. Persons skilled in the art will readily appreciate that variations and modifications may be made without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention.

Claims

1. A lysosomal targeting chimera based on Sortilin, characterized in that include: A ligand molecule of a lysosome targeting receptor and a ligand molecule targeting a protein to be degraded, connected by a linker; the lysosome targeting receptor is Sortilin, and the ligand molecule of the lysosome targeting receptor is a polypeptide targeting Sortilin; The linker is a polypeptide chain composed of a PEG chain, a carbon chain, and amino acids; The protein to be degraded is a membrane protein or an extracellular protein.

2. The lysosomal targeting chimera based on Sortilin according to claim 1, characterized in that The polypeptide targeting Sortilin is neurotensin or its analogs.

3. The lysosomal targeting chimera based on Sortilin according to claim 2, characterized in that The amino acid sequence of the neurotensin is shown in SEQ ID No.

1.

4. The lysosomal targeting chimera based on Sortilin according to claim 1, characterized in that The linker is an SMCC linker, a polyethylene glycol linker, a C2-C100 carbon chain, or a peptide chain composed of different numbers of amino acids, and the number of ethylene glycol units in the polyethylene glycol linker is 2-100.

5. The lysosomal targeting chimera based on Sortilin according to claim 1, characterized in that The polypeptide targeting Sortilin is a polypeptide targeting Sortilin with biological response, and an enzyme response sequence and an integrin receptor targeting sequence are connected to the polypeptide targeting Sortilin.

6. The lysosomal targeting chimera based on Sortilin according to claim 5, characterized in that The enzyme response sequence is matrix metalloproteinase, and the integrin receptor targeting sequence is RGD sequence.

7. The lysosomal targeting chimera based on Sortilin according to claim 5, characterized in that The amino acid sequence of the polypeptide targeting Sortilin connected with the enzyme response sequence and the integrin receptor targeting sequence is shown in SEQ ID No.

2.

8. Use of the lysosome-targeting chimera according to any one of claims 1 to 7 in the preparation of anti-tumor drugs or drugs for treating autoimmune system diseases.

9. The use according to claim 8, characterized in that The tumor is liver cancer, osteosarcoma, colon cancer, lung cancer, melanoma or breast cancer.

10. The use according to claim 8, characterized in that The autoimmune system disease is psoriasis.

Images