Chimeric polypeptide programmed targeted protein degradation technology

Abstract

Provided are a chimeric polypeptide used for degrading a target protein, and a method for treating a disease using the chimeric polypeptide. The chimeric polypeptide comprises a target protein binding motif, a lysosome transport receptor binding motif, and optionally a linker linking the motifs.

Description

Targeted protein degradation technology programmed by chimeric peptides Technical Field

[0001] The present invention belongs to the field of biomedicine. It relates to targeted protein degradation technology programmed by chimeric polypeptides. Specifically, the present invention provides a chimeric polypeptide for degrading a target protein, comprising a target protein binding motif, a lysosomal transport receptor binding motif, and optionally, a linker connecting the motifs.

[0002] Background of the Invention

[0003] Alzheimer's disease (AD) is the most common cause of dementia, affecting approximately 57 million people worldwide. 4 AD is characterized by the pathological features of senile plaques and neurofibrillary tangles in the brain, leading to progressive cognitive impairment. The discovery of β-amyloid (Aβ) peptide as the core component of AD senile plaques led to the development of the "amyloid cascade hypothesis", which proposes that abnormal deposition of Aβ in the brain is the core driver of AD pathogenesis. 5 Therefore, targeting Aβ has long been a key approach to developing AD. 6-9 Recently, anti-Aβ immunotherapy trials have provided clear evidence that reducing the Aβ load in patients' brains can slow the progression of early AD. 1,2 Despite clinical benefits, anti-Aβ immunotherapy in AD patients and mouse models often exacerbates Fc receptor (FcR)-induced inflammation and cerebral amyloid angiopathy (CAA) as side effects. 3 However, the remarkable beneficial effects of anti-Aβ immunotherapy clearly indicate that improving the clearance of brain Aβ has become a promising therapeutic strategy for AD.

[0004] In fact, the clearance of Aβ in the brain involves multiple pathways, including phagocytosis by microglia and astrocytes, proteolytic degradation by enzymes, and peripheral Aβ clearance. 10 Recent studies have found that high levels of Aβ can flow from the brain to the periphery. Methods to enhance peripheral Aβ clearance can help significantly reduce the Aβ burden in the brain and help alleviate AD pathology. 11-17 Furthermore, peripheral Aβ clearance is considered to be an easier and safer treatment approach than traditional central clearance methods that involve introducing agents into the brain. 10,18 However, peripheral Aβ-targeted clearance-mediating approaches are still under development and far from clinical translation.

[0005] Including LYTAC (lysosome-targeting chimera) 19、20 、MoDE-A (extracellular protein molecule degrader via asialoglycoprotein receptor) 21、KineTAC (cytokine receptor targeting chimera) 22 , integrin-mediated lysosomal degradation (IFLD) 23 Extracellular targeted protein degradation (eTPD) has recently been developed as a novel pharmaceutical approach for the selective removal of extracellular proteins, in which bispecific antibodies, conjugates, or small molecules recruit the extracellular protein of interest to cell surface endocytic receptors, thereby transporting it to the lysosome for degradation. 24 In addition, LYTAC has been used to clear pathologically relevant plasma PCSK9 protein in mice, which provides proof of concept that the eTPD method can effectively clear plasma proteins. 25 Despite these remarkable advances, current eTPD platforms still suffer from significant drawbacks, including complex chemical synthesis processes, heterogeneity of antibody conjugates, complex antibody engineering, and difficulty in genetic encoding.

[0006] Summary of the Invention

[0007] Here, the inventors developed a modular eTPD platform, termed synthetic peptide-programmed lysosomal targeting chimeras (SPYTACs), in which synthetic peptides mediate the formation of a ternary protein complex between low-density lipoprotein receptor-related protein 1 (LRP1) and a target protein. The synthetic peptides are highly modular and can universally and programmably capture a variety of pathogenic proteins. Following capture, the target protein is endocytosed via the LRP1 pathway and transported to the lysosome, where it is degraded by lysosomal proteases.

[0008] To validate the inventors' technology, the inventors focused on Aβ degradation in cells and mice. The inventors found that soluble proteins and cell surface proteins can be effectively endocytosed and degraded by cells expressing LRP1. In addition, the inventors found that SPYTAC-mediated eTPD acts mainly in a liver-specific manner in mice. Using the 5xFAD mouse model of AD, the inventors demonstrated that SPYTAC treatment can achieve comparable beneficial effects compared to early anti-Aβ antibody (lecanemab) treatment, but with minimal side effects. More importantly, the inventors found that SPYTAC can also improve cognitive function and learning behavior in late-stage 5xFAD mice. The inventors have developed a versatile, programmable platform for targeted degradation of extracellular and cell surface proteins under the guidance of modular synthetic peptides, and applied it to peripheral Aβ clearance using the hepatic lysosomal pathway to treat Alzheimer's disease.

[0009] Therefore, the present invention provides at least the following embodiments:

[0010] Embodiment 1. A chimeric polypeptide for degrading a target protein, comprising a target protein binding motif and a lysosomal transport receptor binding motif.

[0011] Embodiment 2. The chimeric polypeptide of embodiment 1, wherein the target protein binding motif is located at the N-terminus of the lysosomal transport receptor binding motif.

[0012] Embodiment 3. The chimeric polypeptide of embodiment 1 or 2, wherein the target protein binding motif and the lysosomal transport receptor binding motif are connected via a linker, such as a flexible peptide linker.

[0013] Embodiment 4. The chimeric polypeptide of embodiment 3, wherein the flexible peptide linker comprises (GSS) n wherein n is an integer selected from 1-10, preferably n is 1.

[0014] Embodiment 5. The chimeric polypeptide of any one of embodiments 1-4, wherein the target protein is a pathogenic polypeptide.

[0015] Embodiment 6. The chimeric polypeptide of embodiment 5, wherein the pathogenic polypeptide is an Aβ peptide, such as the Aβ shown in SEQ ID NO: 49. 1-42 .

[0016] Embodiment 7. The chimeric polypeptide of embodiment 6, wherein the target protein binding motif is an Aβ peptide binding motif, for example, the Aβ peptide binding motif comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-7, preferably, the Aβ peptide binding motif comprises the amino acid sequence shown in SEQ ID NO: 1.

[0017] Embodiment 8. The chimeric polypeptide of any one of embodiments 1-7, wherein the lysosomal trafficking receptor is selected from LRP1, ASGPR and CI-M6PR19, preferably, the lysosomal trafficking receptor is LRP1.

[0018] Embodiment 9. The chimeric polypeptide of embodiment 8, wherein the lysosomal transport receptor binding motif is an LRP1 binding motif, for example, the LRP1 binding motif comprises an amino acid sequence selected from any one of SEQ ID NOs: 9-16, preferably, the LRP1 binding motif comprises the amino acid sequence shown in SEQ ID NO: 9.

[0019] Embodiment 10. The chimeric polypeptide of any one of embodiments 6-9, wherein the chimeric polypeptide comprises the amino acid sequence of one of SEQ ID NOs: 17-32, preferably, the chimeric polypeptide comprises the amino acid sequence shown in SEQ ID NO: 17.

[0020] Embodiment 11. The chimeric polypeptide of any one of embodiments 1-10, wherein the chimeric polypeptide is a synthetic polypeptide.

[0021] Embodiment 12. An expression vector comprising a nucleotide sequence encoding the chimeric polypeptide of any one of embodiments 1-11 operably linked to an expression control sequence.

[0022] Embodiment 13. A pharmaceutical composition comprising the chimeric polypeptide of any one of embodiments 1-10 and / or the expression vector of embodiment 12, and a pharmaceutically acceptable carrier or excipient.

[0023] Embodiment 14. A method of treating a disease, comprising administering a therapeutically effective amount of the chimeric polypeptide of any one of embodiments 1-10 or a therapeutically effective amount of the pharmaceutical composition of embodiment 13 to a subject in need thereof.

[0024] Embodiment 15. The method of embodiment 14, wherein the target protein is Aβ peptide, and the disease is a disease caused by or associated with amyloid protein, such as Alzheimer's disease.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1: Development of the LRP1-mediated SPYTAC platform. a, Experimental workflow. c, Direct interaction of FITC-conjugated Aβ with SP1 measured by fluorescence polarization binding assay (n = three independent experiments). d, Binding of LRP1 to FITC-SP1 or FITC-SP4 measured by solid-phase binding (SPB) assay (n = 5 replicates). e, Immunoprecipitation using MOAB-2 (left) and LRP1 (right) antibodies in AD mouse brain and WT mouse liver lysates confirmed the ternary interaction of SPYTAC with Aβ and LRP1. f, Targeted delivery of LRP1-specific siRNA into HPG2 cells demonstrated reduced binding affinity between LRP1 and SPYTAC by immunoprecipitation. All data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

[0027] Figure 2: SPYTAC in vitro assay. a, Live-cell imaging of HepG2 and HeLa cells incubated with 1 μM FITC-streptavidin (FITC-SA) and 12 μM biotin-SP1 or biotin-SP3 at 37°C for 10 minutes. Scale bar, 10 μm. b, Quantification shows that the amount of internalized FITC-SA in HepG2 cells increased relative to HeLa cells after treatment with biotin-SP1 or biotin-SP3 (n = 30, ****p < 0.0001, unpaired two-tailed t-test). c, Live-cell imaging of HepG2 and HeLa cells incubated with 1 μM FITC-amyloid-β1-42 (FITC-Aβ1-42) and 12 μM SP1 or SP3 at 37°C for 10 minutes. Scale bar, 10 μm. d, Quantification shows an increase in the amount of FITC-Aβ1-42 internalized in HepG2 cells relative to HeLa cells after treatment with SP1 or SP3 (n = 30, ****p < 0.0001, unpaired two-tailed t-test). e, (Top) Schematic diagram of the designed minimal LRP1 (miniLRP1) construct. (Bottom) Quantification of FITC-SA internalization in HeLa cells ectopically expressing miniLRP1 after treatment with 12 μM biotin-SP2 (n = 30; ns, not significant; ****p < 0.0001, unpaired two-tailed t-test). f, Conformational representation of the SP2-miniLRP1 interaction using a pull-down assay. g, Confocal microscopy imaging of cell surface Aβ (csAβ) degradation in HepG2csAβ and HeLacsAβ cells following treatment with 12 μM SP1 or SP3 for 24 hours. h, Live-cell imaging of HepG2 cells incubated with 1 μM Cy5-anti-Myc-tag antibody and 12 μM SP21 or SP22 at 37°C for 10 minutes. Scale bar, 10 μm. i, Quantification shows an increase in the amount of Cy5-anti-Myc-tag antibody internalized in HepG2 cells treated with SP21 relative to SP22 (n = 30, ****p < 0.0001, unpaired two-tailed t-test). j, Live-cell imaging of HepG2 cells expressing secreted SP1 (sSP1) or sSP21 incubated with 1 μM FITC-Aβ1-42 at 37°C for 10 minutes. Scale bar, 10 μm. k, Quantification shows an increase in the amount of FITC-Aβ1-42 internalized in HepG2 cells transfected with sSP1 relative to sSP21 (n = 30, ****p < 0.0001, unpaired two-tailed t-test).

[0028] Figure 3: SPYTAC hijacks the liver for in vivo target protein degradation. a, Experimental workflow. b, c, Distribution (b) and determination (c) of Cy5.5-labeled Aβ42 in various organs by in vivo near-infrared imaging 15 minutes after injection (n=3 mice). d, Representative in vivo confocal images of Cy5.5-labeled SP systemically present in the liver, hepatocytes (green), and SP (red) of eGFP mice (n=3 mice). e, f, Representative images (e) and quantification (f) of Cy5.5-labeled SP (red) bound to Aβ (green) and targeted to hepatocytes (DAPI, blue). n=33 fields of view analyzed per condition, scale bar, 5 μm. g, Representative images of Cy5.5-labeled SP (red) bound to Aβ (green) and colocalized with lysosomes (Lamp1, white) in hepatocytes (DAPI, blue). Scale bars, 5 μm (top) and 1 μm (bottom). All data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.

[0029] Figure 4: SPYTAC is effective and safe in treating early-stage AD in 5×FAD mice. a, 5×FAD Veh , 5×FAD Lecam and 5×FAD SP Representative images of sulfo-S staining of mouse brains. Scale bar: 800 μm. b, Quantification of the area and number of Aβ plaques in each group (n = 5 mice per group). c, Representative images of 3 types of plaques. Scale bar: 6 μm. d, Quantification of the proportion of Aβ plaque types in each group (n = 5 mice, 100 plaques analyzed for each condition). e, Levels of Aβ42 in the plasma of 5×FAD mice after SPYTAC treatment (n = 4 mice). f, g, h, Levels of Aβ42 in the plasma of 5×FAD mice in the Morris water maze test. SP The cognitive impairment of mice was significantly improved. SPMice showed improved latency to locate the platform (f) and increased number of target crossings (g), without changes in swimming speed (g) (n = 9 to 11 mice per group). (h) Representative images of trajectories of mice on day 6 of the probe test. i, Representative images of trajectories during the novel object recognition (NOR) test (left), and percentage of time spent exploring familiar and novel objects during the NOR test (right). (n = 9 to 11 mice per group). j, Representative images and quantification of CAA pathology in 5×FAD mice expressing Aβ (Thio-S, green) and CD31 (red). n = 5 mice; scale bar, 20 μm. k, Levels of proinflammatory proteins in the brains of 5×FAD mice after SPYTAC or antibody treatment. n = 5 mice. All data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

[0030] Figure 5: SPYTAC can improve AD-type pathology and cognitive impairment in late-stage 5×FAD mice. a, Plasma Aβ42 levels in 5×FAD mice after SPYTAC treatment (n=3 mice). b, 5×FAD Veh , 5×FAD Lecam and 5×FAD SP Representative images of sulfo-S staining of mouse brains. Scale bar: 500 μm. c, Quantification of the area and number of Aβ plaques in each group (n = 5 mice per group). d, f, Quantification of Golgi staining (d) and the number of hippocampal spines (f) (n = 3 mice per group). One-way ANOVA was performed using Dunnett's multiple comparison test (P = 0.0099). **P = 0.0067, *P = 0.0359. Scale bar, 500 μm (upward direction), 10 μm (downward direction). g, h, i, In the Morris water maze test, 5×FAD SP The cognitive dysfunction of mice was significantly improved. SP Mice showed improved latency to locate the platform (g) and increased number of target crossings (h), without changes in swimming speed (h) (n = 8 to 10 mice per group). (i) Representative trajectory images of mice on day 6 of the probe test. j, Representative images of movement trajectories in the novel object recognition (NOR) test (left), and percentage of time spent exploring familiar and novel objects in the NOR test (right). (n = 8 to 10 mice per group). All data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

[0031] Figure 6: SPYTACs designed to bind LRP1 and Aβ in vitro. a, Binding of candidate peptides to Aβ42 (left) or LRP1 (right) was determined by SPB assay (three independent experiments; n = 6, *p < 0.05, ****p < 0.0001, unpaired two-tailed t-test). b, Representative dot blot analysis of LRP1 binding to SP1, SP2, SP3, and SP4, with BSA as a negative control. c, Representative dot blot analysis of Aβ42 binding to SP1 and SP4, with BSA as a negative control. d, Immunoprecipitation using HepG2, Huh-7, and HeLa cell lysates confirmed the binding of SP1 to LRP1, but not to ASGPR and CI-M6PR. e, Representative dot blot analysis showing the lack of binding of SP1, SP2, SP3, and SP4 to APSGPR1 and APSGPR2, with Aβ42 and BSA as positive and negative controls, respectively. f, Western blot evaluation of LRP1 protein expression after knockdown using siRNA in HepG2 cells.

[0032] Figure 7: SPYTAC delivers extracellular proteins to cells expressing LRP1. a, Live-cell imaging of HepG2 and HeLa cells incubated with 1 μM FITC-SA and 12 μM biotin-SP1, biotin-SP2, biotin-SP3, or biotin-SP4 at 37°C for 10 minutes. Scale bar, 10 μm. b, Quantification of internalized FITC-SA in HepG2 and HeLa cells treated with SP. (n = 30; ns, not significant; ****p < 0.0001, unpaired two-tailed t-test). c, Live-cell imaging of HepG2 and HeLa cells incubated with 1 μM FITC-Aβ1-42 and 12 μM SP1, SP2, SP3, or SP4 at 37°C for 10 minutes. Scale bar, 10 μm. d, Quantification of internalized FITC-Aβ1-42 in HepG2 and HeLa cells treated with SP. (n = 30, ****p < 0.0001, unpaired two-tailed t-test). e, Live-cell imaging of Huh-7 cells incubated with 1 μM FITC-SA or FITC-Aβ1-42 and 12 μM biotin-SP1, biotin-SP2, biotin-SP3, or biotin-SP4 for 10 minutes at 37°C. Scale bar, 10 μm. f, Quantification of internalized FITC-SA or FITC-Aβ1-42 in Huh-7 cells treated with biotin-SP. (n = 30, ****p < 0.0001, unpaired two-tailed t-test). g, Live-cell imaging of AML12 cells incubated with 1 μM FITC-SA or FITC-Aβ1-42 and 12 μM biotin-SP1 or biotin-SP3 for 10 minutes at 37°C. Scale bar, 10 μm. h, Quantification of internalized FITC-SA or FITC-Aβ1-42 in AML12 cells treated with SP (n=30, ****p<0.0001, unpaired two-tailed t-test).

[0033] Figure 8: Mini-LRP1-mediated protein uptake in HeLa cells. a, Schematic representation of six constructs of the human LRP1 component domains and Myc-tagged miniLRP1. b, Live-cell imaging of HeLa cells transiently expressing miniLRP1 after treatment with 1 μM FITC-SA and 12 μM biotin-SP2. Scale bar, 10 μm.

[0034] Figure 9: SPYAC promotes degradation of cell-surface Aβ42 in HepG2 cells. a, Schematic diagram of the lentiviral csAβ construct and the establishment of HepG2 and HeLa cell lines stably expressing csAβ. b, Plasma membrane localization of csAβ in HepG2csAβ and HeLacsAβ cells visualized using confocal microscopy. Scale bar, 10 μm. c, Time course of csAβ degradation in HepG2csAβ and HeLacsAβ cells incubated with 12 μM SP1 for 0 hour, 10 minutes, 1 hour, and 24 hours. Scale bar, 10 μm. d, Schematic diagram of the lenti-sSP construct. e, Target proteins added to the culture medium can be endocytosed by HepG2 cells and targeted to lysosomes for degradation mediated by secreted SP (sSP). f, Live cell imaging of HepG2 cells transfected with sSP1 or sSP21 and incubated with 1 μM Cy5 anti-Myc-tag antibody. Scale bar, 10 μm. g, Quantification of internalized Cy5-anti-Myc-tag antibody in HepG2 cells transfected with sSP1 or sSP21. (n = 30, ****p < 0.0001, unpaired two-tailed t-test). h, Time course of csAβ degradation in HepG2 csAβ cells transfected with sSP1 or sSP21, as assessed by confocal microscopy. Scale bar, 10 μm.

[0035] Figure 10: In vivo protein targeting of the liver by SPYTAC. a, b, Visualization (a) and quantification (b) of Cy5.5-SA in various organs by near-infrared imaging 15 minutes after SPYTAC injection (n = 3 mice). c, Representative confocal images showing the distribution of Cy5.5-SA in various organs of SPYTAC-treated EGFP mice. Scale bar, 800 μm. d, Representative in vivo confocal images showing the distribution of Cy5.5-SA in various organs of EGFP mice after SYPTAC treatment. Scale bar, 20 μm. e, Representative in vivo confocal images showing the distribution of Cy5.5-SA in the brain of EGFP mice after SPYTAC administration. Scale bar, 10 μm. f, Hepatocytes target Cy5.5-Aβ42 via SP1 but not SP4. Scale bar, 20 μm.

[0036] Figure 11: SPYTAC is effective and safe for treating early-stage AD in 5×FAD mice. a, Plasma Aβ42 levels in 5×FAD mice within 3 hours before and after SPYTAC treatment (n=5 mice, *p<0.05, unpaired two-tailed t-test). b, Plasma Aβ42 levels in 5×FAD mice within 3 hours before and after SPYTAC treatment (n=5 mice, *p<0.05, unpaired two-tailed t-test). veh , 5×FAD Lecam and 5×FAD SPRepresentative images of mouse brains stained with IHC (n = 5 mice). Scale bar, 800 μm. c, d, Open field test results of the three groups showed no difference in movement speed between the groups (n = 8 to 11 mice per group). e, 5× FAD veh , 5×FAD Lecam and 5×FAD SP Representative confocal images of abnormal activation of microglia (Iba1, red) in mouse brain (n = 5 mice). Scale bar, 20 μm.

[0037] Figure 12: SPYTAC ameliorates AD-type pathology and cognitive impairment in late-stage 5×FAD mice. a, MOAB-2 was used to treat 5×FAD veh and 5×FAD SP Representative images of mouse brains stained with IHC (n = 5 mice). Scale bar, 500 μm. b, 5× FAD veh , 5×FAD Lecam and 5×FAD SP Representative images of IHC staining of mouse brains (n ​​= 5 mice). Scale bar, 20 μm. c, Representative and quantitative blots of Aβ pathology-related markers from SYTAC-treated mice, normalized to GAPDH (n = 5 mice). d, Schematic diagram of the Y-maze (left), number of arm entries (middle), and percentage of alternations (calculated as (actual alternations / maximum alternations-2) × 100) (right) (n = 8 to 10 mice per group). e, f, Open field test results for the three groups showed no difference in movement speed between the groups (n = 8 to 10 mice per group).

[0038] Figure 13: a, b, Serum biochemical parameters and complete blood routine examination after 4 weeks of continuous SPYTAC treatment (n = 3 mice). c, H&E staining showed no difference in liver, spleen, lung, and heart after 4 weeks of SPYTAC treatment (n = 3 mice). d, 5×FAD after 4 weeks of continuous SPYTAC treatment ve h and 5×FAD SP Body weight of mice (n=8 to 10 mice per group).

[0039] Figure 14: a, Immunofluorescence staining of LRP1 in HeLa, HepG2, Huh-7, and AML12 cells. Scale bar, 10 μm.

[0040] Figure 15: SPYTAC can cross the blood-brain barrier (BBB) ​​to promote the degradation of extracellular proteins in the brain. (A) Visualization of the subcellular localization of FITC-SP1, FITC-SP4, and AF594-Aβ42 in mouse brain. Scale bar, 3 μm. (B) Quantification of the SPYTAC fluorescence signal shown in (A). (n=24; ****p<0.0001, one-way ANOVA with Tukey's correction). (C) pHrodotin was measured by live cell flow cytometry in mouse brain endothelial cells (bEnd.3) using biotin-SP1-4. TM Endocytosis of red avidin (10 μg / mL). (D) Endocytosis of red avidin (10 μg / mL) with biotin-SP1-4 and pHrodo TM Median fluorescence intensity (MFI) of red-affinity-treated bEnd.3 cells. MFI was determined by live cell flow cytometry. (n = 3; *p < 0.05, ****p < 0.0001, one-way ANOVA with Tukey's correction). (E) In U251 cells, the median fluorescence intensity (red is LysoTracker) of the bEnd.3 cells was determined by colocalization (red is LysoTracker). TM ) Live cell imaging showing SPYTAC-mediated FITC-SA uptake associated with lysosomal degradation. Scale bar, 10 μm. (F) SPYTAC-mediated pHrodo uptake in U251 cells as determined by flow cytometry analysis. TM Red avidin endocytosis. (n = 3; ****p < 0.0001, one-way ANOVA with Tukey's correction). (G) (Top) Schematic diagram of SPYTAC transmembrane transport in an in vitro BBB model, with bEnd.3 cells seeded in the apical chamber and U251 cells seeded in the basolateral chamber. SPYTAC in the apical chamber is able to cross the BBB and be internalized by U251 cells in the basolateral chamber. (Bottom) Live cell imaging of SPYTAC-mediated FITC-SA uptake by U251 cells in the basolateral chamber. (H) MFI of U251 cells treated with biotin-SP1, biotin-SP4, and PBS (negative control) in an in vitro BBB model. MFI was determined by live cell flow cytometry. (n = 3; ****p < 0.0001, one-way ANOVA with Tukey's correction).

[0041] Figure 16: SPYTAC exhibits LRP1-dependent blood-brain barrier (BBB) ​​penetration. (A) Representative intravital confocal images show the distribution of Cy5.5-SA in the brain of EGFP mice after SPYTAC administration. Scale bar, 10 μm. (B) (Top) Subcellular distribution of FITC-SP1 in microglia (Iba1), astrocytes (GFAP), and neurons (NeuN) in the mouse brain. (Bottom) Immunofluorescence staining of LRP1 with Iba1, LRP1 with GFAP, and LRP1 with NeuN in microglia, astrocytes, and neurons in the mouse brain, respectively. Scale bar, 5 μm. (C) Immunofluorescence staining of LRP1 in bEnd.3, U251, C8-D1A, BV2, and N2A cell lines. Scale bar, 10 μm. (DI) Endocytosis of FITC-SA in C8-D1A (D), BV2 (E), and N2A (F) cells using biotin-SP1-4. C8-D1A, BV2, and N2A cells were treated with 12 μM biotin-SP1-4 and 1 μM FITC-SA at 37°C for 10 min and then stained with LysoTracker. TM (Red) Marker. Scale bar, 10 μm. Incubated at 37°C with biotin-SP1-4 and pHrodo TM Median fluorescence intensity (MFI) of C8-D1A (G), BV2 (H), and N2A (I) cells incubated with Red Avidin for 30 minutes. MFI was determined by live cell flow cytometry. (n = 3; ns, not statistically significant, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Tukey's correction). (J) (Top) Schematic illustration of SPYTAC-mediated Aβ uptake in an in vitro BBB model, in which bEnd.3 cells are seeded in the apical chamber and U251 cells are seeded in the basolateral chamber. SPYTAC in the apical chamber crosses the BBB and targets Aβ to U251 cells in the basolateral chamber. (Bottom) U251 cells in the basolateral chamber respond to SPYTAC-mediated biotin-Aβ42 / pHrodo TM MFI of Red Avidin uptake. MFI was determined by live cell flow cytometry. (n = 3; ****p < 0.0001, one-way ANOVA with Tukey's correction).

[0042] Detailed Description of the Invention

[0043] definition

[0044] In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the corresponding fields and are common procedures. All patent and non-patent literature described herein are incorporated by reference in their entirety.

[0045] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0046] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded polymers of RNA or DNA that optionally contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single-letter designations as follows: "A" for adenosine or deoxyadenosine (RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide. Although nucleotide sequences herein may be presented as DNA sequences (including T), when reference is made to RNA, one skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0047] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0048] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0049] Chimeric polypeptides for degradation of target proteins

[0050] In some aspects, the present invention provides a chimeric polypeptide for degrading a target protein, comprising a target protein binding motif and a lysosomal transport receptor binding motif.

[0051] In some embodiments, the target protein binding motif is located at the N-terminus of the lysosomal transport receptor binding motif.

[0052] In some embodiments, the target protein binding motif can be directly linked to the lysosomal transport receptor binding motif.

[0053] In some embodiments, the target protein binding motif and the lysosomal transport receptor binding motif can be connected via a linker.

[0054] The linker described herein can be a flexible peptide linker. The flexible peptide linker can connect different functional parts / motifs in a fusion protein / fusion polypeptide so that the different functional parts / motifs retain their respective functions / activities. A variety of suitable such flexible peptide linkers are known in the art, and those skilled in the art can easily obtain and / or determine suitable flexible peptide linkers.

[0055] In some embodiments, the flexible peptide linker comprises (GSS) n wherein n is an integer selected from 1 to 10, preferably n is 1. In some specific embodiments, the flexible peptide linker is GSS.

[0056] As used herein, a "target protein" refers to any protein or polypeptide that is desired to be degraded via a proteolytic pathway within a cell. The target protein may be a free protein or polypeptide, such as a protein or polypeptide secreted outside the cell, or a cellular protein fragment. Alternatively, the target protein may be a protein or polypeptide expressed on the cell surface, such as a membrane protein.

[0057] In some preferred embodiments, the target protein is a pathogenic polypeptide.

[0058] Examples of pathogenic peptides include, but are not limited to, Aβ peptide. β-Amyloid (Aβ) peptide is a 39-43 amino acid peptide produced by the hydrolysis of amyloid precursor protein (APP) by β- and γ-secretases. The presence of Aβ plaques in the brain is a hallmark of Alzheimer's disease.

[0059] In some embodiments, the Aβ peptide is a soluble Aβ peptide. In some embodiments, the Aβ peptide is an aggregated Aβ peptide. In some embodiments, the target protein is Aβ 1-42 In some embodiments, the Aβ 1-42 Comprising the amino acid sequence shown in SEQ ID NO:49.

[0060] As used herein, a "target protein binding motif" refers to a polypeptide that specifically binds to a target protein of interest. In some embodiments, the target protein binding motif does not substantially bind to proteins other than the target protein. In some embodiments, the target protein binding motif is expressed in a number of molecules less than 1×10 -7 M, preferably less than 1×10 -8 M, more preferably less than 1×10 -9 M, more preferably less than 1×10 -10 M, more preferably less than 1×10 -11 M, more preferably less than 1×10 -12 The KD value of M specifically binds to the target protein.

[0061] The target protein binding motif may be about 4 to about 100 amino acids in length. Preferably, the target protein binding motif is no more than 100 amino acids in length, no more than 50 amino acids in length, no more than 40 amino acids in length, no more than 30 amino acids in length, no more than 20 amino acids in length, no more than 10 amino acids in length, or no more than 5 amino acids in length.

[0062] In some embodiments, the target protein binding motif is an Aβ peptide binding motif. In some embodiments, the Aβ peptide binding motif comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-7. Preferably, the Aβ peptide binding motif comprises the amino acid sequence shown in SEQ ID NO: 1.

[0063] As used herein, "lysosomal transport receptor" refers to a receptor on the cell surface that, after specifically binding to a corresponding ligand (e.g., a polypeptide ligand), can cause the ligand or a protein / polypeptide comprising the ligand to be endocytosed into the interior of the cell and transported to the intracellular lysosome for degradation. Examples of lysosomal transport receptors include, but are not limited to, LRP1, ASGPR, and CI-M6PR19. In some preferred embodiments, the lysosomal transport receptor is LRP1.

[0064] As used herein, "lysosomal transport receptor binding motif" refers to a polypeptide that can specifically bind to a lysosomal transport receptor, which can be internalized into the cell and transported to the intracellular lysosome after specifically binding to the lysosomal transport receptor, and then degraded. In some embodiments, the lysosomal transport receptor binding motif does not substantially bind to proteins other than the lysosomal transport receptor. In some embodiments, the lysosomal transport receptor binding motif is expressed in less than 1×10 -7 M, preferably less than 1×10 -8 M, more preferably less than 1×10 -9 M, more preferably less than 1×10 -10 M, more preferably less than 1×10 -11M, more preferably less than 1×10 -12 The KD value of M specifically binds to lysosomal trafficking receptors.

[0065] The length of the lysosomal transport receptor binding motif can be about 4 to about 100 amino acids. Preferably, the length of the lysosomal transport receptor binding motif is no more than 100 amino acids, no more than 50 amino acids, no more than 40 amino acids, no more than 30 amino acids, no more than 20 amino acids, no more than 10 amino acids, or no more than 5 amino acids.

[0066] In some embodiments, the lysosomal transport receptor binding motif is an LRP1 binding motif. In some embodiments, the LRP1 binding motif comprises an amino acid sequence selected from any one of SEQ ID NOs: 9-16. Preferably, the LRP1 binding motif comprises the amino acid sequence shown in SEQ ID NO: 9.

[0067] In some embodiments, the chimeric polypeptide is a synthetic polypeptide. In some embodiments, the chimeric polypeptide can be produced by chemical synthesis. Methods for producing polypeptides by chemical synthesis are known in the art. The short length of the chimeric polypeptides of the present invention is particularly suitable for chemical synthesis and is advantageous for pharmaceutical preparation.

[0068] In some embodiments, the chimeric polypeptide can be produced recombinantly. The chimeric polypeptide can be recombinantly produced by introducing an expression vector containing a coding sequence encoding the chimeric polypeptide into a suitable host cell. To facilitate secretory expression, isolation, purification, or identification of the fusion polypeptide, a suitable signal peptide or tag can also be added to the chimeric polypeptide. The sequences of such signal peptides or tags are known in the art and can be used as needed by those skilled in the art.

[0069] An exemplary signal peptide comprises the amino acid sequence shown in MNSFSTSAFGPVAFSLGLLLVLPAAFPAP (IL6_sp, SEQ ID NO: 50). Exemplary tags include, but are not limited to, Myc tags, His tags, and the like.

[0070] In some specific embodiments, the chimeric polypeptide of the present invention comprises the amino acid sequence of one of SEQ ID NOs: 17 to 34. In some preferred embodiments, the chimeric polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 17.

[0071] In another aspect, the present invention provides a method for degrading a target protein, the method comprising contacting a chimeric polypeptide of the present invention, the target protein, and a cell expressing the lysosomal transport receptor, thereby causing the target protein to be internalized into the cell and degraded by the lysosome. In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method.

[0072] 3. Polynucleotides, Expression Vectors, and Host Cells

[0073] In another aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a chimeric polypeptide of the present invention.

[0074] In another aspect, the present invention provides an expression vector comprising the polynucleotide of the present invention operably linked to an expression control sequence.

[0075] The vectors used in the expression constructs of the present invention include those that replicate autonomously in the host cell, such as plasmid vectors; and also include vectors that can be integrated into the host cell DNA and replicated together with the host cell DNA. Many vectors suitable for the present invention are commercially available.

[0076] "Expression control sequence" and "expression control element" are used interchangeably to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that affect the transcription, RNA processing or stability, or translation of the sequence of interest. Expression control sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0077] As used herein, the term "operably linked" refers to a regulatory sequence that is linked to a nucleotide sequence of interest such that transcription of the nucleotide sequence of interest is controlled and regulated by the regulatory sequence. Techniques for operably linking regulatory sequences to nucleotide sequences of interest are known in the art.

[0078] In another aspect, the present invention provides a host cell comprising a polynucleotide of the present invention or transformed with an expression vector of the present invention, wherein the host cell is capable of expressing the chimeric polypeptide of the present invention. Preferably, the host cell is a recombinant host cell.

[0079] Host cells that can be used to express the polypeptides or fusion proteins of the present invention include prokaryotes, yeast, and higher eukaryotic cells.

[0080] The recombinant expression vectors of the present invention can be introduced into host cells by any of a number of well-known techniques, including but not limited to heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, liposome-mediated transfection, calcium phosphate precipitation, protoplast fusion, microprojectile bombardment, viral transformation, and the like.

[0081] In another aspect, the present invention provides a method for recombinantly producing a chimeric polypeptide of the present invention, comprising:

[0082] a) culturing the host cell of the present invention under conditions allowing expression of the chimeric polypeptide of the present invention;

[0083] b) obtaining the chimeric polypeptide expressed by the host cell from the culture obtained in step a); and

[0084] c) optionally further purifying the chimeric polypeptide obtained from step b).

[0085] Pharmaceutical compositions and disease treatments

[0086] In another aspect, the present invention also provides a pharmaceutical composition comprising a chimeric polypeptide of the present invention for degrading a target protein and / or an expression vector of the present invention, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is used to treat a disease in a subject. The specific disease treated depends on the target protein. For example, when the target protein is an Aβ polypeptide, the disease may include Alzheimer's disease (as described below).

[0087] Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants, or emulsifiers, or combinations thereof. The amount of a pharmaceutically acceptable carrier in a pharmaceutical composition can be determined experimentally based on the activity of the carrier and the desired properties of the formulation, such as stability and / or minimal oxidation.

[0088] In another aspect, the present invention provides a method for treating a disease using a chimeric polypeptide of the present invention for degrading a target protein, the method comprising administering a therapeutically effective amount of a chimeric polypeptide of the present invention or a pharmaceutical composition of the present invention to a subject in need thereof. The specific disease treated depends on the target protein. For example, when the target protein is an Aβ polypeptide, the disease may include Alzheimer's disease (as described below).

[0089] As used herein, "treating" a subject having a disease means that the subject's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or the development of the disease.

[0090] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition. As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates, or improves the symptoms of a disease or condition, or cures the disease or condition.

[0091] The dosage regimen for utilizing the chimeric polypeptide is selected based on a variety of factors, including, for example, the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the specific chimeric polypeptide or salt thereof used. An ordinarily skilled physician can readily determine and prescribe the effective amount of the composition required to prevent, counter, or inhibit the progression of the condition.

[0092] Exemplary treatment regimens require administration once a day, once every two days, once a week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, once every three to six months, or a slightly shorter initial dosing interval (e.g., once a week to once every three weeks) followed by a longer dosing interval (e.g., once a month to once every three to six months). The frequency and interval of administration can be determined by those skilled in the art based on the pharmacokinetic parameters of the chimeric polypeptide.

[0093] The "subject" herein can be a mammal, including but not limited to a rodent such as a mouse or a rat, a primate such as a monkey or a human. Preferably, the subject is a human.

[0094] Targeted degradation of Aβ peptide

[0095] In some aspects, the present invention provides a chimeric polypeptide for degrading Aβ peptide, comprising an Aβ peptide binding motif and a lysosomal trafficking receptor binding motif.

[0096] In some embodiments, the Aβ peptide binding motif is located at the N-terminus of the lysosomal transport receptor binding motif. In some embodiments, the Aβ peptide binding motif can be directly linked to the lysosomal transport receptor binding motif.

[0097] In some embodiments, the Aβ peptide binding motif and the lysosomal transport receptor binding motif can be connected via a linker, such as a flexible peptide linker.

[0098] In some embodiments, the flexible peptide linker comprises (GSS) n wherein n is an integer selected from 1 to 10, preferably n is 1. In some specific embodiments, the flexible peptide linker is GSS.

[0099] In some embodiments, the Aβ peptide is a soluble Aβ peptide. In some embodiments, the Aβ peptide is an aggregated Aβ peptide. In some embodiments, the Aβ peptide is an Aβ peptide. 1-42 In some embodiments, the Aβ 1-42 Comprising the amino acid sequence shown in SEQ ID NO:49.

[0100] In some embodiments, the Aβ peptide binding motif comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 7. Preferably, the Aβ peptide binding motif comprises the amino acid sequence shown in SEQ ID NO: 1.

[0101] In some embodiments, the lysosomal transport receptor binding motif is an LRP1 binding motif. In some embodiments, the LRP1 binding motif comprises an amino acid sequence selected from any one of SEQ ID NOs: 9-16. Preferably, the LRP1 binding motif comprises the amino acid sequence shown in SEQ ID NO: 9.

[0102] In some embodiments, the chimeric polypeptide is a synthetic chimeric polypeptide. In some embodiments, the chimeric polypeptide can be produced recombinantly. In some specific embodiments, the chimeric polypeptide of the present invention comprises the amino acid sequence of one of SEQ ID NOs: 17-32. In some preferred embodiments, the chimeric polypeptide of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0103] In another aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a chimeric polypeptide for degrading Aβ peptide of the present invention.

[0104] In another aspect, the present invention provides an expression vector comprising a polynucleotide operably linked to an expression control sequence, wherein the polynucleotide comprises a nucleotide sequence encoding a chimeric polypeptide for degrading Aβ peptide of the present invention.

[0105] In another aspect, the present invention also provides a pharmaceutical composition for reducing Aβ peptide levels in a subject, comprising a chimeric polypeptide for degrading Aβ peptide of the present invention and / or an expression vector comprising a nucleotide sequence encoding a chimeric polypeptide for degrading Aβ peptide of the present invention, and a pharmaceutically acceptable carrier or excipient.

[0106] In another aspect, the present invention provides a method for reducing Aβ peptide levels in a subject, comprising administering to the subject a therapeutically effective amount of a chimeric polypeptide for degrading Aβ peptide of the present invention, or a therapeutically effective amount of a pharmaceutical composition for reducing Aβ peptide levels in a subject of the present invention.

[0107] In some embodiments, the pharmaceutical composition or method is for reducing Aβ peptide levels in peripheral blood of a subject. In some embodiments, the pharmaceutical composition or method is for reducing Aβ peptide levels in the brain of a subject.

[0108] In some embodiments, Aβ peptide levels may be reduced by at least 10%, at least 25%, at least 50%, at least 75%, or by at least 1-fold, at least 2-fold, at least 3-fold, or more compared to a control.

[0109] In another aspect, the present invention provides a method for treating a disease caused by or associated with amyloid protein in a subject, the method comprising administering to the subject a therapeutically effective amount of the chimeric polypeptide for degrading Aβ peptide of the present invention, or a therapeutically effective amount of the pharmaceutical composition for reducing Aβ peptide levels in a subject of the present invention.

[0110] In another aspect, the present invention provides use of the chimeric polypeptide for degrading Aβ peptide of the present invention, or the pharmaceutical composition for reducing Aβ peptide levels in a subject of the present invention in the preparation of a medicament for treating diseases caused by or associated with amyloid protein.

[0111] Diseases caused by or associated with amyloid as described herein include, but are not limited to, amyloidosis; neurological disorders such as Alzheimer's disease (AD); diseases characterized by loss of cognitive memory ability, such as mild cognitive impairment (MCI), Lewy body dementia, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson-dementia syndrome; and other diseases based on or associated with amyloid, such as progressive supranuclear palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and macular degeneration. In some specific embodiments, the disease is Alzheimer's disease (AD). Example

[0112] The present invention can be further understood by reference to the specific embodiments described herein, which are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Obviously, those skilled in the art will appreciate that various modifications and variations can be made to the present invention without departing from the spirit of the present invention, and such modifications and variations also fall within the scope of the present invention.

[0113] Methods and Materials:

[0114] peptides

[0115] Peptides (Table 1) were synthesized by TGpeptide (Nanjing, China). For biotinylated peptides, biotin was conjugated to the N-terminal residue of the peptide. Peptide purity was assessed by mass spectrometry to ensure ≥95%. All peptides were dissolved in phosphate-buffered saline (PBS) or dimethyl sulfoxide (DMSO) and stored at -80°C.

[0116] Table 1. Synthetic peptides used in the SPP-LYTAC test

[0117] # Insoluble in DMSO and PBS.

[0118] Plasmid construction and lentivirus production

[0119] DNA sequences encoding cell-surface Aβ (csAβ), secreted SPYTAC peptides, and other sequences were designed and synthesized de novo using oligonucleotides and constructed into the pLVX-mTagBFP2-CMV-pA backbone by seamless in vitro assembly. Lentivirus was produced by co-transfecting HEK293T cells with the pLVX plasmid and vectors encoding packaging proteins (pMD2.G and psPAX2). Viral supernatant was collected 48 hours after transfection, filtered through a 0.45 μm filter, and used immediately for transduction.

[0120] animal

[0121] C57BL / 6J mice were purchased from SPF Biotechnology Co., Ltd., and 5xFAD transgenic mice (B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / Mmjax,34840-JAX) were purchased from The Jackson Laboratory. Given the sex differences in AD neuropathology, only male C57BL / 6 and 5xFAD mice were used in this experiment. All mice were housed at the Laboratory Animal Center of the Institute of Zoology under identical conditions (12-h light-dark cycle, five mice per cage, free access to food and water). All experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences.

[0122] Cell culture and transfection

[0123] All cell lines were grown and maintained at 37°C and 5% CO2. HEK293T, HeLa, HepG2, and Huh-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin / streptomycin (Gibco). AML12 cells were cultured in DMEM / F12 (Gibco) supplemented with 10% FBS, 10 μg / mL insulin (Roche), 5.5 μg / mL transferrin (Sigma), 5 ng / mL selenium (Sigma), 40 ng / mL dexamethasone (Sigma), and 1% penicillin / streptomycin. Mycoplasma negative results were routinely ensured. Cells were transiently transfected with plasmids using Lipofectamine LTX (Invitrogen) according to the manufacturer's instructions.

[0124] Western blotting

[0125] Cultured cells or mouse tissues were homogenized in ice-cold RIPA lysis and extraction buffer (89901; Thermo) supplemented with a protease inhibitor cocktail (78439; Thermo). Protein concentration was determined using a BCA assay kit (23250; Thermo). Protein samples were separated by 10%–12% SDS-PAGE, blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore), blocked in 5% milk for 60 min, and incubated with mouse monoclonal anti-β-actin (1:4,000; A5441; Sigma), rabbit polyclonal anti-Iba1 (1:3,000; 019-19741; Wako), rabbit polyclonal anti-ASGPR1 (1:3,000; ab127896; Abcam), and rabbit monoclonal anti-LRP1 (1:4,000; A5441; Sigma). The membranes were incubated with monoclonal anti-CI-M6PR (1:6000; ab92544; Abcam), rabbit monoclonal anti-CI-M6PR (1:1000; ab134153; Abcam), mouse monoclonal anti-β-amyloid Moab-2 (1:3000; ab126649; Abcam), mouse monoclonal anti-NeuNm (1:3000; ab104224; Abcam), and mouse monoclonal anti-GAPDH (1:4000; AF0006; Beyotime) antibodies at 4°C overnight. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies for 2 hours at room temperature. Immunoreactive bands were detected using enhanced chemiluminescence reagent (ECL, Pierce) and quantified using ImageJ software.

[0126] Enzyme-linked immunosorbent assay (ELISA)

[0127] Plasma Aβ1-42 levels in mice were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, USA) according to the manufacturer's instructions. Levels of proinflammatory cytokines TNF-α, IFN-γ, IL-1β, and IL-6, as well as anti-inflammatory cytokines IL-4 and IL-10, were measured in brain homogenates and blood using ELISA kits (eBioscience, USA). Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and soluble LRP-1 (sLRP-1) were measured using ELISA kits (Jiangsu Jingmei Biotechnology Co., Ltd., China).

[0128] Solid phase binding assay

[0129] Solid-phase binding was performed using an enzyme-linked immunosorbent assay (ELISA) in 96-well plates. Streptavidin-coated 96-well plates (ACROBiosystems, SP-11-5 plates) were washed three times with 0.05% PBST (PBS containing 0.1% Tween 20) and blocked with 1% BSA for 1 hour at 37°C. Subsequently, purified SPYTAC peptide (synthetic, 50 mg / ml) was added and incubated at 37°C for 1 hour. After washing, LRP1 (KMD, KMH265) or Aβ protein (Anaspec, AS-20276) was added and protein binding was allowed to proceed at 37°C for 1 hour. After washing, the plates were incubated with primary antibodies against LRP1 (1:2000; ab53444; Abcam) or MOAB-2 (1:2000; ab126649; Abcam) for 2 hours at 37°C. After washing, each secondary antibody (1:2000) was added and incubated at 37°C for 2 hours. Subsequently, streptavidin-Poly-HRP40 conjugate was incubated at 37°C for 30 minutes. Finally, TMB reagent was used for color development, and the absorbance at 450 nm was recorded.

[0130] Co-immunoprecipitation (Co-IP)

[0131] The interaction between SPYTAC and endogenous Aβ or LRP1 was determined using lysates from 5×FAD mouse brain tissue, wild-type (WT) mouse liver tissue, or cell lines. Tissues or cells were lysed in Tris-buffered saline (TBS) containing 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Nonidet P40 surrogate (T-DET O-9), and protease inhibitors (Roche), and the lysates were incubated overnight at 4°C. Biotinylated candidate peptides / BeyoMag TMStreptavidin magnetic beads. The beads were washed four times in TBS containing Tween 20 (TBST) and boiled in SDS-PAGE loading buffer or a buffer containing 0.2% SDS and 0.1% Tween-20. Samples were analyzed by immunoblotting.

[0132] Dot blot

[0133] SP1, SP2, SP3, and SP4 peptides diluted in PBS were spotted onto PVDF membranes using a dot blot manifold (GE Healthcare). The membrane strips were blocked with 5% milk in PBS for 2 hours at room temperature and incubated with tissue lysis buffer (20 mg / mL in PBS, BSA as a control) at 4°C overnight. After washing three times with 0.1% PBST, bound peptides were detected using primary antibodies against human LRP1 (1:2000; ab53444; Abcam) and MOAB-2 (1:2000; ab126649; Abcam), followed by incubation with streptavidin-polyHRP40 conjugate (1:2000). Signals were detected using ECL reagents.

[0134] Confocal microscopy imaging and analysis

[0135] Images were acquired in Z-scan format using a ZEISS 780 / 880 confocal laser scanning microscope equipped with x20 and x40 objectives. For Aβ plaque quantification, at least 18 views in the cortex or hippocampus were counted. Images were processed and analyzed using ImageJ software or Imaris software (Bitplane, Switzerland). Imaris was used for 3D rendering of confocal images for colocalization and quantification analysis.

[0136] In vivo SPYTAC administration

[0137] SPYTAC, anti-Aβ monoclonal antibodies, and corresponding mIgG controls were administered via intraperitoneal (ip) injection at a dose of 40 mg / kg.

[0138] Hematoxylin and eosin (H&E) staining

[0139] Paraffin-embedded tissue was cut into 5 mm thick sections using a rotary microtome. Sections were dewaxed in xylene and rehydrated in graded alcohols (100%, 100%, 95%, 80%, 70%), incubated in hematoxylin solution, rinsed in running water to remove excess hematoxylin, differentiated in 1% acid alcohol for 5-10 seconds, and then rinsed in running water for 10 minutes. Finally, sections were stained with eosin, dehydrated in graded ethanol and xylene, and mounted with Cytoseal-60 (Stephens Scientific).

[0140] Thioflavin-S staining

[0141] For Aβ plaque detection, brain sections were pre-incubated with 0.1% Thioflavin S (Sigma) in 50% ethanol in the dark for 5 minutes, then washed twice with 50% ethanol and three times with PBS before antibody staining.

[0142] Golgi staining

[0143] Golgi staining was performed according to the manufacturer's protocol (Hito Golgi-Cox OptimStain TM The density of spines in CA1 pyramidal neurons was assessed using a 40x kit (see kit, USA). Dendritic spine counts were estimated blindly at high magnification (40x). Pyramidal neurons were selected from three fields scattered across the CA1 region within each brain slice. Three consecutive brain slices encompassing the CA1 region were selected from each mouse.

[0144] Immunohistochemistry (IHC)

[0145] Mice were anesthetized with 2.5% avertin (200 mg / kg body weight), then perfused with cold PBS, and then perfused with 4% paraformaldehyde (PFA). Subsequently, the mouse brain was taken out and post-fixed overnight with 4% PFA, then dehydrated with 30% sucrose. Finally, the brain was coronally sliced ​​into 40 μm thickness using a cryostat (Leica SM2010R) and stored in a cryoprotectant storage solution (125 mL ethylene glycol, 125 mL glycerol, and 150 mL 0.1 M PB) at -20 ° C until use. For immunohistochemical staining, the sections were washed 3 times with PBS, then blocked at room temperature for 2 hours with 5% BSA, 1% TritonX-100 in PBS, and then incubated overnight at 4 ° C with a primary antibody (in a PBS solution of 1% BSA, 0.2% TritonX-100). The primary antibodies used included rabbit polyclonal anti-Iba1 (1:1000; 019-19741; Wako), goat rabbit polyclonal anti-Iba1 (1:500; ab5076; abcam), rabbit monoclonal anti-GFAP (1:1000; ab209064; abcam), mouse monoclonal anti-NeuN (1:500; 14-0112; eBioscience), rabbit polyclonal anti-CD31 (1:500; ab8021; abcam), mouse monoclonal anti-MOAB-2 (1:2000; ab126649; abcam), and rat monoclonal anti-LPR1 (1:500; ab53444; abcam). After washing, sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 / 568 / 594 / 647 in blocking solution containing DAPI (Invitrogen).

[0146] Morris water maze test

[0147] The water maze is a circular pool of water (120 cm in diameter and 60 cm in height) with a white inner surface. The escape platform is fixed in the center of a quadrant and submerged 1 cm below the water surface. During training, mice are allowed to navigate in the water tank to find the hidden platform. If the mouse fails to find the platform within 60 seconds, it is gently guided to the platform and allowed to stay there for 25 seconds. Each mouse undergoes eight training trials starting from a different quadrant every day for 5 days. The test is performed 24 hours after the last training trial. The test process is a single probe test in which the platform is removed and the mouse is allowed to swim in the water tank for 60 seconds. Behavior is analyzed by video tracking software (EthoVision, Noldus, the Netherlands). The delay in finding the platform during the training trial and the time spent in each quadrant during the test are recorded.

[0148] Open field test

[0149] The open field test reflects the exploration and anxiety behavior of the new environment and is based on the idea that mice naturally prefer to be close to the protective wall rather than being exposed to the danger of the open field. The test is carried out in a square open field box, which includes an inner square (150mm×150mm) as the "center area" and an outer square as the "surrounding area". Each mouse is gently placed on the floor and allowed to freely explore the area for 10 minutes to study their spontaneous movement activity. The total time spent, the distance traveled, and the movement trajectory of the center and surrounding areas are all measured by the tracking system.

[0150] Y-maze test

[0151] The Y-maze apparatus consists of three 30 cm long radial arms (called the starting arm, the new arm, and the other arms) that form a "Y" shape starting from a central space. Based on the rodents' innate curiosity to explore new areas, mice were placed in the starting arm to explore the maze. In brief, mice were placed in the starting arm for exploration and allowed 5 minutes to freely locate the new arm using spatial cues (training period). After a 2-hour interval, the mice were placed in the Y-maze again as part of a training period protocol to assess spatial memory. Time, distance, entry time, and movement trajectory were recorded by an automatic video tracking system.

[0152] Statistical analysis

[0153] Data are presented as mean ± SEM (standard error of the mean). Statistical significance was determined by two-tailed Student's t-test or one-way ANOVA followed by Dunn's test, and p < 0.05 was considered statistically significant.

[0154] Example 1: LRP1-mediated SPYTAC platform

[0155] SPYTAC is a synthetic peptide-programmed TPD system consisting of an endogenous lysosomal transport receptor and a synthetic peptide (Figure 1a). The synthetic peptide acts as a bispecific linker and contains three motifs: an N-terminal target protein binding motif, a C-terminal receptor binding motif, and a GSS linker for increased peptide flexibility (Figure 1a and Table 1). To facilitate tracking of the peptide, a biotin or fluorescein isothiocyanate (FITC) moiety is conjugated to the N-terminal residue.

[0156] The SPYTAC of this embodiment is designed to selectively bind to Aβ while targeting LRP1, a well-known lysosomal transport receptor. (Figure 1a). The inventors selected a group of Aβ-binding peptides and LRP1-binding peptides and created a series of different chimeras (Table 1). In vitro binding assays determined the relative binding abilities of these chimeras and showed various affinities for Aβ peptides and LRP1 proteins, except that some chimeras were insoluble (Figure 6a and Table 1). The inventors selected synthetic peptide 1 (SP1) for further characterization because SP1 outperformed other chimeras in the inventors' tests (Figure 6a), while SP2, SP3, and SP4 served as negative controls because they are mutants designed to eliminate the ability to bind to Aβ or / and LRP1 (Figure 1b). In vitro fluorescence polarization assays showed that FITC-Aβ and FITC-SP1 fluorescence polarization increased in an S-shape with increasing amounts of SP1 and LRP1, respectively (Figure 1c).

[0157] To further validate the ternary interaction of SPYTAC with Aβ and LRP1, the present inventors performed solid phase binding assays, pull-down, and dot blot analysis of SPYTAC by using immobilized biotinylated SP to capture LRP1 in liver lysates expressing LRP1 and Aβ in brain lysates expressing amyloid precursor protein (APP), and demonstrated that only SP1 could simultaneously target LRP1 and Aβ (Figure 1d and Figure 6b-d). In addition, the present inventors used cell lysates or purified proteins to test the binding of SPYTAC to LRP1 and other well-known receptors such as asialoglycoprotein receptor (ASGPR). 20、21 and cation-independent mannose 6-phosphate receptor (CI-M6PR) 19 Repeated pull-down assays (Fig. 6d) (Fig. 6e) were performed and demonstrated that SP1 targets LRP1 28 Instead of ASGPR and CI-M6PR, SPYTAC should act through the LRP1 pathway (Figure 1e). In addition, the present inventors knocked down LRP1 using siRNA in HepG2 cells (Figure 6f) and observed a decrease in LRP1 enrichment in SPYTAC pull-down assays using cell lysates after LRP1 knockdown (Figure 1f).

[0158] Example 2: SPYTAC targeted degradation of soluble proteins in vitro

[0159] Next, the inventors tested the ability of SPYTAC to mediate endocytosis and lysosome-dependent degradation of extracellular proteins in cultured cells. LRP1-positive HepG2 and LRP1-negative HeLa cells ( FIG. 14 ) were co-incubated with biotinylated SP1, SP2, SP3, or SP4, respectively, and fluorescently labeled streptavidin (FITC-SA) was used as an indicator. After 10 minutes, the SP1 and SP2 groups were incubated with LysoTracker. TM A large number of punctate FITC-SA signals were found in the co-localized HepG2 cells, while fewer punctate signals (about 1-fold) were detected in the SP3 and SP4 groups (Figures 2a, b and 7a, b). The inventors next investigated whether SP1 could mediate the degradation of soluble Aβ peptides in HepG2 cells and incubated HepG2 or HeLa cells with FITC-Aβ and SP1, SP2, SP3 or SP4, respectively. This result was further confirmed by confocal microscopy, where only in HepG2 cells, SP1 treatment resulted in the degradation of soluble Aβ peptides compared with SP2, SP3 and SP4. TM The colocalized FITC-Aβ signal was higher (Fig. 2c, d and Fig. 7c, d). These results were also confirmed in other cell lines expressing LRP1, including Huh7 and AML12 cells (Fig. 7e-h and Fig. 14).

[0160] To further confirm that SPYTAC's TPD function is dependent on LRP1, a series of minimal LRP1 (miniLRP1) receptor constructs were ectopically expressed in HeLa cells (Figure 8a) to assess the ability of a single miniLRP1 to affect Aβ absorption. The inventors found that miniLRP1 receptors containing full-length β chains can mediate Aβ uptake in HeLa cells (Figure 2d and Figure 8b). In addition, in a pull-down assay using SP2, miniLRP1_v0 but not miniLRP1_v1 was enriched, confirming the interaction between the two (Figure 2d). These results indicate that SPYTAC acts in an LRP1-dependent manner.

[0161] To directly visualize SPYTAC's targeted degradation of Aβ, the inventors used lentivirus to construct cell lines stably expressing Aβ1-42 on the plasma membrane in HepG2 (HepG2csAβ) and HeLa cells (HeLacsAβ) (Figures 9a, b and Supplementary Sequences). The inventors then treated HepG2csAβ and HeLacsAβ cells with SP1 and observed extensive degradation of cell-surface Aβ in HepG2 cells after 24 hours, as evidenced by the disappearance of membrane Aβ signals (Figures 2g and 9c). These results not only demonstrate that SPYTAC can achieve specific degradation of Aβ but also suggest the expanded application of SPYTAC in the targeted degradation of cell-surface proteins.

[0162] In addition, the modularity of SPYTAC enables high programmability, thereby enabling targeted degradation of various pathogenic proteins. To establish the promotion of SPYTAC, the inventors chose to target the degradation of anti-Myc tag antibodies as a proof of concept. By changing the N-terminal Aβ binding motif to the Myc tag sequence (Table 1), the inventors demonstrated that Cy5-Myc tag antibodies can be captured into lysosomes for degradation, such as the Cy5 punctate signal in HepG2 and the LysoTracker TM The co-localization of SPYTAC with the engineered cells was shown in Figure 2h, i. Simultaneously, the inventors ectopically expressed a gene cassette encoding a secreted SP to demonstrate the genetic encodeability of SPYTAC (Figure 9d, e). The inventors' results showed that SPYTAC secreted by the engineered cells can also promote the degradation of extracellular and membrane proteins (Figure 2j, k and Figure 8f-h).

[0163] Example 3: SPYTAC hijacks the liver for targeted protein degradation in vivo

[0164] Since LRP1 is not a tissue-specific receptor (Figure 14b, c), the inventors attempted to determine the biodistribution of SPYTAC in mice. A biotin-SP1 / Cy5.5-SA mixture or Cy5.5-SA was intravenously injected into wild-type mice, and organs were isolated 15 minutes after injection (pi) and subjected to near-infrared fluorescence imaging (Figure 3a). After administration of SP1, a high fluorescent signal was detected in the liver, but not when Cy5.5-SA was used alone (Figure 10a, b). Biodistribution assessments were repeated using SP1 / Cy5.5-Aβ1-42, SP4 / Cy5.5-Aβ1-42 mixture, or Cy5.5-Aβ1-42, and the results showed that SP1 primarily targeted Aβ to the liver (Figure 3b, c).

[0165] To further understand the subcellular distribution of SPYTAC in vivo, freshly resected organs from biotin-SP1 / Cy5.5-SA and biotin-SP4 / Cy5.5-SA-treated GFP-expressing mice were analyzed using confocal microscopy. This revealed that SP1 was primarily localized in the liver, with sporadic distribution in the kidney and brain (Figure 10c). In vivo observation of small animals using an in vivo microscopy system allows for a comprehensive understanding of the complex aspects of peptide distribution, tissue targeting, and cellular delivery in vivo. More detailed analysis revealed that the biotin-SP1 / Cy5.5-SA fluorescence signal was primarily present in hepatocytes, while the biotin-SP4 / Cy5.5-SA remained extracellular (Figure 3d). Regarding the biotin-SP1 / Cy5.5-SA fluorescence signal in the kidney and brain, it was retained in the vasculature (Figures 10d, e). Therefore, biodistribution and LRP1 expression jointly contribute to the ultimate subcellular localization of SP in vivo, allowing SPYTAC to preferentially target extracellular proteins for degradation in a liver-specific manner.

[0166] The inventors next tested whether SPYTAC could target soluble Aβ to liver lysosomes for degradation in vivo. In wild-type mice, biotin-SP1 / FITC-Aβ significantly accumulated in hepatocytes 15 minutes after injection, but not biotin-SP3 / FITC-Aβ or FITC-Aβ alone (Figures 3c, d). Similarly, SP1 increased Aβ's lysosomal localization by 5-fold compared to SP4 (Figures 3e, f). Thus, SPYTAC is capable of capturing plasma Aβ for targeted degradation in the liver.

[0167] Example 4: SPYTAC has blood-brain barrier (BBB) ​​permeability: promoting the degradation of extracellular proteins in the brain

[0168] First, high-resolution confocal imaging was used to analyze the brains of green fluorescent protein (GFP)-expressing mice treated with biotin-SP1 / Cy5.5-SA. Cy5.5-SA-derived signals were detected (Figure 16A), demonstrating that SPYTACs can cross the blood-brain barrier (BBB). To obtain more quantitative data, wild-type (WT) mice were intraperitoneally injected with FITC-SP1 / AF594-Aβ42, FITC-SP4 / AF594-Aβ42, or AF594-Aβ42 and analyzed for fluorescence signals from SPYTAC and Aβ. Signals from SP1 were 6.5-fold and 10.6-fold stronger than those from SP4 and Aβ42, respectively (Figures 15A and 15B). Furthermore, SP1 was shown to be internalized by low-density lipoprotein receptor-related protein 1 (LRP1)-positive microglia, astrocytes, and neurons in the brain (Figure 16B). These findings suggest that SPYTACs are able to cross the blood-brain barrier and promote protein degradation in the brain.

[0169] Inspired by the high efficiency of SP1 in crossing the blood-brain barrier (BBB), we further evaluated the endocytosis and transcellular transport of SPYTAC in an in vitro BBB model. Using flow cytometry, we detected the endocytosis and transcellular transport of SPYTACs into the mouse brain endothelial cell line bEnd.3 (LRP1 + ) cellular uptake ( FIG16C ). As expected, SPYTACs containing LRP1-binding motifs (SP1 and SP2) showed significantly higher fluorescence intensity than SPYTACs lacking LRP1-binding motifs (SP3 and SP4) ( FIG15C and FIG15D ).

[0170] We also used LRP1 + U251 (glioblastoma), C8-D1A (astroglial cells), BV2 (microglia), and N2A (neuroblastoma) cell lines were used to study SPYTAC-mediated endocytosis and lysosomal degradation in astrocytes, microglia, and neurons (Figure 16C). In the presence of biotin-SP1 and biotin-SP2, FITC-SA and LysoTracker TM Colocalization was observed in the presence of biotin-SP3 and biotin-SP4 (Figure 15E and Figures 16D-16F). TM An increase in cellular fluorescence was observed during Red Avidin uptake ( FIG. 15F and FIG. 16G-16I ).

[0171] These results indicate that SPYTAC can mediate LRP1-dependent endocytosis and lysosomal trafficking of targeted proteins, demonstrating its ability to target protein degradation (TPD) in brain cells.

[0172] We further evaluated the blood-brain barrier (BBB) ​​penetration ability of SPYTAC using an in vitro blood-brain barrier (BBB) ​​Transwell model, in which bEnd.3 cells were seeded in the apical chamber and U251 cells were seeded in the basolateral chamber (Figure 15G and Figure 16J). TM Red Avidin fluorescence signal showed that biotin-SP1 had higher cellular uptake than biotin-SP4 or negative control (Figure 15G and Figure 15H), indicating that SP1 can promote LRP1-mediated trans-blood-brain barrier transport. In addition, after introducing biotin-Aβ42 into the basolateral chamber, it was found that SP1 in the apical chamber was able to penetrate the blood-brain barrier and deliver Aβ42 to U251 cells, which was confirmed by a significant increase in median fluorescence intensity (MFI) (Figure 16J).

[0173] Taken together, these results demonstrate that SPYTAC can efficiently cross the blood-brain barrier via LRP1-mediated transcellular transport and promote the degradation of extracellular proteins in the brain via LRP1-mediated endocytosis.

[0174] Example 5: SPYTAC is effective and safe in treating AD in mild to moderate 5xFAD mice

[0175] To verify whether SPYTAC treatment in vivo can reduce brain Aβ burden in AD mouse models, thereby improving cognitive impairment, the inventors tested the efficacy of SPYTAC in mild to moderate (5 months) 5xFAD mice. In the inventors' preliminary tests, 5xFAD mice were given a single dose of SYPYAC or vehicle, and the results showed that plasma Aβ levels decreased rapidly 30 minutes after injection (Figure 11a). In the following experiments, early (3 months) 5xFAD mice were treated with SPYTAC (5xFAD SP ) Repeat treatment for 1 month, and choose lecanemab (5xFAD Lecam ) and vehicle(5xFAD veh ) treated 5xFAD mice as positive and negative controls, respectively. After treatment, the inventors analyzed the effect of SPYTAC in improving Aβ-related brain lesions in 5xFAD mice and observed that 5xFAD SP Aβ burden in the mouse cerebral cortex (CTX) and hippocampus (Hippo) was significantly reduced, including the number and size of Aβ plaques (average >40%), even lower than 5xFAD Lecammice, but not significantly (Figure 4a, b and Figure 11b). A more detailed evaluation revealed a different Aβ plaque pattern in 5xFAD mice after treatment. veh 、5xFAD Lecam and 5xFAD SP These patterns were designated as type I, II, and III (Figure 4c). Further quantitative analysis showed that 5xFAD SP The proportion of type III plaques is increased in mice, 5xFAD Lecam The proportion of type II plaques increased in mice (Figure 4d). veh and 5xFAD Lecam Compared with mice, 5xFAD SP The total level of brain Aβ in mice was significantly reduced (Figure 4e), suggesting that the peptide can clear circulating Aβ, thereby reducing the Aβ burden in the brain.

[0176] Inspired by the significantly reduced brain Aβ load, cognitive tests were performed on the treated 5xFAD mice. In the open field test, all treated mice showed no obvious signs of anxiety and no significant differences in motor ability compared with wild-type mice (Figure 11c, d). In the Morris water maze test, the 5xFAD mice showed no significant differences in motor ability compared with wild-type mice. Lecam Compared with mice, 5xFAD SP Mice showed significantly improved spatial learning, as evidenced by shorter escape latencies in the platform test, and better memory consolidation, as evidenced by longer time in the target quadrant and more platform areas traversed in the probe test (Fig. 4f-h). These improvements were consistent with those observed in the 5× FAD. Lecam In the novel object recognition (NOR) test, the improvement was comparable to that observed in 5xFAD mice (Fig. 4f-h). veh Compared with mice, 5xFAD SP Mice and 5xFAD Lecam Mice spent more time exploring novel objects than familiar objects, indicating improved short-term memory (Figure 4i). These results suggest that SPYTAC treatment can rescue cognitive impairment in early-stage 5xFAD mice, achieving beneficial effects comparable to those of Lecanemab treatment.

[0177] The use of anti-Aβ antibodies may significantly increase the risk of ARIA (amyloid-associated imaging abnormalities). The inventors detected and quantified the deposition of Aβ in the walls of brain blood vessels. The inventors observed symptoms similar to cerebral amyloid angiopathy (CAA) in some mice treated with Lecanemab. veh Compared with mice, 5xFAD SPMice showed background levels of vessel-associated Aβ, while 5xFAD Lecam Mice showed a significantly increased CAA phenotype, i.e., accumulated Aβ deposits in blood vessels (Figure 4j). Considering that SPYTAC promotes liver-targeted peripheral Aβ clearance, treatment with SPYTAC can avoid ARIA that occurs during anti-Aβ monoclonal antibody treatment. In addition, when the antibody binds to Aβ plaques, it induces microglial activation, promotes the decomposition of Aβ plaques, and promotes brain tissue repair. However, this process also triggers the decomposition of Aβ in the blood vessel wall, leading to inflammation and causing side effects. Compared with 5xFAD veh and 5xFAD Lecam Compared with mice, the present inventors detected 5xFAD SP The proinflammatory response and microglial activation in mice were significantly downregulated (Figure 4k and Figure 11e). The inventors' results show that SPYTAC minimizes the side effects associated with CAA and proinflammatory cytokines, while effectively eliminating the brain Aβ burden.

[0178] Example 6: SPYTAC ameliorates AD-type pathology and cognitive impairment in late-stage 5xFAD mice

[0179] Next, the inventors tested whether SPYTAC could improve AD-type pathology in the late stage of AD. In 9-month-old 5xFAD mice, SPYTAC treatment reduced the level of Aβ in the blood (Figure 5a). Repeated use of SPYTAC for one month significantly reduced the brain Aβ burden, as shown by a decrease in the number and size of Aβ plaques in the hippocampus and cerebral cortex after SPYTAC treatment (Figures 5b, c and Figure 12a). In addition, compared with 5xFAD vehicle Compared with mice, 5xFAD SP In the mouse brain, microglia and astrocyte overactivation, axonal degeneration, neuronal damage, and loss of dendritic spines and synapses were alleviated (Figure 5d, f and Figure 12b, c). These results indicate that targeting peripheral Aβ in the liver with SPYTAC significantly reduced Aβ burden, neuroinflammation, and neurodegeneration in the brain.

[0180] Improvements in behavioral tests reflected improvements in cognitive deficits, consistent with a reduction in brain Aβ burden. vehicle Compared with mice, 5xFAD SP Mice showed improved spatial learning and better memory consolidation, as shown by the results of the Morris water maze test (Figure 5g-i). SPYTAC treatment also significantly improved spatial recognition memory, as shown by longer time spent in the novel arm and increased percentage of spontaneous alternations in the Y-maze test (Figure 12d). veh Compared with mice, 5xFADSP Mice showed enhanced recognition memory, spending more time exploring the novel object than the familiar object in the NOR test (Figure 5j). veh Compared with mice, 5xFAD SP The mice showed no obvious anxiety (Fig. 12e, f).

[0181] Finally, the inventors evaluated liver toxicity in mice based on the clearance protocols for these site-specific and nonspecific SPYTAC peptides. Liver function tests in mouse serum demonstrated that SPYTAC administration did not cause liver toxicity (Figures 13a, b). Histological analysis of organs such as the liver, lungs, kidneys, and heart also showed no significant toxicity between treated and control mice (Figure 13c). Body weights also showed no difference (Figure 13d). The inventors' results demonstrate that SPYTAC treatment has good efficacy and safety in an AD mouse model.

[0182] Here, the inventors have developed a SPYTAC platform for the degradation of extracellular and membrane proteins. These bispecific peptides form a ternary protein complex between the target protein and LRP1 on the cell surface and mediate the endocytosis of the target protein and subsequent lysosomal degradation. The bispecific peptides are highly modular, programmable, and genetically encodeable. By replacing the N-terminal target protein binding motif, SPYTAC can capture a variety of proteins, enabling targeted degradation of a range of pathogenic proteins. In theory, by changing the C-terminal receptor binding motif, SPYTAC can be modified to utilize other endocytic receptors, including the well-documented ASGPR 20、21 and CI-M6PR19. A recent study using a de novo protein design approach 26 The pioneering work of also confirms the inventors' discovery that synthetic peptides can facilitate the design, development and application of lysosomal targeting chimeras. In addition, unlike another recently designed LYTAC platform, this platform uses short chemically conjugated peptides that utilize the transglutaminase 2 (TG2) / LRP1 pathway. 27 The inventors' SPYTAC platform utilizes fully synthetic peptides that can be genetically encoded and secreted from engineered cells, enabling the advancement of engineered cell and gene therapies. Importantly, SPYTAC is independent of the extracellular TG2 enzyme, significantly simplifying its implementation.

[0183] In addition to the above advantages, SPYTAC has several advantages over traditional LYTAC: the synthetic peptide is relatively small in size and has low immunogenicity. Moreover, it is a chimera of chemically conjugated antibodies. 20、21 or protein-antibody chimeras 29In contrast, synthetic peptides can be fully synthesized and have a relatively simple production process and low cost. At the same time, compared with the recently developed DNA-based aptamers 30、31 In contrast, synthetic peptides are much safer than DNA administration, which may lead to potential gene integration or recombination.

[0184] Recent breakthroughs in anti-Aβ immunotherapy in clinical trials suggest that Aβ clearance is a promising strategy for treating AD if brain Aβ can be reduced to a certain low level. 1,2,32,33 Furthermore, peripheral clearance of plasma Aβ can enhance Aβ efflux across the blood-brain barrier (BBB), thereby reducing the brain Aβ burden. 13-17 . Based on these findings, the inventors attempted for the first time to use SPYTAC to treat AD in 5xFAD model mice. In the inventors' research, the inventors demonstrated that SPYTAC treatment can significantly alleviate AD-type pathology and cognitive impairment, and its improvement effect on early-stage 5xFAD mice is comparable to that of Lecanemab treatment approved by the U.S. Food and Drug Administration (FDA). In addition, SPYTAC can effectively reduce the accumulation of Aβ in the brain by purifying plasma Aβ. Importantly, SPYTAC uses the TPD mechanism for Aβ clearance, which is completely different from traditional antibody therapy and therefore does not usually induce microbleeds and pro-inflammatory responses similar to CAA. In addition, the inventors verified that in later treatments, SPYTAC also helps to reduce brain Aβ aggregation and improve cognitive impairment without affecting liver function. To the best of the inventors' knowledge, SPYTAC technology has demonstrated for the first time that synthetic peptides can effectively mediate the degradation of extracellular pathogenic proteins through the LRP1 pathway in vitro and in vivo.

[0185] In summary, the inventors have developed a SPYTAC system that selectively accelerates the degradation of extracellular and cell surface proteins in vitro and in vivo. The inventors' SPYTAC shows great promise in the treatment of AD by targeting peripheral Aβ degradation in a liver-specific manner. The high modularity, programmability, and genetic encodeability of SPYTAC hold promise as a universal therapeutic platform for clearing pathogenic proteins in a variety of diseases, including neurodegenerative disorders and cancer.

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Claims

1. A chimeric polypeptide for degrading a target protein, comprising a target protein binding motif and a lysosomal transport receptor binding motif.

2. The chimeric polypeptide of claim 1, wherein the target protein binding motif is located at the N-terminus of the lysosomal trafficking receptor binding motif.

3. The chimeric polypeptide of claim 1 or 2, wherein the target protein binding motif and the lysosomal transport receptor binding motif are connected via a linker, such as a flexible peptide linker.

4. The chimeric polypeptide of claim 3, wherein the flexible peptide linker comprises (GSS) n wherein n is an integer selected from 1-10, preferably n is 1.

5. The chimeric polypeptide of any one of claims 1 to 4, wherein the target protein is a pathogenic polypeptide.

6. The chimeric polypeptide of claim 5, wherein the pathogenic polypeptide is an Aβ peptide, such as Aβ shown in SEQ ID NO:

49. 1-42 .

7. The chimeric polypeptide of claim 6, wherein the target protein binding motif is an Aβ peptide binding motif, for example, the Aβ peptide binding motif comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-7, preferably, the Aβ peptide binding motif comprises the amino acid sequence shown in SEQ ID NO:

1.

8. The chimeric polypeptide of any one of claims 1 to 7, wherein the lysosomal trafficking receptor is selected from the group consisting of LRP1, ASGPR, and CI-M6PR19, preferably, the lysosomal trafficking receptor is LRP1.

9. The chimeric polypeptide of claim 8, wherein the lysosomal transport receptor binding motif is an LRP1 binding motif, for example, the LRP1 binding motif comprises an amino acid sequence selected from any one of SEQ ID NOs: 9-16, preferably, the LRP1 binding motif comprises the amino acid sequence shown in SEQ ID NO:

9.

10. The chimeric polypeptide according to any one of claims 6 to 9, wherein the chimeric polypeptide comprises the amino acid sequence of one of SEQ ID NOs: 17 to 32, preferably, the chimeric polypeptide comprises the amino acid sequence shown in SEQ ID NO:

17.

11. The chimeric polypeptide of any one of claims 1 to 10, wherein the chimeric polypeptide is a synthetic polypeptide.

12. An expression vector comprising a nucleotide sequence encoding the chimeric polypeptide according to any one of claims 1 to 11 operably linked to an expression control sequence.

13. A pharmaceutical composition comprising the chimeric polypeptide according to any one of claims 1 to 10 and / or the expression vector according to claim 12, and a pharmaceutically acceptable carrier or excipient.

14. A method for treating a disease, comprising administering a therapeutically effective amount of the chimeric polypeptide of any one of claims 1 to 10 or a therapeutically effective amount of the pharmaceutical composition of claim 13 to a subject in need thereof.

15. The method of claim 14, wherein the target protein is Aβ peptide and the disease is a disease caused by or associated with amyloid protein, such as Alzheimer's disease.

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