Targeting liposome for protein degrader delivery
Patent Information
- Authority / Receiving Office
- EP · EP
- Patent Type
- Applications
- Current Assignee / Owner
- EMD MILLIPORE CORP
- Filing Date
- 2024-07-26
- Publication Date
- 2026-06-10
AI Technical Summary
Current protein degraders face challenges with poor cell permeability, especially for larger molecules, and lack tissue-specific recognition, leading to adverse side effects due to broad expression profiles of E3 ligases.
Development of targeting liposomes comprising phospholipids, cholesterol or cholesterol derivatives, PEG lipids, targeting lipids, and either protein degraders or pre-fused protein degrader-ligase complexes, which enhance cell permeability and tissue specificity.
The targeting liposomes improve the cell uptake and targetability of protein degraders, achieving enhanced protein degradation with reduced side effects and increased efficacy at lower drug concentrations.
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Abstract
Description
TARGETING LIPOSOME FOR PROTEIN DEGRADER DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 517,174, filed August 2, 2023, the entire content of which is incorporated herein by reference.BACKGROUND
[0002] Protein degradation induced by proteolysis-targeting chimera systems has attracted extensive attentions in drug discovery, protein engineering and chemical biology. It could reach to undruggable drug targets and expand the scope of curable diseases. Protein degraders are bifunctional small molecule which can connect E3 ligase ligand to protein of interests (POI). Their action relies on the capability to bring E3 ligase and POI into proximity, trigger directed polyubiquitination and subsequent proteasome-mediated degradation of the POI. Although numerous studies have reported highly efficient protein degraders, three major challenges still exist. First, the cell permeability for protein degrader is poor especially for ones with large molecular weight and multiple hydrogen bond donors / acceptors. Second, the degradation requires the formation of an active ternary complex, which, however, would decrease the degradation ability dramatically at a high concentration due to "hook effect" of the three-component complexation. Third, the protein degraders lack of tissue-specific recognition which would induce adverse side effects, as the E3 ligase has broad expression profiles.
[0003] Current studies have focused on antibody conjugation methods which could improve the cell permeability and targetability of small molecular protein degraders. However, the challenges to release conjugated but also active protein degrader within target cells and avoid overdosage-induced "hook effects" are not addressed.
[0004] While Lipid nanoparticles including liposome have proven effective to transport small molecules and proteins into cells and / or intracellular compartments, a need exists for new lipid nanoparticles that can effectivelydeliver cargos such as protein degraders and pre-fused protein degrader complexes into target cells.SUMMARY
[0005] A targeting liposome for protein degrader delivery is provided. In some embodiments, the targeting liposome including a phospholipid, a cholesterol or cholesterol derivative, a PEG lipid, a targeting lipid; and a protein degrader. In other embodiments, the targeting liposome including a phospholipid, a cholesterol or cholesterol derivative, a PEG lipid, a targeting lipid; and a protein degrader-ligase complex.
[0006] In various embodiments, the molar ratio of phospholipid:cholesterol:PEG lipid:targeting lipid is in the range from (40- 65):(25-45):(0-7):(0.5-5).
[0007] The phospholipid is generally a phosphatidylcholine (PC). In various embodiments, the phospholipid may be egg PC, 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.
[0008] In various embodiments, the cholesterol derivative may be cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.
[0009] In various embodiments, the PEG lipid may be PE-PEG500, PE- PEG1000, PE-PEG2000, PE-PEG5000, wherein PE is selected from 1 ,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1 ,3-Dipalmitoyl- glycero-2-phosphoethanolamine (DSPE).
[0010] In various embodiments, the targeting lipid may be DSPE-PEG2000 folate, cholesterol-PEG2000-folate DSPE-PEG2000-Biotin, DSPE-PEG2000- DBCO, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD, DSPE-PEG- peptide, and DSPE-PEG-antibody.
[0011] In various embodiments, the protein degrader may be ARV-771 , MZ1 , ARV-825, ARV-471 , ARV-110, ARV-766, FHD609, KT474, NX2127,CFT8634, CFT1946, AT1 , BSJ-03-204, BSJ-04-132, BSJ-4-116, CG-428, CG-858, CRBN-6-5-5-VHL, dBET6, DD 03-171 , or LC2.
[0012] In embodiments including a protein degrader-ligase complex, the protein degrader-ligase complex may be ARV-771 -VHL, MZ1 -VHL, ARV-825- CRBN, ARV-471 -CRBN, ARV-110-CRBN, ARV-766-CRBN, FHD609-CRBN, KT474-CRBN, NX2127-CRBN, CFT8634-CRBN, CFT1946-CRBN, AT1 -VHL, BSJ-03-204-CRBN, BSJ-04-132-CRBN, BSJ-4-116-CRBN, CG-428-CRBN, CG-858-CRBN, CRBN-6-5-5-VHL, dBET6-CRBN, DD 03-171 -CRBN, and LC2-VHL.
[0013] Also provided is a kit for preparing a targeting liposome for protein degrader delivery, the kit including a phospholipid, a cholesterol or cholesterol derivative, a PEG lipid, and a targeting lipid.BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The drawings are provided to illustrate one or more versions of the present invention and are not to be construed as limiting the scope of the claims.
[0015] FIG. 1 illustrates the scheme of targeting liposomes for protein degrader delivery.
[0016] FIG. 2 shows size characterization of liposomes which did encapsulate or did not encapsulate protein degrader ARV-771 .
[0017] FIG. 3 shows polydispersity of liposome which did encapsulate or not encapsulate protein degraders (ARV-771 ).
[0018] FIG. 4 shows the targetability of liposomes in HeLa and HEK 293 cells.
[0019] FIG. 5 shows the biocompatibility of liposome carriers.
[0020] FIG. 6 shows enhanced protein degradation achieved using the targeting liposome delivery systems described herein compared with free protein degrader ARV-771 and non-targeting liposome.
[0021] FIG. 7 shows enhanced targetability and cytotoxicity of the targeting liposome delivery systems described herein compared with non-targeting liposomes.DETAILED DESCRIPTION
[0022] Combining the advances of targeting lipid nanoparticles, we herein describe the synthesis of targeting lipid nanoparticles to selectively deliver protein degrader or pre-fused protein degrader complex into target cells.
[0023] In one aspect, the provided herein are a nanoparticle compositions including (1) liposome containing phospholipid, which may be saturated or unsaturated, PEG lipids, cholesterol or a cholesterol derivative and targeting lipids; and (2) protein degraders or pre-fused complexes of protein degrader and ligase.
[0024] In another aspect, a method of delivering protein degraders into target cells is provided, which involves encapsulating the protein degraders into targeting nanoparticles. In addition, we also discovered that pre-fused protein degrader and E3 ligase complex could significantly enhance protein degradation and exhibit higher targetability compared to free protein degrader.
[0025] Advantages of the lipid nanoparticles provided herein include (1 ) increased cell permeability of protein degrader and prefused protein complex; (2) Cell-selective protein degradation; and (3) Enhanced protein degradation with a low drug concentration.
[0026] In a first embodiment, the targeting liposome for protein degrader delivery provided herein include a phospholipid, a cholesterol or cholesterol derivative, a PEG lipid, a targeting lipid; and a protein degrader.
[0027] In various embodiments, the molar ratio of phospholipid:cholesterol:PEG lipid:targeting lipid is in the range from (40- 65):(25-45):(0-7):(0.5-5). In some preferred embodiments, the molar ratio of phospholipid:cholesterol:PEG lipid:targeting lipid is in the range from 57:38:4.5:0.5 to 57:38:1 :4.
[0028] In a preferred embodiment, the phospholipid is a phosphatidylcholine (PC). In various embodiments, the phospholipid may be selected from egg PC, 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and combinations thereof.
[0029] In various embodiments, the cholesterol or cholesterol derivative is selected from cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.
[0030] The PEG lipid may be selected from PE-PEG500, PE-PEG1000, PE- PEG2000, PE-PEG5000, wherein PE is selected from 1 ,2-dioleoyl-sn-glycero- 3-phosphoethanolamine (DOPE) and 1 ,3-Dipalmitoyl-glycero-2- phosphoethanolamine (DSPE), and is preferably PE-PEG2000.
[0031] In various embodiments, the targeting lipid is selected from DSPE- PEG2000 folate, cholesterol-PEG2000-folate DSPE-PEG2000-Biotin, DSPE- PEG2000-DBCG, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD, DSPE- PEG-peptide, and DSPE-PEG-antibody. In some preferred embodiments, the targeting lipid is selected fromDSPE-PEG2000 folate and cholesterol- PEG2000-folate.
[0032] The protein degrader may be virtually any protein degrader. Exemplary protein degraders include, for example, ARV-771 , MZ1 , ARV-825, ARV-471 , ARV-110, ARV-766, FHD609, KT474, NX2127, CFT8634, CFT1946, AT1 , BSJ-03-204, BSJ-04-132, BSJ-4-116, CG-428, CG-858, CRBN-6-5-5-VHL, dBET6, DD 03-171 , and LC2. Some preferred protein degraders include ARV- 771 , MZ1 , ARV-825, ARV-471 and ARV-110.
[0033] In a second embodiment, the targeting liposome for protein degrader delivery includes a phospholipid, a cholesterol or cholesterol derivative, a PEG lipid, a targeting lipid; and a protein degrader-ligase complex.
[0034] In this embodiment, molar ratio of phospholipid:cholesterol:PEG lipid:targeting lipid may be in the range from (40-65) :(25-45):(0-7): (0.5-5). In some preferred embodiments, the molar ratio is in the range from 57:38:4.5:0.5 to 57:38:1 :4.
[0035] In preferred embodiments, the phospholipid is a phosphatidylcholine (PC). In various embodiments, the phospholipid may be selected from egg PC, POPC, DOPC and combinations thereof.
[0036] In various embodiment, the cholesterol or cholesterol derivative is selected from cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.
[0037] In various embodiments, the PEG lipid is selected from PE-PEG500, PE-PEG1000, PE-PEG2000, PE-PEG5000, wherein PE is selected from 1 ,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1 ,3-Dipalmitoyl- glycero-2-phosphoethanolamine (DSPE). In one preferred embodiment, the PEG lipid is PE-PEG2000.
[0038] In various embodiments, the targeting lipid is selected from DSPE- PEG2000 folate, cholesterol-PEG2000-folate DSPE-PEG2000-Biotin, DSPE- PEG2000-DBCO, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD, DSPE- PEG-peptide, e.g., GALA, TAT, and so forth, and DSPE-PEG-antibody, e.g., transferrin. In some preferred embodiments, the targeting lipid is DSPE- PEG2000 folate or cholesterol-PEG2000-folate.
[0039] The targeting nanolipid is not limited to specific protein degrader-ligase complexes. Some exemplary protein degrader-ligase complexes include ARV- 771 -VHL, MZ1 -VHL, ARV-825-CRBN, ARV-471 -CRBN, ARV-1 10-CRBN, ARV-766-CRBN, FHD609-CRBN, KT474-CRBN, NX2127-CRBN, CFT8634- CRBN, CFT1946-CRBN, AT1 -VHL, BSJ-03-204-CRBN, BSJ-04-132-CRBN, BSJ-4-116-CRBN, CG-428-CRBN, CG-858-CRBN, CRBN-6-5-5-VHL, dBET6-CRBN, DD 03-171 -CRBN, and LC2-VHL.
[0040] Further provided is a kit for preparing a targeting liposome for protein degrader delivery, the kit including a phospholipid, a cholesterol or cholesterol derivative, a PEG lipid, and a targeting lipid. Each of the components can be as described above.
[0041] In some embodiments, the included phospholipid is a PC, e.g., egg PC, 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof. The cholesterol or cholesterol derivative may be, e.g., cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate or combinations thereof. The included PEG lipid may be, e.g., PE-PEG500, PE-PEG1000, PE-PEG2000, PE-PEG5000, wherein PE is selected from 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1 ,3-Dipalmitoyl-glycero-2-phosphoethanolamine (DSPE). The targeting lipid included with the kit may be, e.g., DSPE-PEG2000 folate, cholesterol-PEG2000-folate DSPE-PEG2000-Biotin, DSPE-PEG2000-DBCO, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, DSPE-PEG-antibody and combinations thereof. In some embodiments, the kit includes instructions.
[0042] In some embodiments, the kit may include a single phospholipid, a single cholesterol or cholesterol derivative, a single PEG lipid and a single targeting lipid. In other embodiments, the kit may include more than one of any of the components. In still other embodiments, the kit may further include one or more protein degraders and / or one or more protein degrader-ligase complex.EXAMPLES
[0043] A folate-modified targeting liposome is used to deliver protein degraders, as well as pre-fused protein degrader and E3 ligase complexes. The targeting liposome selectively targets certain cells, enhancing the cell uptake of loaded protein degraders, and results in more efficient protein degradation-induced cell death.
[0044] 1 . Formulation preparation
[0045] The drug-loaded liposome was prepared by extrusion method. Lipids (10mg) were weighted and dissolved with CHOIs in round flask. A lipid thin film was formed by using rotary evaporators to remove CHCh. The lipid film was dried in vacuum oven overnight. The protein degrader or prefused protein degrader complex dispersed in PBS buffer (1 mL) was added into lipid thin film and gently vortex to dissolve the lipid film and obtain a milk-like solution. The solution was extruded through nucleopore membrane with pore size of 100nm using mini hand extruder with thermojacket (Genizer,CA, USA). After 21- times extrusion, the lipid solution was collected and stored at 4 °C prior to use.
[0046] 2. Characterization
[0047] The hydrodynamic diameter and the diameter distribution of obtained liposome were characterized with a Zetasizer (Nano-ZS90, Malvern, UK). In a sample cuvette, the lipid dispersion was diluted with PBS buffer to 0.5 mg / ml. The data were collected at room temperature with three individual scans.
[0048] Size characterization of liposome which did encapsulate or not encapsulate protein degraders (ARV-771) is shown in FIG. 2. The liposome with and without drug loading showed very close (100-120nm) size distribution, which suggested the encapsulation of protein degraders did not influence the surface properties of liposome.
[0049] Polydispersity of liposome which did encapsulate or not encapsulate protein degraders (ARV-771) is illustrated in FIG. 3. The narrow distribution of liposome size suggested the stability of liposome after drug encapsulation.
[0050] 3.Targetability
[0051] To evaluate if the targeting liposome could selectively improve the cell uptake of target cell lines, the targeting liposome were loaded with fluorescent probes. Dil is a lipophilic dye which only shows fluorescence in lipid bilayer and represented the presence of liposome. To prepare Dil-loaded liposomes, Dil in ethanol was mixed with lipid dispersions to achieve a final Dil-to-lipid molar ratio of 8%. Dil solution was directly mixed with lipid dispersions, followed by microfluidic-based two-phase mixing to form 100 nm liposome.
[0052] Approximately 200,000 receptor-positive cells (e.g., HeLa cells) or receptor-negative cells (e.g., HEK 293 cells) in fetal bovine serum (FBS)- supplemented medium were seeded into a 96-well microplate and incubated overnight. The cells were washed with medium once and incubated with Dil- loaded liposome for 48 h. Subsequently, the cells were washed with sterilePBS four times. The fluorescence of obtained cells was measured by microplate reader (Varioskan LUX, Thermo Fisher Scientific) with excitation wavelength of 550 nm and emission wavelength of 585 nm.
[0053] Targetability of liposomes is illustrated in FIG. 4. The liposome was loaded with hydrophobic dyes which only show fluorescence within lipid bilayers. The amount of dye-loaded liposome suggests the cell uptake capability of liposome and drug-loaded liposome. The target cell model used here is Hela cell which overexpressed folate receptors. In contrast, HEK 293 cell which expresses less folate receptors was used as control. The results showed Hela cells uptake 4 times of dye-loaded folate liposome than dye- loaded plain liposome. In contrast, the untargeted HEK 293 cells uptake almost same amount of dye-loaded folate liposome and dye-loaded plain liposome. This data suggested the targetability of obtained folate liposome.
[0054] 4. Cytotoxicity
[0055] The cytotoxicity experiment to evaluate the biocompatibility of liposome and the cancer cell killing efficiency of protein degrader-loaded liposome was performed with Cell Counting Kit-8 assay (catalog no. 96992, Sigma-Aldrich, MO, US). Receptor-positive cells (e.g., HeLa cell) were used as a representative mammalian cell. Approximately, 40,000 cells in FBS- supplemented cell medium were seeded into a 96-well microplate and incubated overnight. The medium was removed and replaced with liposomes which were diluted with FBS-supplemented cell medium at desired concentration. After 24 h incubation, the liposome dispersion was removed and washed with PBS buffer. Followed by the addition of Cell Counting Kit-8 reagents and incubation in 37°C incubator for 1 h, the absorption at 450 nm was read with a microplate reader (Varioskan LUX, Thermo Fisher Scientific).
[0056] FIG. 5 shows the x cells maintained high cell viability while in contact with the folate-liposomes, which suggests the biocompatibility of our delivery system.
[0057] In one aspect, when using liposome to deliver protein degrader (ARV- 771 ), the degrader showed enhanced protein degradation at sameconcentration as free ones, which suggests the enhanced cell permeability by liposome delivery systems. Meanwhile, drug-loaded folate-liposome showed slightly higher cell killing effects compared to drug-loaded plain liposome, which suggests the targetability of drug-loaded folate-liposome. This is illustrated in FIG. 6
[0058] As illustrated in FIG. 7, in another aspect, it was found that the targeting liposome system can also deliver pre-fused protein degrader and E3 ligase complex into target cells (i.e., Hela cells) with enhanced targetability and cell killing effects. The cytotoxicity data shows folate liposome loaded with pre-fused complex could kill more cells than plain liposome that is similarly loaded with pre-fused complex. At the concentration of 2.1 ug / mL of prefused complex, which corresponded to 0.2 ug / ml protein degrader, the drug-loaded folate liposome killed more than 70% of target cells. In contrast, the drug- loaded plain liposome could only kill 30% of target cells. Therefore, preformation of protein degrader and E3 ligase complex is shown to (1 ) decrease the amount of protein degrader in order to be efficient, and (2) significantly increase the targetability of drug-loaded folate liposome.
[0059] The Examples provided herein are illustrative in nature and are not meant to limit the scope of the invention as defined by the claims.
Claims
CLAIMSWhat is claimed is:1 . A targeting liposome for protein degrader delivery comprising: a phospholipid, a cholesterol or cholesterol derivative, a PEG lipid, a targeting lipid; and a protein degrader.
2. The targeting liposome of claim 1 wherein the molar ratio of phospholipid:cholesterol:PEG lipid:targeting lipid is in the range from (40- 65):(25-45):(0-7): (0.5-5).
3. The targeting liposome of claim 2 wherein the molar ratio of phospholipid:cholesterol:PEG lipid:targeting lipid is in the range from 57:38:4.5:0.5 to 57:38:1 :4.
4. The targeting liposome any of claims 1 to 3 wherein the phospholipid is a phosphatidylcholine (PC).
5. The targeting liposome of claim 4 wherein the phospholipid is selected from the group consisting of egg PC, 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.
6. The targeting liposome of any of claims 1 to 5, wherein the cholesterol or cholesterol derivative is selected from the group consisting of cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.
7. The targeting liposome of any of claims 1 to 6 wherein the PEG lipid is selected from the group consisting of PE-PEG500, PE-PEG1000, PE-PEG2000, PE-PEG5000, wherein PE is selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1 ,3-Dipalmitoyl- glycero-2-phosphoethanolamine (DSPE), and is preferably PE-PEG2000.
8. The targeting liposome of any of claims 1 to 7 wherein the targeting lipid is selected from the group consisting of DSPE-PEG2000 folate, cholesterol-PEG2000-folate DSPE-PEG2000-Biotin, DSPE-PEG2000-DBCO, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, and DSPE-PEG-antibody, and is preferably selected from the group consisting of DSPE-PEG2000 folate and cholesterol-PEG2000-folate.
9. The targeting liposome of any of claims 1 to 8 wherein the protein degrader is selected from the group consisting of ARV-771 , MZ1 , ARV-825, ARV-471 , ARV-1 10, ARV-766, FHD609, KT474, NX2127, CFT8634, CFT1946, AT1 , BSJ-03-204, BSJ-04-132, BSJ-4-116, CG-428, CG-858, CRBN-6-5-5-VHL, dBET6, DD 03-171 , and LC2, and is preferably selected from the group consisting of ARV-771 , MZ1 , ARV-825, ARV-471 and ARV- 1 10.
10. A targeting liposome for protein degrader delivery comprising: a phospholipid, a cholesterol or cholesterol derivative, a PEG lipid, a targeting lipid; and a protein degrader-ligase complex.1 1 . The targeting liposome of claim 10 wherein the molar ratio of phospholipid:cholesterol:PEG lipid:targeting lipid is in the range from (40- 65):(25-45):(0-7): (0.5-5), and is preferably in the range from 57:38:4.5:0.5 to 57:38:1 :4.
12. The targeting liposome of claim 10 or 1 1 wherein the phospholipid is a phosphatidylcholine (PC).
13. The targeting liposome of claim 12 wherein the phospholipid is selected from the group consisting of egg PC, POPC, DOPC and combinations thereof.
14. The targeting liposome of any of claims 10 to 13, wherein the cholesterol or cholesterol derivative is selected from the group consisting of cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.
15. The targeting liposome of any of claims 10 to 14 wherein the PEG lipid is selected from the group consisting of PE-PEG500, PE-PEG1000, PE- PEG2000, PE-PEG5000, wherein PE is selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1 ,3-Dipalmitoyl- glycero-2-phosphoethanolamine (DSPE), preferably PE-PEG2000.
16. The targeting liposome of any of claims 10 to 15 wherein the targeting lipid is selected from the group consisting of DSPE-PEG2000 folate, cholesterol-PEG2000-folate DSPE-PEG2000-Biotin, DSPE-PEG2000-DBCO, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, and DSPE-PEG-antibody, and is preferably selected from the group consisting of DSPE-PEG2000 folate and cholesterol-PEG2000-folate.
17. The targeting liposome of any of claims 10 to 16 wherein the protein degrader-ligase complex is selected from the group consisting of ARV-771 - VHL, MZ1 -VHL, ARV-825-CRBN, ARV-471 -CRBN, ARV-110-CRBN, ARV- 766-CRBN, FHD609-CRBN, KT474-CRBN, NX2127-CRBN, CFT8634-CRBN, CFT1946-CRBN, AT1 -VHL, BSJ-03-204-CRBN, BSJ-04-132-CRBN, BSJ-4- 116-CRBN, CG-428-CRBN, CG-858-CRBN, CRBN-6-5-5-VHL, dBET6- CRBN, DD 03-171-CRBN, and LC2-VHL.
18. A kit for preparing a targeting liposome for protein degrader delivery, the kit comprising: a phospholipid, a cholesterol or cholesterol derivative,a PEG lipid, and a targeting lipid.
19. The kit of claim 18 wherein the phospholipid is a PC, preferably selected from the group consisting of egg PC, 1 -palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.
20. The kit of any of claim 18 or 19 wherein the cholesterol is selected from the group consisting of cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof I.21 . The kit of any of claims 18 to 20 wherein the PEG lipid is selected from the group consisting of PE-PEG500, PE-PEG1000, PE-PEG2000, PE- PEG5000, wherein PE is selected from the group consisting of 1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE) and 1 ,3-Dipalmitoy l-g lycero-2- phosphoethanolamine (DSPE) and combinations thereof, and is preferably PE-PEG2000.
22. The kit of any of claims 18 to 21 wherein the targeting lipid is selected from the group consisting of DSPE-PEG2000 folate, cholesterol-PEG2000- folate DSPE-PEG2000-Biotin, DSPE-PEG2000-DBCO, DSPE-PEG2000- mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, DSPE-PEG-antibody and combinations thereof, and is preferably selected from the group consisting of DSPE-PEG2000 folate and cholesterol-PEG2000-folateand combinations thereof.
23. The kit of claim 17 further comprising instructions.