Cyclopeptide-based lysosomal targeted degradation agent

Abstract

Provided herein is a bifunctional lysosomal targeted degradation agent comprising: a peptide ligand that binds to an RGD binding integrin as a shuttle molecule for lysosomal degradation; and a protein binding agent that binds to a membrane of interest or an extracellular protein. The bifunctional degradation agents are useful, for example, for selective targeted degradation of membranes and extracellular proteins through endosomal / lysosomal pathways. Also provided herein are compositions comprising the bifunctional degradation agents and methods of using the bifunctional degradation agents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Priority is hereby claimed to U.S. Provisional Application No. 63 / 482,445, filed on January 31, 2023, which is incorporated herein by reference in its entirety.

[0003] Federal Funding Statement

[0004] This invention was made with government support under Grants GM120357 and GM148266 from the National Institutes of Health. The government has certain rights in this invention.

[0005] Sequence Listing

[0006] This application contains a sequence listing that has been submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on January 25, 2024, is named PCT--09824495-P220339WO01--APP--SEQ_LIST and is 14,322 bytes in size. Background Art

[0007] Targeted protein degradation (TPD) is emerging as an exciting therapeutic option to combat diseases involving abnormally expressed or mutated pathogenic proteins by engaging our body's natural protein disposal system. TPD via chimeric molecules is a novel therapeutic approach (Deshaies, 2020, Nature 580:329–338). These chimeras are heterobifunctional molecules in which one end binds to the protein of interest (POI) and the other end directs the resulting complex to a specific degradation pathway. To date, proteolysis-targeting chimeras (PROTACs) have received the most attention. (See Sakamoto et al., 2001, Proc. Natl. Acad. Sci. 98:8554-8559; Luh et al., 2020, Angew. Chem. Int. Ed. 59:15448-15466; Wu et al., 2020, Nat. Struct. Mol. Biol. 27:605–614.) PROTACs contain an E3 ligase ligand that directs the targeted protein to the proteasome for degradation. (Lai and Crews, 2017, Nat. Rev. Drug Discovery. 16:101-114; Salami and Crews, 2017, Science 355:1163-1167; Cromm and Crews, 2017, Cell Chem. Biol. 24:1181-1190; Toure and Crews, 2016, Angewandte Chemie Int. 55:1966-1973.) However, PROTACs can only deplete intracellular proteins. Many disease targets are membrane or extracellular proteins.

[0008] To expand the scope of targets, researchers report a method for labeling extracellular protein targets with ligands that are involved in the active transport of molecules into membrane receptors in cells. The labeled proteins are naturally shuttled to lysosomes in the cell, where they are degraded. Bifunctional lysosomal-targeted degraders are typically produced by conjugating ligands for lysosomal targeting receptors (LTRs) on the cell surface to ligands that can bind to extracellular protein targets. The LTRs used in previous studies are carbohydrate-binding proteins, including the cation-independent mannose 6-phosphate receptor (CIM6PR or insulin-like growth factor II receptor) (Banik et al., 2020, Nature. 584:291-297) and the asialoglycoprotein receptor (ASGPR) (Zhou et al., 2021, ACS Cent. Sci. 7:499-506; Ahn et al., 2021, Nat. Chem. Biol. 17:937-946; Caianiello et al., 2021, Nat. Chem. Biol. 17:947-953). Receptor-ligand interactions trigger the internalization of extracellular proteins through receptor-mediated endocytosis, further inducing degradation of targets in lysosomes.

[0009] By conjugating the ligand of CIM6PR on the cell surface to a molecule that binds to an extracellular protein target, a bifunctional lysosomal targeted degrader has been developed (Banik et al., 2020, Nature 584: 291-297). This bifunctional lysosomal targeted degrader that recruits CIM6PR is also known as a lysosomal targeting chimera (LYTAC). CIM6PR is widely expressed in most cell types. Receptor-ligand interactions trigger the internalization of extracellular proteins through receptor-mediated endocytosis, further inducing the degradation of targets in lysosomes. CIM6PR is a transmembrane receptor that transports proteins carrying N-glycans capped with mannose 6-phosphate (M6P) residues to lysosomes (Ghosh et al., 2003, Nat. Rev. Mol. Cell Biol. 4:202-213; Coutinho et al., 2012, Mol. Genet. Metab. 105:542-550). Early studies have shown that M6P-modified albumin increases cellular uptake (Beljaars et al., 1999, Hepatology 29:1486-1493). Subsequently, CIM6PR was used to deliver therapeutic drugs conjugated to M6P derivatives for lysosomal enzyme replacement therapy and cancer treatment (Ghosh et al., 2003, Nature Reviews Mol Cell Biol 4:202-213; Gary-Bobo et al., 2007, Curr. Med. Chem. 14:2945-2953). Various molecules (such as peptides, proteins, or liposomes) are covalently linked to M6P or its analogs to achieve targeted drug delivery (Hoogendoorn et al., 2014, Angewandte Chem. Int. Ed. 53:10975-10978; Crucianelli et al., 2014, RSC Adv. 4:58204-58207; Das et al., 2016, Acs Macro Letters 5:809-813; Agarwal et al., 2016, Chem. Commun. 52:327-330; Hyun et al., 2018, Cell Chem. Biol. 25:1255-1267). To extend the use of the CIM6PR / M6P system to targeted protein degradation, LYTACs were constructed by conjugating a mixture of PEGylated peptides containing 20-40 units of M6P analogs to antibodies against the POI. Unlike the drug delivery process that involves internalization of a covalently linked M6P protein target, LYTACs allow for the transport of complexes formed by non-covalent interactions between the protein target and the LYTAC.It has been shown that LYTAC can successfully degrade secretory proteins and membrane proteins in lysosomes through CIM6PR (Banik et al., 2020, Nature 584: 291-297). However, the challenges associated with the synthesis of heterogeneous mixtures of polymeric glycopeptides with 20-40 units of M6P analogs and the antibodies used in the LYTAC system limit its practicality in drug development. In addition, since CIM6PR is ubiquitously expressed in most cell types, POI is non-selectively delivered to all cell types.

[0010] The present disclosure addresses the unmet need for selective means of degrading membrane and extracellular proteins. By developing new LTRs, different selectivities can be achieved for the degradation of certain membrane or extracellular protein targets depending on the expression profile of the LTR. Summary of the Invention

[0011] Provided herein is a bifunctional lysosomal-targeted degrader comprising: a peptide ligand configured to act as a shuttle molecule to bind to RGD-binding integrin for lysosomal degradation; and a protein binder configured to bind to a preselected membrane or extracellular protein.

[0012] In some versions, the peptide ligand is configured to act as a shuttle molecule to specifically bind to an RGD-binding integrin for lysosomal degradation. In some versions, the protein binder is configured to specifically bind to a preselected membrane or extracellular protein.

[0013] In one version, the peptide ligand is a cyclic peptide.An exemplary peptide ligand disclosed herein is cyclo(Arg-Gly-Asp-D-Phe-Lys) (SEQ ID NO: 1).

[0014] In some embodiments, the protein binder of the bifunctional degrader binds to a membrane protein. The membrane protein can be a membrane receptor. For example, the membrane receptor can be, by way of example and not limitation, epidermal growth factor receptor (EGFR).

[0015] In some embodiments, the protein binding agent of the bifunctional degrader binds to an extracellular protein.

[0016] The protein-binding agent of the bifunctional degrader can be any type of moiety capable of binding to the membrane or extracellular protein to be targeted for degradation via the endosomal / lysosomal pathway. For example, the protein-binding agent is a polypeptide, a ligand, an aptamer, a nanoparticle, or a small molecule.

[0017] In some embodiments, the protein binder of the bifunctional degrader is a polypeptide. For example, the protein binder can be an antibody (whole antibody or antibody fragment), wherein the fragment retains protein binding activity. In a specific version of the bifunctional lysosomal targeted degrader, the antibody is configured to bind to the EGFR protein. In one embodiment, the antibody is cetuximab.

[0018] The bifunctional lysosomal targeted degradation agent may further comprise one or more linkers to facilitate the connection of the peptide ligand to the protein binding agent. In some embodiments, the linker is poly (ethylene glycol).

[0019] Also provided herein is a pharmaceutical composition comprising any of the bifunctional lysosomal targeted degraders disclosed herein. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0020] Also provided herein is a method for degrading a membrane or extracellular protein, the method comprising contacting the membrane or the extracellular protein with any of the bifunctional lysosome-targeted degraders disclosed herein, wherein the bifunctional lysosome-targeted degrader shuttles the membrane or the extracellular protein to a lysosome for degradation.

[0021] Also provided herein is a method comprising administering to an individual in need thereof a therapeutically effective amount of any of the pharmaceutical compositions of the present disclosure. In some embodiments, the individual is a human. In some embodiments, the individual suffers from cancer.

[0022] The objects and advantages of the present disclosure will become more fully apparent from the following detailed description of the preferred embodiments of the present disclosure made in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shown are gel fluorescence analyses of Neutravidin-650 (NA-650) uptake by Huh7 and MCF7 cells treated for 6 hours with 2 μM cRGD-biotin and 500 nM NA-650, compared to treatment without cRGD-biotin.

[0024] Figure 2 The fluorescence absorption of NA-650 in B16F10, Huh7 and MCF7 cells treated with 2 μM cRGD-biotin (blue bars) or triGalNAc(GN)-biotin (black bars) and 500 nM NA-650 for 24 hours is shown. Treatment without any degrading agent (yellow bars) is a negative control.

[0025] Figure 3 Shown is the colocalization of NA-650 with lysosomal tracers in cells treated with 2 μM cRGD-biotin and 500 nM NA-650 for 4 hours (left panel) and 24 hours (right panel).

[0026] Figure 4 Western blot of EGFR in HepG2 cells treated for 24 hours with 10 nM or 100 nM cRGD (cRGD-PEG3-Ctx and cRGD-PEG12-Ctx) linked to cetuximab (Ctx) using different length PEG linkers. The treatment was compared with the folate-based degrader Ctx-FA and the transferrin-based degraders TF-P7-PEG12-Ctx, TF-P9-PEG12-Ctx, and TF-P12-PEG12-Ctx. Actin was used as a loading control. "-" indicates a negative control.

[0027] Figure 5 Western blot of EGFR in MCF7 cells treated for 24 hours with 10 nM or 100 nM cRGD (cRGD-PEG3-Ctx and cRGD-PEG12-Ctx) linked to cetuximab (Ctx) using different length PEG linkers is shown. The treatment was compared with the folic acid-based degradation agent Ctx-FA and the transferrin-based degradation agents TF-P7-PEG12-Ctx, TF-P9-PEG12-Ctx, and TF-P12-PEG12-Ctx. Actin was used as a loading control. "-" indicates a negative control.

[0028] Figure 6 Western blot of EGFR in MCF7 cells treated for 24 hours with 10 nM cRGD (cRGD-PEG3-Ctx and cRGD-PEG12-Ctx) linked to cetuximab (Ctx) using different length PEG linkers is shown. The treatment was compared with the folate-based degradation agent Ctx-FA and the transferrin-based degradation agents TF-P7-PEG12-Ctx, TF-P9-PEG12-Ctx, and TF-P12-PEG12-Ctx. Actin was used as a loading control. "-" indicates a negative control.

[0029] Figure 7Western blot of EGFR in HeLa cells treated for 24 hours with 10 nM cRGD linked to cetuximab (Ctx) using PEG linkers of varying lengths (cRGD-PEG3-Ctx and cRGD-PEG12-Ctx). The treatment was compared with the folate-based degrader Ctx-FA and the transferrin-based degraders TF-P7-PEG12-Ctx, TF-P9-PEG12-Ctx, and TF-P12-PEG12-Ctx. Actin was used as a loading control. "-" indicates a negative control.

[0030] Figure 8 Shown are the MCF dose responses of Ctx-cRGD at 0 nM (control), 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM.

[0031] Figure 9 Shown is the MCF time course response of Ctx-cRGD over 48 hours.

[0032] Figure 10 are fluorescence micrographs showing colocalization of EGFR and lysosomal markers for Ctx and Ctx-cRGD.

[0033] Figure 11 Shown are individual and merged fluorescence micrographs showing colocalization of EGFR, LAMP1, DAPI, as well as a merged image of all three.

[0034] Figure 12 is a gel depicting the degradation of PDL1 by Atz-PEG3-cRGD and Atz-PEG12-cRGD.

[0035] Figure 13 is a gel depicting the competition of 6 μM cRGD-azide on Ab-cRGD-induced anti-biotin-647 uptake.

[0036] Figure 14 Is a gel depicting the inhibition of lysosomal degradation of anti-biotin-647 by 50 nM Bafilomycin A1 (BAF1).

[0037] Figure 15 is a gel depicting the inhibition of EGFR degradation by increasing concentrations of cRGD-azide.

[0038] Figure 16 Gels depicting the inhibition of EGFR degradation by the lysosomal degradation inhibitors bafilomycin A1 (BAF1, 50 nM) and chloroquine (CQ, 10 uM).

[0039] Figure 17Shown are gels (A, C, E) and gel quantification (B, D, F), which depict that Ctx-cRGD has a higher degradation efficacy against cancer cells (Hela (A, B) and HepG2 (C, D)) than against normal cells (HACAT (E, F)). DETAILED DESCRIPTION

[0040] Provided herein are bifunctional lysosomal targeted degraders comprising: (a) a peptide ligand that acts as a shuttle molecule to bind to an RGD-binding integrin for lysosomal degradation; and (b) a protein binder that binds to a membrane or extracellular protein of interest. The bifunctional degraders disclosed herein can be used, for example, to selectively target and degrade membrane and extracellular proteins through the endosomal / lysosomal pathway. In one aspect, the bifunctional degraders induce specific degradation of oncogenic proteins in cancer cells through RGD-binding integrins. Also provided herein are compositions comprising the bifunctional degraders and methods of using the bifunctional degraders to inhibit disease states, including cancer.

[0041] It should be understood that the bifunctional degraders, compositions, and methods disclosed herein are not limited to the specific embodiments described, as such embodiments may, of course, vary. The bifunctional degraders, compositions, and methods disclosed herein may comprise, consist of, or consist essentially of the various elements or steps disclosed herein. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0042] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "or" means "and / or." Recitation of ranges of values ​​is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included in the stated ranges and are independently combinable.

[0043] All patents and publications referenced or mentioned herein are indicative of the level of skill of those skilled in the art to which the present disclosure pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been individually incorporated by reference in its entirety or set forth herein in its entirety. Applicants reserve the right to actually incorporate into this specification any and all materials and information from any such referenced patent or publication.

[0044] It should be understood that certain features of bifunctional degraders, compositions, and methods described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of bifunctional degraders, compositions, and methods described for clarity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments are expressly contemplated by this disclosure and are disclosed herein as if each combination were individually and expressly disclosed, provided such combinations comprise operable processes and / or compositions. In addition, all subcombinations listed in the embodiments describing these variables are also expressly contemplated by the present bifunctional degraders, compositions, and methods and are disclosed herein as if each such subcombination were individually and expressly disclosed herein.

[0045] Bifunctional lysosomal-targeted degraders

[0046] Provided herein are bifunctional lysosomal-targeted degraders comprising: a peptide ligand that acts as a shuttle molecule to bind to RGD-binding integrins for lysosomal degradation; and a protein binder that binds to a membrane or extracellular protein of interest.

[0047] Integrins are heterodimeric transmembrane glycoproteins consisting of an α subunit and a β subunit. The α and β subunits bind to ligand binding sites at the interface to form a non-covalent complex. Integrins act as adhesion receptors, capable of signaling in two directions across the plasma membrane. These events, referred to as "inside-out" and "outside-in" signaling, are either due to binding to extracellular ligands or through interactions between the integrin intracellular domain and the cytoskeleton. Thus, integrins can enable human cells to respond to changes in the extracellular environment (via outside-in signaling) and can influence the extracellular environment itself (via inside-out signaling). See Hynes, 2002, Cell, 110:673–687; Zhu et al., 2007, Blood, 110:2475–2483; and Slack et al., 2022, Nature Reviews Drug Discovery, 21:60–78. Of the 24 human integrin subtypes known to date, eight integrin dimers, namely αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, α8β1, and αIIbβ3, recognize the tripeptide Arg-Gly-Asp (RGD) motif within extracellular matrix proteins.

[0048] Peptide ligands that can bind to integrins have been widely studied for delivering anticancer drugs. See Nieberler et al., 2017, Cancers (Basel) 9:116; Hatley et al., 2018, Chem. Int. Ed. 57:3298-3321; Sani et al., 2021, Chembiochem 22:1151-1160; and Ludwig et al., 2021, Cancers (Basel) 13:1711. In the present disclosure, it is shown that peptide ligands that bind to RGD-binding integrins can be used to develop lysosomal targeted degraders. As disclosed herein, peptide ligands are connected to binding agents for membrane or extracellular proteins of interest to produce lysosomal targeted degraders. Once inside the lysosome, the target protein is released from the molecule and degraded. The shuttle molecule is released back outside the cell where it can bind to another target protein.

[0049] Any peptide ligand (including linear and cyclic peptides) that binds to RGD-binding integrins is contemplated herein.

[0050] In one version of the present disclosure, the peptide ligand is a cyclic peptide that binds to the RGD-binding integrin. Cyclization of the peptide can increase stability and reduce conformational space to improve the biological efficacy of the molecule. Non-limiting examples of cyclic peptides that bind to the RGD-binding integrin include c(RGDfK) (SEQ ID NO: 1), c(RGDfV) (SEQ ID NO: 2), c(RGDfE) (SEQ ID NO: 3), c(RGDyK) (SEQ ID NO: 4), c(RGDfC) (SEQ ID NO: 5), c(phgisoDGRk) (SEQ ID NO: 6), c(RGDf(NMe)V) (SEQ ID NO: 7), and c(FRGDLAFp(NMe)K) (SEQ ID NO: 8). See Kapp et al., 2017, Sci. Rep. 7: 39805.

[0051] In one embodiment, the cyclic peptide is c(RGDfK)=cyclo(Arg-Gly-Asp-D-Phe-Lys) (SEQ ID NO: 1). The key residues are Arg-Gly-Asp or RGD, and Lys is part of the linker. This cyclic peptide has strong affinity for the αvβ3 isoform (2 nM), moderate binding to αvβ5, αvβ6, and α5β1 (50-350 nM), and weak binding to αvβ8 and αllbβ3 (>5,000 nM) (Kapp et al., 2017, Scientific Reports 7:39805).

[0052] As disclosed herein, bifunctional lysosomal targeted degraders include protein binding agents that bind to membrane or extracellular proteins of interest.

[0053] In some embodiments, the protein binding agent binds to a membrane protein.

[0054] In certain embodiments, the membrane protein is a membrane receptor. The membrane receptor of interest includes, but is not limited to, stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, receptors in the epidermal growth factor receptor (EGFR) family (e.g., HER2 (human epidermal growth factor receptor 2), etc.), receptors in the fibroblast growth factor receptor (FGFR) family, receptors in the vascular endothelial growth factor receptor (VEGFR) family, receptors in the platelet-derived growth factor receptor (PDGFR) family, receptors in the rearranged during transfection (RET) receptor family, receptors in the Eph receptor family, receptors in the lectin domain receptor (DDR) family, and mucins (e.g., MUC1).

[0055] In one particular version, the membrane receptor is EGFR, which is known to be frequently mutated or overexpressed in different types of human cancers (Yarden and Pines, 2012, Nat Rev Cancer. 12:553–563; Sigismund et al., 2018, Mol. Oncol. 12:3–20).

[0056] Membrane proteins can be immunosuppressive receptors. As used herein, "immunosuppressive receptors" are receptors that negatively regulate immune responses present on immune cells. Examples of inhibitory immunoreceptors include immunosuppressive receptors of the Ig superfamily, including but not limited to: CD200R, CD300a (IRp60; mouse MAIR-1), CD300f (IREM-1), CEACAM1 (CD66a), FcγRIIb, ILT-2 (LIR-1; LILRB1; CD85j), ILT-3 (LIR-5; CD85k; LILRB4), ILT-4 (LIR-2; LILRB2), ILT-5 (LIR-3; LILRB3; mouse PIR-B); LAIR-1, PECAM-1 (CD31), PILR-α (FDF03), SIRL-1, and SIRP-α. Additional examples of immunosuppressive receptors include sialic acid-binding Ig-like lectin (Siglec) receptors, e.g., Siglec 7, Siglec 9, etc. Additional examples of immunosuppressive receptors include C-type lectins, which include, but are not limited to, CLEC4A (DCIR), Ly49Q, and MICL. Detailed information on immunosuppressive receptors can be found, for example, in Steevels et al., 2011, Eur. J. Immunol. 4: 575-587.

[0057] The membrane protein may optionally be a ligand for an immunosuppressive receptor, an example of which is CD47, which binds to SIRP-α to prevent phagocytosis and is known to be overexpressed in cancer cells (Eladl et al., 2020, J. Hematol. Oncol. 13:96).

[0058] Membrane proteins can also be immune checkpoint molecules, which include immune checkpoint proteins and ligands. Non-limiting examples of immune checkpoint molecules include members of the PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT and B7 families. In one embodiment, the membrane protein is PD-L1 (programmed cell death ligand 1), which binds to PD-1 (programmed cell death 1) to inhibit apoptosis and is known to be overexpressed in cancer cells (Yi et al., 2021, Journal of Hematology and Oncology 14:10).

[0059] In some embodiments, the protein binding agent binds to an extracellular protein.

[0060] The extracellular protein can be a ligand for a membrane receptor. Membrane receptor ligands of interest include, but are not limited to, growth factors (e.g., epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), etc.), cytokines (e.g., interleukins, interferons, tumor necrosis factor (TNF), transforming growth factor β (TGF-β), including any specific subtypes of such cytokines), hormones, etc.

[0061] Alternatively, the extracellular protein can be an antibody, for example, an antibody bound to a membrane protein or different extracellular proteins. The antibody can be an autoantibody. "Autoantibody" means an antibody against one or more proteins in the protein of the individual self produced by the immune system. Cancer cells can induce an immune response, thereby producing tumor-associated autoantibodies. The limiting examples of autoantibodies include rheumatoid factor (RF), antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (ANCA), anti-double-stranded DNA (anti-dsDNA), anticentromere antibodies (ACA), anti-cyclic citrullinated peptide antibodies (anti-CCP), extractable nuclear antigen antibodies (ENA), anti-cardiolipin antibodies, beta-2 glycoprotein 1 antibodies, antiphospholipid antibodies (APA), lupus anticoagulant (LA), anti-tissue transglutaminase (anti-tTG), anti-gliadin antibodies (AGA), intrinsic factor antibodies, parietal cell antibodies, thyroid antibodies, smooth muscle antibodies (SMA), anti-mitochondrial antibodies (AMA), anti-glomerular basement membrane (GBM), acetylcholine receptor (AChR) antibodies, etc.

[0062] The extracellular protein may be a secreted protein, including but not limited to secreted growth factors, extracellular matrix degrading proteases, cell motility factors, and immunomodulatory cytokines or other biologically active molecules.

[0063] The extracellular protein may also be a mutein.

[0064] When the protein binder of the bifunctional degrader binds to a membrane or extracellular protein, the membrane or extracellular protein can be present on or produced by a cancer cell. A "cancer cell" refers to a cell that exhibits a tumor cell phenotype, which can be characterized by, for example, abnormal cell growth, abnormal cell proliferation, loss of density-dependent growth inhibition, anchorage-independent growth potential, the ability to promote tumor growth and / or development in an immunocompromised non-human animal model, and / or any appropriate cell transformation indicator. "Cancer cell" can be used interchangeably herein with "tumor cell," "malignant cell," "neoplastic cell," or "cancerous cell," and encompasses cancer cells of solid tumors, semi-solid tumors, hematologic malignancies (e.g., leukemia cells, lymphoma cells, myeloma cells, etc.), primary tumors, metastatic tumors, and the like. In some embodiments, the membrane protein present on the cancer cell is a tumor-associated antigen or a tumor-specific antigen.

[0065] The protein-binding agent of the bifunctional degrader can be any type of moiety capable of binding to the membrane or extracellular protein to be targeted for degradation via the endosomal / lysosomal pathway. In certain aspects, the protein-binding agent is selected from a polypeptide, a ligand (e.g., a ligand of a membrane receptor, wherein the membrane receptor is targeted for degradation), an aptamer, a nanoparticle, and a small molecule.

[0066] Protein binding agents can be small molecules. "Small molecule" means a compound with a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less.

[0067] The protein binding agent can be a polypeptide, such as an antibody. The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.); complete antibodies (e.g., antibodies composed of tetramers, which in turn are composed of two dimers of heavy and light chain polypeptides); single-chain antibodies; antibody fragments that retain specific binding to membrane or extracellular proteins (e.g., full-chain or single-chain antibody fragments), including but not limited to Fv, single-chain Fv (scFv), Fab, F(ab')2, Fab', (scFv')2, bifunctional antibodies, and nanobodies; chimeric antibodies; monoclonal antibodies; fully human antibodies; humanized antibodies (e.g., humanized complete antibodies, humanized antibody fragments, etc.); and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein or fragment thereof. The antibody can be detectably labeled with, for example, an in vivo imaging agent. The antibody can be further conjugated to other moieties, such as polyethylene glycol (PEG), etc. Fusion to the antibody Fc region (or fragment thereof), conjugation to PEG, etc. can be used, for example, to increase the serum half-life of the antibody when administered to a subject.

[0068] In some versions, the antibody is configured to specifically bind to a cancer antigen.

[0069] Antibodies can also be configured to bind to whole complement or a fragment thereof. In certain embodiments, the antibodies bind to one or more immunodominant epitopes within whole complement or a fragment thereof.

[0070] Alternatively, the antibody may bind to a membrane receptor or a membrane receptor ligand. Or the antibody may bind to an epidermal growth factor (EGF) protein (eg, human EGF) or one or more immunodominant epitopes within the EGF protein.

[0071] In certain embodiments, the antibody binds to the EGFR protein. In certain embodiments, the antibody binds to one or more immunodominant epitopes within the EGFR protein. In a certain embodiment, the antibody comprises the CDRs present in cetuximab (Ctx). In another specific embodiment, the antibody comprises the variable light chain and variable heavy chain present in cetuximab. In a specific embodiment, the antibody is cetuximab.

[0072] In certain embodiments, the antibodies bind to an immunosuppressive receptor. In certain embodiments, the antibodies bind to one or more immunodominant epitopes within an immunosuppressive receptor.

[0073] In certain embodiments, the antibodies bind to a ligand of an immunosuppressive receptor. In certain embodiments, the antibodies bind to one or more immunodominant epitopes within a ligand of an immunosuppressive receptor. In certain embodiments, the antibodies bind to a CD47 protein. In certain embodiments, the antibodies bind to one or more immunodominant epitopes within a CD47 protein.

[0074] In certain embodiments, the antibody binds to an immune checkpoint molecule. In certain embodiments, the antibody binds to one or more immunodominant epitopes within an immune checkpoint molecule. In certain embodiments, the antibody binds to a PD-L1 protein. In certain embodiments, the antibody binds to one or more immunodominant epitopes within a PD-L1 protein. In a certain embodiment, the antibody comprises the CDRs present in atezolizumab (Atz). In another specific embodiment, the antibody comprises the variable light chain and variable heavy chain present in atezolizumab. In a specific embodiment, the antibody is atezolizumab.

[0075] The bifunctional lysosomal-targeted degraders disclosed herein can be in any suitable form. In some embodiments, the bifunctional degraders are conjugates. Thus, in certain embodiments, the bifunctional degraders disclosed herein comprise an RGD-binding integrin peptide ligand conjugated to a protein-binding agent. In some embodiments, the protein-binding agent is a polypeptide, and the bifunctional molecule is a fusion protein comprising the RGD-binding integrin peptide ligand fused to the protein-binding agent.

[0076] In certain embodiments, one or more linkers can be used to facilitate the connection of the RGD binding integrin peptide ligand to the protein binder. Non-limiting examples of such linkers include ester linkers (e.g., N-hydroxysuccinimide (NHS) esters, sulfo-NHS esters, or PFP esters or thioesters), amide linkers, maleimide or maleimide-based linkers; valine-citrulline linkers; hydrazone linkers; N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linkers; succinimidyl-4-(A '-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linkers; vinyl sulfone-based linkers; linkers including polyethylene glycol (PEG), such as, but not limited to, tetraethylene glycol; linkers including propionic acid; linkers including decenoic acid, and linkers including any combination thereof. In one embodiment, the linker is PEG. PEGs of different lengths can be used as linkers, such as PEG3, PEG 12, and the like.

[0077] In some aspects, the linker is a chemically unstable linker, such as an acid-cleavable linker that is stable at neutral pH (blood stream pH 7.3-7.5) but undergoes hydrolysis when internalized into the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of target cells (e.g., cancer cells). Chemically unstable linkers include but are not limited to hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, etc. According to certain embodiments, the linker is an enzyme-unstable linker, such as an enzyme-unstable linker that is stable in the blood stream but undergoes enzymatic cleavage when internalized into target cells (e.g., by lysosomal proteases (such as cathepsin or plasmin) in the lysosomes of target cells (e.g., cancer cells)). Enzyme-labile linkers include, but are not limited to, linkers comprising peptide bonds, e.g., dipeptide-based linkers such as valine-citrulline linkers, such as maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linkers, valyl-alanyl-p-aminobenzyloxy (Val-Ala-PAB) linkers, etc. Chemically labile linkers, enzyme-labile and non-cleavable linkers are known and described, e.g., in Ducry and Stump, 2010, Bioconjugate Chem. 21: 5-13.

[0078] In certain aspects, the bifunctional degrader enhances the degradation of the membrane or extracellular protein relative to the degradation of the membrane or extracellular protein in the presence of the protein binder alone. According to some embodiments, the bifunctional degrader enhances the degradation of the membrane or extracellular protein relative to the degradation of the membrane or extracellular protein in the presence of the RGD-binding integrin peptide ligand or protein binder alone. In this context, "enhanced degradation" means that under the same conditions, the membrane or extracellular protein is degraded in the presence of the bifunctional degrader and is not degraded in the presence of the protein binder alone or the RGD-binding integrin peptide ligand or protein binder alone; or under the same conditions, the membrane or extracellular protein is degraded to a greater extent in the presence of the bifunctional degrader than in the presence of the protein binder alone or the RGD-binding integrin peptide ligand or protein binder alone. Under the same conditions, when the extent of degradation of a membrane or extracellular protein in the presence of a bifunctional degrader is greater than the degradation of a membrane or extracellular protein in the presence of a protein binder alone or an RGD-binding integrin peptide ligand or protein binder alone, the degradation in the presence of the bifunctional degrader can be 1.2-fold or greater, 1.4-fold or greater, 1.6-fold or greater, 1.8-fold or greater, 2-fold or greater, 2.5-fold or greater, 3-fold or greater, 3.5-fold or greater, 4-fold or greater, 4.5-fold or greater, 5-fold or greater, 5.5-fold or greater, 6-fold or greater, 6.5-fold or greater, 7-fold or greater, 7.5-fold or greater, 8-fold or greater, 8.5-fold or greater, 9-fold or greater, 9.5-fold or greater, or 10 or greater.

[0079] Composition

[0080] Disclosed herein are compositions comprising any of the bifunctional lysosomal targeted degraders of the present disclosure.

[0081] The composition may optionally include the bifunctional degradation agent of the present disclosure in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffer solution, or the like. One or more additives, such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), protease inhibitors, glycerol, etc. may be present in such compositions.

[0082] Also disclosed herein are pharmaceutical compositions comprising any of the bifunctional lysosomal targeted degraders of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutical compositions typically include a therapeutically effective amount of a bifunctional degrader. A "therapeutically effective amount" means a dose sufficient to produce a desired result, for example, an amount sufficient to produce a beneficial or desired therapeutic (including preventive) result, such as reducing cell proliferation in an individual suffering from a cell proliferative disorder (e.g., cancer) associated with a membrane or extracellular protein bound by a protein binder of the bifunctional degrader. The effective amount can be administered in one or more administrations.

[0083] The bifunctional degraders disclosed herein can be incorporated into various formulations for therapeutic administration. More specifically, the bifunctional degraders can be formulated into pharmaceutical compositions by combining with appropriate pharmaceutically acceptable excipients or diluents, and can be formulated into formulations in solid, semisolid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants, and aerosols.

[0084] Formulations of the disclosed bifunctional degraders suitable for administration to an individual (eg, suitable for human administration) are generally sterile and, depending on the route of administration chosen, may further be free of detectable pyrogens or other contaminants that would render administration to an individual inappropriate.

[0085] In terms of pharmaceutical dosage forms, the bifunctional degraders can be administered alone or in appropriate association and combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are by no means limiting.

[0086] For oral preparations, the bifunctional degraders can be used alone or in combination with appropriate additives to prepare tablets, powders, granules or capsules, for example, with conventional additives (such as lactose, mannitol, corn starch or potato starch); with binders (such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatin); with disintegrants (such as corn starch, potato starch or sodium carboxymethylcellulose); with lubricants (such as talc or magnesium stearate); and, if desired, with diluents, buffers, wetting agents, preservatives and flavorings.

[0087] The bifunctional degraders can be formulated into preparations for injection by dissolving, suspending or emulsifying the compound in an aqueous or non-aqueous solvent (such as vegetable oil or other similar oils), synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol; and if desired, using conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0088] The pharmaceutical composition can be in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form, wherein the lyophilized formulation is reconstituted with a sterile solution prior to administration. The standard procedure for reconstitution of a lyophilized composition is to add back a volume of purified water (usually equivalent to the volume removed during lyophilization); however, a solution containing an antibacterial agent can be used to produce a pharmaceutical composition for parenteral administration.

[0089] Aqueous formulations of bifunctional degradants can be prepared in a pH buffered solution, for example, at a pH range of about 4.0 to about 8.0 (e.g., about 4.5 to about 7.5, for example, about 5.0 to about 7.0). Examples of buffers suitable for pH in this range include phosphate-, histidine-, citrate-, succinate-, acetate-, and other organic acid buffers. The buffer concentration can be about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on, for example, the desired tonicity of the buffer and formulation.

[0090] How to use

[0091] Disclosed herein are methods of using the bifunctional lysosomal targeted degraders of the present disclosure.

[0092] Provided herein is a method for degrading membrane or extracellular protein. Such methods are included in the case where a bifunctional lysosomal targeted degradation agent shuttles membrane or extracellular protein to lysosome for degradation, and contacts membrane or extracellular protein with any bifunctional lysosomal targeted degradation agent in the bifunctional lysosomal targeted degradation agent of the present disclosure. Such methods can be used in various applications. In some aspects, the method is carried out in vitro (for example, in a tube, cell culture plate or well, etc.), and can be used for example, testing and / or research applications. In other aspects, the method is carried out in vivo (for example, in an individual administering a bifunctional degradation agent), and can be used for example, clinical / therapeutic applications.

[0093] Also provided are methods comprising administering a therapeutically effective amount of any of the bifunctional degraders of the present disclosure or any of the pharmaceutical compositions of the present disclosure to an individual in need thereof. Various individuals can be treated according to the subject methods. Generally, such subjects are "mammals" or "mammals," where these terms are broadly used to describe organisms within the mammalian class, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the individual is a human.

[0094] An effective amount of a bifunctional degrader (or a pharmaceutical composition comprising the same) is an amount that, when administered alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), at one or more doses, is effective to reduce a medical condition (e.g., cancer) in a subject by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, as compared to the symptoms in a subject not treated with the bifunctional degrader or pharmaceutical composition.

[0095] The method comprises administering a therapeutically effective amount of any of the bifunctional degraders of the present disclosure or any of the pharmaceutical compositions of the present disclosure to an individual suffering from cancer. According to such methods, the protein binder of the bifunctional degrader binds to a membrane or extracellular protein that at least contributes to the individual's cancer, and wherein the targeted degradation of the membrane or extracellular protein of the bifunctional degrader is used to treat the individual's cancer. In certain aspects, the protein binder binds to a protein selected from the group consisting of: a membrane receptor, a ligand of a membrane receptor, an immunosuppressive receptor, a ligand of an immunosuppressive receptor, an immune checkpoint molecule, an autoantibody, a secreted protein, and a mutant protein.

[0096] For example, the individual to be treated may have a cancer characterized by the presence of solid tumors, semi-solid tumors, primary tumors, metastatic tumors, etc. In some embodiments, the individual has a cancer selected from the group consisting of breast cancer, melanoma, lung cancer, colorectal cancer, prostate cancer, glioma, bladder cancer, endometrial cancer, kidney cancer, leukemia (e.g., acute myeloid leukemia (AML)), liver cancer (e.g., hepatocellular carcinoma (HCC), such as primary or recurrent HCC), non-Hodgkin lymphoma, pancreatic cancer, thyroid cancer, any combination thereof, and any subtype thereof.

[0097] In any of the methods of using the bifunctional degraders of the present disclosure, the bifunctional degraders generally enhance the degradation of membrane or extracellular proteins relative to the degradation of membrane or extracellular proteins in the presence of the protein-binding agent alone. Similarly, in any of the methods of using the bifunctional degraders of the present disclosure, according to some embodiments, the bifunctional degraders enhance the degradation of membrane or extracellular proteins relative to the degradation of membrane or extracellular proteins in the presence of the RGD-binding integrin peptide ligand or the protein-binding agent alone.

[0098] "Treat," "treating," or "treatment" means at least an improvement in the symptoms associated with a medical condition (e.g., a cell proliferative disorder, e.g., cancer) in a subject, where improvement is used broadly to refer to at least a decrease in the magnitude of a parameter, e.g., a symptom associated with the medical condition being treated. Thus, treatment also includes situations in which the medical condition, or at least the symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated), such that the subject no longer suffers from the medical condition or at least the symptoms characterizing the medical condition.

[0099] The bifunctional degradant or pharmaceutical composition can be administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local administration routes. Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical, ocular, intravenous, intraarterial, nasal, oral and other enteral and parenteral routes of administration. In some embodiments, administration is performed by parenteral administration. If desired, the routes of administration can be combined or adjusted according to the bifunctional degradant and / or the desired effect. The bifunctional degradant or pharmaceutical composition can be administered in a single dose or in multiple doses. In some embodiments, the bifunctional degradant or pharmaceutical composition is administered intravenously. In some embodiments, the bifunctional degradant or pharmaceutical composition is administered by injection, for example, for systemic delivery (e.g., intravenous infusion) or delivery to a local site.

[0100] Examples

[0101] Cyclic peptides were tested as binders to RGD-binding integrins for the development of lysosomal-targeted degraders. The cyclic peptide cRGD = cyclo(Arg-Gly-Asp-D-Phe-Lys) (SEQ ID NO: 1), also known as c(RGDfK) (SEQ ID NO: 1) (Kapp et al., 2017, Scientific Reports 7: 39805). The key residues are Arg-Gly-Asp or RGD, while Lys is part of the linker. This cyclic peptide has a strong affinity for the αvβ3 isoform (2 nM), moderate binding to αvβ5, αvβ6, and α5β1 (50-350 nM), and weak binding to αvβ8 and αIIbβ3 (> 5,000 nM) (Kapp et al., 2017, Scientific Reports 7: 39805).

[0102] A conjugate of c(RGDfK) (SEQ ID NO: 1) and biotin (cRGD-biotin, Vivitide, PCI-3697-PI-1MG) can bind to the fluorescent model target protein Neutravidin-650 (NA-650). The uptake of the model target protein NA-650 by cRGD-biotin was tested by treating Huh7 cells and MCF7 cells with 2 μM cRGD-biotin and 500 nM NA-650 for 6 hours. Figure 1 It is shown that in the presence of cRGD-biotin, uptake of NA-650 protein was observed in both Huh7 cells and MCF7 cells.

[0103] The uptake of NA-650 was also compared to triGalNAc(GN)-biotin, one of the most effective lysosomal-targeted degraders for the uptake of secretory proteins into hepatocytes (e.g., Huh7) (Zhou et al., 2021, ACS Central Science 7:499). Huh7 cells were treated with 2 μM cRGD-biotin or GN-biotin and 500 nM NA-650 for 24 hours, and the results were compared with the treatment of non-hepatocytes B16F10 and MCF7. Figure 2 As shown, in the presence of GN-biotin, only Huh7 cells took up NA-650. In the presence of cRGD-biotin, all three cell types took up NA-650.

[0104] Colocalization of NA-650 protein with lysosomal tracers, such as Figure 3 The results indicate that cyclic peptide-based lysosomal-targeted degraders can promote the uptake of soluble model target proteins into lysosomes.

[0105] The cRGD peptide was also linked to cetuximab (Ctx), an antibody that binds to the membrane target protein EGFR. Lysosomal-targeted degraders were prepared and tested for their ability to degrade EGFR. Two linkers were used between the cyclic peptide binder to the receptor and the antibody (EGFR binder). The linkers were polyethylene glycol 3 (PEG3; H-(O-CH2-CH2)3-OH) and polyethylene glycol 12 (PEG12; H-(O-CH2-CH2)3-OH). 12-OH). A construct of cetuximab conjugated to folic acid via a PEG3 linker (Ctx-FA) was used as a positive control. To prepare cRGD-cetuximab conjugates (cRGD-PEG3-Ctx, cRGD-PEG12-Ctx), cetuximab was reacted with DBCO-PEG3-NHS or DBCO-PEG12-NHS ester at a molar ratio of 1:25 at room temperature on a rotator overnight in 200 μL PBS at a concentration of 1.8 mg / ml. The mixture was then purified 5 times with 500 μL PBS using a 10 kDa Amicon centrifugal filter, the concentration of the DBCO-labeled antibody was subsequently measured by BCA assay, and the reaction was continued overnight with cRGD-N3 on a rotator at room temperature. The resulting antibody conjugate was then purified 5 times with 500 μL PBS using a 10 kDa Amicon centrifugal filter. The purified antibody conjugate was then conjugated to cRGD via the NHS moiety. To prepare Ctx-FA, cetuximab was reacted with DBCO-PEG3-NHS ester at a molar ratio of 1:25 at a concentration of 1.8 mg / ml in 200 μL PBS on a rotator at room temperature overnight. The mixture was then purified five times with 500 μL PBS using a 10 kDa Amicon centrifugal filter, followed by measurement of the concentration of DBCO-labeled antibody by BCA assay, and reacted with folic acid-N3 on a rotator at room temperature overnight. The resulting antibody conjugate was then purified five times with 500 μL PBS using a 10 kDa Amicon centrifugal filter. Degradation results ( Figure 4-7 ) showed that EGFR protein levels were significantly reduced in cells treated with antibodies labeled with the cyclic peptide cRGD, indicating that EGFR was degraded in the cells. In the cancer cells tested, degraders with longer PEG linkers (PEG12) had better degradation activity against EGFR. The degradation effect observed at a concentration of 100 nM was less than that at a concentration of 10 nM, which may be due to the hook effect (Douglass et al., 2013, Journal of the American Chemical Society (J.Am.Chem.Soc.) 135:6092).

[0106] An MCF dose response experiment was performed using the DBCO-PEG12-NHS construct (also referred to herein as Ctx-cRGD) at 0 nM (control), 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM. MCF7 cells were seeded in 24-well plates at 70% confluence and incubated overnight in 350 μL complete medium. Cells were then treated with Ctx-PEG12-cRGD at various concentrations in 50 μL of medium for 24 hours as indicated in the figure before being collected for Western blot analysis. The results are shown in Figure 2. Figure 8 Shown in.

[0107] The MCF time course response to Ctx-cRGD over 48 hours was determined. MCF7 cells were seeded at 70% confluence in 24-well plates and incubated overnight in 350 μL complete medium. Cells were then treated with Ctx-PEG12-cRGD at 10 nM in 50 μL medium for various periods of time as indicated before collection for Western blot analysis. Figure 9 Shown in.

[0108] Figure 10 Figure 2 is a fluorescence micrograph showing the colocalization of EGFR and lysosomal markers for Ctx and Ctx-cRGD. MCF7 cells were plated on 8-well chamber slides at a density of 20,000 cells / well in 200 μL of complete culture medium. The cells were treated with 10 nM Ctx-PEG12-cRGD at 37°C for 24 hours and subsequently washed three times with PBS. The cells were then fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton-100 for 5 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were then incubated with an anti-EGFR antibody in 1% BSA overnight at 4°C. The next day, the cells were incubated with an anti-rabbit 594 secondary antibody for 1 hour at room temperature. After each antibody incubation, the cells were washed three times with PBS. The cells were then mounted with a slow-fade-anti-fade mounting medium containing DAPI. Images were acquired with a Leica SP8 3x STED super-resolution microscope at 60x magnification using a 10x objective and analyzed using ImageJ.

[0109] Figure 11 Shown are single and merged fluorescence micrographs showing the colocalization of EGFR, LAMP1, DAPI, and a merged image of all three. MCF7 cells at a density of 20,000 cells / well were distributed on 8-well chamber slides in 200 μL complete medium. The cells were treated with 10nM Ctx-cRGD at 37°C for 24 hours and then washed three times with PBS. The cells were then fixed with 4% paraformaldehyde for 15 minutes, then permeabilized with 0.5% Triton-100 for 5 minutes and blocked with 5% BSA for 1 hour at room temperature. The cells were then incubated with anti-EGFR and anti-LAMP1 antibodies in 1% BSA at 4°C overnight. The next day, the cells were incubated with anti-mouse 488 and anti-rabbit 594 secondary antibodies at room temperature for 1 hour. After each antibody incubation, the cells were washed three times with PBS. The cells were then mounted with a slow-fading-anti-fading mounting medium containing DAPI. Images were acquired with a Leica SP8 3x STED super-resolution microscope at 60x magnification using a 10x objective and analyzed using ImageJ.

[0110] Figure 12 Shown is a gel depicting the degradation of PDL1 with Atz-PEG3-cRGD and Atz-PEG12-cRGD. The Atz-PEG3-cRGD and Atz-PEG12-cRGD constructs are atz (Atz) connected to cRGD via a PEG3 or PEG12 linker. Atz at a concentration of 1.8 mg / ml in 200 μL PBS was reacted with DBCO-PEG3-NHS or DBCO-PEG12-NHS ester at a molar ratio of 1:25 on a rotator at room temperature overnight. The mixture was then purified 5 times with 500 μL PBS using a 10kDa Amicon centrifugal filter, the concentration of the DBCO-labeled antibody was subsequently measured by BCA assay, and the reaction was continued overnight on a rotator at room temperature with cRGD-N3. The resulting antibody conjugate was then purified 5 times with 500 μL PBS using a 10kDa Amicon centrifugal filter. MCF7 cells were seeded in 24-well plates at 70% confluence and incubated overnight in 350 μL of complete culture medium. Cells were then treated with various concentrations of Atz-PEG3-cRGD or Atz-PEG12-cRGD in 50 μL of culture medium for 24 hours, as indicated, before being harvested for Western blot analysis. Atz-PEG3-FA, treated at 10 nM, served as a positive control.

[0111] Figure 13Figure 1 is a gel depicting the competition of 6 μM cRGD-azide (Vivitide, RGD-3749-PI-5MG) against Ab-cRGD-induced uptake of anti-biotin-647 antibody (Jackson ImmunoResearch, 200-602-211). Ab-cRGD is a goat anti-mouse IgG antibody (Ab) linked to cRGD via a PEG12 linker. Ab at a concentration of 1.8 mg / ml in 200 μL PBS was reacted with DBCO-PEG12-NHS ester at a molar ratio of 1:25 on a rotator at room temperature overnight. The mixture was then purified five times with 500 μL PBS using a 10 kDa Amicon centrifugal filter, followed by measurement of the concentration of DBCO-labeled antibody by BCA assay, and reacted with cRGD-N3 on a rotator at room temperature overnight. The resulting antibody conjugate was then purified five times with 500 μL PBS using a 10 kDa Amicon centrifugal filter. One day before treatment, cells were seeded in 48-well plates at 70% confluence in 200 μL of complete medium. Cells were then sequentially treated with 25 μL of medium containing 50 nM anti-biotin-647 and 25 μL of medium containing 25 nM Ab-cRGD and incubated at 37°C for the indicated periods, followed by two washes with PBS before collection for in-gel fluorescence analysis. Cells were pre-incubated with a free excess of 6 μM cRGD-azide for 1 hour at 4°C for competition.

[0112] Figure 14 This is a gel depicting the inhibition of lysosomal degradation of anti-biotin-647 by 50nM bafilomycin A1 (BAF1). MCF7 cells were seeded in 48-well plates at 70% confluence and maintained in 200 μL complete medium. The next day, cells were incubated with 25nM Ab-cRGD and 50nM anti-biotin-647 for 3 hours and then washed three times with PBS. The cells were then maintained in fresh medium with or without 50nM bafilomycin A1 (BAF1) for another 3 hours before being collected for in-gel fluorescence analysis.

[0113] Figure 15 This is a gel depicting the inhibition of EGFR degradation by increasing concentrations of cRGD-azide. MCF7 cells were seeded in a 24-well plate at 70% confluence and incubated overnight in 350 μL of complete culture medium. Cells were pretreated with free excess cRGD-azide at the indicated concentrations for 1 hour at 4°C, followed by incubation with 10 nM Ctx-PEG12-cRGD for 8 hours. Cells were then harvested for Western blotting.

[0114] Figure 16Figure 2 is a gel depicting the inhibition of EGFR degradation by the lysosomal degradation inhibitors bafilomycin A1 (BAF1, 50nM) and chloroquine (CQ, 10uM). MCF7 cells were seeded in 24-well plates at 70% confluence and incubated overnight in 350μL complete medium. In the presence of 10nM Ctx-PEG12-cRGD, cells were treated with the lysosomal degradation inhibitors bafilomycin A1 (BAF1) at 50nM and chloroquine (CQ) at 10uM for 6 hours.

[0115] Figure 17 Gels (A, C, E) and gel quantification (B, D, F) are shown, which depict that Ctx-cRGD has a higher degradation efficacy against cancer cells (Hela (A, B) and HepG2 (C, D)) than against normal cells (HACAT (E, F)). Hela, HepG2, and HACAT cells were seeded in 24-well plates at 70% confluence and incubated overnight in 350 μL complete medium. Cells were treated with 10 nM Ctx or Ctx-PEG12-cRGD for 24 hours before collection for Western blot analysis.

Claims

1. A bifunctional lysosomal targeted degradation agent comprising: a peptide ligand configured to act as a shuttle molecule to bind to the RGD-binding integrin for lysosomal degradation; and A protein binding agent is configured to bind to a preselected membrane or extracellular protein.

2. The bifunctional lysosomal targeted degrader according to claim 1, wherein the peptide ligand is configured to act as a shuttle molecule to specifically bind to RGD-binding integrin for lysosomal degradation.

3. The bifunctional lysosomal targeted degrader according to claim 1, wherein the protein binding agent is configured to specifically bind to a pre-selected membrane or extracellular protein. The bifunctional lysosomal targeted degrader according to claim 1 , wherein the peptide ligand is a cyclic peptide.

5. The bifunctional lysosomal targeted degrader according to claim 1, wherein the peptide ligand is cyclo(Arg-Gly-Asp-D-Phe-Lys) (SEQ ID NO: 1). The bifunctional lysosomal targeted degrader according to claim 1 , wherein the protein binding agent binds to a membrane protein.

7. The bifunctional lysosomal targeted degrader according to claim 6, wherein the protein binding agent binds to a membrane receptor.

8. The bifunctional lysosomal targeted degrader according to claim 7, wherein the protein binding agent binds to epidermal growth factor receptor (EGFR).

9. The bifunctional lysosomal targeted degrader according to claim 1, wherein the protein binding agent binds to extracellular proteins.

10. The bifunctional lysosomal targeted degrader according to claim 1, wherein the protein binding agent is a polypeptide, a ligand, an aptamer, a nanoparticle or a small molecule. The bifunctional lysosomal targeted degrader according to claim 1 , wherein the protein binding agent is a polypeptide.

12. The bifunctional lysosomal targeted degrader according to claim 1, wherein the protein binding agent is an antibody.

13. The bifunctional lysosomal targeted degrader according to claim 12, wherein the antibody is configured to bind to EGFR protein. The bifunctional lysosomal targeted degrader according to claim 13 , wherein the antibody is cetuximab.

15. The bifunctional lysosomal targeted degrader according to claim 1, further comprising one or more linkers to facilitate the connection between the peptide ligand and the protein binding agent.

16. The bifunctional lysosomal targeted degrader according to claim 15, wherein the linker is poly (ethylene glycol).

17. A pharmaceutical composition comprising the bifunctional lysosomal targeted degrader according to claim 1. The pharmaceutical composition according to claim 17 , further comprising a pharmaceutically acceptable carrier.

19. A method for degrading membrane or extracellular proteins, the method comprising: contacting the membrane or the extracellular protein with the bifunctional lysosomal targeted degradation agent according to claim 1; The bifunctional lysosomal targeted degrader shuttles the membrane or extracellular protein to the lysosome for degradation.

20. A method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 17 to an individual in need thereof.

21. The method of claim 20, wherein the individual is a human.

22. The method of claim 20, wherein the individual has cancer.

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