Cyclic peptide-based lysosome-targeted degradative agents

Abstract

Provided herein is a bifunctional lysosome-targeting degradative 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 protein or extracellular protein of interest. The bifunctional degradative agent is useful, for example, for the selective targeted degradation of membrane proteins and extracellular proteins via the endosomal / lysosomal pathway. Also provided herein are compositions comprising the bifunctional degradative agent and methods for using the bifunctional degradative agent.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] Priority is claimed to U.S. Provisional Patent Application No. 63 / 482,445, filed January 31, 2023, which is incorporated herein by reference in its entirety.

[0002] [Federal Funding Statement] This invention was made with government support under grants GM120357 and GM148266 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] [Sequence table] This application contains a Sequence Listing which has been submitted in XML format and is incorporated herein by reference in its entirety. The XML copy was created on January 25, 2024, is named PCT--09824495-P220339WO01--APP--SEQ_LIST, and is 14,322 bytes in size. [Background technology]

[0004] Targeted protein degradation (TPD) has emerged as an exciting therapeutic option for addressing diseases involving aberrantly expressed or mutated pathogenic proteins by engaging our body's natural protein disposal system. TPD using chimeric molecules is a novel therapeutic modality (Deshaies, 2020, Nature. 580:329-338). These chimeras are heterobifunctional molecules, with one end binding to a protein of interest (POI) and the other directing the resulting complex to a specific degradation pathway. Protein degradation targeting chimeras (PROTACs) have attracted the most attention to date. (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 to target proteins 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, Angew. Chem. Int. Ed. 55:1966-1973.) However, PROTACs can only deplete intracellular proteins. There are many disease targets that are membrane or extracellular proteins.

[0005] To broaden the targeting range, researchers have reported methods for tagging extracellular protein targets with ligands for membrane receptors involved in the active transport of molecules into cells. The tagged proteins are then naturally shuttled to lysosomes within the cell, where they are degraded. Bifunctional lysosome-targeting degraders are generally created by conjugating a ligand for a cell surface lysosome-targeting receptor (LTR) with a ligand capable of binding to the extracellular protein target. The long-term receptors (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 interaction leads to the internalization of extracellular proteins via receptor-mediated endocytosis, which further induces target degradation in lysosomes.

[0006] One type of bifunctional lysosome-targeted degrader was developed by conjugating a ligand for cell surface CIM6PR with a molecule that binds to an extracellular protein target (Banik et al., 2020, Nature. 584:291-297). This type of bifunctional lysosome-targeted degrader that recruits CIM6PR was also called a lysosome-targeted chimera (LYTAC). CIM6PR is ubiquitously expressed in most cell types. Receptor-ligand interaction triggers internalization of the extracellular protein via receptor-mediated endocytosis, further inducing degradation of the target within the lysosome. CIM6PR is a transmembrane receptor that transports proteins with 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 showed that M6P-modified albumin increased its uptake into cells (Beljaars et al., 1999, Hepatology. 29:1486-1493). CIM6PR was subsequently used to deliver therapeutic agents conjugated with M6P derivatives for lysosomal enzyme replacement therapy and cancer treatment (Ghosh et al., 2003, Nat. Rev. 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, have been covalently attached to M6P or its analogs to achieve targeted drug delivery (Hoogendoorn et al., 2014, Angew. 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 polyglycopeptides containing 20–40 units of M6P analogs to an antibody of a POI. Unlike drug delivery processes that involve internalization of covalently bound M6P-protein targets, LYTACs enable the transport of complexes formed through noncovalent interactions between the protein target and LYTAC. It has been shown that LYTACs can successfully degrade both secreted and membrane proteins in lysosomes via CIM6PR (Banik et al., 2020, Nature. 584:291–297). However, challenges associated with synthesizing and conjugating a heterogeneous mixture of polymeric glycopeptides containing 20–40 units of M6P analogs to the antibody used in the LYTAC system limited its usefulness in drug development. Additionally, because CIM6PR is ubiquitously expressed in most cell types, the POI is delivered nonselectively to all cell types.

[0007] This disclosure addresses an unmet need for selective means of degrading membrane and extracellular proteins. By developing new LTRs, different selectivities for degradation of certain membrane or extracellular protein targets can be achieved depending on the expression profile of the LTR. Summary of the Invention

[0008] Provided herein are bifunctional lysosome-targeted degradative agents, the bifunctional lysosome-targeted degradative agents comprising a peptide ligand configured to bind to an RGD-binding integrin as a shuttle molecule for lysosomal degradation, and a protein binding agent configured to bind to a preselected membrane protein or extracellular protein.

[0009] In some variations, the peptide ligand is configured to specifically bind to an RGD-binding integrin as a shuttle molecule for lysosomal degradation, hi some variations, the protein-binding agent is configured to specifically bind to a preselected membrane protein or extracellular protein.

[0010] In one variation, 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).

[0011] In some embodiments, the protein binding agent of the bifunctional degrading agent binds to a membrane protein. The membrane protein can be a membrane receptor. For example, the membrane receptor can be (by way of example, but not limitation) the epidermal growth factor receptor (EGFR).

[0012] In some embodiments, the protein binding agent of the bifunctional degradative agent binds to an extracellular protein.

[0013] The protein-binding agent of the bifunctional degrading agent can be any type of moiety that can bind to a membrane protein or extracellular protein targeted for degradation via the endosomal / lysosomal pathway. For example, the protein-binding agent can be a polypeptide, a ligand, an aptamer, a nanoparticle, or a small molecule.

[0014] In some embodiments, the protein binding agent of the bifunctional degrading agent is a polypeptide. For example, the protein binding agent can be an antibody, either a whole antibody or a fragment of an antibody, where the fragment retains protein binding activity. In a specific variation of the bifunctional lysosome-targeted degrading agent, the antibody is configured to bind to the EGFR protein. In one embodiment, the antibody is cetuximab.

[0015] The bifunctional lysosome-targeted degradation agent may further comprise one or more linkers to facilitate attachment of the peptide ligand to the protein-binding agent. In some embodiments, the linker is poly(ethylene glycol).

[0016] Also provided herein are pharmaceutical compositions comprising any of the bifunctional lysosome-targeted degradative agents of the present disclosure. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0017] Also provided herein are methods for degrading membrane or extracellular proteins, comprising contacting the membrane or extracellular protein with any of the bifunctional lysosome-targeted degradative agents of the present disclosure, wherein the bifunctional lysosome-targeted degradative agent shuttles the membrane or extracellular protein to lysosomes for degradation.

[0018] Also provided herein are methods 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 has cancer.

[0019] The objects and advantages of the present disclosure will become more apparent from the following detailed description of preferred embodiments of the present disclosure, taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0020] [Figure 1] 1 shows gel fluorescence analysis of neutravidin-650 (NA-650) uptake by Huh7 and MCF7 cells treated with 2 μM cRGD-biotin and 500 nM NA-650 for 6 hours compared to treatment without cRGD-biotin. [Figure 2]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. Treatment without degrading agent (yellow bars) serves as a negative control. [Figure 3] Colocalization of NA-650 with a lysosomal tracker in cells treated with 2 μM cRGD-biotin and 500 nM NA-650 for 4 hours (left panel) and 24 hours (right panel) is shown. [Figure 4] Western blots of EGFR in HepG2 cells treated for 24 hours with 10 or 100 nM cRGD conjugated to cetuximab (Ctx) using PEG linkers of different lengths (cRGD-PEG3-Ctx and cRGD-PEG12-Ctx). Treatments were compared with folate-based degraders (Ctx-FA) and 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. [Figure 5] Western blots of EGFR in MCF7 cells treated for 24 hours with 10 or 100 nM cRGD conjugated to cetuximab (Ctx) using PEG linkers of different lengths (cRGD-PEG3-Ctx and cRGD-PEG12-Ctx). Treatments were compared with folate-based degraders (Ctx-FA) and 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. [Figure 6]Western blots of EGFR in MCF7 cells treated for 24 hours with 10 nM cRGD conjugated to cetuximab (Ctx) using PEG linkers of different lengths (cRGD-PEG3-Ctx and cRGD-PEG12-Ctx). Treatments were compared with folate-based degraders (Ctx-FA) and 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. [Figure 7] Western blots of EGFR in Hela cells treated for 24 hours with 10 nM cRGD conjugated to cetuximab (Ctx) using PEG linkers of different lengths (cRGD-PEG3-Ctx and cRGD-PEG12-Ctx). Treatments were compared with folate-based degraders (Ctx-FA) and 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. [Figure 8] MCF dose response to 0 (control), 0.01, 0.1, 1, 10, and 100 nM Ctx-cRGD is shown. [Figure 9] Shown is the MCF time course response of Ctx-cRGD over 48 hours. [Figure 10] Fluorescence micrographs showing co-localization of EGFR with lysosomal markers for Ctx and Ctx-cRGD. [Figure 11] Individual and merged fluorescence micrographs are shown showing the co-localization of EGFR, LAMP1, DAPI, and merged images of all three. [Figure 12] 1 is a gel showing the degradation of PDL1 by Atz-PEG3-cRGD and Atz-PEG12-cRGD. [Figure 13] 1 is a gel showing competition of Ab-cRGD-induced anti-biotin-647 uptake by 6 μM cRGD-azide. [Figure 14]1 is a gel showing the inhibition of lysosomal degradation of anti-biotin-647 by 50 nM bafilomycin A1 (BAF1). [Figure 15] 1 is a gel showing inhibition of EGFR degradation by increasing concentrations of cRGD-azide. [Figure 16] 1 is a gel showing inhibition of EGFR degradation by the lysosomal degradation inhibitors bafilomycin A1 (BAF1, 50 nM) and chloroquine (CQ, 10 μM). [Figure 17] Gels (A, C, E) and gel quantification (B, D, F) are shown, demonstrating that Ctx-cRGD has higher degradation efficacy against cancer cells (Hela (A, B) and HepG2 (C, D)) than normal cells (HACAT (E, F)). DETAILED DESCRIPTION OF THE INVENTION

[0021] Provided herein are bifunctional lysosome-targeting degradative agents, comprising (a) a peptide ligand that binds to an RGD-binding integrin as a shuttle molecule for lysosomal degradation, and (b) a protein-binding agent that binds to a membrane protein or extracellular protein of interest. The bifunctional degradative agents disclosed herein are useful, for example, for the selectively targeted degradation of membrane proteins and extracellular proteins via the endosomal / lysosomal pathway. In one aspect, the bifunctional degradative agent induces the degradation of oncogenic proteins specifically in cancer cells via RGD-binding integrins. Also provided herein are compositions comprising the bifunctional degradative agents, as well as methods of using the bifunctional degradative agents to inhibit disease states, including cancer.

[0022] It should be understood that the bifunctional decomposition agents, compositions, and methods disclosed herein are not limited to the specific embodiments described, and as such may, of course, vary. The bifunctional decomposition agents, compositions, and methods disclosed herein may comprise, consist of, or consist essentially of 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.

[0023] It should be noted that as used in this specification and 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." The recitation of ranges of values ​​is merely intended to serve as a shorthand method of referring individually to each separate value falling within that range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All range endpoints are included within the range and are independently combinable.

[0024] All patents and publications referenced or mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains, and each such referenced patent or publication is specifically incorporated by reference to the same extent as if it were individually incorporated by reference in its entirety or as if set forth in its entirety herein. Applicants reserve the right to physically incorporate into this specification all materials and information from any such cited patent or publication.

[0025] For clarity, it is understood that certain features of the bifunctional decomposition agents, compositions, and methods that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the bifunctional decomposition agents, compositions, and methods that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed, to the extent that such combinations encompass operable processes and / or compositions. In addition, all subcombinations listed in the embodiments describing such variables are also specifically embraced by the bifunctional decomposition agents, compositions, and methods of the present invention and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0026] Bifunctional lysosome-targeted degradative agents Provided herein are bifunctional lysosomal-targeted degradation agents that include 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 protein or extracellular protein of interest.

[0027] Integrins are heterodimeric transmembrane glycoproteins containing one α- and one β-subunit. The α- and β-subunits are bound in a noncovalent complex with a ligand-binding site at the interface. Integrins function as adhesion receptors and have the ability to signal in both directions across the plasma membrane. These events, termed "inside-out" and "outside-in" signaling, result from either binding to extracellular ligands or interaction with the cytoskeleton via the integrin intracellular domain. Thus, integrins can enable human cells to respond to changes in the extracellular environment (via outside-in signaling) or 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, Nat. Rev. Drug Discov. 21:60-78. Of the 24 known human integrin subtypes, eight integrin dimers (i.e., αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, α8β1, and αIIbβ3) recognize the tripeptide Arg-Gly-Asp (RGD) motif in extracellular matrix proteins.

[0028] Peptide ligands capable of binding to integrins have been widely studied for the delivery of anti-cancer 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. This disclosure demonstrates that peptide ligands that bind to RGD-binding integrins can be used to develop lysosomal-targeted degradative agents. As disclosed herein, the peptide ligands are conjugated to binders of membrane or extracellular proteins of interest to generate lysosomal-targeted degradative agents. Once inside the lysosome, the target protein is released from the molecule and degraded. The shuttle molecule is released extracellularly, where it can bind to another target protein.

[0029] Any peptide ligand that binds to RGD-binding integrins is contemplated as useful herein, including linear and cyclic peptides.

[0030] In one variation of the present disclosure, the peptide ligand is a cyclic peptide that binds to RGD-binding integrins. Cyclization of the peptide can increase stability and reduce conformational space, improving the biological efficacy of the molecule. Non-limiting examples of cyclic peptides that bind to RGD-binding integrins 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.

[0031] 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, while 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 αIIbβ3 (>5,000 nM) (Kapp et al., 2017, Sci. Rep. 7:39805).

[0032] As disclosed herein, bifunctional lysosome-targeted degradative agents include protein-binding agents that bind to membrane or extracellular proteins of interest.

[0033] In some embodiments, the protein binding agent binds to a membrane protein.

[0034] In certain embodiments, the membrane protein is a membrane receptor. Membrane receptors of interest include, but are not limited to, stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, receptors of the epidermal growth factor receptor (EGFR) family (e.g., HER2 (human epidermal growth factor receptor 2)), receptors of the fibroblast growth factor receptor (FGFR) family, receptors of the vascular endothelial growth factor receptor (VEGFR) family, receptors of the platelet-derived growth factor receptor (PDGFR) family, receptors of the transfection-rearranged (RET) receptor family, receptors of the Eph receptor family, receptors of the discoidin domain receptor (DDR) family, and mucin proteins (e.g., MUC1).

[0035] In a particular variation, the membrane receptor is EGFR, which is known to be frequently mutated or overexpressed in different types of human cancer (Yarden and Pines, 2012, Nat Rev Cancer. 12:553-563; Sigismund et al., 2018, Mol. Oncol. 12:3-20).

[0036] The membrane protein may be an immunoinhibitory receptor. As used herein, an "immunoinhibitory receptor" refers to a receptor present on immune cells that negatively regulates immune responses. Examples of inhibitory immunoreceptors include immunoinhibitory receptors of the Ig superfamily, including, but not limited to, CD200R, CD300a (IRp60; mouse MAIR-I), CD300f (IREM-1), CEACAM1 (CD66a), FcyRIIb, 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. Further examples of immunoinhibitory receptors include sialic acid-binding Ig-like lectin (Siglec) receptors, such as Siglec7 and Siglec9. Further examples of immunoinhibitory receptors include C-type lectins, including, but not limited to, CLEC4A (DCIR), Ly49Q, and MICL. Further details regarding immunoinhibitory receptors can be found, for example, in Steevels et al., 2011, Eur. J. Immunol. 4:575-587.

[0037] The membrane protein may optionally be a ligand for an immunoinhibitory receptor, one example of which is CD47, which binds to SIRP-α, prevents phagocytosis, and is known to be overexpressed in cancer cells (Eladl et al., 2020, J. Hematol. Oncol. 13:96).

[0038] The membrane protein may be an immune checkpoint molecule, including immune checkpoint proteins and ligands. Non-limiting examples of immune checkpoint molecules include PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, and members of the B7 family. In one embodiment, the membrane protein is PD-L1 (programmed cell death ligand 1), which binds to PD-1 (programmed cell death-1) to suppress apoptosis and is known to be overexpressed in cancer cells (Yi et al., 2021, J. Hematol. Oncol. 14:10).

[0039] In some embodiments, the protein binding agent binds to an extracellular protein.

[0040] The extracellular protein may 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 particular subtype of such cytokines), hormones, etc.

[0041] Alternatively, the extracellular protein may be an antibody, for example, an antibody that binds to a membrane protein or a different extracellular protein. The antibody may be an autoantibody. "Autoantibody" refers to an antibody produced by the immune system that is directed against one or more of an individual's own proteins. Cancer cells can induce an immunological response, resulting in the production of tumor-associated autoantibodies. Non-limiting examples of autoantibodies include rheumatoid factor (RF), antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (ANCA), anti-double-stranded DNA (anti-dsDNA), anti-entelomere antibodies (ACA), anti-cyclic citrullinated peptide antibodies (anti-CCP), soluble nuclear antigen antibodies (ENA), anticardiolipin 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), antimitochondrial antibodies (AMA), anti-glomerular basement membrane (GBM), acetylcholine receptor (AChR) antibodies, and the like.

[0042] The extracellular protein may be a secreted protein, including, but not limited to, secreted growth factors, extracellular matrix-degrading proteinases, cell motility factors and immunomodulatory cytokines, or other bioactive molecules.

[0043] The extracellular protein may also be a mutant protein.

[0044] When the protein-binding agent of the bifunctional degrading agent binds to a membrane protein or an extracellular protein, the membrane protein or extracellular protein may be present on or produced by a cancer cell. "Cancer cell" refers to a cell that exhibits a neoplastic cell phenotype, which may be characterized by, for example, one or more of: aberrant cell growth, aberrant cellular proliferation, loss of density-dependent growth inhibition, anchorage-independent growth ability, the ability to promote tumor growth and / or development in an immunocompromised non-human animal model, and / or any suitable indicator of cellular transformation. "Cancer cell" may be used interchangeably herein with "tumor cell," "malignant cell," "neoplastic cell," or "cancerous cell," and includes cancer cells of solid tumors, semi-solid tumors, hematological malignancies (e.g., leukemia cells, lymphoma cells, myeloma cells, etc.), primary tumors, metastatic tumors, etc. In some embodiments, the membrane protein present on a cancer cell is a tumor-associated antigen or a tumor-specific antigen.

[0045] The protein-binding agent of the bifunctional degradation agent can be any type of moiety that can bind to a membrane protein or extracellular protein targeted for degradation via the endosomal / lysosomal pathway. In certain embodiments, the protein-binding agent is selected from a polypeptide, a ligand (e.g., a ligand of a membrane receptor, where the membrane receptor is targeted for degradation), an aptamer, a nanoparticle, and a small molecule.

[0046] A protein binding agent may be a small molecule. By "small molecule" is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 50 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less.

[0047] The protein binding agent may 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.); whole antibodies (e.g., antibodies consisting of a tetramer consisting of two dimers of a heavy and a light chain polypeptide); single-chain antibodies; fragments of antibodies (e.g., whole antibody fragments or single-chain antibodies) that retain specific binding to a membrane protein or extracellular protein, including, but not limited to, Fv (scFv), Fab, F(ab')2, Fab', (scFv')2, diabodies, and nanobodies; chimeric antibodies; monoclonal antibodies; fully human antibodies; humanized antibodies (e.g., humanized whole antibodies, humanized antibody fragments, etc.); and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein or fragment thereof. An antibody may be detectably labeled, for example, with an in vivo imaging agent. The antibody may be further conjugated to other moieties, such as, for example, polyethylene glycol (PEG). Fusion to the Fc region (or fragment thereof) of the antibody, conjugation to PEG, etc. may be useful, for example, to increase the serum half-life of the antibody upon administration to a subject.

[0048] In certain variations, the antibody is configured to specifically bind to a cancer antigen.

[0049] The antibody may also be configured to bind to intact complement or a fragment thereof, hi certain embodiments, the antibody binds to one or more immunodominant epitopes within intact complement or a fragment thereof.

[0050] Alternatively, the antibody may bind to a membrane receptor or membrane receptor ligand, or the antibody may bind to an epidermal growth factor (EGF) protein, e.g., human EGF, or one or more immunodominant epitopes within the EGF protein.

[0051] 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 certain embodiments, the antibody comprises CDRs present in cetuximab (Ctx). In another certain embodiment, the antibody comprises the variable light chain and variable heavy chain present in cetuximab. In a specific embodiment, the antibody is cetuximab.

[0052] In certain embodiments, the antibody binds to an immunoinhibitory receptor. In certain embodiments, the antibody binds to one or more immunodominant epitopes within the immunoinhibitory receptor.

[0053] In certain embodiments, the antibody binds to a ligand of an immunoinhibitory receptor. In certain embodiments, the antibody binds to one or more immunodominant epitopes within the ligand of an immunoinhibitory receptor. In certain embodiments, the antibody binds to the CD47 protein. In certain embodiments, the antibody binds to one or more immunodominant epitopes within the CD47 protein.

[0054] 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 certain embodiments, the antibody comprises CDRs present in atezolizumab (Atz). In another certain embodiment, the antibody comprises the variable light chain and variable heavy chain present in atezolizumab. In a specific embodiment, the antibody is atezolizumab.

[0055] The bifunctional lysosome-targeting degrading agent disclosed herein can be in any suitable form.In some embodiments, the bifunctional degrading agent is a conjugate.Therefore, in certain embodiments, the bifunctional degrading agent disclosed herein comprises 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 an RGD-binding integrin peptide ligand fused to a protein binding agent.

[0056] In certain embodiments, one or more linkers can be used to facilitate the attachment of the RGD-binding integrin peptide ligand to a protein binder. Non-limiting examples of such linkers include ester linkers (e.g., N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, or PFP ester, or thioester), 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, polyethylene glycol (PEG)-containing linkers (e.g., but not limited to, tetraethylene glycol), propanoic acid-containing linkers, and linkers containing any combination thereof. In one embodiment, the linker is PEG. PEGs of various lengths, such as PEG3, PEG12, etc., may also be used as linkers.

[0057] In certain aspects, the linker is a chemically labile linker, such as an acid-cleavable linker, that is stable at neutral pH (pH 7.3-7.5 in the bloodstream) but undergoes hydrolysis upon internalization 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 labile linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, and the like. According to certain embodiments, the linker is an enzyme-labile linker, e.g., an enzyme-labile linker that is stable in the bloodstream but undergoes enzymatic cleavage upon internalization into target cells (e.g., by lysosomal proteases (e.g., cathepsin or plasmin) in the lysosomes of target cells (e.g., cancer cells)). Enzyme-labile linkers include, but are not limited to, linkers containing peptide bonds, for example, dipeptide-based linkers such as valine-citrulline linkers, for example, maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linker, valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, etc. Chemically labile linkers, enzyme-labile linkers, and non-cleavable linkers are known and are described in detail in, for example, Ducry and Stump, 2010, Bioconjugate Chem. 21:5-13.

[0058] In certain aspects, the bifunctional degrading agent enhances the degradation of the membrane protein or extracellular protein compared to the degradation of the membrane protein or extracellular protein in the presence of the protein-binding agent alone. According to some embodiments, the bifunctional degrading agent enhances the degradation of the membrane protein or extracellular protein compared to the degradation of the membrane protein or extracellular protein in the presence of the RGD-linked integrin peptide ligand or protein-binding agent alone. In this context, "enhancing degradation" means that the membrane protein or extracellular protein is degraded in the presence of the bifunctional degrading agent and not in the presence of the protein-binding agent alone or the RGD-linked integrin peptide ligand or protein-binding agent alone under the same conditions, or that the membrane protein or extracellular protein is degraded to a greater extent in the presence of the bifunctional degrading agent than the membrane protein or extracellular protein is degraded in the presence of the protein-binding agent alone or the RGD-linked integrin peptide ligand or protein-binding agent alone under the same conditions. If a membrane protein or extracellular protein is degraded to a greater extent in the presence of a bifunctional degrading agent than the membrane protein or extracellular protein is degraded in the presence of a protein-binding agent alone, or in the presence of an RGD-binding integrin peptide ligand or a protein-binding agent alone, under the same conditions, the degradation can be 1.2-fold or more, 1.4-fold or more, 1.6-fold or more, 1.8-fold or more, 2-fold or more, 2.5-fold or more, 3-fold or more, 3.5-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 5.5-fold or more, 6-fold or more, 6.5-fold or more, 7-fold or more, 7.5-fold or more, 8-fold or more, 8.5-fold or more, 9-fold or more, 9.5-fold or more, or 10-fold or more in the presence of a bifunctional degrading agent.

[0059] composition Disclosed herein are compositions comprising any of the bifunctional lysosome-targeted degradative agents of the present disclosure.

[0060] The composition may optionally include a bifunctional degrading agent of the present disclosure present in a liquid medium. The liquid medium may be an aqueous liquid medium such as water or a buffer solution. Such compositions may contain one or more additives, such as salts (e.g., NaCl, MgCl, KCl, MgSO), 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.

[0061] Also disclosed herein are pharmaceutical compositions comprising any of the bifunctional lysosome-targeted degrading agents of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutical compositions generally contain a therapeutically effective amount of the bifunctional degrading agent. A "therapeutically effective amount" refers to an amount sufficient to produce a desired result, e.g., a beneficial or desired therapeutic (including prophylactic) result, e.g., a reduction in cell proliferation in an individual with a cell proliferative disorder (e.g., cancer) associated with a membrane protein or extracellular protein to which the protein-binding agent of the bifunctional depolymerizing agent binds. An effective amount can be administered in one or more administrations.

[0062] The bifunctional decomposition agent of the present disclosure can be incorporated into various formulations for therapeutic administration.More specifically, the bifunctional decomposition agent can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable excipient or diluent, and can be formulated into a solid, semi-solid, liquid, or gaseous preparation such as a tablet, capsule, powder, granule, ointment, solution, injection, inhalant, and aerosol.

[0063] Formulations of the bifunctional degrading agents of the present disclosure suitable for administration to an individual (e.g., suitable for administration to a human) are generally sterile and, depending on the selected route of administration, may further be free of detectable pyrogens or other contaminants that would contraindicate administration to an individual.

[0064] In pharmaceutical dosage forms, the bifunctional degrading agent may be administered alone or in appropriate association and combination with other pharmaceutically active compounds. The following methods and excipients are merely examples and are in no way limiting.

[0065] For oral preparations, the bifunctional disintegrants can be used alone or in combination with suitable additives to prepare tablets, for example, with conventional additives such as lactose, mannitol, corn starch, or potato starch, with binders such as crystalline cellulose, cellulose derivatives, acacia, 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, humectants, preservatives, and flavoring agents.

[0066] The bifunctional degradants can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent such as vegetable oil or other similar oil, synthetic fatty acid glyceride, ester of higher fatty acid, or propylene glycol, together with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives, as needed.

[0067] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, and the lyophilized preparation is reconstituted with a sterile solution before administration. The standard procedure for reconstituting a lyophilized composition is to add a volume of pure water (typically equal to the volume removed during lyophilization), but solutions containing antimicrobial agents may also be used to produce pharmaceutical compositions for parenteral administration.

[0068] Aqueous formulations of bifunctional degrading agents can be prepared in a pH buffer solution, for example, at a pH ranging from about 4.0 to about 8.0, for example, from about 4.5 to about 7.5, for example, from about 5.0 to about 7.0. Examples of buffers suitable for a pH within this range include phosphate, histidine, citrate, succinate, acetate, and other organic acid buffers. The concentration of the buffer can be, for example, from about 1 mM to about 100 mM or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0069] How to use Disclosed herein are methods of using the bifunctional lysosome-targeted degradative agents of the present disclosure.

[0070] Provided herein are methods for degrading membrane proteins or extracellular proteins. Such methods include contacting a membrane protein or extracellular protein with any of the bifunctional lysosome-targeted degradative agents disclosed herein under conditions in which the bifunctional lysosome-targeted degradative agent shuttles the membrane protein or extracellular protein to lysosomes for degradation. Such methods can be useful for a variety of applications. In certain embodiments, the methods are performed in vitro (e.g., in a tube, cell culture plate, or well, etc.), and are useful, for example, for testing and / or research applications. In other embodiments, the methods are performed in vivo (e.g., in an individual to whom the bifunctional degradative agent is administered), and are useful, for example, for clinical / therapeutic applications.

[0071] Also provided are methods comprising administering a therapeutically effective amount of any of the bifunctional degradation products or pharmaceutical compositions of the present disclosure to an individual in need thereof. A variety of individuals can be treated according to the methods of the present invention. Generally, such subjects are "mammals" or "mammals," terms used broadly to describe organisms within the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the individual is a human.

[0072] An effective amount of a bifunctional degrading agent (or pharmaceutical composition comprising same) is an amount that, when administered in one or more doses alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is effective to reduce the symptoms of a medical condition (e.g., cancer) in an individual 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, compared to the symptoms of an individual not being treated with the bifunctional degrading agent or pharmaceutical composition.

[0073] The methods include administering to an individual with cancer a therapeutically effective amount of any of the disclosed bifunctional degrading agents or pharmaceutical compositions. According to such methods, the protein-binding agent of the bifunctional degrading agent binds to at least a membrane protein or extracellular protein that contributes to the individual's cancer, and targeted degradation of the membrane protein or extracellular protein using the bifunctional degrading agent treats the individual's cancer. In certain aspects, the protein-binding agent binds to a protein selected from a membrane receptor, a ligand of a membrane receptor, an immunoinhibitory receptor, a ligand of an immunoinhibitory receptor, an immune checkpoint molecule, an autoantibody, a secreted protein, and a mutant protein.

[0074] For example, the individual being treated may have a cancer characterized by the presence of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, etc. In some embodiments, the individual has a cancer selected from breast cancer, melanoma, lung cancer, colon 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's lymphoma, pancreatic cancer, thyroid cancer, any combination thereof, and any subtype thereof.

[0075] In any of the methods using the bifunctional degrading agents of the present disclosure, the bifunctional degrading agent generally enhances the degradation of the membrane protein or extracellular protein compared to the degradation of the membrane protein or extracellular protein in the presence of a protein-binding agent alone. Similarly, in any of the methods using the bifunctional degrading agents of the present disclosure, according to some embodiments, the bifunctional degrading agent enhances the degradation of the membrane protein or extracellular protein compared to the degradation of the membrane protein or extracellular protein in the presence of an RGD-linked integrin peptide ligand or a protein-binding agent alone.

[0076] "Treate," "treating," or "treatment" refers to at least an amelioration of symptoms associated with an individual's medical condition (e.g., a cell proliferative disorder, e.g., cancer), where amelioration 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 a medical condition, or at least a symptom associated therewith, is completely inhibited (e.g., prevented from occurring or halted (e.g., terminated)) such that an individual no longer suffers from the medical condition, or at least a symptom characterizing the medical condition.

[0077] The bifunctional depolymerization agent 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, and systemic and local administration routes. Conventional and pharmaceutically acceptable administration routes include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical, intraocular, intravenous, intraarterial, nasal, oral, and other enteral and parenteral routes of administration. In some embodiments, administering is by parenteral administration. If necessary, administration routes may be combined or tailored depending on the bifunctional depolymerization agent and / or the desired effect. The bifunctional depolymerization agent or pharmaceutical composition may be administered in a single dose or multiple doses. In some embodiments, the bifunctional depolymerization agent or pharmaceutical composition is administered intravenously. In some embodiments, the bifunctional depolymerization agent or pharmaceutical composition is administered, for example, by injection (e.g., intravenous infusion) for systemic delivery or to a local site. [Example]

[0078] We tested a cyclic peptide as a binder for 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, Sci. Rep. 7:39805). The key residues are Arg-Gly-Asp or RGD, while 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 αIIbβ3 (>5,000 nM) (Kapp et al., 2017, Sci. Rep. 7:39805).

[0079] A biotin conjugate of c(RGDfK) (SEQ ID NO: 1) (cRGD-biotin, Vivitide, PCI-3697-PI-1MG) can bind to the fluorescent model target protein, neutravidin-650 (NA-650). cRGD-biotin was tested for uptake of the model target protein, NA-650, by treating Huh7 and MCF7 cells with 2 μM cRGD-biotin and 500 nM NA-650 for 6 hours. Figure 1 shows that in the presence of cRGD-biotin, uptake of NA-650 protein was observed in both Huh7 and MCF7 cells.

[0080] We also compared the uptake of NA-650 with triGalNAc(GN)-biotin, one of the most efficient lysosome-targeted degradation agents for the uptake of secreted proteins into hepatocytes (e.g., Huh7) (Zhou et al., 2021, ACS Cent. Sci. 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 those of non-hepatocyte cells B16F10 and MCF7. As shown in Figure 2, in the presence of GN-biotin, only Huh7 cells internalized NA-650. In the presence of cRGD-biotin, all three cell types internalized NA-650.

[0081] Furthermore, colocalization of the NA-650 protein with the lysosomal tracker was observed, as shown in the images in Figure 3. The results demonstrate that a cyclic peptide-based lysosomal-targeting degrader can promote the uptake of a soluble model target protein into lysosomes.

[0082] We also conjugated the cRGD peptide to cetuximab (Ctx), an antibody capable of binding to the membrane-targeting protein EGFR. Lysosome-targeted degraders were prepared and tested for EGFR degradation. Two linkers were used between the receptor cyclic peptide binder and the EGFR antibody binder: polyethylene glycol 3 (PEG3; H-(O-CH2-CH2)3-OH) and polyethylene glycol 12 (PEG12; H-(O-CH2-CH2) 12The cetuximab conjugates (cRGD-PEG3-Ctx, cRGD-PEG12-Ctx) were prepared by reacting cetuximab at a concentration of 1.8 mg / ml in 200 μL of PBS with DBCO-PEG3-NHS or DBCO-PEG12-NHS ester at a molar ratio of 1:25 overnight on a rotator at room temperature. The mixture was then purified five times with 500 μL of PBS using a 10 kDa Amicon centrifugal filter. The concentration of the DBCO-labeled antibody was then measured by BCA assay and reacted with cRGD-N3 overnight on a rotator at room temperature. The resulting antibody conjugates were then purified five times with 500 μL of 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 in 200 μL of PBS at a concentration of 1.8 mg / ml overnight on a rotator at room temperature. The mixture was then purified five times with 500 μL of PBS using a 10 kDa Amicon centrifugal filter. The concentration of the DBCO-labeled antibody was then measured by BCA assay, and the antibody conjugate was reacted with folate-N3 overnight on a rotator at room temperature. The resulting antibody conjugate was then purified five times with 500 μL of PBS using a 10 kDa Amicon centrifugal filter. The degradation results (Figures 4-7) showed a significant decrease in EGFR protein levels in cells treated with the cyclic peptide-cRGD-labeled antibody, suggesting that EGFR was degraded within the cells. Degraders with longer PEG linkers (PEG12) have better degradative activity against EGFR in the cancer cells tested. Less degradative activity was observed at concentrations of 100 nM than at 10 nM, likely due to the hook effect (Douglass et al., 2013, J. Am. Chem. Soc. 135:6092).

[0083] MCF7 dose-response experiment using 0 (control), 0.01, 0.1, 1, 10, and 100 nM of the DBCO-PEG12-NHS construct (also referred to herein as Ctx-cRGD). MCF7 cells were seeded into 24-well plates at 70% confluence and incubated overnight in 350 μL of complete culture medium. Cells were then treated with various concentrations of Ctx-PEG12-cRGD in 50 μL of medium, as indicated, for 24 hours before being harvested for Western blot analysis. The results are shown in Figure 8.

[0084] The MCF time course response of Ctx-cRGD over 48 hours was determined. MCF7 cells were seeded into 24-well plates at 70% confluence and incubated overnight in 350 μL of complete culture medium. Cells were then treated with 10 nM Ctx-PEG12-cRGD in 50 μL of medium as indicated and then harvested for Western blot analysis. The results are shown in Figure 9.

[0085] Figure 10 shows fluorescence micrographs showing colocalization of EGFR and lysosomal markers for Ctx and Ctx-cRGD. MCF7 cells were distributed at a density of 20,000 cells / well in 200 μL of complete culture medium into 8-well chamber slides. Cells were treated with 10 nM Ctx-PEG12-cRGD for 24 hours at 37°C and subsequently washed three times with PBS. 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. Cells were then incubated with anti-EGFR antibody in 1% BSA overnight at 4°C. The next day, cells were incubated with anti-rabbit-594 secondary antibody for 1 hour at room temperature. After each antibody incubation, cells were washed three times with PBS. Cells were then mounted with slow-fade-anti-fade mounting medium containing DAPI. Images were acquired with a Leica SP8 3x STED super-resolution microscope at 10x eyepiece, 60x magnification, and analyzed with ImageJ.

[0086] Figure 11 shows individual and merged fluorescence micrographs demonstrating the colocalization of EGFR, LAMP1, and DAPI, as well as a merged image of all three. MCF7 cells were distributed at a density of 20,000 cells / well in 200 μL of complete culture medium into 8-well chamber slides. Cells were treated with 10 nM Ctx-cRGD at 37°C for 24 hours and subsequently washed three times with PBS. Cells were then fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton-100 for 5 minutes, and blocked with 5% BSA at room temperature for 1 hour. Cells were then incubated overnight with anti-EGFR and anti-LAMP1 antibodies in 1% BSA at 4°C. The following day, cells were incubated with anti-mouse-488 and anti-rabbit-594 secondary antibodies at room temperature for 1 hour. After each antibody incubation, cells were washed three times with PBS. The cells were then mounted with slow-fade-antifade mounting medium containing DAPI. Images were acquired using a Leica SP8 3x STED super-resolution microscope at 10x eyepieces and 60x magnification, and analyzed using ImageJ.

[0087] Figure 12 shows a gel demonstrating the degradation of PDL1 by Atz-PEG3-cRGD and Atz-PEG12-cRGD. The Atz-PEG3-cRGD and Atz-PEG12-cRGD constructs are atezolizumab (Atz) conjugated to cRGD via a PEG3 or PEG12 linker. Atezolizumab at a concentration of 1.8 mg / ml in 200 μL of PBS was reacted with DBCO-PEG3-NHS or DBCO-PEG12-NHS ester at a molar ratio of 1:25 overnight at room temperature on a rotator. The mixture was then purified five times with 500 μL of PBS using a 10 kDa Amicon centrifugal filter. The concentration of the DBCO-labeled antibody was then measured by BCA assay and reacted with cRGD-N3 overnight at room temperature on a rotator. The resulting antibody conjugate was then purified five times with 500 μL of PBS using a 10 kDa Amicon centrifugal filter. MCF7 cells were seeded into 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 medium, as indicated, for 24 hours and then harvested for Western blot analysis. Treatment with 10 nM Atz-PEG3-FA was used as a positive control.

[0088] Figure 13 shows a gel showing the competition of Ab-cRGD-induced anti-biotin-647 antibody (Jackson ImmunoResearch, 200-602-211) uptake by 6 μM cRGD-azide (Vivitide, RGD-3749-PI-5MG). Ab-cRGD is a goat anti-mouse IgG antibody (Ab) conjugated to cRGD via a PEG12 linker. Ab at a concentration of 1.8 mg / ml in 200 μL of PBS was reacted with DBCO-PEG12-NHS ester at a 1:25 molar ratio overnight at room temperature on a rotator. The mixture was then purified five times with 500 μL of PBS using a 10 kDa Amicon centrifugal filter. The concentration of the DBCO-labeled antibody was then measured by BCA assay and reacted with cRGD-N3 overnight at room temperature on a rotator. The resulting antibody conjugate was then purified five times with 500 μL of PBS using a 10 kDa Amicon centrifugal filter. Cells were seeded into 48-well plates at 70% confluence in 200 μL of complete culture medium one day before treatment. Cells were then sequentially treated with 25 μL of medium containing 50 nM anti-biotin-647 and 25 μL of culture medium containing 25 nM Ab-cRGD, incubated at 37°C for the indicated times, and subsequently washed twice with PBS before harvesting for gel fluorescence analysis. Cells were preincubated with 6 μM excess free cRGD-azide for 1 hour at 4°C for competition.

[0089] Figure 14 is a gel showing the inhibition of lysosomal degradation of anti-biotin-647 by 50 nM bafilomycin A1 (BAF1). MCF7 cells were seeded into 48-well plates at 70% confluence and maintained in 200 μL of complete culture medium. The next day, cells were incubated with 25 nM Ab-cRGD and 50 nM anti-biotin-647 for 3 hours, followed by washing three times with PBS. Cells were then maintained in fresh medium with or without 50 nM bafilomycin A1 (BAF1) for an additional 3 hours before being harvested for gel fluorescence analysis.

[0090] Figure 15 is a gel showing the inhibition of EGFR degradation by increasing concentrations of cRGD-azide. MCF7 cells were seeded into 24-well plates at 70% confluence and incubated overnight in 350 μL of complete culture medium. Cells were pretreated with excess free 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.

[0091] Figure 16 is a gel showing inhibition of EGFR degradation by the lysosomal degradation inhibitors bafilomycin A1 (BAF1, 50 nM) and chloroquine (CQ, 10 μM). MCF7 cells were seeded into 24-well plates at 70% confluence and incubated overnight in 350 μL of complete culture medium. Cells were treated with 50 nM bafilomycin A1 (BAF1) and 10 μM chloroquine (CQ), lysosomal degradation inhibitors, in the presence of 10 nM Ctx-PEG12-cRGD for 6 hours.

[0092] Figure 17 shows gels (A, C, E) and gel quantification (B, D, F) demonstrating that Ctx-cRGD has higher degradation efficacy against cancer cells (Hela (A, B) and HepG2 (C, D)) than normal cells (HACAT (E, F)). Hela, HepG2, and HACAT cells were seeded into 24-well plates at 70% confluence and incubated overnight in 350 μL of complete culture medium. Cells were treated with 10 nM Ctx or Ctx-PEG12-cRGD for 24 hours and then harvested for Western blot analysis.

Claims

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

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

3. 2. The bifunctional lysosome-targeted degradative agent of claim 1, wherein the protein-binding agent is configured to specifically bind to a preselected membrane protein or extracellular protein.

4. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the peptide ligand is a cyclic peptide.

5. The bifunctional lysosome-targeted degradative agent of claim 1, wherein the peptide ligand is cyclo(Arg-Gly-Asp-D-Phe-Lys) (SEQ ID NO: 1).

6. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the protein-binding agent binds to a membrane protein.

7. The bifunctional lysosome-targeted degradative agent of claim 6 , wherein the protein-binding agent binds to a membrane receptor.

8. The bifunctional lysosome-targeted degradation agent of claim 7 , wherein the protein-binding agent binds to epidermal growth factor receptor (EGFR).

9. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the protein-binding agent binds to an extracellular protein.

10. The bifunctional lysosome-targeted degradation agent of claim 1 , wherein the protein-binding agent is a polypeptide, a ligand, an aptamer, a nanoparticle, or a small molecule.

11. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the protein-binding agent is a polypeptide.

12. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the protein-binding agent is an antibody.

13. The bifunctional lysosome-targeted degradative agent of claim 12, wherein the antibody is configured to bind to an EGFR protein.

14. The bifunctional lysosome-targeted degradative agent of claim 13, wherein the antibody is cetuximab.

15. The bifunctional lysosome-targeted degradative agent of claim 1, further comprising one or more linkers to facilitate attachment of the peptide ligand to the protein-binding agent.

16. The bifunctional lysosome-targeted degradative agent of claim 15, wherein the linker is poly(ethylene glycol).

17. A pharmaceutical composition comprising the bifunctional lysosome-targeted degrading agent of claim 1.

18. 18. The pharmaceutical composition of claim 17, further comprising a pharmaceutically acceptable carrier.

19. 1. A method for degrading membrane or extracellular proteins, comprising: contacting the membrane protein or extracellular protein with the bifunctional lysosome-targeted degradation agent of claim 1; The method, wherein the bifunctional lysosome-targeted degradative agent shuttles the membrane protein or extracellular protein to lysosomes for degradation.

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

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

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

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