Folate-antibody conjugates as lysosome-targeted degraders.

Abstract

Provided herein is a bifunctional lysosome-targeted degradative agent, which comprises a folate or folic acid analog configured to bind to a folate receptor as a shuttle molecule for lysosomal degradation, and a protein-binding moiety configured to bind to a membrane protein or extracellular protein of interest. The bifunctional degradative agent is used, for example, for selectively targeted degradation of membrane proteins and extracellular proteins via the endosomal / lysosomal pathway. Also provided herein are methods of using the bifunctional degradative agent, as well as compositions comprising the bifunctional degradative agent.

Description

[Technical field]

[0001] [Federal Government Funding Statement] This invention was made with Government support under award GM120357 from the National Institutes of Health. The Government has certain rights in this invention.

[0002] [CROSS-REFERENCE TO RELATED APPLICATIONS] Priority is claimed to Provisional Patent Application No. 63 / 322,426, filed March 22, 2022, the contents of which are incorporated herein by reference. [Background technology]

[0003] Targeted protein degradation (TPD) has emerged as an attractive therapeutic option to address diseases involving aberrantly expressed or mutated pathogenic proteins by engaging the body's natural protein processing systems. TPD with chimeric molecules is a novel therapeutic approach (Deshaies, 2020, Nature. 580: 329-338). These chimeras are heterobifunctional molecules with one end that binds to the protein of interest (POI) and the other end that directs the resulting complex to a specific degradation pathway. PROteolysis 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 E3 ligase ligands to route 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. Many disease targets are also membrane or extracellular proteins.

[0004] To broaden the scope of targets, researchers have reported a method to label extracellular protein targets with ligands for membrane receptors involved in the active transport of molecules into cells. The labeled proteins are naturally delivered to lysosomes in the cell, where they are degraded. Bifunctional lysosome-targeted degraders are generally created by conjugating a ligand for a lysosome-targeting receptor (LTR) on the cell surface with a ligand that can bind to the extracellular protein target. This process is shown diagrammatically in Figure 1. The LTRs utilized 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 induces internalization of extracellular proteins via receptor-mediated endocytosis and further induces degradation of targets in lysosomes.

[0005] One type of bifunctional lysosome-targeted degraders was developed by conjugating the ligand of CIM6PR on the cell surface with a molecule that binds to an extracellular protein target (Banik et al., 2020, Nature. 584: 291-297). This type of bifunctional lysosome-targeted degraders that recruit CIM6PR was also named LYsosome TArgeting Chimeras (LYTACs). CIM6PR is ubiquitously expressed in most cell types. The receptor-ligand interaction triggers the internalization of extracellular proteins via receptor-mediated endocytosis, which further induces the degradation of the target in 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 albumin modified with M6P had increased cellular uptake (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 conjugated to M6P or its analogues 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 analogues to an antibody of the POI. Unlike drug delivery processes that involve internalization of covalently bound M6P protein targets, LYTAC allows for the transport of complexes formed by non-covalent interactions between protein targets and LYTAC. It has been shown that LYTAC successfully degrades both secreted and membrane proteins in lysosomes via CIM6PR (Banik et al., 2020, Nature. 584: 291-297). However, its utility in drug development has been limited by challenges associated with the synthesis and attachment of a heterogeneous mixture of polymeric glycopeptides with 20-40 units of M6P analogs to the antibodies utilized in the LYTAC system. Furthermore, because CIM6PR is ubiquitously expressed in most cell types, the POI is delivered non-selectively to all cell types. There remains an unmet need for selective means to degrade extracellular proteins. Summary of the Invention

[0006] Provided herein are bifunctional lysosome-targeted degradative agents that include a folate moiety or an analog thereof configured to bind to a folate receptor as a shuttle molecule for lysosomal degradation, and a protein-binding moiety configured to bind to a membrane protein or an extracellular protein.

[0007] In one version, the protein-binding portion of the bifunctional degrader is configured to bind to a membrane protein. The membrane protein can be a membrane receptor. For example, the membrane receptor can be (by way of example and without limitation) epidermal growth factor receptor (EGFR). The membrane protein can be an immunoinhibitory receptor or a ligand of an immunoinhibitory receptor, such as CD47. The membrane protein can be an immune checkpoint molecule. For example, the immune checkpoint molecule can be programmed cell death ligand 1 (PD-L1).

[0008] In another version, the protein-binding portion of the bifunctional degrading agent is configured to bind to an extracellular protein, which can be (by way of example and without limitation) a ligand for a membrane receptor, an autoantibody, a secreted protein, a mutated protein, etc.

[0009] The protein-binding moiety of the bifunctional degrading agent can be any type of moiety capable of binding to a membrane protein or extracellular protein targeted for degradation via the endosomal / lysosomal pathway, hi certain embodiments, the protein-binding moiety is selected from a polypeptide, a ligand, an aptamer, a nanoparticle, and a small molecule.

[0010] In one version, the protein-binding portion of the bifunctional degrading agent is a small molecule.

[0011] In another version, the protein-binding portion of the bifunctional degrading agent is a polypeptide.

[0012] The protein-binding moiety may be an antibody, either a whole antibody or a fragment of an antibody, said fragment retaining protein-binding activity. The antibody or fragment thereof is preferably configured to bind to 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, or a mutated protein. In a particular version of the bifunctional lysosomal targeted degrader, the antibody is configured to bind to the EGFR protein. In one embodiment, the antibody is cetuximab. In another embodiment, the antibody is configured to bind to the CD47 protein. The antibody may be configured to bind to the PD-L1 protein. In one embodiment, the antibody is atezolizumab.

[0013] The bifunctional degrading agent may further comprise a linker connecting the folate moiety or analogue thereof to the protein binding moiety. Preferred linkers include poly(ethylene glycol) ("PEG"), N-hydroxysuccinimide ("NHS"), and the like, either alone or in combination.

[0014] The bifunctional lysosome-targeted degradative agents disclosed herein selectively target cells that express the folate receptor by binding to the folate receptor, including cancer cells and other neoplastic cells that express the folate receptor.

[0015] Also provided herein are pharmaceutical compositions comprising any of the bifunctional lysosomally targeted degradative agents of the present disclosure, which may be administered neat or in combination with a pharma- ceutical acceptable carrier configured for any selected route of administration (e.g., enteral, parenteral, topical, rectal, etc.).

[0016] Also provided herein are methods for degrading membrane or extracellular proteins, the methods comprising contacting the membrane or extracellular protein with any of the bifunctional lysosomally targeted degradative agents disclosed herein, which shuttle the membrane or extracellular protein to lysosomes for degradation.

[0017] Also provided herein are methods comprising administering a therapeutically effective, neoplastic cell proliferation inhibiting amount of any of the pharmaceutical compositions of the present disclosure to an individual in need thereof. In some embodiments, the individual is a human. In some embodiments, the individual has cancer.

[0018] The objects and advantages of the present disclosure will become more apparent from the following detailed description of preferred embodiments thereof, taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0019] [Figure 1] 1 shows chimeric degrader-induced degradation of an extracellular protein of interest (POI) via a lysosomal targeting receptor (LTR). [Figure 2A] The chemical structure of FA-PEG-NHS is shown. [Figure 2B] The chemical structures of DBCO-PEG3-NHS and N3-PEGn-FA are shown. [Diagram 3] A set of three graphs showing fluorescence intensity indicating uptake of NA-650 in HeLa cells (left graph), HepG2 cells (middle graph), and MCF7 cells (right graph) treated with FA-PEG3-biotin ("NA-650+FA-biotin") or without FA-PEG3-biotin ("NA-650"). [Figure 4A] 1 shows MALDI-MS spectra of anti-mouse IgG Fab fragments with ("Fab-FA") or without ("Fab") folate labeling. [Figure 4B]1 shows MALDI-MS spectra of full-size anti-mouse IgG with ("Ab-FA") or without ("Ab") folate labeling. [Figure 5A] 1 shows a gel fluorescence analysis of mouse anti-biotin IgG-647 protein uptake by HeLa cells treated with folate-labeled antibody ("Ab-FA" and "Fab-FA") or antibody alone ("Ab" and "Fab"). [Figure 5B] 1 shows a gel fluorescence analysis of mouse anti-biotin IgG-647 protein uptake by HeLa cells treated with folate-labeled antibody ("Ab-FA" and "Fab-FA") or antibody alone ("Ab" and "Fab"). [Figure 5C] FIG. 5C is a histogram of the relative fluorescence intensity of the gel bands shown in FIG. 5B. [Figure 6] 1 shows a gel fluorescence analysis of mouse anti-biotin IgG-594 protein uptake by A549, HepG2, FaDu, and Hela cells treated with folate-labeled antibody ("Ab-FA") or antibody alone ("Ab"). [Figure 7] MALDI-MS spectra of Ctx, Ctx-FA1, Ctx-FA2, and Ctx-FA3 are shown. [Figure 8] Western blots of EGFR in Hela cells treated with Ctx, Ctx-FA1, and Ctx-FA2 at 3 nM, 10 nM, and 30 nM for 48 h (upper panel) and 72 h (lower panel). Actin is used as a loading control. "-" represents the negative control. [Figure 9] Figure 1 shows Western blots of EGFR in Hela cells treated with Ctx and Ctx-FA at 0.1 nM, 1 nM, and 10 nM for 6 h and 48 h. Actin is used as a loading control. "-" represents the negative control. [Figure 10]Western blot of EGFR in multiple cancer cell lines treated with Ctx and Ctx-FA ("degrader") at 10 nM for 48 hours. Actin is used as a loading control. "-" represents the negative control. [Figure 11] Western blot of EGFR and LTR (folate receptor) in multiple cell lines treated with Ctx-FA at 10 nM for 48 hours. Actin is used as a loading control. [Figure 12] Normalization of EGFR and LTR to actin based on the gel image in FIG. 11 is shown. [Figure 13] 12. EGFR degradation rate and LTR / EGFR ratio in multiple cell lines calculated based on the data in FIG. [Figure 14] Western blots of phosphorylated EGFR (pEGFR) and phosphorylated MAPK (pMAPK) in cells pretreated with 10 nM Ctx-FA ("degrader") for 24 h and then incubated with 100 ng / ml EGF for 10 or 60 min. Actin is used as a loading control. "-" represents the negative control. [Figure 15] Western blot of EGFR in HepG2 hepatocytes treated with Ctx, Ctx-oligomeric M6P, Ctx-tri-GalNAc(GN), and Ctx-FA. Actin is used as a loading control. [Figure 16] Figure 1 shows western blots of EGFR, p-EGFR, pAkt, and pMAPK in Cal27 head and neck cancer cells treated with M6P-Ctx and Ctx-FA at 10 nM and 300 nM. Actin is used as a loading control. "-" represents the negative control. [Figure 17]Figure 1 shows western blots of EGFR, p-EGFR, pAkt, and pMAPK in FaDu head and neck cancer cells treated with M6P-Ctx and Ctx-FA at 10 nM and 300 nM. Actin is used as a loading control. "-" represents the negative control. [Figure 18A] Western blots of EGFR, pAkt, and pMAPK in HeLa cells treated with M6P-Ctx and Ctx-FA at 0 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM. Actin is used as a loading control. [Figure 18B] Western blots of EGFR, pAkt, pMAPK in HeLa cells treated with M6P-Ctx and Ctx-FA at 0, 2, 4, 6, 8, 16, 24, 48, and 72 hours are shown. Actin is used as a loading control. [Figure 19] Western blots of EGFR in FaDu cells (upper panel) and HepG2 cells (lower panel) co-treated with 10 nM Ctx-FA ("degrader") and 200 nM bafilomycin ("BAF") or 3 mM folic acid ("ligand") for 6 hours are shown compared to cells treated with Ctx-FA ("degrader") alone. Actin is used as a loading control. "-" denotes negative control. [Figure 20A] Confocal microscopy images of EGFR and a marker for lysosomal membranes (LAMP1) in FaDu head and neck cancer cells are shown. Figure 20A shows two images of the same well of FaDu cells treated with 10 nM Ctx-FA for 24 hours. [Figure 20B] Figure 20B shows confocal microscopy images of EGFR and lysosomal membrane marker (LAMP1) in FaDu head and neck cancer cells. Figure 20B shows a comparison of FaDu cells treated with 10 nM Ctx-FA degrading agent for 24 hours (upper panel) and FaDu cells not treated with Ctx-FA degrading agent (lower panel). [Figure 21]1 shows MALDI-MS spectra of Atz with folate labeling ("Atz-FA") or without folate labeling ("Atz"). [Figure 22] Western blot of PD-L1 in multiple cancer cell lines treated with 10 nM Atz-FA ("Degrader") or Atz alone ("Atz") for 24 hours. Actin was used as a loading control. "-" represents the negative control. [Figure 23] Western blots of CD47 in HepG2 and MDA-MB-231 cells treated with 10 nM folate-labeled CD47 antibody ("anti-CD47 degrader") or antibody alone ("anti-CD47"). Actin was used as a loading control. "-" represents the negative control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Provided herein is a bifunctional lysosome-targeted degrading agent, which comprises (a) a folate moiety or folate analog configured to bind to a folate receptor as a shuttle molecule for lysosomal degradation, and (b) a protein-binding moiety (or referred to herein as a "protein-binding agent") configured to bind to a membrane or extracellular protein of interest. The bifunctional degrading agents disclosed herein are used, for example, for selectively targeted degradation of membrane and extracellular proteins via the endosomal / lysosomal pathway. In one aspect, the bifunctional degrading agent induces degradation of oncogenic proteins specifically in cancer cells via the folate receptor, which is overexpressed in many cancer cells. Also provided herein are compositions comprising the bifunctional degrading agent, as well as methods of using the bifunctional degrading agent to inhibit disease states, including cancer.

[0021] It should be understood that the bifunctional decomposition agents, compositions, and methods disclosed herein are not limited to the particular embodiments described, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0022] It is 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 the range, unless otherwise stated herein, and each separate value is incorporated herein as if it were individually set forth herein. All range endpoints are included within the range and are independently combinable.

[0023] For clarity, it is understood that certain features of the bifunctional decomposition agents, compositions, and methods described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the bifunctional decomposition agents, compositions, and methods described in the context of a single embodiment may be provided separately or in any suitable subcombination. All combinations of the embodiments are specifically encompassed by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed, to the extent that such combinations encompass operable processes and / or compositions. Furthermore, all subcombinations listed in the embodiments describing such variables are also specifically encompassed by the bifunctional decomposition agents, compositions, and methods of the present invention and are disclosed herein as if each and every subcombination were individually and expressly disclosed herein. It is also noted that the bifunctional decomposition agents, compositions, and methods provided herein may be suitably practiced in the absence of any element not specifically disclosed herein.

[0024] Bifunctional lysosome-targeted degraders Provided herein is a bifunctional lysosome-targeted degradative agent, comprising a folate or an analog thereof configured to bind to a folate receptor as a shuttle molecule for lysosomal degradation, operably linked to a protein-binding moiety ("protein-binding agent") configured to bind to a membrane protein or extracellular protein of interest. As used herein, "operably linked" means that the folate moiety (or folate analog moiety) can be directly or indirectly linked to the protein-binding agent. Thus, a direct linkage would include a direct (no intervening atom) chemical bond between the two moieties, or there may be any type of linking moiety ("linker") between the folate moiety (or folate analog moiety) and the protein-binding moiety.

[0025] Folic acid is a small molecule that passes through cell membranes through binding to folic acid receptor.As disclosed herein, folic acid is attached to the binding agent of target membrane protein or extracellular protein to generate folic acid protein binding degrader.When folic acid is in the cell, it enters endosomes, where it deposits the target protein as payload, and the target protein is degraded.

[0026] The term "folate analog" is used herein to mean a moiety that is structurally similar to folate and is configured to bind to the folate receptor. The term "folate analog" includes, but is not limited to, folic acid, pteropolyglutamic acid, and folate receptor-binding pteridines, such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid, and their deaza and dideaza analogs. The terms "deaza" and "dideaza" analogs refer to art-recognized analogs that have carbon atoms in place of one or two nitrogen atoms in the naturally occurring folic acid structure, or analogs or derivatives thereof. For example, deaza analogs include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of folate. Dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs of folate. Other folate analogues useful as complexing ligands include folate receptor binding analogues: aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deaminohydroxyfolate, deaza analogues such as 1-deazamethopterin or 3-deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).These folate analogues are conventionally called "folates", reflecting their ability to bind to folate receptors.Further analogues of folate that bind to folate receptors are described in US Patent Publication Nos. 2005 / 0227985 and 2004 / 0242582, which are incorporated herein by reference.

[0027] Folate receptors, or folate-binding proteins, comprise single-chain glycoproteins that bind and contribute to the uptake of folates and other compounds in vivo (Elwood, 1989, J. Biol. hem. 264:14893-14901). Certain folate receptors are single-chain glycoproteins with high-affinity binding sites for folates and other compounds, such as methotrexate. The mature folate receptor glycoprotein has a size of approximately 42 kDa and has been observed to be involved in the internalization of folates and antifolate drugs into cells (Elwood et al., 1997, Biochemistry 36:1467-1478). The folate receptor gene family comprises four members, FRα or FOLR1, FRβ or FOLR2, FRγ or FOLR3, and FRδ or FOLR4 (Leamon and Jackman, 2008, Vitam Horm. 79:203-33).

[0028] Folate receptor alpha (FRα) is a glycosylphosphatidylinositol-linked cell surface glycoprotein with high affinity for folate. Most normal tissues do not express FRα, except at low levels in the kidney and lung. However, high levels of FRα have been found in epithelial ovarian cancer, endometrial adenocarcinoma, non-small cell lung cancer (NSCLC) of the adenocarcinoma subtype, and triple-negative breast cancer (TNBC) (Scaranti et al., 2020, Nat. Rev. 17:349-359). FRα expression is maintained in metastatic and recurrent cancers of ovarian cancer patients, and FRα expression has been observed after chemotherapy in epithelial ovarian and endometrial cancers. Folate receptor beta (FRβ), originally thought to be present only in the placenta, is also detected in bone marrow cells and acute myeloid leukemia (Pan et al., 2002, Blood. 100:594-602). Because expression of the folate receptor is restricted, the bifunctional degraders disclosed herein confer selectivity to cells expressing the folate receptor to degrade membrane and extracellular proteins of interest.

[0029] As disclosed herein, the bifunctional lysosome-targeted degradative agent comprises a protein-binding agent configured to bind to a membrane protein or extracellular protein of interest. In some embodiments, the protein-binding agent binds to a membrane protein.

[0030] 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 epidermal growth factor receptor (EGFR) family (such as HER2 (human epidermal growth factor receptor 2)), receptors of fibroblast growth factor receptor (FGFR) family, receptors of vascular endothelial growth factor receptor (VEGFR) family, receptors of platelet-derived growth factor receptor (PDGFR) family, receptors of rearranged during transfection (RET) receptor family, receptors of Eph receptor family, receptors of discoidin domain receptor (DDR) family, and mucin proteins (such as MUC1).

[0031] In a particular version, the membrane receptor is EGFR, which is known to be frequently mutated or overexpressed in various types of human cancers (Yarden and Pines, 2012, Nat Rev Cancer. 12:553-563; Sigismund et al., 2018, Mol. Oncol. 12:3-20).

[0032] The membrane protein may be an immunoinhibitory receptor. As used herein, an "immunoinhibitory receptor" is a receptor present on immune cells that negatively regulates immune response. Examples of immunoinhibitory receptors include, but are not limited to, the immunoinhibitory receptors of the Ig superfamily, including 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 immunosuppressive receptors include sialic acid-binding Ig-like lectin (Siglec) receptors, such as Siglec 7, Siglec 9, etc. Further examples of immunosuppressive receptors include C-type lectins, including but not limited to CLEC4A (DCIR), Ly49Q, and MICL. Details regarding immunosuppressive receptors can be found, for example, in Steevels et al., 2011, Eur. J. Immunol. 4:575-587.

[0033] Membrane proteins may optionally be ligands for immunoinhibitory receptors, one example being CD47, which is known to bind SIRP-α to prevent phagocytosis and is overexpressed in cancer cells (Eladl et al., 2020, J. Hematol. Oncol. 13:96).

[0034] 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 inhibit apoptosis and is known to be overexpressed in cancer cells (Yi et al., 2021, J. Hematol. Oncol. 14:10).

[0035] As disclosed herein, the bifunctional lysosome-targeted degradative agent comprises a protein-binding agent configured to bind to a membrane protein or extracellular protein of interest. In some embodiments, the protein-binding agent binds to an extracellular protein.

[0036] 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 particular subtype of such cytokines), hormones, and the like.

[0037] Alternatively, the extracellular protein may be an antibody, such as an antibody that binds to a membrane protein or a different extracellular protein. The antibody may be an autoantibody. By "autoantibody" is meant an antibody generated by the immune system that is directed against one or more of an individual's own proteins. Cancer cells may induce an immune 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), anticentromere antibodies (ACA), anti-cyclic citrullinated peptide antibodies (anti-CCP), extracted nuclear antigen antibodies (ENA), anticardiolipin antibodies, beta2 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.

[0038] 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.

[0039] The extracellular protein may be a mutated protein.

[0040] When the protein binding agent of the bifunctional degrader binds to a membrane protein or extracellular protein, the membrane protein or extracellular protein may be present on or produced by a cancer cell. By "cancer cell" is meant a cell that exhibits a neoplastic cell phenotype, which may be characterized by, for example, one or more of abnormal cell growth, abnormal cell proliferation, loss of density-dependent growth inhibition, anchorage-independent growth ability, ability to promote tumor growth and / or development in immunocompromised non-human animal models, and / or any suitable indicator of cell transformation. "Cancer cell" may be used interchangeably herein with "tumor cell", "malignant cell", "neoplastic cell", or "cancerous cell", and includes cancer cells such as 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.

[0041] The protein-binding agent of the bifunctional degrading agent can be any type of moiety capable of binding to a membrane protein or extracellular protein that is 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 for a membrane receptor, which is targeted for degradation), an aptamer, a nanoparticle, and a small molecule.

[0042] The protein binding agent can be a small molecule. By "small molecule" is meant 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. In one embodiment, the small molecule is biotin.

[0043] 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 comprised of a tetramer, which in turn is composed of two dimers of a heavy and a light chain polypeptide); single chain antibodies, fragments of antibodies (e.g., full chain antibodies or single chain antibody fragments) that retain specific binding to a membrane protein or extracellular protein, including, but not limited to, Fv, single chain Fv (scFv), Fab, F(ab')2, Fab', (scFv')2, bispecific antibodies, 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 antibodies may be further conjugated to other moieties, such as, for example, polyethylene glycol (PEG). Fusion to the Fc region (or fragment thereof) of an antibody, conjugation to PEG, etc., may be used, for example, to extend the serum half-life of the antibody upon administration to a subject.

[0044] In certain versions, the antibody is configured to bind to a cancer antigen.

[0045] The antibody may be configured to bind to intact complement or a fragment thereof. In certain embodiments, the antibody binds to one or more immunodominant epitopes within intact complement or a fragment thereof.

[0046] Alternatively, the antibody may bind to a membrane receptor or a membrane receptor ligand.

[0047] Alternatively, the antibody may bind to an epidermal growth factor (EGF) protein, for example, human EGF, or to one or more immunodominant epitopes within the EGF protein.

[0048] In certain embodiments, the antibody binds to EGFR protein. In certain embodiments, the antibody binds to one or more immunodominant epitopes in EGFR protein. In certain embodiments, 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 certain embodiments, the antibody is cetuximab.

[0049] In certain embodiments, the antibody binds to an immunoinhibitory receptor. In certain embodiments, the antibody binds to one or more immunodominant epitopes within an immunoinhibitory receptor.

[0050] In certain embodiments, the antibody binds to the ligand of the immunoinhibitory receptor. In certain embodiments, the antibody binds to one or more immunodominant epitopes in the ligand of the immunoinhibitory receptor. In certain embodiments, the antibody binds to the CD47 protein. In certain embodiments, the antibody binds to one or more immunodominant epitopes in the CD47 protein.

[0051] In certain embodiments, the antibody binds to an immune checkpoint molecule. In certain embodiments, the antibody binds to one or more immunodominant epitopes in 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 in a PD-L1 protein. In certain embodiments, the antibody comprises CDRs present in atezolizumab (Atz). In another particular embodiment, the antibody comprises a variable light chain and a variable heavy chain present in atezolizumab. In certain embodiments, the antibody is atezolizumab.

[0052] The bifunctional lysosomal targeting degrading agent disclosed herein can be in any suitable form.In some embodiments, the bifunctional degrading agent is a complex.Thus, in certain embodiments, the bifunctional degrading agent disclosed herein comprises folate bound to protein binding agent.In certain embodiments, one or more linkers can be utilized to facilitate the binding of folate to protein binding agent. 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; vinylsulfone-based linkers; linkers including, but not limited to, polyethylene glycol (PEG), such as tetraethylene glycol; linkers including propanoic acid; linkers including caproleic acid, and linkers including any combination thereof. In one embodiment, the linker is PEG. In another embodiment, the linker is PEG and NHS.

[0053] In certain aspects, the linker is a chemically labile linker, e.g., a linker that is stable at neutral pH (bloodstream pH 7.3-7.5) but is hydrolytically cleavable 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., a linker that is stable in the bloodstream but is enzymatically cleaved by lysosomal proteases (e.g., cathepsin or plasmin) in the lysosomes of target cells (e.g., cancer cells) upon internalization into target cells. Enzyme-labile linkers include, but are not limited to, linkers that contain peptide bonds, including dipeptide-based linkers, such as valine-citrulline linkers, such as maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linker, valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, etc. Chemically unstable linkers, enzyme-labile linkers, and non-cleavable linkers are known and are described in detail, for example, in Ducry and Stump, 2010, Bioconjugate Chem. 21:5-13.

[0054] Folate-protein binder conjugates can be formed by covalently linking folate to a protein binder via one or more functional groups, either directly or via one or more linker molecules to form a covalent conjugate. Exemplary methods for preparing folate and antibody conjugates are provided in the Examples section below.

[0055] In certain aspects, the bifunctional degrading agent enhances the degradation of the membrane protein or extracellular protein relative 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 relative to the degradation of the membrane protein or extracellular protein in the presence of folate or the 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 in the presence of folic acid or the 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 in the presence of the protein-binding agent alone or in the presence of folic acid or the protein-binding agent alone under the same conditions. If a membrane protein or extracellular protein is degraded in the presence of a bifunctional degrader to a greater extent than it is degraded in the presence of a protein binder alone or in the presence of folic acid or a protein binder alone under the same conditions, 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 greater in the presence of a bifunctional degrader.

[0056] composition Disclosed herein are compositions comprising any of the bifunctional lysosomally targeted degradative agents disclosed herein.

[0057] The composition may optionally include one in which the bifunctional decomposition agent of the present disclosure is present in a liquid medium. The liquid medium may be an aqueous liquid medium such as water, a buffer solution, etc. In such a composition, one or more additives may be present, such as salts (e.g., NaCl, MgCl2, KCI, 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.

[0058] Also disclosed herein are pharmaceutical compositions comprising any of the bifunctional lysosome-targeted degrading agents of the present disclosure and a pharma- ceutically acceptable carrier. The pharmaceutical composition generally comprises a therapeutically effective amount of the bifunctional degrading agent. By "therapeutically effective amount" is meant a dosage sufficient to produce a desired result, such as a reduction in cell proliferation in an individual with a cell proliferative disease (e.g., cancer) associated with a membrane protein or extracellular protein to which the protein binding agent of the bifunctional degrading agent or the like binds, e.g., an amount sufficient to effect a beneficial or desired therapeutic result (including prevention). An effective amount can be administered in one or more doses.

[0059] The bifunctional decomposition agent of the present disclosure can be incorporated into various preparations for therapeutic administration.More specifically, the bifunctional decomposition agent can be made into a pharmaceutical composition by combining with suitable pharma-ceutically acceptable excipient or diluent, and can be made into a solid, semi-solid, liquid or gas form preparation, such as tablet, capsule, powder, granule, ointment, solution, injection, inhalant and aerosol.

[0060] 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 be free of detectable pyrogens or other contaminants that would contraindicate administration to an individual.

[0061] In pharmaceutical dosage forms, the bifunctional degrading agent can be administered alone or in appropriate association or combination with other pharma- ceutical active compounds. The following methods and excipients are merely exemplary and in no way limiting.

[0062] For oral preparations, the bifunctional disintegrants can be used alone or in combination with suitable additives, such as conventional additives such as lactose, mannitol, corn starch, or potato starch; binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if desired, diluents, buffers, wetting agents, preservatives, and flavorings, to produce tablets, powders, granules, or capsules.

[0063] The bifunctional decomposition agent can be formulated into an injectable preparation by dissolving, suspending, or emulsifying in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oil, synthetic fatty acid glyceride, higher fatty acid ester, or propylene glycol, together with conventional additives, such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, preservatives, etc., if desired.

[0064] The pharmaceutical composition can be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, where lyophilized formulation is reconstituted with sterile solution before administration.The standard procedure for reconstituting lyophilized composition is to add back a certain amount of pure water (typically the same amount as removed during lyophilization), but solutions containing antibacterial agents can be used to produce pharmaceutical compositions for parenteral administration.

[0065] Aqueous formulations of bifunctional degraders can be prepared in pH buffer solutions at pHs ranging from about 4.0 to about 8.0, such as from about 4.5 to about 7.5, e.g., from about 5.0 to about 7.0. Examples of buffers suitable for pHs within this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. Buffer concentrations 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.

[0066] How to use Disclosed herein are methods of using the bifunctional lysosome-targeted degradative agents of the present disclosure.

[0067] Provided herein are methods for degrading membrane or extracellular proteins. Such methods include contacting a membrane or extracellular protein with any of the bifunctional lysosome-targeted degradative agents disclosed herein under conditions in which the bifunctional lysosome-targeted degradative agent delivers the membrane or extracellular protein to lysosomes for degradation. Such methods are used in a variety of applications. In certain embodiments, the methods are performed in vitro (e.g., in a tube, cell culture plate or well, etc.), 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), for example, for clinical / therapeutic applications.

[0068] Also provided are methods that include administering a therapeutically effective amount of any of the bifunctional degrading agents or pharmaceutical compositions of the present disclosure to an individual in need thereof. A variety of individuals can be treated according to the subject methods. Generally, such subjects are "mammals" or "mammals", which terms are used broadly to describe organisms belonging to the class Mammalia, including 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.

[0069] An effective amount of a bifunctional resolving 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 an individual's disease condition (e.g., cancer) 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 individual's symptoms in the absence of treatment with the bifunctional resolving agent or pharmaceutical composition.

[0070] The method comprises administering a therapeutically effective amount of any of the bifunctional degrading agents or any of the pharmaceutical compositions of the present disclosure to an individual having cancer. According to such a method, the protein-binding agent of the bifunctional degrading agent binds to a membrane protein or extracellular protein that at least contributes to the individual's cancer, wherein the individual's cancer is treated by targeted degradation of the membrane protein or extracellular protein using the bifunctional degrading agent. In certain aspects, the protein-binding agent binds to a protein selected from a membrane receptor, a ligand for a membrane receptor, an immunoinhibitory receptor, a ligand for an immunoinhibitory receptor, an immune checkpoint molecule, an autoantibody, a secreted protein, and a mutant protein.

[0071] 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.

[0072] In any of the methods of using the bifunctional degradation agents of the present disclosure, the bifunctional degradation agents generally enhance the degradation of the membrane or extracellular protein relative to the degradation of the membrane or extracellular protein in the presence of a protein-binding agent alone. Similarly, in any of the methods of using the bifunctional degradation agents of the present disclosure, according to some embodiments, the bifunctional degradation agents enhance the degradation of the membrane or extracellular protein relative to the degradation of the membrane or extracellular protein in the presence of folate or a protein-binding agent alone.

[0073] "Treat," "treating," or "treatment" refers to at least an amelioration of symptoms associated with a medical condition (e.g., a cell proliferative disorder, e.g., cancer) in an individual, where amelioration is used in a broad sense to refer to at least a reduction in a parameter associated with the medical condition being treated, e.g., the magnitude of the symptoms. Thus, treatment also includes situations in which the medical condition, or at least the symptoms associated therewith, are completely suppressed, e.g., prevented from developing, or arrested, e.g., terminated, such that the individual is no longer afflicted by the medical condition, or at least the symptoms characterizing the medical condition.

[0074] The bifunctional degrading agent or pharmaceutical composition may be administered to an individual using any available method and route suitable for drug delivery, including systemic and local routes of administration, as well as in vivo and ex vivo methods. Conventional pharma- ceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intraocular, intravenous, intraarterial, nasal, oral, and other enteral and parenteral routes of administration. In some embodiments, administration is by parenteral administration. Routes of administration may be combined, if desired, or tailored depending on the bifunctional degrading agent and / or the desired effect. The bifunctional degrading agent or pharmaceutical composition may be administered in a single dose or multiple doses. In some embodiments, the bifunctional degrading agent or pharmaceutical composition is administered intravenously. In some embodiments, the bifunctional degrading agent or pharmaceutical composition is administered by injection, for example, for systemic delivery (e.g., intravenous infusion), or to a localized site. EXAMPLES

[0075] method Antibody labeling with FA-PEG-NHS: To label the antibody with folate (FA), 100 μL of antibody (at a concentration of 1.8 mg / mL) in PBS was mixed with folate-NHS ester at a molar ratio of 1:25. The reaction was incubated overnight at room temperature on a rotator and subsequently filtered five times with 500 μL of PBS using a 10 kDa Amicon Centrifugal Filter. See Figure 2A for the chemical structure of FA-PEG-NHS.

[0076] Antibody labeling with DBCO-PEG3-NHS and N3-PEGn-FA: To label the antibody with DBCO, 200 μL of antibody in PBS (at a concentration of 1.8 mg / mL) was mixed with DBCO-PEG3-NHS ester at a molar ratio of 1:25. The reaction was incubated overnight at room temperature on a rotator and then filtered five times with 500 μL of PBS using a 10 kDa Amicon Centrifugal Filter. The concentration of DBCO-labeled antibody was then determined by BCA assay and mixed with N3-PEGn-FA at a molar ratio of 1:25. The reaction was incubated overnight at room temperature on a rotator and then filtered five times with 500 μL of PBS using a 10 kDa Amicon Centrifugal Filter. See Figure 2B for the chemical structures of DBCO-PEG3-NHS and N3-PEGn-FA.

[0077] MALDI-MS α-Cyano-4-hydroxycinnamic acid (HCCA) was dissolved in 50% acetonitrile / water to obtain a 10 mg / mL solution as matrix solution. Samples were absorbed onto Omix C4 pipette tips and washed three times with 0.1% TFA before elution with 20 μL of 75% acetonitrile / water. 1 μL of sample solution and 1 μL of HCCA solution were spotted onto the MALDI target plate, mixed thoroughly, and the spot was allowed to dry at room temperature. MALDI-MS spectra were acquired on a Bruker UltraFlex MALDI-TOF / TOF mass spectrometer operated in linear positive ion mode. Masses were calculated from windowed raw data in Sigmaplot 13.0 by fitting a Gaussian curve, with a constant baseline as an additional free parameter. Starting values ​​of parameters were the program's default values, and fits were obtained automatically 200 iterations. Plots were generated in Origin 2020, where a 100-point windowed FFT filter was used to remove high frequency noise.

[0078] Mouse IgG uptake experiment: Cells were plated at 70% confluence in 24-well plates. Complete growth medium supplemented with 50 nM mouse anti-biotin IgG-647 and 25 nM FA-labeled goat anti-mouse IgG was added sequentially. Cells were incubated at 37°C for 24 hours, after which they were lysed and subjected to gel fluorescence analysis.

[0079] Targeted Protein Degradation: Cells were seeded in 24-well plates at 70% confluence. The next day, cells were treated with 10 nM degraders for 24 or 48 h, then harvested for Western blot analysis. For EGFR degradation, 100 ng / ml EGF was added and incubated for 10 or 60 min after 24 h degradation to stimulate the EGFR signaling pathway.

[0080] Western blotting: Cells were lysed in 1X RIPA lysis buffer containing 25 mM Tris, pH 7-8, 150 mM NaCl, 0.1% (w / v) sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% (v / v) Triton X-100, protease inhibitor cocktail (Roche, 1 tablet per 10 mL), and 1 mM phenylmethylsulfonyl fluoride for 10 min on ice. Lysates were then centrifuged at 16000g for 15 min at 4°C, and the supernatants were collected before measuring protein concentration using the BCA assay. Lysates were mixed with 4x Laemmli Loading Dye after adjusting to equal volumes and heated at 99°C for 5 min. After cooling, samples were subjected to 7.5% or 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were first blocked with 5% (w / v) nonfat milk in TBS-T wash buffer (137 mM NaCl, 20 mM Tris, 0.1% (v / v) Tween) and then incubated with primary antibodies overnight at 4°C. Membranes were washed three times with TBST, then incubated with secondary HRP-linked antibodies for 1 h and then washed three times with TBST. Membranes were then incubated in Clarity ECL substrate for 3–5 min before immunoblotting by ChemiDoc MP Imaging Systems.

[0081] Confocal Microscopy: FaDu cells were seeded on 8-well chamber slides at a density of 20,000 cells / well in 200 μL of complete culture medium. After attachment, cells were treated with 10 nM Ctx-FA for 24 h at 37 °C. Cells were then washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton-100 for 5 min. After blocking with 5% BSA for 1 h at room temperature (RT), cells were co-incubated with anti-EGFR and anti-LAMP1 or EEA1 antibodies in 1% BSA overnight at 4 °C. The next day, cells were washed with PBS and incubated with anti-mouse 488 or anti-rabbit 594 secondary antibodies for 1 h at room temperature. Cells were then mounted with slowfade antifade mounting medium containing DAPI. Images were acquired using a Leica SP8 3x STED super-resolution microscope at 10x eyepiece and 100x magnification and analyzed with ImageJ.

[0082] Competitive assay: FaDu or HepG2 cells were seeded in 24-well plates at 70% confluence. The next day, cells were co-treated with 10 nM of degradant and 200 nM of Bafilomycin A1 or 3 mM of folic acid for 6 h, then harvested for Western blot analysis.

[0083] Example 1: Uptake mediated by small molecule degraders and antibody-based degraders In this example, a small molecule degrader, Folate-PEG3-Biotin (Nanocs), was tested, having the following chemical structure:

[0084] [ka]

[0085] Folate-PEG3-biotin degrader contains biotin as a protein binder, which binds to avidin and streptavidin with high specificity and affinity. We observed that Hela, HepG2, and MCF7 cells were treated with or without folate-PEG3-biotin degrader, and that the degrader-treated cells showed higher uptake of NA-650 (NeutrAvidin-650, a fluorescently labeled protein that strongly binds biotin) than cells treated with NA-650 alone (Figure 3).

[0086] Next, an antibody-based degrader was tested as a protein binder, comprising an antibody labeled with folate-PEG10-NHS having the following chemical structure:

[0087] [ka]

[0088] Anti-mouse IgG antibodies (Ab 150 kDa, Fab 50 kDa) were labeled with folate-PEG10-NHS and the uptake of the fluorescent protein target mouse anti-biotin IgG-647 was monitored in Hela cells treated with or without folate-labeled antibodies. Figures 4A and 4B show MALDI-MS spectra of anti-mouse IgG Fab fragments and full-size anti-mouse IgG with or without folate labeling. Figures 5A-5C show gel fluorescence analysis suggesting that there was a higher uptake of the protein target in cells treated with folate-labeled anti-mouse IgG antibodies compared to cells treated with antibody alone. The uptake of the protein target was higher in Hela cells treated with folate-labeled full-size anti-mouse IgG than with anti-mouse IgG Fab monomers (Figure 5A).

[0089] In general, the uptake mediated by small molecule degraders is moderate compared to antibody-based degraders. Consistent uptake was observed upon 24-hour treatment of antibody-based degraders in HeLa cells (Figures 5B and 5C). As shown in Figure 6, we also observed uptake of anti-biotin IgG-594 protein by antibody-based degraders in multiple cell lines.

[0090] Example 2: EGFR Degradation Cetuximab (Ctx) is an approved antibody drug that blocks the function of the cell surface epidermal growth factor receptor (EGFR) for the treatment of metastatic colorectal and head and neck cancer. (Ctx is commercially available from ImClone LLC, New York, New York under the registered trademark "ERBITUX"®; USNDC Codes 66733-948-23 and 66733-958-23.) The main side effects of Ctx are related to blocking EGFR in normal tissues. Selective degradation of EGFR in cancer cells provides significant therapeutic benefit.

[0091] In this example, Ctx was labeled with folate-PEG10-NHS from two different vendors (Nanocus and Ruixibiotech) to generate degradants Ctx-FA1 and Ctx-FA2. Ctx was also labeled with folate-PEG3-NHS to generate degradant Ctx-FA3. Figure 7 shows the MALDI-MS spectra of Ctx, Ctx-FA1, Ctx-FA2, and Ctx-FA3.

[0092] The results suggest that Ctx-FA2 was able to induce the degradation of EGFR in Hela cells at 48 and 72 hours of treatment, as shown in Figure 8. The results are reproducible at different concentrations of the Ctx-FA degrading agent, as shown in Figure 9. As shown in Figure 10, the degradation of EGFR was also observed in multiple cancer cell lines treated with the Ctx-FA degrading agent.

[0093] We observed more efficient degradation of the membrane protein EGFR by the Ctx-FA degrader compared to uptake of the anti-biotin protein target in the medium, which was the exact opposite result compared to our previous study on GalNAc-labeled antibodies, where uptake of the protein target in the medium was much more efficient than degradation of EGFR (Zhou et al., 2021, ACS Cent. Sci. 7:499-506).

[0094] Therefore, the correlation between the degradation efficiency and the ratio of LTR (folate receptor) / EGFR expression level was also examined. Figure 11 shows the expression levels of EGFR and LTR in multiple cell lines treated with 10 nM Ctx-FA for 48 hours. Based on the gel images in Figure 11, the expression levels of EGFR and LTR were normalized to actin (Figure 12). The EGFR degradation rate and the LTR / EGFR ratio were calculated (Figure 13), but no obvious correlation was found between the EGFR degradation rate and the LTR / EGFR ratio.

[0095] The effect of Ctx-FA degrading agent was also tested by EGFR signaling. Cells were pretreated with 10 nM Ctx-FA for 24 h, followed by incubation with 100 ng / ml EGF for 10 or 60 min to stimulate EGFR signaling pathway. Proteins were collected and subjected to Western blotting for phosphorylated EGFR (pEGFR) and MAPK (pMAPK). The results showed that pEGFR was barely detectable in cells treated with Ctx or Ctx-FA degrading agent (Figure 14), suggesting that both Ctx and Ctx-FA effectively blocked EGFR signaling.

[0096] Therefore, the degradation efficiency of the Ctx-FA decomposition agent was compared with other EGFR decomposition agents. As shown in Figure 15, HepG2 liver cells treated with the Ctx-FA decomposition agent showed better degradation of EGFR compared with three LTR-derived decomposition agents, including Ctx alone, Ctx-oligomer M6P, and Ctx-tri-GalNAc (GN). It also shows that the Ctx-FA decomposition agent had better degradation efficiency of EGFR compared with M6P-Ctx in Cal27 head and neck cancer cells (Figure 16), Fadu head and neck cancer cells (Figure 17), and Hela cells (Figures 18A and 18B).

[0097] To study the mechanism of Ctx-FA degrading agent, Fadu cells and HepG2 cells were co-treated with Ctx-FA degrading agent and lysosomal inhibitors folic acid and bafilomycin (BAF). Co-treatment effectively reduced the degradation of EGFR (Figure 19), suggesting that the degradation of EGFR in the presence of Ctx-FA degrading agent is via the lysosomal degradation pathway. As shown by the confocal microscopy image in Figure 20A, we observed co-localization of EGFR with a marker for lysosomal membrane (LAMP1) in Fadu head and neck cancer cells treated with 10 nM Ctx-FA degrading agent for 24 hours, while in the absence of Ctx-FA degrading agent, EGFR is localized on the cell membrane, as shown by the confocal microscopy image in Figure 20B.

[0098] Example 3: Degradation of other cancer-associated targets (PD-L1 and CD47) via cancer-specific LTRs The PD-L1 degraders are based on atezolizumab (Atz), the first FDA-approved PD-L1 antibody. Atezolizumab is a commercially available monoclonal antibody sold under the trade name "TECENTRIQ" (registered trademark) (Genentech, San Francisco, USA;). Figure 21 shows the MALDI-MS spectra of Atz with and without folate labeling. We investigated the degradation of PD-L1 in multiple cancer cell lines treated with or without the folate-labeled Atz degrader. The results showed effective degradation of PD-L1 by the folate-labeled Atz degrader in most of the cells tested (Figure 22).

[0099] We also investigated a CD47 degrading agent based on a folate-labeled CD47 antibody, and Figure 23 shows the degradation of CD47 in HepG2 and MDA-MB-231 cells treated with the degrading agent.

Claims

1. A bifunctional lysosome-targeted degradative agent, comprising: The bifunctional lysosome-targeted degradative agent comprises a folate moiety or an analog thereof configured to bind to a folate receptor as a shuttle molecule for lysosomal degradation, and a protein-binding moiety configured to bind to a membrane protein or an extracellular protein; A bifunctional lysosome-targeted degradative agent, wherein the folate moiety or analogue thereof is operably linked to the protein-binding moiety.

2. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the protein-binding portion is configured to bind to a membrane protein.

3. The bifunctional lysosome-targeted degradative agent of claim 2 , wherein the protein-binding portion is configured to bind to a membrane receptor.

4. The bifunctional lysosome-targeted degradative agent of claim 3 , wherein the protein-binding moiety is configured to bind to epidermal growth factor receptor (EGFR).

5. The bifunctional lysosome-targeted degradative agent of claim 2 , wherein the protein-binding portion is configured to bind to an immunosuppressive receptor.

6. The bifunctional lysosome-targeted degradative agent of claim 2 , wherein the protein-binding portion is configured to bind to a ligand of an immunoinhibitory receptor.

7. The bifunctional lysosome-targeted degradative agent of claim 6 , wherein the protein-binding portion is configured to bind to CD47.

8. The bifunctional lysosome-targeted degradative agent of claim 2 , wherein the protein-binding moiety is configured to bind to an immune checkpoint molecule.

9. The bifunctional lysosome-targeted degradative agent of claim 8, wherein the protein-binding moiety is configured to bind to programmed cell death ligand 1 (PD-L1).

10. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the protein-binding moiety is configured to bind to an extracellular protein.

11. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the protein-binding moiety is selected from a polypeptide, a ligand, an aptamer, a nanoparticle, and a small molecule.

12. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the protein-binding moiety is a non-protein small molecule.

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

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

15. The bifunctional lysosome-targeted degrading agent of claim 14 , wherein the antibody is configured to bind to the EGFR protein.

16. The bifunctional lysosome-targeted degrading agent of claim 15, wherein the antibody is cetuximab.

17. The bifunctional lysosome-targeted degrading agent of claim 14 , wherein the antibody is configured to bind to CD47 protein.

18. The bifunctional lysosome-targeted degrading agent of claim 14, wherein the antibody is configured to bind to PD-L1 protein.

19. The bifunctional lysosome-targeted degrading agent of claim 18, wherein the antibody is atezolizumab.

20. The bifunctional lysosome-targeted degradative agent of claim 1 , further comprising a linker connecting the folate moiety or analog thereof to the protein-binding moiety.

21. The bifunctional lysosome-targeted degradative agent of claim 20 , wherein the linker is PEG.

22. The bifunctional lysosome-targeted degradative agent of claim 20 , wherein the linker is PEG and NHS.

23. The bifunctional lysosome-targeted degradative agent of claim 1 , wherein the bifunctional lysosome-targeted degradative agent is configured to selectively target cells that express a folate receptor.

24. The bifunctional lysosome-targeted degradative agent of claim 23, wherein the cell expressing a folate receptor is a cancer cell.

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

26. 26. The pharmaceutical composition of claim 25, further comprising a pharma- ceutically acceptable carrier.

27. 1. A method for degrading a membrane protein or an extracellular protein, comprising: The method comprises contacting the membrane protein or the extracellular protein with a bifunctional lysosome-targeted degradative agent according to claim 1, The method, wherein the bifunctional lysosome-targeted degradative agent shuttles the membrane protein or the extracellular protein to lysosomes for degradation.

28. 26. A method comprising administering to an individual in need thereof a therapeutically effective, neoplastic cell proliferation inhibiting amount of the pharmaceutical composition of claim 25.

29. 30. The method of claim 28, wherein the individual is a human.

30. 30. The method of claim 28, wherein the individual has cancer.

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