Ketoboronate tag and methods of use thereof for cytosolic delivery and targeting proteins for degradation

Abstract

Disclosed is a ketoboronate compound for use as a covalent-reversible, lysine-reactive cellular uptake tag that enable cytosolic delivery of agents such as small organic molecules, peptides, and proteins such as antibodies and lysosomal delivery and degradation of plasma membrane proteins. Methods of using constructs including the ketoboronate compound are provided.

Description

KETOBORONATE TAG AND METHODS OF USE THEREOF FOR CYTOSOLIC DELIVERY AND TARGETING PROTEINS FOR DEGRADATIONREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Provisional Application Serial Number 63 / 562,071, filed March 6, 2024, the contents of which are incorporated herein by reference in their entireties.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (name: UIC0111WO_ST26.xml; size: 5,388 bytes; and date of creation: February 24, 2025) is herein incorporated by reference in its entirety .BACKGROUND OF THE INVENTION

[0003] Administered drugs often enter the cell by passive diffusion, a process mostly governed by the size and lipophilicity of the small molecule. Although therapeutic biologics have demonstrated remarkable success, constituting 30 percent of all FDA-approved therapies from 2015 to 2018, the large size and poor cell-permeability of peptide-, protein-, and antibody-drugs limits their targets to extracellular and plasma membrane proteins. Thus, technologies that enable cellular delivery of otherwise impermeable biologics and improve their bioavailability are highly desired.

[0004] Cell-penetrating peptides (CPP) are a specific class of peptides reported to cross the cell membrane. Despite attempts at prediction and rational design, the mechanisms of how CPPs traverse the cell membrane remain unclear and heavily debated, since they likely differ among peptides. Successful uptake typically requires relatively high concentrations of CPPs. Additionally, CPPs present several other limitations includinghigh molecular weight, cytotoxicity, entrapment in endosomes, and poor reliability. Cell-penetrating poly(disulfide)s (CPDs) have overcome some of the aforementioned limitations. CPDs are synthesized via disulfide-exchange polymerization reactions, where an initiator thiol reacts with a strained disulfide monomer containing a guanidinium cation before termination with a iodoacetamide group linked to the cargo. CPDs have been mostly used to translocate proteins attached to the polymer. While CPDs offer several advantages over CPPs including the ease of preparation, improved reliability, and reduced toxicity, the mechanism of how CPDs enters the cell is not well established, as the reactive thiols / cysteines in the membrane have not been identified, limiting further development of this technology. Furthermore, the full scope of compatible cargos remains to be determined.

[0005] It has been demonstrated that attachment of a single cyclic disulfide, asparagusic acid (AspA), enables cellular uptake of up to 20-mer, fully unprotected peptides via the transferrin receptor (Abegg et al. (2017) J. Am. Chem. Soc. 139:231-238). The AspA tag is easily installed by treating the unfunctionalized and unprotected peptides with asparagusic acid NHS ester to modify the TV-terminal amine. To demonstrate the utility of this approach, proapoptotic peptides Bak BH3 and Bim BH3 were labeled with an N-terminal AspA tag. Only cells treated with AspA-tagged Bak and Bim BH3 peptides became apoptotic. Transferrin receptor protein 1 (TRFC) was identified by chemoproteomics as the main target responsible for the AspA tag uptake. The advantages of the AspA tag over existing techniques include the small tag size, ease of preparation, no toxicity in non-cancerous cells, and highly efficient translocation via TFRC and clathrin-mediated endocytosis (Abegg et al. (2017) J. Am. Chem. Soc. 139:231-238). However, the uptake rate stronglydepends on the native expression level of the transferrin receptor.

[0006] Many chemical strategies have been developed to target proteins of interest for degradation but the vast majority of these methods rely on recruitment of intracellular degradation machinery and are therefore not applicable to degradation of membrane-associated proteins with extracellular domains. A series of glycoconjugates have been reported that are composed of an antibody targeting the extracellular domain on a protein of interest (POI) and either a large (9.7 to 37.1 kDa) oligomeric mannose-6-phosphonate glycopeptide (M6P) or a triantenerrary N- acetylgalactosamine (tri-GalNAc, 3.4 kDa)(Fan et al. (2014) Nat. Neurosci. 17(3):471-480; Ahn et al. (2021) Nat. Chem. Biol. 17:937-946). Termed LYTACs (lysosome targeting chimeras), these molecules enable endocytic uptake and lysosomal degradation of the POI via the cation-independent mannose-6-phosphate receptor (IGF2R) or the liver-specific asialoglycoprotein receptor (ASGPR). Another approach, named AbTACs, uses a bifunctional antibody recognizing a POI and a cell surface E3 ubiquitin ligase resulting in intracellular ubiquitination and degradation of the POI (Cotton et al. (2021) J. Am. Chem. Soc. 143(2):593-598; Gramespacher et al. (2002) ACS Chem Biol. 17(5):1259-1268). Moreover, a method called SignalTACs uses a fusion between the signaling motif from the cation-independent mannose-6-phosphate receptor (CI-M6PR) to an antibody recognizing a POI (Yu et al. (2023) J. Am. Chem. Soc. 145(34):19107-19119). These strategies were successfully applied to degrade several proteins of therapeutic interest in cell culture, including the epidermal growth factor receptor (EGFR), TFRC, epidermal growth factor receptor 2 (HER2), and PD-L1 and in vivo for the extracellular cytokine MIF and HER2 (Caianiello et al. (2021) Nat. Chem. Biol. 17(9):947-953) . However, the efficiency of this strategy depends on the native expression levels of a single receptor, IGF2R forM6P, ASGPR for tri-GalNAc, HER2 for SignalTACs, or a specific E3 ligase. Yet another approach, dubbed GlueTACs (Zhang et al. (2021) J. Am. Chem. Soc. 143 (40):16377-16382), uses an engineered nanobody with an unnatural amino acid covalently reacts with a POI followed by internalization via a conjugated CPP and lysosomal sorting sequence. This strategy enabled the degradation of PD-L1 both in cell culture and in vivo. The major drawback of this technology is the laborious engineering of a nanobody bearing an unnatural amino acid with a covalent reactive group (fluorosulfate or bromine) positioned in the close proximity to a nucleophilic amino acid near the POI epitope .

[0007] Many approaches exist to facilitate cellular uptake and cytosolic delivery of peptides and proteins (e.g., CPPs, CPDs, and single-residue uptake tags such as AspA) and independent approaches have been reported for targeted lysosomal delivery of plasma membrane proteins for degradation (LYTAC), AbTACs, and GlueTACs. Unfortunately, these technologies bear significant limitations, thus creating an unmet demand for a small, single residue tag that can enter the cells via several membrane receptors and is able to facilitate both the cytosolic delivery of peptides, proteins, and drugs and lysosomal delivery of endogenous plasma membrane proteins for degradation. The present invention addresses this need in the art.SUMMARY OF THE INVENTION

[0008] This invention provides a construct comprising an agent covalently linked to a 2-ketoboronate compound of Formula IFormula I

[0009] Also provided is a method of facilitating entry of an agent into a cell by contacting the cell with the construct of Formula I.

[0010] Further provided is a method of facilitating degradation of a plasma membrane protein or secreted protein by contacting a cell with the construct of Formula I, wherein the agent of the construct is a ligand that binds the plasma membrane protein or secreted protein.

[0011] A method of preventing or treating a disease or condition is also provided, which comprises administering to a subject in need thereof an effective amount of a construct of Formula I, wherein the agent of the construct is a therapeutic agent for preventing or treating the disease or condition.

[0012] Also provided is a 2-ketoboronate compound having the structure:BRIEF DESCRIPTION OF DRAWINGS

[0013] FIG. 1. Schematic representation of lysine-mediated uptake of a ketoboronate (KB)-modified drugs and macromolecules. Shown is iminoboronate formation between a solvent-exposed lysine on the plasma membrane and a KB tag.

[0014] FIGS. 2A-2B. Schematics of different approaches for cargo delivery. FIG. 2A. Lysine of the transferrin receptor protein 1 (TFRC) and HLA class I histocompatibility antigen A, B and C (HLA-ABC) on the extracellular surface react with the KB tagged drugs and peptides and the covalently bound cargo is taken up by the cell through clathrin-mediated endocytosis (CME) or clathrin-dependent endocytosis (CIE) followed by endosomal escape to biologically act. FIG. 2B. KB-antibody conjugates bind to TFRC or HLA-ABC and the targeted membrane protein and the KB tag mediates its endocytosis to be addressed to lysosomes for degradation.

[0015] FIG. 3. Cellular uptake of Boc-Fluorescein (FITC), AspA- FITC and ketoboronate (KB)-FITC derivatives. HeLa Kyoto cells were treated with 10 pM compounds for 1 hour. Fluorescence microscopy images were quantified (shown are raw values and SEM; n = 3).

[0016] FIG. 4. Quantification of the cellular uptake of KB-FITC 6 at 37°C or 4°C (shown are relative values and SEM; n = 3). HeLa cells were treated with 10 pM compounds (Boc-FITC or KBFITC) for 1 hour.

[0017] FIG. 5. Quantification of KB-FITC 6 (100 pM) uptake in HeLa Kyoto cells for 6 hours in the presence of albumin (40 mg / mL) (shown are raw values and SEM; n = 3).

[0018] FIG. 6. Quantification of KB-FITC 6 (10 pM) uptake in HeLa Kyoto cells for 1 hour in the absence and presence of various concentrations of serum (shown are relative values and SEM; n = 3).

[0019] FIG. 7. Quantification of Boc-FITC or KB-FITC (100 pM) uptake in HeLa Kyoto cells for 6 hours in the presence of albumin (40 mg / mL) (shown are relative values and SEM; n = 3).

[0020] FIG. 8. Cellular uptake of KB-FITC 6 in HeLa Kyoto cells overexpressing Arf6Q67L, a constitutively active form of Arf6 that is locked in its GTP-bound state, or pre-treated with 10pM of dyngo-4a for 30 minutes or 50 pM of LY294002 for 5 hours followed by treatment with 10 pM KB-FITC for 1 hour at 37°C.

[0021] FIG 9. HeLa cells were treated with IKK-NBD, KB conjugated-IKK-NBD, or Antp (antennapedia) conjugated-IKK-NBD for 3 hours, then stimulated with 10 ng / mL TNFa(±)for 30 minutes. Nuclear translocation of RELA by fluorescence microscopy was performed and quantified (shown are relative values and SD; n = 3, ***P<0.005 by two-sided Student's t-test).

[0022] FIG 10. Cellular uptake of KB-FITC-BLM-A512, and FITC- BLM-A5 11. HT-29 and HCT-116 cells were treated with 10 pM compounds for 6 hours. Fluorescence microscopy images were taken and quantified (shown are raw values and SEM; n = 3).

[0023] FIG 11. Cellular uptake of complex 15 and complex 16. HeLa Kyoto cells were treated with 5 pM compounds for 9 hours with 10% FCS or without FCS and then labeled with LYSOTRACKER® Green for 1 hour. Fluorescence microscopy images are taken and quantified (shown are raw values and SEM; n = 3).

[0024] FIG. 12. Quantification of western blot analysis of pHER2 Tyrl248, HER2, pERKl / 2 Tyr202 / Thr204, ERK, pAKT Ser473, AKT and [3-Tubulin upon 48-hour treatment of BT-474 cells with 30 nM IgG, 30 nM Trastuzumab (TTZ) or 30 nM KB-PEG15-TTZ.

[0025] FIG. 13. Cell viability of BT-474 cells treated with the IgG, TTX, KB-PEG15-TTZ or compound 9 at the indicated concentrations for 144 hours (shown are relative values and SD; n = 3).

[0026] FIG. 14. Cell viability of SK-BR-3 cells treated with the IgG, TTX, KB-PEG15-TTZ or compound 9 at the indicated concentrations for 144 hours (shown are relative values and SD; n = 3).

[0027] FIG. 15. Cell viability of KPL-4cells treated with the IgG, TTX, KB-PEG15-TTZ or compound 9 at the indicated concentrations for 144 hours (shown are relative values and SD; n = 3).

[0028] FIG 16. Quantification of HER2 recovery over time upon treatment with indicated compounds. BT-474 cells were reverse transfected with either mock or HER2 siRNA or treated with 100 nM of TTZ and KB-TTZ in full FCS-containing culture media. Cells were collected every day and day 1 corresponds to +1 day after the start of the transfection or the treatment. The medium was replaced at day 4 and day 8.

[0029] FIG 17. Measurement of BT-474 xenograft tumor volume upon treatment with indicated compounds at 3 mg / mL. BT-474 cells were injected into the mammary fat pad of NOG mice and tumors were allowed to grow for 70 days. Vehicle (PBS), TTZ (3 mg / kg), or KBTTZ (3 mg / kg) were injected intraperitoneal every 3-4 days (n - 3 mice per group).

[0030] FIG. 18. Quantification of OVCAR-3 cells that migrated during a 24-hour Transwell™ assay following treatment with PBS, Bevacizumab (Bev) or KB-PEG23-Bev (KB-Bev) for 48 hours (24 hours before and 24 hours during Transwell™ assay).

[0031] FIG. 19. Quantification of OVCAR-3 cells that migrated during a 24-hour Transwell™ assay following treatment with PBS, Bevacizumab (Bev) or KB-PEG23-Bev (KB-Bev) for 48 hours (24 hours before and 24 hours during Transwell™ assay with or without double knockdown of TFRC and HLA-ABC proteins).

[0032] FIG. 20. Quantification of OVCAR-3 cells that invaded through Matrigel™ during a 24-hour Transwell™ assay following treatment with PBS, Bevacizumab (Bev) or KB-PEG23-Bev (KB-Bev) for 48 hours (24 hours before and 24 hours during Transwell™ assay).DETAILED DESCRIPTION OF THE INVENTION

[0033] A covalent-reversible lysine-reactive tag, termed 2- ketoboronate (KB), is described, which enables superior uptake efficiency and cytosolic delivery of various protein-based therapeutics and for lysosomal delivery of endogenouslyexpressed plasma membrane and secreted proteins of interest for later degradation. The new uptake tag fulfills the unmet demand for a small, versatile cellular delivery technology for impermeable macromolecules. The KB tag was shown to provide a strong improvement in the cellular uptake of fluorescein (FITC)- conjugates in different cell lines. Combining biochemical and cell biology methods, the importance of the iminoboronate bond formation was established including its reversibility (FIG. 1). Further, it was determined that the transferrin receptor protein 1 (TFRC) and HLA class I histocompatibility antigen A, B and C (HLA-ABC) mediate the cellular uptake at the plasma membrane via a dual mechanism; binding and endocytosis of TFRC and HLA-ABC with a combination of CME and CIE pathways (FIG. 2A). In addition to delivery of FITC reagents, the inventive tag enabled superior uptake efficiency and cytosolic delivery of cytotoxic agents such as BLM-A5 and BH3 domain peptides. Moreover, it was demonstrated that a KB-IKK-NBD peptide conjugate could be delivered into the cytoplasm and act as a protein-protein interaction inhibitor against the activation and nuclear translocation of RELA. With streptavidin as model a protein, co- localization with the late endosomal and lysosomal protein LAMP1 was shown. KB-antibody conjugates were also produced that enabled internalization of endogenously expressed plasma membrane proteins, EGFR and HER2, and targeting of these proteins to lysosomes for degradation (FIG. 2B). Among the three HER2-positive cell lines tested (BT-474, SK-BR-3 and KPL-4), HER2 was efficiently degraded even in the trastuzumab-resistant KPL-4 cell line. Further, compared to antibody alone, the KB- antibody conjugate decreased cell viability of the three cell lines tested. Notably, HER2 was also shown to be degraded in vivo in a BT-474 tumor xenograft model with a significant effect on the tumor volumes compared to antibody alone and vehicle samples .

[0034] Accordingly, provided herein is a 2-ketoboronate compound of Formula I, pharmaceutically acceptable salts thereof, and constructs and methods of use of the same,whereinrepresents a phenyl or a 5-membered heteroaryl;L is absent or -0-;R1is selected from alkyl, haloalkyl, and a 5-membered heteroaryl; andR2is selected from hydrogen, alkyl, halo, and haloalkyl.

[0035] In some aspects, is phenyl. In other aspectsisa 5-membered heteroaryl. In one aspect, the 5-membered heteroaryl is pyrazolyl.

[0036] In some aspects, R1is methyl. In other aspects, R1is trifluoromethyl. In another aspect, R1is a 5-membered heteroaryl. In a further aspect, R1is thiophene.

[0037] In some aspects, R2is H. In other aspects, R2is F.

[0038] In some aspects, the 2-ketoboronate compound has the structure:where R is selected from alkyl, haloalkyl, and a 5-membered heteroaryl.

[0039] In some aspects, the 2-ketoboronate compound has the structure:

[0040] Terms used herein may be preceded and / or followed by a single dash, "-,"or a double dash, "=," to indicate the bond order of the bond between the named substituent and its parent moiety; a single dash indicates a single bond and a double dash indicates a double bond. In the absence of a single or double dash it is understood that a single bond is formed between the substituent and its parent moiety. Substituents are intended to be read "left to right" unless a dash indicates otherwise. For example, Ci-Cc-alkoxycarbonyloxy and -OC(O)Ci-Ce alkyl indicate the same functionality; similarly arylalkyl and -alkylaryl indicate the same functionality.

[0041] "Alkyl" means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms unless otherwise specified. The alkyl may be substituted with one or more groups selected from halo, hydroxy, alkoxy, cyano, amino, carboxyl and carboxyalkyl. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3- dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. In some aspects, "alkyl" refers to methyl.

[0042] "Halo" or "halogen" means -Cl, ~Br, -I, or -F. In some aspects, "halo" or "halogen" refers to -Cl or -F. In some aspects, "halo" or "halogen" refers to -F.

[0043] "Haloalkyl" means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl. In some aspects, each "haloalkyl" is a fluoroalkyl, for example, a polyfluoroalkyl such as a substantially perfluorinated alkyl. In some aspects, "haloalkyl" is trifluoromethyl.

[0044] "5-Membered heteroaryl" means a monocyclic 5-membered heteroaromatic ring. The 5-membered ring consists of two double bonds and one, two, three, or four nitrogen atoms, oxygen atoms, sulfur atoms, or a combination thereof. The 5-membered heteroaryl may be connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl. Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5~membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. In some aspects, the 5-membered heteroaryl group is pyrazolyl. In some aspects, the 5-membered heteroaryl group is thiophenyl .

[0045] "Saturated," as used herein, means the referenced chemical structure does not contain any multiple carbon-carbon bonds. For example, a saturated cycloalkyl group as defined herein includes cyclohexyl, cyclopropyl, and the like.

[0046] "Unsaturated" means the referenced chemical structure contains at least one multiple carbon-carbon bond, but is not aromatic. For example, a unsaturated cycloalkyl group as defined herein includes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like.

[0047] "Pharmaceutically acceptable salts" refers to salts or zwitterionic forms of the present compounds. Salts of the present compounds may be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation. The pharmaceutically acceptable salts of the present compounds can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, tartaric, and citric. Nonlimiting examples of salts of compounds of the disclosure include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the disclosure can bequaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds herein is intended to include the present compounds as well as pharmaceutically acceptable salts thereof.

[0048] In one aspect, a construct is provided, which comprises an agent covalently attached or linked to at least one 2- ketoboronate compound of Formula I. In aspects where the agent is a macromolecule, more than one 2-ketoboronate compound of Formula I may be covalently attached to the agent, e.g., 2, 3, 4, 5, 6 or more 2-ketoboronate compounds of Formula I. In aspects where more than one 2-ketoboronate compounds of Formula I is linked to an agent, the 2-ketoboronate compounds of Formula I may be the same or different.

[0049] In some aspects, an agent is covalently attached or linked directly to the 2-ketoboronate compound of Formula I. In some aspects, an agent is covalently attached or linked to the 2-ketoboronate compound of Formula I via at least one linker, e.g., 1, 2, 3, 4, 5, 6 or more linkers. In some aspects, when more than one linker is used, the linkers may be the same or different. In some aspects, the linker may have the structure:or wherein n may be in the range of 2 to 30, e.g., in the range of 2 to 24 or 4 to 23; and each of x and y may be in the range of 2 to 10, e.g., in the range of 3 to 10 or 3 to 8. In some aspects, x + y may be in the range of 4 to 16, e.g., in the range of 5 to 15 or 4 to 14. In some aspects, the linker may have one or more of the following structures:

[0050] Exemplary constructs may have a general structing including, for example:

[0051] In some aspects, the 2-ketoboronate compound of Formula I, linker, and agent by be covalently linked by click chemistry.

[0052] In some aspects, the agent is a small organic molecule, peptide, or protein. In some aspects, the agent is a non- therapeutic agent. In some aspects, the agent is a therapeutic agent. In some aspects, the agent is a selectable marker or an imaging marker of use in the diagnosis of a disease or condition. In some aspects, the agent is a non-therapeutic small organic molecule such as a label or dye, e.g., FITC. In some aspects, the agent is a non-therapeutic protein, e.g., a luminescent or fluorescent protein {e.g., green fluorescent protein). In some aspects, the agent is a therapeutic small organic molecule {e.g., a drug known for use in the prevention or treatment of a disease or condition) . In some aspects, the agent is a therapeutic peptide or protein, e.g., a cytokine, chemokine,growth factor, hormone, ligand, or antibody (e.g., IgG or IgA) known for use in the prevention or treatment of a disease or condition.

[0053] In another aspect is provided a method of facilitating, improving or increasing entry of an agent into a cell by contacting the cell with a construct comprising an agent covalently attached or linked to a 2-ketoboronate compound of Formula I. "Entry of an agent into a cell," refers to transport of the agent from the exterior of the cell (i.e., extracellular) into the interior of the cellfi.e., intracellular). In some aspects, entry of an agent into a cell is by endocytosis. In some aspects, entry of an agent into a cell is by clathrin- mediated endocytosis. In some aspects, entry of an agent into is by clathrin-independent endocytosis. In some aspects, entry of the agent is increased by at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 3.0-, 4.0-, 5.0-, 6.0- , 7.0-, 8.0-, 9.0-, or 10-fold or more as compared to the cell contacted with the same agent that is not covalently attached or linked to the 2-ketoboronate compound of Formula I.

[0054] In another aspect is provided a method of facilitating degradation of a plasma membrane protein or secreted by contacting the cell with a construct comprising an agent covalently attached or linked to a 2-ketoboronate compound of Formula I, wherein the agent of the construct is a ligand that binds the plasma membrane protein or secreted protein. In some aspects, degradation of the plasma membrane protein or secreted protein refers to lysosomal degradation of the plasma membrane protein or secreted protein. In some aspects, the ligand covalently linked to a 2-ketoboronate compound of Formula I binds to the plasma membrane protein or secreted protein and the 2-ketoboronate directs uptake and lysosomal degradation of the plasma membrane protein or secreted protein. In some aspects, degradation of a plasma membrane protein or secreted protein isincreased by at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 3.0-, 4.0-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0- , or 10-fold or more as compared to the cell contacted with the same ligand that is not covalently attached or linked to the 2- ketoboronate compound of Formula I.

[0055] Examples of ligands that may be covalently attached or linked to the 2-ketoboronate compound of Formula I include those that bind to plasma membrane receptors for growth factors such as Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Tumor Necrosis Factor alpha (TNF-a), Transforming growth factor beta (TGF-pi), Fibroblast Growth Factor 1 (FGF1), Fibroblast Growth Factor 2 (FGF2); plasma membrane receptors for cytokines such as Interferon gamma (IFN-y) and interleukins (IL), e.g., IL-1, IL-2, IL-6, IL-5, IL-8, IL-10, IL-21, and IL- 22; and plasma membrane proteins such as Carboxypeptidase M, members of the IKB kinase (IKK) complex, insulin-like growth factor 2 receptor, and matrix metalloproteinase-1. A ligand may be, e.g., a small organic molecule, peptide, or protein, e.g., antibody that binds to the plasma membrane protein.

[0056] Examples of ligands that may be covalently attached or linked to the 2-ketoboronate compound of Formula I include, but are not limited to, a PDL1 antibody, HER2 antibody, platelet- derived growth factor antibody, protein tyrosine kinase 7 antibody, mesenchymal epithelial transition factor antibody, G protein coupled receptor 65 antibody, CD47 antibody, CD71 antibody, EGFR antibody, VEGFA antibody and ABCG2 antibody.

[0057] Exemplary ligands include that may be covalently attached or linked to the 2-ketoboronate compound of Formula I include, but are not limited to, Abciximab, Adalimumab, Aducanumab, Alemtuzumab, Alirocumab, Amivantamab, Anifrolumab, Ansuvimab, Anrukinzumab, Apolizumab, Aprutumab ixadotin, Ascrinvacumab, Atezolizumab, Avelumab, Avelumab, Axatilimab, Bamlanivimab, Basiliximab, Bedinvetmab, Belimumab,Benralizumab, Bevacizumab, Bimekizumab, Brentuximab vedotin, Brodalumab, Brolucizumab, Burosumab, Canakinumab, Caplacizumab, Capromab, Catumaxomab, Cemiplimab, Certolizumab pego, Cetuximab, Concizumab, Cosibelimab, Crizanlizumab, Crovalimab, Daclizumab, Daratumumab, Denosumab, Dinutuximab (beta), Divozilimab, Domagrozumab, Donanemab, Dostarlimab, Dupilumab, Durvalumab, Eculizumab, Elotuzumab, Emapalumab, Emicizumab, Enfortumab vedotin, Epcoritamab, Eptinezumab, Erenumab, Ertumaxomab, Etaracizumab, Evinacumab, Evolocumab, Faricimab, Fremanezumab, Frunevetmab, Galcanezumab, Garadacimab, Gemtuzumab, Glofitamab, Golimumab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Idarucizumab, Inebilizumab, Infliximab, Inotuzumab, Ipilimumab, Isatuximab, Isatuximab, Lanadelumab, Lecanemab, Loncastuximab tesirine, Lokivetmab, Margetuximab, Marstacimab, Mepolizumab, Mirikizumab; Mirvetuximab, Mogamulizumab, Mosunetuzumab, Moxetumomab, Natalizumab, Naxitamab, Necitumumab, Netakimab, Nimotuzumab, Nivolumab, Obiltoxaximab, Obinutuzumab, Ocrelizumab, Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab, Pozelimab, Polatuzumab, Racotumomab, Ramucirumab, Raxibacumab, Ravulizumab, Regdanvimab, Reslizumab, Retifanlimab, Risankizumab, Rituximab, Romosozumab, Rovelizumab, Rozanolixizumab, Ruplizumab, Sacituzumab, Sarilumab, Satralizumab, Secukinumab, Siltuximab, Sipavibart, Sotrovimab, Spesolimab, Sutimlimab, Tafasitamab, Talquetamab, Tarlatamab, Teclistamab, Teplizumab, Teprotumumab, Tezepelumab, Tildrakizumab, Tisotumab, Tocilizumab, Tositumomab, Tralokinumab, Trastuzumab, Tremelimumab, Ublituximab, □stekinumab, Vedolizumab, Vilobelimab, Zanidatamab, Zenocutuzumab, and Zolbetuximab.

[0058] In some aspects is provided a method of preventing or treating a disease or condition by administering to a subject in need thereof an effective amount of a construct comprisingan agent covalently attached or linked to a 2-ketoboronate compound of Formula I, wherein the agent of the construct is a therapeutic agent for preventing or treating the disease or condition. Diseases or conditions that may be prevented or treated in accordance with the method therein include, but are not limited to, inflammatory diseases or conditions (e.g., rheumatoid arthritis, asthma), cancer (e.g., renal cell carcinoma, large-cell lung carcinoma, ovarian cancer, breast cancer, prostate cancer, melanoma, osteosarcoma, thyroid cancer), autoimmune conditions (e.g., psoriasis, alopecia areata, vitiligo, Sjogren's syndrome, systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, inflammatory bowel disease), cardiovascular diseases (e.g., atherosclerosis), neurodegenerative diseases (e.g., Alzheimer's disease, Huntington's disease) and other conditions such as chronic obstructive pulmonary disease, acute lung injury, and cystic fibrosis.

[0059] In some aspects, the construct is administered in the form of a pharmaceutical composition comprising the construct in combination with a pharmaceutically acceptable carrier. '''Pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner. "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, latest edition. For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used.

[0060] "Treating" or "treatment" includes the treatment of a disease or condition described herein, in a subject, preferably a human, and includes inhibiting a disease or condition, i.e.farresting its development; relieving a disease or condition, i.e., causing regression of the disorder; slowing progression of the disease or condition; and / or inhibiting, relieving, or slowing the onset or progression of one or more symptoms of the disease or condition.

[0061] "Prevent" or "preventing" refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0062] "Subject" refers to a warm blooded animal such as a mammal, preferably a human, or a human child, which is afflicted with, or has the potential to be afflicted with one or more diseases and conditions described herein.

[0063] "Effective amount" refers to the amount of a disclosed construct or pharmaceutical composition comprising the same that is nontoxic and sufficient to effect beneficial or desired biological, emotional, medical, or clinical response of a cell, tissue, system, animal, or human. This may include a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

[0064] An effective amount may be administered in one or more administrations, applications, or dosages. The term may also include within its scope amounts effective to enhance or restore to substantially normal physiological function. It is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective dailydose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. The dosage may be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a subject may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0065] A response to an effective amount of a disclosed construct and / or pharmaceutical composition, for example, may be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and may be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed construct and / or pharmaceutical composition, by changing the disclosed construct and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products .

[0066] In some aspects, a pharmaceutical composition is provided comprising an effective amount of one or more constructs described herein and one or more pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers,adjuvants, excipients, or carriers. The pharmaceutical composition may be used, for example, for treating diseases or conditions. Suitable carriers for non-liquid formulations are also known to those of skill in the art. A thorough discussion of pharmaceutically acceptable excipients and salts is available in Remington's Pharmaceutical Sciences, latest edition. Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, may be present in such vehicles. A biological buffer may be any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank's buffered saline, and the like.

[0067] Depending on the intended mode of administration, the pharmaceutical compositions may be in the form of solid, semi- solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include an effective amount of the selected construct in combination with a pharmaceutically acceptable carrier and, in addition, may include other pharmaceutical agents, adjuvants, diluents, buffers, and the like .

[0068] In general, the compositions of the disclosure may be administered in any effective amount by any of the accepted modes of administration. Suitable dosage ranges depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, the indication towards which the administration is directed, and the preferences and experience of the medical practitioner involved.One of ordinary skill in the art of treating such diseases will be able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this application, to ascertain an effective amount of the compositions of the disclosure for a given disease.

[0069] Thus, the compositions of the disclosure may be administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.

[0070] For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, and the like, an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, referenced above.

[0071] Yet another aspect is the use of permeation enhancer excipients including polymers such as: polycations (chitosan and its quaternary ammonium derivatives, poly-L-arginine, aminatedgelatin); polyanions (N-carboxymethyl chitosan, poly-acrylic acid); and thiolated polymers (carboxymethyl cellulose- cysteine, polycarbophil-cysteine, chitosan-thiobutylamidine, chitosanthioglycolic acid, chitosan-glutathione conjugates).

[0072] For oral administration, the composition may generally take the form of a tablet, capsule, a softgel capsule or may be an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules are preferred oral administration forms. Tablets and capsules for oral use can include one or more commonly used carriers such as lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. Typically, the compositions of the disclosure can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents may also be incorporated into the mixture.

[0073] Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0074] When liquid suspensions are used, the construct may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like and with emulsifying and suspending agents. If desired, flavoring, coloring and / or sweetening agents can be added as well. Other optional components for incorporation into an oral formulationherein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like.

[0075] Parenteral formulations may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solubilization or suspension in liquid prior to injection, or as emulsions. Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.

[0076] Parenteral administration includes intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and include aqueous and non-aqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Administration via certain parenteral routes can involve introducing the formulations of the disclosure into the body of a patient through a needle or a catheter, propelled by a sterile syringe or some other mechanical device such as a continuous infusion system. A formulation provided by the disclosure may be administered usinga syringe, injector, pump, or any other device recognized in the art for parenteral administration.

[0077] Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration may involve the use of a slow release or sustained release system such that a constant level of dosage is maintained .

[0078] Preparations according to the disclosure for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms can also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They can be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.

[0079] Sterile injectable solutions may be prepared by incorporating one or more of the constructs herein in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions may be preparedby incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Thus, for example, a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

[0080] Alternatively, the pharmaceutical compositions of the disclosure may be administered in the form of suppositories for rectal administration. These may be prepared by mixing the agent with a suitable nonirritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0081] Preferred formulations for topical drug delivery may be ointments and creams. Ointments are semisolid preparations which are typically based on petrolatum or other petroleum derivatives. Creams containing the selected construct are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. The specific ointment or cream base to be used, aswill be appreciated by those skilled in the art, is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing.

[0082] Formulations for buccal administration include tablets, lozenges, gels and the like. Alternatively, buccal administration can be effected using a transmucosal delivery system as known to those skilled in the art. The compounds of the disclosure can also be delivered through the skin or mucosal tissue using conventional transdermal drug delivery systems, i.e., transdermal "patches" wherein the agent is typically contained within a laminated structure that serves as a drug delivery device to be affixed to the body surface. In such a structure, the drug composition is typically contained in a layer, or "reservoir," underlying an upper backing layer. The laminated device can contain a single reservoir, or it can contain multiple reservoirs. In one aspect, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, can be either a polymeric matrix as described above, or it can be a liquid or gel reservoir, or can take some other form. The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing layer should be substantiallyimpermeable to the active agent and any other materials that are present.

[0083] The pharmaceutical compositions of the disclosure may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, propellants such as fluorocarbons or nitrogen, and / or other conventional solubilizing or dispersing agents.

[0084] The compositions of the disclosure may be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other conventional solubilizing or dispersing agents. The construct may generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFG) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol conveniently may also contain a surfactant such as lecithin. The dose of drug may be controlled by a metered valve. Alternatively, the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition can be presented inunit dose form for example in capsules or cartridges of, e.g.rgelatin or blister packs from which the powder can be administered by means of an inhaler.

[0085] The disclosed compounds, constructs and / or pharmaceutical compositions comprising the compounds or constructs may conveniently be presented as a kit, whereby two or more components, which may be active or inactive ingredients, carriers, diluents, and the like, are provided with instructions for preparation of the actual dosage form by the patient or person administering the drug to the patient. Such kits may be provided with all necessary materials and ingredients contained therein, or they may contain instructions for using or making materials or components that must be obtained independently by the patient or person administering the drug to the patient.

[0086] In further aspects, a kit may include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, a kit may contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple patients ("bulk packaging"). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like .

[0087] In a further aspect, the disclosed kits may be packaged in a daily dosing regimen (e.g., packaged on cards, packaged with dosing cards, packaged on blisters or blow-molded plastics, etc.). Such packaging promotes products and increases patient compliance with drug regimens. Such packaging can also reducepatient confusion. The present invention also features such kits may further containing instructions for use.

[0088] In a further aspect, the present disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein. Associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0089] In various aspects, the disclosed kits may also comprise compounds and / or products co-packaged, co-formulated, and / or co- delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and / or product and another component for delivery to a patient.

[0090] It is contemplated that the disclosed kits may be used in connection with the disclosed methods of making, the disclosed methods of using or treating, and / or the disclosed compositions .

[0091] The following non-limiting examples are provided to further illustrate the present invention.Example 1: Cellular uptake screen of KB probes

[0092] To identify a new tag for cellular uptake, screen of cellular uptake was performed using Boc-FITC 1 and AspA-FITC 2 as negative and positive controls, respectively.

[0093] A series of KB tags were prepared as shown in Schemes 1- .N-phenyl-bisSCHEME 1SCHEME 25,60%SCHEME 36,85%SCHEME 4SCHEME 5

[0094] The KB tags were FITC labeled as shown in Scheme 6.SCHEME 6

[0095] The screen of cellular uptake was performed in a HeLa Kyoto cell line at 37°C, with a one hour treatment. As expected, no significant uptake was detected with the tagless derivative, Boc-FITC, while the KB-conjugate derivatives demonstrated various levels of cellular uptake, including a strong effect forKB tags 3, 4 and 6, which exceeded that AspA-FITC (FIG. 3). After quantification, KB tags 3 and 4 showed similar uptake and the parafluoro containing KB tag 6, was identified as the best tag with a 3.5-fold increase in quantified FITC signal compared to the AspA-FITC. Notably, the cellular uptake observed in HeLa Kyoto cells was reproducible in three additional cell lines, i.e., the invasive breast carcinoma cell line MCF7, the epithelial colorectal adenocarcinoma cell line Caco-2, and the mouse embryonic fibroblast cell line NIH3T3. Accordingly, KB tag 6 was selected as the KB tag scaffold for further analysis.

[0096] Various environmental conditions were examined to determine whether they had any effect on cellular uptake of KB tag 6 labeled with FITC (KB-FITC 6) including temperature (FIG. 4), albumin (FIG. 5), and serum (FIGS. 6-7).Example 2: Evaluation of KB-FITC structure mediating cellular uptake

[0097] To investigate the nature of the iminoboronate formation that mediates the cellular uptake, three new reagents were

[0098] Compounds 8 and 10 were synthesized as shown in Schemes7 and 8, respectively.SCHEME 8

[0099] Probe 8 was designed without the ketone which is mechanistically essential for the reaction with primary amines to form the iminoboronate and to confirm the lack of cellular uptake without ketone. Compound 9 should be a direct and reversible competitor for lysines engaged by the tag due to theKB moiety. The sulfotetrafluorophenyl reagent 10 is a cell- impermeable, lysine-reactive probe that was designed based on a known lysine-specific chemoproteomic probe. Commercially available phenylboronic acid (PBA) was used as control to show that the cellular uptake relies on the iminoboronate formation and not on the boronate ester formation with glycans on the cell surface. Indeed, boronic acids are known to bind diols. HeLa Kyoto cells were treated with 10 pM compounds for 1 hour or co- treated with 10 pM KB-FITC and competitor and incubated for 1 hour.

[0100] This analysis indicated that treatment of HeLa Kyoto cells with the probe 8 showed almost no signal (< 3% based on FITC signal of the KB tag 6). HeLa Kyoto cells were co-treated with a 100-fold excess of various compounds prior to incubation with KB-FITC. Co-treatment of live HeLa Kyoto cells with either 9 or 10 showed -75% reduction in the cellular uptake of KB-FITC. Treatment with PBA or removal of N-linked glycans by PNGase F treatment did not result in a decreased internalization of KB- FITC. These data confirmed the lysine-driven cellular uptake of the KB tag.Example 3: Proteins Mediating Cellular Uptake of KB-FITC

[0101] To identify the proteins involved in the uptake of the probes, a classical chemical proteomics strategy was used. The identified in situ proteomic targets of KB-FITC were filtered using three selection parameters; (i) intensity > 5-fold in control-treated sample (Boc-FITC), (ii) plasma membrane localization annotated by GO term, and (iii) identified in proteomes from both tested cell lines. This selection resulted in a short list of only two proteins: transferrin receptor protein 1 (TFRC) and HLA class I histocompatibility antigen A, B and C (HLA-A, HLC-B and HLA-C). TFRC is a highly abundant transmembrane glycoprotein responsible for the import oftransferrin-bound iron into cells by clathrin-mediated endocytosis (CME). This efficient cellular uptake pathway has been extensively studied for drug delivery purposes. HLA-A, HLA- B and HLA-C (HLA-ABC) are transmembrane proteins that are part of the major histocompatibility complex I (MHC-I) responsible for presenting peptide fragments to cytotoxic T cells. HLA-ABC are known to shuttle between the plasma membrane and endosomes via clathrin-independent endocytosis (CIE). To confirm the observed binding to TFRC and HLA-ABC, TFRC-V5 and HLA-A-V5 were overexpressed in HeLa Kyoto cells, the cells were lysed, and lysates were treated with KB-FITC and Boc-FITC. Subsequently, the lysates were reduced with NaBHaCN, derivatized by CuAAC with biotin azide, enriched over streptavidin beads, eluted, and analyzed by western blot. The membrane images showed labeling of both overexpressed proteins by KB-FITC but not by Boc-FITC.

[0102] To understand the contribution of TFRC and HLA-ABC in the uptake of KB-FITC 6, these proteins were knocked down using siRNA. Indeed, the TFRC knockdown led to a ~50% decrease and the HLA-ABC knockdown led to a ~40% decrease for KB-FITC 6 compared to mock cells (P<0.005 by two sided Student's t-test). Moreover, the knockdown of both proteins led to a ~75% decrease for KB- FITC 6 (P<0.005 by two sided Student's t-test). Conversely, transient overexpression of TFRC-mCherry in HeLa Kyoto cells resulted in an increase in fluorescent punctae, presumably representing endosomal vesicles filled with KB-FITC 6. Likewise, a similar increase in punctae was observed with KB-FITC 6 upon overexpression of HLA-A-mCherry.

[0103] To further assess the contribution of HLA-ABC and TFRC in the uptake of KB-FITC 6, cells were cotreated with KB-FITC 6 and an anti-HLA-ABC antibody or an anti-TFRC antibody. Treatment of cells with either antibody resulted in a ~35% decrease in uptake of KB-FITC 6 as measured by fluorescent microscopy (P<0.005 by two-sided Student's t-test). When the cells weretreated with both the anti-TFRC antibody and anti-HLA-ABC antibody, an even greater reduction, -55% decrease, in fluorescent intensity due to KB-FITC 6 uptake was observed (P<0.005 by two-sided Student's t-test). No reduction in FITC signal intensity was observed in cells treated with an anti- rabbit IgG control antibody.

[0104] Target engagement of the KB tag 6 for TFRC and HLA-ABC was subsequently analyzed using a competitive pulldown assay. This analysis revealed a micromolar range occupancy of the KB tag 6 with EC$o of 7.4 pM and 20.2 pM for TFRC and HLA-A, respectively. To assess the reversibility of the binding of KB- FITC with its targets, the pH was modified after the interaction to mimic the endosomal pH that would occur during the trafficking process and the possible release of KB-FITC 6. The pH was changed before the enrichment over streptavidin beads. This analysis showed a pH-dependent decrease of the pull-downed TFRC-V5 and HLA-A-V5 proteins (Table 1), validating the covalent-reversible nature of the interaction with KB-FITC 6 that is emphasized in low pH conditions. More generally these data demonstrate the large contribution of TFRC and HLA endocytosis pathways to mediate an efficient cellular uptake and delivery into the cytoplasm.TABLE 1

[0105] To identify a binding site of the KB tag 6 on the TFRC protein, a classical chemical proteomics site identification pull-down assay was used. TFRC-V5 was overexpressed, soluble and pellet fractions were separated using ultracentrifugation thereby enriching TFRC-V5 in the pellet fraction, and the pelletfraction was treated with a desthiobiotin reagent bearing the KB tag 6, which was prepared as shown in Scheme 9.SCHEME 9

[0106] This analysis identified lysine K193 as the binding site. Thus, this site was mutated to an alanine, which reduced the ability to pull-down the TFRC protein (loss of about 70% of binding capacity) with the pull-down assay followed by western blot analysis. Collectively, these results demonstrate that the KB-mediated cellular uptake relies on two endocytic pathways through TFRC and HLA-ABC.

[0107] To further confirm the KB-FITC 6 uptake via CME and CIE, a series of imaging experiments with KB-FITC 6 was performed. HLA proteins have been shown to internalize via the GTPase Arf6- mediated pathway and are internalized into vesicles that then either fuse with or mature into Rab5-associated endosomes. Thus, Arf6Q67L, a constitutively active form of Arf6 that is locked in its GTP-bound state, was transiently overexpressed and a global and more diffuse uptake of KB-FITC 6 with some vacuolar-type structures was observed. This mutant is known to lead to a stimulation of the CIE, resulting in an enhanced trafficking of HLA. HeLa Kyoto were also treated with LY294002, a small moleculePI3K inhibitor and subsequently treated with KB--FITC 6. Inhibition of Arf6 activation by LY294002 significantly reduced the cellular uptake of KB-FITC 6. TFRC-mediated uptake via CME and HLA-mediated uptake via CIE can be dynamin-dependent (dynamin 1 and / or 2). Using dyngo-4a, a more potent small molecule derivative of dynasore, inhibitor of dynamin, it was shown that KB-FITC 6 internalization could be further inhibited (FIG. 8).Example 4: Conjugation of apoptotic BH3 peptides to KB tag leads to cytotoxicity in cancer cells

[0108] It was determined whether unprotected and unfunctionalized BH3 peptides, Bak BH3 (GQVGRQLAIIGDDINR; SEQ ID N0:l) and Bim BH3 (MRPEIWIAQELRRIGDEFNA; SEQ ID NO:2) could be delivered to cells by appending KB tag 6 thereto. The resulting constructs, referred to as KB-Bak BH3 and KB-Bim BH3, were prepared by standard peptide coupling of the KB tag 6 to the N-terminus of the peptides. For comparison, AspA-Bak BH3 and AspA-Bim BH3 peptides were also prepared. The ability of these conjugates to induce apoptosis in HeLa cells was determined. HeLa cells were treated 24 hours with compound 9, free asparagusic acid, Bak BH3 peptide, Bim BH3 peptide, AspA- conjugated Bak and Bim BH3 peptides, KB-Bak BH3 peptide, KB-Bim BH3 peptide, or staurosporine (STS) as a positive control. Annexin V staining was performed to detect cells that had externalized phosphatidylserine, a signature of apoptosis. Strong annexin V staining was observed in HeLa cells treated with the AspA- and KB-tagged peptides, but not in cells treated with the untagged BH3 peptides, compound 9, or free asparagusic acid. Thus, even peptides as long as 20 amino acid residues could be internalized into cells using KB tag 6.

[0109] Viability curves of HeLa and Caco-2 cells treated with each of the above-reference compounds at various concentrationswere assessed for 36 hours. Both AspA-tagged and KB-tagged BH3 peptides exhibited strong cytotoxicity at much lower concentrations in Caco-2 cells than HeLa cells. However, the KB- tagged peptides displayed a significantly higher cytotoxicity compared to their AspA counterpart (Table 2).TABLE 2

[0110] In view of the foregoing, a construct such as KB-Bak BH3 and / or KB-Bim BH3 may be of particular use in the treatment of inflammatory conditions that BH3 mimetics are known to treat, e.g., malignancies such as chronic lymphocytic leukemia.Example 5: KB-IKK-NBD conjugate inhibits the activation and nuclear translocation of RELA

[0111] It was subsequently determined whether protein-protein interaction inhibitors could be delivered to cells using KB tag 6. Accordingly, KB tag 6 was covalently linked to the N-terminus of the IKK NEMO Binding Domain (IKK-NBD) peptide (TALDWSWLQTE; SEQ ID NO:3), which comprises residues 735-745 of IKK(3 and is known to block the interaction between NEMO (an IKB complex regulatory protein) with the IKB kinase complex in cells thereby inhibiting the NF-KB pathway.

[0112] Uptake and biological activity of this peptide was assessed using electrophoretic mobility shift assay (EMSA) based on the nuclear translocation and DNA binding of RELA (NF-KB p65; Tan et al. (2015) Sci. Rep. 10(5):12116; Chen (2016) Cold Spring harb. Perspect. Med. 6(3):a026104). For this analysis, HeLa cells were treated with IKK-NBD peptide, compound 9, DMSO, KB tag 6 conjugated-IKK-NBD, or Antp (antennapedia,DRQIKIWFQNRRMKWKK; SEQ ID NO:4) conjugated-IKK-NBD for 3 hours, then stimulated with 10 ng / mL TNFot(±)for 30 minutes. Nuclear extracts were prepared, incubated with a double-stranded DNA oligonucleotide (5'-AGAGGGGACTTTCCGAGG-3'; SEQ ID NO:5) conjugated to ALEXA FLUOR® 488, separated by native-PAGE and examined for fluorescence. In control cells, EMSA showed the expected binding of RELA to its fluorescent DNA oligomeric substrate upon stimulation with TNFa (Table 3). However, cells treated with KB tag 6 conjugate showed a concentration-dependent decrease in RELA-nuclear translocation and DNA binding activity. Remarkably, the KB-IKK-NBD conjugate inhibited the nuclear translocation of RELA more potently than the commonly used cell- penetrating Antp peptide conjugated to IKK-NBD. Free KB tag 9 and the unconjugated IKK-NBD peptide did not show any inhibition of the nuclear translocation. These results were also confirmed by fluorescence microscopy, RELA immunostaining and quantification (FIG. 9). Altogether, these data further demonstrate the advantages of the KB tag compared to established cellular uptake technologies.TABLE 3*Relative values; n = 3

[0113] In view of the foregoing, a construct such as KB-IKK-NBD may be of particular use in the treatment of inflammatory conditions that IKK-NBD is known to treat, e.g., inflammation in the lungs, ear inflammation, and neurological deficits caused by cerebral ischemia.Example 6: Conjugation of BLM-A5 to KB tag leads to improved cellular uptake and cytotoxicity in cancer cells

[0114] Bleomycin A5 (BLM-A5) is part of the BLM antitumor antibiotics family produced by the bacteria Streptomyces verticillus . Bleomycins induce DNA single- and double-strand breaks and are clinically used to treat lymphomas, testicular, ovarian, and cervical cancers, typically in combination with other chemotherapy drugs. Unfortunately, BLMs cannot be administered orally due to their poor colon membrane permeability. It was hypothesized that attaching a KB tag would greatly improve the membrane permeability and thus the cellular uptake of BLM-A5. Thus, the FITC- 11 and KB-FITC-conjugated 12 derivatives of BLM-A5 were prepared and tested in two different colon cancer cell lines for cellular uptake and BLM-A5 and KB tagged BLM-A5 13 were prepared and tested in the same colon cancer cell lines for altering cell viability. For viability, HT-29 and HCT116 cells treated with BLM-A5, compound 9, or KB- BLM-A5 13 for 48 hours (n = 3).

[0115] A significantly stronger uptake of KB-FITC-BLM-A512 was observed in both HCT 116 and HT-29 cancer cells (FIG. 10). In addition, 5.6-fold and 3.5-fold stronger cytotoxicity was observed for KB-BLM-A5 13 in HCT 116 and HT-29 cells, respectively, as compared to BLM-A5 (Table 4). No loss in viability was observed in cells treated with compound 9.TABLE 4

[0116] In view of the foregoing, a construct such as KB-BLM-A5 may be of particular use in the treatment of cancers that Bleomycin A5 is known to treat, e.g., lymphomas, squamous cell carcinomas, and testicular cancer.Example 7: Example of protein cargo delivery

[0117] Cellular uptake of a larger molecule was subsequently assessed. In this analysis, the cellular uptake of KB taggedtetravalent streptavidin (Sav), a ~60 kDa protein, was determined. A tetramethylrhodamine (TMR)-conjugated Sav was prepared. Tetrameric protein complexes were assembled. One tetrameric complex, complex 15, was composed of TMR-conjugated Sav without a KB tag. A second tetrameric complex, complex 16, was prepared by mixing imino-protected TMR-conjugated Sav with biotinylated KB tag 14 in a 1:4 ratio. The biotinylated KB tag 14, prepared as shown in Scheme 10, enabled cellular uptake of TMR-conjugated Sav both in the presence or absence of 10% serum(FIG. 11).SCHEME 10

[0118] Similar to what was seen in the FITC studies herein, fluorescent puncta were observed. Accordingly, a lysosomal dye, LysoTracker™, was used to see if the internalized proteincomplex 16 was accumulating in lysosomes and co-localizing with the lysosomal dye. Indeed, the fluorescent signals of the LysoTracker™ and the TMR from complex 16 were colocalized, indicating that KB-protein conjugates are being taken up by the cell and targeted to lysosomes.Example 8: KB-mediated degradation of Epidermal Growth Factor (EGFR)

[0119] KB tag 6 was also conjugated to Cetuximab (CTX), a Food and Drug Administration (FDA)-approved monoclonal antibody against EGFR. KB-Antibody conjugate 17 was prepared by modifying free lysines of the antibody using NHS-TCO (trans-cyclooctene) in PBS (step (i), Scheme 12). The TCO-antibody was then subjected to tetrazine-TCO ligation in PBS (step (ii), Scheme 12) to conjugate and add the KB tag with PEG linkers of different lengths 15 (n=9), 16 (n=15) or 17 (n=23) (Scheme 11).SCHEME 11SCHEME 12

[0120] PEG linkers of different length (9, 15 or 23) were screened for optimal EGER degradation. The resulting KB- conjugated antibodies were referred to as KB-PEG9-CTX, KB-PEG15- CTX and KB-PEG23-CTX to reflect the different PEG linkers. The epidermoid carcinoma A431 cell line, which is known to express high levels of EGFR, was treated with a control immunoglobulin (IgG), epidermal growth factor (EGF), unconjugated CTX and KB- conjugated CTX with different PEG linkers for 48 hours in 10% FCS-containing culture medium. Degradation of EGFR was observed by western blot analysis with all three PEG linkers with the KB- PEG15-CTX showing the best activity (Table 5). Indeed, 66% of EGFR was degraded with 10 nM KB-PEG15-CTX, and 100 nM KB-PEG15- CTX achieved >80% degradation. CTX treatment alone did not degrade EGFR.TABLE 5

[0121] EGFR is targeted to the lysosome as observed by imaging and it co-localizes with the late endosomal and lysosomal protein LAMP1. EGFR co-localized with LAMPl-positive compartments following a 6-hour treatment with 30 nM KB-PEG15- CTX. Longer incubation times (24 hours and 48 hours) resulted in a marked decrease in EGFR staining both at the plasma membrane and in endocytic vesicles indicating successful degradation. These results demonstrate the use of KB-antibody conjugates as a new tool for mediating lysosomal protein degradation. In addition, a construct such as KB-PEG15-CTX may be of particular use in the treatment of cancers that Cetuximab is known to treat, e.g., bowel cancer and head and neck cancers.Example 9: KB-mediated degradation of HER2

[0122] Using the same approach in described in Example 8, KB tag 6 was covalently linked to Trastuzumab (TTZ). Degradation of Her2 was analyzed in three HER2-positive breast cancer cell lines which are commonly used as models for TTZ treatment: BT- 474 and SK-BR-3 (trastuzumab-sensitive cell lines) and KPL-4 (a trastuzumab-resistant cell line) . The three different constructs, KB-PEG9-TTZ, KB-PEG15-TTZ, and KB-PEG23-TTZ, were screened for optimal HER2 degradation. Cells were treated with a control IgG, unconjugated TTZ or with 10 nM KB-TTZ and 100 nM KB-TTZ for each PEG linker for 48 hours in 10% FCS-containing culture media. The highest degradation activity of HER2 wasobserved with PEG15 (Table 6). Indeed, >70% of HER2 was degraded with 10 nM KB-TTZ, and 100 nM KB-TTZ achieved >85% degradation in BT-474 cells. Similarly, KB-PEG15-TTZ at 100 nM achieved 80% and 90% degradation of HER2 in SK-BR-3 and KPL-4 cells, respectively. TTZ treatment alone did not degrade HER2. These results were further confirmed via immunofluorescence microscopy in all three cell lines where it was observed that HER2 was depleted from the plasma membrane over time, re-localized to LAMPl-positive vesicles with a clear co-localization of LAMP1 / HER2 staining after a 6-hour treatment. The HER2 signal for IgG and TTZ treated cells was not affected after 48-hour treatment .TABLE 6

[0123] Encouraging, HER2 degradation was observed in the presence of 10% FCS as well as 40 mg / mL albumin (Table 7), thereby demonstrating the applicability of the KB conjugate for targeted degradation of a POI in vivo. In particular, HER2 expression levels in BT-474, SK-BR-3, and KPL-4 cells was determined upon treatment with IgG, TTZ, or KB-PEG15-TTZ for 48 hours in media containing 10% FCS or 10% FCS with 40 mg / mL albumin (Full FCS). Greater than 60% of the activity was retained in these cells. One suggested mechanism of resistance of KPL-4 is the overexpression of p95HER2, a truncated version of HER2 that is missing the extracellular domain but maintains kinase activity. However, these cells still express HER2 full-length, which was degraded via targeting with KB-antibody conjugates.TABLE 7^relative values; n = 3.

[0124] The degradation of HER2 through the lysosome degradation pathway was further confirmed using chloroquine, a known lysosome inhibitor. Cells were treated with indicated concentrations of TTZ or PEG15 KB-TTZ for 48 hours in presenceor absence of chloroquine in 10% FCS-containing culture media. Indeed, treatment with KB-TTZ in presence of chloroquine prevented HER2 degradation in BT-474, SK-BR-3 and KPL-4 cell lines (Table 8).TABLE 8

[0125] To fully characterize the relation between TFRC / HLA-ABC and HER2 degradation, an immunofluorescence study in BT-474 cells was performed to analyze HER2 degradation in cells treated with TFRC and HLA-ABC siRNA. As observed with the uptake of KB- FITC in knock-down conditions, here, HER2 levels were differently affected depending on the silenced targets after KB- TTZ treatment. HER2 degradation was clearly observed in the mock siRNA treated sample. In comparison, HLA-ABC siRNA showed a greater level of HER2 degradation, followed by TFRC siRNA, and the double knock-down showed nearly an identical level of HER2 as that of the IgG or TTZ controls. These results could indicate a higher involvement of TFRC in cellular uptake of HER2. HER2 levels by western blot analysis further characterized therelation between TFRC / HLA-ABC and HER2 degradation using KB- PEG15-TTZ. Western blot analysis showed that silencing of TFRC, HLA-ABC, or both simultaneously does not affect HER2 levels.

[0126] It was further examined whether the observed degradation of HER2 using KB-TTZ decreases HER2 activation and signaling in BT-474 cells. For this analysis, the phosphorylation status of HER2 (Tyrl248) and downstream proteins involved in HER2 signaling were analyzed. These downstream proteins included extracellular signal-regulated kinase (ERK) 1 (Thr202) and ERK2 (Tyr204) and RAC-alpha serine / threonine-protein kinase (AKT1) (Ser473). TTZ alone lead to a decrease in phosphorylation of HER2, ERK1 / 2 and AKT (FIG. 12). Notably, treatment with KB- PEG15-TTZ further decreased HER2 signaling as a result of a combined effect of receptor binding and degradation. Taken together, these results indicate that treatment with KB-TTZ allowed for efficient HER2 degradation.

[0127] Cell viability was also analyzed in each of the BT-474, SK-BR-3 and KPL-4 cell lines. KB-PEG15-TTZ treatment of BT-474 cells induced a shift in the inhibition of cell viability compared to TTZ. A plateau of 60% cell viability was observed for TTZ and KB-PEG15-TTZ at both 30 nM and 100 nM in BT-474 cells (FIG. 13). However, only KB-PEG15-TTZ was able to reduce cell viability at 10 nM (« 70% cell viability) in BT-474 cells. Treatments with IgG and 9 did not have any effect on cell viability .

[0128] For the SK-BR-3 cell line, a similar shift in effective does was observed. Both KB-PEG15-TTZ and TTZ at 100 nM reduced cell viability to about 50% (FIG. 14). However, 30 nM TTZ had only a marginal effect on cell viability, whereas 30 nM KB- PEG15-TTZ effectively reduced cell viability to about 60%.

[0129] In the trastuzumab-resistant cell line KPL-4, as expected, TTZ had no effect on cell viability. Notably, a moderate response was observed at 300 nM for KB-PEG15-TTZ (FIG.15). These data indicate that the effective concentration of TTZ can be lowered by conjugation to KB.

[0130] To confirm that the degradation of HER2 is mediated by complex formation between HER2 and the KB-conjugate antibody recruiting TFRC and HLA, BT-474 cells were treated with compound 9 at 0.001, 0.01, 0.1, 1, 10 and 100 pM for 48 hours in 10% FCS- containing culture media. This analysis indicated that at even the high concentrations, compound 9 could not affect HER2 levels.

[0131] As a first step toward in vivo analysis, in situ HER2 protein level recovery was determined to estimate the timeline and repetition of injections needed to efficiently degrade HER2. BT-474 cell line was selected based on the efficiency of HER2 degradation and the degree of sensitivity to KB-PEG15-TTZ (and TTZ) during cell viability measurements. HER2 siRNA silencing was also included in this study. BT-474 cells were treated at day 0 and then cells were collected every day up to day 9. The highest degradation levels of HER2 were reached at days 2 and 3 for the siRNA knockdown and KB-PEG15-TTZ-treated cells, with HER2 recovering shortly thereafter (FIG. 16; Table 9). The knockdown lasted longer for the HER2 siRNA treated cells because « 90% recovery was reached after 7 days compared to 5 days after KB-PEG15-TTZ treatment. This difference could be explained by the upstream mechanism of the knock-down for the siRNA silencing compared to the lysosomal degradation as well as the need to replace the medium during the experiment.TABLE 9

[0132] The stability of the KB-conjugate antibody was assessed in human plasma. Plasma was mixed with TTZ or KB-PEG15-TTZ at a final concentration of 0.1 mg / mL and an aliquot was removed from each tube every day for 5 days. This analysis showed similar stability of TTZ and KB-PEG15-TTZ in human plasma with more than 70% of the antibodies left after 5 days (Table 10).TABLE 10

[0133] The activity of the KB-PEG15-TTZ construct was analyzed in vivo in BT-474 tumor xenografts. BT-474 cells were injected into the mammary fat pad of NOG mice and tumors were allowed to grow for 70 days. Vehicle (PBS), TTZ (3 mg / kg), or KB-PEG15-TTZ (3 mg / kg) were injected intraperitoneal every 3-4 days (n = 3 mice per group). Additional mice were treated with KB-PEG15-TTZ at 0.3 mg / mL, 1 mg / mL and 10 mg / mL to study the effect of the concentration on HER2 degradation. The excised tumors at day 8 were subjected to anti-HER2 western blot analysis. The KB-PEG15- TTZ construct provided for a significant level of HER2 degradation in this tumor xenograft model in a concentration dependent manner (Table 11). A slight level of HER2 degradation by TTZ was also observed.TABLE 11

[0134] At the end of the study, at day 78, a clear and significative difference in tumor volumes between TTZ- and KB- PEG15-TTZ-treated mice was observed with a decrease in volume of about 2.5-fold in KB-PEG15-TTZ-treated xenografts compared to TTZ (FIG. 17). Altogether, these results demonstrate the use the TFRC / HLA-ABC membrane proteins as entry for cellular uptake with KB conjugates as an efficient method for in situ and in vivo lysosomal protein degradation strategy. In addition, a construct such as KB-PEG15-TTZ may be of particular use in the treatment of cancers that Trastuzumab is known to treat, e.g., HER2-positive early-stage breast cancer, HER2-positive metastatic breast cancer, and HER2-positive gastric cancer.Example 10: KB-mediated degradation of VEGFA

[0135] Using the same approach in described in Example 8, KB tag 6 was covalently linked to Bevacizumab (Bev). Degradation of VEGFA in the high-grade serous ovarian adenocarcinoma cell line OVCAR-3 was analyzed. Cells were treated a control IgG, with 300 nM unconjugated Bev, and with 10, 30, 100, 300 nM KB- Bev with different linker lengths for 48 hours in media containing 5% FCS. Notably, a remarkable concentration effect on VEGFA degradation and a correlation between the PEG length and efficiency of degradation was observed (Table 12). The longer the PEG, the higher level of degradation. For instance, at 30 nM of KB-Bev, 10%, >25% and >75% of VEGFA was degraded with PEG9, PEG15, and PEG23 linkers, respectively. Bev treatment alone did affect VEGFA expression levels. A maximum degradation of >90% was achieved at 300 nM KB-PEG15-Bev and 100 nM and 300 nM KB-PEG23-Bev.TABLE 12^relative values; n = 3

[0136] In light of its activity, KB-PEG23-Bev (KB-Bev) was used to determine cellular uptake. Cellular uptake of VEGFA for lysosomal targeted degradation was confirmed by incubating HeLa cells with Alexa Fluor® 647-labeled VEGFA (VEGFA647) and cotreated with 300 nM IgG, KB-PEG23-Tetrazine linker (KB-Tz), Bev, and KB-Bev for 6 hours with 5% FCS. Following treatment, cells were extensively washed and co-stained with LysoTracker™ for 1 hour. A similar staining pattern between VEGFA647 and LysoTracker™ dyes was observed in KB-Bev treated condition, indicating that the complex of KB-Bev with VEGFA647 is located in the endosomal pathways ultimately reaching the lysosome for degradation. This immunofluorescence microscopy confirmed that the enhanced cellular fluorescence resulted from intracellular uptake of VEGFA647 rather than surface binding.

[0137] Combined knockdown of TFRC and HLA-ABC led to an almost complete recovery of VEGFA levels compared to mock cells in KB- Bev-treated conditions (Table 13) after a 48-hour treatment with Bevacizumab (100 nM), or KB-PEG23-Bev (100 nM) in media containing 5% FCS.TABLE 13*relative values; n = 3.

[0138] Bevacizumab has been shown to inhibit to some extent migration and invasion of aggressive cancer cell lines. Therefore, it was determined whether to KB-PEG23-Bev construct could boost migration and invasion inhibition. Transwell™ migration assays were carried out using OVCAR-3 cells treated with PBS, 100 and 300 nM of Bev alone or KB-PEG23-Bev. Cells were allowed to migrate across the Transwell™ chamber for 24 hours. There was no major difference between the two concentrations of KB-PEG23-Bev. However, the KB-tagged antibody inhibited migration 2-fold more than Bev alone with about 40% migrated cells after 24 hours in the KB-PEG23-Bev-treated cells compared to 70% for Bev alone (FIG. 18). Moreover, it was confirmed that VEGFA-mediated migration inhibition was driven by TFRC and HLA-ABC mediated uptake. Double knockdown of TFRC and HLA-ABC in OVCAR-3 cells treated with KB-PEG23-Bev led to similar migration results as those of Bev alone (FIG. 19). Therefore, binding of KB-PEG23-Bev to its VEGFA target without the additional uptake and degradation cycle promoted more migration inhibition.

[0139] KB-PEG23-Bev also improved the inhibition of OVCAR-3 cellular invasion through Matrigel™ using the Transwell™ assay (FIG. 20). Collectively, these results demonstrate that KB tag conjugates may be used broadly to study various pathologies involving not only dysregulation on cell surface proteins but also on extracellular proteins. In addition, a construct such as KB-PEG23-Bev may be of particular use in the treatment ofcancers that Bevacizumab is known to treat, e.g., colon and rectal cancer, non-small cell lung cancer, breast cancer, renal cell carcinoma, glioblastoma, ovarian cancer, fallopian tube cancer, and primary peritoneal cancer.Example 11: KB-mediated inhibition of TNF signaling

[0140] UCB-9260, an orally active compound, which inhibits TNF signaling by stabilizing an asymmetric form of the trimer. UCB- 9260 is selective for TNF over other superfamily members.UCB-9260

[0141] Several approaches may be taken to covalently attach one or more KB tags to UCB-9260. In one approach, a KB tag is attached via a linker to the pyridine ring.

[0142] In another approach, a KB tag is attached via a linker to the pyrazole ring.

[0143] In another approach, a KB tag is attached via a linker to the phenyl ring.

[0144] Using any one of these constructs, it is expected that the cellular uptake of UCB-9260 will be enhanced compared to unmodified UCB-9260 thereby increasing inhibition of TNF signaling and / or decreasing the dose necessary to achieve the effect of inhibiting TNF signaling. Similar strategies may be used to KB tag TNF binders such as C87 and SPD340. Such constructs are of use in the treatment of autoimmune diseases such as rheumatoid arthritis and Crohn's disease.Example 12: Additional Linkers for covalently attaching a

Claims

WHAT IS CLAIMED IS:

1. A construct comprising an agent covalently linked to a 2- ketoboronate compound of Formula IFormula I wherein (''A-J] represents a phenyl or a 5-membered heteroaryl;L is absent or -0-;R1is selected from alkyl, haloalkyl, and a 5-membered heteroaryl; andR2is selected from hydrogen, alkyl, halo, and haloalkyl.

2. The construct of claim 1, whereinis phenyl.

3. The construct of claim 1, whereinis a 5-membered heteroaryl .

4. The construct of claim 3, wherein the 5-membered heteroaryl is pyrazolyl.

5. The construct of any one of claims 1-4, wherein R2is hydrogen .

6. The construct of any one of claims 1-4, wherein R2is F.

7. The construct of any one of claims 1-6, wherein R1is methyl.

8. The construct of any one of claims 1-6, wherein R1is trifluoromethyl .

9. The construct of any one of claims 1-6, wherein R1is a 5-membered heteroaryl.

10. The construct of claim 9, wherein the 5-membered heteroaryl is thiophene.

11. The construct of any one of claims 1-10, wherein the agent is a small organic molecule, peptide, or protein.

12. The construct of claim 11, wherein the protein is an antibody .

13. The construct of any one of claims 1-12, wherein the agent is a therapeutic agent.

14. The construct of any one of claims 1-13, wherein the agent is covalently linked to the 2-ketoboronate compound via a linker.

15. The construct of any one of claims 1-14 wherein the linker has the structure:or wherein n may be in the range of 2 to 30; and each of x and y may be in the range of 2 to 10.

16. A method of facilitating entry of an agent into a cell comprising contacting the cell with a construct of claim 1 thereby facilitating entry of the agent into the cell.

17. A method of facilitating degradation of a plasma membrane protein comprising contacting a cell with a construct of claim 1, wherein the agent of the construct is a ligand that binds the plasma membrane protein, thereby facilitating degradation of the plasma membrane protein.

18. A method of preventing or treating a disease or condition comprising administering to a subject in need thereof an effective amount of a construct of claim 1, wherein the agent of the construct is a therapeutic agent for preventing or treating the disease or condition.

19. A 2-ketoboronate compound having the structure:where R is selected from alkyl, haloalkyl, and a 5-membered heteroaryl .

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