Biomolecular condensates as protein degradation tools for intracellular targets

Abstract

The present disclosure generally relates to technologies for intracellular targeted protein degradation, methods for intracellular targeted protein degradation in a subject in need thereof. The method includes condensing antibodies within biomolecular condensates using liquid-liquid phase separation and administering to the subject a therapeutically effective amount of the biomolecular condensates.

Description

Atty. Dkt. No.: 121384-0302BIOMOLECULAR CONDENSATES AS PROTEIN DEGRADATION TOOLS FOR INTRACELLULAR TARGETS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No.63 / 777,366, filed March 25, 2025, the entire contents of which are incorporated herein by reference.BACKGROUND[00021 The present invention relates generally to the field of intracellular targeted protein degradation (TPD), particularly to methods of condensing antibodies within biomolecular condensates (BMCs) using liquid-liquid phase separation (LLPS) and administering a therapeutically effective amount of BMCs to a subject. The present disclosure also provides methods of increasing cellular uptake of BMCs and facilitating mutant specific degradation via antibody-directed binding.

[0003] There is a need for improved methods of treating intracellular pathogenic and undruggable proteins.SUMMARY OF THE INVENTION

[0004] The present disclosure provides, among other things, methods for targeted protein degradation, methods of increasing cellular internalization rates of biomolecular condensates, methods of selectively inducing targeted protein degradation, and methods of selectively inhibiting proliferation of cells containing the targeted proteins, as well as a novel biomolecular condensate comprising an antibody and a semi-permeable shell coated around the biomolecular condensate.

[0005] In one aspect, the present disclosure provides a method for intracellular targeted protein degradation in a subject in need thereof comprising (a) condensing antibodies within biomolecular condensates using liquid-liquid phase separation (b) coating the biomolecularAtty. Dkt. No.: 121384-0302condensates with semi-permeable protective shells and (c) administering to the subject a therapeutically effective amount of the biomolecular condensates.

[0006] In some embodiments, the semi-permeable protective shells are metal-phenolic networks.

[0007] In some embodiments, coating the biomolecular condensates with semi-permeable protective shells increases the cellular internalization rate of biomolecular condensates relative to the cellular internalization rate of biomolecular condensates in the absence of semi-permeable protective shells.

[0008] In some embodiments, coating the biomolecular condensates with semi-permeable protective shells decreases the size of the biomolecular condensates relative to the size of biomolecular condensates in the absence of semi-permeable protective shells.

[0009] In some embodiments, coating the biomolecular condensates with semi-permeable protective shells increases the colloidal stability of the biomolecular condensates relative to the colloidal stability of biomolecular condensates in the absence of semi-permeable protective shells.

[0010] In some embodiments, biomolecular condensates selectively induce targeted protein degradation of a pathogenic protein without affecting a wild-type variant.[00.11] In some embodiments, biomolecular condensates selectively inhibit proliferation of cells containing a pathogenic protein.

[0012] In some embodiments, administering to the subject the biomolecular condensates increases apoptosis of cells containing a pathogenic protein relative to apoptosis of cells containing the pathogenic protein in absence of administering the biomolecular condensates to the subject.

[0013] In some embodiments, the subject has an established tumor.

[0014] In some embodiments, the biomolecular condensates are administered intratum orally.Atty. Dkt. No.: 121384-0302[0015| In some embodiments, administering the biomolecular condensates to the subject with the established tumor increases tumor growth inhibition relative to tumor growth inhibition in the subject in the absence of administering biomolecular condensates.[0016J In some embodiments, the targeted protein is at least one of a -synuclein or mutant KRAS.

[0017] In some embodiments, the antibodies are at least one recombinant immunoglobulin Gs, anti -pan KRAS, anti -KRAS G12V, or anti-a-synuclein.10018] In some embodiments, the biomolecular condensates are peptide-based condensates.10019] In some embodiments, the method includes integrating a proteasome-targeting motif into a precursor of the liquid-liquid phase separation.

[0020] In some embodiments, integrating the proteasome targeting motif into the precursor increases the recruitment of proteasome and increases proteasome mediated degradation relative to proteasome recruitment in the absence of integrating the proteasome targeting motif.

[0021] In some embodiments, the subject has at least one of cancer, neurodegeneration.

[0022] In some embodiments the antibodies are interchangeable.

[0023] In some embodiments, the method includes monitoring the subject for one or more toxicities.10024] In some embodiments, the biomolecular condensates are between 200 nanometers and 600 nanometers.

[0025] In another aspect, the present disclosure provides a method to generate a biomolecular condensate comprising (a) condensing antibodies within biomolecular condensates using liquid-liquid phase separation and (b) coating the biomolecular condensates with semi-permeable protective shells.Atty. Dkt. No.: 121384-0302BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG 1A-1B demonstrate a design of IgG-BMC@MPNs for cytosolic entry and proteasome targeting. FIG. 1A shows an illustration of LLPS precursors. An arginine-rich precursor (C-cRs-C-CG-RRRG, top) was cyclized via two cysteine residues to enhance cell penetration and fused with an RRRG motif for proteasome recruitment, separated by a GG spacer (top). The poly-aspartic-acid precursor Dio, carrying complementary negative charges, served as the counterion for inducing LLPS (bottom). FIG. IB shows a schematic of the protein degradation process by IgG-BMC@MPNs. IgG first complexes with arginine-rich precursors via electrostatic interactions. The subsequent addition of pAsp precursor induces formation of IgG-enriched BMCs, which are then coated with a semipermeable metal phenolic network (MPN) shell. The MPN coating stabilizes the condensates, prevents coalescence, and reduces their size to the nanometer scale. The resulting BMC@MPNs enter cells facilitated by the reduced size and cRs domain. After release into cytosol, the antibodies bind to the target proteins through antibody -target interaction (ATIs), while the proteasome-targeting motif recruits proteasome to initiate protein degradation. FIG. 2A-2P demonstrates engineering of IgG-BMC@MPNs with enhanced cell uptake. FIG. 2A shows a schematic of IgG-BMC@MPNs fabrication. IgGs were incubated with arginine-rich precursor to allow electrostatic binding, followed by pAsp precursor addition to induce LLPS. The resulting micro-BMCs were coated with EGCG and FeCh. FIG. 2B UV-vis spectra of arginine-rich precursor, pAsp precursor, EGCG, Fe3+, and their combinations during BMC@MPNs assembly. Increased ODeoo indicates BMC scattering; red shift at 280 nanometers (nm) (characteristic phenol peak) reflects MPN formation. FIG. 2C shows phase contrast microscopy images of IgG-BMC@MPNs in water and after being air-dried. FIG. 2D shows EDX mapping of IgG-BMC@MPNs showing the MPN composition. FIG. 2E droplet size was reduced after MPN coating, measured by DLS. FIG. 2F shows an IgG loading curve in 100 pg IgG-BMC@MPNs. IgG-BMC@MPNs demonstrated a 3-time loading capability of IgG in weight. FIG. 2G shows pH-dependent release profile of IgG from IgG-BMC@MPNs in 48 hrs. Mean ±SD shown (N=3). FIG. 2H shows fluorescence-activated cell sorting (FACS) analysis of SW480 cells transfected with AF488-anti-KRAS existing in its free form, Pro-Iect, Xfect, BMCs, and BMC@MPN. NC, non-treated, control. FIG. 21 shows fluorescence micrographs of SW480 cells incubated with AF488- IgG- BMC@MPNs. FIG.Atty. Dkt. No.: 121384-03022J shows a FACS analysis of SW480 cells treated with cell uptake inhibitors before incubation with AF488-IgG-BMC@MPNs for 4 hours. FIG. 2K shows representative SEM (upper) and TEM (lower) images of BMC@MPNs interacting with SW480 cell surfaces reveal CPP -mediated BMC-cell interaction. FIG. 2L shows zeta potential of IgG increased with positively-charged arginine-rich precursor addition, indicating IgG-polyarginine complexation; negatively-charged pAsp precursor served as a negative non-binding control.FIG. 2M shows turbidity of BMCs decreased rapidly without MPN coating, indicating coalescence and phase merging of uncoated BMCs; in contrast, IgG-BMC@MPNs remained stable as droplets.FIG. 2N shows FACS analysis of SW480 cells transfected with AF488-anti-KRAS existing in its free form, Pro-Ject, Xfect, BMCs, and BMC@MPN. NC, non-treated control. FIG.20 shows FACS analysis of SW480 cells pretreated with cell uptake inhibitors before incubation with AF488-IgG-BMC@MPNs or BMCs. Data normalized to no-inhibitor-treated group. MFI, mean fluorescence intensity. FIG.2P shows TEM images of BMC@MPNs after being internalized by SW480 cells. BMC@MPNs were enclosed within intracellular vesicles. The electron-sparse inner core corresponds to the BMC, while the electron-dense outer shell, rich in iron, represents the MPN coating.

[0027] FIG. 3A-3N demonstrate antibody-guided targeting enabledKRASG12V-selective degradation. FIG. 3A shows immunoprecipitation of KRAS from SW480 cells showed interaction with anti -KRAS, but not isotype controls, following 12 hour (hr) treatment with 5 pM antibody-loaded BMC@MPNs. cRafRBD band indicates residual active KRAS. FIG. 3B shows representative 2-day KRAS degradation blotting and grayscale quantification histogram. FIG. 3C shows a 6-day time-dependent KRAS degradation curve of SW480 cells treated with anti-KRAS-BMC@MPNs. Non-specific IgG isotype was included as the negative control. FIG. 3D shows a schematic of orthogonal-antibody blotting. Cells were treated with anti-pan-KRAS or anti-KRASG12V-BMC@MPNs. The residual total KRAS or KRASG12Vwas resolved using detection antibodies that bind to distinct epitopes. immunoblots showing variant-selective KRAS degradation. Wild-type, heterozygous, or homozygous mutant KRAS-expressing cells were treated for 2 days with either anti-pan-KRAS-BMC@MPNs or anti-KRASG12V-BMC@MPNs. FIG. 3E shows an immunoblot of mutant-selective KRAS degradation. Wild-type, heterozygous, or homozygous mutant KRAS-expressing cell lines were treated for 2 days with either anti-KRAS-BMC@MPNs or anti-KRASG12V-BMC@MPNs. FIG. 3F shows a grayscale quantification of immunoblots. TheAtty. Dkt. No.: 121384-0302anti-KRASWTtreatment degraded both KRAS types across all cell lines, whereas the anti-KRASG12Vtreatment selectively reduced KRASG12Vlevels in heterozygous and homozygous mutant cells. FIG. 3G shows a schematic of co-culture assay. FIG.3H-3I shows confocal images show SW480 cells (red) dominated under NC conditions (FIG. 3g) but were suppressed after anti-KRASG12V-BMC@MPN treatment, while HT29 cells (blue) remained unaffected (FIG. 3h). CFSE signal (green) was weak and uniform in NC (FIG. 3g), but colocalized with SW480 cells post-treatment, indicating reduced cell division upon treatment. (FIG. 3h). FIG. 3J-3K shows a flow cytometry analysis showed that SW480 cells dominated in the co-culture under NC conditions (FIG. 3 J) but were suppressed with anti-KRASG12V-BMC@MPNs treatment, whereas HT29 cell growth remained unaffected (FIG. 3K). FIG. 3L-3M shows a Ki-67 proliferation assay revealed significant suppression of SW480 cell proliferation (FIG. 3L), with HT29 cell proliferation unaffected (FIG. 3M). FIG. 3N shows 6-day KRAS degradation blot and grayscale quantification show the degradation profile following a single anti-KRAS-BMC@MPN treatment.[0028| FIG. 4A-4K demonstrates that targeted degradation of KRASG12Vinhibits tumor growth in vivo. FIG. 4A shows a schematic of dosing regimen and anti-tumor effect assessment of anti-KRAS-BMC@MPNs in KRASG12VSW480 cell line-derived (CDX)-bearing nude mice, s.c., subcutaneous. FIG. 4B shows tumor growth curves in response to intratumoral injection of specified antibodies at the indicated dose three times a week for a total of six doses (n = 8 per group). FIG. 4C shows representative tumor images at the endpoint. From top to bottom: PBS, BMC@MPN, isotype-BMC@MPN, and anti-KRAS-BMC@MPNs-treated groups. FIG. 4D shows endpoint tumor volume. FIG. 4E shows endpoint tumor weight. FIG. 4F shows representative images of mice at endpoint. FIG. 4G shows H&E staining results showed increased necrosis and reduced matrix density in the anti-KRAS-BMC@MPNs treatment group compared to the isotype and vector control groups, indicating reduced tumor aggressiveness and enhanced immune infiltration. Nuclei appear blue; cytoplasm and extracellular matrix appear red. Black arrow: tumor heterogeneity; green arrow: tumor matrix; red arrow: blooding point. FIG. 4H shows a TUNEL assay showed elevated apoptosis in the anti-KRASG12V-BMC@MPNs treatment group (stained in AF488). FIG. 41 shows immunohistochemical staining of Ki-67, caspase-3, and KRAS as indicators of proliferation, apoptosis, and the target, respectively. The antiAtty. Dkt. No.: 121384-0302KRAS-BMC@MPNs treated group showed reduced proliferation, increased apoptosis, and reduced KRAS levels compared to the control groups. FIG. 4J shows body weight change curves during the treatment period. FIG. 4K shows serum biochemistry analysis of nude mice at the endpoint. ALP, alkaline phosphatase; ALT, alanine transaminase; AMY, amylase; ALB, albumin; TBIL, total bilirubin; TP, total protein; BUN, blood urea nitrogen; CRE, creatinine; GLOB, globulin; Glu, glucose; Ca, calcium; PHOS, phosphorus; Cl, chloride; K, potassium; NA, sodium, n = 5 per group. FIG. 4L shows a TUNEL assay showed elevated apoptosis in the anti-KRASG12V-BMC@MPNs treatment group (stained in AF488).

[0029] FIG. 5A-5P demonstrate proteasome-mediated degradation enables the disruption of intracellular targets. FIG. 5A shows representative western blot (WB) analysis of KRAS degradation in SW480 cells pretreated with MG132 or choloroquine followed by anti-KRAS-BMC@MPNs treatment. KRAS levels were reduced to 27% in untreated cells, reduced by 91% with MG132 by not by chloroquine. FIG. 5B shows a grayscale quantification histogram. FIG. 5C shows a representative pose of the interaction between PTM and the 7UJD proteasome regulatory subunits, shown in a space-filling representation. FIG. 5D shows Confocal microscopy showed reduced a-syn (Texas Red channel) after 48-hr incubation with anti-a-syn-BMC@MPNs. FIG. 5E shows a 48-hr quantitative a-syn degradation analysis using SDS-PAGE and greyscale analysis. FIG. 5F shows a viral construct for generating a-syn overload cell model. FIG. 5G shows a Orthogonal cell viability, and LDH release study showing anti-a-syn-BMC@MPN treatment attenuated neuro cytotoxicity due to a-syn overload. FIG. 5H shows a schematic of pull-down strategy for LFQ. FIG. 5I-5J shows pull-down protein differential expression analysis in SW480 cells (FIG. 51) and SK-N-SH cells (FIG. 5 J) both showed enrichment of PSMD family subunits and reduced levels of targets. FIG. 5K-5L shows a KEGG pathway enrichment analysis indicated the proteasome pathway as one of the top 10 hits for both cell types, along with the RAS signaling (SW480 cells) (FIG. 5K) or neuro-disease pathways (PD, HD, AD) (SK-N-SH cells) (FIG. 5L). FIG. 5M shows a grayscale quantification histogram. FIG. 5N shows WB analysis of KRAS degradation using RRRG (R) or AAAG (A) negative control. Only the RRRG motif induced KRAS degradation, demonstrating that electrostatic interaction with PSMD9 is essential for proteasome recruitment. FIG. 50 shows a grayscale quantification histogram.Atty. Dkt. No.: 121384-0302[0O3O| FIG. 6A-6B demonstrates optimization of MPN coating. FIG. 6A shows a 1 mg / mL EGCG and 1 mg / mL FeCh varying in volume added to 10 pL pArg condensates system are investigated for complex condensation. Empty dots: BMC disappeared after adding FeCh; red dots: aggregation occurred; blue dots: BMC remained after adding FeCh, but disappeared after being centrifuged; green dots: BMC remained after adding FeCh and being centrifuged. Grey dots: data not collected due to aggregation induced by over-dose EGCG. Optimized BMC@MPN is highlighted in the dashed red box. A dashed grey box designates representative condensates for turbidity analysis. FIG. 6B shows a turbidity analysis of representative condensates. Missing data points indicate aggregation or condensates disassembly. Data are shown as the mean ±SD (N=3).[00311 FIG. 7A-7C demonstrates improved colloidal stability of IgG-BMC@MPNs. FIG.7A-7B shows room temperature (FIG. 7A) and 37°C (FIG. 7B) turbidity change in solutions varying in osmotic pressure in 24 hours indicated improved osmotic stability of BMC@MPN compared to BMC. Data are shown as the mean ±SD (N=3). FIG. 7C shows zeta potential data of BMC and BMC@MPN indicating improved colloidal stability after MPN coating.[00321 FIG. 8A-8G Demonstrate characterization of IgG-BMC@MPNs. FIG. 8A-8B shows SEM (FIG. 8A) and TEM (FIG. 8B) images of IgG BMC@MPN. FIG. 8C-8D shows nanomechanical properties mapping of BMC@MPN using AFM at low (FIG. 8C) and high mag (FIG. 8D). FIG. 8E-8F shows 3D height profile (FIG. 8E) and line profile (FIG. 8F) of BMC@MPN by AFM at indicated position (red dot line). FIG. 8G shows TEM 3D images of COA@MPN.[00331 FIG. 9A-9L demonstrates optimization of MPN coating. FIG. 9A-9L show SEM (FIG. 9A-9D), TEM (FIG. 9E-9H), and EDX (FIG. 9I-9L) images of IgG-BMC@MPN using metal ions of difference valance (Mg2+, Cu2+, Zr4+, Cs+).

[0034] FIG. 10A-10B demonstrates cytoprotective function of MPN. FIG. 10A shows an EDX line profile of optimized IgG-BMC@MPN. FIG. 10B shows EDS images of MPNs after core removal using dimethylformamide.Atty. Dkt. No.: 121384-0302

[0035] FIG. 11A-11B demonstrates IgG loading capacity quantification. FIG. 11A shows a UV-vis spectra of 10 nm colloidal gold nanoparticles (cAuNP), BMC@MPN, and cAuNP IgG-loaded BMC@MPN. FIG. 11B shows bright field and annular dark field TEM images of cAuNP IgG-BMC@MPN.

[0036] FIG. 12A-12D demonstrates saturation concentration for cellular uptake of IgG-BMC@MPNs. FIG. 12A shows representative NTA (nanoparticle tracking analysis) image of IgG-BMC@MPNs inflow tubing. FIG. 12B-12D shows uptake saturation curve quantified using FACS of TMR-IgG-BMC@MPNs in SW480 cells when incubated with different amounts of TMR-IgG-BMC@MPN.

[0037] FIG. 13A-13B demonstrates a cell uptake efficacy study of IgG-BMC@MPNs. FIG.13A shows a FACS analysis. FIG. 13B shows microscopy images of cells transfected with Alexa Fluor 488 IgG by its free form, Pro-Ject, X-Fect or BMC@MPN in SW480 cells.[0038| FIG. 14A-14D demonstrates a time-dependent uptake curve of IgG-BMC@MPNs in SW480 cells. FIG. 14A-14B shows FACS analysis of AF488-conjugated non-specific IgG, denoted as isotype control. FIG. 14C-14D shows AF488-conjugated anti-KRAS.

[0039] FIG. 15A-15B demonstrates microscopy study of bio-nano interaction. FIG. 15A shows fluorescence microscopy images of cell uptake of AF488-IgG-BMC@MPNs within 12 hr in SW480 cells. FIG. 15B shows SEM images of AF488-IgG-BMC@MPNs interact with cell membrane within 20 min in SW480 cells.

[0040] FIG. 16A-16B demonstrates a cell internalization mechanism study. FIG. 16A shows a FACS analysis. FIG. 16B shows microscopy images of SW480 cells treated with various inhibitors before incubation with AF488-IgG-BMC@MPN for 4 h. Two groups were included as control: non-treated cells (blank) and cells treated with AF488-IgG-BMC@MPN without inhibitors (NC, non-treated control).

[0041] FIG. 17A-17C demonstrates a cell internalization mechanism study by electron microscope. FIG. 17A-17B shows TEM images showing different stages during cell internalization in SW480 cells. FIG. 17C shows SEM images showing different stages when IgG-BMC@MPNs interact with cell membranes.Atty. Dkt. No.: 121384-0302[0042| FIG. 18A-18B demonstrates cytosol localization analysis of IgG-BMCs@MPNs. FIG. 18A shows fluorescence microscopy images of SW480 cells treated with AF488-anti-KRAS-BMC@MPN at log magnification. FIG. 18B shows a Pearson colocalization analysis between the cell membrane and native KRAS as control.

[0043] FIG. 19A-19B demonstrates KRAS degradation kinetics induced by anti-KRAS-BMC@MPNs in SW480 cells. FIG. 19A shows a 6-day FACS analysis of KRAS level, stained by AF555 anti-KRAS as detecting antibody, after being treated by anti-KRAS-BMC@MPN or isotype anti-KRAS-BMC@MPN in HT29 cells. FIG. 19B shows an AF555 isotype staining was used as baseline control.

[0044] FIG. 20 shows orthogonal validation of mutant-selective KRAS degradation. Wildtype, heterozygous, or homozygous mutant KRAS-expressing cell lines were treated with anti-KRAS-BMC@MPNs or anti-KRASG12V-BMC-@MPNs for 2 days. Cell lysates were resolved using SDS-PAGE and probed with pan-KRAS or G12V-selective antibodies to quantify the degradation level of each KRAS type individually. The anti-KRAS-treated group showed degradation of both KRAS types in all three cell lines, while the anti-KRASG12V-treated group affected only the KRASG12V level in mutant heterozygous and homozygous cells. This indicates that the selectivity of degradation originates from antibody-antigen interaction. Isotype-BMC@MPNs and NC were used as negative controls.

[0045] FIG. 21A-21B demonstrates a six-day coculture cell growth assay. FIG. 21A-21B shows flow cytometry results of representative SW480 and HT29 subpopulations in coculture, either untreated (FIG. 21A) or treated with anti-KRASG12V (FIG. 21B).

[0046] FIG. 22 demonstrates a six-day coculture proliferation assay. FIG. 22 shows after 6 days of treatment with anti-KRASG12V-BMC@MPNs, the proliferative peak (Ki-67) of SW480 cells decreased compared to the non-treated group, while the proliferation level of HT29 cells was not significantly impacted.

[0047] FIG. 23 shows in vivo tumor growth images.

[0048] FIG. 24 shows an anti-proliferation assay of dissociated cells from tumors.Atty. Dkt. No.: 121384-0302[0049| FIG. 25 shows H&E staining of kidney and liver. No abnormal histology was observed in all treated groups.

[0050] FIG. 26A-26B demonstrates lysosome inhibitor could not rescue degradation effect.FIG. 26A shows immunoblot showed that when pretreated with MG132, KRAS degradation was inhibited, while such effect was not observed using Bafilomycin Al, a lysosome inhibitor. FIG. 26B shows a grayscale quantification.[00511 FIG. 27A-27D shows molecular docking between proteasome-targeting motifs and proteasomes. FIG. 27A shows RRRG and 26S. FIG. 27B shows RRRG and 19S. FIG. 27C shows AAAG and 26 S. FIG. 27D shows AAAG and 19S.

[0052] FIG. 28 demonstrates IgG-BMC@MPNs showed nuclear entry capability. Anti-a-syn-BMC@MPNs have high nucleus delivery efficacy in SK-N-SH cells.10053] FIG. 29 demonstrates establishment of a-syn overload cells. Post-transfection of SK-N-SH cells with pLVX-IRES-ZsGreenl / a-syn. ZsGreen / a-syn signal reaches the peak value around 48 hr after transfection.DETAILED DESCRIPTION

[0054] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0055] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. All the various embodiments of the present disclosure will not be described herein. Many modifications and variations of the disclosure can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appendedAtty. Dkt. No.: 121384-0302claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.Definitions

[0056] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0057] As used herein, the single forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0058] As used herein, the term “about,” when used to modify a numerical value, indicates that deviations of up to 10% above and below the numerical value, including the numerical value, remain within the intended meaning of the recited value. For example, “about 10” should be understood as both “10” and “9-11”.

[0059] As used herein, the term “optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0060] As used herein, the term “administering” of an agent to a subject includes any route of introducing or delivering the agent to the subject to perform its intended function.Administration can be carried out by any suitable route, including, but not limited to, intravenously, intramuscularly, intraperitoneally, subcutaneously, and other suitable routes as described herein. Administration includes self-administration and the administration by another.Atty. Dkt. No.: 121384-0302[0061| As used herein, the term “antibody” generally refers to an antibody comprising two light-chain polypeptides and two heavy-chain polypeptides (unless the context in which this term is used suggests otherwise). Antibodies include different antibody isotypes including IgM, IgG, IgA, IgD, and IgE antibodies. The term “antibody” includes, without limitation, a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a privatized antibody, a deimmunized antibody, and a fully human antibody. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, llama, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody[00621 As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method. “Consisting of’ shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).[00631 As used herein, the term “effective amount” or “therapeutically effective amount” refers to a quantity of an agent sufficient to achieve a beneficial or desired clinical result upon treatment. In the context of therapeutic applications, the amount of a therapeutic agent administered to the subject can depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, sex, body weight, effective concentration of the therapeutic agent administered, and tolerance to drugs. It can also depend on the degree, severity, and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progressionAtty. Dkt. No.: 121384-0302of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art.

[0064] As used herein, the term “reduce” or “decrease” means to alter negatively by at least about 5% including, but not limited to, alter negatively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.[0065 { Treatment of cancer, autoimmune disorder, or neurodegeneration can be at any time during cancer or an infection, autoimmune disorder, or neurodegeneration. Certain embodiments of the present disclosure can be administered as a combination (e.g., with a second active), or separately concurrently or in sequence (sequentially) in accordance with the methods described herein as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly, or annually or between about 1 to 10 week, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by cancer or an infection, autoimmune disorder, or neurodegeneration. Thus, a method can be practiced by one or more times (e.g., 1-10, 1-5, or 1-3 times) an hour, day, week, moth, or year. The skilled artisan will know when it is appropriate to delay or discontinue administration. A nonlimiting dosage schedule is 1-7 times per week, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or range or value within such ranges.Subject Population and Methods of Administration

[0066] As used herein “patient” and “subject” are used interchangeably.

[0067] In the methods described herein, the term “subject” includes but is not limited to a subject at risk of cancer, an autoimmune disorder, or neurodegeneration, as well as a subject that has already developed cancer, an autoimmune disorder, or neurodegeneration. Such subjects include mammalian animals (mammals), such as non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans. Subjects include animal disease models,Atty. Dkt. No.: 121384-0302for example, mouse or other animal models of cancer or an infection, autoimmune disorders, or neurodegeneration known in the art.

[0068] In some embodiments, the methods described herein, the subject is an adult human. In other embodiments, the subject is a juvenile human.

[0069] In methods described herein, administering an agent, and / or composition can be accomplished by any method known in the art suitable for the particular type of formulation selected. Suitable routes of administration include without limitation oral, intratumoral, parenteral (including intramuscular, subcutaneous, intradermal, intravascular, intravenous, intraarterial, intrarticular, intramedullary, and intrathecal), intraperiotenal, and topical (including dermal / epicutaneous, transdermal, mucosal, transmucosal, intranasal (e.g., by nasal spray or drop), intraocular (e.g., by eye drop), pulmonary (e.g., by inhalation), buccal, sublingual, rectal, and vaginal).[0070| In some aspects, in the methods described herein, the administering to a subject in need can be done intratum orally.

[0071] Doses in the methods described herein can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials. Initial study doses can be based upon animal studies, e.g. a mouse, and the amount of treatment or agent disclosed herein administered in an amount that is determined to be effective.

[0072] Exemplary non-limiting amounts (doses) are in a range of about 0.1 mg / kg to about 100 mg / kg, and any numerical value or range or value within such ranges. Greater or lesser amount (doses) can be administered, for example 0.01-500 mg / kg, and any numerical value or range or value within such ranges. The dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1-10 ug / kg, 10-25 ug / kg, 50-100 ug / kg, 100-500 ug / kg, 500-1,000 ug / kg, 1-5 mg / kg, 5-10 mg / kg, 10-20 mg / kg, 20-50 mg / kg, 20-50 mg / kg, 20-50 mg / kg, 50-100 mg / kg, 100-250 mg / kg, 200-500 mg / kg, or more, two, three, four, or more times per hour, day, week, month, or annually. A typical range will be fromAtty. Dkt. No.: 121384-0302about 0.3 mg / kg to about 50 mg / kg, 0-25 mg / kg, or 1.0-10 mg / kg, or any numerical value or range or value within such ranges.

[0073] The maximum tolerable dose of the methods described herein can be readily established, and the effective amount providing a detectable therapeutic benefit to the patient may also be determined, as can the temporal requirement for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regiments are exemplified herein, these examples in no way limit the dose and administration regiment that may be provided to a patient in practicing the present disclosure.

[0074] In the methods described herein, the route, dose, number and frequency of administrations, treatments, and timing / intervals between treatment and disease development can be modified. In certain embodiments, a desirable treatment of the present disclosure will elicit robust, long-lasting immunity against cancer or an infection, autoimmune disorders, orneurodegeneration. Thus, in certain embodiments, disclosure methods, uses and compositions provide long-lasting immunity to cancer or an infection, autoimmune disorders, or neurodegenerationTargeted Protein Degradation

[0075] Targeted protein degradation (TPD) is often used as a therapeutic strategy that leverages a cell’s degradation system to eliminate pathogenic proteins. Molecules like proteolysis-targeting chimeras (PROTACS) (Nguyen, T. M. et al. Nat. Chem. 16, 218-228 (2024); Bondeson, D. P. etal. Nat. Chem. Biol. 11, 611-617 (2015)), lysosome targeting chimeras (LyTACS) (Banik, S. M. etal. Nature 584, 291-297 (2020); Marei, H. et al. Nature 610, 182-189 (2022); Pance, K. et al. Nat. Biotechnol. 41, 273-281 (2023)), and molecular glues (Mercer, J. A. etal. Science 383, eadk4422 (2024); Lu, G. etal. Science 343, 305-309 (2014); Han, T. etal.. Science 356, eaal3755 (2017)) facilitate the binding between proteins and degradation systems, initiating the breakdown of these pathogenic protein targets.

[0076] Selectivity of TPD depends on precise binding to targets. Antibodies are shown to be ideal for achieving this precision due to their increased affinity and specificity for antigen recognition (Ahn, G. et al. Nat. Chem. Biol. 17, 937-946 (2021); Ehlinger, A. & Walters, K.Atty. Dkt. No.: 121384-0302J. Biochem. 52, 3618-3628 (2013)). Antibody-based targeting enables the degradation of pathogenic proteins and has been shown to be effective in degrading receptor proteins such as programmed death-ligand 1 (PD-L1) (Marei, H. et al. Nature 610, 182-189 (2022); Cotton, A. D., Nguyen, D. P., Gramespacher, J. A., Seiple, I. B. & Wells, J. A. J. Am. Chem. Soc.143, 593-598 (2021)). Although this has been shown to be effective at degrading extracellular proteins, antibody based TPD is limited due to antibodies large size and other properties that render them impermeable to cell membranes (Chamberlain, P. P. & Hamann, L. G. Nat. Chem. Biol. 15, 937-944 (2019)).

[0077] Many conditions such as cancers, autoimmune disorders, and neurodegeneration are associated with intracellular pathogenic proteins such as KRAS, BCL-2, and Tau (Gregory, J. A., Hickey, C. M., Chavez, J. & Cacace, A. M. Cell Chem. Biol. 31, 1688-1698 (2024); Dale, B. et al. Nat. Rev. Cancer 21, 638-654 (2021)). These diseases may benefit from adaptable antibody-based therapies designed to target and degrade pathogenic intracellular proteins. However, while several intracellular delivery methods have been tested, such as therapies using single-chain variable fragment (Tanaka, T. & Rabbitts, T. H. J. Mol. Biol. 376, 749-757 (2008); Colby, D. W. et al. Proc. Natl. Acad. Sci. 101, 17616-17621 (2004)) or conjugating naked antibodies with cell-penetrating peptides, these approaches remain limited due to issues including shortened half-lives and undesirable immune response.

[0078] In one aspect, the present disclosure provides methods of intracellular TPD comprising condensing antibodies within biomolecular condensates using liquid-liquid phase separation (LLPS). The pathogenic proteins that are targeted are proteins directly related to cancers, autoimmune disorders, and / or neurodegeneration as mentioned above. These pathogenic proteins include for example and without limitation KRAS and a-synuclein.

[0079] KRAS is a 21 kDa cytoplasmic protein devoid of druggable binding sites. Mutant KRAS which is implicated in several cancers, is considered undruggable due to the absence of binding sites for conventional degraders (Huang, L., Guo, Z., Wang, F. & Fu, L. Signal Transduct. Target. Ther. 6, 386 (2021); Parikh, K. etal. J. Hematol. Oncol. 15, 152 (2022)). Oncogenic KRAS (e.g., KRASG12V) is characterized by a single amino acid mutation that current degraders are unable to selectively target under their native expression conditionsAtty. Dkt. No.: 121384-0302(Huang, L., Guo, Z., Wang, F. & Fu, L. Signal Transduct. Target. Ther. 6, 386 (2021);Martin, A. et al. bioRxiv (2024)).

[0080] a-synuclein (a-syn) is a 17 kDa nuclear protein associated with neurodegenerative diseases such as Parkinson’s (PD), Alzheimer’s (AD), and Huntington’s (HD) (Goedert, M. Nat. Rev. Neurosci. 2, 492-501 (2001)). Targeting a-synuclein is challenging due to its predominant nuclear localization at high expression levels. In addition, the autophagy-lysosomal pathway (ALP) is often impaired under these conditions, making proteasomal degradation a vital alternative (Qu, J. et al. Cell Chem. Biol. 27, 751-762. e4 (2020)).

[0081] In some embodiments, the pathogenic protein targeted by the targeted protein degradation is at least one of mutant KRAS or a-syn.Biomolecular Condensates

[0082] According to one embodiment, provided herein is a biomolecular condensate comprising an antibody. The antibodies condensed into biomolecular condensates are antibodies directly related to the treatment of cancers, autoimmune disorders, and neurodegeneration as discussed above. In some embodiments the antibodies are at least one of anti-KRASG12V, recombinant immunoglobulin Gs (IgGs), anti-a-syn, and / or anti-KRAS configured to target the corresponding pathogenic protein. In some embodiments, the antibodies are interchangeable. For example, when targeting the pathogenic protein a-syn the antibody anti-a-syn can be used for biomolecular condensate formation. For example, when targeting the pathogenic protein KRAS, the antibody anti-KRAS can be used for biomolecular condensate formation.

[0083] LLPS-based materials are used as delivery vehicles due to their biocompatible precursors and aqueous assembly which preserves the bioactivity of guest molecules (e.g., antibodies) and minimizes the toxicity (Li, P. etal. Nature 483, 336-340 (2012); Aumiller, W. M. & Keating. Nat. Chem. 8, 129-137 (2016); Liang, T. et al. Nat. Biomed. Eng. 8, 1469- 1482 (2024); Strom, A. R. et al. Cell 187, 5282-5297.e20 (2024)). LLPS-based materials include for example and without limitation peptide-based condensates, histone- and DNA-based condensates. According to one embodiment, provided herein a LLPS precursorAtty. Dkt. No.: 121384-0302comprises at least one of an arginine rich peptide (e.g., C-cRs-C-GG-RRRG) and an aspiratic-acid decapeptide (e.g., Dio) with complementary charge valency. Electrostatic properties between these peptides drive BMC formation by inducing LLPC and concomitantly condensing the antibodies through coulombic interactions.

[0084] Peptide-based condensates are known to direct cytosolic delivery and trafficking of macromolecular therapeutics without compromising their bioactivity (Sun, Y. et al. Nat. Chem. 14, 274-283 (2022); Liu, J., Spruijt, E., Miserez, A. & Langer, R. Nat. Rev. Mater. 8, 139-141 (2023)). In some embodiments, the biomolecular condensates are peptide-based condensates, meaning they are formed primarily through the self-assembly or phase separation of synthetic or recombinant peptides. These peptides are typically designed to contain modular sequence motifs that promote multivalent interactions — such as electrostatic, hydrophobic, it-it stacking, cation-7t interactions, or hydrogen bonding — driving spontaneous condensation under physiological or tunable conditions (Visser, B. S. Nat. Rev. Chem. 8.9 686-700. (2024)).

[0085] In some aspects, the biomolecular condensates can also be formed from non-peptidic components, which may include a wide variety of biologically or synthetically derived macromolecules capable of undergoing phase separation or coacervation. These may include nucleic acids, polysaccharides, and proteins, etc. (Gao, Y, et al. Nat. Chem. Biol.18.12 1307-1318. (2022)).

[0086] According to one embodiment, provided herein is a biomolecular condensate comprising a proteasome-targeting motif (PTM) incorporated into the LLPS precursor. The proteasome-targeting motif may be incorporated as an RRRG fragment for proteasome engagement.Semi-Permeable Shell

[0087] In an embodiment, provided herein, the BMCs are coated with a semi-permeable shell. In one embodiment, the semi-permeable shell is a metal-phenolic network (MPN). In one embodiment, the BMCs are coated with epigallocatechin-3 -gallate (ECCG) / FeCls to form the MPN. In addition to MPNs, other shell materials may be used to encapsulate theAtty. Dkt. No.: 121384-0302BMCs depending on the desired stability, permeability, targeting capability, and / or responsiveness to environmental cues. Alternatives include (1) Lipid bilayers or liposomelike coatings: providing a biomimetic surface and biocompatibility for intracellular delivery (Zhang, Q. et al. Nat. Nanotechnol. 13.12 1182-1190 (2018)), or (2) Synthetic polymer shells (e.g., PEG-based hydrogels): PEGylation offers stealth properties and controlled degradation function (Gillich, T. et al. ACS Nano 7.1 316.329. (2013)). MPNs deposition includes incubation with precursors of the shell material (i.e., EGCG) under controlled conditions (i.e., pH >8.5), followed by adding Fe3+ for the formation of the network.

[0088] In some aspects, provided herein, adding the semi-permeable shell reduces the size of the biomolecular condensates. In one aspect, the size of the biomolecular condensate may be between about 200 nanometers (nm) to about 600 nm. In some embodiments, the size of the biomolecular condensate is about 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, or 600 nm.Methods of Use10089] According to one embodiment, described herein is a method for intracellualr targeted protein degradation in a subjet in need thereof. The method comprises, or consists essentially of, or yet further consists of condensing an antibody within a biomolecular condensate using LLPS, coating the biomolecular condensate with a semi-permeable protective shell, and administering to the subject a therapeutically effective amount of the biomolecular condensate. In some aspects, the subject has at least of cancer or a neurodegenerative disorder.

[0090] According to one embodiments, described herein is a method for generating a biomolecular condensate for TPD. The method comprises, or consists esentailly of, or yet further consistes of condensing an antibody within a biomolecular condensate using LLPS and coating the biomolecular condensate with a semi-permeable protective shell.

[0091] In some aspects, the methods further consists of integrating a proteasome-targeting motif into a precursor of the LLPS. In some aspects, integrating the proteasome-targeting motif into the precursor increases a recruitment of proteasome and increases proteasomeAtty. Dkt. No.: 121384-0302mediated degradation relative to proteasome recruitment and proteasome mediated degradation in the absence of integrating the proteasome-targeting motif.

[0092] In some aspects, the semi-permeable shell is a metal-phenolic network. In some aspects, coating the biomolecular condensate with a semi-permeable protective shell increases a cellular internalization rate, decreases the size, and / or increases collodial stability of of the biomolecular condensate reltaive to cellular internalization rate, the size, and / or the collodial stability of the same biomolecular condensate lacking a semi-permeable protective shell.

[0093] In some aspects, the biomolecular condensate selectively inhibits proliferation of cells containing a pathogenic protein. In some aspects, the biomolecular condensate selectively induces targeted protein degradation of the pathogenic protein without affecting a wild-type variant. In some aspects, the pathogenic protein is at least one of mutant KRAS or a-synuclein. In some aspects, the antibody is at least one of recombinant immunoglobulin Gs, anti-KRAS, or anti-a-syn.

[0094] In some aspects, the subject has an established tumor. In some aspects, administering the biomolecular condensate to the subject with the established tumor increases tumor growth inhibition relative to tumor growth inhibition in a subject in the absence of administering a biomolecular condensate.

[0095] In some aspects, administration of the biomolecular condensates is performed intratum orally.

[0096] In some aspects, the subject is monitored for one or more toxi cities following the administration of BMCs.Kits

[0097] As set forth herein, the present disclosure provides methods for administering biomolecular condensates and compositions to a tumor or subject in need. In one aspect, the present disclosure provides kits for performing these methods as well as instructions for carrying out the methods of the present disclosure.Atty. Dkt. No.: 121384-0302

[0098] In one aspect, the kit comprises, or alternatively consists essentially of, or yet further consists of, any one or more of: a biomolecular condensate as disclosed herein, or composition as disclosed herein, or an optional instruction of use. Such a kit may also comprise, or alternatively consist essentially of, or yet further comprise media and other reagents appropriate for the methods disclosed herein.

[0099] The kits of this disclosure can also comprise e.g., a buffering agent, a preservative, or a protein-stabilizing agent. The kits can further comprise components necessary for detecting the detectable-label, e.g., an enzyme or a substrate. The kits can also contain a control sample or series of control samples, which can be assayed and compared to the test sample. Each component of a kit can be enclosed within an individual container and all of the various containers can be within a single packaging, along with instructions for interpreting results of the assays performed using the kit. The kits of the present disclosure may container a written product on or in the kit container. The written product describes how to use the reagents contained in the kit.

[0100] As amenable, these suggested kit components may be packaged in a manner customary for use by those skilled in the art. For example, these suggested kit components may be provided in solution or as a liquid dispersion or the like.

[0101] The following examples illustrate selected embodiments, but in no way limit the scope of the invention.ExperimentalExample 1: Materials and Methods

[0102] The following materials and methods were used for the following Examples.

[0103] Materials: Applicant used the following materials. Epigallocatechin gallate (EGCG), iron(III) chloride hexahydrate (FeCh 6H2O), 2-amino-2- (hydroxymethyl)- 1,3 -propanediol (Tris), dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), nitric acid (HN03), hydrochloric acid (HC1), fluorescein isothiocyanate (FITC), Duolink® Proximity Litigation Assay kit (DUO92101) were purchased from MilliporeSigma. Minimum essential mediumAtty. Dkt. No.: 121384-0302(MEM), Dulbecco’s modified Eagle medium (DMEM), RPMI-1640 medium, Iscove's modified Dulbecco's Medium (IMDM), McCoy's 5A medium, Opti-MEM™ reduced serum medium, buffer saline (PBS), fetal bovine serum (FBS), penicillin / streptomycin, and 0.25% trypsin-ethylenediaminetetraacetic acid, Pierce Pro-Ject™ protein transfection reagent, Ni-NTA His bind resin, Texas Red™-X phalloidin (#T7471), QTracker 625 (#A10198) and QTracker 800 (#Q25071MP) were purchased from Thermo Fisher. Xfect™ protein transfection reagent was purchased from Takarabio. Oligopeptides of arginine and aspartic acid were purchased from GenScript. The Annexin V-FITC / PI apoptosis kit (#KA3805) was purchased from Abnova. Carboxyfluorescein succinimidyl ester (CFSE) was purchased from BD Bioscience (#565082). Cell Count Kit 8 (CCK8) and lactate dehydrogenase (LDH) cytotoxicity kit were purchased from Dojindo. Rabbit and mouse-specific HRP / DAB Detection IHC kit and AF488-TUNEL kit were purchased from Abeam. High-purity Milli-Q water with a resistivity of 18.2 MQ cm was obtained from an inline Millipore water purification system.[01041 Applicant conducted UV-Vis absorption and fluorescence spectra measurements using a BioTek Cytation 3 cell imaging multi-mode reader. Dynamic light scattering (DLS) and (^-potential measurements were performed on a Malvern Zetasizer Ultra. Phase contrast images were captured using an EVOS XL Core inverted microscope. Confocal laser scanning microscopy (CLSM) images were acquired using a Nikon SoRa Spinning Disk Microscope. TEM images and EDX profiles were obtained using a JEOL ARM200CF instrument operated at 200 kV. SEM images were acquired using a JEOL JSM-7900FLV operated at 10 kV; samples were coated with 10 nm osmium tetroxide using a Filgen OPC60A osmium coater. Atomic force microscopy (AFM) images were obtained using a Bruker ICON. Nanoparticle tracking analysis (NTA) was performed using NanoSight. Isothermal titration calorimetry (ITC) analysis was conducted using an Affinity ITC from TA Instruments. ICP-MS was performed on a Thermo iCAP Q. FACS was performed on a BD FACSymphony™ A5 Cell Analyzer.

[0105] Cell Culture: Applicant obtained HT29 (human colorectal adenocarcinoma, RRID: CVCL 0320), NCI-H441 (human lung adenocarcinoma, RRID: CVCL 1561), SW480 (human colorectal adenocarcinoma, RRID: CVCL 0546), SK-N-SH (human neuroblastoma,Atty. Dkt. No.: 121384-0302RRID: CVCL 0531), and HEK293T (human embryonic kidney, RRID: CVCL 0063) cell lines from the American Type Culture Collection (ATCC). Applicant cultured HT29, SW480, and HEK293 cells in DMEM, NCI-H441 cells in RPML1640, and SK-N-SH cells in IMDM. All media were supplemented with 10% FBS and 1% penicillin / streptomycin and maintained at 37 °C in a humidified 5% CO2-humidified incubator.[0106| Molecular docking: Applicant employed a semi-flexible shape-complementary docking approach to predict peptide-proteasome binding sites and interaction modes. The peptides AAAG and RRRG were docked with the 26S proteasome complex (PDB ID:5VY3), the 19S subunit (extracted from 26S), and PSMD2 (extracted from 19S) utilizing AutoDock Vina 1.1.2. Initially, RRRG and AAAG were docked with the 26S complex.Subsequently, the 19S subunit exhibiting the most favorable docking position was extracted for further analysis. PSMD2 was then selected as the final docking subunit based on optimized docking poses. Applicant conducted protein preprocessing, encompassing the removal of water molecules and redundant ligands, as well as the addition of hydrogen atoms, using PyMol 2.4. The 3D peptide structures were generated, and energy was minimized using ChemDraw 20.0. PDBQT files for docking simulations were created with AutoDock Tools 1.5.6. For the 26S proteasome, the docking box dimensions were set to 120 A x 124 A x 126 A with a grid spacing of 1.00 A, centered at coordinates x: 116.487, y: 116.484, z: 116.481. For the 19S subunit, the docking box dimensions were 72 A x 114 A x 116 A with a grid spacing of 1.00 A, centered at coordinates x: 107.899, y: 0.658, z: 24.371. For PSMD2, the docking box dimensions were 74 A x 60 A x 74 A with a grid spacing of 1.00 A, centered at coordinates x: 209.927, y: 210.964, z: 195.545. All other docking parameters were maintained at default values. The docking protocol generated the top nine binding poses, with the conformation exhibiting the lowest binding energy and highest cluster frequency designated as the most probable binding mode between the ligand and protein. Finally, PLIP and PyMol 2.4 were used to visualize the docking results (Trott, O. & Olson, A. J. J. Comput. Chem. 31, 455-461 (2010)).

[0107] Fabrication of IgG condensates: To form the IgG condensates Applicant dissolved cRs in water (1.4 mg / mL), while D10 was initially dissolved in DMSO (10 mg / mL) before dilution in Tris buffer (5 mM, pH 8.5) to a final concentration of 1.0 mg / mL. IgG-BMCsAtty. Dkt. No.: 121384-0302were formed by mixing c Rs and DIO in water (200 pL) at varying charge ratios (0.1, 0.5, 1, 2, 5, 10, 20) while maintaining a constant total charge of 1.2 mM; the volume ratio was standardized to 1:5. IgG-BMC@MPNs were prepared by mixing c Rs and D10 in water (200 pL) at a 1:5 volume ratio, followed by the addition of EGCG (0.5 mg / mL) and FeCh 6H2O (1.0 mgmL-1) at varying volume ratios (0.1, 0.5, 1, 2, 5, 10) to a final volume of 300 pL, maintaining a 1:3 volume ratio. Applicant added tris buffer (10 mM, pH 8.5, 350 pL) to adjust pH, and the resulting IgG-BMC@MPNs were collected by centrifugation (900 ref, 5 min), washed with water, and stored at 4 °C. IgG-IgG-BMC@MPNs were prepared by premixing c Rs with IgG (1 mg / mL) overnight at 4 °C before proceeding with the steps.

[0108] Applicant monitored the phase separation behavior between c Rs and D10, along with MPN deposition, through turbidity measurements utilizing a UV-Vis spectrometer within glass-bottom 384-well plates. The extinction at 600 nm (OD600) of BMC samples, encompassing pure peptides, MPN precursors, and their mixtures, was employed to compute relative turbidity (see Jin, Z. el al. ACS Nano 17, 16980-16992 (2023)).

[0109] Applicant determined the IgG loading efficiency by collecting and disassembling the IgG-COA@MPN using ultra high-speed centrifugation at 30,000 ref, subsequently quantifying the encapsulated IgG amount in supernatant via enzyme-linked immunosorbent assay (ELISA). Applicant calculated loading capacity (LC%) and encapsulation efficacy (EE%) based on the following equations:Weight of IgG encapsulatedLD% = x 100% Weight of C0A@MPNs formed by 100 pg cR8Weiqht of IqG encapsulatedEE% = - - -7 r r— - x 100%Weight of feeding IgG

[0110] Characterization of IgG-BMC@MPNs: To characterize IgG-BMC@MPNs applicant used Hydrodynamic size (DH) and zeta potential: The DH / PDI (Vtot = 50 pL) and zeta potential (Vtot = 800 pL) of BMCs or BMC@MPNs samples using a compact Zetasizer after 1 minute of thorough mixing.Atty. Dkt. No.: 121384-0302[01111 Isothermal Titration Calorimetry (ITC) analysis'. The Applicant performed isothermal titration calorimetry by filling the sample cell with an EGCG solution (1 mg / mL, 350 pL), while the reference cell contained double distilled water. The calorimeter was equipped with an injection syringe loaded with c Rs solution. To initiate the experiment, an initial 0.4 pL injection of cRs (1.0 mg / mL) was followed by 14 subsequent 2 pL injections until thermal equilibrium was reached. The sample cell contents were continuously stirred at 1000 rpm and maintained at 298 K. Applicant found that raw calorimetric data, represented as a plot of heat change (pcal / s) against injection time, exhibited a series of peaks corresponding to each injection. These peaks were processed by the instrument's software to generate a plot of enthalpy change per mole of inj ectant (kcal / mol) against the molar ratio.[01121 Colloidal stability. Applicant measured thermo stability by mixing, BMCs or BMC@MPNs samples in water at the concentration of 1,020 particles / mL and shaken at room temperature or 37 °C at the rate of 300 rpm using Eppendorf ThermoMixer. Applicant measured osmotic stability by mixing BMCs or BMC@MPNs samples at solutions varying in osmotic pressure (normal saline; 100 mM or IM sodium chloride; IM PBS at pH of 5.5, 7.4, and 8.5; DMEM and DMEM supplemented with 10% FBS) at the same particle concentration and shaken at room temperature or 37 °C at the rate of 300 rpm using Eppendorf ThermoMixer. Applicant determined droplet coalescence levels turbidimetrically using the previously described equation.

[0113] Release profile '. Applicant generated time-dependent release curves for IgG-BMC@MPNs under pH conditions simulating the stomach (pH 5.5), cellular fluid (pH 7.4), and intestinal environment (pH 8.5). To quantify the release of gold nanoparticles (AuNPs), which were employed as cargo by labeling IgG with 10 nm AuNPs, samples were subjected to ICP-MS. IgG-BMC@MPNs were dispersed in 1 M PBS solutions at the indicated pH values to achieve a concentration of 1050 particles / mL. Applicant loaded these suspensions were into the upper compartments of CostarR Spin-XR centrifuge filter tubes equipped with 0.22 um pore size cellulose acetate (CA) membranes, while the lower compartments were filled with 1 mL of the corresponding PBS solution. The tubes were subjected to shaking at 300 rpm and 37 °C using an Eppendorf ThermoMixer. Applicant extracted 50 uL aliquots were extracted from the lower compartment, digested with aqua regia, diluted 1 : 10,000 in 4%Atty. Dkt. No.: 121384-0302nitric acid, and analyzed by inductively coupled plasma mass spectrometry (ICPMS), at predetermined time points. Applicant determined the total gold content, representing the initial loading amount, from unprocessed IgG-BMC@MPNs in the respective PBS solutions. Applicant calculated the cumulative release percentage based on the following equation:Cumulative release % = —Au’releasedx100%tot.[0114| Morphology study. Applicant obtained bright-field TEM, HADDF, and EDX spectroscopy images using a JEOL JEM-ARM200CF microscope operated at 200 kV in scanning transmission electron microscopy (STEM) mode. Additional TEM images of BMC@MPNs at various orientations were captured on a JEOL JEM-3200FS microscope at 300 kV. SEM images were acquired with a JEOL JSM-7900FLV microscope at an accelerating voltage of 10 kV following deposition of a 10 nm osmium tetroxide (OsO4) coating using a Filgen OPC60A osmium coater. Applicant determined nanomechanical properties by atomic force microscopy (AFM) in contact mode using a Bruker ICON instrument.

[0115] Cell uptake efficacy study: For cell transfection, Applicant seeded SW480 cells at 2.5 x 105 cells / well in 12-well plates overnight. Cells were washed with reduced serum Opti-MEM media to remove FBS. AF488-IgG-BMC@MPNs were pre-mixed with reduced serum Opti-MEM media at a parti cle-to-cell ratio of 1000: 1, then added to the cells and incubated for 4 hours. Commercial protein transfection reagents, Pro-Ject and Xfect, were applied according to the manufacturer's protocols.

[0116] For flow cytometry analysis, Applicant enzymatically dissociated SW480 cells using trypsin, washed twice with PBS, and resuspended in PBS at a concentration of 1 million cells per milliliter prior to analysis.

[0117] For both TEM and SEM analyses, Applicant seeded SW480 cells on 12 millimeter (mm) coverslips and processed as previously described. For TEM, monolayers were fixed in 2.5% glutaraldehyde / 2% paraformaldehyde / 0.1 M cacodylate buffer (pH 7.4) at 4 °C, followed by post-fixation in 1% osmium tetroxide and 2% uranyl acetate staining. ApplciantAtty. Dkt. No.: 121384-0302dehydrated samples through a graded ethanol series and embedded in Durcupan™ ACM resin. Applicant cut ultrathin sections (70 or 100 nm) using a Leica ultramicrotome and imaged on a JEOL JEM-3200FS electron microscope operating at 300 kV. For SEM, cells were processed identically until dehydration, followed by critical point drying and coating with 10 nm osmium tetroxide. SEM images were acquired using a JEOL JSM-7900FLV at 5 kV acceleration voltage.

[0118] Applicant employed confocal microscopy to investigate the intracellular uptake and distribution of BMC@MPNs in cultured cells. Following fixation with 4% paraformaldehyde, cells were immunostained with Texas Red™-X Phalloidin for 45 minutes at room temperature to visualize actin filaments, followed by Hoechst 33342 nuclear staining for 15 minutes. Cellular association and localization of the capsules were assessed using a Nikon SoRa spinning disk confocal microscope, with subsequent image analysis and colocalization quantification performed using NIS-Elements and Fiji software.10119] Cell internalization study: To investigate the pathway of condensate internalization, Applicant treated SW480 cells with various inhibitors including chlorpromazine (30 pM), 5-(N-Ethyl-N-isopropyl) amiloride (50 pM), sodium azide (100 mM), or methyl-P-cyclodextrin (2.5 mM) for 1 hour, followed by the addition of 100 pl AF488-loaded-IgG-BMC@MPNs for an additional 4 hours. Applicant washed cells twice subsequently washed with PBS and analyzed via fluorescence microscopy or flow cytometry after trypsinization. A control group exposed to AF488-loaded-IgG-BMC@MPNs without inhibitors was included, as well as an untreated negative control. Additionally, Applicant pre-incubated a group of cells for 1 hour at 4 °C and maintained at this temperature during the 4-hour uptake process to assess temperature-dependent internalization.|0120] To investigate endosomal escape of BMC@MPNs, Applicant seeded SW480 cells in 12-well plates at 5xl05cells / well in 1 mL of DMEM with 10% FBS and incubated overnight. The next day, media were replaced with 500 pL fresh DMEM to minimize nanoparticle sedimentation distance. Calcein (1.5 mg / mL in DPBS) and bafilomycin Al (for the inhibition group) were added to achieve final concentrations of 150 pg / mL and 100 nM, respectively, in the presences or absence of nanoparticles (untreated, BMCs, or BMC@MPNs). Cells wereAtty. Dkt. No.: 121384-0302incubated for 2 hours. Non-fluorescent BMCs and BMS@MPNs were used to prevent signal interference. After incubation, cells were washed three times with DPBS to remove extracellular calcein and BMCs, then imaged by confocal microscopy.

[0121] Protein of interest degradation study: To assess the inhibitory effect of IgG-BMC@MPNs on KRASG12V or a-synuclein, Applicant seeded 2.5 * 105 SW480 cells per well in 12-well plates and incubated with IgG-BMC@MPNs for varying durations prior to cell lysis or flow cytometry analysis (FACS). For western blotting, Applicant subjected cell lysates to SDS-PAGE, followed by immunoblotting with specific antibodies (Table 1) and band quantification using FIJI software normalized to P-actin. FACS was employed as a complementary method to evaluate protein degradation. Cells were trypsinized, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with AF555-conjugated anti-KRAS of a different isotype than the cargo antibody to prevent binding site competition before FACS analysis (Table 1).Table 1: Therapeutic antibody cargo listsAntibodies ConcentrationAnti-RAS 0.5 mg / mLRabbit IgG, isotype Ctrl 0.5 mg / mLAF488 Anti-Ras 0.5 mg / mLAnti-RAS (G12B mutant) 0.365 mg / mLAnti-a-synuclein 0.645 mg / mL

[0122] Protein-Protein Interaction and proteasome engagement study: To validate the intracellular interaction between IgG and the POI, Applicant performed a concentrationdependent co-immunoprecipitation (co-IP) assay. Applicant exposed SW480 cells (2.5 x 106 per well) to cell-to-particle ratios of 1 : 100, 1 :200, and 1 :500 for 24 hours within a 12-well plate format, followed by cell lysis. Active GTP-bound RAS proteins were immunoprecipitated using cRAFRBD-immobilized agarose beads for 2 hours at 4°C.Atty. Dkt. No.: 121384-0302Following washes with lysis buffer, equal amounts of immunoprecipitated products were subjected to Western blot analysis. Free anti -KRAS, empty BMC@MPNs, and isotype-loaded BMC@MPNs served as controls.

[0123] To investigate the potential involvement of proteasomes in the degradation process, Applicant pretreated cells with 10 nM MG132, 20 pM chloroquine, 25 nM bortezomib, 500 nM TAK-243, or pepstatin A / leupeptin (10 pM / 20 pM) for 4 hours before being intubated with IgG-BMC@MPNs at a 1:200 cell-to-particle ratio for 24 hours before lysis. Residual protein levels were then determined by Western blot analysis of SDS-PAGE resolved samples.

[0124] Label-free quantification (LFQ) proteomics: Applicant modified the Rs precursor with a 6-amino acid His tag for pulling down protein complexes. SW480 or SK-N-SH cells were incubated with anti-KRAS or anti-a-syn-BMC@MPNs for 48 hours before lysis. Cell lysates were incubated with Ni-NTA His-Bind overnight according to the manufacturer's protocol. The eluent was processed for label-free affinity purification-MS (AP-MS) using an Orbitrap Exploris 240. Data was processed using MaxQuant and Perseus at Northwestern University Proteomics Center of Excellence Core Facility (PCE).

[0125] Proximity litigation assay (PLA) for evaluating BMC@MPNs binding to endogenous targets: Applicant performed PLA to evaluate intracellular binding of IgG-BMS@MPNs to endogenous KRAS or a-syn using Duolink® Proximity Litigation Assay Kit (Sigma Aldrich). Applicant seeded cells on glass coverslips, treated with anti-KRAS-BMC@MPNs or controls for 12 hours, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. After blocking, cells were incubated with rabbit anti-KRAS and mouse anti-human Fc primary antibodies overnight at 4 °C. PLA probes were applied, followed by ligation, amplification, and detection per manufacturer’s instructions.Fluorescent PLA puncta were visualized by confocal microscopy.

[0126] CHX-chase assay for KRAS degradation: Applicant seeded SW480 cells in 6-well plates and allowed to adhere overnight. The following day, Applicant treated the cells with either non-specific isotype-BMC@MPNs or anti-KRAS-BMC@MPNs for 2 hours to allow intracellular uptake and engagement of KRAS targets. After treatment, Applicant replacedAtty. Dkt. No.: 121384-0302the culture medium with fresh medium containing 50 pg / mL CHX to inhibit de novo protein synthesis. Applicant harvested the cells at multiple time points following CHX addition (0, 4, 8, and 12 hours). At each time point, cells were washed with cold PBS and lysed using RIPA buffer supplemented with protease inhibitors. Protein concentrations were calibrated by BCA assay, and equal amounts of total protein were subjected to SDS-PAGE, followed by WB using anti-KRAS and anti-GAPDH. The remaining KRAS protein levels at each time point were quantified by densitometry using ImageJ software and normalized to GAPDH. A reduction in KRAS half-life following anti-KRAS-BMC@MPN treatment compared to controls was interpreted as evidence of targeted KRAS degradation.

[0127] Co-culture anti-proliferation study: Applicant labeled HT29 cells and SW480 cells with QTracker 800 and QTracker 625 according to the manufacturer's protocol. Both cell lines were then stained with CFSE and mixed at a 1 : 1 ratio. Applicant seeded cell mixtures in 12-well plates at 2.5 * 105 cells / well in complete DMEM. After being incubated with anti-KRASG12V-BMC@MPNs (cell-to-particle ratio = 1:2000) for different durations, Applicant dissociated cells using trypsin, washed with PBS twice, and proceeded to FACS.

[0128] Orthogonal degradation validation: Applicant plated HT29, NCI-H441, and SW480 cells in 12-well plates at a density of 2.5 x 105 cells / well overnight. After being incubated with a blank medium, BMC@MPNs, anti-KRAS-BMC@MPNs, or anti-KRASG12V-BMC@MPNs at a cell-to-particle ratio of 1 :2000 for 48 hr, cells were lysed and proceeded to Western blot to quantify the total level of remaining KRAS and the percentage of KRASG12V. Antibodies used in this assay are listed in Table 1.

[0129] Construct of a-synuclein overload model SK-N-SH Cells: To generate lentiviral particles expressing human a-synuclein, Applicant cloned the a-synuclein gene into the lentiviral vector pLVX-IRES-ZsGreenl, creating pLVX-IRES-ZsGreenl / a-syn3. HEK 293T cells were co-transfected with pLVX-IRES-ZsGreenl / a-syn (or control empty vector), the packaging plasmid psPAX2, and the envelope plasmid pMD2G. After 72 hours, the culture medium containing lentiviral particles (LV-a-syn) was collected, centrifuged at 3000 rpm for 20 minutes, and determined to have titers of up to 3-4* 106infectious units per milliliter.Atty. Dkt. No.: 121384-0302[013O| Cell toxicity study: Applicant assessed the cell viability of HT29, SW470, NCI-H441, or SK-N-SH cells using either the CCK8 or LDH assay according to the manufacturer’s protocol. Briefly, SK-N-SH cells were seeded in 48-well plates and incubated for 24 hours before transfection with either pLVX-IRES-ZsGreenl or pLVX-IRES-ZsGreenl / a-syn for an additional 24 hours, followed by experimental treatments. For the CCK8 assay, absorbance was measured at 490 nm. For the LDH assay, the absorbance of the supernatant was measured at 490 nm.

[0131] In vivo tumor treatment efficacy: Applicant subcutaneously injected the SW480 cells (5 x 106 cells / mouse) into athymic nude mice. After one week of tumor cell inoculation, mice were randomly divided into four groups (n=8 / group) and treated with PBS, BMC@MPN, isotype-BMC@MPN, or anti-KRAS-BMC@MPN (10 mpk) via intratumoral injection thrice weekly. Tumor volumes were measured every two days throughout the treatment period. At the study endpoint (day 21), mice were euthanized, and tumor tissues were collected for subsequent analyses.

[0132] Applicant assessed cell apoptosis and proliferation using TUNEL, capsase-3, and Ki67 immunohistochemical staining, respectively. Isolated tumor tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned to a thickness of 5 um, and subjected to immunohistochemistry according to standard protocols. Quantitative analysis was performed by microscopic examination of five representative fields per section.

[0133] Tumor specimens were sectioned into 5 pm thick slices, stained with hematoxylin and eosin (H&E), and examined under an optical microscope for histological evaluation.

[0134] Tumor growth inhibition index (TGI %) was calculated based on the following equations:TGI % =x 10Q% (Ep,endVlsotype,EP Vlsotype.dayO

[0135] Animal Care: Applicant housed mice were housed in an animal facility at Northwestern University (NU) under federal, state, local, and National Institutes of HealthAtty. Dkt. No.: 121384-0302(NIH) guidelines. All animal experiments were performed in accordance with NIH guidelines and approved by the Institutional Animal Care and Use Committee (IACUC).

[0136] In Vivo safety studies: Applicant collected blood samples for the evaluation of serum chemistry, without using anticoagulant and left to clot for 30 minutes at room temperature. The samples were then centrifuged at 5,000g to separate the serum. All analyses were conducted by IDEXX Bioanalytics Services.Example 2: Engineering of IgGBMCs with robust cytosolic entry

[0137] The Applicants representative LLPS precursor consists of arginine- rich peptide (C-CRs-C-GG-RRRG) and an aspartic-acid decapeptide (Dio) with complementary charge valency (FIG. 1A). Electrostatic interactions between these peptides drive complex BMC formation, with molecular charge on the backbone attributed to the positive guanidinium and negative carboxylate groups, thereby inducing LLPS and concomitantly condensing IgGs through coulombic interactions (Jin, Z. et al. ACS Nano 17, 16980-16992 (2023)). Applicant cyclized the octaargine (cRs) moiety of arginine-rich precursor via a disulfide bond between two cysteine residues to serve as the cell-penetrating domain (CPD) and incorporated a RRRG fragment as the proteasome-targeting motif (PTM) (Bonger, K. M., Chen, L.-c., Liu, C. W. & Wandless, T. J. Nat. Chem. Biol. 7, 531-537 (2011); Qu, J. et al. Cell Chem. Biol.27, 751-762. e4 (2020)). Applicant introduced a GG spacer between the two domains, promoting an extended PTM conformation for optimal proteasome engagement. Applicant hypothesized the integration of cell-penetrating and proteasome-recruiting elements into LLPS precursors would facilitatelgG-BMCs in (1) condensing and transporting of IgG to bind the cytosol, and (2) variant- selective binding between IgG and intracellular targets, and (3) recruiting the proteasome to the IgG-target complex (FIG. IB).

[0138] Applicant first incubated IgG with the the arginine-rich precursor, followed by adding Dio to induce LLPS, and then coated with epigallocatechin-3 -gallate (EGCG) / FeC13 to form the MPN shell (FIG. 2A). A one-pot strategy was used to fabricate BMCs through sequential mixing of IgG, LLPS precursors, epigallocatechin gallate (EGCG), and FeCL (FIG. 2A, FIG. 6A-6B). Applicant characterized the non-covalent complexation between IgG and the arginine precursor by increased zeta potential (Q with an apparent KD of 4.3 ± 1.8 pM,Atty. Dkt. No.: 121384-0302indicating sufficient binding affinity to support IgG-precursor complex for target engagement and proteasome recruitment in cells (FIG. 2B). Applicant characterized the coacervation and coating process using UV-vis spectroscopy, with increased ODeoo indicating BMC@MPNs scattering and a red shift of 40 nm at A280 suggesting MPN formation (FIG. 2B). At a physiological temperature (37°C) and varying osmotic pressures, purified IgG-BMC@MPNs remained as monodisperse, stable droplets after air-drying, demonstrating enhanced colloidal stability conferred by the semipermeable MPN coating, in contrast to the rapid coalescence seen in uncoated BMCs (FIG. 2C). Applicant confirmed the elemental composition of BMC@MPNs using high-angle annular dark field (HAADF) imaging and energy-dispersive X-ray spectroscopy (EDX) (FIG. 3F). Applicant used EDX mapping to show co-localized C, N, O, and Fe signals within individual BMC@MPNs, indicating successful MPN deposition (Fig. 3F). The coated particles measured approximately 300 nm, encapsulating 289.4 pg of IgG per 100 pg of pArg, enabling robust IgG trafficking compared to larger uncoated BMCs (~2 pm) or simple arginine / IgG mixtures.

[0139] The resulting IgG-BMC@MPNs were purified through iterative centrifugation and redispersion, demonstrating enhanced colloidal stability. Unlike uncoated BMCs that coalesce after formation, IgG-BMCs remain dispersible in the buffer of choice even after airdrying (FIG. 2C). When exposed to 37 °C or aqueous solutions with different osmotic pressures for up to 48 hr (FIG. 10A-10B), turbidity remained stable for IgG-BMC@MPNs but decreased significantly in uncoated IgG-BMCs due to coalescence, indicating enhanced colloidal stability following the coating. This stability was further validated by an increased zeta (Q potential after coating (FIG. 7A-7C). The monodisperse core-shell structure, along with the distribution of C, O, N, and Fe in the coating, was confirmed through analysis by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray (EDX) elemental mapping. (FIG. 2D, FIG. 8A-8G, FIG. 9A-9L, FIG. 10A-10B). Remarkably, IgG-BMC@MPNs displayed a reduced size of 400 nm, making them better candidates for cellular uptake compared to the undecorated BMCs at about 2 pm (FIG. 2E)

[0140] Applicant hypothesized that the guest molecules could be released from BMCs into the cytosol, where cellular macromolecules and ions might disrupt LLPS through competitiveAtty. Dkt. No.: 121384-0302multivalent interactions, with the MPN semipermeable coating preserving condensate integrity and regulating IgG release. To test this, Applicant monitored the turbidity of BMCs with or without MPN coatings in DMEM supplemented with 10% FBS. Applicant found that uncoated BMCs showed rapid turbidity loss, indicating droplet fusion and subsequent IgG release into the dilute phase. In contrast, MPN-coated BMCs remained stable droplets, confirming the MPN’s protective role in preserving droplet integrity and regulating IgG release from the condensed phase (FIG. 2D). Applicant further evaluated the role of the MPN dissociation in regulating IgG-BMC@MPN release kinetics at pH 5.4, 7.0, and 8.2, which simulate lysosome, cytosol, and mitochondrial environments, respectively. IgGs labeled with 10 nm colloidal gold nanoparticles (cAuNPs) were quantified by ICP-MS (FIG. 2G). IgGs labeled with 10 nm colloidal gold nanoparticles (cAuNPs) were released from BMC@MPNs via dialysis and quantified by ICP-MS (FIG. 2G). Release was most rapid at pH 5.4, reaching 80% within 15 hours, and progressively slower at neutral and basic pH. These results highlight the role of MPN in stabilizing BMCs and enabling environmentally responsive IgG release, two aspects critical for cytosolic delivery.

[0141] IgG loading capacity of IgG-BMC@MPNs was quantified using an enzyme-linked immunosorbent assay (ELISA). IgG-BMC@MPNs produced with 100 pg of pArg encapsulated 289.4 pg of IgG, resulting in a high loading capacity with a 3:1 mass ratio (FIG.2M). We then tested the in vitro release kinetics of IgG-BMCs-BMCs at pH 5.4, 7.0, and 8.2, corresponding to the stomach, extracellular fluid, and intestinal environments. The IgGs were tagged with 10 nm colloidal gold nanoparticles (cAuNPs) and quantified via inductively coupled plasma mass spectrometry (ICP-MS) (FIG. 2G, FIG. 11A-11B). The accumulative release profile revealed the highest and fastest IgG release under acidic conditions at 80% in 15 hr, followed by neutral and basic environments. This observation is likely linked to the pH-dependent dissociation rates of MPNs (Guo, J. etal. Angew. Chem., Int. Ed. 53, 5546-5551 (2014)).

[0142] Applicant assessed the cellular internalization of IgG-BMC@MPNs. Applicant quantified particle concentration (particles / mL) by nanoparticle tracking analyzer (NTA) and established the cellular uptake saturation curve by varying particle concentration with a fixed cell number (FIG. 12A-12D). A 1:1000 cell-to-particle ratio was selected for furtherAtty. Dkt. No.: 121384-0302experiments based on the uptake saturation curve. Subsequently, Applicant compared the delivery efficiency of Alexa Fluor 488 (AF488)-labeled IgG-BMC@MPNs in SW480 cells with macro IgG-BMCs, commercial protein transfection reagents (Pro-Ject, lipid-based; Xfect, CPP -based), and using free AF488-IgG as a control. IgG-BMC@MPNs exhibited superior internalization rate, with fFluorescence-activated cell sorting (FACS) analysis confirming 65% IgG delivery efficiency, outperforming Pro-Ject (9%), Xfect (47%), and uncoated IgG-BMCs (27%) (FIG.2H; FIG 13A-13B). Fluorescence-activated cell sorting (FACS) results revealed that IgG-BMC@MPNs achieved a level of 99% positive cells after 4-hours of incubation, significantly outperforming Pro-Ject (32%), Xfect (42%), and uncoated macro IgG-BMCs (54%) (FIG.2N). Applicant used fluorescence microscopy to further validate rapid intracellular localization, with update detectable within 5 minutes and maximal accumulation at 4 hours post-transfection (FIG.21). Applicant demonstrated the enhanced cell entry performance of IgG-BMC@MPNs over conventional synthetic carriers and micro BMCs. Applicant confirmed the internalization of IgG-BMC@MPNs into the cells, opposed to cell surface adherence (FIG.31). Notably, IgG-BMC@MPNs uptake occurred within 5 minutes after addition to the cell culture medium, with fluorescence intensity peaking at 4-hr post-transfection (FIG. 14A-14D; FIG. 15A-15B).

[0143] Applicant investigated the mechanism underlying the high internalization efficiency, by pretreating SW480 cells with a panel of inhibitors before incubating with IgG-BMC@MPNs or IgG-BMCs. Inhibitors targeting passive or non-specific uptake included methyl-P-cyclodextrin (MpCD, lipid raft depletion), cytochalasin D (CyD, actin polymerization inhibitor), and 5-(N-ethyl-N-isopropyl)amiloride (EIP A, pinocytosis inhibitor), while sodium azide (NaNs, ATP depletion) and chlorpromazine (CPM, clathrin-mediated endocytosis inhibitor) were used to block active or receptor-mediated endocytosis. The pinocytosis inhibitor amiloride (AM) did not significantly impact the uptake of AF488-IgG-BMC@MPNs. In contrast, treatment with methyl-P-cyclodextrin (MpCD) reduced nano BMC@ MPN update to 43% and micro BMC uptake to 32%. CyD and EIPA decreased micro BMC uptake to 42% and 36%, and nano BMC@MPN uptake to 72% and 62%, respectively. NaNs and CPM significantly inhibited nano BMC@MPN uptake (15% and 23%), while micro BMCs remained largely unaffected. Applicants found that BMC@MPNs exploit energy-dependent, clathrin-mediated endocytosis, likely enabled by their smaller size,Atty. Dkt. No.: 121384-0302which allows them to interact with receptors and engage vesicles for entering cells (FIG. 20). Applicant found that BMC@MPNs produced a more uniform intracellular fluorescence distribution, suggesting that nano-BMCs enable a homogeneous cellular uptake, which is important for precise dosing control. These findings are consistent with TEM observations showing nano BMC@MPNs enclosed within intracellular vesicles, a characteristic of active transport (FIG. 2P). Following uptake, BMC@MPNs achieve endosomal escape via the proton pump effect of the MPN coating, enabling cytosolic delivery. Applicant found sodium azide (NaNs), which deplete cholesterol and ATP, respectively, reduced uptake by 67% and 52%, indicating a cholesterol-dependent, energy-driven endocytic process was operative similar to cell-penetrating peptide (CPP) mechanisms (Futaki, S. & Nakase, I. Acc. Chem. Res. 50, 2449-2456 (2017)). Chlorpromazine (CPM), a clathrin inhibitor, reduced uptake by 27%, also suggesting clathrin-mediated endocytosis involvement, possibly due to nanoparticle size reduction (Rennick, J. J., Johnston, A. P. R. & Parton, R. G. Nat.NanotechnoL 16, 266-276 (2021)). Uptake occurred at low temperatures (4 °C), implying direct penetration through cell membranes did not require endocytosis, ascribed to the function of the CPP domain (FIG. 2 J) (Futaki, S. & Nakase, I. Acc. Chem. Res. 50, 2449-2456 (2017)). These results were cross-validated by AF488 signal visualization via fluorescence microscopy (FIG. 16A-16B). Electron microscopy images further illustrated the interaction between IgG-BMC@MPNs with the cell membrane (FIG. 2K; FIG. 17A-17C).These findings highlight multiple modes of both active and passive uptake contributing to IgG-BMC@MPNs’ internalization, a feature not observed in current micro-condensates (Sun, Y. et a!. Nat. Chem. 14, 274-283 (2022); Shebanova, A. et al. Adv. Set. 11, 2402652 (2024)).Example 3: IgG-BMC@MPNs induce mutant-specific KRASG12Vdegradation

[0144] Applicant examined whether the condensed IgG could engage intracellular targets via antigen recognition using KRAS, a 21 kDa cytoplasmic protein lacking druggable pockets, was selected as the target. Mutant KRAS, which is implicated in several cancers, is considered 'undruggable' due to the absence of binding sites for conventional degraders (Huang, L., Guo, Z., Wang, F. & Fu, L. Signal Transduct. Target. Ther. 6, 386 (2021);Parikh, K. et al. J. HematoL Oncol. 15, 152 (2022)). Applicant hypothesized that IgG-BMC@MPNs could enable selective degradation through antibody-guided recognition. TheAtty. Dkt. No.: 121384-0302ATI-driven targeting of IgG-BMC@MPNs could overcome this limitation to achieve effective KRAS degradation. Applicant treated SW480 cells with free anti-KRAS, empty BMC@MPNs, or BMC@MPNs loaded with 5um isotype of anti-KRAS antibodies for 23 hours followed by immunoprecipitation (IP) using the cRafRBD domain, which binds KRAS at a non-overlapping site relative to the antibody epitope. Pull-down blots revealed strong binding between endogenous KRAS and anti-KRAS, with negligible binding to isotype-BMC@MPNs, as indicated by heavy chain (HC) band intensity. Applicant found that the HC signal increased with anti-KRAS-BMC@MPN concentration, confirming dose-dependent intracellular target engagement. Correspondingly, Applicant observed a reduction in active KRAS pulled down by cRafRBD only with anti-KRAS-BMC@MPNs, not with isotype controls, indicating that target recognition is essential for degradation. (FIG. 3A). Western blot (WB) analysis revealed KRAS degradation started within 8 hours, with a maximum reduction to 67% at 48 hours, followed by partial recovery to -70% by day 6. Applicant observed no degradation in cells treated with isotype-BMC@MPNs, serving as the negative control . Upon inhibition of protein synthesis, cycloheximide (CHX) chase assay showed that anti-KRAS-BMC@MPNs reduced the KRAS half-life to less than 12 hours, compared to 22-24 hours in isotype control and CHX-only groups, indicating that IgG-BMC@MPNs accelerate KRAS degradation. These results demonstrate that IgG-BMC@MPNs achieve intracellular target engagement, induce and accelerate KRAS degradation upon internalization. The assays using the cRaf domain, selected because of its non-competitive binding with cargo antibodies. Pull-down blot analysis revealed concentration-dependent IgG heavy-chain (HC) bands, confirming that the cargo antibody binds KRAS (FIG. 3A; FIG. 18A-18B)[01451 Applicant hypothesized that mutant-specific degradation would selectively inhibit mutant cell proliferation. Applicant evaluated the proliferation inhibition effect of KRAS degradation in a co-culture of HT29WT / WTand SW480G12V / G12Vcells (FIG. 3G). Applicant barcoded HT29 and SW480 cells with Qtracker 800 and 625, respectively. Both cell types were mixed at equal proportions and labeled with carboxyfluorescein succinimidyl ester (CFSE) as an indicator of cell division, where fluorescence signal dilution reflects cell division. After six days of treatment with anti-KRASG12V-BMC@MPNs, Applicant found that HT29 cells became dominant (FIG. 31), in contrast to the untreated group, where SW480Atty. Dkt. No.: 121384-0302cells slightly outcompeted HT29 due to their faster doubling time (FIG. 3H). Applicant found that CFSE fluorescence in the untreated group was uniformly weak, indicating high proliferation (FIG. 3H), while strong CFSE signals in the treated group co-localized with SW480 cells, reflecting suppressed division (FIG. 31). Flow cytometry quantification reinforced this trend when barcode-labeled cell populations were sorted and ratios measured. Applicant found that the results indicated dominance of HT29 cells in the treated group due to growth suppression of SW480 cells at the ratio of 82% to 18% (FIG. 3K). Applicant found that in the untreated group, SW480 cells slightly outcompeted HT29 at the ratio of 58% to 42% due to their shorter doubling time (FIG. 3J). Applicant observed significant suppression of proliferative activity was observed in SW480 subpopulation (17% proliferating cells with anti-KRASG12V-BMC@MPNs vs. 62% in NC, (FIG. 3M), whereas HT29 cells remained unaffected (40% with anti-KRASG12V-BMC@MPNs vs. 42% in NC, FIG. 3L).

[0146] KRAS level changes were monitored using western blot (WB) after treatment with anti-KRAS-BMC@MPNs. Efficient degradation of the mutated protein was observed, with WB results indicating an average degradation level of 67% within 48 hrs (FIG. 3B). To assess the half-life of KRAS degradation, residual KRAS was stained with APC-conjugated antibodies after treatment and subsequently quantified using flow cytometry. Analysis of the degradation curve indicated 59.3% degradation within two days, with levels returning to baseline by six days post-treatment, while the isotype-BMC@MPNs control group showed no changes in KRAS level (FIG. 3C; FIG. 19A-19B). These findings suggest a 2-day functional lifespan of the cargo IgG, aligning with WB results.

[0147] Oncogenic KRAS is characterized by a single amino acid mutation that current degraders are unable to selectively target under their native expression conditions40,42(Huang, L., Guo, Z., Wang, F. & Fu, L. Signal Transduct. Target. Ther. 6, 386 (2021); Martin, A. et al. biaRxiv (2024)). The use of mutant-specific antibodies would enable selective degradation of the KRASG12Vsubtype without affecting the wild-type, which is crucial for normal cell survival (FIG. 3D; Huang, L., Guo, Z., Wang, F. & Fu, ^.Signal Transduct. Target. Ther. 6, 386 (2021)). To test this, HT29 (wild-type homozygous), SW480 (G12V-mutant homozygous), and NCI-H441 (wild-type / G12V-mutant heterozygous) cells were treated with either anti-pan-KRAS-BMC@MPNs, which bind all KRAS subtypes, or anti-KRASG12V-Atty. Dkt. No.: 121384-0302BMC@MPNs, which only bind the KRASG12Vmutant. Residual wild-type or KRASG12Vlevels were then resolved using orthogonal immunoblotting. Results showed that while total KRAS levels were reduced in all cell lines treated with anti-pan-KRAS-BMC@MPNs, only NCI-H441WT / G12Vand SW480G12V / G12Vcells responded to anti-KRASG12V-BMC@MPNs treatment, aligning with their expression of the mutant subtype (FIG. 3E). This suggests the anti-KRASG12Vconstruct is essential for mutant-specific degradation (FIG. 3E-3F; FIG. 20).In NCI-H441WT / G12Vcells, the remaining total KRAS levels were similar for both treatments (63% for anti-pan-KRAS input vs. 72% for anti-KRASG12Vinput). However, exposure to anti-KRASG12Vreduced the KRASG12V / total KRAS ratio (0.58 for anti-pan-KRAS vs. 0.26 for anti-KRASG12V), whereas total KRAS levels remained stable compared to the untreated group (56% for NC vs. 48% for anti-pan-KRAS; FIG. 3F; FIG. 20). This finding is consistent with the result that HT29 wildtype homozygous cells did not respond to anti-KRASG12V-BMC@MPN treatment (FIG. 3F, left). Collectively, these results demonstrated that anti-KRASG12VBMCs selectively induce degradation of the mutant KRAS without affecting the wild-type variant.

[0148] Mutant-specific degradation would selectively inhibit mutant cell proliferation. To investigate, the proliferation inhibition effect of KRAS degradation in a co-culture of HT29 and SW480 cells was assessed (FIG. 3G). HT29 cells were labeled with Qtracker 800, while SW480 cells were with Qtracker 625 for identification. Both cell types were mixed at equal proportions and labeled with carboxyfluorescein succinimidyl ester (CFSE) as an indicator of cell division, where signal decay reflects mitotic activity. After six days of treatment with anti-KRASG12V-BMC@MPNs, HT29 cells predominated in the co-culture, while under the non-treated (NC) condition, SW480 cells slightly outcompeted HT29 due to their shorter doubling time. The CFSE signal in the NC condition was homogeneous and weak, indicating high proliferation levels (FIG. 4H). Conversely, strong CFSE signals were observed in the anti-KRASG12V-BMC@MPN group, co-localizing with SW480 cells, indicating mutantspecific growth suppression by anti-KRASG12V-BMC@MPNs (FIG. 4K). This trend was quantified using flow cytometry, where each cell population was sorted and the ratios quantified. The results indicated a significant dominance of HT29 cells in the treated group due to growth suppression of SW480 cells at the ratio of 82% to 18% (FIG. 4K; FIG. 21 A-21B). In the untreated group, SW480 cells slightly outcompeted HT29 due to their shorterAtty. Dkt. No.: 121384-0302doubling time at the ratio of 58% to 42% (FIG. 4J). Additionally, significant suppression of proliferative activity was observed in SW480 cells (17% proliferating cells with anti-KRASG12V-BMC@MPNs VS. 52% in NC), whereas HT29 cells remained unaffected (40% with anti-KRASG12V-BMC@MPNs vs. 40% in NC) (FIG. 4L-4M; FIG. 22). Collectively, these results demonstrate the mutant-specific KRAS degradation effect at the cellular level.Example 4: Targeted degradation of KRASG12Vinhibited tumor growth in vivo[014 | Applicant evaluated the in vivo TPD efficacy of IgG-BMC@MPNs using a KRASG12Vxenograft model in athymic nude mice. Applicant established subcutaneous tumors with SW480 cells, followed by intratumoral injections of empty, isotype, or anti-KRAS-BMC@MPNs (8 mice per group, 10 mg / kg, 8-12 pL per dose), administered three times per week for a total of six doses (FIG. 4A). Applicant found that at endpoint, the anti-KRAS-BMC@MPN group exhibited strong tumor suppression, with approximately 80-fold and 50-fold reductions in tumor volume and weight, respectively, compared to the isotype group. Notably, two of eight tumors were eliminated entirely (FIG. 4B-F). Applicant used hematoxylin and eosin (H&E) staining to show increased necrosis (red arrows) and reduced matrix density (green arrows) in the anti-KRAS-BMC@MPN group. Applicant found that these findings align with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay results showing elevated apoptosis in the treated tumors (FIG. 4L).Applicant found that, Immunohistochemistry (IHC) staining for Ki-67, caspase-3, and KRAS demonstrated reduced proliferation, enhanced apoptosis, and significant degradation of KRAS in the anti-KRAS-BMC@MPN-treated group (FIG. 41). These results demonstrate that BMC@MPNs enable selective in vivo degradation of KRASG12V, suppressing tumor growth and modulating apoptosis and proliferation, highlighting their therapeutic potential in KRAS-driven cancers.

[0150] Applications of BMCs have been underexplored in vivo due to their vulnerability to coalescences or aggregation under normal conditions (Jiang, L. et al. J. Am. Chem. Soc. 143, 24108-24115 (2023)). With improved colloidal stability and robust in vitro TPD capability demonstrated for IgG-BMC@MPNs, their in vivo TPD efficacy was evaluated for the first time using a KRASG12Vxenograft athymic nude mouse model. Nude mice were implantedAtty. Dkt. No.: 121384-0302subcutaneously with SW480 cells to establish tumor, followed by intratumoral injections of empty, isotype, or anti-KRAS-BMC@MPNs at a dosage of 10 mg per kg (mpk), administered three times per week for a total of six doses (FIG. 4A). At the study endpoint, the anti-KRAS-BMC@MPN-treated group exhibited significant inhibition of tumor growth compared to the isotype group, with an average 80-fold and a 50-fold reduction in tumor volume and weight, respectively (FIG. 4B-4F; FIG. 23). Hematoxylin and eosin (H&E) staining demonstrated significant necrosis (red arrow) in the anti-KRAS-BMC@MPN-treated group, which was absent in control groups, and reduced tumor matrix density (green arrow), indicating lower tumor aggressiveness and enhanced immune cell infiltration (FIG. 4G) (Galon, J. et al. Science 313, 1960-1964 (2006); Fabiano, E., Zhang, J. & Reinhart-King, C.Curr. Opin. Biomed. Eng. 22, 100383 (2022)). The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay indicated increased apoptosis in the anti-KRAS-BMC@MPN-treated group (FIG. 4H). IHC staining for Ki-67, p53, and KRAS demonstrated reduced proliferation, enhanced apoptosis, and significant degradation of KRAS in the anti-KRAS-BMC@MPN-treated group (FIG. 41; FIG. 24).

[0151] Applicant found that throughout the treatment, anti-KRAS-BMC@MPN nanoformulations did not impact the body weight of the mice, indicating a favorable safety profile (FIG. 4 J). Serum biochemistry analysis of 15 biomarkers related to osmotic balance (PO43", Na+, K+, Ca2+), metabolic homeostasis (alkaline phosphatase, alanine transaminase, amylase, albumin, total bilirubin, total protein, blood urea nitrogen, glucose), and immune function (creatinine, globulin) revealed no significant abnormalities, suggesting no acute systemic toxicity or hemolysis after repeated dosing (FIG. 4K). Histopathological analysis of major organs, including the heart, liver, spleen, lung, and kidney, showed no histological changes or tissue damage, further confirming the tolerability of both the treatment and the BMC@MPN material at the tested dose. Collectively, Applicant found that these findings demonstrate that IgG-BMC@MPNs enable effective mutant-specific degradation with a favorable in vivo safety profile, highlighting their potential as a targeted protein degradation strategy for KRAS-driven cancers. Blood chemistry analysis showed that all parameters in mice treated with drug-loaded nanoparticles remained consistent with those of control mice, with no evidence of hemolysis (FIG. 4K). Histological examination of liver and kidney exhibited no abnormalities in either organ, indicating acceptable tolerability at specifiedAtty. Dkt. No.: 121384-0302dosage (FIG. 25). These findings indicate that IgG-BMC@MPNs is safe and effective TPD modalities for in vivo anti-tumor therapy.Example 5: Proteasome-mediated degradation enables the degradation of intracellular targets

[0152] Applicant investigated the protein degradation mechanism triggered by BMC-mediated antibody delivery, by pretreating SW480 cells with MG132 (proteasome inhibitor) or chloroquine (autophagy -lysosomal pathway inhibitor) and then incubated with anti-KRAS-BMC@MPNs. Applicant observed a marked reduction in KRAS (residual 27%) was in cells lacking inhibitor, which was rescued by 91% with MG132 but not by chloroquine, indicating that degradation proceeds with the proteasome rather than the lysosome (FIG. 5A; FIG. 5M). This result was further supported by experiments using additional inhibitors for the proteasome and autophagy-lysosome pathway (bortezomib and pepstatin A, respectively. Applicant observed no ubiquitinated KRAS bands, and TAK-243 (a ubiquitin-activating enzyme inhibitor) failed to block degradation, supporting a ubiquitin-independent process. Applicant observed no rescue with the no-PTM control (lacking proteasome-targeting) or the isotype control (lacking KRAS-binding), suggesting that both KRAS engagement and proteasome recruitment are required for degradation.

[0153] To assess proteasome-dependent degradation, KRAS levels in SW480 cells pretreated with MG132, a proteasome inhibitor, against untreated cells. WB results revealed a significant decrease in KRAS levels (54%) in the NC group; this effect was inhibited by 89% if pretreated with MG132 (FIG. 5A-5B). The absence of ubiquitination bands validated the hypothesis that anti-KRAS-BMC@MPNs-induced proteolysis is initiated by direct proteasome recruitment via the PTM domain. As a crosslink validation, no degradation rescue was observed with the lysosome inhibitor Bafilomycin Al or the control carrier Xfect reagent, which lacks proteasome-targeting properties (FIG. 26A-26B).

[0154] Applicant reasoned that polyarginine mediates charge based interfactions with the proteason and this reaction correlates with degradation activity. Applicant performed molecular docking using PTM, RRRG, as the test peptide and AAAG as a length-matched, electrically neutral control. Docking analysis revealed that, PSMD9, a 19S regulatory subunit involved in proteolytic activation40, was a preferred binding site for RRRG, with a bindingAtty. Dkt. No.: 121384-0302free energy of -7.7 kcal / mol compared to -4.6 kcal / mol for AAAG (threshold: - 5.0 kcal / mol). RRRG formed more hydrogen bonds and docked in closer proximity to the proteasome than AAAG, supporting a charge-mediated targeting mechanism (FIG. 5C; Table 2). Applicant performed experimental validation using RRRG or AAAG as PTMs showed that only the RRRG fusion induced degradation, as shown by immunoblot (FIG. 5N-50). Applicant demonstrated that electrostatic interaction with PSMD9 is essential for proteasome recruitment and highlight the importance of rational PTM design in enabling ubiquitin-independent degradation through direct proteasome targeting.

[0155] Although previous studies suggested that polyarginine mediates charge-based interactions with the proteasome (Bonger, K. M., Chen, L.-c., Liu, C. W. & Wandless, T. J. Nat. Chem. Biol. 7, 531-537 (2011); Qu, J. etal. Cell Chem. Biol. 27, 751-762.e4 (2020); Kudriaeva, A., Kuzina, E. S., Zubenko, O., Smirnov, I. V. & Belogurov Jr., FASEB J. 33, 6852-6866 (2019)), it was confirmed that binding correlates with degradation activity.Molecular docking was performed to identify PTM binding sites on the 26S proteasome, using AAAG as an electrically neutral control. PMSD9, a subunit of the 19S regulatory core involved in proteolytic activation (Bashore, C. etal. Nat. Chem. Biol. 19, 55-63 (2023)), was the preferred binding site for RRRG, with a binding free energy of -7.7 kcal / mol compared to -4.6 kcal / mol for AAAG (standard affinity threshold: -5.0 kcal / mol) (Trott, O. & Olson, A. J. J. Comput. Chem. 31, 455-461 (2010)). The RRRG formed more hydrogen bonds and was closer to the proteasome compared to AAAG, suggesting a charge-mediated targeting mechanism (FIG. 5C; Table 2; FIG.27A-27D).

[0156] Applicant hypothesized that this proteasome-dependent degradation strategy could be extended to other intracellular targets by incorporating target-specific antibodies. Applicant selected a-synuclein (a-syn), a 17 kDa nuclear protein implicated in many neurodegenerative diseases. Applicant treated SK-N-SH cells with anti-a-syn-BMC@MPNs, resulting in a 65% reduction of a-syn within 48 hours, as confirmed by WB and confocal imaging (FIG. 5P). To assess proteasome engagement, Applicant established an a-syn overexpression model in SK-N-SH cells, which increased a-syn levels threefold and induced over 60% cell death within 48 hours. Applicant evaluated the neuroprotective effect of anti-a-syn-BMC@MPNs by measuring cell viability and apoptosis, with or without MG132 pretreatment. Treatment withAtty. Dkt. No.: 121384-0302anti-a-syn-BMC@MPNs significantly reduced neurotoxicity, increasing cell viability to 79% (vs. 32% untreated) and decreasing lactate dehydrogenase (LDH) release by 28%, restoring levels to those of healthy controls. These results highlight the adaptability of this proteasome-targeting platform for degrading diverse intracellular proteins.

[0157] Applicant compared the degradation process across cell types, by using label-free quantitative proteomics (LFQ) using his-tagged cationic peptides as pull-down anchors to reveal target-specific degradation profiles (FIG. 5H). Applicant found in SW480 cells, anti-KRASG12V-BMC@MPN treatment downregulated 17 Ras-related proteins, including 12 in the active GTP -bound form, indicating selective degradation of active Ras (FIG. 51).Applicant found in SK-N-SH cells, 10 neurodegeneration-associated proteins were similarly reduced (FIG. 5 J). Enrichment of 19S proteasome subunits (PSMD) in both models confirmed PTM-mediated proteasome recruitment, consistent with docking predictions.Applicant performed KEGG analysis further highlighting RAS signaling (SW480), PD / HD / AD pathways (SK-N-SH), and proteasome pathways as the top-enriched. Applicant further evaluated engagement of PSMD9 during KRAS and a-syn degradation using a proximity ligation assay (PLA). Applicant observed distinct punctate PLA in the cytosol of SW480 cells and the nucleus of SK-N-SH cells following treatment with anti-KRASG12V-BMC@MPNs and anti-a-syn-BMC@MPNs, respectively, confirming spatially resolved interactions between PSMD9 and each target. Applicant showed that these findings suggest that degradation occurs within the native subcellular compartment of each protein, with PSMD9 acting as a shared proteasomal subunit for initiating degradation. Collectively, Applicant showed that these results confirm that IgG-BMC@MPNs enable target-specific, proteasome-mediated degradation across distinct subcellular locations and disease-relevant cellular contexts, demonstrating the adaptability of this platform.Table 2: Molecular docking interaction analysisDocking Binding Hydrogen Bonds Hydrophobic Salt Bridges 7t-Cation Pair Energy Interaction Interaction (kcal / mol)Aty. Dkt. No.: 121384-0302AAAG- . . -4.0. Glu 65W (3.44A) Arg 86W (3.69A) Lys19S Glu 65W (3.58 A) Phe 149Y(3.72A) 65W(2.94A)Gin 68W(3.11 A) Glu 159Y(3.99A)Thr78W (4.01 A)Glu HOY (3.03 A)Lys 114Y (3.89A). -4.3. Gin 885 (3.62 A) . ''iie'i'3660 . 7. . 7.26S Gin 885 (1.99 A)Gin 885 (2.54 A)Thr 895 (2.69 A)Ser 1566 (3.08 A)Ser 1566 (2.39 A)Gin 1586 (2.25 A)AAAG- -4.6 Phe 304A (2.87 A) Glu 306A (3.54A) His 301 A / PSMD9 Phe 304A (2.09 A) Asn 493 A (3.76A) (5.26 A)Gly 491 A (2.26 A)Gly 491A (1.80 A)Gly 491 A (3.38 A)Asn 493 A (3.09 A).. -7.1. Phe 3^ . Glu 77V . . 7.19S Asn 3 OX (2.16 A) Arg 57X (3.70 A) (3.65 A)Arg 57X (2.75 A)Arg 57X (2.53 A)Arg 57X (2.79 A)Glu 77X (2.37 A)Thr 81V (2.76 A)Lys 100W (2.65 A)Ser 119V (2.52 A)Ser 119V (2.74 A)Glu 121V (1.95 A)Aty. Dkt. No.: 121384-0302RRRG- Thr 341 (3.53 A) Ala 821 (3.71 A) 26S Thr 341 (3.54 A) Tyr 1671 (3.71 A)Thr 341 (2.29A) Thr 341 (1.95 A) Thr 551 (2.40 A) Gly 561 (2.49 A) Ser 811 (2.92 A) Ser 811 (2.42 A) Ala 821 (1.96 A) Ala 831 (2.86 A) Asp 841 (2.73 A) Tyr 1671 (2.78 A) Ser 2021 (3.23 A) Ser 2021 (2.84 A) Ser 2021 (2.20 A) Ser 2031 (2.12 A) Ser 2031 (3.15 A) Ser 2031 (3.36 A) Ser 2031 (2.18 A)RRRG- 7.7 Asn 273A(2.47A) His 301 A PSMD9 His 301A(2.67A) (4.08 A)Phe 304A(2.08A) Phe 304A(2.06A) Gly 491A(2.50A) Asn 493A(2.50A) Asn 493A(2.53A) Asn 493A (2.33 A) Gly 528A (2.48 A) Gly 528A (2.58 A) Ser 529A (2.04 A) Ser529A (3.13 A) Lys 569A (3.07 A) Ser789A (3.15 A)Atty. Dkt. No.: 121384-0302> J Gin 790A (2.80 A) J | Vai 791 A (2.72 A) |Example 6: Adaptation of approach to a-synuclein

[0158] Applicant shows that IgG-BMC@MPNs can be adapted to targeting various targets by incorporating antibodies tailored to the corresponding targets. As a proof-of-concept, a-synuclein (a-syn), a 17 kDa nuclear protein associated with neurodegenerative diseases such as Parkinson’s (PD), Alzheimer’s (AD), and Huntington’s (HD) (Goedert, M. Nat. Rev.Neurosci. 2, 492-501 (2001)) was used by the Applicant. Targeting a-synuclein is challenging due to its predominant nuclear localization at high expression levels. In addition, the autophagy-lysosomal pathway (ALP) is often impaired under these conditions, making proteasomal degradation a vital alternative (Qu, J. et al. Cell Chem. Biol. 27, 751-762. e4 (2020). To test the effect of anti-a-syn-BMC@MPNs for a-syn degradation, Applicant treated SK-N-SH cells with anti-a-syn-BMC@MPNs. Confocal microscopy and WB analysis showed significant a-syn reduction within 2 days (54.8%; FIG. 5D-5E; FIG. 28). To demonstrate the activity of BMCs as a universal TPD tool, neurotoxicity rescue in an a-syn overload model (FIG. 5F; FIG. 29) was assessed by the Applicant. Cell counting (CCK-8) and lactate dehydrogenase (LDH) release assays were used concurrently for orthogonal validation. 48 hr post-transfection, a-syn-overloaded cells exhibited a 62% reduction in cell viability with a 128% increase in LDH release, indicating significant neurotoxicity, indicating that neurotoxicity was mitigated following anti-a-syn-BMC@MPNs treatment. The rescue effect was inhibited by MG132, indicating that the proteasomal pathway mediates the degradation process is shared in both a-syn and KRAS breakdown (FIG. 5G).

[0159] To elucidate the underlying degradation machinery, quantitative mass spectrometry to perform label-free quantification (LFQ) of pull-down cationic peptides was used by the Applicant, enabling a comprehensive analysis of IgG-BMC@MPN-mediated degradation across the proteome in cells. To prevent competitive binding at antibody-occupied sites, His-tagged cationic peptides as pull-down anchors were used (FIG. 5H). Differential expression analysis in SW480 cells showed downregulation of 17 Ras-related proteins, including 12Atty. Dkt. No.: 121384-0302proteins in the RAS-GTP binding form, indicating targeted degradation of active Ras after anti-KRASG12V-BMC@MPN treatment (FIG. 51). In SK-N-SH cells, 10 proteins associated with neurodegenerative diseases were downregulated (FIG. 5J). Enrichment of multiple proteasome 19S subunits (PSMD) in both cell types indicated proteasome involvement in the degradation process. This finding aligns with molecular docking results, which demonstrated PTM interactions that directly recruit the 19S complex, thereby initiating degradation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis further validated that the RAS signaling pathway in SW480 cells (FIG. 5K), PD / HD / AD pathways in SK-N-SH cells, (FIG. 5L), and the proteasomal pathway were among the top 10 enriched pathways, highlighting their key roles in the degradation of corresponding targets.Equivalents[016(>| Unless otherwise defined, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.10161] Although the foregoing refers to preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill yin the art that various modifications may be made to the disclosed embodiments and that such modification are intended to be within the scope of the present invention.

[0162] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there in no intention in use of such terms and expressions of excluding any equivalents of the features shown and described or portion thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.Atty. Dkt. No.: 121384-0302[01631 Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0164] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0015] In addition, where features of the present technology are described in terms of Markush groups. Those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of member of the Markush group.]0166[ All publications, patent applications, GenBank citations, ATCC citations, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the specification, including definitions, will control.

Claims

1. Atty. Dkt. No.: 121384-0302WHAT IS CLAIMED IS:

1. A method for intracellular targeted protein degradation in a subject in need thereof comprising:(a) condensing an antibody within a biomolecular condensate using liquid-liquid phase separation;(b) coating the biomolecular condensate with a semi-permeable protective shell; and (b) administering to the subject a therapeutically effective amount of the biomolecular condensate.

2. The method of claim 1, wherein the semi-permeable protective shell is a metal-phenolic network.

3. The method of claim 1, wherein coating the biomolecular condensate with the semi-permeable protective shell increases a cellular internalization rate of the biomolecular condensate relative to the cellular internalization rate of the same biomolecular condensate lacking the semi-permeable protective shell.

4. The method of claim 1, wherein coating the biomolecular condensate with a semi-permeable shell decreases a size of the biomolecular condensate relative to the size of the same biomolecular condensate lacking a semi-permeable protective shell.

5. The method of claim 1, wherein coating the biomolecular condensate with the semi-permeable shell increases a colloidal stability of the biomolecular condensate relative to the colloidal stability of the same biomolecular condensate lacking a semi-permeable protective shell.

6. The method of claim 1, wherein the biomolecular condensate selectively induces targeted protein degradation of a pathogenic protein without affecting a wild-type variant.Atty. Dkt. No.: 121384-03027. The method of claim 1, wherein the biomolecular condensate selectively inhibits proliferation of cells containing a pathogenic protein.

8. The method of claim 1, wherein administering to the subject the biomolecular condensate increases apoptosis of cells containing a pathogenic protein relative to apoptosis of cells containing the pathogenic protein in absence of administering the biomolecular condensate to the subject.

9. The method of claim 1, wherein the subject has an established tumor.

10. The method of claim 9, wherein the biomolecular condensate is administered intratum orally.

11. The method of claim 10, wherein administering the biomolecular condensate to the subject with the established tumor increases tumor growth inhibition relative to tumor growth inhibition in an subject in the absence of administering a biomolecular condensate.

12. The method of claim 1, wherein a pathogenic protein is at least one of mutant KRAS or a-synuclein.

13. The method of claim 1, wherein the antibody is at least one of recombinant immunoglobulin Gs, anti-KRAS, or anti-a-synuclein.

14. The method of claim 1, wherein the biomolecular condensate is a peptide-based condensate.Atty. Dkt. No.: 121384-030215. The method of claim 1, further comprising integrating a proteasome-targeting motif into a precursor of the liquid-liquid phase separation.

16. The method of claim 15, wherein integrating the proteasome-targeting motif into the precursor increases a recruitment of proteasome and increases proteasome mediated degradation relative to proteasome recruitment and proteasome mediated degradation in an absence of integrating the proteasome-targeting motif.

17. The method of claim 1, wherein the subject has at least one of cancer or a neurodegenerative disorder.

18. The method of claim 1, further comprising monitoring the subject for one or more toxicities.

19. The method of claim 1, wherein the biomolecular condensate is between 200 nanometers and 600 nanometers.

20. A method to generate a biomolecular condensate for intracellular targeted protein degradation comprising:(a) condensing an antibody within a biomolecular condensate using liquid-liquid phase separation; and(b) coating the biomolecular condensate with a semi-permeable protective shell.

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