Modular platforms for targeted protein degradation

Abstract

The present disclosure provides recombinant peptides, compositions, and methods thereof for targeting proteins for degradation while bypassing ubiquitination mechanisms.

Description

[0001] PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0002] Docket No. 10046-609W01

[0003] MODULAR PLATFORMS FOR TARGETED PROTEIN DEGRADATION

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] 5 This invention was made with Government Support under Grant No. R01 GM124501 awarded by the National Institutes of Health. The Government has certain right in the invention.

[0006] RELATED APPLICATION

[0007] This PCT application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 695,889, filed September 18, 2024, entitled “MODULAR PLATFORMS FOR TARGETED PROTEIN DEGRADATION,” which is incorporated by reference herein in its entirety.

[0008] REFERENCE TO SEQUENCE LISTING

[0009] 15 The sequence listing submitted on September 18, 2025, as an .XML file entitled “10046-609W01_ST26” created on September 18, 2025, and having a file size of 20,595 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0010] FIELD

[0011] 20 The present disclosure provides recombinant peptides, compositions, and methods thereof for targeting proteins for degradation while bypassing ubiquitination mechanisms.

[0012] BACKGROUND

[0013] Proteolysis targeting chimeras (PROTACs) and molecular glues (MGs) target proteins for destruction by channeling them into the Ubiquitin-Proteasome-System (UPS), which is one of the two cellular protein removal pathways. In the UPS pathway, target proteins are first tagged with the small protein ubiquitin. The tagged proteins are then recognized by a large proteolytic machine, the proteasome, and digested into peptides.

[0014] PROTACs and MGs aim to induce target protein ubiquitination by inducing their

[0015] 30 binding to Ubiquitin Ligase enzymes (E3s). However, ubiquitination is regulated in a complex manner and not fully understood. Thus, it is difficult to ensure that ubiquitination occurs efficiently. In addition, the ubiquitination machinery is easily affected by mutations, allowing resistance to develop relatively easily; and the ubiquitin mediated degradation pathway has PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0016] Docket No. 10046-609W01 been shown to be attenuated by proteostasis, stress, and aging. Finally, and most importantly, it is well established that ubiquitination alone is not sufficient to target proteins for destruction physiologically. Thus, it is not surprising that current TPD approaches, which are based on induced ubiquitination, fail frequently. What is needed in the art is a compound, composition, 5 method, or a combination thereof capable of withstanding mutations and resistance to proteolytic mechanisms.

[0017] The compounds, compositions, and methods disclosed herein address these and other needs.

[0018] 10 SUMMARY

[0019] The present disclosure provides a recombinant polypeptide, or pharmaceutical compositions thereof, for bypassing the ubiquitination step and targeting proteins to the proteosome. The present disclosure also provides nucleic acids (including but not limited to deoxyribose nucleic acid (DNA) and ribose nucleic acid (RNA)), expression vectors, and cells 15 for expressing said recombinant polypeptide. The present disclosure also provides methods using said recombinant polypeptide to degrade a target polypeptide in a cell, to treat a subject in need thereof.

[0020] In some aspects, disclosed herein is a recombinant polypeptide that binds to both a target polypeptide and a proteasome, the recombinant polypeptide comprising: (a) a targetbinding domain, wherein said target-binding domain binds to the target polypeptide; (b) a proteasome-binding domain that binds to the proteasome; and (c) a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker facilitates the proteasome recognizing an initiation region of the target polypeptide.

[0021] In some embodiments, the linker is designed such that a region of the target polypeptide comes in contact with the proteasome in a manner which allows the proteasome to initiate degradation of the target polypeptide. In some embodiments, the linker is designed such that one or more amino acids of the target polypeptide that are recognized by the proteasome are presented to the proteasome in order to initiate degradation of the target polypeptide. In some embodiments, the linker is 10-100 amino acids long. In some embodiments, the linker comprises one or more a -helix spacers.

[0022] In some embodiments, the target polypeptide is a polypeptide associated with a disease. In some embodiments, the target-binding domain is positioned N-terminally relative to the proteasome binding domain. In some embodiments, the target-binding domain is positioned C- terminally relative to the proteasome binding domain. PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0023] Docket No. 10046-609W01

[0024] In some embodiments, the proteasome-binding comprises a ubiquitin-like domain (UBL). In some embodiments, the proteasome-binding domain comprises a domain from HPV E7, gankyrin, Rad23a, Rad23b, UBL domain of midnolin, a-Helix-c domain of midnolin, or a coiled-coil domain.

[0025] In some embodiments, the recombinant polypeptide further comprises a cellpenetrating peptide (CPP) sequence or a cellular receptor-binding sequence. In some embodiments, the target-binding domain comprises a nanobody. In some embodiments, the target-binding domain comprises an engineered protein interaction domain. In some embodiments, the target-binding domain comprises a monobody. In some embodiments, the target-binding domain binds to a prion, a viral polypeptide, a disease-associated protein, a cellular polypeptide having a disease-associated mutation, or a product of an oncogene. In some embodiments, the target-binding domain binds to a product of an oncogene. In some embodiments, the oncogene comprises Abl or Shp2. In some embodiments, the oncogene comprises Abl and Shp2.

[0026] In some embodiments, the target binding domain binds to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, or C-terminal SH2 domain of Shp2. In some embodiments, the target binding domain binds to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, and C-terminal SH2 domain of Shp2. In some embodiments, the target-binding domain binds to misfolded beta-amyloid. In some embodiments, the target-binding domain binds to Tau protein. In some embodiments, the polypeptide comprises a subcellular localization signal. In some embodiments, the subcellular localization signal is a nuclear localization signal.

[0027] In some embodiments, the target binding domain comprises at least 90% identity to any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 10. In some embodiments, the proteasome-binding domain comprises at least 90% identity to any one of SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 14. In some embodiments, the linker comprises at least 90% identity to any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, the linker comprises an alpha helix of SEQ ID NO: 12.

[0028] In some aspects, disclosed herein is a nucleic acid molecule (including, but not limited to DNA and RNA) encoding the polypeptide according to any preceding aspect.

[0029] In some aspects, disclosed herein is an expression vector comprising the nucleic acid of any preceding aspect, wherein the nucleic acid is operably linked to a promoter. In some embodiments, the promoter is an inducible promoter, a repressible promoter, or a constitutive promoter. In some embodiments, the promoter is a tissue or cell type specific promoter. In some embodiments, the vector is a plasmid, a viral vector, or an episomal vector. PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0030] Docket No. 10046-609W01

[0031] In some aspects, disclosed herein is a cell comprising the vector of any preceding aspect.

[0032] In some aspects, disclosed herein is a recombinant protein comprising a targeting binding domain operably linked to a proteasome binding domain by a linker peptide, wherein the targeting binding domain comprises at least 90% sequence identity to SEQ ID NOs: 3, 4, 5, or 10; wherein the proteosome binding domain comprises at least 90% sequence identity to SEQ ID NOs: 11 or 14; and wherein the linker peptide comprises 90% or more identity to SEQ ID NOs: 7, 8, or 9.

[0033] In some aspects, disclosed herein is a pharmaceutical composition comprising the recombinant protein of any preceding aspect and a pharmaceutically acceptable carrier.

[0034] In some aspects, disclosed herein is a pharmaceutical composition comprising the polypeptide of any preceding aspect and a pharmaceutically acceptable carrier.

[0035] In some aspects, disclosed herein is a method of degrading a target polypeptide in a cell, the method comprising: (a) exposing the protein to a recombinant polypeptide that binds to both a target polypeptide and a proteasome, wherein the recombinant polypeptide comprises:

[0036] (i) a target-binding domain, wherein said target binding domain binds to the target polypeptide;

[0037] (ii) a proteasome-binding domain that binds to the proteasome; and (iii) a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker facilitates the proteasome recognizing an initiation region of the target polypeptide; and (b) allowing the recombinant polypeptide to bring the target polypeptide into contact with the proteasome, wherein the proteasome degrades the target polypeptide.

[0038] In some embodiments, the method of the preceding aspect comprises the linker being designed such that a region of the target polypeptide comes in contact with the proteasome in a manner which allows the proteasome to initiate degradation of the target polypeptide. In some embodiments, the method of the preceding aspect comprises the linker being designed such that one or more amino acids of the target polypeptide which are recognized by the proteasome in order to initiate degradation of the target polypeptide are presented to the proteasome. In some embodiments, the method of the preceding aspect comprises the linker being 10-100 amino acids long. In some embodiments, the method of the preceding aspect comprises the linker comprising one or more a-helix spacers.

[0039] In some embodiments, the method of the preceding aspect comprises the target polypeptide being a polypeptide associated with a disease. In some embodiments, the method of the preceding aspect comprises the proteasome-binding comprising a ubiquitin-like domain, PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0040] Docket No. 10046-609W01 including but not limited to a domain from HPV E7, gankyrin, Rad23a, Rad23b, UBL domain of midnolin, a-Helix-c domain of midnolin, or a coiled-coil domain.

[0041] In some embodiments, the method of any preceding aspect further comprises a cellpenetrating peptide (CPP) sequence or a cellular receptor-binding sequence. In some embodiments, the method of the preceding aspect comprises the target-binding domain comprising a nanobody. In some embodiments, the method of the preceding aspect comprises the target-binding domain comprising an engineered protein interaction domain. In some embodiments, the method of the preceding aspect comprises the target-binding domain comprising a monobody. In some embodiments, the method of the preceding aspect comprises the target-binding domain binding to a prion, a viral polypeptide, a disease-associated protein, a cellular polypeptide having a disease- associated mutation or the product of an oncogene. In some embodiments, the method of the preceding aspect comprises the target-binding domain binding to the product of an oncogene, wherein the oncogene includes but is not limited to Abl, Shp2, or a combination thereof. In some embodiments, the method of the preceding aspect comprises the target binding domain binding to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, or C-terminal SH2 domain of Shp2. In some embodiments, the method of the preceding aspect comprises the target binding domain binding to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, and C-terminal SH2 domain of Shp2. In some embodiments, the method of the preceding aspect comprises the target-binding domain binding to misfolded beta-amyloid. In some embodiments, the method of the preceding aspect comprises the targetbinding domain binding to Tau protein.

[0042] In some embodiments, the method of the preceding aspect comprises the polypeptide comprising a subcellular localization signal, including but not limited to a nuclear localization signal.

[0043] In some aspects, disclosed herein is a method of treating a subject in need of degradation of a target polypeptide, the method comprising: (a) exposing the protein to a recombinant polypeptide that binds to both a target polypeptide and a proteasome, wherein the recombinant polypeptide comprises: (i) a target-binding domain, wherein said target binding domain binds to the target polypeptide; (ii) a proteasome-binding domain that binds to the proteasome; and (iii) a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker facilitates the proteasome recognizing an initiation region of the target polypeptide; and (b) allowing the recombinant polypeptide to bring the target polypeptide into contact with the proteasome, wherein the proteasome degrades the target polypeptide. PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0044] Docket No. 10046-609W01

[0045] In some embodiments, the subject has cancer, a neurogenerative disorder, an autoimmune disease, an age-related degenerative disease, a viral disease, or an inflammatory disorder. In some embodiments, the subject has been exposed to a substance which causes accumulation of unwanted target polypeptide in the subject.

[0046] In some embodiments, the method of treating comprises the linker being designed such that a region of the target polypeptide comes in contact with the proteasome in a manner which allows the proteasome to initiate degradation of the target polypeptide. In some embodiments, the method of treating comprises the linker being designed such that one or more amino acids of the target polypeptide which are recognized by the proteasome in order to initiate degradation of the target polypeptide are presented to the proteasome. In some embodiments, the method of treating comprises the linker being 10-100 amino acids long. In some embodiments, the method of treating comprises the linker comprises one or more a-helix spacers.

[0047] In some embodiments, the method of treating comprises the target polypeptide being a polypeptide associated with a disease. In some embodiments, the method of treating comprises the proteasome-binding comprising a ubiquitin-like domain. In some embodiments, the method of treating comprises the proteasome-binding domain comprising a domain from HPV E7, gankyrin, Rad23a, Rad23b, UBL domain of midnolin, a-Helix-c domain of midnolin, or a coiled coil domain.

[0048] In some embodiments, the method of treating further comprises a cell-penetrating peptide (CPP) sequence or a cellular receptor-binding sequence. In some embodiments, the method of treating comprises the target-binding domain comprising a nanobody. In some embodiments, the method of treating comprises the target-binding domain comprising an engineered protein interaction domain. In some embodiments, the method of treating comprises the target-binding domain comprising a monobody. In some embodiments, the method of treating comprises the target-binding domain binding to a prion, a viral polypeptide, a disease- associated protein, a cellular polypeptide having a disease- associated mutation or the product of an oncogene. In some embodiments, the method of treating comprises the target-binding domain binding to the product of an oncogene, including but not limited to Abl, Shp2, or a combination thereof. In some embodiments, the method of treating comprises the target binding domain binding to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, or C- terminal SH2 domain of Shp2. In some embodiments, the method of treating comprises the target binding domain binding to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, and C-terminal SH2 domain of Shp2. In some embodiments, the method of treating comprises PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0049] Docket No. 10046-609W01 the target-binding domain binding to misfolded beta-amyloid. In some embodiments, the method of treating comprises the target-binding domain binding to Tan protein.

[0050] In some embodiments, the method of treating comprises the polypeptide comprising a subcellular localization signal, including but not limited to a nuclear localization signal.

[0051] BRIEF DESCRIPTION OF FIGURES

[0052] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0053] 5 Figure 1 shows that an adaptor with a UBL domain and a monobody specific to the SH2 domain of Abl induces concentration-dependent proteasome-mediated degradation of a model protein fused to the SH2 domain of AbL in vitro.

[0054] Figures 2A and 2B show that the expression of the adaptor with UBL and target-specific monobody in situ (HEK293 cells) induces the degradation in a target-specific manner. Flow cytometry-based data shows as compared to a nonbinding adapter about 70% of the target protein degraded by a target-specific adapter.

[0055] Figure 3 shows that the expression of the adaptor with UBL and target-specific monobody in situ (HEK293 cells) induces target-specific degradation.

[0056] Figure 4 shows that the degradation of the target human proteins can be tuned by a

[0057] 15 linker connecting the UBL and target- specific domain. Expression of adapters in situ (HEK 293 cells) with three linkers of varying lengths and sequences connecting the UBL and targetspecific nanobody, degrades target human proteins differently.

[0058] Figure 5 shows the quantitation of the effect linker on the adapter function. Depending on the target protein, adapter-mediated degradation can be tuned by a sequence and the length

[0059] 20 of the linker connecting the UBL and target-specific domain (n=2, SD+ / -).

[0060] Figure 6 shows the adapters and target protein binding. As compared to a mutant adapter with a reduced affinity towards the target protein FLAG-tagged human Abl kinase targeting adapters (I, II, III, IV) can pull down Abl protein from human cells, (in I, II, III, IV - UBL and Abl targeting monobody are in different orientations and connected with varying linkers of size).

[0061] Figure 7 shows Abl targeting adapters can degrade Abl proteins from human cells. As compared to a mutant adapter with a reduced affinity towards the target protein two FLAG- tagged human Abl kinase targeting adapters (I, II) were able to deplete 50% of overexpressed Abl proteins from the human cells (in I, II, III, IV - UBL and Abl targeting monobody are in

[0062] 30 different orientations and connected with varying linkers of size, n=4 SE+ / -). PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0063] Docket No. 10046-609W01

[0064] Figure 8 shows the degradation of Myc and Stat3 proteins by nanobody-based adapters. Compared to mutant adapters with reduced target specificity Myc or Stat3 targeting adapters can induce the degradation of the native proteins from the breast cancer cells.

[0065] Figure 9 shows the degradation of Myc by a nanobody-based adapter. Compared to

[0066] 5 mutant adapters with reduced target specificity Myc targeting adapters can induce the degradation of over 60% native proteins from the breast cancer cells.

[0067] Figure 10 shows the degradation of Stat3 by a nanobody-based adapter. Compared to mutant adapters with reduced target specificity Stat3 targeting adapters can induce the degradation of over 60% of native proteins from the breast cancer cells.

[0068] Figure 11 shows the degradation of cellular target proteins by two alternate direct proteasome targeting tags. The expression of an adapter containing two different proteasome targeting domains of mandolin (UBL and C-alpha helix) and protein target-specific affinity molecular induces efficient degradation of target proteins.

[0069] Figure 12 shows the degradation of cellular target proteins by two alternate direct

[0070] 15 proteasome targeting tags. The mandolin’s proteasome targeting domains UBL and C-alpha helix induces the degradation of target proteins to different extents (n=2, SD + / -). Nuclear proteins like p27 are almost completely degraded by NLS -containing proteasome targeting tag (MID-C).

[0071] Figure 13 shows improved and modular protein degraders. It has a broader target

[0072] 20 spectrum and improvements are in, proteasome binding tags, likers, and target-specific affinity molecule.

[0073] DETAILED DESCRIPTION

[0074] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing

[0075] 30 other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof. PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0076] Docket No. 10046-609W01

[0077] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0078] 5

[0079] Terminology

[0080] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”,

[0081] 15 include plural referents unless the context clearly dictates otherwise.

[0082] The following definitions are provided for the full understanding of terms used in this specification.

[0083] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be

[0084] 20 within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0085] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0086] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or

[0087] 30 other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0088] Docket No. 10046-609W01 be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0089] "Comprising" is intended to mean that the compositions, methods, etc. include the

[0090] 5 recited elements, but do not exclude others. "Consisting essentially of’ when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0091] Reference also is made herein to peptides, polypeptides, proteins, and compositions

[0092] 15 comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999,

[0093] 20 Brooks / Cole, 110).

[0094] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4- dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation

[0095] 30 lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0096] Docket No. 10046-609W01 addition of a glycosyl group to either asparagine, hydroxy lysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of

[0097] 5 thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine).

[0098] The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety).

[0099] 15 A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known

[0100] 20 amino acid sequence with other amino acids sequences from a variety of databases.

[0101] Percent identity may be measured over the length of an entire defined polypeptide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0102] “Proteolysis” refers to the breakdown of proteins or polypeptides into smaller polypeptides or amino acids. This process, if uncatalyzed, can be extremely slow, taking hundreds of years. Proteolysis typically is a catalyzed process enforced by cellular enzymes

[0103] 30 called proteases. This also encompasses the term(s) “cleave”, “cleavage”, and “cleavable”, which all refer to breaking of peptide bonds or bonds between amino acids.

[0104] A “nucleic acid” is a chemical compound that serves as the primary informationcarrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0105] Docket No. 10046-609W01 a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.

[0106] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences,

[0107] 5 refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite

[0108] 15 includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp

[0109] 20 (discussed above).

[0110] Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0111] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0112] 30 A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information’s website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0113] Docket No. 10046-609W01 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater

[0114] 5 sequence identity over a certain defined length relative to a reference polynucleotide.

[0115] The term “recombinant” describes any DNA, proteins, cells, or organisms that are made by combining genetic material from two different sources. For example, a bacterial gene being inserted into a human plasmid, or human DNA construct, to create another construct that would not otherwise be found in either genome.

[0116] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or

[0117] 15 indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the subject matter described herein are used to delay development of a disease or to slow the progression of a disease.

[0118] A drug that is administered “concurrently” with one or more other drugs is administered

[0119] 20 during the same treatment cycle, on the same day of treatment as the one or more other drugs, and, optionally, at the same time as the one or more other drugs. For instance, for cancer therapies given every 3 weeks, the concurrently administered drugs are each administered on day-1 of a 3 -week cycle.

[0120] An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. For example, an effective amount of the drug for treating cancer may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth;

[0121] 30 and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. The effective amount may extend progression free survival (e.g. as measured by Response Evaluation Criteria for Solid Tumors, RECIST, or CA-125 changes), result in an objective response (including a partial response, PR, or complete response, CR), increase PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0122] Docket No. 10046-609W01 overall survival time, and / or improve one or more symptoms of cancer (e.g. as assessed by FOSI).

[0123] As used herein, the term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in treatment

[0124] 5 of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. For use in therapy, therapeutically effective amounts, as well as salts thereof, may be administered as the raw chemical. Additionally, the active ingredient may be presented as a pharmaceutical composition.

[0125] As used herein, unless defined otherwise in a claim, the term “optionally” means that the subsequently described event(s) may or may not occur, and includes both event(s) that occur and event(s) that do not occur.

[0126] As used herein, unless defined otherwise, the phrase “optionally substituted”, “substituted” or variations thereof denote an optional substitution, including multiple degrees

[0127] 15 of substitution, with one or more substituent group, for example, one, two or three. The phrase should not be interpreted as duplicative of the substitutions herein described and depicted.

[0128] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which

[0129] 20 the formulation would be administered.

[0130] A “pharmaceutically acceptable excipient” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable excipient includes, but is not limited to, a buffer, carrier, stabilizer, or preservative.

[0131] The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a molecule. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate,

[0132] 30 ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1 , 1'-methylene-bis- (2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0133] Docket No. 10046-609W01 structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion.

[0134] Other salts, which are not pharmaceutically acceptable, may be useful in the preparation

[0135] 5 of compounds of described herein and these should be considered to form a further aspect of the subject matter. These salts, such as oxalic or trifluoroacetate, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable salts.

[0136] As used herein, the term “plurality” refers to two or more conjugates. Each conjugate can be the same or different from any other conjugate in the plurality.

[0137] A “small molecule” or “small molecular compound” generally refers to an organic molecule that is less than about 5 kilodaltons (Kd) in size. In some embodiments, the small molecule is less than about 4 Kd, 3 Kd, about 2 Kd, or about 1 Kd. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D,

[0138] 15 about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, small molecules are non-polymeric. Small molecules are not proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, etc. A derivative of a small

[0139] 20 molecule refers to a molecule that shares the same structural core as the original small molecule, but which can be prepared by a series of chemical reactions from the original small molecule.

[0140] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0141] 30 The term “vector” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0142] Docket No. 10046-609W01

[0143] “expression vectors”. In some embodiments, the vector can comprise DNA or RNA for delivery.

[0144] Recombinant Polypeptides

[0145] 5 Herein, the present disclosure provides several advantages including: (1) the ability to bypass the ubiquitination step and instead shunt proteins directly to the proteasome without the need for ubiquitination; and (2) the UBLs offer a privileged pathway to degradation via the proteasome, including resistance to proteostasis, stress, and aging.

[0146] The present disclosure provides a recombinant polypeptide, or pharmaceutical

[0147] 10 compositions thereof, for bypassing the ubiquitination step and targeting proteins to the proteosome. The present disclosure also provides nucleic acids, expression vectors, and cells for expressing said recombinant polypeptide. The present disclosure also provides methods using said recombinant polypeptide to degrade a target polypeptide in a cell, to treat a subject in need thereof.

[0148] In some aspects, disclosed herein is a recombinant polypeptide that binds to both a target polypeptide and a proteasome, the recombinant polypeptide comprising: (a) a targetbinding domain, wherein said target-binding domain binds to the target polypeptide; (b) a proteasome-binding domain that binds to the proteasome; and (c) a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker

[0149] 20 facilitates the proteasome recognizing an initiation region of the target polypeptide.

[0150] In some aspects, disclosed herein is a recombinant protein comprising a targeting binding domain operably linked to a proteasome binding domain by a linker peptide. Any of these three components can be any protein which is capable of 1) binding a target; 2) linking the target binding domain to the proteasome binding domain; and 3) binding a proteasome.

[0151] In some aspects, disclosed herein is a recombinant protein comprising a targeting binding domain operably linked to a proteasome binding domain by a linker peptide, wherein the targeting binding domain comprises at least 90% sequence identity to SEQ ID NOs: 3, 4, 5, or 10; wherein the proteosome binding domain comprises at least 90% sequence identity to SEQ ID NOs: 11, 13, or 14, ; and wherein the linker peptide comprises 90% or more identity to SEQ ID NOs: 7, 8, 9, or 12.

[0152] In some aspects, disclosed herein is a ribose nucleic acid (RNA), including but not limited to a messenger RNA (mRNA), encoding a recombinant polypeptide or a recombinant protein that binds to both a target polypeptide and a proteasome, the recombinant polypeptide comprising: (a) a target-binding domain, wherein said target-binding domain binds to the target PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0153] Docket No. 10046-609W01 polypeptide; (b) a proteasome-binding domain that binds to the proteasome; and (c) a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker facilitates the proteasome recognizing an initiation region of the target polypeptide.

[0154] In some aspects, disclosed herein is a pharmaceutical composition comprising the recombinant protein or the recombinant polypeptide of any preceding aspect and a pharmaceutically acceptable carrier.

[0155] In some aspect, disclosed herein is a pharmaceutical composition comprising the RNA of any preceding aspect and a pharmaceutically acceptable carrier.

[0156] In some embodiments, the linker is designed such that a region of the target polypeptide comes in contact with the proteasome in a manner which allows the proteasome to initiate degradation of the target polypeptide. In some embodiments, the linker is designed such that one or more amino acids of the target polypeptide that are recognized by the proteasome are presented to the proteasome in order to initiate degradation of the target polypeptide. In some embodiments, the linker is 10-100 amino acids long. In some embodiments, the linker comprises one or more a -helix spacers.

[0157] In some embodiments, the target polypeptide is a polypeptide associated with a disease. In some embodiments, the target-binding domain is positioned N-terminally relative to the proteasome binding domain. In some embodiments, the target-binding domain is positioned C- terminally relative to the proteasome binding domain.

[0158] In some embodiments, the proteasome-binding comprises a ubiquitin-like domain (UBL). In some embodiments, the proteasome-binding domain comprises a domain from HPV E7, gankyrin, Rad23a, Rad23b, UBL domain of midnolin, a-Helix-c domain of midnolin, or a coiled-coil domain.

[0159] In some embodiments, the target-binding domain comprises a nanobody. In some embodiments, the target-binding domain comprises an engineered protein interaction domain. In some embodiments, the target-binding domain comprises a monobody. In some embodiments, the target-binding domain binds to a prion, a viral polypeptide, a disease- associated protein, a cellular polypeptide having a disease-associated mutation, or a product of an oncogene. In some embodiments, the target-binding domain binds to a product of an oncogene. In some embodiments, the oncogene comprises Abl or Shp2. In some embodiments, the oncogene comprises Abl and Shp2.

[0160] In some embodiments, the target binding domain binds to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, or C-terminal SH2 domain of Shp2. In some embodiments, PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0161] Docket No. 10046-609W01 the target binding domain binds to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, and C-terminal SH2 domain of Shp2. In some embodiments, the target-binding domain binds to misfolded beta-amyloid. In some embodiments, the target-binding domain binds to Tan protein. In some embodiments, the polypeptide comprises a subcellular localization signal. In some embodiments, the subcellular localization signal is a nuclear localization signal.

[0162] In some embodiments, the target binding domain comprises at least 90% identity to any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 10. In some embodiments, the target binding domain comprises 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID

[0163] 5 NO: 5, SEQ ID NO: 6, or SEQ ID NO: 10.

[0164] In some embodiments, the proteasome-binding domain comprises at least 90% identity to any one of SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 14. In some embodiments, the proteasome-binding domain comprises 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to any one of SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 14.

[0165] 10 In some embodiments, the linker comprises at least 90% identity to any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, the linker comprises 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, the linker comprises an alpha helix of SEQ ID NO: 12. In some embodiments, the linker comprises 5%, 6%, 7%, 8%, 9%, 10%,

[0166] 15 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 12.

[0167] In some embodiments, the target-binding domain is positioned N-terminally relative to the proteasome binding domain. In some embodiments, the target-binding domain is positioned C-terminally relative to the proteasome binding domain.

[0168] 20 In some embodiments, the recombinant polypeptide further comprises a cellpenetrating peptide (CPP) sequence or a cellular receptor-binding sequence. As used herein the terms “cell penetrating peptide” refers to segments of polypeptide sequence that allow a polypeptide to cross the cell membrane (e.g., the plasma membrane in the case a eukaryotic cell). In some embodiments, the recombinant polypeptide further comprises a Human

[0169] 25 Immunodeficiency TAT CPP, a VP22 CPP, and any variants thereof.

[0170] Target Binding Domains

[0171] The target binding domain of the recombinant polypeptide disclosed herein can bind any target which is for direct delivery to the proteasome. A molecule which can bind to a target binding domain and be acted on or degraded by the proteasome is a target protein according to PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0172] Docket No. 10046-609W01 the present application. In general, target proteins may include, for example, structural proteins, receptors, enzymes, cell surface proteins, proteins pertinent to the integrated function of a cell, including proteins involved in catalytic activity, aromatase activity, motor activity, helicase activity, metabolic processes (anabolism and catabolism), antioxidant activity, proteolysis,

[0173] 5 biosynthesis, proteins with kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulator activity, signal transducer activity, structural molecule activity, binding activity (protein, lipid carbohydrate), receptor activity, cell motility, membrane fusion, cell communication, regulation of biological processes, development, cell differentiation, response to stimulus, behavioral proteins, cell adhesion proteins, proteins involved in cell death, proteins involved in transport (including protein transporter activity, nuclear transport, ion transporter activity, tumor associated antigens, channel transporter activity, carrier activity, permease activity, secretion activity, electron transporter activity, pathogenesis, chaperone regulator activity, nucleic acid binding activity, transcription regulator activity, extracellular organization and

[0174] 15 biogenesis activity, translation regulator activity. Targets can include proteins from eurkaryotes and prokaryotes including humans as targets for drug therapy, other animals, including domesticated animals, microbials for the determination of targets for antibiotics and other antimicrobials and plants, and even viruses, among numerous others.

[0175] In some embodiments, the target-binding domain binds to a prion, a viral polypeptide,

[0176] 20 a disease-associated protein, a cellular polypeptide having a disease-associated mutation, or a product of an oncogene. In some embodiments, the target-binding domain binds to a product of an oncogene. In some embodiments, the oncogene includes, but is not limited to Abl, Shp2, Myc (or c-Myc), and Stat3.

[0177] One of skill in the art can readily ascertain what target binding domains can be used with the desired target to be bound. Examples of such target binding domains include, but are not limited to, antibodies or fragments thereof, such as monoclonal antibodies, single chain antibodies (scFvs), Fv fragments or Fab fragments. Monoclonal antibodies to a target molecule can be made using standard methods such as, for example, hybridoma-based methods, genetically altered and transgenic mouse-based methods, recombinant methods, and display

[0178] 30 methods. Human antibodies can be made using methods such as, for example, transgenic mice comprising human heavy chain and light chain loci, human B -lymphocytes, recombinant methods, and display methods. In certain embodiments, monoclonal antibodies may be manipulated by recombinant techniques. In certain such embodiments, nucleic acid(s) encoding the heavy chain and light chain of the monoclonal antibody chains may be isolated PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0179] Docket No. 10046-609W01 and cloned from the cell expressing the antibody. For example, RNA can be prepared from cells expressing the desired antibody, such as mature B-cells or hybridoma cells, using standard methods. The RNA can then be used to make cDNA using standard methods, and the cDNA can be amplified, for example, by PCR, using specific oligonucleotide primers.

[0180] 5 In certain embodiments, human or non-human antibodies can be chimerized. In certain embodiments, mouse monoclonal antibodies are chimerized by replacing the mouse Fc with a human Fc. In certain embodiments, human monoclonal antibodies are chimerized by replacing the human Fc with a non-human animal Fc. In certain embodiments, the human Fc is replaced with a mouse Fc. Certain exemplary methods for making chimeric antibodies are provided, for example, in Morrison et al. (1984) Proc. Nat’l Acad. Sci. USA 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; Takeda et al. (1985) Nature 314:452-454; and U.S. Pat. Nos. 6,075,181 and 5,877,397.

[0181] In certain embodiments, non-human antibodies are “humanized.” As a non-limiting example, a mouse monoclonal antibody that specifically binds the target molecule may be

[0182] 15 humanized in order to reduce immunogenicity (e.g., reduced human anti-mouse antibody (HAMA) response) when administered to a human. In certain embodiments, a humanized antibody has a similar binding affinity for the target molecule as the non-humanized parent antibody. In certain embodiments, a humanized antibody has increased binding affinity for the target molecule when compared to the non-humanized parent antibody. Certain exemplary

[0183] 20 humanization methods include, but are not limited to, CDR grafting and human engineering, as described in detail below.

[0184] In certain embodiments, one or more complementarity determining regions (CDRs) from the light chain and / or heavy chain variable regions of an antibody with the desired binding specificity (the “donor” antibody) are grafted onto human framework regions (FRs) of the light and / or heavy chain of an “acceptor” antibody in order to create a humanized antibody with the binding specificity of the donor antibody.

[0185] In certain embodiments, cDNA encoding a heavy chain and / or light chain can be modified in order to modify the expressed heavy and / or light chain. For example, in certain embodiments, the constant region of a mouse heavy or light chain can be replaced with the

[0186] 30 constant region of a human heavy or light chain. In this manner, in certain embodiments, a chimeric antibody can be produced which possesses human antibody constant regions but retains the binding specificity of a mouse antibody. Alternatively, the constant region of a human heavy or light chain can be replaced with the constant region of a non-human animal heavy or light chain. In this case, a chimeric antibody can be produced which possesses non- PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0187] Docket No. 10046-609W01 human animal antibody constant regions, e.g., for expression in a non-human animal model, but retains the binding specificity of the human antibody.

[0188] The target binding domain can also be a monobody or nanobody, or comprise a engineered protein interaction domain. Monobodies of the disclosure are small proteins

[0189] 5 comparable in size to a single chain antibody. These genetically engineered proteins specifically bind target sequences including antigens. Monobodies of the disclosure may specifically target one or more distinct proteins or target sequences. Nanobody® (Ablynx N. V) molecules (Nbs), also referred to as single domain-based VHHs, are antibody fragments derived from heavy-chain only IgG antibodies found in the Camelidae family.

[0190] The target binding domain can also be a small peptide. Peptides that can bind to specific targets have been obtained by structure free screening or structure based design. Structure free techniques include phage display, RNA display and other screening methods. Also disclosed are the use of synbodies (synthetic antibodies). Two examples of small peptides which can bind a target include the small peptide ligand QLLRHLILH (SEQ ID NO: 6) and

[0191] 15 GQVGRQLAIIGDAINR (SEQ ID NO: 5), which are described in more detail in Example 1. It should be understood that the targeting domain can also be any interaction domain known in the art including, but not limited to Leucine (Leu) Rich Repeat domains, affinity domains, naturally occurring domains, artificially designed domains (such as those designed by artificial intelligence), and interaction domains derived by selections.

[0192] 20 In some embodiments, the target binding domain binds to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, and / or C-terminal SH2 domain of Shp2. In some embodiments, the target-binding domain binds to misfolded beta-amyloid. In some embodiments, the target-binding domain binds to Tau protein. In some embodiments, the polypeptide comprises a subcellular localization signal. In some embodiments, the subcellular localization signal is a nuclear localization signal, including but not limited to a Abl nuclear localization signal, a Shp2 nuclear localization signal, a Myc (or c-Myc) nuclear localization signal and a Stat3 nuclear localization signal.

[0193] The target binding domain disclosed herein can be optimized (engineered) to best interact with the target. These modifications can include, but are not limited to, chemically

[0194] 30 modified or biologically modified forms of the peptide. Such modifications are described in the definitions section above.

[0195] Linkers

[0196] The linker disclosed herein can be any amino acid sequence which can join together the target binding moiety and the proteasome-binding domain. In some embodiments, the linker is PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0197] Docket No. 10046-609W01 designed such that a region of the target polypeptide comes in contact with the proteasome in a manner which allows the proteasome to initiate degradation of the target polypeptide. In some embodiments, the linker is designed such that one or more amino acids of the target polypeptide that are recognized by the proteasome are presented to the proteasome in order to

[0198] 5 initiate degradation of the target polypeptide.

[0199] In some embodiments, the linker is 10-100 amino acids long. In some embodiments, the linker comprises one or more a-helix spacers. In some embodiments, the linker is an unstructured amino acid sequence. In some embodiments, the linker is a stiff structure. In some embodiments, the linker is a flexible structure. In some embodiments, the linker is resistant to protein targeted degradation. In some embodiments, the linker is resistant to the proteosome, one or more proteases, and / or any known proteolytic mechanism. In some embodiments, the target polypeptide is a polypeptide associated with a disease. Specific examples of linkers include, but are not limited to, those found in SEQ ID NOS: 7, 8, 9, 12, or any fragments thereof. It should be understood that the linker of the present disclosure includes, but is not

[0200] 15 limited to an alpha helix fragment of SEQ ID NO: 12, wherein said alpha helix fragment comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% sequence identity to SEQ ID NO: 12. These specific linkers are described in more detail below.

[0201] Proteasome Binding Domain

[0202] 20 The proteasome binding domain of the recombinant polypeptide disclosed herein can comprise any molecule which is capable of binding to the proteasome and delivering the recombinant polypeptide and its associated target to the proteasome. One example is the mandolin protein which regulates the ubiquitin-independent degradation of transcription regulators in the nucleus of the cell. This is described in more detail in Example 1. In another example, the proteasome binding domain comprises a ubiquitin-like domain (UBL). In some embodiments, the proteasome-binding domain comprises a domain from HPV E7, gankyrin, Rad23a, Rad23b, UBL domain of midnolin, a-Helix-c domain of midnolin, or a coiled-coil domain.

[0203] 30 Methods of Use

[0204] In some aspects, disclosed herein is a pharmaceutical composition comprising the recombinant polypeptide or recombinant protein of any preceding aspect and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0205] Docket No. 10046-609W01 pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil 5 emulsion) and / or various types of wetting agents.

[0206] In some aspects, disclosed herein is a method of degrading a target polypeptide in a cell, the method comprising: (a) exposing the protein to a recombinant polypeptide that binds to both a target polypeptide and a proteasome, wherein the recombinant polypeptide comprises: (i) a target-binding domain, wherein said target binding domain binds to the target polypeptide;

[0207] 10 (ii) a proteasome-binding domain that binds to the proteasome; and (iii) a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker facilitates the proteasome recognizing an initiation region of the target polypeptide; and (b) allowing the recombinant polypeptide to bring the target polypeptide into contact with the proteasome, wherein the proteasome degrades the target polypeptide.

[0208] In some aspects, disclosed herein is a method of treating a subject in need of degradation of a target polypeptide, the method comprising: (a) exposing the protein to a recombinant polypeptide that binds to both a target polypeptide and a proteasome, wherein the recombinant polypeptide comprises: (i) a target-binding domain, wherein said target binding domain binds to the target polypeptide; (ii) a proteasome-binding domain that binds to the proteasome; and (iii) a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker facilitates the proteasome recognizing an initiation region of the target polypeptide; and (b) allowing the recombinant polypeptide to bring the target polypeptide into contact with the proteasome, wherein the proteasome degrades the target polypeptide.

[0209] In some aspects, disclosed herein is a method of treating a subject in need of degradation of a target polypeptide, the method comprising: (a) administering to the subject a nucleic acid including, but not limited to DNA or RNA (such as, for example mRNA), encoding a recombinant polypeptide that binds to both a target polypeptide and a proteasome, wherein the recombinant polypeptide comprises: (i) a target-binding domain, wherein said target binding domain binds to the target polypeptide; (ii) a proteasome-binding domain that binds to the proteasome; and (iii) a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker facilitates the proteasome recognizing an initiation region of the target polypeptide; and (b) allowing the recombinant polypeptide to PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0210] Docket No. 10046-609W01 bring the target polypeptide into contact with the proteasome, wherein the proteasome degrades the target polypeptide.

[0211] In some embodiments, the method of the preceding aspect comprises the linker being designed such that a region of the target polypeptide comes in contact with the proteasome in a manner which allows the proteasome to initiate degradation of the target polypeptide. In some embodiments, the method of the preceding aspect comprises the linker being designed such that one or more amino acids of the target polypeptide which are recognized by the proteasome in order to initiate degradation of the target polypeptide are presented to the proteasome. In some embodiments, the method of the preceding aspect comprises the linker being 10-100 amino acids long. In some embodiments, the method of the preceding aspect comprises the linker comprising one or more a-helix spacers.

[0212] In some embodiments, the method of the preceding aspect comprises the target polypeptide being a polypeptide associated with a disease. In some embodiments, the method of the preceding aspect comprises the proteasome-binding comprising a ubiquitin-like domain, including but not limited to a domain from HPV E7, gankyrin, Rad23a, Rad23b, UBL domain of midnolin, a-Helix-c domain of midnolin, or a coiled-coil domain.

[0213] In some embodiments, the method of any preceding aspect further comprises a cellpenetrating peptide (CPP) sequence or a cellular receptor-binding sequence. In some embodiments, the method of the preceding aspect comprises the target-binding domain comprising a nanobody. In some embodiments, the method of the preceding aspect comprises the target-binding domain comprising an engineered protein interaction domain. In some embodiments, the method of the preceding aspect comprises the target-binding domain comprising a monobody. In some embodiments, the method of the preceding aspect comprises the target-binding domain binding to a prion, a viral polypeptide, a disease-associated protein, a cellular polypeptide having a disease- associated mutation or the product of an oncogene. In some embodiments, the method of the preceding aspect comprises the target-binding domain binding to the product of an oncogene, wherein the oncogene includes but is not limited to Abl, Shp2, or a combination thereof. In some embodiments, the method of the preceding aspect comprises the target binding domain binding to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, or C-terminal SH2 domain of Shp2. In some embodiments, the method of the preceding aspect comprises the target binding domain binding to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, and C-terminal SH2 domain of Shp2. In some embodiments, the method of the preceding aspect comprises the target-binding domain binding to misfolded PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0214] Docket No. 10046-609W01 beta-amyloid. In some embodiments, the method of the preceding aspect comprises the targetbinding domain binding to Tan protein.

[0215] In some embodiments, the method of the preceding aspect comprises the polypeptide comprising a subcellular localization signal, including but not limited to a nuclear localization signal.

[0216] In some embodiments, the subject has cancer, a neurogenerative disorder, an autoimmune disease, an age-related degenerative disease, a viral disease, or an inflammatory disorder. In some embodiments, the subject has been exposed to a substance which causes accumulation of unwanted target polypeptide in the subject.

[0217] 5 For the prevention or treatment of disease, the appropriate dosage of the disclosed recombinant polypeptide (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of recombinant polypeptide use, the severity and course of the disease, whether the recombinant polypeptide is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical

[0218] 10 history and response to the recombinant polypeptide, and the discretion of the attending physician. The recombinant polypeptide is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (e.g. 0.1 mg / kg-10 mg / kg) of a recombinant polypeptide can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate

[0219] 15 administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of a recombinant polypeptide would be in the range from about 0.05 mg / kg to about 10

[0220] 20 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful.

[0221] 25 The progress of this therapy is easily monitored by conventional techniques and assays.

[0222] Nucleic Acids, Vectors, and Cells PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0223] Docket No. 10046-609W01

[0224] In some aspects, disclosed herein is a nucleic acid molecule, wherein the nucleic acid includes, but is not limited to DNA and RNA, encoding a recombinant polypeptide of any preceding aspect.

[0225] In some aspects, disclosed herein is an expression vector encoding a recombinant

[0226] 5 polypeptide of any preceding aspect, wherein said the expression vector is operably linked to a promoter. In some embodiments, the vector is a plasmid, a viral vector, or an episomal vector. A plasmid or a viral vector can be capable of extrachromosomal replication or, optionally, can integrate into the host genome. As used herein, the term "integrated" used in reference to an expression vector (e.g., a plasmid or viral vector) means the expression vector, or a portion thereof, is incorporated (physically inserted or ligated) into the chromosomal DNA of a host cell. As used herein, a “viral vector” refers to a virus-like particle containing genetic material which can be introduced into a eukaryotic cell without causing substantial pathogenic effects to the eukaryotic cell. A wide range of viruses or viral vectors can be used for transduction but should be compatible with the cell type the virus or viral vector are transduced into (e.g., low

[0227] 15 toxicity, capability to enter cells). Suitable viruses and viral vectors include adenovirus, lentivirus, retrovirus, among others. In some embodiments, the expression vector encoding a recombinant polypeptide is a naked DNA or is comprised in a nanoparticle (e.g., liposomal vesicle, porous silicon nanoparticle, gold-DNA conjugate particle, polyethyleneimine polymer particle, cationic peptides, etc.). In some embodiments, the expression vector encoding the

[0228] 20 recombinant polypeptide comprises RNA, such as for example mRNA.

[0229] The vector of any preceding aspect can be delivered with a high degree of specificity to a particular tissue type. Accordingly, a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al., Proc. Natl. Acad. Sci. 92, (1995):9747-9751, reported that Moloney murine leukemia virus can be modified to express human neuregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein

[0230] 30 comprising a ligand for the cell-surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells. PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0231] Docket No. 10046-609W01

[0232] Vectors can be delivered in vivo by administration to an individual subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, intrathecal, intratracheal, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an

[0233] 5 individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.

[0234] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing nucleases and / or donor constructs can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Alternatively, RNA, such as for example mRNA, can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and,

[0235] 15 although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0236] A ) Retroviral Vectors

[0237] A retrovirus is an animal virus belonging to the virus family of Retro viridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by

[0238] 20 Verma, I.M., Retroviral vectors for gene transfer.

[0239] A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse

[0240] 30 transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5’ to the 3’ LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. The removal of the PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0241] Docket No. 10046-609W01 gag, pol, and env genes allows for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative

[0242] 5 selectable markers along with other genes in the insert.

[0243] Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

[0244] B) Adenoviral Vectors

[0245] 15 The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61:1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj- Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome- mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of

[0246] 20 the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994);

[0247] 30 Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0248] Docket No. 10046-609W01

[0249] Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0250] A viral vector can be one based on an adenovirus which has had the El gene removed

[0251] 5 and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome.

[0252] C) Adeno-asscociated viral vectors

[0253] Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding

[0254] 15 the green fluorescent protein, GFP.

[0255] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.

[0256] 20 Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and sitespecific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United states Patent No. 6,261,834 is herein incorporated by reference for material related to the AAV vector.

[0257] D) Large payload viral vectors

[0258] Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter and Robertson,. Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses (herpes

[0259] 30 simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B -cells as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, constitutively expressed during infection PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0260] Docket No. 10046-609W01 with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes.

[0261] 5 Other useful systems include, for example, replicating and host-restricted nonreplicating vaccinia virus vectors.

[0262] In some embodiments, the promoter is an inducible promoter, a repressible promoter, or a constitutive promoter. In some embodiments, the promoter is a tissue or cell type specific promoter. Non-limiting examples of tissue or cell type specific promoter include but are not limited to surfactant protein B promoter (SP-B in lung), B29 promoter (B cells), CD14 promotor (monocytic cells), CD43 promoter (leukocytes and platelets), CD68 promoter (macrophages), Desmin promoter (muscle), Elastase- 1 promoter (pancreatic acinar cells), endoglin promoter (endothelial cells), Fibronectin promoter (differentiating cells and healing tissues), Flt-1 promoter (endothelial cells), GFAP promoter (astrocytes), Mb promoter

[0263] 15 (muscle), SYN1 promoter (neurons), SV40 / bAlb promoter (Liver)) and cancer specific promoters (including, but not limited to carcinoembryonic antigen (CEA) promoter, hTERT promoter, epidermal growth factor receptor (EGFR) promoter, human epidermal growth factor receptor / neu (HER2 / NEU) promoter, vascular endothelial growth factor receptor (VEGFR) promoter, folate receptor (FR) promoter, transferrin receptor (CD71) promoter, mucines

[0264] 20 promoters, tumor resistance antigen 1-60 (TRA-1-60) promoter, cyclooxygenase (COX) promoter, cytokeratin 18 promoter, cytokeratin 19 promoter, surviving promoter, and chimeric antigen receptor (CAR) promoters, alpha-fetoprotein (AFP) promoter, thyroid transcription factor 1 (TTF-1) promoter, glypican-3 protein (GPC3) promoter, human secretory leukocyte protease inhibitor (hSLPI) promoter, ERBB2 promoter, Mucin 1 (MUC1) promoter, L-plastin promoter, alpha-lactalbumin (LALBA) promoter, cyclooxygenase 2 (COX2) promoter, epithelial glycoprotein (EPG2) promoter, A33 promoter, uPAR promoter, breast cancer 1 (BRCA1) and BRCA2 promoters.

[0265] In some aspects, disclosed herein is a cell comprising the expression vector of any preceding aspect. In some embodiments, the cell is a eukaryotic cell. In some embodiments,

[0266] 30 the cell is a prokaryotic cell.

[0267] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0268] Docket No. 10046-609W01 scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0269] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0270] 5

[0271] EXAMPLES

[0272] The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0273] Example 1: Improved Modular Platforms for Targeted Protein Degradation

[0274] The present technology provides several advantages including the ability to bypass the ubiquitination step and instead shunt proteins directly to the proteasome without the need for

[0275] 15 ubiquitination. Adaptors were developed that simultaneously bind target protein and proteasome, feeding the target directly into the proteasome for destruction.

[0276] The direct proteasome targeting technology was improved in three ways:

[0277] 1. Additional direct proteasome-targeting signals were tested for efficient degradation of previously hard-to-be-degraded substrates. Midnolin protein regulates the

[0278] 20 ubiquitin-independent degradation of transcription regulators in the nucleus of the cell. Midnolin has three domains, it can bind to the proteasome via its c-terminal a- helix and a ubiquitin-like domain (UBL), and it uses its Catch domain to bind a region within substrates (ref2). It has been shown that the c-terminal a-helix and a ubiquitin-like domain of midnolin can be fused to target affinity molecules and degrade some of the target proteins much more efficiently than Rad23 UBL. Another added feature of midnolin’ s c-terminal a-helix is that it has an inbuilt nuclear localization signal that can be beneficial for aiming at nuclear target protein.

[0279] 2. A more efficient linker was discovered that improved the degradation of previously hard-to-degrade target proteins.

[0280] 30 3. Currently, this technology relies on target affinity molecules like monobodies and nanobodies which are short, soluble, and stable affinity molecules. Other affinity molecules were tested based on the protein-protein interaction surface of the target protein. For example, a small peptide ligand (QLLRHLILH (SEQ ID NO: 6)) has PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0281] Docket No. 10046-609W01 been shown to bind ERa protein a therapeutic target for breast cancer, and BCL-xl associates with a small peptide GQVGRQLAIIGDAINR (SEQ ID NO: 5).

[0282] These improvements provide added advantages over other proteasome-based direct proteasome targeting platforms. These improvements are modular with each component being 5 modulated based on the structure, cellular localization, and the binding partners of the target protein.

[0283] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the 10 invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0284] PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0285] Docket No. 10046-609W01

[0286] SEQUENCE LISTINGS

[0287] 1. SEQ ID NO: 1 - Proteosome Targeting Degrader with MID-N

[0288] MSLAIHSTTGTRYDLAVPPDETVEGLRKRLSQRLKVPKERLALLHKDTRLSSGKLQE

[0289] FGVGDGSKLTLVPTVEAGVDGGSGGGSVQLVESGGALVQPGGSLRLSCAASGFPVN

[0290] RYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNS LKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGGHHHHHH

[0291] 2. SEQ ID NO: 2 - Proteosome Targeting Degrader with MID-C

[0292] MSASLLQGQSQIRMCKPPGDRLRQTENRATRCKVERLQLLLQQKRLRRKARRDARG

[0293] PYHWSPSRKAGRSDSSSSVDGGSGGGSVQLVESGGALVQPGGSLRLSCAASGFPVNR

[0294] YSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSL KPEDTAVYYCNVNVGFEYWGQGTQVTVSSGGHHHHHH

[0295] 3. SEQ ID NO : 3 - Myd88 Death Domain

[0296] ALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLE LGPSIEEDCQKYILKQ

[0297] 4. SEQ ID NO: 4 - IRAK4 Death Domain

[0298] MNKPITPSTYVRCLNVGLIRKLSDFIDPQEGWKKLAVAIKKPSGDDRYNQFHIRRFEA

[0299] LLQTGKSPTSELLFDWGTTNCTVGDLVDLLIQNEFFAPASLLLPDAVPKTANTLPSKE

[0300] 5. SEQ ID NO: 5 - BCL-xL peptide ligand

[0301] GQVGRQLAIIGDAINR

[0302] 6. SEQ ID NO: 6 - Era peptide ligand

[0303] QLLRHLILH

[0304] 7. SEQ ID NO: 7 - GS Linker (GS)

[0305] VDGGSGGGS

[0306] 8. SEQ ID NO: 8 - Linker 1 (LI)

[0307] VDGGSANGTSGASGPAGGS

[0308] 9. SEQ ID NO: 9 - Linker 2 (L2) PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0309] Docket No. 10046-609W01

[0310] VDGGSANGTSGASGPAGGGSANGTSGASGPAGGS

[0311] 10. SEQ ID NO: 10 - Nanobody peptide (from Vicugna pacos) GenBank: QBF76413.1 - QVQLQESGGGSVQAGGSLRLSCVASRRTFSNYADAMGWFRQAPGKEREFVAAISW NSATTRYLDSVKARFTISRVNANNTVYLQMNSLKPEDTAVYYCAAKPTGSFPPVEEE KYN YWGQGTQ VT VS S

[0312] 11. SEQ ID NO: 11 - Dictyostelium discoideum DNA for cyclic nucleotide phosphodiesterase inhibitor (Gene ID 7314) - MAKIIISLILLLSLFSFSYGAYNCNKLNCSSKNTKCRTFSCTSVVGCYYTDKCTSPDLC HNSACNASTGNCTLTTISCNDNNPCTDDFCHPGYGCYSVPNSCDPGVICQQNCNDND PCTYDFCDALNICRHSETYCNDGDACTLNTCGVNGCNFTKISCDDNDPCTADYCSTL YGCYHEPIECSIKVPCNIDSDCNRNNGCETFTCNLSTNTCDYYAKNCGGWPCINNQC TTGSISN

[0313] 12. SEQ ID NO: 12 - Myosin VI (from Homo sapiens) GenBank: AAK00229.1 - MEDGKPVWAPHPTDGFQMGNIVDIGPDSLTIEPLNQKGKTFLALINQVFPAEEDSKK DVEDNCSLMYLNEATLLHNIKVRYSKDRIYTYVANILIAVNPYFDIPKIYSSEAIKSYQ GKSLGTRPPHVFAIADKAFRDMKVLKMSQSIIVSGESGAGKTENTKFVLRYLTESYG TGQDIDDRIVEANPLLEAFGNAKTVRNNNSSRFGKFVEIHFNEKSSVVGGFVSHYLLE KSRICVQGKEERNYHIFYRLCAGASEDIREKLHLSSPDNFRYLNRGCTRYFANKETDK QILQNRKSPEYLKAGSMKDPLLDDHGDFIRMCTAMKKIGLDDEEKLDLFRVVAGVL HLGNIDFEEAGSTSGGCNLKNKSAQSLEYCAELLGLDQDDLRVSLTTRVMLTTAGGT KGTVIKVPLKVEQANNARDALAKTVYSHLFDHVVNRVNQCFPFETSSYFIGVLDIAG FEYFEHNSFEQFCINYCNEKLQQFFNERILKEEQELYQKEGLGVNEVHYVDNQDCID LIEAKLVGILDILDEENRLPQPSDQHFTSAVHQKHKDHFRLTIPRKSKLAVHRNIRDDE GFIIRHFAGAVCYETTQFVEKNNDALHMSLESLICESRDKFIRELFESSTNNNKDTKQ KAGKLSFISVGNKFKTQLNLLLDKLRSTGASFIRCIKPNLKMTSHHFEGAQILSQLQCS GMVSVLDLMQGGYPSRASFHELYNMYKKYMPDKLARLDPRLFCKALFKALGLNEN DYKFGLTKVFFRPGKFAEFDQIMKSDPDHLAELVKRVNHWLTCSRWKKVQWCSLS VIKLKNKIKYRAEACIKMQKTIRMWLCKRRHKPRIDGLVKVGTLKKRLDKFNEVVS VLKDGKPEMNKQIKNLEISIDTLMAKIKSTMMTQEQIQKEYDALVKSSEELLSALQK KKQQEEEAERLRRIQEEMEKERKRREEDEKRRRKEEEERRMKLEMEAKRKQEEEER KXREDDEKRIQAEVEAQLARQKEEESQQQAVLEQERRDRELALRIAQSEAELISDEA PCT / US25 / 46935 18 September 2025 (18.09.2025)

[0314] Docket No. 10046-609W01

[0315] QADLALRRNDGTRPKMTPEQMAKEMSEFLSRGPAVLATKAAAGTKKYDLSKWKY

[0316] AELRDTINTSCDIELLAACREEFHRRLKVYHAWKSKNKKRNTETEQRAPKSVTDYDF

[0317] APFLNNSPQQNPAAQIPARQREIEMNRQQRFFRIPFIRPADQYKDPQSKKKGWWYAH

[0318] FDGPWIARQMELHPDKPPILLVAGKDDMEMCELNLEETGLTRKRGAEILPRQFEEIW

[0319] ERCGGIQYLQNAIESRQARPTYATAMLQSLLK

[0320] 13. SEQ ID NO: 13 - MID-N

[0321] MSLAIHSTTGTRYDLAVPPDETVEGLRKRLSQRLKVPKERLALLHKDTRLSSGKLQE

[0322] FGVGDGSKLTLVPTVEAG

[0323] 14. SEQ ID NO: 14 - MID-C

[0324] MSASLLQGQSQIRMCKPPGDRLRQTENRATRCKVERLQLLLQQKRLRRKARRDARG

[0325] PYHWSPSRKAGRSDSSSS

Claims

PCT / US25 / 46935 18 September 2025 (18.09.2025)Docket No. 10046-609W01CLAIMSWhat is claimed is:

1. A recombinant polypeptide that binds to both a target polypeptide and a proteasome, the recombinant polypeptide comprising: a. a target-binding domain, wherein said target-binding domain binds to the target polypeptide; b. a proteasome-binding domain that binds to the proteasome; and c. a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker facilitates the proteasome recognizing an initiation region of the target polypeptide.

2. The polypeptide of claim 1, wherein the linker is designed such that a region of the target polypeptide comes in contact with the proteasome in a manner which allows the proteasome to initiate degradation of the target polypeptide.

3. The polypeptide of claim 1 or 2, wherein the linker is designed such that one or more amino acids of the target polypeptide that are recognized by the proteasome are presented to the proteasome in order to initiate degradation of the target polypeptide.

4. The polypeptide of any one of claims 1-3, wherein the linker is 10-100 amino acids long.

5. The polypeptide of any one of claims 1-4, wherein the linker comprises one or more a- helix spacers.

6. The polypeptide of any one of claims 1-5, wherein the target-binding domain is positioned N-terminally or C-terminally_relative to the proteasome binding domain.

7. The polypeptide of any one of claims 1-6, wherein the proteasome-binding comprises a ubiquitin-like domain (UBL).

8. The polypeptide of any one of claims 1-7, wherein the proteasome-binding domain comprises a domain from HPV E7, gankyrin, Rad23a, Rad23b, UBL domain of midnolin, a- Helix-c domain of midnolin, or a coiled-coil domain.PCT / US25 / 46935 18 September 2025 (18.09.2025)Docket No. 10046-609W019. The polypeptide of any one of claims 1-8, further comprising a cell-penetrating peptide (CPP) sequence or a cellular receptor-binding sequence.

10. The polypeptide of any one of claims 1-9, wherein the target-binding domain comprises a nanobody, an engineered protein interaction domain, and / or a monobody.

11. The polypeptide of any one of claims 1-10, wherein the target- binding domain binds to a prion, a viral polypeptide, a disease-associated protein, a cellular polypeptide having a disease-associated mutation, or a product of an oncogene.

12. The polypeptide of claim 11, wherein the oncogene comprises Abl or Shp2.

13. The polypeptide of any one of claims 1-12, wherein the target-binding domain binds to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, or C-terminal SH2 domain of Shp2.

14. The polypeptide of any one of claims 1-13, wherein the target- binding domain binds to misfolded beta-amyloid or a Tau protein.

15. The polypeptide of any one of claims 1-14, wherein the polypeptide comprises a subcellular localization signal.

16. The polypeptide of any one of claims 1-15, wherein the target binding domain comprises at least 90% identity to any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 10.

17. The polypeptide of any one of claims 1-16, wherein the proteasome-binding domain comprises at least 90% identity to any one of SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 14.

18. The polypeptide of any one of claims 1-17, wherein the linker comprises at least 90% identity to any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.PCT / US25 / 46935 18 September 2025 (18.09.2025)Docket No. 10046-609W0119. The polypeptide of any one of claims 1-18, wherein the linker comprises an alpha helix of SEQ ID NO: 12.

20. A nucleic acid molecule encoding the recombinant polypeptide according to any one of claims 1-19.

21. An expression vector comprising the nucleic acid of claim 20, wherein the nucleic acid is operably linked to a promoter.

22. A cell comprising the vector of claim 21.

23. A pharmaceutical composition comprising the recombinant polypeptide of any one of claims 1-19 and a pharmaceutically acceptable carrier.

24. A method of degrading a target polypeptide in a cell, the method comprising: a. exposing the protein to a recombinant polypeptide that binds to both a target polypeptide and a proteasome, wherein the recombinant polypeptide comprises: i. a target-binding domain, wherein said target binding domain binds to the target polypeptide; ii. a proteasome-binding domain that binds to the proteasome; and iii. a linker, wherein the linker has been designed such that, when the target polypeptide is bound to the proteasome, the linker facilitates the proteasome recognizing an initiation region of the target polypeptide; and b. allowing the recombinant polypeptide to bring the target polypeptide into contact with the proteasome, wherein the proteasome degrades the target polypeptide.

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