Cytoplasmic delivery technology

Abstract

The invention provides protein conjugates for cytoplasmic delivery of cargos (e.g., polypeptide cargos). The invention further provides pharmaceutical compositions comprising the protein conjugates, as well as methods of using and producing the protein conjugates.

Description

Attorney Docket No: 243735.000431 CYTOPLASMIC DELIVERY TECHNOLOGY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 675,498, filedJuly 25, 2024, the contents of which is incorporated by reference herein in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submittedelectronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 23, 2025, is named 243735_000431_SL.xml and is 70,348 bytes in size. FIELD OF THE INVENTION

[0003] The invention relates to protein conjugates for cytoplasmic delivery of cargos (e.g.,polypeptide cargos). The invention further relates to pharmaceutical compositions comprising the protein conjugates as well as methods of producing and using the protein conjugates. BACKGROUND OF THE INVENTION

[0004] The vast majority of cellular functions are controlled by interactions amongbiomacromolecules inside cells, and dysregulation of such interactions can lead to diseases. Consequently, selectively controlling macromolecular interactions within cells has been a central strategy for perturbing cellular functions to address basic scientific questions and can be the foundation of therapeutic development.

[0005] Gene-directed technologies such as gene knockout and knockdown are powerful, butthey affect the abundance of a protein of interest. They cannot assess the role of interactions involving a specific segment of a protein (e.g., a domain, and a peptide motif). Genetically modifying a target, such as site-directed mutations, can offer higher-resolution information than altering the target abundance, but substantial prior knowledge is required to design such mutations. Moreover, such modifications are not portable, requiring separate manipulations of each cell that one wishes to study, and they are not usually suitable for temporal control.

[0006] Small molecule compounds binding to intracellular targets, often referred to as toolcompounds, can be made cell-permeable and have been widely used as research tools andAttorney Docket No: 243735.000431 therapeutics. Unfortunately, it is challenging to develop selective and potent tool compounds, and the vast majority of intracellular targets remain “undruggable” by small molecules.

[0007] Hence, there has been intense interest and effort to exploit protein-based reagents as“tool biologics”. Modern engineered binding proteins, such as monobodies, can achieve high selectivity and potency more readily than small molecules. However, delivery of such tool biologics into the cytoplasm has been a major bottleneck, because the cell membrane blocks the transport of large, polar molecules such as water-soluble proteins. Although genetically encoded tool biologics have demonstrated their utility as research tools in precisely controlling intracellular targets and their interactions, they have some of the same limitations as genetic modifications, in particular the lack of portability, i.e., the need to engineer each cell and limited temporal control.

[0008] Delivering protein reagents efficiently into the cytoplasm and nucleus has been the holygrail in protein engineering and drug discovery. This is an area of intense efforts by many investigators utilizing diverse methods over the last 30 years. Still, there are no FDA-approved drugs of this type, despite the fact that many tool biologics against prominent undruggable targets have been developed, indicating the extraordinary demand for such agents. Existing methods do not effectively deliver protein cargos at levels that can achieve stoichiometric inhibition of intracellular targets. Furthermore, existing methods often trigger aggregation of reagents and / or are highly toxic to cells.

[0009] Therefore, there is a pressing need in the biomedical community for an effectivetechnology to safely deliver biologics into the cytoplasm. SUMMARY OF THE INVENTION

[0010] As specified in the ‘Background’ section above, there is a need in the art to exploitprotein-based reagents as tool biologics (e.g., a cargo) and develop an efficient technology for cytoplasmic delivery of biologics reagents (e.g., a delivery vehicle, or a carrier) including methods, components, and / or compositions thereof. The present application addresses these and other needs.

[0011] In one aspect, provided herein is a protein conjugate comprising(i) a protein carrier conjugated to a cationic polymer; and (ii) a polypeptide cargo,Attorney Docket No: 243735.000431 wherein the protein carrier is linked to the polypeptide cargo via a peptide bond to form a contiguous polypeptide.

[0012] In some embodiments, the contiguous polypeptide is recombinantly produced.

[0013] In some embodiments, the contiguous polypeptide is chemically or enzymaticallyproduced.

[0014] In some embodiments, the protein carrier is linked to the polypeptide cargo via aproteolytic cleavable linker.

[0015] In some embodiments, the proteolytic cleavable linker is recognized by an intracellularprotease.

[0016] In some embodiments, the protein carrier comprises an N-terminal polypeptide or a C-terminal polypeptide.

[0017] In some embodiments, the protein carrier comprises an N-terminal polypeptide.

[0018] In some embodiments, the protein carrier and / or the polypeptide cargo do not containnative cysteine residue(s) or have been engineered to remove native cysteine residue(s).

[0019] In some embodiments, the protein carrier is modified to introduce one or more cysteineresidues for site-specific conjugation with the cationic polymer.

[0020] In some embodiments, the protein carrier is modified to introduce one, two, three, orfour cysteine residues.

[0021] In some embodiments, the one or more cysteine residues are introduced to the proteincarrier outside the interface between the protein carrier and the intracellular protease.

[0022] In some embodiments, the one or more cysteine residues are introduced into the N-terminal polypeptide of the protein carrier.

[0023] In some embodiments, the one or more cysteine residues are introduced into the N-terminal polypeptide as a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide.

[0024] In some embodiments, the protein carrier is conjugated to one or more cationicpolymers at the one or more cysteine residues.

[0025] In some embodiments, the protein carrier comprises a ubiquitin-like protein (UBL), afunctional fragment, or derivative thereof.

[0026] In some embodiments, the one or more cysteine residues are introduced to the UBL.Attorney Docket No: 243735.000431

[0027] In some embodiments, the N-terminal polypeptide of the protein carrier is located atamino terminus of the UBL.

[0028] In some embodiments, the intracellular protease is a UBL-specific protease.

[0029] In one aspect, provided herein is a protein conjugate comprising(i) a protein carrier conjugated to a cationic polymer; and (ii) a cargo, wherein the protein carrier is operably linked to the cargo, and wherein the protein carrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof.

[0030] In some embodiments, the cargo is a polypeptide cargo.

[0031] In some embodiments, the protein carrier is linked to the cargo via a peptide bond toform a contiguous polypeptide.

[0032] In some embodiments, the cargo is linked to the UBL via a proteolytic cleavable linker.

[0033] In some embodiments, the UBL is cleaved from the cargo by a UBL-specific protease.

[0034] In some embodiments of the protein conjugate comprising a protein carrier comprisinga UBL, the protein carrier further comprises an N-terminal polypeptide or a C-terminal polypeptide.

[0035] In some embodiments of the protein conjugate comprising a protein carrier comprisinga UBL, the protein carrier comprises an N-terminal polypeptide.

[0036] In some embodiments of the protein conjugate comprising a protein carrier comprisinga UBL, the N-terminal polypeptide of the protein carrier is located at amino terminus of the UBL.

[0037] In some embodiments, the UBL has been engineered to remove native cysteineresidue(s).

[0038] In some embodiments of the protein conjugate comprising a protein carrier comprisinga UBL, the protein carrier is modified to introduce one, or more cysteine residues.

[0039] In some embodiments, the UBL is modified to introduce one or more cysteine residuesoutside the interface between the UBL and its cognate UBL specific protease.

[0040] In some embodiments, one or more cysteine residues are introduced into the N-terminalpolypeptide of the protein carrier.Attorney Docket No: 243735.000431

[0041] In some embodiments, the one or more cysteine residues are introduced into the N-terminal polypeptide as a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide.

[0042] In some embodiments, the protein carrier is conjugated to one or more cationicpolymers at the one or more cysteine residues.

[0043] In some embodiments, the ubiquitin-like protein (UBL) is selected from ubiquitin, asmall ubiquitin-like modifier (SUMO) protein, neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8), autophagy-related protein 8 (ATG8), autophagy-related 12 (ATG12), ), human leukocyte antigen (HLA)-F adjacent transcript 10 or Ubiquitin D (FAT10), interferon (IFN)-stimulated gene 15 (ISG15), ubiquitin related modifier 1 (URM1), or ubiquitin fold modifier 1 (UFM1).

[0044] In some embodiments, the UBL comprises or consists of an amino acid sequence setforth in any one of the SEQ ID NOs: 1-8, 53-60, and 72 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in any one of the SEQ ID NOs: 1-8, 53-60, and 72.

[0045] In some embodiments, the SUMO protein is SUMO1, SUMO2, SUMO3, or SUMO4.

[0046] In some embodiments, the SUMO protein comprises or consists of an amino acidsequence set forth in any one of the SEQ ID NOs: 1-8 and 53-55 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in any one of the SEQ ID NOs: 1-8 and 53-55.

[0047] In some embodiments, the SUMO protein is SUMO1.

[0048] In some embodiments, SUMO1 comprises or consists of an amino acid sequence setforth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53, or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53.

[0049] In some embodiments, the cysteine residue at position 52 of SUMO1 has been mutatedto a non-cysteine residue.

[0050] In some embodiments, the cysteine residue at position 52 of SUMO1 has been mutatedto an alanine.

[0051] In some embodiments, SUMO1 is modified to introduce one or more cysteine residuesby mutating the residue at the amino acid position 33, amino acid position 46, amino acid position 53, amino acid position 85, and / or amino acid position 50 to cysteine.Attorney Docket No: 243735.000431

[0052] In some embodiments, SUMO1 comprises or consists of an amino acid sequence setforth in SEQ ID NO: 8 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 8.

[0053] In some embodiments, the C-terminal or proximal to the C-terminal sequence of theSUMO protein comprises Gly-Gly.

[0054] In some embodiments, the UBL-specific protease is a ubiquitin-specific protease (USP)or SUMO-specific protease (SENP).

[0055] In some embodiments, the SENP is SENP1, SENP2, SENP3, SENP5, SENP6, and / orSENP7.

[0056] In some embodiments, the cationic polymer is a polyamine.

[0057] In some embodiments, the cationic polymer is selected from polyethylenimine (PEI),poly(L-lysine) (PLL), poly(D-lysine) (PDL), polyamidoamine (PAA), Poly(amino-co-ester)s (PAEs), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), poly(propylene imine) (PPI), poly-L-arginine (PLA), poly-guanidine, poly-D-arginine, poly(D,L-lysine hydrobromide), poly-L-ornithine, PEI-Gua, PEI-Arg, PEI-His, PEI-Trp, or derivatives and combinations thereof.

[0058] In some embodiments, the cationic polymer is PEI.

[0059] In some embodiments, the PEI is a linear PEI or branched PEI.

[0060] In some embodiments, the branched PEI is a short-chain PEI.

[0061] In some embodiments, the PEI has a molecular weight of less than 2000 Da.

[0062] In some embodiments, the PEI has a molecular weight ranging from about 600 Da toabout 1800Da.

[0063] In some embodiments, the PEI has a molecular weight of about 600 Da (PEI600) or1200 Da (PEI1200).

[0064] In some embodiments, the PEI has a molecular weight of about 600 Da (PEI600).

[0065] In some embodiments, the PEI is branched PEI600 (bPEI600) or branched PEI1200(bPEI1200).

[0066] In some embodiments, the PEI is branched PEI600 (bPEI600).

[0067] In some embodiments, the protein carrier is conjugated to the cationic polymer via alinker.

[0068] In some embodiments, the linker is a heterobifunctional crosslinker.Attorney Docket No: 243735.000431

[0069] In some embodiments, the linker comprises an electrophile selected fromhaloacetophenone, epoxide, haloacetamide, maleimide, isothiocyanate, vinyl sulfone, nitrile, iminonitrile, hydrazone, butynoate, acrylester, acrylonitrile, acrylamide, benzisothiazolone, and N-hydroxy(methyl)phthalimide.

[0070] In some embodiments, the linker comprises a N-hydroxysuccinimide (NHS) ester and amaleimide group.

[0071] In some embodiments, the linker is selected from N-alpha-maleimidoacetoxylsuccinimide ester (AMAS), N-beta-maleimidopropyl-oxysuccinimide ester (BMPS), N-gamma- maleimidobutyryl-oxysuccinimide ester (GMBS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N- epsilon-malemidocaproyl-oxysuccinimide ester (EMCS), succinimidyl 4-(p- maleimidophenyl)butyrate (SMPB), succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC- SMCC), N-(11-maleimidoundecanoyloxy)succinimide (KMUS), or derivatives and combinations thereof.

[0072] In some embodiments, the linker is N-alpha-maleimidoacetoxyl succinimide ester(AMAS).

[0073] In some embodiments, the protein conjugate is capable of penetrating into thecytoplasm of a mammalian cell.

[0074] In some embodiments, the cargo interacts with an intracellular target.

[0075] In some embodiments, the intracellular target is selected from rat sarcoma virus (RAS),Src homology 2 domain-containing phosphatase 2 (SHP2), Aurora A kinase, Src-family kinases, signal transducer and activator of transcription 3 (STAT3), PR domain containing 14 (PRDM14), WD repeat domain 5 (WDR5), mixed lineage kinase domain-like protein (MLKL), p53, and ABL kinase.

[0076] In some embodiments, RAS is Kirsten rat sarcoma viral oncogene homolog (KRAS),neuroblastoma RAS viral oncogene homolog (NRAS), or Harvey rat sarcoma viral oncogene homolog (HRAS).

[0077] In some embodiments, the polypeptide cargo is selected from a monobody (Mb), adesigned ankyrin repeat protein (DARPin), an Anticalin, an affilin, an affibody, a peptide, an antibody or antigen-binding fragment, an enzyme, or derivatives and combinations thereof.Attorney Docket No: 243735.000431

[0078] In some embodiments, the polypeptide cargo is selected from a monobody, a designedankyrin repeat protein (DARPin), or derivatives thereof.

[0079] In some embodiments, the polypeptide cargo is tandem monobodies comprising two ormore monobody units.

[0080] In some embodiments, the monobody or DARPin targets KRAS, HRAS, NRAS,Aurora A kinase, ABL kinase, a Src-family kinase, SHP2, STAT3, PRDM14, WDR5, MLKL, or p53.

[0081] In some embodiments, the monobody is selected from 12VC1, NS1, 12D3, 12D4,12D5, AS25, AS27, HA4, 7C12, Nsa1, NSa5, CS3, S14, S4, Mb1, Mb2, Mb3, Mb4, Mb5, Mb6, Mb19, Mb20, Mb24, Mb27, Mb32, Mb33, MS3-6, Mb13, R15, Fgr_1, Fgr_2, Hck_1, Hck_2, Lck_1, Lck_3, Lyn_2, Lyn_4, Src_2, Yes_1, Yes_3, MLKL_26, MLKL_27, MLKL_32, MLKL_33, and MLKL_37.

[0082] In some embodiments, the monobody comprises or consists of an amino acid sequenceset forth in any one of the SEQ ID NOs:14-52 and 69-70 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of the SEQ ID NOs:14-52 and 69-70.

[0083] In some embodiments, the DARPin is K27, C10, or C10-H82R.

[0084] In some embodiments, the antibody or antigen-binding fragment thereof is selectedfrom a single chain antibody, a single domain antibody, a variable heavy homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, a monoclonal antibody, a bispecific antibody or antigen-binding fragment thereof, a bi-epitopic antibody or antigen-binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, an scFv, a VH domain, a monovalent antibody or a bivalent antibody.

[0085] In some embodiments, the peptide is a stapled peptide or bicyclic peptide.

[0086] In some embodiments, the protein conjugate further comprises one or more additionalmoieties.

[0087] In some embodiments, the additional moiety is a His6-tag (SEQ ID NO: 12).

[0088] In some embodiments, the N-terminal polypeptide of the protein carrier comprises theHis6-tag (SEQ ID NO: 12).Attorney Docket No: 243735.000431

[0089] In some embodiments, the additional moiety is a V5 tag, wherein the V5 tag is at a C-terminus of the polypeptide cargo.

[0090] In some embodiments, the additional moiety is a cell-targeting moiety, wherein the N-terminal polypeptide of the protein carrier comprises the cell-targeting moiety.

[0091] In some embodiments, the cell-targeting moiety is a polypeptide that binds specificallyto a cell-surface protein.

[0092] In some embodiments, the protein conjugate further comprises an albumin-bindingdomain.

[0093] In some embodiments, the albumin-binding domain comprises or consists of an aminoacid sequence set forth in SEQ ID NO:66 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of the SEQ ID NO:66.

[0094] In some embodiments, the contiguous polypeptide comprises or consists of an aminoacid sequence set forth in any one of the SEQ ID Nos: 9, 11, 62-65, and 71 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of the SEQ ID NOs: 9, 11, 62-65, and 71.

[0095] In some embodiments, the contiguous polypeptide comprises or consists of an aminoacid sequence set forth in SEQ ID NO: 62 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62.

[0096] In one aspect, provided herein is a nucleic acid molecule encoding the contiguouspolypeptide in the protein conjugate of the present disclosure.

[0097] In some embodiments, the nucleic acid molecule comprises (i) a nucleotide sequenceencoding the protein carrier; and (ii) a nucleotide sequence encoding the polypeptide cargo.

[0098] In some embodiments, the nucleic acid molecule comprises or consists of the sequenceset forth in SEQ ID NO: 10 or a sequence having at least 80% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 10.

[0099] In one aspect, provided herein is a nucleic acid molecule comprising a nucleotidesequence encoding the protein carrier of the protein conjugate of the present disclosure.

[0100] In some embodiments, the nucleic acid molecule comprises the nucleotide sequenceencoding the protein carrier comprises or consists of the sequence set forth in SEQ ID NO: 61 or a sequence having at least 80% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 61.Attorney Docket No: 243735.000431

[0101] In one aspect, provided herein is a nucleic acid molecule comprising a nucleotidesequence encoding the polypeptide cargo of the protein conjugate of the present disclosure.

[0102] In some embodiments, the nucleic acid molecule of the present disclosure is a DNAmolecule.

[0103] In some embodiments, the nucleic acid molecule of the present disclosure is an RNAmolecule.

[0104] In one aspect, provided herein is a polypeptide encoded by the nucleic acid molecule ofthe present disclosure.

[0105] In one aspect, provided herein is a recombinant vector comprising the nucleic acidmolecule of the present disclosure.

[0106] In one aspect, provided herein is an isolated host cell comprising the nucleic acidmolecule of the present disclosure or the vector of the present disclosure.

[0107] In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or amammalian cell.

[0108] In some embodiments, the bacterial cell is Escherichia coli (E.coli).

[0109] In one aspect, provided herein is a method of producing a protein conjugate, comprisinga) generating a contiguous polypeptide comprising (i) a protein carrier and (ii) apolypeptide cargo; and b) conjugating a cationic polymer to the protein carrier to form a protein conjugate.

[0110] In some embodiments, the contiguous polypeptide is generated recombinantly.

[0111] In some embodiments, the contiguous polypeptide is generated by culturing a host cellcomprising the nucleic acid molecule of the present disclosure or the vector of the present disclosure to express the contiguous polypeptide.

[0112] In some embodiments, the contiguous polypeptide is generated by attaching thepolypeptide cargo to the protein carrier chemically or enzymatically.

[0113] In some embodiments of the method of producing a protein conjugate, the methodfurther comprises purifying the contiguous polypeptide from the host cell after step a).

[0114] In one aspect, provided herein is a method of producing a protein conjugate, comprisinga) conjugating a cationic polymer to a protein carrier; andb) attaching a polypeptide cargo to the protein carrier chemically or enzymatically toform a contiguous polypeptide, thereby forming a protein conjugate.Attorney Docket No: 243735.000431

[0115] In some embodiments of the method of producing a protein conjugate, the methodfurther comprises purifying the protein conjugate after step b).

[0116] In some embodiments of the method of producing a protein conjugate, the proteincarrier is linked to the polypeptide cargo via a proteolytic cleavable linker.

[0117] In some embodiments of the method of producing a protein conjugate, the proteolyticcleavable linker is recognized by an intracellular protease.

[0118] In some embodiments of the method of producing a protein conjugate, the proteincarrier comprises an N-terminal polypeptide or a C-terminal polypeptide.

[0119] In some embodiments of the method of producing a protein conjugate, the proteincarrier comprises an N-terminal polypeptide.

[0120] In some embodiments of the method of producing a protein conjugate, the proteincarrier and / or the polypeptide cargo do not contain native cysteine residue(s) or have been engineered to remove native cysteine residue(s).

[0121] In some embodiments of the method of producing a protein conjugate, the proteincarrier is modified to introduce one or more cysteine residues for site-specific conjugation with the cationic polymer.

[0122] In some embodiments of the method of producing a protein conjugate, the proteincarrier is modified to introduce one, two, three or four cysteine residues.

[0123] In some embodiments of the method of producing a protein conjugate, the one or morecysteine residues are introduced to the protein carrier outside the interface between the protein carrier and the intracellular protease.

[0124] In some embodiments of the method of producing a protein conjugate, the one or morecysteine residues are introduced into the N-terminal polypeptide of the protein carrier.

[0125] In some embodiments of the method of producing a protein conjugate, the one or morecysteine residues are introduced into the N-terminal polypeptide of the protein carrier as a serine- cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide.

[0126] In some embodiments of the method of producing a protein conjugate, the proteincarrier is conjugated to one or more cationic polymers at the one or more cysteine residues.

[0127] In some embodiments of the method of producing a protein conjugate, the proteincarrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof.Attorney Docket No: 243735.000431

[0128] In some embodiments of the method of producing a protein conjugate, the one or morecysteine residues are introduced to the UBL.

[0129] In some embodiments of the method of producing a protein conjugate, the ubiquitin-like protein (UBL) is selected from ubiquitin, a small ubiquitin-like modifier (SUMO) protein, neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8), autophagy- related protein 8 (ATG8), autophagy-related 12 (ATG12), human leukocyte antigen (HLA)-F adjacent transcript 10 or Ubiquitin D (FAT10), interferon (IFN)-stimulated gene 15 (ISG15), ubiquitin related modifier 1 (URM1), or ubiquitin fold modifier 1 (UFM1).

[0130] In some embodiments of the method of producing a protein conjugate, the UBLcomprises or consists of an amino acid sequence set forth in any one of the SEQ ID NOs: 1-8, 53-60, and 72 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in any one of the SEQ ID NOs: 1-8, 53-60, and 72.

[0131] In some embodiments of the method of producing a protein conjugate, the SUMOprotein is SUMO1, SUMO2, SUMO3, or SUMO4.

[0132] In some embodiments of the method of producing a protein conjugate, the SUMOprotein comprises or consists of an amino acid sequence set forth in any one of the SEQ ID NOs: 1-8 and 53-55 or at least 70 % sequence identity to an amino acid sequence set forth in any one of the SEQ ID NOs: 1-8 and 53-55.

[0133] In some embodiments of the method of producing a protein conjugate, the proteincarrier comprises SUMO1, a functional fragment, or derivative thereof.

[0134] In some embodiments of the method of producing a protein conjugate, SUMO1comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53, or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53.

[0135] In some embodiments of the method of producing a protein conjugate, the cysteineresidue at position 52 of SUMO1 has been mutated to a non-cysteine residue.

[0136] In some embodiments of the method of producing a protein conjugate, the cysteineresidue at position 52 of SUMO1 has been mutated to an alanine.

[0137] In some embodiments of the method of producing a protein conjugate, SUMO1 ismodified to introduce one or more cysteine residues at the amino acid position 33, amino acid position 46, amino acid position 53, amino acid position 85, and / or amino acid position 50.Attorney Docket No: 243735.000431

[0138] In some embodiments of the method of producing a protein conjugate, SUMO1comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8 or at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 8.

[0139] In some embodiments of the method of producing a protein conjugate, the C-terminalor proximal to the C-terminal sequence of the SUMO protein comprises Gly-Gly.

[0140] In some embodiments of the method of producing a protein conjugate, the intracellularprotease is a UBL-specific protease.

[0141] In some embodiments of the method of producing a protein conjugate, the UBL-specific protease is a ubiquitin-specific protease (USP) or SUMO-specific protease (SENP).

[0142] In some embodiments of the method of producing a protein conjugate, the SENP isSENP1, SENP2, SENP3, SENP5, SENP6, and / or SENP7.

[0143] In some embodiments of the method of producing a protein conjugate, the cationicpolymer is a polyamine.

[0144] In some embodiments of the method of producing a protein conjugate, the cationicpolymer is selected from polyethylenimine (PEI), poly(L-lysine) (PLL), poly(D-lysine) (PDL), polyamidoamine (PAA), Poly(amino-co-ester)s (PAEs), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), poly(propylene imine) (PPI), poly-L-arginine (PLA), poly- guanidine, poly-D-arginine, poly(D,L-lysine hydrobromide), poly-L-ornithine, PEI-Gua, PEI- Arg, PEI-His, PEI-Trp, or derivatives and combinations thereof.

[0145] In some embodiments of the method of producing a protein conjugate, the cationicpolymer is PEI.

[0146] In some embodiments of the method of producing a protein conjugate, the PEI is alinear PEI or branched PEI.

[0147] In some embodiments of the method of producing a protein conjugate, the branchedPEI is a short-chain PEI.

[0148] In some embodiments of the method of producing a protein conjugate, the PEI has amolecular weight of less than 2000 Da.

[0149] In some embodiments of the method of producing a protein conjugate, the PEI has amolecular weight ranging from about 600 Da to about 1800Da.

[0150] In some embodiments of the method of producing a protein conjugate, the PEI has amolecular weight of about 600 Da (PEI600) or 1200 Da (PEI1200).Attorney Docket No: 243735.000431

[0151] In some embodiments of the method of producing a protein conjugate, the PEI has amolecular weight of about 600 Da (PEI600).

[0152] In some embodiments of the method of producing a protein conjugate, the PEI isbranched PEI600 (bPEI600) or branched PEI1200 (bPEI1200).

[0153] In some embodiments of the method of producing a protein conjugate, the PEI isbranched PEI600 (bPEI600).

[0154] In some embodiments of the method of producing a protein conjugate, the proteincarrier is conjugated to the cationic polymer via a linker.

[0155] In some embodiments of the method of producing a protein conjugate, the linker is aheterobifunctional crosslinker.

[0156] In some embodiments of the method of producing a protein conjugate, the linkercomprises an electrophile selected from haloacetophenone, epoxide, haloacetamide, maleimide, isothiocyanate, vinyl sulfone, nitrile, iminonitrile, hydrazone, butynoate, acrylester, acrylonitrile, acrylamide, benzisothiazolone, and N-hydroxy(methyl)phthalimide.

[0157] In some embodiments of the method of producing a protein conjugate, the linkercomprises a N-hydroxysuccinimide (NHS) ester and a maleimide group.

[0158] In some embodiments of the method of producing a protein conjugate, the linker isselected from N-alpha-maleimidoacetoxyl succinimide ester (AMAS), N-beta-maleimidopropyl- oxysuccinimide ester (BMPS), N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS), m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-epsilon-malemidocaproyl- oxysuccinimide ester (EMCS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH), succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), N-(11- maleimidoundecanoyloxy)succinimide (KMUS), or derivatives and combinations thereof.

[0159] In some embodiments of the method of producing a protein conjugate, the linker is N-alpha-maleimidoacetoxyl succinimide ester (AMAS).

[0160] In some embodiments of the method of producing a protein conjugate, the cationicpolymer is conjugated to the protein carrier at a pH of 6-8.

[0161] In some embodiments of the method of producing a protein conjugate, the polypeptidecargo interacts with an intracellular target.Attorney Docket No: 243735.000431

[0162] In some embodiments of the method of producing a protein conjugate, the intracellulartarget is selected from rat sarcoma virus (RAS), Src homology 2 domain-containing phosphatase 2 (SHP2), Aurora A kinase, Src-family kinases, signal transducer and activator of transcription 3 (STAT3), PR domain containing 14 (PRDM14), WD repeat domain 5 (WDR5), mixed lineage kinase domain-like protein (MLKL), p53, and ABL kinase.

[0163] In some embodiments of the method of producing a protein conjugate, RAS is Kirstenrat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS), or Harvey rat sarcoma viral oncogene homolog (HRAS).

[0164] In some embodiments of the method of producing a protein conjugate, the polypeptidecargo is selected from a monobody, a designed ankyrin repeat protein (DARPin), an Anticalin, an affilin, an affibody, a peptide, an antibody or antigen-binding fragment, an enzyme, or derivatives and combinations thereof.

[0165] In some embodiments of the method of producing a protein conjugate, the polypeptidecargo is selected from a monobody, a designed ankyrin repeat protein (DARPin), or derivatives thereof.

[0166] In some embodiments of the method of producing a protein conjugate, the polypeptidecargo is tandem monobodies comprising two or more monobody units.

[0167] In some embodiments of the method of producing a protein conjugate, the monobodyor DARPin targets KRAS, HRAS, NRAS, Aurora A kinase, ABL kinase, a Src-family kinase, SHP2, STAT3, PRDM14, WDR5, p53, or MLKL.

[0168] In some embodiments of the method of producing a protein conjugate, the monobody isselected from 12VC1, NS1, 12D3, 12D4, 12D5, AS25, AS27, HA4, 7C12, Nsa1, NSa5, CS3, S14, S4, Mb1, Mb2, Mb3, Mb4, Mb5, Mb6, Mb19, Mb20, Mb24, Mb27, Mb32, Mb33, MS3-6, Mb13, R15, Fgr_1, Fgr_2, Hck_1, Hck_2, Lck_1, Lck_3, Lyn_2, Lyn_4, Src_2, Yes_1, Yes_3, MLKL_26, MLKL_27, MLKL_32, MLKL_33, and MLKL_37.

[0169] In some embodiments of the method of producing a protein conjugate, the monobodycomprises or consists of an amino acid sequence set forth in any one of the SEQ ID NOs:14-52 and 69-70 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of the SEQ ID NOs:14-52 and 69-70.

[0170] In some embodiments of the method of producing a protein conjugate, the DARPin isK27, C10, or C10-H82R.Attorney Docket No: 243735.000431

[0171] In some embodiments of the method of producing a protein conjugate, the antibody orantigen-binding fragment thereof is selected from a single chain antibody, a single domain antibody, a variable heavy homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, a monoclonal antibody, a bispecific antibody or antigen-binding fragment thereof, a bi- epitopic antibody or antigen-binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, an scFv, a VH domain, a monovalent antibody or a bivalent antibody.

[0172] In some embodiments of the method of producing a protein conjugate, the peptide is astapled peptide or bicyclic peptide.

[0173] In some embodiments of the method of producing a protein conjugate, the contiguouspolypeptide comprises or consists of the sequence set forth in any one of the SEQ ID Nos: 9, 11, 62-65, and 71 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of the SEQ ID NOs: 9, 11, 62-65, and 71.

[0174] In some embodiments of the method of producing a protein conjugate, the contiguouspolypeptide comprises or consists of the sequence set forth in SEQ ID NO: 62 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62.

[0175] In one aspect, provided herein is a protein conjugate produced by the method of thepresent disclosure.

[0176] In one aspect, provided herein is a pharmaceutical composition comprising the proteinconjugate of the present disclosure, the nucleic acid molecule of the present disclosure, the vector of the present disclosure, or the host cell of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient.

[0177] In one aspect, provided herein is a method of delivering a cargo into the cytoplasm of acell, comprising a) producing a protein conjugate comprising the cargo according to the method of the present disclosure; and b) contacting the cell with the produced protein conjugate.

[0178] In one aspect, provided herein is a method of targeting an intracellular target in a cell,comprising a) producing a protein conjugate comprising a cargo that interacts with the intracellular target according to the method of the present disclosure; and b) contacting the cell with the produced protein conjugate.

[0179] In some embodiments, the cell is a mammalian cell.

[0180] In some embodiments, the mammalian cell is an adherent cell.Attorney Docket No: 243735.000431

[0181] In some embodiments, the mammalian cell is a non-adherent cell.

[0182] In some embodiments, the method occurs ex vivo.

[0183] In some embodiments, the method occurs in vivo.

[0184] In one aspect, provided herein is a method of preventing, treating, or diagnosing adisease in a subject in need thereof, comprising administering to the subject the protein conjugate of the present disclosure, or the pharmaceutical composition of the present disclosure, wherein the cargo interacts with a target associated with disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0185] Figs. 1A-1B depict the design and characterization of cytosolic delivery reagents (PEI-SUMO-Monobody (Mb) reagents). A scheme of the reagent design and the cleavage upon cytoplasmic delivery by endogenous SENP proteases is shown in Fig.1A. An alternate design of the recombinant fusion protein in which Cys is introduced in a segment N-terminal to and outside SUMO is shown in Fig.1B. CysNindicates multiple Cys residues. Figures 1A-1B disclose “His6” as SEQ ID NO: 12.

[0186] Figs. 2A-2C depict PEI conjugation and purification of cytosolic delivery reagents. Thecrystal structure of SUMO1–SENP1 complex with the initially selected conjugation sites of SUMO1 indicated in circles and labeled is shown in Fig.2A. Examples of positions of SUMO1 to which Cys can be introduced is illustrated in Fig.2A. Four positions in SUMO1 are indicated in circles and labeled in the crystal structure of SUMO1–SENP1 complex (Protein Data Bank (PDB) entry 2IY1). These positions are exposed to the solvent in the complex with a cognate protease, SENP1 and thus the introduction of Cys and subsequent chemical conjugation are not expected to inhibit the cleavage at the C-terminal Gly-Gly sequence, marked with “GG” and indicted in square. SDS-PAGE showing purification of the SUMO1-12VC1 fusion (His6- SUMO1(C52A; K46C; Q53C)-12VC1-V5) using Ni-affinity chromatography is shown in Fig. 2B. e1-e4 denote elution fractions (e3 was skipped). Size-exclusion chromatograph using a Superdex 75 column of PEI2-SUMO-12VC1 is shown in Fig.2C, demonstrating its monodispersed nature. PEI conjugates often do not elute at a position expected for the molecular weights.

[0187] Figs. 3A-3C depict cytosolic delivery of the reagents assessed using the SUMO-cleavage assay to detect the reagent delivered in the cytoplasm. The experimental scheme isAttorney Docket No: 243735.000431 shown in Fig.3A. Upon cytosolic entry, the peptide bond between the carrier and cargo, at the C terminus of the GG sequence, is cleaved by endogenous SENPs. For the PEI-SUMO control without a cargo, the GG sequence was mutated to Ala-Ala to prevent the cleavage of the V5 tag so that it can be detected in immunoblots. Immunoblotting of HEK293T cells treated with the indicated reagents at 1 µM is shown in Fig.3B. Cells were washed after harvesting and subjected to immunoblotting. Only lane 4, with His6-SUMO1(C52A;K46C;Q53C)-12VC1-V5 conjugated with PEI600, shows a band corresponding to cleaved monobody. Lane 6 is the purified sample without incubation with cells. Results of immunoblotting from an experiment with higher concentrations of the reagents (5 µM) than B (1 µM) are shown in Fig.3C. The uncleaved reagents are detected here. Figure 3A discloses “His6” as SEQ ID NO: 12.

[0188] Figs. 4A-4C depict the biological effects of delivery reagents including selectiveinhibition of KRAS(G12C)- and (G12V)-dependent cells with PEI-SUMO-12VC1. The experimental scheme is shown in Fig.4A. Dose-dependent cytotoxic effects of His6- SUMO1(C52A;K46C;Q53C)-12VC1-V5 conjugated with PEI600 or PEI1200 on NCI-H358 lung cancer cells are shown in Fig.4B. Dose response curves of SUMO-12VC1 conjugated with PEI600 and PEI1200 (1 µM) on the viability of H358 after 72 hours treatment, measured using Prest Blue are shown in Fig.4B. The bottom panel of Fig.4B shows a titration of His6- SUMO1(C52A;K46C;Q53C)-12VC1-V5 conjugated with PEI600 at a low concentration range, showing an IC50 value of approximately 50 nM. Cytotoxic effects of His6- SUMO1(C52A;K46C;Q53C)-12VC1-V5 (and controls) on the indicated cells with different oncogenic drivers are shown in Fig.4C.Viability of indicated cell lines treated with a MEK inhibitor tramenitib (1 nM; positive control for all these cell lines), PEI600-SUMO (1 µM), and PEI600-SUMO-12VC1 (1 µM) for 72 hours is shown in Fig.4C. Tramenitib is a MEK inhibitor that is expected to be cytotoxic to all the cell lines. PEI-SUMO is a negative control that lacks the 12VC1 cargo. Figure 4A discloses “His6” as SEQ ID NO: 12.

[0189] Figs. 5A-5B depict cell-signaling effects of the delivery reagents. Bright field imagesand apoptosis (probed with a caspase-3 / 7 cleavage dye (darker stain; Incucyte)) of H358 cells treated with the indicated reagents for 24 hours are shown in Fig.5A. Inhibition of RAS- mediated signaling resulted in changes in cell morphology and caspase-3 / 7 cleavage that are clearly visible in cells treated with His6-SUMO1(C52A;K46C;Q53C)-12VC1-V5 conjugated with PEI600 and with sotorasib (positive control) but not in the other conditions (left panels).Attorney Docket No: 243735.000431 Inhibition of RAS-mediated signaling in H358 cells by His6-SUMO1(C52A;K46C;Q53C)- 12VC1-V5 conjugated with PEI600, as probed using immunoblotting for phosphor-ERK is shown in Fig.5B. Sotorasib is a small-molecule inhibitor of KRAS(G12C), serving as a positive control. The lane marked with M shows molecular weight standards.

[0190] Figs. 6A-6B depict the preparation of a reagent with another monobody, NS1, and itsefficacy. The elution profile from size-exclusion chromatography of His6- SUMO1(C52A;K46C;Q53C)-NS1-V5 conjugated with PEI600 is shown in Fig.6A. The cytotoxicity (inhibitory effects) of the reagent PEI-SUMO-NS1 (1 µM) to a panel of cells is shown in Fig.6B.

[0191] Figs. 7A-7D depict characterization of a TAT-12VC1 fusion protein. TAT-12VC1fusion has poor biophysical properties and is generally toxic. A scheme showing the construct design is shown in Fig.7A. SDS-PAGE showing purification of the protein samples taken at different points is shown in Fig.7B. The SEC elution profile of the purified reagent from size- exclusion chromatography is shown in Fig.7C. The sample did not elute as a peak, and instead as an extremely broad “band” with low signal intensities (note the small vertical axis range). Cytotoxicity of TAT-12VC1 with controls for the indicated cell lines is shown in Fig.7D. Trametinib is a MEK inhibitor, serving as a positive control for all the cell lines. Figure 7A discloses “His6” as SEQ ID NO: 12.

[0192] Fig. 8 depicts the comparison of the folds and sizes of monobodies (top) and DARPin(bottom). The two monobody-RAS complexes were superposed using RAS as the reference. The molecules are drawn to the same scale.

[0193] Fig. 9 depicts the challenges for targeting intracellular proteins. Biologics have beenused mostly to target extracellular proteins. Mutated intracellular proteins often drive diseases and remain undruggable.

[0194] Fig. 10 depicts various approaches to target intracellular disease drivers usingbiologics. If there is a mechanistic link between an intracellular disease-driver and a surface protein, one can target the surface protein as a surrogate target (Oury et al. (2021), Nature, 595, pages 404–408). The MHC-presented peptides can be targeted by the HapImmune Concept (Hattori et al. (2023), Cancer Discov.,13:132–45).

[0195] Fig. 11 depicts a flowchart that illustrates rapid drug prototyping with intracellularmonobodies.Attorney Docket No: 243735.000431

[0196] Fig. 12 depicts examples of monobody-target complex structures in PDB.

[0197] Figs. 13A-13B depict an example of targeting a single member of a large protein family(e.g., SH2 domain-containing phosphatase 2 (SHP2) or SH2ome (122)) using monobodies.

[0198] Figs. 14A-14B depict an exemplary monobody, 12VC1, that selectively andnoncovalently binds to both KRAS(G12C) and KRAS(G12V).

[0199] Fig. 15 depicts that noncovalent inhibition of KRAS(G12V) blocks signaling andinhibits tumor growth in mouse xenograft.

[0200] Fig. 16 depicts ribbon diagrams of FN3, VHH, and Fab proteins.

[0201] Fig. 17 depicts the development of monobodies with exquisite specificity.

[0202] Figs. 18A-18C depict inhibition of BCR-ABL with a cell-deliverable monobody. Fig.18A shows a size-exclusion chromatograph (SEC) profile of PEI-SUMO-AS25 showing mono- dispersity. Fig.18B shows that PEI-SUMO-AS25 (10 µM) selectively inhibited K562 cells that depends on BCR-ABL (left). KG1a is an off-target cell line that is not dependent on BCR-ABL. Fig.18C shows time course of the induction of apoptosis measured by caspase 3 / 7 cleavage with PEI-SUMO-AS25. The bottom images show representative snapshots at 12 hours.

[0203] Fig. 19 depicts inhibition of SHP2 with a cell-deliverable monobody. Inhibition ofKYSE520 cells by PEI-SUMO-NSa1 (1 µM). KYSE520 depends on SHP2 activity. A375 is an off-target cell line.

[0204] Fig. 20 depicts results of immunoblotting of PEI-SUMO-12VC1 incubated in cellculture media (24-72 hours at 37 °C) and in mouse plasma (24 hours at 37 °C with three different plasma samples).

[0205] Figs. 21A-21B depict the effectiveness of PEI-SUMO-12VC1 that is conjugated via aCys residue in an N-terminal extension of SUMO. Fig.21A shows cell viability, normalized to no treatment, of the indicated cell lines with the indicated reagents. The leftmost bar (A) represents the result with the PEI-SUMO-12VC1 reagent and B-F are controls. Fig.21B shows dose-dependent effect of the reagent on the indicated cell lines. The IC50 values were derived from curve fitting of a 1:1 binding model (dashed lines).

[0206] Figs. 22A-22B depict that low-molecular weight (MW) PEIs are nontoxic and do notaggregate with serum albumin. Fig.22A shows effects of PEI on the viability of the indicated cells measured using PrestBlue after 72-hour culture in standard media. Fig.22B shows SECAttorney Docket No: 243735.000431 profiles of BSA (20 µM) mixed with the indicated PEIs (100 µM), showing no effects of low- MW PEIs. DETAILED DESCRIPTION

[0207] Molecular interactions inside cells govern cellular decision making. To understandhow cells execute such decision making and to manipulate cellular behavior, one needs to be able to precisely control molecular interactions within cells. However, a limited ability to do so is a major void in basic science and in therapeutic intervention.

[0208] As already described in the ‘Background’ Section above, gene-directed technologiesare powerful, but they have many shortcomings as tools for mechanistic studies of intracellular proteins and their interactions.

[0209] Small molecule tool compounds have been impactful in advancing mechanisticknowledge of molecules that they target, mostly proteins, and interactions involving the targeted molecules. However, the inherent challenge in developing selective and potent small molecules, particularly against flat surfaces devoid of deep pockets that are common in protein-protein interaction interfaces, has not been solved. Compared with the size of the intracellular proteome, only a small number of high-quality tool compounds have been developed.

[0210] Protein-based reagents such as antibodies and synthetic binding proteins can, unlikesmall molecule compounds, achieve high levels of selectivity and potency to most biomacromolecules. For example, the monobody system that produces synthetic binding proteins with high selectivity and high potency to challenging targets has been developed (5-7). Therefore, engineered binding proteins, or “tool biologics”, can overcome the challenge of developing ligands to the majority of intracellular targets.

[0211] However, tool biologics create a new challenge. It has been extraordinarilychallenging to deliver protein-based reagents into the cytoplasm of mammalian cells, because the plasma membrane presents a formidable barrier for entry of polar molecules like proteins. Still, the attractiveness of the high performance of tool biologics has stimulated the development of technologies for effective delivery of tool biologics into the cytoplasm.

[0212] Although genetically encoded tool biologics have been powerful in mechanisticstudies and in discovering new targets and new sites for therapeutic targeting, they have major limitations. Usually, only a subset of cells in culture expresses the genetically encoded tool andAttorney Docket No: 243735.000431 at heterogenous levels, which necessitates the isolation of a desired sub-population of cells for further analysis. Such manipulation in turn can introduce bias, such as enrichment of cells resistant to the perturbation caused by the expressed tool biologic. Furthermore, the temporal resolution of the perturbation with the reagent is limited even using an inducible promoter. Moreover, their use in in vivo studies in animal models is limited due to the difficulty in introducing them systemically or in a sufficiently high fraction of targeted cells.

[0213] Ideal reagents for intracellular targets should have the specificity and potency ofbiologics reagents and the portability and temporal control of cell-permeable small molecules. The cytoplasmic delivery of biologics of the present disclosure would fulfill these requirements.

[0214] Currently available cell-penetrating reagents do not deliver a cargo to the cytoplasmwith efficiency that is high enough for most biomedical investigation. A closer inspection of these reagents using assays that strictly measure reagents in the cytoplasm revealed that they are mostly trapped in the endosomal vesicles that are still topologically outside, with only small fractions of the reagents reaching the cytoplasm (13). Therefore, although cytoplasmic delivery of biologics is an area of intense research activities, there remains a major void in the current capabilities. Clearly, innovative approaches are needed to make a breakthrough in this challenging but potentially transformative technology.

[0215] Furthermore, the conjugation of a cell-penetrating reagent often leads to the formationof large assemblies (“soluble aggregates”) of an otherwise highly soluble, monomeric cargo. Aggregation makes it difficult to estimate the concentration of the active species in a reagent. Clearly, aggregating materials cannot be used as therapeutics without extensive formulation studies.

[0216] Ideal cytoplasmic delivery technology should have high delivery efficiency; minimalperturbation of the cargo function or modification; and produce well behaving reagents free of aggregation. As described below, the technology of the present disclosure has high potential to satisfy these requirements.

[0217] In one aspect, the present disclosure provides a technology that can efficiently delivera polypeptide cargo, such as monobodies, into the cytoplasm of mammalian cells. In various embodiments, the present technology utilizes a protein conjugate comprising (i) a protein carrier conjugated to a cationic polymer; and (ii) a polypeptide cargo. The cargo can be released from the protein conjugate in the cytoplasm by endogenous proteases. This “space rocket”-likeAttorney Docket No: 243735.000431 mechanism ensures that the biological functions of the cargo in the cytoplasm are not affected by the delivery vehicle. Thus, the technology disclosed herein is substantially more effective and also has substantially less toxicity than existing technologies for the same purpose. Indeed, as demonstrated in the Examples section below, the technology described herein was used to deliver a KRAS-targeting monobody into KRAS-driven cancer cells. The monobody inhibited cell growth with IC50 values in the mid-nM range, with no toxicity to cells not harboring the KRAS mutants that the monobody targets.

[0218] The technology described herein can have application in virtually all areas ofbiomedical research including drug discovery.

[0219] To extend the cytoplasmic delivery technology described herein, installing cell-typespecificity to the reagents can be achieved. This function can be implemented by attaching a targeting moiety, such as another monobody, specific to a cell-surface marker. Such reagents can improve the attachment to cells that display the surface antigen, which in turn can improve the overall delivery efficiency. The use of the cytoplasmic delivery technology described herein in vivo, can dramatically expand the scope of cell type-specific inhibition of intracellular targets, a therapeutic area currently explored mainly with antibody-drug conjugates.

[0220] Combined with the availability of many monobodies that have been developedagainst intracellular targets (and other synthetic binding proteins), the cytoplasmic delivery technology of the present disclosure can be readily applied to start addressing pressing biological questions at the cellular level.

[0221] The cytoplasmic delivery technology of the present disclosure can also be translatedtoward clinical applications. For example, potent monobody cargos against “undruggable” targets can be delivered to inhibit disease cells or potentiate (or suppress) immune responses.

[0222] Together, the technology of the present disclosure has virtually endless potential inbiomedical applications and inspires new types of basic research investigations and transformative therapeutic approaches. Definitions

[0223] Unless otherwise defined herein, scientific and technical terms used in connection withthe present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall includeAttorney Docket No: 243735.000431 pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well-known and commonly used in the art.

[0224] The methods and techniques of the present invention are generally performed accordingto conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0225] The following terms, unless otherwise indicated, shall be understood to have thefollowing meanings:

[0226] The terms “polypeptide” and “protein” used interchangeably herein encompass nativeor artificial proteins, protein fragments, and polypeptide analogs or derivatives of a protein sequence. The terms include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. A polypeptide or protein may be monomeric or polymeric. A polypeptide or protein typically has an N-terminus and a C-terminus. The N-terminus is the start of an amino acid chain of a protein, terminated by an amino acid with a free amine group (-NH2). The C-terminus is the end of an amino acid chain of a protein, terminated by a free carboxyl group (-COOH).

[0227] The term “fragment” in regard to polypeptides refers to a polypeptide that has anamino-terminal and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the full-length naturally-occurring sequence. Also,Attorney Docket No: 243735.000431 fragments according to the invention may be made by truncation, e.g., by removal of one or more amino acids from the N and / or C-terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and / or C terminal in this way. Fragments may also be generated by one or more internal deletions. In some embodiments, fragments are at least 5, 6, 8 or 10 amino acids long. In other embodiments, the fragments are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, 200, or 400 amino acids long. In some embodiments, fragments encompass derivatives of the polypeptides. In some embodiments, a fragment may be a functional fragment. In one embodiment, the functional fragment is to a fragment of a polypeptide that retains a particular binding or catalytic activity characteristic of the entire polypeptide.

[0228] The terms “polynucleotide” and “nucleic acid molecule” used interchangeably hereinmean a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The terms include single and double stranded forms.

[0229] As used herein, the term “derivative” includes a chemical modification of a referencepolypeptide, polynucleotide, or other molecule such as chemical compound. A “derivative” of a polypeptide further includes those polypeptides that are “derived” from a reference polypeptide by having, for example, amino acid substitutions, deletions, or insertions relative to a reference polypeptide. A polypeptide may be “derived” from a wild-type polypeptide or from any other polypeptide.

[0230] In certain embodiments, amino acid substitutions of a protein or portion thereof arethose which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, or (4) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the normally- occurring sequence.

[0231] A conservative amino acid substitution should not substantially change the structuralcharacteristics of the parent sequence. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C.Attorney Docket No: 243735.000431 Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991), which are each incorporated herein by reference.

[0232] As used herein, the twenty naturally occurring amino acids and their abbreviationsfollow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0233] Similarly, a “derivative” of a polynucleotide further includes those polynucleotides thatare “derived” from a reference polynucleotide by having, for example, nucleotide substitutions, deletions, or insertions relative to a reference polypeptide. Non-limiting examples of chemical modifications involving nucleic acids include, for example, modifications to the base moiety, sugar moiety, phosphate moiety, phosphate-sugar backbone, or a combination thereof.

[0234] The term “monobody” as used herein refers to an antibody mimetic or syntheticbinding protein that is constructed using a fibronectin type III domain (FN3) as a molecular scaffold.

[0235] The term “antibody” refers to an immunoglobulin molecule capable of specificbinding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region(s) of the immunoglobulin molecule. As used herein, the term “antibody”, encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multi-specific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulinsAttorney Docket No: 243735.000431 are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.

[0236] A typical antibody molecule comprises a heavy chain variable region (VH) and alight chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, the EU definition, the “Contact” numbering scheme, the “IMGT” numbering scheme, the “Aho” numbering scheme, and / or the contact definition, all of which are well known in the art. (See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol.196:901-917, Al-lazikani et al (1997) J. Molec. Biol.273:927-948; Edelman et al., Proc Natl Acad Sci U S A.1969 May;63(1):78-85; and Almagro, J. Mol. Recognit.17:132-143 (2004); MacCallum et al., J. Mol. Biol.262:732-745 (1996), Lefranc M P et al., Dev Comp Immunol, 2003 January; 27(1):55-77; and Honegger A and Pluckthun A, J Mol Biol, 2001 Jun.8; 309(3):657-70. See also hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0237] In some embodiments, the antibody described herein is a full-length antibody, whichcontains two heavy chains and two light chains, each including a variable domain and a constant domain. Alternatively, the antibody can be an antigen-binding fragment of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab’)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retainsAttorney Docket No: 243735.000431 functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0238] Any of the antibodies described herein, can be either monoclonal or polyclonal. A“monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

[0239] As used herein, “isolated” means a biological component (such as a nucleic acid,polypeptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and polypeptides that have been “isolated” thus include nucleic acids and polyproteins purified by standard purification methods. “Isolated” nucleic acids or polypeptides can be part of a composition and still be isolated if such composition is not part of the native environment of the nucleic acid or polypeptide. The term also embraces nucleic acids and polypeptides prepared by recombinant DNA technology or recombinant expression in a host cell as well as chemically synthesized nucleic acids or polypeptides.

[0240] As used herein, the term “operably linked” refers to a first molecule joined to a secondmolecule, wherein the molecules are in an arrangement permitting them to operate in the intended manner. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, a protein carrier is operably linked to a cargo if the protein carrier mediates the delivery of the cargo into a cell.

[0241] Operably linked sequences also include both expression control sequences that arecontiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” as used herein means polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmicAttorney Docket No: 243735.000431 mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0242] The term “vector”, as used herein, means a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded DNA loop into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).

[0243] The term “recombinant host cell” (or simply “host cell”), as used herein, means a cellinto which an exogenous nucleic acid and / or recombinant vector has been introduced. It should be understood that “recombinant host cell” and “host cell” mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0244] The term “percent sequence identity” means a ratio, expressed as a percent of thenumber of identical residues over the number of residues compared.Attorney Docket No: 243735.000431

[0245] Sequence identity for nucleic acid sequences may be analyzed over a stretch of at leastabout nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol.183:63-98 (1990); Pearson, Methods Mol. Biol.132:185- 219 (2000); Pearson, Methods Enzymol.266:227-258 (1996); Pearson, J. Mol. Biol.276:71-84 (1998); herein incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.

[0246] A reference to a nucleotide sequence encompasses its complement unless otherwisespecified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.

[0247] Sequence identity for polypeptides, is typically measured using sequence analysissoftware. Protein analysis software matches sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters, as specified with the programs, to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, see GCG Version 6.1. (University of Wisconsin Wis.) FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol.183:63- 98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000)). Another preferred algorithm whenAttorney Docket No: 243735.000431 comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters, as supplied with the programs. See, e.g., Altschul et al., J. Mol. Biol.215:403-410 (1990); Altschul et al., Nucleic Acids Res.25:3389-402 (1997).

[0248] The length of polypeptide sequences compared for homology will generally be at leastabout 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences.

[0249] The term “substantial similarity” or “substantial sequence similarity,” when referring toa nucleic acid or fragment thereof, means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 80%, preferably at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

[0250] As applied to polypeptides, the term “substantial identity” means that two peptidesequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, as supplied with the programs, share at least 70%, 75%, 80%, or 85% sequence identity, preferably at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0251] As used herein, “conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol.243:307-31 (1994). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine,Attorney Docket No: 243735.000431 alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine- glutamine.

[0252] Alternatively, a conservative substitution or replacement, as the terms are usedinterchangeably herein, is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256:1443-45 (1992), herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0253] The term “potency” is a measurement of biological activity and may be designated asIC50, or effective concentration of a compound needed to inhibit 50% of a biological activity in a cell which activity is mediated by the compound.

[0254] The phrase “effective amount” or “therapeutically effective amount” as used hereinrefers to an amount necessary (at dosages and for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount is at least the minimal amount, but less than a toxic amount, of an active agent which is necessary to impart therapeutic benefit to a subject.

[0255] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical acceptableexcipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject’s immune system. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington’s Pharmaceutical Sciences, 18thedition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20thEd. Mack Publishing, 2000). As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally regarded as physiologically tolerable.

[0256] The term “treating”, as used herein, unless otherwise indicated, means reversing,alleviating, inhibiting the progress of, delaying the progression of, delaying the onset of, or preventing the disease, disorder, or condition to which such term applies, or one or moreAttorney Docket No: 243735.000431 symptoms of such disease, disorder or condition. The term “treatment”, as used herein, unless otherwise indicated, refers to the act of treating as “treating” is defined immediately above. The term “treating” also includes adjuvant and neo-adjuvant treatment of a subject. For the avoidance of doubt, reference herein to “treatment” includes reference to curative, palliative and prophylactic treatment.

[0257] The terms “patient”, “individual”, “subject”, and “animal” are used interchangeablyherein and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, goats, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.

[0258] The term “about” or “approximately” means within a statistically meaningful range of avalue. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0259] The terms “a,” “an”, and “the” do not denote a limitation of quantity, but rather denotethe presence of “at least one” of the referenced item.

[0260] The practice of the present invention employs, unless otherwise indicated, conventionaltechniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual.3rded. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g., in U.S. Patent No.7,912,698 and U.S. Patent Appl. Pub. Nos.2011 / 0202322 and 2011 / 0307437.Attorney Docket No: 243735.000431 Protein Conjugates

[0261] In some aspects, the present disclosure provides a protein conjugate comprising (i) aprotein carrier described herein conjugated to a cationic polymer and (ii) a cargo described herein. In some embodiments, the cargo is a polypeptide cargo or a chemical compound (e.g., a small molecule) that can interact with a target such as, but not limited to, an intracellular target. In some embodiments, the protein conjugate comprises a protein carrier genetically fused to and / or chemically or enzymatically linked to a cargo.

[0262] In some embodiments, the protein carrier is linked to the polypeptide cargo via apeptide bond to form a contiguous polypeptide. In some embodiments, the contiguous polypeptide is produced by a recombinant expression method.

[0263] In some embodiments of the protein conjugate, the protein carrier and the polypeptidecargo are linked chemically or enzymatically to form a contiguous polypeptide. Non-limiting examples of techniques to prepare contiguous polypeptides include native chemical ligation, sortase, or subtiligase reactions. Any of these reactions may be employed for the formation of the peptide bond between the protein carrier and the polypeptide cargo. In certain embodiments, the contiguous polypeptide comprises the protein carrier and the polypeptide cargo linked via a peptide bond.

[0264] In some embodiments of the protein conjugate, the protein carrier is fused to thepolypeptide cargo to form a fusion protein. In some embodiments, the fusion protein is produced by a recombinant expression method. In some embodiments, the contiguous polypeptide is cleaved by a protease such as, but not limited to, an intracellular protease to release the polypeptide cargo from the protein carrier. In some embodiments, the intracellular protease is a cysteine protease. In some embodiments, the intracellular protease is a ubiquitin-like proteins (UBL)-specific protease. The UBL-specific protease includes a ubiquitin-specific protease (USP) or Small Ubiquitin-like Modifier (SUMO)-specific protease (SENP). In some embodiments, the intracellular proteases are deubiquitinating enzymes (DUBs). Deubiquitinating enzymes (DUBs)are described in Cruz et al Pharmaceuticals 2021, 14, 848; which is incorporated herein byreference in its entirety. In some embodiments, the intracellular protease is a sentrin / SUMO - specific protease (SENP). In some embodiments, the sentrin / SUMO-specific protease (SENP) is SENP1, SENP2, SENP3, SENP5, SENP6, and / or SENP7.Attorney Docket No: 243735.000431

[0265] In some embodiments of the protein conjugate, the protein carrier is linked to thepolypeptide cargo via a proteolytic cleavable linker. In some embodiments of the protein conjugate, the proteolytic cleavable linker is a peptide linker.

[0266] In some embodiments of the protein conjugate, the protein carrier comprises aterminal peptide. In some embodiments of the protein conjugate, the terminal polypeptide is an N-terminal polypeptide or a C-terminal polypeptide. In certain embodiments of the protein conjugate, the terminal polypeptide is an N-terminal polypeptide. The terminal polypeptide of the protein carrier generally exhibits a disordered structure compared to the other domain(s) of the protein carrier, such as the UBL domain. This disordered structure can confer flexibility to the terminal polypeptide and facilitate attachment of other molecules such as, but not limited to, cationic polymers, with minimal effects on the overall structure or stability of the carrier.

[0267] In some embodiments of the protein conjugate, the protein carrier and / or thepolypeptide cargo do not contain native cysteine residue(s) or have been engineered to remove native cysteine residue(s). In some embodiments of the protein conjugate, the protein carrier and / or the polypeptide cargo are modified to substitute or remove cysteine residue(s) prior to conjugation to the cationic polymer. In some embodiments of the protein conjugate, the protein carrier is modified to introduce cysteine residue(s) prior to conjugation. Cysteine residue(s) can be introduced by the substitution or addition to the native structure of the protein carrier. In some embodiments of the protein conjugate, the protein carrier is modified to introduce one or more cysteine residues outside the interface between the protein carrier and the intracellular protease. In some embodiments of the protein conjugate, the protein carrier is modified to introduce one or more cysteine residues within the interface between the protein carrier and the intracellular protease. In some embodiments of the protein conjugate, one or more cysteine residue(s) are introduced outside of the protein carrier at the N-terminus segment of the protein carrier. In one embodiment of the protein conjugate, one or more cysteine residue(s) are introduced into the N- terminal polypeptide of the protein carrier. In some embodiments of the protein conjugate, one or more cysteine residue(s) are introduced into the N-terminal polypeptide of the protein carrier as a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide. The protein conjugate design representing the introduction of the Cys residue within the N- terminal polypeptide of the protein carrier is illustrated in Fig.1B and as SEQ ID NOs: 11, 62, 65, and 71 with the SUMO protein. In some embodiments of the protein conjugate, one or moreAttorney Docket No: 243735.000431 cysteine residue(s) are introduced within the UBL protein. The protein conjugate design representing the introduction of the Cys residue within the SUMO protein is shown in SEQ ID NOs: 63 and 64.

[0268] In some embodiments, the protein conjugate comprises any protein carrier describedherein. In some embodiments, the protein conjugate comprises the cargo described herein.

[0269] In certain embodiments, the protein conjugate comprises (i) a protein carrierconjugated to a cationic polymer; and (ii) a cargo, wherein the protein carrier is operably linked to the cargo, and wherein the protein carrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof. In some embodiments, the protein carrier comprises a UBL and one or more terminal polypeptides. In one embodiment, the protein carrier comprises a UBL together with an N-terminal polypeptide and / or a C-terminal polypeptide. In some embodiments of the protein conjugate, the cargo is a polypeptide cargo and the protein carrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof. In certain embodiments, the polypeptide cargo is monobody, a designed ankyrin repeat protein (DARPin), or derivatives thereof; and the protein carrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof. In certain embodiments, the polypeptide cargo is monobody, a designed ankyrin repeat protein (DARPin), or derivatives thereof; and the protein carrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof and an N-terminal polypeptide. In certain embodiments, the polypeptide cargo is monobody, a designed ankyrin repeat protein (DARPin), or derivatives thereof; and the protein carrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof and an N- terminal polypeptide at the amino-terminus of the UBL, said functional fragment, or derivative thereof. Protein Carrier

[0270] The present disclosure provides protein carriers that can function as delivery vehiclesupon chemical conjugation to cationic polymer(s). In some embodiments, the protein carriers comprise terminal polypeptides. In some embodiments, the protein carriers deliver cargos of the present disclosure into the cytoplasm of the target cells.

[0271] In some embodiments, the protein carrier is modified for conjugation to a cationicpolymer. In some embodiments, the cationic polymer is conjugated to one or more specific sitesAttorney Docket No: 243735.000431 in the protein carrier. The site-specific conjugation can eliminate the possibility of chemical modification of the cargo that could be detrimental to the cargo function.

[0272] In some embodiments, the protein carrier is engineered to remove native cysteine(Cys) residue(s). Removal of native cysteine (Cys) residue(s) can include substitution or deletion of native cysteine (Cys) residue(s). In some embodiments, the protein carrier does not contain native cysteine residue(s). In some embodiments, the protein carrier is cysteine free prior to conjugation to the cationic polymer(s).

[0273] In some embodiments, the protein carrier is modified to introduce one or morecysteine residues. The cysteine residues can be introduced by substitution or addition to the native sequence of the protein carrier. The cysteine residues can be introduced to the terminal polypeptide of the protein carrier. The terminal polypeptide of the protein carrier can be an N- terminal polypeptide or a C-terminal polypeptide. In certain embodiments, cysteine residues are introduced into the N-terminal polypeptide of the protein carrier. In some embodiments, the protein carrier is modified to introduce one or more cysteine residues outside the interface between the protein carrier and its cognate intracellular protease. In some embodiments, the protein carrier is modified to introduce one or more cysteine residues within the interface between the protein carrier and its cognate intracellular protease. Positions that can be potentially used for introducing Cys for conjugation can be deduced by analyzing the exposure of the amino acid side chains using programs such as PDBePISA (ebi.ac.uk / pdbe / pisa / pistart.html)

[0274] In some embodiments, the protein carrier is modified to introduce up to eight cysteineresidues. In some embodiments, the protein carrier is modified to introduce up to seven cysteine residues. In some embodiments, the protein carrier is modified to introduce up to six cysteine residues. In some embodiments, the protein carrier is modified to introduce up to five cysteine residues. In some embodiments, the protein carrier is modified to introduce up to four cysteine residues. In some embodiments, the protein carrier is modified to introduce one, two, three, or four cysteine residues. In some embodiments, the protein carrier is modified to introduce one cysteine residue. In some embodiments, the protein carrier is modified to introduce two cysteine residues. In some embodiments, the protein carrier is modified to introduce three cysteine residues. In some embodiments, the protein carrier is modified to introduce four cysteine residues.Attorney Docket No: 243735.000431

[0275] In some embodiments, the protein carrier is conjugated to one or more cationicpolymers at the one or more cysteine residues of the protein carrier. In some embodiments, the protein carrier is conjugated to one or more cationic polymers at the one or more cysteine residues present in the N-terminal polypeptide of the protein carrier.

[0276] In some embodiments, the protein carrier is genetically fused to and / or chemically orenzymatically linked to the cargo of the present disclosure. In some embodiments, the protein carrier is operably linked to the cargo of the present disclosure. In some embodiments, when the cargo is the polypeptide cargo, the protein carrier is chemically or enzymatically linked to a polypeptide cargo to form a contiguous polypeptide. In some embodiments, when the cargo is the polypeptide cargo, the protein carrier is fused to the polypeptide cargo using a recombinant expression method to form a fusion protein.

[0277] In some embodiments, the protein carrier is linked to the polypeptide cargo via aproteolytic cleavable linker. In some embodiments, the proteolytic cleavable linker is a peptide linker. Such peptide linker can be rigid or flexible in nature. In certain embodiments, the protein carrier is linked to the polypeptide cargo via a peptide bond.

[0278] In some embodiments, the linker comprises glycine repeats through which the proteincarrier links to the polypeptide cargo. In some embodiments, the linker comprises diglycine (Gly-Gly) through which the protein carrier links to the polypeptide cargo. In some embodiments, the protein carrier comprises diglycine (Gly-Gly) to which the polypeptide cargo links to form a contiguous polypeptide. In some embodiments, the protein carrier comprises diglycine (Gly-Gly) at C-terminus of the protein carrier. In some embodiments, the protein carrier comprises diglycine (Gly-Gly) proximal to the C-terminus of the protein carrier. In some embodiments, proximal is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of the C-terminus of the protein carrier. The Gly-Gly portion of the protein carrier may be altered as described in Drag et al, Biochem J.2008 Jan 15;409(2):461-9; which is incorporated herein by reference in its entirety.

[0279] In some embodiments, the linker comprises an intracellular protease cleavage site. Insome embodiments, the intracellular protease cleavage site is recognized by a ubiquitin-like protein (UBL)-specific protease. In some embodiments, the intracellular protease cleavage site is recognized by a ubiquitin-specific protease (USP) or SUMO-specific protease (SENP). In some embodiments, the intracellular protease cleavage site is recognized by a sentrin / SUMO-specificAttorney Docket No: 243735.000431 protease (SENP). In some embodiments, the sentrin / SUMO-specific protease (SENP) is SENP1, SENP2, SENP3, SENP5, SENP6, and / or SENP7. For example, SENP1 can cleave short peptides in the absence of the globular domain of SUMO protein. (See Drag et al, Biochem J.2008 Jan 15;409(2):461-9; which is incorporated herein by reference in its entirety.)

[0280] In some embodiments, the ability of a protein carrier to be cleaved by an intracellularprotease makes it suitable for delivering a cargo into the cytoplasm, as such protein carriers do not remain attached to the cargo and therefore would not perturb the function of the delivered cargo in the cytoplasm. The intracellular protease mediated cleavage may occur exclusively in the cytoplasm.

[0281] In some embodiments, the intracellular protease is a cysteine protease. In someembodiments, the intracellular protease is a UBL (ubiquitin-like proteins)-specific protease. The UBL-specific protease includes a ubiquitin-specific protease (USP) or SUMO-specific protease (SENP). In some embodiments, the intracellular proteases are deubiquitinating enzymes (DUBs).Deubiquitinating enzymes (DUBs) are described in Cruz et al Pharmaceuticals 2021, 14, 848;which is incorporated herein by reference in its entirety. In some embodiments, the intracellular protease is a sentrin / SUMO-specific protease (SENP). In some embodiments, the sentrin / SUMO- specific protease (SENP) is SENP1, SENP2, SENP3, SENP5, SENP6, and / or SENP7.

[0282] In some embodiments, the protein carrier comprises a ubiquitin-like protein (UBL), afunctional fragment, or derivative thereof. In some embodiments, the protein carrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof and a terminal polypeptide. In some embodiments, the terminal polypeptide is an N-terminal polypeptide or a C-terminal polypeptide. In certain embodiments, the protein carrier comprising a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof further comprises an N-terminal polypeptide. In some embodiments, the N-terminal polypeptide is located at amino terminus of the UBL.

[0283] In some embodiments, ubiquitin-like proteins (UBLs) are selected from ubiquitin(e.g., ubiquitin B), small ubiquitin-like modifier (SUMO), neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8), autophagy-related protein 8 (ATG8), autophagy-related 12 (ATG12), ubiquitin related modifier 1 (URM1), ubiquitin fold modifier 1 (UFM1), human leukocyte antigen (HLA)-F adjacent transcript 10 or Ubiquitin D (FAT10), and interferon (IFN)-stimulated gene 15 (ISG15). Examples of UBLs include, but are not limited to,Attorney Docket No: 243735.000431 SUMO1, SUMO2, SUMO3, SUMO4, NEDD8, light chain 3 alpha (LC3A), light chain 3 beta (LC3B), light chain 3 beta 2 (LC3B2), light Chain 3 Gamma (LC3C), gamma-aminobutyric acid receptor-associated protein (GABARAP), gamma-aminobutyric acid receptor-associated protein- like 1 (GABARAPL1), gamma-aminobutyric acid receptor-associated protein-like 2 (GABARAPL1; also known as GATE-16), autophagy-related protein 8 (ATG8), autophagy- related protein 12 (ATG12), FAT10, ISG15, URM1, and UFM1 (See Cappadocia et al Chem Rev.2018,118, 889–918; which is incorporated herein by reference in its entirety), or a functional fragment, or derivative thereof. Any member protein of the ubiquitin family can be employed as the protein carrier according to the present disclosure.

[0284] In some embodiments, the UBL has been engineered to remove native cysteineresidue(s). In some embodiments, the UBL is engineered to substitute native cysteine residue(s) with the non-cysteine amino acid residue(s).

[0285] In some embodiments, the protein carrier comprising a UBL, a functional fragment,or derivative thereof is modified to introduce one, or more cysteine residues. In some embodiments, the one or more cysteine residues are introduced within the UBL, a functional fragment, or derivative thereof. In some embodiments, the one or more cysteine residues are introduced at the amino terminus and / or at the carboxy terminus of UBL. In some embodiments, the one or more cysteine residues are introduced at the amino terminus of UBL. In some embodiments, the protein carrier comprising a UBL is modified to introduce one or more cysteine residues outside the interface between the UBL and its cognate UBL-specific protease.

[0286] In some embodiments, the UBL is modified to introduce one or more cysteineresidues for site-specific conjugation to the cationic polymer. In some embodiments, the UBL is modified to introduce one or more cysteine residues outside the interface between the UBL and its cognate UBL specific protease. In some embodiments, the UBL is modified to introduce one or more cysteine residues within the interface between the UBL and its cognate UBL specific protease.

[0287] In some embodiments, the UBL comprises an amino acid sequence set forth in anyone of SEQ ID NOs: 1-8, 53-60, and 72 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72. In some embodiments, the UBL comprises an amino acid sequence set forth in any one of the SEQAttorney Docket No: 243735.000431 ID NOs: 1-8, 53-60, and 72 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72. In some embodiments, the UBL comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72. In some embodiments, the UBL comprises a functional fragment of SEQ ID NOs: 1-8, 53-60, and 72 as indicated with underline here in the ‘List of Sequences’ Section.

[0288] In some embodiments, the UBL protein is linked to the cargo via a linker. In someembodiments, the UBL protein is linked to the cargo via a linker comprising a diglycine (Gly- Gly). In certain embodiments, the UBL protein is linked to the cargo via a diglycine peptide present in the UBL protein.

[0289] In certain embodiments, the protein carrier comprises a small ubiquitin-like modifier(SUMO) protein, a functional fragment, or derivative thereof. In some embodiments, the SUMO protein is SUMO1, SUMO2, SUMO3, or SUMO4. In certain embodiments, the SUMO protein is SUMO1. In some embodiments, the SUMO1 protein is engineered to substitute native cysteine residue with the non-cysteine amino acid residue. In some embodiments, the SUMO1 protein is modified to substitute native cysteine amino acid at position 52 with alanine amino acid. Such modified SUMO1 protein is further conjugated to the cationic polymer and / or linked to a cargo as illustrated herein.

[0290] In some embodiments, the SUMO1 protein is modified to introduce one or morecysteine residues at the amino acid position 20, amino acid position 21, amino acid position 23, amino acid position 25, any amino acid position from 27 to 33, amino acid position 35, amino acid position 37, amino acid position 39, any amino acid position from 41 to 43, amino acid position 45, amino acid position 46, amino acid position 49, amino acid position 50, amino acid position from 52 to 58, amino acid position 73, amino acid position 78, amino acid position 79, and / or any amino acid position from 83 to 85. In some embodiments, the SUMO1 protein is modified to introduce one or more cysteine residues at the amino acid position 33, amino acid position 46, amino acid position 53, and / or amino acid position 85. In some embodiments, the SUMO1 protein is modified to introduce one or more cysteine residues at the amino acid position 33 and / or amino acid position 50. The positions of the amino acid residues are according to residue numbering of the SUMO protein having a Uniprot Accession No.63165 (SEQ ID NO: 1). In some embodiments, the SUMO1 protein is modified to introduce one or more cysteine residues outside the interface between the SUMO1 protein and its cognate intracellular protease.Attorney Docket No: 243735.000431 In some embodiments, the SUMO1 protein is modified to introduce one or more cysteine residues within the interface between the SUMO1 protein and its cognate intracellular protease.

[0291] In some embodiments, the SUMO protein is linked to the cargo via a linker. In someembodiments, the SUMO protein is linked to the cargo via a linker comprising a diglycine (Gly- Gly). In certain embodiments, the SUMO protein is linked to the cargo via a diglycine peptide present in the SUMO protein.

[0292] In some embodiments, the SUMO protein linked to the cargo is cleaved by a SUMOspecific protease to release the cargo in the cytoplasm. In some embodiments, the SUMO specific protease is sentrin-specific protease (SENP). In some embodiments, the SENP protein is SENP1. Any of the SENPs in the cells may release the cargo into the cytoplasm from the SUMO protein. In some embodiments, the sentrin / SUMO-specific protease (SENP) is SENP1, SENP2, SENP3, SENP5, SENP6, and / or SENP7. In some embodiments, the SUMO protein is SUMO1 protein and the SUMO specific protease is SENP1. In some embodiments, the SUMO is produced with a precursor protein and efficiently cleaved with SUMO-specific proteases, SENPs, in the cytoplasm and nucleus of cells.

[0293] In some embodiments, the SUMO1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 75 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1.

[0294] In some embodiments, the SUMO1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequenceAttorney Docket No: 243735.000431 identity to an amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 75 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 2. SEQ ID NO:2 represents an exemplary functional fragment of the SUMO1 protein. Amino acid sequences of SEQ ID NO: 2-8 represent the mutated variants of the SUMO1 protein. These sequences can be further mutated to introduce cysteine residue(s) at one or more positions as described herein.

[0295] In some embodiments, the SUMO1 protein is engineered to remove cysteine residueat position 52. In some embodiments, the SUMO1 protein comprises Cys52Ala (C52A) amino acid substitution. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 75 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 3.

[0296] In some embodiments, the SUMO1 protein is modified to introduce cysteineresidue(s) at position 33, position 46, position 53, and / or position 85. In some embodiments, the SUMO1 protein comprises Glu33Cys (E33C), Lys46Cys (K46C), Gln53Cys (Q53C), and / orAttorney Docket No: 243735.000431 Glu85Cys (E85C) amino acid substitutions. In some embodiments, the SUMO1 protein comprises (i) Cys52Ala (C52A) and (ii) Glu33Cys (E33C), Lys46Cys (K46C), Gln53Cys (Q53C), and / or Glu85Cys (E85C) amino acid substitutions. The amino acid sequences of the modified SUMO1 protein are provided herein with the substitutions indicated with an underline (SEQ ID Nos: 3-8).

[0297] In some embodiments, the SUMO1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 75 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 4.

[0298] In some embodiments, the SUMO1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 75 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5.Attorney Docket No: 243735.000431

[0299] In some embodiments, the SUMO1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 6 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having at least 75 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6.

[0300] In some embodiments, the SUMO1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 75 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 7.

[0301] In some embodiments, the SUMO1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8 orAttorney Docket No: 243735.000431 an amino acid sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 75 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8.

[0302] In some embodiments, the SUMO1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 53 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 53, or a functional fragment, or derivative thereof. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 53 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 53, or a functional fragment, or derivative thereof. In some embodiments, the SUMO1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 53, or a functional fragment, or derivative thereof.

[0303] In some embodiments, the SUMO2 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 54 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 54, or a functional fragment, or derivative thereof. In some embodiments, the SUMO2 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 54 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 54, or a functional fragment, or derivative thereof. In some embodiments, the SUMO2 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 54, or a functional fragment, or derivative thereof.

[0304] In some embodiments, the SUMO3 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 55 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequenceAttorney Docket No: 243735.000431 identity to an amino acid sequence set forth in SEQ ID NO: 55, or a functional fragment, or derivative thereof. In some embodiments, the SUMO3 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 55 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the SUMO3 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 55, or a functional fragment, or derivative thereof.

[0305] In some embodiments, the UBB protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 56 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 56, or a functional fragment, or derivative thereof. In some embodiments, the UBB protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 56 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 56, or a functional fragment, or derivative thereof. In some embodiments, the UBB protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 56, or a functional fragment, or derivative thereof.

[0306] In some embodiments, the NEDD8 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 57 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 57, or a functional fragment, or derivative thereof. In some embodiments, the NEDD8 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 57 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 57, or a functional fragment, or derivative thereof. In some embodiments, the NEDD8 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 57, or a functional fragment, or derivative thereof.

[0307] In some embodiments, the ISG15 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 58 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 58, or a functional fragment, or derivative thereof. In some embodiments, the ISG15 protein comprises or consists of an aminoAttorney Docket No: 243735.000431 acid sequence set forth in SEQ ID NO: 58 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 58, or a functional fragment, or derivative thereof. In some embodiments, the ISG15 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 58, or a functional fragment, or derivative thereof.

[0308] In some embodiments, the URM1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 59 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 59, or a functional fragment, or derivative thereof. In some embodiments, the URM1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 59 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 59, or a functional fragment, or derivative thereof. In some embodiments, the URM1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 59, or a functional fragment, or derivative thereof.

[0309] In some embodiments, the UFM1 protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 60 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 60, or a functional fragment, or derivative thereof. In some embodiments, the UFM1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 60 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 60, or a functional fragment, or derivative thereof. In some embodiments, the UFM1 protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 60, or a functional fragment, or derivative thereof.

[0310] In some embodiments, the ubiquitin protein comprises or consists of an amino acidsequence set forth in SEQ ID NO: 72 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 72, or a functional fragment, or derivative thereof. In some embodiments, the ubiquitin protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 72 or an amino acid sequence having at least 80 %Attorney Docket No: 243735.000431 sequence identity to an amino acid sequence set forth in SEQ ID NO: 72, or a functional fragment, or derivative thereof. In some embodiments, the ubiquitin protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 72, or a functional fragment, or derivative thereof. Cationic Polymers

[0311] The present disclosure provides cationic polymers to facilitate delivery of the proteinconjugates of the present disclosure into the cytoplasm of the target cells.

[0312] In some embodiments, the cationic polymer undergoes ionization in a solution havinga pH value below the pKa value of the amine group of the cationic polymer. In some embodiments, the cationic polymer is positively charged in a solution having a pH value below the pKa value of the amine group of the cationic polymer comprises at least 50%, 55%, 60%, 65%, 70%, 75%.80%, 85%, 90%, 95%, or 100% by weight of the cationic polymer. In some embodiments, the cationic polymer undergoes protonation in a solution having a pH value below the pKa value of the amine group of the cationic polymer. In some embodiments, the protonation (e.g., multiple protonation) of the cationic polymer results in a plurality of positive surface charges.

[0313] In some embodiments, the cationic polymers are polyamines.

[0314] Examples of cationic polymers suitable for use in the protein conjugates of thepresent disclosure include, but are not limited to, polyethylenimine (PEI), poly(L-lysine) (PLL), poly(D-lysine) (PDL), polyamidoamine (PAA), Poly(amino-co-ester)s (PAEs), poly[2-(N,N- dimethylamino)ethyl methacrylate] (PDMAEMA), poly(propylene imine) (PPI), poly-L-arginine (PLA), poly-guanidine, poly-D-arginine, poly(D,L-lysine hydrobromide), poly-L-ornithine, conjugates of PEI (such as PEI-Gua, PEI-Arg, PEI-His, PEI-Trp), or derivatives and combinations thereof.

[0315] In some embodiments, the cationic polymer is polyethylenimine (PEI). The PEI canbe a linear PEI or a branched PEI. The branched PEI can include a short-chain PEI. In some embodiments, the branched PEI is a branched PEI600 (bPEI600) or a branched PEI1200 (bPEI1200).

[0316] In some embodiments, the PEI has a molecular weight of less than about 2000 Da. Insome embodiments, the PEI has a molecular weight of less than about 200Da, about 300Da,Attorney Docket No: 243735.000431 about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900Da, about 1000Da, about 1200Da, about 1300Da, about 14100Da, about 1500Da, about 1600Da, about 1700Da, about 1800Da, about 1900Da, or about 2000 Da. In some embodiments, the PEI has a molecular weight ranging from about 100 Da to about 2000Da. In some embodiments, the PEI has a molecular weight ranging from about 100 Da to about 500Da, about 500 Da to about 1000Da, about 1000 Da to about 1500Da, or about 1500 Da to about 2000Da. In some embodiments, the PEI has a molecular weight ranging from about 600 Da to about 1800Da. In some embodiments, the PEI has a molecular weight of about 600 Da or 1200 Da. The term molecular weight as used herein refers to an average molecular weight of the cationic polymer such as PEI.

[0317] In some embodiments, the protein conjugate comprises a plurality of cationicpolymers (e.g., PEI) conjugated to the protein carrier. In some embodiments, the plurality of the cationic polymers (e.g., PEI) depends on the number of cysteine residues present in the protein carrier of the protein conjugate. In some embodiments, the protein conjugate comprises one or more cationic polymers (e.g., PEI) conjugated to the protein carrier. In some embodiments, the plurality of cationic polymers (e.g., PEI) is in the range of 1 to 8 molecules.

[0318] In some embodiments, the protein conjugate comprises one cationic polymer (e.g.,PEI) conjugated to the protein carrier. In some embodiments, the protein conjugate comprises two cationic polymers (e.g., PEI) conjugated to the protein carrier. In some embodiments, the protein conjugate comprises three cationic polymers (e.g., PEI) conjugated to the protein carrier. In some embodiments, the protein conjugate comprises four cationic polymers (e.g., PEI) conjugated to the protein carrier. In some embodiments, the protein conjugate comprises five cationic polymers (e.g., PEI) conjugated to the protein carrier. In some embodiments, the protein conjugate comprises six cationic polymers (e.g., PEI) conjugated to the protein carrier. In some embodiments, the protein conjugate comprises seven cationic polymers (e.g., PEI) conjugated to the protein carrier. In some embodiments, the protein conjugate comprises eight cationic polymers (e.g., PEI) conjugated to the protein carrier. Linkers Conjugating the Cationic Polymers to the Protein Carriers

[0319] In some embodiments, the protein carrier is conjugated to the cationic polymer via alinker. As used herein, the term “conjugated” refers to covalent attachment of one molecule to aAttorney Docket No: 243735.000431 second molecule. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker comprises two reactive groups having ability to bind to two same or different molecules (e.g., a protein carrier and a cationic polymer). In some embodiments, the linker is a cleavable linker or non-cleavable linker. In some embodiments, the terminal polypeptide of the protein carrier is conjugated to the cationic polymer via the linker. In one embodiment, the N-terminal polypeptide of the protein carrier is conjugated to the cationic polymer via the linker. In some embodiments, the UBL of the protein carrier is conjugated to the cationic polymer via the linker.

[0320] In some embodiments, the linker conjugating the protein carrier to the cationicpolymer is a heterobifunctional crosslinker. In some embodiments, the linker binds to the protein carrier via a thioester or a disulfide bond. In some embodiments, the linker comprises an electrophile selected from haloacetophenone, epoxide, haloacetamide, maleimide, isothiocyanate, vinyl sulfone, nitrile, iminonitrile, hydrazone, butynoate, acrylester, acrylonitrile, acrylamide, benzisothiazolone, and N-hydroxy(methyl)phthalimide (See Petri et al Eur J Med Chem.2018 Dec 5:160:94-107; which is incorporated herein by reference in its entirety).

[0321] In some embodiments, the heterobifunctional crosslinker comprises a N-hydroxysuccinimide (NHS) ester and a maleimide group. Examples of linkers include, but are not limited to, N-alpha-maleimidoacetoxyl succinimide ester (AMAS), N-beta-maleimidopropyl- oxysuccinimide ester (BMPS), N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS), m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-epsilon-malemidocaproyl- oxysuccinimide ester (EMCS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH), succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), N-(11- maleimidoundecanoyloxy)succinimide (KMUS), or derivatives and combinations thereof. In some embodiments, the linker is N-alpha-maleimidoacetoxyl succinimide ester (AMAS).

[0322] The linker may be attached to the protein carrier at various positions, depending onthe type of the link. In some embodiments, the linker is attached to the cysteine residue(s) of the protein carrier. Any linker having an ability to link to a cysteine residue(s) of the peptide or protein may be employed for the present disclosure to conjugate the protein carrier to the cationic polymer.Attorney Docket No: 243735.000431 Cargos

[0323] In some embodiments, the present disclosure provides cargos capable of interactingwith an intracellular target. The cargo can be genetically fused and / or chemically or enzymatically linked to the protein carrier of the present disclosure to facilitate delivery of the cargo into the cytoplasm. The cargo suitable for genetical fusion and / or chemical linkage with the protein carrier of the present disclosure includes a chemical cargo (e.g., a small molecule chemical compound) or a polypeptide cargo.

[0324] In some embodiments, the cargo is selected from a chemical compound, a protein, apeptide, a peptidomimetic, an antibody, an antibody fragment, an antibody mimetic, a monobody, a fusion protein comprising a monobody domain, an aptamer, a ribozyme, a small molecule chemical compound, a short hairpin RNA (shRNA), an antisense nucleic acid molecule, a small interfering RNA (siRNA), a micro RNA (miRNA), a nucleic acid encoding an antisense nucleic acid molecule such as an antisense oligonucleotide (ASO), or a nucleic acid sequence encoding a protein.

[0325] In some embodiments, the cargo that interacts with an intracellular target includes acompound that can bind specifically to the intracellular protein to, e.g., enhance or reduce an activity of the said intracellular protein. In some embodiments, the cargo is agonistic or antagonistic. Mutated intracellular proteins often drive diseases and remain “undruggable” due to their limited druggable sites (Fig.9). Various approaches that can be employed for targeting intracellular proteins using biologic agents are shown in Fig.10.

[0326] In some embodiments, the intracellular target is selected from rat sarcoma virus(RAS) kinase (e.g., Kirsten rat sarcoma viral oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene homolog (HRAS), or neuroblastoma RAS viral oncogene homolog (NRAS)), Aurora A kinase, ABL kinase, Src-family kinases (e.g., Fgr, Hck, Lck, Lyn, Src, Yes), Src homology 2 domain-containing phosphatase 2 (SHP2), signal transducer and activator of transcription 3 (STAT3), PR domain containing 14 (PRDM14), WD repeat domain 5 (WDR5), p53, or mixed lineage kinase domain-like protein (MLKL). In some embodiments, the RAS kinase is KRAS, NRAS, or HRAS. In some embodiments, the intracellular target is the SH2 domain of ABL1. In some embodiments, the intracellular target is the SH2 domain of SHP2 or SHP2 phosphatase.Attorney Docket No: 243735.000431

[0327] In certain embodiments, the cargo is a polypeptide cargo. In some embodiments, thepolypeptide cargo does not contain cysteine residue(s). In some embodiments, the polypeptide cargo is engineered to remove native cysteine residue(s).

[0328] In some embodiments, the polypeptide cargo is selected from a monobody, adesigned ankyrin repeat protein (DARPin), an affilin, an affimer, an affitin, an alphabody, an Anticalin, an avimer, a knottin, an armadillo repeat proteins, an affibody, a fynomer, a gastrobody, a clostridal antibody mimetic protein (nanoCLAMPs), an optimer, a repebody, a recombinant fibronectins (e.g., Pronectin™ and the like), a centyrin, and an obody, a peptide, an antibody or antigen-binding fragment, or derivatives and combinations thereof.

[0329] In some embodiments, the antibody or antigen binding fragments includes a singlechain antibody, a single domain antibody, a variable heavy homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, a monoclonal antibody, a bispecific antibody or antigen- binding fragment thereof, a bi-epitopic antibody or antigen-binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, an scFv, a VH domain, a monovalent antibody or a bivalent antibody.

[0330] In some embodiments, the polypeptide cargo is an immunoglobulin. In oneembodiment, the immunoglobulin (Ig) is a protein containing one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins may have a number of structural forms, including, but not limited to, full length antibodies, antibody fragments, and individual immunoglobulin domains.

[0331] In some embodiments, the polypeptide cargo is an IgG. In one embodiment, the IgGis a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, the IgG class comprises IgG1, IgG2, IgG3, and IgG4. In mice, the IgG class comprises IgG1, IgG2a, IgG2b, IgG3.

[0332] In one embodiment, polypeptide cargo includes an immunoglobulin (Ig) domainwhich is a region of an immunoglobulin that exists as a distinct structural entity as ascertained by one skilled in the art of protein structure. Ig domains typically have a characteristic folding topology. The known Ig domains in the IgG class of antibodies are the variable heavy chain domain (VH), the heavy chain constant domains—Cγ1, Cγ2, Cγ3—together comprising the Cγ domain which includes the hinge region between Cγ1 and Cγ2, the variable domain of the lightAttorney Docket No: 243735.000431 chain (VL), and the constant domain of the light chain (CL), which in humans comprises either the kappa (CO or lambda (CA) light chain constant domain.

[0333] In one embodiment, the polypeptide cargo includes an Fc region. The “Fc region”(also known as the “fragment crystallizable” or “tail” region) may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. For all heavy chain constant region amino acid positions discussed in the present invention, numbering is according to the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85, describing the amino acid sequence of myeloma protein EU, which is the first human IgG1 sequenced. The EU index of Edelman et al. is also set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. Thus, the “EU index as set forth in Kabat” or “EU index of Kabat” refers to the amino acid residue numbering system based on the human IgG1 EU antibody of Edelman et al. as set forth in Kabat 1991.

[0334] The Fc region of an immunoglobulin generally comprises two constant domains, CH2and CH3. Typically, an “Fc polypeptide,” as the term is used herein, comprises a CH2 and a CH3 domain and can include at least a portion of the hinge domain, but does not usually include the entire CH1 domain. In some embodiments, an Fc region can be present in dimeric or monomeric form.

[0335] In some embodiments, the peptide is a stapled peptide or bicyclic peptide.

[0336] In certain embodiments, the polypeptide cargo is monobody, a designed ankyrinrepeat protein (DARPin), or derivatives thereof.

[0337] In certain embodiments, the polypeptide cargo is monobody. In some embodiments,the monobodies comprise two or more monobody units.

[0338] Monobodies are small (~90 aa; 10 kDa), simple and modular proteins. Monobodiesare stable without disulfide bonds and are also suitable for intracellular expression (compared to <50% of nanobodies expressed in HEK cells). Synonyms of monobodies are Adnectin orfibronectin domain / scaffold. See Koide et al. (1998) J Mol Biol 284:1141; (2002) PNAS99:1253; (2007) PNAS 104:6632; (2012) J Mol Biol.415:393; Gilbreth et al. (2008) J Mol Biol, 381:407; Wojcik et al. (2010) Nature Struct Mol Biol 17:519;Sha et al. (2013) PNAS 110:14924;Attorney Docket No: 243735.000431 Sha et al. (2017) Protein Sci, 26:910; Hantschel et al. (2020) Current Op Struct Biol, 60:167; and Dingus et al. bioRxiv doi.org / 10.1101 / 2021.04.06.438746; each incorporated herein by reference in its entirety. Centyrin derived from a consensus sequence of 14 FN3 domains and Tn3 (enascin-FN3 derived monobody) are homologs of monobodies that are under clinical development. Ribbon diagrams of FN3, VHH, and Fab protein structures are shown in Fig.16. The non-limiting method that can be employed for the development of monobodies is shown in Fig.17.

[0339] The term “monobody” as used herein encompasses a polypeptide which includes a β-strand domain lacking in disulfide bonds and containing a plurality of β-strands, two or more loop regions each connecting one β-strand to another β-strand, and optionally an N-terminal tail, a C-terminal tail, or both, wherein at least one of the two or more loop regions, the N-terminal tail, or the C-terminal tail is characterized by activity in binding a target protein or molecule. More specifically, such monobodies of the present disclosure can include three or more loop regions or, even more specifically, four or more loop regions.

[0340] In some embodiments, the size of a monobody cargo is less than about 30 kDa. Insome embodiments, the size of a monobody cargo is less than about 20 kDa.

[0341] To achieve the specificity in the binding of a monobody to an intracellular target, theamino acid sequence of the monobody may be modified relative to the scaffold used for its construction. Scaffolds for formation of a monobody should be highly soluble and stable. An ideal scaffold for formation of a monobody should be small enough for structural analysis, yet large enough to accommodate multiple binding domains so as to achieve tight binding and / or high specificity for its target.

[0342] An exemplary scaffold for formation of a monobody is the fibronectin type IIIdomain (Fn3). Fibronectin is a large protein which plays essential roles in the formation of extracellular matrix and cell-cell interactions; it consists of many repeats of three types (types I, II, and Ill) of small domains (Baron et al., 1991).

[0343] Fn3 itself is the paradigm of a large subfamily (Fn3 family or s-type Ig family) of theimmunoglobulin superfamily. The Fn3 family includes cell adhesion molecules, cell surface hormone and cytokine receptors, chaperonins, and carbohydrate-binding domains (for reviews, see Bork Doolittle, 1992; Jones, 1993; Bork et al., 1994; Campbell Spitzfaden, 1994; Harpez Chothia, 1994).Attorney Docket No: 243735.000431

[0344] Fn3 is small (about 94 residues), monomeric, soluble, and stable. It is one of fewmembers of immunoglobulin superfamily (IgSF) that do not have disulfide bonds and, therefore, is stable under reducing conditions. The tenth type III module of fibronectin has a fold similar to that of immunoglobulin domains, with seven β strands forming two antiparallel β sheets, which pack against each other. The structure of the type H module includes seven β strands, which form a sandwich of two antiparallel sheets, one containing three strands (ABE) and the other four strands (C’CFG) (Williams Barclay, 1988). The β sheet contains residues Glu-9-Thr-14 Ser-17- Asp-23 and Thr-56-Ser-60. The majority of the conserved residues contribute to the hydrophobic core, with the invariant hydrophobic residues Trp-22 and Try-68 lying toward the N-terminal and C-terminal ends of the core, respectively. The β strands are much less flexible and appear to provide a rigid framework upon which functional, flexible loops can be built. The topology is similar to that of immunoglobulin C domains.

[0345] In some embodiments, monobodies described herein are fibronectin type III (Fn3)-derived polypeptide monobodies. In some embodiments, the Fn3-derived monobodies include at least two Fn3 β-strand domain sequences with a loop region sequence linked between adjacent β- strand domain sequences and optionally, an N-terminal tail of at least about 2 amino acids, a C- terminal tail of at least about 2 amino acids, or both.

[0346] The loop region sequence, the N-terminal tail, or the C-terminal tail, or combinationsthereof include an amino acid sequence which has binding specificity for an intracellular target. To render a loop region sequence, N-terminal tail, or C-terminal tail capable of binding to an intracellular target, either the loop region sequence, the N-terminal tail, the C-terminal tail, or a combination thereof varies by deletion, insertion, or replacement of at least two amino acids from a corresponding loop region, N-terminal tail, or C-terminal tail in a wild-type or mutant Fn3 scaffold.

[0347] In one embodiment, an Fn3 scaffold is the tenth Fn3 domain of human fibronectin(FNfnl0). In one embodiment, an Fn3 scaffold is the tenth Fn3 domain of human fibronectin which has a modified Asp7, which is replaced by a non-negatively charged amino acid residue such as Asn or Lys.

[0348] In one embodiment, the monobodies comprise seven β-strand domain sequences(designated A through G) and six loop regions (AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop) which connect the seven β-strand domain sequences.Attorney Docket No: 243735.000431

[0349] In some embodiments, the monobody can be prepared by recombinant techniques,thereby affording the deletion, insertion, or replacement of at least two amino acids from a corresponding loop region, N-terminal tail, or C-terminal tail in a wild-type or mutant Fn3 scaffold. Deletions can be a deletion of at least two amino acid residues up to substantially all but one amino acid residue appearing in a particular loop region or tail. Insertions can be an insertion of at least two amino acid residues up to about 25 amino acid residues, preferably at least two up to about 15 amino acid residues. Replacements can be replacements of at least two up to substantially all amino acid residues appearing in a particular loop region or tail. Alternatively, the monobody of the present disclosure can be prepared as described in described in U.S. Pat. No.9,512,199B2 (Loew et al.), the contents of which are incorporated herein in their entirety.

[0350] The deletions, insertions, and replacements (relative to wild-type or previously knownmutant) on Fn3 scaffolds can be achieved using recombinant techniques beginning with a known nucleotide sequence. Desired mutations can be introduced to the Fn3 gene using either cassette mutagenesis, oligonucleotide site-directed mutagenesis techniques (Deng Nickoloff, 1992), or Kunkel mutagenesis (Kunkel et al., 1987).

[0351] Both cassette mutagenesis and site-directed mutagenesis can be used to preparespecifically desired nucleotide coding sequences. Cassette mutagenesis can be performed using the same protocol for gene construction described above and the double-stranded DNA fragment coding a new sequence can be cloned into a suitable expression vector. Many mutations can be made by combining a newly synthesized strand (coding mutations) and an oligonucleotide used for the gene synthesis.

[0352] Regardless of the approach utilized to introduce mutations into the monobodynucleotide sequence, sequencing can be performed to confirm that the designed mutations (and no other mutations) were introduced by mutagenesis reactions.

[0353] Rapid drug prototyping with intracellular monobodies is shown here in Fig. 11 andalso described in Gupta et al. (2018) Nature Chem Biol 14:895; Sha et al. (2017) Protein Sci, 26:910; Hantschel et al. (2020) Curr Op Struct Biol 60:167; and Akkapeddi, Teng, Koide (2021) RSC Med Chem 12:1839. Fig.12 shows the PDB structures of exemplary monobody-target complexes. Targeting a single member of a large protein family is shown in Figs.13A-13B. (Sha, Gencer et al. (2013) PNAS 110:14924; Liu et al. Mol Cell 2006).Attorney Docket No: 243735.000431

[0354] In some embodiments, the monobody or DARPin described herein targets RASkinase (e.g., KRAS, HRAS, or NRAS), Aurora A kinase, ABL kinase, Src-family kinases (e.g., Fgr, Hck, Lck, Lyn, Src, Yes), SHP2, STAT3, PRDM14, WDR5, p53, or MLKL.

[0355] Non-limiting examples of monobodies include RAS-targeting monobodies (e.g.,12VC1, NS1, 12D3, 12D4, 12D5) (60-61), NRAS-targeting monobodies (e.g., Mb19, Mb20,Mb24) (WO 2023 / 192915 A1), apoRAS-targeting monobodies (e.g., R15) (62), ABL1 SH2- targeting monobodies (e.g., AS25, AS27, HA4, 7C12) (52, 54-55), SHP2 SH2-targeting monobodies (e.g., NSa1, NSa5, CS3) (63), SHP2 phosphatase-targeting monobodies (e.g., Mb13) (64), PRDM14-targeting monobody (e.g., S14) (66), WDR5-targeting monobodies (e.g., S4) (67), Aurora kinase A-targeting antibodies monobodies (e.g., Mb1, Mb2, Mb3, Mb4, Mb5, Mb6) (56), MLKL-targeting monobodies (e.g., Mb27,Mb32, Mb33, MLKL_26, MLKL_27, MLKL_32, MLKL_33, and MLKL_37) (57-59), STAT3-targeting monobodies (e.g., MS3-6) (68), FGR SH2-targeting monobodies (e.g., Fgr_1, Fgr_2) (65), HCK SH2-targeting monobodies (e.g., Hck_1, Hck_2) (65), LCK SH2-targeting monobodies (e.g., Lck_1, Lck_3) (65), LYN SH2-targeting monobodies (e.g., Lyn_2, Lyn_4) (65), SRC SH2-targeting monobodies (e.g., Src_2) (65), and YES SH2-targeting monobodies (e.g., Yes_1, Yes_3) (65) (each reference cited above is incorporated herein by references in its entirety). Non-limiting examples of DARPins include p53 targeting DARPins (e.g., C10, C10-H82R) (70).

[0356] In some embodiments, the monobody comprises an amino acid sequence set forth inany one of SEQ ID NOs: 14-52 and 69-70 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 14-52 and 69-70. In some embodiments, the monobody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14-52 and 69-70 or an amino acid sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 14-52 and 69-70. In some embodiments, the monobody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14-52 and 69-70.

[0357] In some embodiments, the monobody targets RAS protein. Table 1 below illustratesnon-limiting examples of RAS-targeting monobodies that can be used as the polypeptide cargo according to the present disclosure.Attorney Docket No: 243735.000431Table 1. RAS-targeting MonobodiesMonobody Isotype specificity Mutation specificity Nucleotide specificity ReferenceNS1 KRAS, NRAS None None 6012VC1 Not Determined G12V, G12C GTP 61R15 None Fast cycling mutants Apo (nucleotide free) 62JAM20 None None GDP 212D1, 12D2, 12D3, 12D4 KRAS G12D GTP 2

[0358] In some embodiments, the monobody cargo is 12VC1. Monobody 12VC1 selectivelyand noncovalently binds to both KRAS(G12C) and KRAS(G12V) as demonstrated in Figs.14A- 14B (61, 69). Noncovalent inhibition of KRAS(G12V) blocks signaling and inhibits tumor growth in a mouse xenograft model (Fig.15) (61). Additional moieties

[0359] In some embodiments, the protein conjugates, contiguous polypeptides, proteincarriers, and / or cargos described herein further comprise one or more additional moieties.

[0360] In some embodiments, the protein conjugates, contiguous polypeptides, and / orprotein carrier further comprises a cell-targeting moiety. In some embodiments, the cell-targeting moiety is a polypeptide (e.g., a monobody, an antibody or antigen-binding fragment) that has binding specificity towards a cell-surface protein (e.g., a PD-L1 protein). The protein carrier comprising the cell-surface protein binding moiety provides cell-type specificity to the protein conjugate and may enable cell type-specific inhibition of intracellular targets.

[0361] In some embodiments, the cell-targeting moiety is a monobody, a designed ankyrinrepeat protein (DARPin), an affilin, an affimer, an affitin, an alphabody, an Anticalin, an avimer, a knottin, an armadillo repeat proteins, an affibody, a fynomer, a gastrobody, a clostridal antibody mimetic protein (nanoCLAMPs), an optimer, a repebody, a recombinant fibronectins (e.g., Pronectin™ and the like), a centyrin, and an obody, a peptide, an antibody or antigen- binding fragment, or derivatives and combinations thereof. In some embodiments, the antibody or antigen binding fragments includes a single chain antibody, a single domain antibody, a variable heavy homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, aAttorney Docket No: 243735.000431 monoclonal antibody, a bispecific antibody or antigen-binding fragment thereof, a bi-epitopic antibody or antigen-binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, an scFv, a VH domain, a monovalent antibody or a bivalent antibody.

[0362] In some embodiments, the protein carrier and / or polypeptide cargo further comprisesone or more affinity tags. In some embodiments, the affinity tag is a His tag, an Avi-tag, a hemagglutinin (HA) tag, a FLAG tag, a Myc tag, a glutathione S-transferase (GST) tag, a maltose binding protein (MBP) tag, a chitin binding protein tag, a calmodulin tag, a V5 tag, a streptavidin binding tag, a green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, or a combination thereof.

[0363] In some embodiments, the protein carrier further comprises a tag at the N-terminus ofthe protein carrier. In some embodiments, the N-terminal polypeptide of the protein carrier comprises a tag. In some embodiments, the tag is a hexahistidine (His6) peptide (SEQ ID NO:12). In some embodiments, the polypeptide cargo further comprises a short peptide tag (e.g., an affinity tag) at the C-terminus of the polypeptide cargo. In some embodiments, the short peptide tag at the C-terminus of the polypeptide cargo is a V5 tag. In some embodiments, the V5 tag comprises the amino acid sequence of SEQ ID NO: 13, or a fragment thereof. The V5 tagged protein conjugate can be analyzed to detect the amount of the cleaved product in the cells, preferably by immunoblotting as illustrated herein.

[0364] In some embodiments, the protein carrier and / or polypeptide cargo further comprisesa moiety to extend the half-life of the polypeptide cargo in circulation system. In some embodiments, the protein carrier and / or polypeptide cargo further comprises an albumin-binding domain. In some embodiments, the N-terminal polypeptide of the protein carrier comprises an albumin-binding domain at the amino terminus of the protein carrier. In some embodiments, the albumin-binding domain comprises the amino acid sequence of SEQ ID NO: 66 or a fragment thereof. In some embodiments, the albumin-binding domain comprises the amino acid sequence of SEQ ID NO: 66 or a sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66.

[0365] In some embodiments, the protein carrier further comprises a serine-cysteine-serine(SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide at the N-terminus of the protein carrier. In some embodiments, the N-terminal polypeptide of the protein carrier furtherAttorney Docket No: 243735.000431 comprises a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide. These peptides are added to introduce one or more cysteine residues to enable conjugation of a protein carrier or a contiguous polypeptide comprising a protein carrier with a cationic polymer.

[0366] In some embodiments, the protein cargo further comprises a small peptide fragmentdesigned to enhance the biophysical properties of the protein cargo. The small peptide fragment designed to enhance the biophysical properties can be derived from the human fibronectin sequence. This small peptide fragment includes a glutamic acid-isoleucine-aspartic acid (EID) sequence, optionally with one or more additional amino acids, such as, but not limited to, lysine and serine. Polypeptides, Polynucleotides and Vectors

[0367] The present disclosure also provides polypeptide sequences of the protein carriers,polypeptide cargos or the contiguous polypeptides of the present disclosure.

[0368] In some embodiments provided herein is a contiguous polypeptide comprising (i) aprotein carrier and (ii) a polypeptide cargo. In some embodiments, the contiguous polypeptide is produced by a recombinant method. In some embodiments, the contiguous polypeptide of the present disclosure can be cleaved by an intracellular protease to release the polypeptide cargo from the protein carrier.

[0369] In some embodiments, provided herein is a contiguous polypeptide of the proteinconjugate described herein. In some embodiments, the contiguous polypeptide is a fusion protein. In some embodiments provided herein is a fusion protein SUMO1-12VC1 comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65. In some embodiments, a fusion protein SUMO1-12VC1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62. In some embodiments provided herein is a fusion protein SUMO1-12VC1 comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65 or a sequenceAttorney Docket No: 243735.000431 having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65. In some embodiments, a fusion protein SUMO1-12VC1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62. In some embodiments provided herein is a fusion protein SUMO1-12VC1 comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65. In some embodiments, a fusion protein SUMO1-12VC1 comprises or consists of the amino acid sequence of SEQ ID NO: 62.

[0370] In some embodiments, a fusion protein ubiquitin-12VC1 comprises or consists of theamino acid sequence set forth in SEQ ID NO: 71 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, a fusion protein ubiquitin-12VC1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, a fusion protein ubiquitin-12VC1 comprises or consists of the amino acid sequence of SEQ ID NO: 71.

[0371] In another aspect, provided herein is a nucleic acid molecule encoding the contiguouspolypeptide in the protein conjugate of the present disclosure.

[0372] In some embodiments, the nucleic acid molecule encoding the contiguouspolypeptide, comprises (i) a nucleotide sequence encoding the protein carrier; and (ii) a nucleotide sequence encoding the polypeptide cargo.

[0373] In some embodiments, provided herein is a nucleic acid molecule encoding theprotein carrier of the protein conjugate described herein. In certain embodiments, provided herein is a nucleic acid molecule encoding a SUMO protein. In certain embodiments, provided herein is a nucleic acid molecule encoding a SUMO protein comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in any one of the SEQ ID NOs: 1-8 and 53. In some embodiments, provided herein is a nucleic acid molecule encoding a SUMO protein comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53 or a sequence having at least 80% sequence identity to an amino acid sequence setAttorney Docket No: 243735.000431 forth in any one of SEQ ID NOs: 1-8 and 53. In some embodiments, provided herein is a nucleic acid molecule encoding a SUMO protein comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53.

[0374] In certain embodiments, the nucleotide sequence encoding the SUMO proteincomprises or consists of a sequence set forth in SEQ ID NO: 61 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 61. In some embodiments, the nucleotide sequence encoding the SUMO protein comprises or consists of a sequence set forth in SEQ ID NO: 61 or a sequence having at least 80 % sequence identity to a nucleotide sequence set forth in SEQ ID NO: 61. In some embodiments, the nucleotide sequence encoding the SUMO protein comprises or consists of a sequence set forth in SEQ ID NO: 61.

[0375] In some embodiments, provided herein is a nucleic acid molecule encoding apolypeptide cargo of the protein conjugate described herein.

[0376] In certain embodiments, provided herein is a nucleic acid molecule encoding amonobody. In certain embodiments, provided herein is a nucleic acid molecule encoding the monobody comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 14-52 and 69-70 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 14-52 and 69-70. In some embodiments, provided herein is a nucleic acid molecule encoding the monobody comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 14-52 and 69-70 or a sequence having at least 80%sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 14- 52 and 69-70. In some embodiments, provided herein is a nucleic acid molecule encoding the monobody comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 14-52 and 69-70.

[0377] In some embodiments, provided herein is a nucleic acid molecule encoding thecontiguous polypeptide of the protein conjugate described herein. In some embodiments, the contiguous polypeptide is a fusion protein. In some embodiments provided herein is a nucleotide sequence encoding the fusion protein SUMO1-12VC1 comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, orAttorney Docket No: 243735.000431 99.9% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65. In some embodiments provided herein is a nucleotide sequence encoding the fusion protein SUMO1-12VC1 comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65. In some embodiments provided herein is a nucleotide sequence encoding the fusion protein SUMO1- 12VC1 comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, and 62-65.

[0378] In some embodiments, provided herein is a nucleotide sequence encoding the fusionprotein SUMO1-12VC1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 62 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, a nucleotide sequence encoding the fusion protein SUMO1-12VC1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 62 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, a nucleotide sequence encoding the fusion protein SUMO1-12VC1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 62.

[0379] In some embodiments, provided herein is a nucleotide sequence encoding the fusionprotein ubiquitin-12VC1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 71 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to an amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, a nucleotide sequence encoding the fusion protein ubiquitin-12VC1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 71 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, a nucleotide sequence encoding the fusion protein ubiquitin-12VC1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 71.

[0380] In some embodiments, the nucleotide sequence encoding the fusion protein SUMO1-12VC1 comprises or consists of the sequence set forth in SEQ ID NO: 10 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%,Attorney Docket No: 243735.000431 or 99.9% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the fusion protein SUMO1-12VC1 comprises or consists of the sequence set forth in SEQ ID NO: 10 or a sequence having at least 80% sequence identity to a nucleotide acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the fusion protein SUMO1-12VC1 comprises or consists of the sequence set forth in SEQ ID NO: 10.

[00381] In some embodiments, the nucleic acid molecule described herein is a DNA moleculeor an RNA molecule. For expression of a polynucleotide described herein, a promoter sequence may be included to position the start site for RNA synthesis. The promoter may be a constitutive promoter or inducible promoter. The polynucleotide may also be operably linked to one or more additional regulatory sequences, such as terminators or enhancers. In one embodiment, the isolated polynucleotide is an mRNA.

[0382] In one aspect, the present disclosure provides a recombinant vector comprising theisolated nucleic acid molecules of the present disclosure. In order to assess the expression of a protein carrier, a polypeptide cargo, or a contiguous polypeptide, a vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.

[00383] In addition, polyadenylation signals suitable for use in a desired host cell can also beemployed to effect appropriate translation of the DNA molecule (encoding the contiguous polypeptide of the present disclosure). Polyadenylation signals suitable for use are well known in the art.

[00384] In one aspect, the present disclosure provides a host cell comprising the isolatednucleic acid molecule(s) or the vector described herein. Non-limiting examples of the host cells that can be used according to the present disclosure includes bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the bacterial cell is E. coli. Upon growing the host cells in a suitable growthAttorney Docket No: 243735.000431 medium, the host cells are encouraged to express the protein carrier, the polypeptide cargo, and / or the contiguous polypeptide of the present disclosure.

[0385] Once a protein carrier, a polypeptide cargo (e.g., a monobody or a DARPin), orcontiguous polypeptide has been produced, it can be used for therapeutic, preventative, or diagnostic purposes as described herein. Preparation of Protein Conjugates

[0386] In one aspect, provided herein is a method of producing a protein conjugate of thepresent disclosure.

[0387] In some embodiments, the method of producing a protein conjugate comprises a)generating a contiguous polypeptide comprising (i) a protein carrier and (ii) a polypeptide cargo of the present disclosure; and b) conjugating a cationic polymer to the protein carrier to form a protein conjugate.

[0388] In some embodiments, the contiguous polypeptide is generated by culturing a hostcell comprising the nucleic acid molecule of the present disclosure or the vector of the present disclosure to express a contiguous polypeptide. Culturing a host cell may include a culture media specified by the respective sources of cell lines (e.g., American Type Culture Collection (ATCC)).

[0389] In some embodiments, the contiguous polypeptide is generated recombinantly as afusion protein. In some embodiments, the contiguous polypeptide is generated by attaching the polypeptide cargo to the protein carrier chemically or enzymatically.

[0390] In some embodiments, the method of producing a protein conjugate comprises a)conjugating a cationic polymer to a protein carrier; and b) attaching a polypeptide cargo to the protein carrier chemically or enzymatically to form a contiguous polypeptide, thereby forming a protein conjugate. In some embodiments, the cationic polymer is conjugated to the UBL of the protein carrier. In some embodiments, the cationic polymer is conjugated to the protein carrier, at a site located externally to the UBL domain. In some embodiments, the cationic polymer is conjugated to the terminal polypeptide of the protein carrier. In one embodiment, the cationic polymer is conjugated to the N-terminal polypeptide of the protein carrier.

[0391] In some embodiments, the contiguous polypeptide or the protein conjugate isproduced in substantially purified form, particularly when their administration to a patient isAttorney Docket No: 243735.000431 contemplated. Purification can be carried out according to previously reported procedures, which involve metal affinity chromatography for monobodies containing a poly-histidine tag (see Koide et al., 1998)

[0392] In some embodiments, the method of producing a protein conjugate further comprisesa step of purification after step a) and / or b). In some embodiments, the method of producing a protein conjugate further comprises purifying the contiguous polypeptide from the host cell after step a). In some embodiments, the method of producing a protein conjugate further comprises purifying the protein conjugate after step b). In some embodiments, the contiguous polypeptide from the host cell after step a) is purified using a chromatography method. Various chromatography and non-chromatography methods may be employed for purification after step a) and / or b) of the method of producing a protein conjugate. In certain embodiments, the fusion protein from the host cell after step a) is purified using nickel affinity chromatography. In certain embodiments, the protein conjugate after step b) is purified using size-exclusion chromatography.

[0393] In some embodiments, the cationic polymer is conjugated to the protein carrier via alinker as described herein. In some embodiments, the cationic polymer is conjugated to the protein carrier at cysteine residue of the protein carrier. In some embodiments, the N-terminal polypeptide of the protein carrier comprises the cysteine residues. In some embodiments, the method of producing a protein conjugate does not require peptide chemical synthesis, precise control of reaction conditions, and / or purification of a specific fraction from heterogenous materials. In some embodiments, provided herein is a protein conjugate produced by the method as described herein.

[0394] In some embodiments, the cationic polymer is conjugated to the protein carrier at apH of about neutral pH. In some embodiments, the cationic polymer is conjugated to the protein carrier at a pH of about 6 to about 8. A skilled person may employ suitable reaction conditions depending on the protein carrier, the linker and / or cationic polymer for conjugation of the protein carrier to the cationic polymer. Pharmaceutical Compositions

[0395] Pharmaceutical compositions comprising the protein conjugate of the presentdisclosure, the nucleic acid molecule of the present disclosure, the vector of the presentAttorney Docket No: 243735.000431 disclosure, or the host cell of the present disclosure are within the scope of the present disclosure. Such pharmaceutical compositions can comprise a therapeutically effective amount of the protein conjugate, the nucleic acid molecule, or the vector, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. Acceptable formulation agents preferably are nontoxic to recipients at the dosages and concentrations employed.

[0396] The pharmaceutical composition can contain formulation agent(s) for modifying,maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation agents include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, methionine or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, histidine, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetra acetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides— preferably sodium or potassium chloride—or mannitol sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants (see, e.g., Remington’s Pharmaceutical Sciences (18thEd., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference for any purpose).Attorney Docket No: 243735.000431

[0397] The optimal pharmaceutical composition can be determined by a skilled artisandepending upon, for example, the intended route of administration, delivery format, and desired dosage (see, e.g., Remington’s Pharmaceutical Sciences, supra). Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the protein conjugate, polynucleotide, or vector of the present disclosure.

[0398] The primary vehicle or carrier in a pharmaceutical composition can be either aqueousor non-aqueous in nature. Suitable pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Histidine or Tris buffer of about pH 6.0 - about 8.5, which can further include sorbitol or a suitable substitute. In one embodiment, protein conjugate compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington’s Pharmaceutical Sciences, supra) in the form of an aqueous solution.

[0399] The pharmaceutical compositions can be selected for parenteral delivery.Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 6 to about 8.

[0400] When parenteral administration is contemplated, the therapeutic compositions for usein this invention can be in the form of a pyrogen-free, parenterally acceptable, aqueous solutionAttorney Docket No: 243735.000431 comprising the desired protein conjugate, polynucleotide, or vector as disclosed herein, in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a protein conjugate, polynucleotide, or vector as disclosed herein, is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

[0401] In one embodiment, a pharmaceutical composition can be formulated for inhalation.For example, the pharmaceutical composition can be formulated as a dry powder for inhalation. Inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions can be nebulized. Pulmonary administration is further described in International Publication No. WO1994020069, which describes the pulmonary delivery of chemically modified proteins.

[0402] It is also contemplated that certain formulations can be administered orally. In oneembodiment, formulations that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre- systemic degradation is minimized. Additional agents can be included to facilitate absorption. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

[0403] Another pharmaceutical composition can involve an effective quantity of a proteinconjugate in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.Attorney Docket No: 243735.000431

[0404] Additional pharmaceutical compositions will be evident to those skilled in the art,including formulations involving protein conjugates, polynucleotides, or vectors, in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art (see, e.g., International Publication No. WO1993015722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions, and Wischke & Schwendeman, 2008, Int. J. Pharm.364: 298-327, and Freiberg & Zhu, 2004, Int. J. Pharm.282: 1-18, which discuss microsphere / microparticle preparation and use). As described herein, a hydrogel is an example of a sustained- or controlled-delivery formulation.

[0405] Additional examples of sustained-release preparations include semipermeablepolymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919 and European Patent No.0058481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et ah, 1983, Biopolymers 22: 547-56), poly(2-hydroxyethyl-methacrylate) (Langer et ah, 1981, J. Biomed. Mater. Res.15: 167-277 and Langer, 1982, Chem. Tech.12: 98-105), ethylene vinyl acetate (Langer et al, supra) or poly-D(−)-3-hydroxybutyric acid (European Patent No. 0133988). Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Epstein et ah, 1985, Proc. Natl. Acad. Sci. U.S.A.82: 3688-92; and European Patent Nos.0036676, 0088046, and 0143949.

[0406] The pharmaceutical composition to be used for in vivo administration typically shouldbe sterile. This can be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The parenteral composition can be diluted into parenteral acceptable diluents (e.g., saline and 5% Dextrose).

[0407] Once the pharmaceutical composition has been formulated, it can be stored in sterilevials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.Attorney Docket No: 243735.000431 Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

[0408] In one embodiment, the pharmaceutical composition comprises a protein conjugateformulated as a powder for injection after reconstitution to a solution for injection.

[0409] Selecting an administration regimen for a therapeutic depends on several factors,including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of conjugate delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, Fc fusion therapeutic proteins, cytokines, and small molecules are available (see, e.g., Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.), 1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al., 2003, New Engl. J. Med.348:601-608; Milgrom, et al., 1999, New Engl. J. Med.341:1966-1973; Slamon, et al., 2001, New Engl. J. Med.344:783-792; Beniaminovitz, et al., 2000, New Engl. J. Med.342:613-619; Ghosh, et al., 2003, New Engl. J. Med.348:24-32; Lipsky, et al., 2000, New Engl. J. Med.343:1594-1602).

[0410] Determination of the appropriate dose is made by the clinician, e.g., using parametersor factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., increased serum phosphate or decreased phosphate excretion.

[0411] Actual dosage levels of the active ingredients in the pharmaceutical compositions ofthe present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration,Attorney Docket No: 243735.000431 the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0412] Compositions comprising the protein conjugates of the disclosure can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, one week, 1-7 times per week, or one month. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intratumorally, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose may be at least 0.05 μg / kg body weight, at least 0.2 μg / kg, at least 0.5 μg / kg, at least 1 μg / kg, at least 10 μg / kg, at least 100 μg / kg, at least 0.2 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, or at least 50 mg / kg (see, e.g., Yang, et al., 2003, New Engl. J. Med.349:427-434; Herold, et al., 2002, New Engl. J. Med.346:1692-1698; Liu, et al., 1999, J. Neurol. Neurosurg. Psych.67:451-456; Portielji, et al., 2003, Cancer. Immunol. Immunother.52: 133-144). The dose may be at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100 μg. The doses administered to a subject may number at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.

[0413] For therapeutic protein conjugates of the disclosure, the dosage administered to apatient may be 0.0001 mg / kg to 100 mg / kg of the patient’s body weight. The dosage may be between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg and 0.25 mg / kg, 0.0001 and 0.15 mg / kg, 0.0001 and 0.10 mg / kg, 0.001 and 0.5 mg / kg, 0.01 and 0.25 mg / kg, or 0.01 and 0.10 mg / kg of the patient’s body weight.

[0414] The dosage of the therapeutic protein conjugate of the disclosure may be calculatedusing the patient’s weight in kilograms (kg) multiplied by the dose to be administered in mg / kg. The dosage of the protein conjugates of the disclosure may be 150 μg / kg or less, 125 μg / kg or less, 100 μg / kg or less, 95 μg / kg or less, 90 μg / kg or less, 85 μg / kg or less, 80 μg / kg or less, 75 μg / kg or less, 70 μg / kg or less, 65 μg / kg or less, 60 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less, 45 μg / kg or less, 40 μg / kg or less, 35 μg / kg or less, 30 μg / kg or less, 25 μg / kg or less, 20Attorney Docket No: 243735.000431 μg / kg or less, 15 μg / kg or less, 10 μg / kg or less, 5 μg / kg or less, 2.5 μg / kg or less, 2 μg / kg or less, 1.5 μg / kg or less, 1 μg / kg or less, 0.5 μg / kg or less, or 0.1 μg / kg or less of a patient’s body weight.

[0415] Unit dose of the therapeutic protein conjugates of the disclosure may be 0.1 mg to 20mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 mg to 2.5 mg, 0.25 mg to 20 mg, 0.25 mg to 15 mg, 0.25 mg to 12 mg, 0.25 mg to 10 mg, 0.25 mg to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0416] The dosage of the therapeutic protein conjugates of the disclosure may achieve aserum titer of at least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 μg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml / ml, or at least 400 μg / ml / ml in a subject. Alternatively, the dosage of the protein conjugate of the disclosure may achieve a serum titer of at least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least, 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 μg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml, or at least 400 μg / ml in the subject.

[0417] Doses of therapeutic protein conjugates of the disclosure may be repeated and theadministrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. Doses of therapeutic protein conjugates of the disclosure may be repeated and administered two times a day (BID), three times a day (TID), or four times a day (QID).

[0418] An effective amount for a particular patient may vary depending on factors such asthe condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects (see, e.g., Maynard, et al., 1996, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent, 2001, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK).Attorney Docket No: 243735.000431

[0419] The route of administration may be by, e.g., topical or cutaneous application,injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, intratumoral, or by sustained release systems or an implant (see, e.g., Sidman et al., 1983, Biopolymers 22:547-556; Langer, et al., 1981, J. Biomed. Mater. Res.15: 167-277; Langer, 1982, Chem. Tech.12:98-105; Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030-4034; U.S. Pat. Nos.6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference their entirety. In one embodiment, an engineered protein conjugate, combination therapy, or a composition of the disclosure is administered using Alkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).

[0420] The frequency of dosing may depend upon the pharmacokinetic parameters of theprotein conjugate in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.

[0421] The route of administration of the pharmaceutical composition is in accord withknown methods, e.g., orally; through injection by subcutaneous, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, intratumoral, or intralesional routes; by sustained release systems (which may also be injected); or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.Attorney Docket No: 243735.000431

[0422] Alternatively or additionally, the composition can be administered locally viaimplantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration. In order to deliver drug, e.g., a protein conjugate as disclosed herein, at a predetermined rate such that the drug concentration can be maintained at a desired therapeutically effective level over an extended period, a variety of different approaches can be employed. In one example, a hydrogel comprising a polymer such as a gelatin (e.g., bovine gelatin, human gelatin, or gelatin from another source) or a naturally- occurring or a synthetically generated polymer can be employed. Any percentage of polymer (e.g., gelatin) can be employed in a hydrogel, such as 5, 10, 15, or 20%. The selection of an appropriate concentration can depend on a variety of factors, such as the therapeutic profile desired and the pharmacokinetic profile of the therapeutic molecule.

[0423] Examples of polymers that can be incorporated into a hydrogel include polyethyleneglycol (“PEG”), polyethylene oxide, polyethylene oxide-co-polypropylene oxide, co- polyethylene oxide block or random copolymers, polyvinyl alcohol, poly(vinyl pyrrolidinone), poly(amino acids), dextran, heparin, polysaccharides, polyethers and the like.

[0424] Another factor that can be considered when generating a hydrogel formulation is thedegree of crosslinking in the hydrogel and the crosslinking agent. In one embodiment, cross- linking can be achieved via a methacrylation reaction involving methacrylic anhydride. In some situations, a high degree of cross-linking may be desirable while in other situations a lower degree of crosslinking is preferred. In some cases, a higher degree of crosslinking provides a longer sustained release. A higher degree of crosslinking may provide a firmer hydrogel and a longer period over which drug is delivered. Any ratio of polymer to crosslinking agent (e.g., methacrylic anhydride) can be employed to generate a hydrogel with desired properties. For example, the ratio of polymer to crosslinker can be, e.g., 8:1, 16:1, 24:1, or 32:1. For example, when the hydrogel polymer is gelatin and the crosslinker is methacrylate, ratios of 8:1, 16:1, 24:1, or 32:1 methyacrylic anhydride:gelatin can be employed.

[0425] One skilled in the art recognizes that different methods of delivery may be utilized toadminister a polynucleotide (e.g., an mRNA or any nucleotide sequence of the present disclosure) or vector into a cell (host cell). Examples include: (1) methods utilizing physicalAttorney Docket No: 243735.000431 means, such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure); and (2) methods wherein the vector is complexed to another entity, such as a liposome, aggregated protein or transporter molecule.

[0426] Furthermore, the actual dose and schedule can vary depending on whether thecompositions are administered in combination with other compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts can vary in in vitro applications depending on the particular cell line utilized Kits

[0427] The present disclosure further provides a kit which may contain any of variouscompositions of the present disclosure, including protein conjugates, polypeptides, polynucleotides, or vectors of the disclosure.

[0428] In some embodiments, a kit may comprise (i) a protein conjugate described herein, apolypeptide described herein, a polynucleotide described herein, and / or a vector described herein, and (ii) packaging for the same.

[0429] In some embodiments, a kit can comprise: (a) a container that contains apharmaceutical composition described herein, for example, a pharmaceutical composition in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; and / or (c) optionally, instructions for (i) use of the solution or (ii) reconstitution and / or use of the lyophilized formulation.

[0430] In some embodiments, a kit may further comprise, one or more of (i) a diluent, (ii) abuffer, (iii) a filter, (iv) a syringe, and / or (v) a needle.

[0431] In some embodiments, the components of the kit may be provided in one or moreliquid solutions. A liquid solution described herein may be an aqueous solution such as a sterile aqueous solution. The components of the kit may also be provided as solids, which may be converted into liquids such as by addition of suitable solvents, which may be provided in another distinct container.

[0432] In some a pharmaceutical composition described herein may be lyophilized.

[0433] In some embodiments, kits may comprise a lyophilized formulation described hereinin a suitable container and instructions for its reconstitution and / or use. Non-limiting examples of suitable containers include, e.g., syringes (such as dual chamber syringes), vials (such as dualAttorney Docket No: 243735.000431 chamber vials), bottles, and test tubes. In various embodiments, a container may be a multi-use container. The container may be formed from a variety of materials such as plastic or glass. The kit and / or container may contain instructions upon or accompanying the container which can denote directions for reconstitution of, e.g., a lyophilized formulation and / or use of the kit. In some embodiments, a label may denote that the lyophilized formulation is to be reconstituted to an appropriate concentration. The label may denote that the formulation is useful or intended for any route of administration disclosed herein.

[0434] The container containing the formulation may be a multi-use vial, which may allowfor repeat administrations (e.g., from 2-6 administrations) of a reconstituted formulation. The kit may further comprise a second container comprising a suitable diluent (e.g., sodium bicarbonate solution).

[0435] Upon mixing of the diluent and a lyophilized formulation, a final concentration in thereconstituted formulation can reached. The kit may further include other materials desirable from a commercial and / or user perspective, including, e.g., other filters, needles, syringes, buffers, diluents, and / or package inserts which may comprise, e.g., instructions for use.

[0436] Kits may contain a single container that contains the formulation of thepharmaceutical composition with or without other components (e.g., other compounds or pharmaceutical compositions of such other compounds) or may have a separate container for each component.

[0437] Kits may include a formulation of the disclosure packaged for use in combinationwith the co-administration of a second compound (such as adjuvants (e.g., GM-CSF, a natural product, a hormone, an antagonist, an anti-angiogenesis agent, an inhibitor, an apoptosis- inducing agent, a chelator, or a chemotherapeutic agent) or a pharmaceutical composition thereof. The components of the kit may pre-mixed and / or pre-complexed or each component of the kit may be in a separate distinct container prior to administration to a patient.

[0438] In some embodiments, the container of a therapeutic kit may be a vial, flask, test tube,bottle, syringe, or any other means of enclosing a solid or liquid. When there is more than one component, the kit may contain a second vial or other container, which may allow for separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid. In some embodiments, a kit may contain an apparatus (e.g., syringes, one or more needles, pipettes,Attorney Docket No: 243735.000431 eye droppers, etc.) which may permit administration of protein conjugates of the disclosure which are components of the kit.

[0439] In one embodiment, provided herein are kits for producing a single-doseadministration unit. The kits can each contain both a first container having a dried protein conjugate and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and dual chamber syringes). Methods of Use

[0440] In one aspect, provided herein is a method of delivering a cargo into the cytoplasm ofa cell.

[0441] The existing methods for cytosolic delivery have some limitations such as lowefficacy, only effective with extremely potent cargos (e.g., toxins and nucleases), toxicity, and poor biophysical properties (e.g., aggregation and precipitation). The delivery methods that include modifying cargos often requires re-engineering of the cargos and may be detrimental to the functions of the cargo. Non-availability of a built-in assay for cytosolic delivery is also a limitation which requires the preparation of a molecule just for assessing delivery. Current methods of delivering cargo also include nanoparticle-based methods. However, nanoparticles have formulation challenges, particularly in in vivo applications. The present disclosure provides protein conjugate-based method that delivers a cargo into the cytoplasm.

[0442] In certain embodiments, the method of delivering a cargo into the cytoplasm of a cellcomprises a) producing a protein conjugate comprising the cargo according to the method of the present disclosure; and b) contacting the cell with the produced protein conjugate.

[0443] In some embodiments, provided herein is a method of targeting an intracellular targetin a cell. In certain embodiments, the method of targeting an intracellular target in a cell comprises a) producing a protein conjugate comprising a cargo that interacts with the intracellular target according to the method of the present disclosure; and b) contacting the cell with the produced protein conjugate.

[0444] In some embodiments of the method of delivering a cargo and / or targeting anintracellular target in a cell, the cell is a mammalian cell. In some embodiments, the mammalian cell is an adherent cell or non-adherent cell. Examples of mammalian cells include, but are notAttorney Docket No: 243735.000431 limited to, epithelial cells, endothelial cells, immune cells, and cancer cells. Non-limiting examples of non-adherent cells include hematopoietic cells.

[0445] In some embodiments, any of the methods of the present disclosure occurs ex vivo orin vivo.

[0446] In some embodiments, provided herein is a method of preventing, treating, ordiagnosing a disease in a subject in need thereof. In certain embodiments, the method of preventing, treating, or diagnosing a disease in a subject in need thereof comprises administering to the subject a protein conjugate or a pharmaceutical composition of the present disclosure, wherein the cargo interacts with a target associated with disease.

[0447] In some embodiments, the target is an intracellular target. In some embodiments, theintracellular target is selected from RAS kinase (e.g., KRAS, HRAS, or NRAS), Aurora A kinase, ABL kinase, Src-family kinases (e.g., Fgr, Hck, Lck, Lyn, Src, Yes), SHP2, STAT3, PRDM14, WDR5, p53, or MLKL. In some embodiments, the RAS kinase is KRAS, NRAS, or HRAS. In some embodiments, the intracellular target is SH2 domain of ABL1. In some embodiments, the intracellular target is SH2 domain of SHP2 or SHP2 phosphatase.

[0448] In addition, the invention provides for use of the protein conjugates, polynucleotides,or vectors, or pharmaceutical compositions thereof, of this disclosure in the manufacture of a medicament for use in treatment or prevention of diseases, disorders, or conditions associated with the intracellular targets.

[0449] In some embodiments, the disease, disorder, or condition is a cancer.

[0450] In some embodiments, the disease, disorder, or condition is an autoimmune disease.

[0451] In some embodiments, a disease, disorder, or condition can be treated byadministering a protein conjugate, polynucleotide, or vector, or a pharmaceutical composition thereof, as described herein, to a patient in need thereof in the amount of a therapeutically effective dose. The administration can be performed as described herein, such as by intravenous injection, intrarectal injection, intraperitoneal injection, intramuscular injection, intratumoral injection, subcutaneous injection, orally in the form of a tablet or liquid formation, or delivery through endoscopy. In most situations, a desired dosage can be determined by a clinician, as described herein, and can represent a therapeutically effective dose of a protein conjugate, polynucleotide, or vector. It will be apparent to those of skill in the art that a therapeutically effective dose will depend, inter alia, upon the administration schedule, the unit dose of agentAttorney Docket No: 243735.000431 administered, whether the composition is administered in combination with other therapeutic agents, and the health of the recipient. The term “therapeutically effective dose,” as used herein, means that amount of protein conjugate, polynucleotide, or vector, that elicits the biological or medicinal response in a tissue system, animal, or human being sought by a researcher, medical doctor, or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.

[0452] In some embodiments, the protein conjugate, polynucleotide, or vector, or apharmaceutical composition thereof, is administered in combination with one or more additional agents suitable for the disease, disorder, or condition being treated. Non-limiting Embodiments of the present invention1. A protein conjugate comprising(i) a protein carrier conjugated to a cationic polymer; and (ii) a polypeptide cargo, wherein the protein carrier is linked to the polypeptide cargo via a peptide bond to form a contiguous polypeptide.2. The protein conjugate of embodiment 1, wherein the contiguous polypeptide isrecombinantly produced.3. The protein conjugate of embodiment 1, wherein the contiguous polypeptide is chemically orenzymatically produced.4. The protein conjugate of any one of embodiments 1-3, wherein the protein carrier is linked tothe polypeptide cargo via a proteolytic cleavable linker.5. The protein conjugate of embodiment 4, wherein the proteolytic cleavable linker isrecognized by an intracellular protease.6. The protein conjugate of any one of embodiments 1-5, wherein the protein carrier comprisesan N-terminal polypeptide or a C-terminal polypeptide.7. The protein conjugate of embodiment 6, wherein the protein carrier comprises an N-terminalpolypeptide.8. The protein conjugate of any one of embodiments 1-7, wherein the protein carrier and / or thepolypeptide cargo do not contain native cysteine residue(s) or have been engineered to remove native cysteine residue(s).Attorney Docket No: 243735.000431The protein conjugate of any one of embodiments 1-8, wherein the protein carrier is modifiedto introduce one or more cysteine residues for site-specific conjugation with the cationic polymer.The protein conjugate of embodiment 9, wherein the protein carrier is modified to introduceone, two, three, or four cysteine residues.The protein conjugate of any one of embodiments 9-10, wherein the one or more cysteineresidues are introduced to the protein carrier outside the interface between the protein carrier and the intracellular protease.The protein conjugate of any one of embodiments 9-11, wherein the one or more cysteineresidues are introduced into the N-terminal polypeptide of the protein carrier.The protein conjugate of embodiment 13, wherein the one or more cysteine residues areintroduced into the N-terminal polypeptide as a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide.The protein conjugate of any one of embodiments 9-13, wherein the protein carrier isconjugated to one or more cationic polymers at the one or more cysteine residues.The protein conjugate of any one of embodiments 1-14, wherein the protein carrier comprisesa ubiquitin-like protein (UBL), a functional fragment, or derivative thereof.The protein conjugate of embodiment 15, wherein the one or more cysteine residues areintroduced to the UBL.The protein conjugate of any one of embodiments 7-15, wherein the N-terminal polypeptideof the protein carrier is located at amino terminus of the UBL.The protein conjugate of any one of embodiments 5-17, wherein the intracellular protease isa UBL-specific protease.A protein conjugate comprising(i) a protein carrier conjugated to a cationic polymer; and (ii) a cargo, wherein the protein carrier is operably linked to the cargo, and wherein the protein carrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof.The protein conjugate of embodiment 19, wherein the cargo is a polypeptide cargo.The protein conjugate of embodiment 20, wherein the protein carrier is linked to the cargovia a peptide bond to form a contiguous polypeptide.Attorney Docket No: 243735.000431The protein conjugate of any one of embodiments 19-21, wherein the cargo is linked to theUBL via a proteolytic cleavable linker.The protein conjugate of embodiment 22, wherein the UBL is cleaved from the cargo by aUBL-specific protease.The protein conjugate of any one of embodiments 19-23, wherein the protein carrier furthercomprises an N-terminal polypeptide or a C- terminal polypeptide.The protein conjugate of embodiment 24, wherein the protein carrier comprises an N-terminal polypeptide.The protein conjugate of embodiment 25, wherein the N-terminal polypeptide is located atthe amino terminus of the UBL.The protein conjugate of any one of embodiments 19-26, wherein the UBL has beenengineered to remove native cysteine residue(s).The protein conjugate of embodiment 19-27, wherein the protein carrier is modified tointroduce one or more cysteine residues.The protein conjugate of any one of embodiments 19-28, wherein the UBL is modified tointroduce one or more cysteine residues outside the interface between the UBL and its cognate UBL specific protease.The protein conjugate of any one of embodiments 19-29, wherein one or more cysteineresidues are introduced into the N-terminal polypeptide of the protein carrier.The protein conjugate of embodiment 30, wherein the one or more cysteine residues areintroduced into the N-terminal polypeptide as a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide.The protein conjugate of any one of embodiments 19-31, wherein the protein carrier isconjugated to one or more cationic polymers at the one or more cysteine residues.The protein conjugate of any one of embodiments 15-32, wherein the UBL is selected fromubiquitin, a small ubiquitin-like modifier (SUMO) protein, neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8), autophagy-related protein 8 (ATG8), autophagy-related 12 (ATG12), ), human leukocyte antigen (HLA)-F adjacent transcript 10 or Ubiquitin D (FAT10), interferon (IFN)-stimulated gene 15 (ISG15), ubiquitin related modifier 1 (URM1), or ubiquitin fold modifier 1 (UFM1).Attorney Docket No: 243735.000431The protein conjugate of any one of embodiments 15-33, wherein the UBL comprises orconsists of an amino acid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72.The protein conjugate of embodiment 33 or embodiment 34, wherein the SUMO protein isSUMO1, SUMO2, SUMO3, or SUMO4.The protein conjugate of embodiment 35, wherein the SUMO protein comprises or consistsof an amino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53-55 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53-55.The protein conjugate of any one of embodiments 33-36, wherein the SUMO protein isSUMO1.The protein conjugate of embodiment 37, wherein SUMO1 comprises or consists of anamino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53.The protein conjugate of embodiment 37 or embodiment 38, wherein the cysteine residue atposition 52 of SUMO1 has been mutated to a non-cysteine residue.The protein conjugate of embodiment 39, wherein the cysteine residue at position 52 ofSUMO1 has been mutated to an alanine.The protein conjugate of any one of embodiments 37-40, wherein SUMO1 is modified tointroduce one or more cysteine residues by mutating the residue at the amino acid position 33, amino acid position 46, amino acid position 53, amino acid position 85, and / or amino acid position 50 to cysteine.The protein conjugate of embodiment 41, wherein SUMO1 comprises or consists of anamino acid sequence set forth in SEQ ID NO: 8 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 8.The protein conjugate of any one of embodiments 33-42, wherein the C-terminal or proximalto the C-terminal sequence of the SUMO protein comprises Gly-Gly.The protein conjugate of any one of embodiments 18 and 23-43, wherein the UBL-specificprotease is a ubiquitin-specific protease (USP) or SUMO-specific protease (SENP).Attorney Docket No: 243735.000431The protein conjugate of embodiment 44, wherein the SENP is SENP1, SENP2, SENP3,SENP5, SENP6, and / or SENP7.The protein conjugate of any one of embodiments 1-45, wherein the cationic polymer is apolyamine.The protein conjugate of any one of embodiments 1-46, wherein the cationic polymer isselected from polyethylenimine (PEI), poly(L-lysine) (PLL), poly(D-lysine) (PDL), polyamidoamine (PAA), Poly(amino-co-ester)s (PAEs), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), poly(propylene imine) (PPI), poly-L-arginine (PLA), poly- guanidine, poly-D-arginine, poly(D,L-lysine hydrobromide), poly-L-ornithine, PEI-Gua, PEI-Arg, PEI-His, PEI-Trp, or derivatives and combinations thereof.The protein conjugate of any one of embodiments 1-47, wherein the cationic polymer is PEI.The protein conjugate of embodiment 48, wherein the PEI is a linear PEI or branched PEI.The protein conjugate of embodiment 49, wherein the branched PEI is a short-chain PEI.The protein conjugate of any one of embodiments 47-50, wherein the PEI has a molecularweight of less than 2000 Da.The protein conjugate of embodiment 51, wherein the PEI has a molecular weight rangingfrom about 600 Da to about 1800Da.The protein conjugate of embodiment 51 or embodiment 52, wherein the PEI has a molecularweight of about 600 Da (PEI600) or 1200 Da (PEI1200).The protein conjugate of embodiment 53, wherein the PEI has a molecular weight of about600 Da (PEI600).The protein conjugate of embodiment 53, wherein the PEI is branched PEI600 (bPEI600) orbranched PEI1200 (bPEI1200).The protein conjugate of embodiment 55, wherein the PEI is branched PEI600 (bPEI600).The protein conjugate of any one of embodiments 1-56, wherein the protein carrier isconjugated to the cationic polymer via a linker.The protein conjugate of embodiment 57, wherein the linker is a heterobifunctionalcrosslinker.The protein conjugate of embodiment 58, wherein the linker comprises an electrophileselected from haloacetophenone, epoxide, haloacetamide, maleimide, isothiocyanate, vinylAttorney Docket No: 243735.000431 sulfone, nitrile, iminonitrile, hydrazone, butynoate, acrylester, acrylonitrile, acrylamide, benzisothiazolone, and N-hydroxy(methyl)phthalimide.The protein conjugate of embodiment 58 or embodiment 59, wherein the linker comprises aN-hydroxysuccinimide (NHS) ester and a maleimide group.The protein conjugate of any one of embodiments 58-60, wherein the linker is selected fromN-alpha-maleimidoacetoxyl succinimide ester (AMAS), N-beta-maleimidopropyl- oxysuccinimide ester (BMPS), N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-epsilon-malemidocaproyl- oxysuccinimide ester (EMCS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH), succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), N-(11- maleimidoundecanoyloxy)succinimide (KMUS), or derivatives and combinations thereof.The protein conjugate of any one of embodiments 58-61, wherein the linker is N-alpha-maleimidoacetoxyl succinimide ester (AMAS).The protein conjugate of any one of embodiments 1-62, wherein the protein conjugate iscapable of penetrating into the cytoplasm of a mammalian cell.The protein conjugate of any one of embodiments 1-63, wherein the cargo interacts with anintracellular target.The protein conjugate of embodiment 64, wherein the intracellular target is selected from ratsarcoma virus (RAS), Src homology 2 domain-containing phosphatase 2 (SHP2), Aurora A kinase, Src-family kinases, signal transducer and activator of transcription 3 (STAT3), PR domain containing 14 (PRDM14), WD repeat domain 5 (WDR5), mixed lineage kinase domain-like protein (MLKL), p53, and ABL kinase.The protein conjugate of embodiment 65, wherein the RAS is Kirsten rat sarcoma viraloncogene homolog (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS), or Harvey rat sarcoma viral oncogene homolog (HRAS).The protein conjugate of any one of embodiments 1-18 and 20-66, wherein the polypeptidecargo is selected from a monobody, a designed ankyrin repeat protein (DARPin), an Anticalin, an affilin, an affibody, a peptide, an antibody or antigen-binding fragment, an enzyme, or derivatives and combinations thereof.Attorney Docket No: 243735.000431The protein conjugate of embodiment 67, wherein the polypeptide cargo is selected from amonobody, a designed ankyrin repeat protein (DARPin), or derivatives thereof.The protein conjugate of embodiment 68, wherein the polypeptide cargo is tandemmonobodies comprising two or more monobody units.The protein conjugate of any one of embodiments 67-69, wherein the monobody or DARPintargets KRAS, HRAS, NRAS, Aurora A kinase, ABL kinase, a Src-family kinase, SHP2, STAT3, PRDM14, WDR5, MLKL, or p53.The protein conjugate of embodiment 70, wherein the monobody is selected from 12VC1,NS1, 12D3, 12D4, 12D5, AS25, AS27, HA4, 7C12, Nsa1, NSa5, CS3, S14, S4, Mb1, Mb2,Mb3, Mb4, Mb5, Mb6, Mb19, Mb20, Mb24, Mb27, Mb32, Mb33, MS3-6, Mb13, R15, Fgr_1, Fgr_2, Hck_1, Hck_2, Lck_1, Lck_3, Lyn_2, Lyn_4, Src_2, Yes_1, Yes_3, MLKL_26, MLKL_27, MLKL_32, MLKL_33, and MLKL_37.The protein conjugate of any one of embodiments 67-71, wherein the monobody comprisesor consists of an amino acid sequence set forth in any one of SEQ ID NOs:14-52 and 69-70 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:14-52 and 69-70.The protein conjugate of embodiment 70, wherein the DARPin is K27, C10, or C10-H82R.The protein conjugate of embodiment 67, wherein the antibody or antigen-binding fragmentthereof is selected from a single chain antibody, a single domain antibody, a variable heavy homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, a monoclonal antibody, a bispecific antibody or antigen-binding fragment thereof, a bi-epitopic antibody or antigen-binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a VH domain, a monovalent antibody or a bivalent antibody.The protein conjugate of embodiment 67, wherein the peptide is a stapled peptide or bicyclicpeptide.The protein conjugate of any one of embodiments 1-75, further comprising one or moreadditional moieties.The protein conjugate of embodiment 76, wherein the additional moiety is a His6-tag (SEQID NO: 12).Attorney Docket No: 243735.000431The protein conjugate of embodiment 77, wherein the N-terminal polypeptide of the proteincarrier comprises the His6-tag (SEQ ID NO: 12).The protein conjugate of embodiment 76, wherein the additional moiety is a V5 tag, whereinthe V5 tag is at a C-terminus of the polypeptide cargo.The protein conjugate of embodiment 76, wherein the additional moiety is a cell-targetingmoiety, wherein the N-terminal polypeptide of the protein carrier comprises the cell-targeting moiety.The protein conjugate of embodiment 80, wherein the cell-targeting moiety is a polypeptidethat binds specifically to a cell-surface protein.The protein conjugate of any one of embodiment 1-81, further comprising an albumin-binding domain.The protein conjugate of embodiment 82, wherein the albumin-binding domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO:66 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in SEQ ID NO:66.The protein conjugate of any one of embodiments 1-18 and 21-83, wherein the contiguouspolypeptide comprises or consists of the sequence set forth in any one of SEQ ID Nos: 9, 11, 62-65, and 71 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, 62-65, and 71.The protein conjugate of embodiment 84, wherein the contiguous polypeptide comprises orconsists of the sequence set forth in SEQ ID NO: 62 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62.A nucleic acid molecule encoding the contiguous polypeptide in the protein conjugate of anyone of embodiments 1-18 and 21-85.The nucleic acid molecule of embodiment 86, comprising (i) a nucleotide sequence encodingthe protein carrier; and (ii) a nucleotide sequence encoding the polypeptide cargo.The nucleic acid molecule of embodiment 86 or embodiment 87, comprising or consistingthe nucleotide sequence set forth in SEQ ID NO: 10 or a sequence having at least 80% sequence identity to a nucleotide acid nucleotide sequence set forth in SEQ ID NO: 10.A nucleic acid molecule comprising a nucleotide sequence encoding the protein carrier of theprotein conjugate of any one of embodiments 1-85.Attorney Docket No: 243735.000431The nucleic acid molecule of embodiment 89, wherein the nucleotide sequence encoding theprotein carrier comprises or consists of the sequence set forth in SEQ ID NO: 61 or a sequence having at least 80% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 61.A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide cargo ofthe protein conjugate of any one of embodiments 1-18 and 20-85.The nucleic acid molecule of any one of embodiments 86-91, wherein the nucleic acidmolecule is a DNA molecule.The nucleic acid molecule of any one of embodiments 86-91, wherein the nucleic acidmolecule is an RNA molecule.A polypeptide encoded by the nucleic acid molecule of any one of embodiments 86-93.A recombinant vector comprising the nucleic acid molecule of any one of embodiments 86-93.An isolated host cell comprising the nucleic acid molecule of any one of embodiments 86-93or the vector of embodiment 95.The isolated host cell of embodiment 96, wherein the isolated host cell is a bacterial cell, ayeast cell, an insect cell, or a mammalian cell.The isolated host cell of embodiment 97, wherein the bacterial cell is Escherichia coli(E.coli).A method of producing a protein conjugate, comprisinga) generating a contiguous polypeptide comprising (i) a protein carrier and (ii) a polypeptide cargo; and b) conjugating a cationic polymer to the protein carrier to form a protein conjugate.. The method of embodiment 99, wherein the contiguous polypeptide is generatedrecombinantly.. The method of embodiment 100, wherein the contiguous polypeptide is generated byculturing a host cell comprising the nucleic acid molecule of any one of embodiments 86-93 or the vector of embodiment 95 to express the contiguous polypeptide.. The method of embodiment 101, wherein the contiguous polypeptide is generated byattaching the polypeptide cargo to the protein carrier chemically or enzymatically.Attorney Docket No: 243735.000431. The method of any one of embodiments 99-102, further comprising purifying thecontiguous polypeptide from the host cell after step a).. A method of producing a protein conjugate, comprisinga) conjugating a cationic polymer to a protein carrier; and b) attaching a polypeptide cargo to the protein carrier chemically or enzymatically to form a contiguous polypeptide, thereby forming a protein conjugate.. The method of any one of embodiments 99-104, further comprising purifying the proteinconjugate after step b).. The method of any one of embodiments 99-105, wherein the protein carrier is linked tothe polypeptide cargo via a proteolytic cleavable linker.. The method of embodiment 106, wherein the proteolytic cleavable linker is recognizedby an intracellular protease.. The method of any one of embodiments 99-107, wherein the protein carrier comprises aterminal polypeptide.. The method of embodiment 108, wherein the protein carrier comprises an N-terminalpolypeptide or a C-terminal polypeptide.. The method of any one of embodiments 99-109, wherein the protein carrier and / or thepolypeptide cargo do not contain native cysteine residue(s) or have been engineered to remove native cysteine residue(s).. The method of any one of embodiments 99-110, wherein the protein carrier is modifiedto introduce one or more cysteine residues for site-specific conjugation with the cationic polymer.. The method of embodiment 111, wherein the protein carrier is modified to introduce one,two, three or four cysteine residues.. The method of embodiment 111 or embodiment 112, wherein the one or more cysteineresidues are introduced to the protein carrier outside the interface between the protein carrier and the intracellular protease.. The method of any one of embodiments 111-113, wherein the one or more cysteineresidues are introduced into the N-terminal polypeptide of the protein carrier.Attorney Docket No: 243735.000431. The method of embodiment 114, wherein the one or more cysteine residues areintroduced into the N-terminal polypeptide as a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide.. The method of any one of embodiments 111-115, wherein the protein carrier isconjugated to one or more cationic polymers at the one or more cysteine residues.. The method of any one of embodiments 99-116, wherein the protein carrier comprises aubiquitin-like protein (UBL), a functional fragment, or derivative thereof.. The method of any one of embodiments 111-113 and 117, wherein the one or morecysteine residues are introduced to the UBL.. The method of embodiment 117 or embodiment 118, wherein the ubiquitin-like protein(UBL) is selected from ubiquitin, a small ubiquitin-like modifier (SUMO) protein, neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8), autophagy- related protein 8 (ATG8), autophagy-related 12 (ATG12), human leukocyte antigen (HLA)-F adjacent transcript 10 or Ubiquitin D (FAT10), interferon (IFN)-stimulated gene 15 (ISG15), ubiquitin related modifier 1 (URM1), or ubiquitin fold modifier 1 (UFM1).. The method of embodiment 119, wherein the UBL comprises or consists of an aminoacid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72.. The method of embodiment 119, wherein the SUMO protein is SUMO1, SUMO2,SUMO3, or SUMO4.. The method of embodiment 120, wherein the SUMO protein comprises or consists of anamino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53-55 or at least 70 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53- 55.. The method of any one of embodiments 118-122, wherein the protein carrier comprisesSUMO1, a functional fragment, or derivative thereof.. The method of embodiment 122, wherein SUMO1 comprises or consists of an amino acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53.Attorney Docket No: 243735.000431. The method of embodiment 123 or embodiment 124, wherein the cysteine residue atposition 52 of SUMO1 has been mutated to a non-cysteine residue.. The method of embodiment 125, wherein the cysteine residue at position 52 of SUMO1has been mutated to an alanine.. The method of any one of embodiments 123-126, wherein SUMO1 is modified tointroduce one or more cysteine residues at the amino acid position 33, amino acid position 46, amino acid position 53, amino acid position 85, and / or amino acid position 50.. The method of embodiment 127, wherein SUMO1 comprises or consists of an amino acidsequence set forth in SEQ ID NO: 8 or at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 8.. The method of any one of embodiments 119-128, wherein the C-terminal or proximal tothe C-terminal sequence of the SUMO protein comprises Gly-Gly.. The method of any one of embodiments 107-129, wherein the intracellular protease is aUBL-specific protease.. The method of embodiment 130, wherein the UBL-specific protease is a ubiquitin-specific protease (USP) or SUMO-specific protease (SENP).. The method of embodiment 131, wherein the SENP is SENP1, SENP2, SENP3, SENP5,SENP6, and / or SENP7.. The method of any one of embodiments 99-132, wherein the cationic polymer is apolyamine.. The method of any one of embodiments 99-133, wherein the cationic polymer is selectedfrom polyethylenimine (PEI), poly(L-lysine) (PLL), poly(D-lysine) (PDL), polyamidoamine (PAA), Poly(amino-co-ester)s (PAEs), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), poly(propylene imine) (PPI), poly-L-arginine (PLA), poly-guanidine, poly-D- arginine, poly(D,L-lysine hydrobromide), poly-L-ornithine, PEI-Gua, PEI-Arg, PEI-His, PEI-Trp, or derivatives and combinations thereof.. The method of any one of embodiments 99-134, wherein the cationic polymer is PEI.. The method of embodiment 135, wherein the PEI is a linear PEI or branched PEI.. The method of embodiment 136, wherein the branched PEI is a short-chain PEI.. The method of any one of embodiments 134-137, wherein the PEI has a molecularweight of less than 2000 Da.Attorney Docket No: 243735.000431. The method of embodiment 138, wherein the PEI has a molecular weight ranging fromabout 600 Da to about 1800Da.. The method of embodiment 138 or embodiment 139, wherein the PEI has a molecularweight of about 600 Da (PEI600) or 1200 Da (PEI1200).. The method of embodiment 140, wherein the PEI has a molecular weight of about 600Da (PEI600).. The method of embodiment 140, wherein the PEI is branched PEI600 (bPEI600) orbranched PEI1200 (bPEI1200).. The method of embodiment 142, wherein the PEI is branched PEI600 (bPEI600).. The method of any one of embodiments 99-143, wherein the protein carrier is conjugatedto the cationic polymer via a linker.. The method of embodiment 144, wherein the linker is a heterobifunctional crosslinker.. The method of embodiment 145, wherein the linker comprises an electrophile selectedfrom haloacetophenone, epoxide, haloacetamide, maleimide, isothiocyanate, vinyl sulfone, nitrile, iminonitrile, hydrazone, butynoate, acrylester, acrylonitrile, acrylamide, benzisothiazolone, and N-hydroxy(methyl)phthalimide.. The method of embodiment 145, wherein the linker comprises a N-hydroxysuccinimide(NHS) ester and a maleimide group.. The method of any one of embodiments 145-147, wherein the linker is selected from N-alpha-maleimidoacetoxyl succinimide ester (AMAS), N-beta-maleimidopropyl- oxysuccinimide ester (BMPS), N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-epsilon-malemidocaproyl- oxysuccinimide ester (EMCS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH), succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), N-(11- maleimidoundecanoyloxy)succinimide (KMUS), or derivatives and combinations thereof.. The method of any one of embodiments 144-148, wherein the linker is N-alpha-maleimidoacetoxyl succinimide ester (AMAS).. The method of any one of embodiments 99-149, wherein the cationic polymer isconjugated to the protein carrier at a pH of 6-8.Attorney Docket No: 243735.000431. The method of any one of embodiments 99-150, wherein the polypeptide cargo interactswith an intracellular target.. The method of embodiment 151, wherein the intracellular target is selected from ratsarcoma virus (RAS), Src homology 2 domain-containing phosphatase 2 (SHP2), Aurora A kinase, Src-family kinases, signal transducer and activator of transcription 3 (STAT3), PR domain containing 14 (PRDM14), WD repeat domain 5 (WDR5), mixed lineage kinase domain-like protein (MLKL), p53, and ABL kinase.. The method of embodiment 152, wherein RAS is Kirsten rat sarcoma viral oncogenehomolog (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS), or Harvey rat sarcoma viral oncogene homolog (HRAS).. The method of any one of embodiments 99-153, wherein the polypeptide cargo isselected from a monobody, a designed ankyrin repeat protein (DARPin), an Anticalin, an affilin, an affibody, a peptide, an antibody or antigen-binding fragment, an enzyme, or derivatives and combinations thereof.. The method of embodiment 154, wherein the polypeptide cargo is selected from amonobody, a designed ankyrin repeat protein (DARPin), or derivatives thereof.. The method of embodiment 155, wherein the polypeptide cargo is tandem monobodiescomprising two or more monobody units.. The method of any one of embodiments 154-156, wherein the monobody or DARPintargets KRAS, HRAS, NRAS, Aurora A kinase, ABL kinase, a Src-family kinase, SHP2, STAT3, PRDM14, WDR5, MLKL, or p53.. The method of embodiment 157, wherein the monobody is selected from 12VC1, NS1,12D3, 12D4, 12D5, AS25, AS27, HA4, 7C12, Nsa1, NSa5, CS3, S14, S4, Mb1, Mb2, Mb3,Mb4, Mb5, Mb6, Mb19, Mb20, Mb24, Mb27, Mb32, Mb33, MS3-6, Mb13, R15, Fgr_1, Fgr_2, Hck_1, Hck_2, Lck_1, Lck_3, Lyn_2, Lyn_4, Src_2, Yes_1, Yes_3, MLKL_26, MLKL_27, MLKL_32, MLKL_33, and MLKL_37.. The method of embodiment 157 or embodiment 158, wherein the monobody comprisesor consists of an amino acid sequence set forth in any one of SEQ ID NOs:14-52 and 69-70 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:14-52 and 69-70.. The method of embodiment 157, wherein the DARPin is K27, C10, or C10-H82R.Attorney Docket No: 243735.000431. The method of embodiment 154, wherein the antibody or antigen-binding fragmentthereof is selected from a single chain antibody, a single domain antibody, a variable heavy homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, a monoclonal antibody, a bispecific antibody or antigen-binding fragment thereof, a bi-epitopic antibody or antigen-binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, an scFv, a VH domain, a monovalent antibody or a bivalent antibody.. The method of embodiment 154, wherein the peptide is a stapled peptide or bicyclicpeptide.. The method of any one of embodiments 99-162, wherein the contiguous polypeptidecomprises or consists of the sequence set forth in any one of SEQ ID Nos: 9, 11, 62-65, and 71 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, 62-65, and 71.. The method of embodiment 163, wherein the contiguous polypeptide comprises orconsists of the sequence set forth in SEQ ID NO: 62 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62.. A protein conjugate produced by the method of any one of embodiments 99-164.. A pharmaceutical composition comprising the protein conjugate of any one ofembodiments 1-85 and 165, the nucleic acid molecule of any one of embodiments 86-93, the vector of embodiment 95, or the host cell of any one of embodiments 96-98, and a pharmaceutically acceptable carrier and / or excipient.. A method of delivering a cargo into the cytoplasm of a cell, comprising a) producing aprotein conjugate comprising the cargo according to the method of any one of embodiments 99-164; and b) contacting the cell with the produced protein conjugate.. A method of targeting an intracellular target in a cell, comprising a) producing a proteinconjugate comprising a cargo that interacts with the intracellular target according to the method of any one of embodiments 99-164; and b) contacting the cell with the produced protein conjugate.. The method of embodiment 167 or embodiment 168, wherein the cell is a mammaliancell.. The method of embodiment 169, wherein the mammalian cell is an adherent cell.Attorney Docket No: 243735.000431171. The method of embodiment 169, wherein the mammalian cell is a non-adherent cell.172. The method of any one of embodiments 167-171, wherein the method occurs ex vivo.173. The method of any one of embodiments 167-171, wherein the method occurs in vivo.174. A method of preventing, treating, or diagnosing a disease in a subject in need thereof,comprising administering to the subject the protein conjugate of any one of embodiments 1- 85 and 165, or the pharmaceutical composition of embodiment 166, wherein the cargo interacts with a target associated with disease. EXAMPLES

[0453] The following examples are provided to further describe some of the embodimentsdisclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.

[0454] In the Examples described below, recombinant proteins are produced from expressionvectors with defined sequences. Protein samples are purified to apparent homogeneity and their biophysical properties are confirmed using size-exclusion chromatography and binding measurements. Biochemical and cell-based experiments are performed with a minimum of three technical replicates, and with a minimum of three biological replicates where applicable. All experiments include positive and negative controls. Cytoplasmic delivery is assessed using the assays described herein, which have substantially higher rigor than image-based assays that are prone to artifacts and misinterpretation. Example 1. Design And Production Of PEI-SUMO-Mb Reagents PEI-modified SUMO as the delivery vehicle.

[0455] The majority of cell-penetrating reagents utilize cationic moieties such as Arg-richpeptides (14). Earlier designs of cell-penetrating peptides utilized linear flexible molecules. More recent designs used more rigid scaffolds for presenting cationic moieties (13, 15, 16). In parallel, cationic polymers such as polyethyleneimine (PEI) have much higher number and density of cationic groups than Arg-based reagents, which may make them great candidates as protein delivery agents.

[0456] The conjugation of short-chain PEI at specific positions in a rigid protein scaffoldintegrates beneficial factors. Specifically, the introduction of Cys residues to an otherwise Cys-Attorney Docket No: 243735.000431 free protein carrier, followed by chemical conjugation of PEI to the Cys sulfhydryl group, enables precise control of the site and number of conjugated PEI molecules (Fig.1A), which in turn enables systematic optimization of reagents. Furthermore, such protein carriers can be first recombinantly fused to a protein cargo that is also devoid of Cys residues, prior to chemical conjugation. This approach would maintain the ability to site-specifically conjugate PEI only to the introduced Cys residues in the protein carrier while fundamentally eliminating the possibility of chemical modification of the cargo that could be detrimental to the cargo function.

[0457] The design as illustrated herein is based on the production of a small fusion proteinusing a standard method followed by site-specific chemical conjugation in a single-pot reaction (Fig.1A). The production does not require peptide chemical synthesis, precise control of reaction conditions, or purification of a specific fraction from heterogenous materials. As such, this approach has low barrier for adaptation, particularly by non-specialist with limited training in protein biochemistry.

[0458] Human small ubiquitin-like modifier (SUMO ) was chosen as a protein carrier for theproof-of-concept studies (Fig.1A). Of SUMO isotypes, SUMO1 was used for studies illustrated herein. SUMO is a small globular protein that is involved in post-translational controls of diverse processes in mammalian cells. Similar to ubiquitin, SUMO is produced with a longer precursor protein and efficiently cleaved with SUMO-specific proteases, SENPs, in the cytoplasm and nucleus of cells. The presence of endogenous proteases localized exclusively in the cytoplasm offers an additional benefit in that the linkage between SUMO and cargo is cleaved after being delivered into the cytoplasm, which regenerates the unmodified cargo and fundamentally eliminates the possibility of the delivery reagents perturbing the function of the delivered cargo in the cytoplasm (Fig.1A). SENP-mediated cleavage also serves as an assay for successful cytoplasmic delivery, which can be utilized in the present disclosure.

[0459] Two sites for PEI conjugation that are exposed on the surface of an alpha-helix inSUMO1 for potential conjugation sites were selected (Fig.2A). These sites are outside the interface of SUMO1 and SENP1 so that modifications at these sites should not affect the cleavage. Indeed, this was the case (Fig.3B). Furthermore, these modifications can block the binding of SUMO-interacting proteins so that the delivered PEI-SUMO minimally perturbs the cellular homeostasis. In summary, the design of the present disclosure enables the production of precisely defined molecules compatible with synthetic binding proteins as cargos. An alternativeAttorney Docket No: 243735.000431 design in which Cys residues are introduced within the carrier but outside the SUMO segment is shown in Fig.1B. Monobodies as ideal cargos for systematic optimization of delivery technology.

[0460] Monobodies, synthetic binding proteins based on the fibronectin type III domain(FN3), are used as the primary cargos for the following reasons. Monobodies are small, Cys-free proteins that can be readily fused to other proteins and peptides. Indeed, SUMO-monobody fusion proteins with additional short peptide tags for purification and detection were produced (Figs.1 and 2). The produced fusion protein had good biophysical properties. The fusion protein produced was soluble and well-behaving. The Cys-free nature of monobodies combined with the use of a Cys-free version of SUMO provides flexibility in introducing Cys residues at desired locations in SUMO-monobody fusion proteins, a feature crucial for optimizing the reagents of the present disclosure.

[0461] A large number of monobodies selective to intracellular targets including RAS,SHP2, and ABL kinase were developed (5). Monobodies lack disulfide bonds and thus they can be readily expressed in the fully functional form in the reducing environment of the cytoplasm of mammalian cells. Their selectivity and potency as genetically encoded (intracellularly expressed) reagents were validated. Thus, the “best case scenario” for effective intracellular delivery of these monobodies was established.

[0462] Together, the physicochemical properties and functions of these monobodies makethem effective and innovative cargos for the development of the present delivery technology.

[0463] The 12VC1 monobody was chosen as the first cargo, because this monobody isknown for its specificity and potency in vitro and in cells has been extensively validated (1). 12VC1 inhibits the interaction of KRAS(G12V) and KRAS(G12C) with RAS effectors, and hence downstream signaling mediated by these KRAS mutants. Cell lines whose growth is inhibited when 12VC1 is expressed in them were identified (1).

[0464] An endogenous Cys52 of human SUMO1 (small ubiquitin-like modifier) was mutatedto Ala to produce Cys-free SUMO1. For brevity, This Cys-free version of human SUMO1 is referred as SUMO hereafter. The gene for the SUMO-12VC1 fusion with two Cys sites located on the helix of SUMO1 was constructed (Fig.2A), expressed and purified the protein from E. coli. The genes encoding His6-SUMO-monobody-V5 (SEQ ID NO: 9) were synthesized and cloned in a pET-based expression vector. The genes were expressed in Escherichia coliAttorney Docket No: 243735.000431 BL21(DE3) and the proteins were purified using Ni Sepharose and Superdex 75 columns (Cytiva). The purified proteins were dialyzed in 100 mM HEPES, 500 mM NaCl, 0.5 mM TCEP, pH 7.5.

[0465] The proteins were chemically coupled with short-chain, branched PEI (bPEI600 andbPEI1200, Polysciences) using a heterobifunctional crosslinker, N-alpha-maleimidoacetoxyl succinimide ester (Thermo Scientific). Branched polyethyleneimine (bPEI) with molecular weight, 600, 1200, or 2000 were dissolved in water at 100 mM, and neutralized with hydrochloric acid. bPEI was diluted to a final concentration of 20 mM in 200 mM HEPES NaOH buffer, pH 7.5, mixed with 10 mM N-α-maleimidoacet-oxysuccinimide ester (AMAS), and incubated for 30 minutes at room temperature. This solution was mixed 1:1 with a protein solution and incubated for 2 hours at room temperature. The sample was dialyzed against 50 mM Tris Cl, 500 mM NaCl, pH7.5 and frozen until used. The modified proteins were water-soluble and monodispersed as tested with size-exclusion chromatography (Fig.2B). Example 2. RAS Inhibition As a Critical Functional Assay For Cytoplasmic Delivery. Integrated optimization of delivery vehicle and cargos.

[0466] Any feature of monobodies can be tailored in such a way that facilitate thedevelopment of the delivery technology of the present disclosure. This integration of cargo and delivery vehicle development can accelerate the optimization of the technology.

[0467] The design of the present disclosure has several advantages as described herein. BothSUMO1 and monobody (built using the 10thFN3 domain of human fibronectin) are human proteins, and thus SUMO1-monobody fusions are expected to have low immunogenicity (21). This human nature of the design contrasts with other approaches that rely on bacterial proteins that are likely to be highly immunogenic (19, 20). Low molecular weight PEI reagents are already used as ingredients of cosmetics for human use and have minimal toxicity. Also, unlike large-molecular weight polyethylene glycols (PEGs) used for half-life extension of biologics that can remain in the blood circulation for extended periods (22), low molecular weight PEI should be cleared rapidly. Finally, the small size of PEI-SUMO-monobody should have short half-life in the blood, which is advantageous in applications where a short exposure is desired. The blood half-life of the reagents of the present disclosure can be increased, as they are compatible with diverse technologies for half-life extension, including an attachment of albumin-binding moiety,Attorney Docket No: 243735.000431 that are already used with monobodies (23, 24), which allows the half-life of the reagent of the present disclosure to be tailored over a wide time frame. Robust and rigorous assays for the delivery efficiency of the actual reagents.

[0468] Artifacts and misinterpretations in measurements of “intracellular delivery” havebeen well documented (13). Thus, the assays that are known to be prone to artifacts, such as those relying solely on microscopic imaging that were commonly used in early studies for assessing the reagents were avoided (13, 25).

[0469] To assess the extent by which the reagents taken up by cells reach the cytoplasm, thecleavage of the SUMO-monobody linkage was probed by endogenous SENPs (Figs.1A and 3A). The feature required for the assay is already built in the reagent itself, and hence, unlike other commonly used assays for cytoplasmic delivery (13), there is no need to modify the reagents of the present disclosure for the purpose of detecting cytoplasmic delivery. Furthermore, the assay does not require engineering of cells, unlike commonly used assays for cytoplasmic delivery such as enzymatic biotinylation, GFP complementation, and CAPA assay (13). These innovative aspects of the reagent design and the assay of the present disclosure enable the critical testing of cytoplasmic delivery of the final reagent designs for applications using unmodified target cells.

[0470] The phenotypes of the monobody cargos produced in the cytoplasm were established,and thus functional assays as a readout for intracellular delivery can be reliably used. A functional assay based on the inhibition of RAS-mediated signaling and simple readout of cytotoxicity was established and used (Fig.4A). Cells were grown with the indicated reagents in vendor-specified media, all containing 10% fetal bovine serum (FBS) and penicillin / streptomycin for 72 hours at 37°C with 5% CO2. Cell viability was assessed using the PrestBlue reagent (ThermoFisher). This type of functional assay is relevant for applications in both mechanistic studies and drug discovery.

[0471] The use of two complementary assays that strictly depend on cytoplasmic delivery isanother innovative and important feature of the disclosure, which also improves the overall rigor.

[0472] The inhibition of proliferation of oncogenic RAS-mediated cells was selected as thereadout for the assay for intracellular delivery. RAS is anchored to the cytoplasmic side of cellular membranes. RAS is an abundant protein with a copy number in the range of ~0.2 million per cells (27). Thus, occupation-based inhibition of RAS requires the delivery of a substantialAttorney Docket No: 243735.000431 number of an inhibitor to the cytoplasm. A panel of monobodies was developed that inhibit oncogenic RAS mutants via different mechanisms (1, 2, 4, 28). The effectiveness of these monobodies as cytoplasmic inhibitors was validated by expressing them as genetically encoded reagents.

[0473] Because of its status as a major “undruggable” cancer driver, oncogenic RAS hasbeen an attractive target for novel therapeutic modalities including cytoplasm-delivered biologics. Recent breakthroughs have resulted in potent and cell-permeable small molecule inhibitors against KRAS(G12C) and KRAS(G12D) (29-31) as well as “pan-RAS(ON)” inhibitors (32), which can serve as positive controls. Thus, cytotoxicity against a KRAS(G12C)- driven cell line, H358, was chosen as the primary assay for the preliminary study.

[0474] Previous studies utilized RAS-mediated signaling as the target of cell-penetratingreagents (33, 34, 18, or WO 2022 / 224257). However, these studies corroborate the challenge of potently inhibiting RAS via cytoplasmic delivery of tool biologics.

[0475] A significant inhibition of cell growth of H358 at sub-µM concentrations of thereagents of the present disclosure, with conjugates with PEI600 was found more potent than those with PEI1200 (Fig.4B). Consequently, the PEI6002 conjugate (comprising two Cys residues conjugated with PEI600) was used for further evaluation. The IC50in the mid- nanomolar range is approximately two orders of magnitude lower (i.e., more efficacious) than values for cell-penetrating reagents typically in the micromolar range. Delivered 12VC1 potently and selectively inhibits its intended targets.

[0476] To test the specificity of inhibition of the reagent, PEI-SUMO-12VC1, it was testedagainst a panel of cell lines with different driver oncogenes. Cells were cultured in media specified by their respective sources, all including 10% fetal bovine serum (FBS).

[0477] The reagents inhibited cell lines driven by KRAS(G12C) and KRAS(G12V), but notthose driven by KRAS(G12D), NRAS(Q61R) BRAF(V600E), or HEK293 cells (Fig.4C). This specificity profile precisely matched with the binding profile of 12VC1 monobody that was established using biochemical assays with purified 12VC1 samples and using cell-based assays with genetically encoded 12VC1 (1). The higher efficacy against H358, a cell line with KRAS(G12C), than PATU8902, a cell line with KRAS(G12V), was also consistent with the higher affinity of 12VC1 to KRAS(G12C) than to KRAS(G12V) (1).Attorney Docket No: 243735.000431

[0478] The cells were also followed using an Incucyte instrument (Fig. 5A, right panels).Cells were grown in RPMI 1640 media with 10% FBS and penicillin / streptomycin 24 hours at 37°C with 5% CO2 with the indicated reagents. Apoptotic cells were detected using the Incucyte caspase-3 / 7 dye. The cell-signaling assay was performed as described in Teng et al Nat Commun.202112(1):2656, which is incorporated herein by reference in its entirety. PEI- SUMO-12VC1 caused apoptosis as probed with caspase-3 / 7 cleavage and a change in the morphology of H358 cells similar to those observed with an FDA-approved inhibitor against KRAS(G12C), sotorasib (Fig.5A). By contrast, no changes were observed in cell growth or morphology when H358 cells were treated with PEI-SUMO, the delivery vehicle without a monobody cargo. His6-SUMO1(C52A; K46C; Q53C)-12VC1-V5 (SEQ ID NO: 9) conjugated with PEI600 induced apoptosis, as expected for the function of the 12VC1 cargo. His6- SUMO1(C52A; K46C; Q53C)-12VC1-V5 conjugated with PEI600 inhibited the MAP kinase signaling, as expected from the inhibition function of 12VC1.

[0479] Together, these results strongly support the conclusion that the observed cytotoxicityis due to the inhibition of RAS-mediated signaling by the binding of delivered 12VC1 to its intended targets, and the delivery vehicle of the present disclosure has minimal toxicity to these cell lines. Most of internalized reagents reach the cytoplasm.

[0480] To further validate cytoplasmic delivery, the cleavage of the fusion protein wasprobed (Fig.3A). The reagent, PEI-SUMO-12VC1 (1 µM, a concentration sufficient for the inhibition of H358 cells (Fig.4C)) and control reagents were added to HEK293T cells and incubated for three hours at 37°C. HEK293T cells were grown in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS). The delivery reagents were added to the media. After incubation, the media were removed, and the cells were washed with phosphate- buffered saline (PBS). The cells were lysed in SDS-PAGE sample buffer and analyzed with immunoblots using an anti-V5 antibody. The V5 tag attached to the C terminus of the monobody cargo was detected by immunoblotting. Remarkably, only the band corresponding to the cleaved product was detected and not the uncleaved from cells that received the reagent (Fig.3B). When the experiment was repeated at a reagent concentration of 5 µM, the reagent still showed no toxicity to HEK293T cells, and the uncleaved reagent was detected (Fig.3C). The cleaved reagents were detected after 3-hour incubation, demonstrating cytosolic delivery of the reagents.Attorney Docket No: 243735.000431 These results indicated that most of the reagent taken up by the cells reached the cytoplasm. In addition, the conjugate with PEI600 was more efficiently cleaved than that with PEI1200 (Fig. 3C) in this assay, which was consistent with the higher efficacy of the PEI600 conjugate in the H358 growth inhibition assay (Fig.4B). Together with the cytotoxicity data, these results demonstrated high efficiency of cytoplasmic delivery achieved with the approach of the present disclosure. TAT-12VC1 triggers nonspecific interactions, and more toxic.

[0481] For comparison, a TAT fusion of 12VC1 monobody was constructed (Fig. 7). Theconstruct was prepared and produced as described for Fig.2. The cytotoxicity assay was performed as described for Fig.4. Although this protein was purified, it showed an extremely strange profile on SEC, suggesting that it interacted strongly with the column matrix. Although such a poorly behaving reagent would not be useful for practical use, it was tested in the cell- based assay anyway. It showed cytotoxicity to negative control cell lines, A375 and HE293T (Fig.7D), indicating general toxicity, consistent with earlier studies (35). In comparison, the reagent of the present disclosure showed no toxicity to these cells at the same concentration (Fig. 4C). The specific cytotoxicity against KRAS(G12C)-dependent cells, H358, was less than the PEI-SUMO-12VC1 reagent (compare Fig.7D with Fig.4C), and the TAT-fusion protein was not detected in the cells by immunoblotting (Fig.3, lane 5).

[0482] The reagents effectively inhibited the growth of NCI-H358, a cell line that dependson KRAS(G12C). His6-SUMO1(C52A; K46C; Q53C)-12VC1-V5 conjugated with PEI600 was more effective than that conjugated with PEI1200. The inhibition was specific to cell lines that depend on KRAS(G12C) and KRAS(G12V), which are the KRAS mutants that the 12VC1 cargo inhibits. The reagent showed no cytotoxicity to other cell lines, suggesting that it is minimally toxic to cell, a desirable property of a delivery reagent.

[0483] The TAT fusion protein can be produced, but it showed an anomalous elution profilefrom the size-exclusion chromatography, suggestive of high levels of nonspecific interactions. The TAT fusion reagent showed significant cytotoxicity for cell lines that the 12VC1 cargo does not inhibit, indicating its general toxicity. The reagent showed higher cytotoxicity to H358 than the negative control cell lines, suggesting that it delivers 12VC1 into the cytosol to some extent. Delivery of a different monobody cargo.Attorney Docket No: 243735.000431

[0484] Next, equivalent conjugates were prepared with another monobodies, NS1, thattargets KRAS and HRAS in a mutant agnostic manner (4).12VC1 and NS1 has 82% sequence identity, a typical level of homology between two monobodies binding to different targets or different epitopes. The reagent was prepared essentially using the methods described for Fig.2. The cytotoxicity assay was performed as described for Fig.4. Similar to the reagent made with the 12VC1 cargo described above, the reagent with NS1 was soluble, and monodispersed in SEC analysis (Fig.6A). This reagent was tested with the same panel of cell lines as for the 12VC1 reagent. The NS1 reagent inhibited the growth of cell lines that depend on oncogenic KRAS mutants (H358, PATU8902, and HPAF-II), the RAS isotypes that NS1 inhibits, demonstrating effective cytosolic delivery (Fig.6B). It showed minimal effects with the other cell lines. The lack of efficacy to SK-ML-147 was consistent with the specificity of NS1. The lower inhibitory efficacy of the NS1 reagent compared with the 12VC1 reagent was expected because, unlike 12VC1, NS1 is not specific to a particular mutant and consequently higher concentrations of NS1 is required to achieve inhibition of a mutant RAS that drives cell proliferation. NS1 binds to KRAS and HRAS in both activated (GTP-bound) and basal (GDP-bound) states, and thus only a fraction of delivered NS1 binds to the oncogenic RAS mutant, and the wild-type alleles present in these cells act as sinks for NS1. By contrast, 12VC1 is exquisitely specific to oncogenic RAS mutants, G12C, and G12V. Thus, these results also highlight the impact of using a cargo that is highly selective to the target of interest on producing high-potency reagent for cytoplasmic delivery, and a potentially greater impact to be achieved when delivery technology and selective cargos are co-developed. Example 3. Refine The Reagent Design Using Monobody Cargos.

[0485] In this Example, the effects of modifying the site and type of chemical conjugation ina vehicle on the efficiency of cargo delivery are tested. The cleavage of the fusion protein by cytoplasmic proteases and functional assays afforded by many validated monobodies against intracellular targets allow for critical evaluation of the delivery efficiency and optimize the design of the reagents.

[0486] Rationale and experimental design: The PEI-SUMO reagents achieved a remarkablelevel of cytoplasmic delivery of 12VC1 monobody with minimal toxicity. The delivery efficiency can be substantially improved by optimizing the reagent design. The locations andAttorney Docket No: 243735.000431 number of PEI conjugation sites, and the size of PEI are tested, and such reagents using cell- based assays are also tested. To ensure the generality of the reagents, two monobody cargos and two cell lines of distinct types are utilized.

[0487] Reagent design and preparation.: SUMO1 has a total of 25 residues whose sidechains are highly solvent accessible in the SUMO1-SENP1 complex (Fig.2A), and thus these positions can potentially be used for introducing Cys residues without compromising the integrity of SUMO1 or its interaction with SENPs. The delivery efficiency can be further optimized by optimizing the conjugation sites.

[0488] An effect of the number of PEI molecules is tested. SUMO containing a single Cysresidue at either position of the original two positions is produced as shown in Fig.2A, and tested their delivery efficiency in comparison with the current PEI2 conjugate. In the other direction, SUMO containing a total of three Cys residues, by adding an additional Cys site to the current Cys2 construct is also produced. A residue 50 located in between the two current positions, i.e., the middle of the helix (Fig.2A), and residue 33 that is located far away from the current positions are tested. Conjugations with PEI600 and PEI1200 that are commercially available are tested. The delivery efficiency is tested using cell-based assays as shown in Figs.3 and 4; also see ‘Cell-based assays’ below). These experiments determine the importance of the number of PEI conjugations.

[0489] If it is found that a single PEI conjugate has the highest efficiency over thosecontaining multiple PEI molecules, The efficiency of reagents containing a single conjugated PEI at different locations can be tested. In addition to the sites that was already selected (see above), additional sites based on the electrostatic characteristics of the surrounding surfaces are selected. A site with additional positive charges in the vicinity can be more effective than that without positive charges.

[0490] An iterative optimization can be performed for a reagent conjugated with multiplePEI molecules. Consequently, sites based on the secondary structure (helix, strand, or loop) and distance between sites are selected. Once the effective designs are identified, a negative-control reagent without the monobody cargo is prepared for use in cell-based assays.

[0491] 12VC1 monobody is used, because a companion assay and its effectiveness as a cargohas been established as illustrated in the Examples. Once representative conjugation designs using 12VC1 as the cargo is identified, such designs with additional monobodies are tested.Attorney Docket No: 243735.000431 Representative designs are selected with different levels of efficiencies to assess whether the reagents have similar delivery efficiency for different monobody cargos.

[0492] SUMO-monobody fusion proteins from E. coli are produced, purified using nickel-affinity chromatography, and conjugated with PEI. The conjugated reagents are purified using size-exclusion chromatography, as described in the Examples (Figs.2B and 2C). All samples are validated with MALDI-MS.

[0493] In parallel, the effect of the His6-tag (SEQ ID NO: 12) at the N terminus of thedesigns are examined as shown in Fig.1A. The His6-tag (SEQ ID NO: 12) is included primarily for purification. The reagents with and without the His6-tag (SEQ ID NO: 12) in cell-based assays are compared. A tobacco etch virus (TEV) protease cleavage site is introduced between the His6-tag (SEQ ID NO: 12) and SUMO as shown in Fig.1A. The fusion protein is purified, cleaved with the TEV protease, and the fusion protein without the His6-tag (SEQ ID NO: 12) is purified. The presence of the His6-tag (SEQ ID NO: 12) is not expected to negatively impact the utility of the reagents of the present disclosure as research tools. For future in vivo applications, the His6-tag (SEQ ID NO: 12) can be eliminated to minimize the immunogenicity of the reagents. Constructs with and without the His6-tag (SEQ ID NO: 12) of representative designs can be characterized.

[0494] Cell-based assays: As shown above, two types of assays can be used: the SUMO-cleavage assay that is universal for all monobody cargos as shown in Fig.3, and functional assays that are tailored depending on the function of the monobody cargo as shown in Fig.4. Because the growth inhibition assay has higher throughput than the SUMO-cleavage assay, the growth inhibition assay can be used as the primary assay for screening different reagents, and the SUMO-cleavage assay can be used as a secondary, confirmation assay.

[0495] A working SUMO-cleavage assay has already been developed as shown in Fig. 3.This assay can be fine-tuned by optimizing methods for sample preparation and detection, and the incubation period and host cells, as well as by expanding controls. The major changes in the overall assay design are not anticipated. The SUMO-cleavage assay is used for cells whose growth is not affected by monobody cargos that have been developed as explained above, such as HEK293T cells. An important advantage of this assay over other common assays for cytoplasmic delivery, such as enzymatic biotinylation, GFP fragment complementation and the CAPA assay (13), is that it does not require engineered cells. SENPs are endogenous proteinsAttorney Docket No: 243735.000431 that are ubiquitously expressed and catalytically efficient (37). Thus, this assay can be readily applied to virtually any cell line of interest.

[0496] The growth inhibition assay of the H358 cell line for 12VC1 is continued to be used,along with positive control drugs and negative control cell lines as shown in Fig.4B. The proliferation of the cells is monitored by measuring the viability at the end-point using the Prest Blue dye as shown in Fig.4C, or by following cell growth and apoptosis using an Incucyte instrument as shown in Fig.5A. To ensure that the delivery technology of the present disclosure is compatible with common cell culture conditions, all cell cultures are performed using standard culture media specified by the respective sources of cell lines (e.g., ATCC). In particular, FBS is not removed from media, a common method to eliminate a major source of inhibition of some cell-penetrating reagents due to their nonspecific binding to albumin present in serum (38, 39). In other words, culture conditions to inflate the apparent efficiency of the reagents of the present disclosure are not manipulated.

[0497] To complement the RAS inhibition assay, an assay for detecting the inhibition ofABL kinase with AS25 monobody is established. AS25 monobody was developed as an allosteric inhibitor of ABL kinase, and its intracellular expression potently inhibits the growth of K562 cells (3). K562 is a non-adherent cell line derived from a chronic myelogenous leukemia (CML) patient, and its growth and survival of K562 cells are exquisitely dependent on BCR- ABL kinase activity and signaling (40). Besides, K562 is a workhorse cell line that is widely used in a broad range of biomedical research including transcriptional regulation, cell signaling, and protein-protein interaction networks, with nearly 18,000 PubMed citations. Thus, establishing a cytoplasmic delivery technology for K562 cells will broadly benefit the biomedical community. PEI-SUMO-AS25 reagents are prepared and monitored for its effect on the proliferation of K562 cells using an Incucyte. An FDA-approved small molecule inhibitor, imatinib is used as a positive control reagent. EXPI293 and OCI-AML3 cell lines, non-adherent cell lines that do not depend on ABL kinase activity are used as negative controls. Finally, the best performing designs are tested against a larger panel of cell lines.

[0498] Anticipated outcomes and potential challenges: The experiments in this Exampledefine the optimal design(s) of PEI-SUMO reagent for the delivery of two different monobodies, 12VC1 targeting KRAS mutants and AS25 targeting ABL kinase. Although the two monobodies have distinct binding interfaces for their respective targets, they still have 80% sequence identity.Attorney Docket No: 243735.000431 Only small differences in the delivery efficiency of PEI-SUMO reagents between these two monobody cargos are anticipated.

[0499] The two-tier assay design allows for rapidly screening of many constructs using twocell lines, H358 (adherent) and K562 (non-adherent) using the growth inhibition assays, followed by testing the most promising candidates with the SUMO-cleavage assay. The SUMO-cleavage assay is used for defining the kinetics of delivery. The systematic optimization of design parameters defines molecular features crucial for effective cytoplasmic delivery. This knowledge helps to advance understanding of the molecular mechanism of protein delivery into the cytoplasm. A growth inhibition assay as described herein is high-throughput. Thus, a large number of designs can be produced and characterized in a timely manner. Example 4. Define Influences Of The Delivery Reagent On Cellular Homeostasis And Host Factors That Influence The Efficiency Of Cytoplasmic Delivery

[0500] In this Example, an effect of the delivery vehicle of the present disclosure on thetranscriptomes of representative cell lines is analyzed to define potential cargo-independent effects. The delivery efficiency of the cargos of the present disclosure in diverse cell types is examined, including cells of epithelial and endothelial origins, cancer cell lines, and immune cells. A genome-wide CRISPR / Cas9 deletion screen is utilized to identify pathways that influence delivery.

[0501] Rationale and experimental design: In order to utilize the delivery technology in aneffective and informed manner, it is important to understand how the delivery reagents, in the absence of a functional monobody cargo, influence the underlying biology of the host cell. Although appropriate controls, such as the delivery vehicle without a functional cargo to account for the influence of the delivery vehicle can be used, ideally, the delivery vehicle should minimally interact with cellular components. The data provided herein show a remarkable lack of toxicity of the reagents of the present disclosure (Fig.4), suggesting that the reagents do not strongly perturb the cellular homeostasis. Still, because SUMO, the protein scaffold for presenting PEI, and SENPs are involved in many cellular processes (42, 43) and inhibition of the SUMO pathway affects transcription of interferon genes (44), it is possible that our reagents without a functional monobody cargo perturb cellular homeostasisAttorney Docket No: 243735.000431

[0502] The extent that the delivered PEI-SUMO can alter the intracellular pool of SUMO1,the parental molecule of the reagents, and whether PEI-SUMO affects cellular homeostasis are determined. SUMO proteins are abundant in human cells, with a copy number estimated to be ~1 million per cells (45). This is ~5-times higher than the abundance as RAS proteins (27). The ability to potently inhibit KRAS(G12C) (Fig.4), which requires stoichiometric binding of delivered 12VC1 monobody to KRAS(G12C), suggests that the number of the delivered reagent in the cytoplasm may approach ~1 million per cell. In addition, the PEI moiety would interact with nucleic acids and other highly negatively charged molecules in the cell. Consequently, the PEI-SUMO portion of the delivered reagent can significantly affect SUMO-mediated cellularprocesses if PEI-SUMO is recognized by the SUMOylation machineries. As the first step todefine potential perturbations, transcriptome analysis of commonly used cell lines is performed, which can provide an unbiased view of effects of the reagents.

[0503] Important features of the host cell required for efficient cytoplasmic delivery aredefined. The delivery efficiency of the reagents against a diverse panel of cell lines is tested, to see whether the reagents show large differences in effectiveness across different cell lines. A genome-wide CRISPR screen on select cell lines is employed to identify genes and pathways that are required for effective delivery of delivery vehicles, which enables the user to predict cell lines that are compatible with the technology of the present disclosure.

[0504] Determine effects of delivered PEI-SUMO reagents: As the first step of investigation,the relative amounts of the delivered PEI-SUMO1 and endogenous SUMO1 in HEK293T cells are assessed using immunoblotting with an anti-SUMO1 antibody (Abcam). The presence of PEI and His6-tag (SEQ ID NO: 12) shifts the position of SUMO1 in SDS-PAGE, enabling the distinction between endogenous SUMO1 and delivered PEI-SUMO1. Cys mutations or PEI- conjugation does not affect the recognition by the anti-SUMO1 antibody is confirmed by performing immunoblotting of purified proteins with and without mutation or PEI modification. Immunoblot images are quantified using fluorescent dye-labeled secondary antibodies on a Li- Cor instrument.

[0505] Effects of the reagents on the transcriptome are assessed. Because SUMO is involvedin transcriptional regulation, transcriptome analysis can reveal potential effects of the reagents in an unbiased manner. Two widely used cell lines that are, HEK293T and K562, are used for this study, because the reagents are expected to often be used with these cells and these cells are goodAttorney Docket No: 243735.000431 representatives of adherent and nonadherent cells from distinct origins. The cells (2 million) are grown using standard culture conditions, on the next day are treated with (and without) a PEI- SUMO-monobody reagent and harvested them after three and 24 hours of incubation. RNAs are extracted immediately afterwards (RNeasy Plus, Qiagen). RNAseq experiments are performed using protocols established (46). RNAseq libraries are constructed using a kit (Illumna), and the samples are analyzed with paired-end sequencing on an Illumina NovaSeq6000. Sequencing results are processed using standard software packages, including bcl2fastq, Trimmomatic and DESeq2.

[0506] Test the efficiency of cytoplasmic delivery against a diverse panel of cells: Cells ofepithelial and endothelial origins, cancer cell lines, and immune cells are used (Table 2). This study is initiated with PEI6002-SUMO-12VC1, and additional reagents that are developed in the experiment of Example 4 are included later. The SUMO-cleavage assay is used to quantify the amounts of reagents that reach the cytoplasm. Table 2. Cell lines Name Characteristics Inhibitory monobody HEK293T Human embryonic kidney cells - K562 Chronic myeloid leukemia AS25 3T3 Mouse fibroblast - HUVEC Human vascular endothelium from umbilical cord - Jurkat Human T cell - NCI-H358 Epithelial-like, from bronchioalveolar carcinoma 12VC1, NS1 OCI-AML3 Acute myeloid leukemia, NRAS(Q61L) - SK-MEL-147 Melanoma NRAS(Q61R) - PATU8902 Pancreatic adenocarcinoma, KRAS(G12V) 12D3, NS1 A-375 Melanoma, BRAF(V600E) - HPAF-II Pancreatic ductal adenocarcinoma, KRAS(G12D) 12D3, NS1 References: 12VC1 (1), 12D3 (2), AS25 (3), NS1 (4); each of which is incorporated herein by reference theirentirety.

[0507] Identify host factors required for efficient cytoplasmic delivery: In order to providethe user mechanistic guidance as to what cell lines are compatible with the reagents, genome- wide CRISPR / cas9 knockout screens are performed to identify genes affecting the delivery efficiency of the reagents. As PEI-SUMO-12VC1 potently and selectively inhibits the growth of H358 cells as illustrated above, genes whose deletion confer resistance to PEI-SUMO-12VC1 are anticipated to be readily identified via positive selection, i.e., growth in the presence of theAttorney Docket No: 243735.000431 reagent. The TKOv3 lentiviral genome-wide CRISPR library is utilized that targets 18,053 protein-coding genes with four guides per gene (48, 49).

[0508] H358 cells are grown and transduced with the library at 1000x representation perreplicate, and grown for 12 doublings, in order to enrich deletions that drive resistance against PEI-SUMO-12VC1 (46). A genomic DNA from the surviving cells is isolated and sequenced, and identified enriched guide RNAs. This screen enriches guide RNAs for genes that are required for cytoplasmic delivery of PEI-SUMO-12VC1 and genes for conferring sensitivity to the inhibition by 12VC1. The latter class likely overlap with genes for conferring sensitivity to small-molecule inhibitors of KRAS(G12C) (50) and also to a small-molecule inhibitor of an upstream molecules, SHP2 (46). The genes known to confer resistance to these inhibitors from the hits are excluded, and a focused library is constructed. A smaller validation screen is performed, first with H358 cells and then with other cells that are sensitive to 12VC1. Enriched genes from each screen (FDR<0.5 to FDR<0.2) are analyzed by KEGG, GSEA, IPA (Ingenuity Pathway Analysis), and STRING (for protein-protein interactions). Other cell lines for which reagents are developed, e.g., K562 and AS25, are tested (see ‘Cell-based assays’), to identify genes that are directly involved in the delivery of the reagents into the cytoplasm.

[0509] Anticipated outcomes and potential challenges: The experiments conducted todetermine effects of delivered PEI-SUMO reagents determine whether PEI-SUMO1 perturbs the biology of the host cell. Given the data of the Examples 1-2 showing minimal effects of the reagent on non-targeted cell lines (Figs.4C and 6) even though substantial concentrations of the reagent delivered in the cytoplasm was detected (Fig.3), effects of the reagents of the present disclosure, if any, are not anticipated to be drastic. A variant of SUMO1 that is incapable of interacting with SUMO-interacting motifs (SIMs) by mutating residues in the binding interface with SIMs can be developed to avoid significant effects (51). Because PEI-SUMO-12VC1 was delivered into the cytoplasm of HEK293T cells (Fig.3), PEI-SUMO up to 10 µM showed no detectable toxicity on HEK293T cells, and analytical procedures including RNAseq are established, technical difficulties in experiments with HEK293T cells are not anticipated. An alternative cell line that is widely used and distinct from HEK293T can be used, based on the results from the testing of the diverse panel of cells described above.

[0510] The experiments described above to determine effects of delivered PEI-SUMOreagents and to test the efficiency of cytoplasmic delivery against a diverse panel of cells willAttorney Docket No: 243735.000431 identify variations of the efficiency of cytoplasmic delivery across different cell lines, which inform the end user, and host pathways critical for effective delivery. The versatility of the SUMO-cleavage assay facilitates experiments to test the efficiency of cytoplasmic delivery against a diverse panel of cells. The high potency of PEI-SUMO-12VC1 against H358 cells facilitates CRISPR / Cas9 deletion screen.

[0511] It is generally accepted that poly-cationic delivery reagents first bind to the cellsurface and endocytosed (15). There are, however, multiple, conflicting mechanisms that have been proposed for the escape of cationic delivery reagents from the endosome, including proton sponge, pore formation, and vesicle budding and collapse (15). It is interesting and important to determine what pathways are crucial for the delivery of PEI-SUMO reagents. The knowledge from these experiments will advance the field of cytoplasmic delivery in general.

[0512] Results from the experiments of the present Example establish efficient cytoplasmicdelivery reagents for a number of commonly used cell lines, including K562, HEK293T, and A375, which can have positive impact on diverse cellular biology investigations by the biomedical research community. Example 5. Characterize The Versatility Of The Technology With Diverse Cargos

[0513] In this Example, the delivery efficiency of cargos that differ in size and topology istested to determine whether the delivery vehicle needs to be tailored for different classes of cargos.

[0514] Rationale and experimental design: A robust reagent for cytoplasmic delivery ofcargos consisting of a single monobody is established in Examples 3 and 4. Here, reagents are examined using different classes of cargos, in order to expand the utility of the delivery reagents.

[0515] New cargos to be used: (i) tandem monobodies consisting of two or three monobodyunits; and (ii) DARPins, another well-established synthetic binding proteins (Fig.8) are tested.

[0516] Tandem monobodies have been used to improve potency and specificity and to createbispecific engagers (52). It remains to be determined whether the same delivery reagent can efficiently deliver a cargo that is twice or thrice as large as the single monobody with which the reagent is being optimized. Tandem monobodies consisting of multiple copies of 12VC1, and those consisting of two or three different monobody clones that bind to the same target, e.g., RAS (Fig. 8) (1, 4, 28) and SHP2 (41), are constructed.Attorney Docket No: 243735.000431

[0517] DARPins represent distinct cargos from monobodies. Both monobodies and DARPinsare stable and free of endogenous Cys residues, and thus both systems are compatible with the reagent preparation procedures (Fig.1A). Monobody has a beta-sandwich fold, whereas DARPin has repeat-protein architecture consisting of predominantly helical units (Fig.8). Also, DARPins are larger in size (~18 kDa) than monobodies (~10 kDa). KRAS-targeting DARPin, K27, that has been rigorously validated as a genetically encoded inhibitor (9, 53) is used.

[0518] Reagent production: The modular nature of the reagent design is exploited to efficientlyproduce different delivery reagents for the new cargos. Several SUMO mutants from the collection developed in Example 4 are selected and combined with the new cargos. SUMO-cargo fusion proteins in E. coli are produced, purified using Ni-affinity chromatography, and conjugated with PEI. The final products are purified using size-exclusion chromatography, as described in the section ‘Reagent design and preparation’ of Example 4.

[0519] Cytoplasmic delivery assays: The SUMO-cleavage assay with HEK293T cells and thegrowth inhibition assay with H358 cells are used to evaluate the reagents produced in this Example. The best-performing reagent with single 12VC1 cargo as a positive control and PEI-SUMO without a cargo as a negative control are included in all measurements.

[0520] The SUMO-cleavage assay with detection using the anti-V5 antibody is not influencedby the cargo size or cargo potency, and thus it is a particularly good assay to compare these distinct reagents. By contrast, the H358 growth assay gives a real-life picture of different reagents, reporting the combination of the efficiency of cytoplasmic delivery and the potency of the cargos.

[0521] Anticipated outcomes and potential challenges: The experiments in this Exampledefine the versatility of the reagents in a dimension orthogonal to those tested in Examples 3 and 4. If significantly reduced efficiency with the larger cargos is observed, the collection of PEIN- SUMO conjugates that are constructed in Example 3 can be screened. The reagents can deliver the larger cargos and DARPins at similar efficiency to that for single monobody cargos, or the reagents can be found suitable for these cargos by systematic screening of the construct collection.

[0522] Experiments with tandem monobodies provide additional information. Tandemmonobodies can have higher inhibitory potency than a single monobody, and thus the same level of target inhibition with fewer molecules that are delivered can be achieved. Tandem monobodies can lead to increased overall efficacy even when the delivery efficiency is reduced.Attorney Docket No: 243735.000431 Example 6. Inhibition of BCR-ABL

[0523] The equivalent reagents were prepared with Mb, AS25, that targets the SH2 domain ofthe ABL kinase and allosterically inhibits the kinase activity as demonstrated as a genetically encoded reagent (3). Although the ABL kinase domain has many FDA-approved inhibitors, the ABL SH2 domain remains undruggable with small molecules. AS25 and 12VC1 has 81% sequence identity, a typical level of homology between two Mbs against distinct targets. Like PEI- SUMO-12VC1, PEI-SUMO-AS25 was soluble, and monodispersed (Fig.18A). PEI-SUMO-AS25 (The amino acid sequence of SUMO-AS25 is designated as SEQ ID NO: 63.) inhibited the growth of K562 cells, nonadherent chronic myelogenous leukemia (CML) cells that depends on the activity of the oncogenic BCR-ABL fusion, and it showed minimal effects on KG1a cells that do not depend on ABL activity (Figs.18B-18C). Example 7. Inhibition of SHP2

[0524] The Mbs that bind to the N-terminal SH2 domain of the SHP2 phosphatase (41, 64)were tested. Although SHP2 is well-established as a drug target (71), the SH2 domains in isolation remain largely “undruggable”. The human proteome contains ≥120 SH2 domains (72), which makes selective targeting of a single SH2 domain particularly challenging. These Mbs showcase ability to selectively target and inhibit currently undruggable proteins. The PEI-SUMO conjugate was developed with Mb NSa1 directed to the N-SH2 domain. NSa1 disrupts the engagement of SHP2 with an upstream binding partner (41). PEI-SUMO-NSa1 (The amino acid sequence of SUMO-NSa1 is designated as SEQ ID NO: 64.) inhibited KYSE520 cells that depend on SHP2activity, whereas it showed no effect on A375 cells (Fig. 19). PEI-SUMO without NSa1 showedno effects on either cell line. Example 8. PEI-SUMO-Mb reagents are stable in culture media and in mouse blood.

[0525] The stability of PEI-SUMO-12VC1 (The amino acid sequence of SUMO-12VC1 isdesignated as SEQ ID NO: 62.) in cell culture media and in mouse plasma was examined. The degradation in culture media was not detected after 72 hours incubation at 37 °C, or in mouse plasma after 24 hours at 37 °C (Fig.20), demonstrating high stability of the reagent in conditions relevant for cell-based and in vivo experiments.Attorney Docket No: 243735.000431 Example 9. PEI conjugation in the N-terminal extension of SUMO produces effective reagents.

[0526] In above-described Examples 6 and 7, the PEI was conjugated to Cys residues that wereintroduced within the SUMO protein. To determine whether PEI was conjugated at a Cys residue in an extension N-terminal to SUMO, a serine-cysteine-serine (SCS) peptide linker was added and conjugated with PEI600 (see SEQ ID NO: 62). This reagent selectively inhibited the proliferation of KRAS(G12C)-dependent cell lines, MIA PaCa-2 and H358, but not other off-target cell lines,A358 and HEK293T (Fig. 21). The reagent had IC50 values of sub-micromolar. These resultsdemonstrate that PEI conjugated to a Cys residue outside SUMO can produce an effective reagent for cytosolic delivery. Example 10. Low-molecular weight (MW) PEIs are nontoxic to cells, and do not form aggregates with serum albumin.

[0527] High-MW PEIs used for nucleic acid delivery, e.g., PEI 25000 (25K), can be toxic tocells (73-75). The cytotoxicity of PEI 25K against cell lines was confirmed (Fig.22A). By contrast, low-MW PEI molecules used for the technology of the present disclosure showed no toxicity even at 100 µM (Fig.22A), eliminating concerns of potential toxicity due to the use of low-MW PEI.

[0528] High-MW PEI-DNA complexes are also known to aggregate in the presence of serumalbumin (75, 76), which can trigger clearance by the immune system. PEI 25K triggeredaggregation of bovine serum albumin (BSA) was confirmed using SEC (Fig. 22B). By contrast,low-MW PEIs did not affect the SEC profile of BSA, with no evidence of aggregates. Thus, these low-MW PEIs only weakly, if at all, interact with BSA. These results are also consistent with the effectiveness of the reagents of the present disclosure in cell culture media containing 10% fetal bovine serum (Figs.4 and 20). References 1. Teng KW, Tsai ST, Hattori T, Fedele C, Koide A, Yang C, Hou X, Zhang Y, Neel BG, O’Bryan JP, Koide S. Selective and noncovalent targeting of RAS mutants for inhibition and degradation. Nat Commun.2021;12(1):2656. Epub 2021 / 05 / 13. doi: 10.1038 / s41467-021-22969- 5. PubMed PMID: 33976200; PMCID: PMC8113534. 2. 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Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell.1988;55(6):1179-88. doi: 10.1016 / 0092-8674(88)90262-0. PubMed PMID: 2849509. 12. Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell.1988;55(6):1189-93. doi: 10.1016 / 0092-8674(88)90263-2. PubMed PMID: 2849510. 13. Deprey K, Becker L, Kritzer J, Pluckthun A. Trapped! A Critical Evaluation of Methods for Measuring Total Cellular Uptake versus Cytosolic Localization. Bioconjug Chem. 2019;30(4):1006-27. Epub 20190405. doi: 10.1021 / acs.bioconjchem.9b00112. PubMed PMID: 30882208; PMCID: PMC6527423. 14. Stanzl EG, Trantow BM, Vargas JR, Wender PA. Fifteen years of cell-penetrating, guanidinium-rich molecular transporters: basic science, research tools, and clinical applications. Acc Chem Res.2013;46(12):2944-54. Epub 20130522. doi: 10.1021 / ar4000554. PubMed PMID: 23697862; PMCID: PMC3796152. 15. Pei D, Buyanova M. Overcoming Endosomal Entrapment in Drug Delivery. Bioconjug Chem.2019;30(2):273-83. Epub 20181219. doi: 10.1021 / acs.bioconjchem.8b00778. PubMed PMID: 30525488; PMCID: PMC6501178. 16. Smith BA, Daniels DS, Coplin AE, Jordan GE, McGregor LM, Schepartz A. Minimally cationic cell-permeable miniature proteins via alpha-helical arginine display. J Am Chem Soc. 2008;130(10):2948-9. Epub 20080214. doi: 10.1021 / ja800074v. PubMed PMID: 18271592. 17. Sonawane ND, Szoka FC, Jr., Verkman AS. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J Biol Chem. 2003;278(45):44826-31. Epub 20030827. doi: 10.1074 / jbc.M308643200. PubMed PMID: 12944394. 18. Zoltek M, Vazquez Maldonado AL, Zhang X, Dadina N, Lesiak L, Schepartz A. HOPS- Dependent Endosomal Escape Demands Protein Unfolding. ACS Cent Sci.2024;10(4):860-70. Epub 20240326. doi: 10.1021 / acscentsci.4c00016. PubMed PMID: 38680556; PMCID: PMC11046473. 19. Schmit NE, Neopane K, Hantschel O. Targeted Protein Degradation through Cytosolic Delivery of Monobody Binders Using Bacterial Toxins. ACS Chem Biol.2019;14(5):916-24. Epub 2019 / 04 / 27. doi: 10.1021 / acschembio.9b00113. PubMed PMID: 31025848.Attorney Docket No: 243735.000431 20. Liao X, Rabideau AE, Pentelute BL. Delivery of antibody mimics into mammalian cells via anthrax toxin protective antigen. Chembiochem.2014;15(16):2458-66. Epub 2014 / 09 / 25. doi: 10.1002 / cbic.201402290. PubMed PMID: 25250705; PMCID: PMC4498471. 21. Tolcher AW, Sweeney CJ, Papadopoulos K, Patnaik A, Chiorean EG, Mita AC, Sankhala K, Furfine E, Gokemeijer J, Iacono L, Eaton C, Silver BA, Mita M. Phase I and pharmacokinetic study of CT-322 (BMS-844203), a targeted Adnectin inhibitor of VEGFR-2 based on a domain of human fibronectin. Clin Cancer Res.2011;17(2):363-71. Epub 2011 / 01 / 13. doi: 10.1158 / 1078-0432.CCR-10-1411. PubMed PMID: 21224368. 22. Bendele A, Seely J, Richey C, Sennello G, Shopp G. Short communication: renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins. Toxicol Sci. 1998;42(2):152-7. doi: 10.1006 / toxs.1997.2396. PubMed PMID: 9579027. 23. Dennis MS, Zhang M, Meng YG, Kadkhodayan M, Kirchhofer D, Combs D, Damico LA. Albumin binding as a general strategy for improving the pharmacokinetics of proteins. J Biol Chem.2002;277(38):35035-43. Epub 2002 / 07 / 18. doi: 10.1074 / jbc.M205854200. PubMed PMID: 12119302. 24. Dinh-Fricke AV, Hantschel O. Improving the pharmacokinetics, biodistribution and plasma stability of monobodies. Front Pharmacol.2024;15:1393112. doi: 10.3389 / fphar.2024.1393112. PubMed PMID: 38617793; PMCID: PMC7615827. 25. Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, Chernomordik LV, Lebleu B. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem.2003;278(1):585-90. Epub 20021030. doi: 10.1074 / jbc.M209548200. PubMed PMID: 12411431. 26. Loison F, Nizard P, Sourisseau T, Le Goff P, Debure L, Le Drean Y, Michel D. A ubiquitin-based assay for the cytosolic uptake of protein transduction domains. Mol Ther. 2005;11(2):205-14. doi: 10.1016 / j.ymthe.2004.10.010. PubMed PMID: 15668132. 27. Mageean CJ, Griffiths JR, Smith DL, Clague MJ, Prior IA. Absolute Quantification of Endogenous Ras Isoform Abundance. PLoS One.2015;10(11):e0142674. Epub 2015 / 11 / 13. doi: 10.1371 / journal.pone.0142674. PubMed PMID: 26560143; PMCID: PMC4641634. 28. Wallon L, Khan I, Teng KW, Koide A, Zuberi M, Li J, Ketavarapu G, Traaseth NJ, O’Bryan JP, Koide S. Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I / II pocket. Proc Natl Acad Sci U S A.Attorney Docket No: 243735.000431 2022;119(43):e2204481119. Epub 20221017. doi: 10.1073 / pnas.2204481119. PubMed PMID: 36252024; PMCID: PMC9618066. 29. Lanman BA, Allen JR, Allen JG, Amegadzie AK, Ashton KS, Booker SK, Chen JJ, Chen N, Frohn MJ, Goodman G, Kopecky DJ, Liu L, Lopez P, Low JD, Ma V, Minatti AE, Nguyen TT, Nishimura N, Pickrell AJ, Reed AB, Shin Y, Siegmund AC, Tamayo NA, Tegley CM, Walton MC, Wang HL, Wurz RP, Xue M, Yang KC, Achanta P, Bartberger MD, Canon J, Hollis LS, McCarter JD, Mohr C, Rex K, Saiki AY, San Miguel T, Volak LP, Wang KH, Whittington DA, Zech SG, Lipford JR, Cee VJ. Discovery of a Covalent Inhibitor of KRAS(G12C) (AMG 510) for the Treatment of Solid Tumors. J Med Chem.2020;63(1):52-65. Epub 2019 / 12 / 11. doi: 10.1021 / acs.jmedchem.9b01180. PubMed PMID: 31820981. 30. Hallin J, Engstrom LD, Hargis L, Calinisan A, Aranda R, Briere DM, Sudhakar N, Bowcut V, Baer BR, Ballard JA, Burkard MR, Fell JB, Fischer JP, Vigers GP, Xue Y, Gatto S, Fernandez-Banet J, Pavlicek A, Velastagui K, Chao RC, Barton J, Pierobon M, Baldelli E, Patricoin EF, 3rd, Cassidy DP, Marx MA, Rybkin, II, Johnson ML, Ou SI, Lito P, Papadopoulos KP, Janne PA, Olson P, Christensen JG. The KRAS(G12C) Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer Discov.2020;10(1):54-71. Epub 2019 / 10 / 30. doi: 10.1158 / 2159-8290.CD-19- 1167. PubMed PMID: 31658955; PMCID: PMC6954325. 31. Wang X, Allen S, Blake JF, Bowcut V, Briere DM, Calinisan A, Dahlke JR, Fell JB, Fischer JP, Gunn RJ, Hallin J, Laguer J, Lawson JD, Medwid J, Newhouse B, Nguyen P, O’Leary JM, Olson P, Pajk S, Rahbaek L, Rodriguez M, Smith CR, Tang TP, Thomas NC, Vanderpool D, Vigers GP, Christensen JG, Marx MA. Identification of MRTX1133, a Noncovalent, Potent, and Selective KRAS(G12D) Inhibitor. J Med Chem.2022;65(4):3123-33. Epub 2021 / 12 / 11. doi: 10.1021 / acs.jmedchem.1c01688. PubMed PMID: 34889605. 32. Holderfield M, Lee BJ, Jiang J, Tomlinson A, Seamon KJ, Mira A, Patrucco E, Goodhart G, Dilly J, Gindin Y, Dinglasan N, Wang Y, Lai LP, Cai S, Jiang L, Nasholm N, Shifrin N, Blaj C, Shah H, Evans JW, Montazer N, Lai O, Shi J, Ahler E, Quintana E, Chang S, Salvador A, Marquez A, Cregg J, Liu Y, Milin A, Chen A, Ziv TB, Parsons D, Knox JE, Klomp JE, Roth J, Rees M, Ronan M, Cuevas-Navarro A, Hu F, Lito P, Santamaria D, Aguirre AJ, Waters AM, Der CJ, Ambrogio C, Wang Z, Gill AL, Koltun ES, Smith JAM, Wildes D, Singh M. Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy. Nature.Attorney Docket No: 243735.000431 2024;629(8013):919-26. Epub 20240408. doi: 10.1038 / s41586-024-07205-6. PubMed PMID: 38589574. 33. Upadhyaya P, Qian Z, Selner NG, Clippinger SR, Wu Z, Briesewitz R, Pei D. Inhibition of Ras signaling by blocking Ras-effector interactions with cyclic peptides. Angew Chem Int Ed Engl.2015;54(26):7602-6. Epub 20150507. doi: 10.1002 / anie.201502763. PubMed PMID: 25950772; PMCID: PMC4591930. 34. Haley RM, Chan A, Billingsley MM, Gong N, Padilla MS, Kim EH, Wang H, Yin D, Wangensteen KJ, Tsourkas A, Mitchell MJ. Lipid Nanoparticle Delivery of Small Proteins for Potent In vivo RAS Inhibition. ACS Appl Mater Interfaces.2023;15(18):21877-92. Epub 20230428. doi: 10.1021 / acsami.3c01501. PubMed PMID: 37115558; PMCID: PMC10727849. 35. Jones SW, Christison R, Bundell K, Voyce CJ, Brockbank SM, Newham P, Lindsay MA. Characterisation of cell-penetrating peptide-mediated peptide delivery. Br J Pharmacol. 2005;145(8):1093-102. doi: 10.1038 / sj.bjp.0706279. PubMed PMID: 15937518; PMCID: PMC1576229. 36. Lo SL, Wang S. An endosomolytic Tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection. Biomaterials. 2008;29(15):2408-14. Epub 20080304. doi: 10.1016 / j.biomaterials.2008.01.031. PubMed PMID: 18295328. 37. Malakhov MP, Mattern MR, Malakhova OA, Drinker M, Weeks SD, Butt TR. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics.2004;5(1-2):75-86. PubMed PMID: 15263846. 38. Erazo-Oliveras A, Najjar K, Dayani L, Wang TY, Johnson GA, Pellois JP. Protein delivery into liv...

Claims

1. Attorney Docket No: 243735.000431 CLAIMS1. A protein conjugate comprising(i) a protein carrier conjugated to a cationic polymer; and (ii) a polypeptide cargo, wherein the protein carrier is linked to the polypeptide cargo via a peptide bond to form a contiguous polypeptide.

2. The protein conjugate of claim 1, wherein the contiguous polypeptide is recombinantlyproduced.

3. The protein conjugate of claim 1, wherein the contiguous polypeptide is chemically orenzymatically produced.

4. The protein conjugate of any one of claims 1-3, wherein the protein carrier is linked to thepolypeptide cargo via a proteolytic cleavable linker.

5. The protein conjugate of claim 4, wherein the proteolytic cleavable linker is recognized by anintracellular protease.

6. The protein conjugate of any one of claims 1-5, wherein the protein carrier comprises an N-terminal polypeptide or a C-terminal polypeptide.

7. The protein conjugate of claim 6, wherein the protein carrier comprises an N-terminalpolypeptide.

8. The protein conjugate of any one of claims 1-7, wherein the protein carrier and / or thepolypeptide cargo do not contain native cysteine residue(s) or have been engineered to remove native cysteine residue(s).

9. The protein conjugate of any one of claims 1-8, wherein the protein carrier is modified tointroduce one or more cysteine residues for site-specific conjugation with the cationic polymer.Attorney Docket No: 243735.00043110. The protein conjugate of claim 9, wherein the protein carrier is modified to introduce one,two, three, or four cysteine residues.

11. The protein conjugate of any one of claims 9-10, wherein the one or more cysteine residuesare introduced to the protein carrier outside the interface between the protein carrier and the intracellular protease.

12. The protein conjugate of any one of claims 9-11, wherein the one or more cysteine residuesare introduced into the N-terminal polypeptide of the protein carrier.

13. The protein conjugate of claim 13, wherein the one or more cysteine residues are introducedinto the N-terminal polypeptide as a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide.

14. The protein conjugate of any one of claims 9-13, wherein the protein carrier is conjugated toone or more cationic polymers at the one or more cysteine residues.

15. The protein conjugate of any one of claims 1-14, wherein the protein carrier comprises aubiquitin-like protein (UBL), a functional fragment, or derivative thereof.

16. The protein conjugate of claim 15, wherein the one or more cysteine residues are introducedto the UBL.

17. The protein conjugate of any one of claims 7-15, wherein the N-terminal polypeptide of theprotein carrier is located at amino terminus of the UBL.

18. The protein conjugate of any one of claims 5-17, wherein the intracellular protease is a UBL-specific protease.

19. A protein conjugate comprising(i) a protein carrier conjugated to a cationic polymer; andAttorney Docket No: 243735.000431 (ii) a cargo, wherein the protein carrier is operably linked to the cargo, and wherein the protein carrier comprises a ubiquitin-like protein (UBL), a functional fragment, or derivative thereof.

20. The protein conjugate of claim 19, wherein the cargo is a polypeptide cargo.

21. The protein conjugate of claim 20, wherein the protein carrier is linked to the cargo via apeptide bond to form a contiguous polypeptide.

22. The protein conjugate of any one of claims 19-21, wherein the cargo is linked to the UBL viaa proteolytic cleavable linker.

23. The protein conjugate of claim 22, wherein the UBL is cleaved from the cargo by a UBL-specific protease.

24. The protein conjugate of any one of claims 19-23, wherein the protein carrier furthercomprises an N-terminal polypeptide or a C- terminal polypeptide.

25. The protein conjugate of claim 24, wherein the protein carrier comprises an N-terminalpolypeptide.

26. The protein conjugate of claim 25, wherein the N-terminal polypeptide is located at aminoterminus of the UBL.

27. The protein conjugate of any one of claims 19-26, wherein the UBL has been engineered toremove native cysteine residue(s).

28. The protein conjugate of claim 19-27, wherein the protein carrier is modified to introduceone, or more cysteine residues.

29. The protein conjugate of any one of claims 19-28, wherein the UBL is modified to introduceone or more cysteine residues outside the interface between the UBL and its cognate UBL specific protease.Attorney Docket No: 243735.00043130. The protein conjugate of any one of claims 19-29, wherein one or more cysteine residues areintroduced into the N-terminal polypeptide of the protein carrier.

31. The protein conjugate of claim 30, wherein the one or more cysteine residues are introducedinto the N-terminal polypeptide as a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide.

32. The protein conjugate of any one of claims 19-31, wherein the protein carrier is conjugated toone or more cationic polymers at the one or more cysteine residues.

33. The protein conjugate of any one of claims 15-32, wherein the UBL is selected fromubiquitin, a small ubiquitin-like modifier (SUMO) protein, neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8), autophagy-related protein 8 (ATG8), autophagy-related 12 (ATG12), ), human leukocyte antigen (HLA)-F adjacent transcript 10 or Ubiquitin D (FAT10), interferon (IFN)-stimulated gene 15 (ISG15), ubiquitin related modifier 1 (URM1), or ubiquitin fold modifier 1 (UFM1).

34. The protein conjugate of any one of claims 15-33, wherein the UBL comprises or consists ofan amino acid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72.

35. The protein conjugate of claim 33 or claim 34, wherein the SUMO protein is SUMO1,SUMO2, SUMO3, or SUMO4.

36. The protein conjugate of claim 35, wherein the SUMO protein comprises or consists of anamino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53-55 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53-55.Attorney Docket No: 243735.00043137. The protein conjugate of any one of claims 33-36, wherein the SUMO protein is SUMO1.

38. The protein conjugate of claim 37, wherein SUMO1 comprises or consists of an amino acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53.

39. The protein conjugate of claim 37 or claim 38, wherein the cysteine residue at position 52 ofSUMO1 has been mutated to a non-cysteine residue.

40. The protein conjugate of claim 39, wherein the cysteine residue at position 52 of SUMO1 hasbeen mutated to an alanine.

41. The protein conjugate of any one of claims 37-40, wherein SUMO1 is modified to introduceone or more cysteine residues by mutating the residue at the amino acid position 33, amino acid position 46, amino acid position 53, amino acid position 85, and / or amino acid position 50 to cysteine.

42. The protein conjugate of claim 41, wherein SUMO1 comprises or consists of an amino acidsequence set forth in SEQ ID NO: 8 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 8.

43. The protein conjugate of any one of claims 33-42, wherein the C-terminal or proximal to theC-terminal sequence of the SUMO protein comprises Gly-Gly.

44. The protein conjugate of any one of claims 18 and 23-43, wherein the UBL-specific proteaseis a ubiquitin-specific protease (USP) or SUMO-specific protease (SENP).

45. The protein conjugate of claim 44, wherein the SENP is SENP1, SENP2, SENP3, SENP5,SENP6, and / or SENP7.Attorney Docket No: 243735.00043146. The protein conjugate of any one of claims 1-45, wherein the cationic polymer is apolyamine.

47. The protein conjugate of any one of claims 1-46, wherein the cationic polymer is selectedfrom polyethylenimine (PEI), poly(L-lysine) (PLL), poly(D-lysine) (PDL), polyamidoamine (PAA), Poly(amino-co-ester)s (PAEs), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), poly(propylene imine) (PPI), poly-L-arginine (PLA), poly-guanidine, poly-D- arginine, poly(D,L-lysine hydrobromide), poly-L-ornithine, PEI-Gua, PEI-Arg, PEI-His, PEI-Trp, or derivatives and combinations thereof.

48. The protein conjugate of any one of claims 1-47, wherein the cationic polymer is PEI.

49. The protein conjugate of claim 48, wherein the PEI is a linear PEI or branched PEI.

50. The protein conjugate of claim 49, wherein the branched PEI is a short-chain PEI.

51. The protein conjugate of any one of claims 47-50, wherein the PEI has a molecular weight ofless than 2000 Da.

52. The protein conjugate of claim 51, wherein the PEI has a molecular weight ranging fromabout 600 Da to about 1800Da.

53. The protein conjugate of claim 51 or claim 52, wherein the PEI has a molecular weight ofabout 600 Da (PEI600) or 1200 Da (PEI1200).

54. The protein conjugate of claim 53, wherein the PEI has a molecular weight of about 600 Da(PEI600).

55. The protein conjugate of claim 53, wherein the PEI is branched PEI600 (bPEI600) orbranched PEI1200 (bPEI1200).Attorney Docket No: 243735.00043156. The protein conjugate of claim 55, wherein the PEI is branched PEI600 (bPEI600).

57. The protein conjugate of any one of claims 1-56, wherein the protein carrier is conjugated tothe cationic polymer via a linker.

58. The protein conjugate of claim 57, wherein the linker is a heterobifunctional crosslinker.

59. The protein conjugate of claim 58, wherein the linker comprises an electrophile selected fromhaloacetophenone, epoxide, haloacetamide, maleimide, isothiocyanate, vinyl sulfone, nitrile, iminonitrile, hydrazone, butynoate, acrylester, acrylonitrile, acrylamide, benzisothiazolone, and N-hydroxy(methyl)phthalimide.

60. The protein conjugate of claim 58 or claim 59, wherein the linker comprises a N-hydroxysuccinimide (NHS) ester and a maleimide group.

61. The protein conjugate of any one of claims 58-60, wherein the linker is selected from N-alpha-maleimidoacetoxyl succinimide ester (AMAS), N-beta-maleimidopropyl- oxysuccinimide ester (BMPS), N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-epsilon-malemidocaproyl- oxysuccinimide ester (EMCS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH), succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), N-(11- maleimidoundecanoyloxy)succinimide (KMUS), or derivatives and combinations thereof.

62. The protein conjugate of any one of claims 58-61, wherein the linker is N-alpha-maleimidoacetoxyl succinimide ester (AMAS).

63. The protein conjugate of any one of claims 1-62, wherein the protein conjugate is capable ofpenetrating into the cytoplasm of a mammalian cell.Attorney Docket No: 243735.00043164. The protein conjugate of any one of claims 1-63, wherein the cargo interacts with anintracellular target.

65. The protein conjugate of claim 64, wherein the intracellular target is selected from ratsarcoma virus (RAS), Src homology 2 domain-containing phosphatase 2 (SHP2), Aurora A kinase, Src-family kinases, signal transducer and activator of transcription 3 (STAT3), PR domain containing 14 (PRDM14), WD repeat domain 5 (WDR5), mixed lineage kinase domain-like protein (MLKL), p53, and ABL kinase.

66. The protein conjugate of claim 65, wherein the RAS is Kirsten rat sarcoma viral oncogenehomolog (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS), or Harvey rat sarcoma viral oncogene homolog (HRAS).

67. The protein conjugate of any one of claims 1-18 and 20-66, wherein the polypeptide cargo isselected from a monobody, a designed ankyrin repeat protein (DARPin), an Anticalin, an affilin, an affibody, a peptide, an antibody or antigen-binding fragment, an enzyme, or derivatives and combinations thereof.

68. The protein conjugate of claim 67, wherein the polypeptide cargo is selected from amonobody, a designed ankyrin repeat protein (DARPin), or derivatives thereof.

69. The protein conjugate of claim 68, wherein the polypeptide cargo is tandem monobodiescomprising two or more monobody units.

70. The protein conjugate of any one of claims 67-69, wherein the monobody or DARPin targetsKRAS, HRAS, NRAS, Aurora A kinase, ABL kinase, a Src-family kinase, SHP2, STAT3, PRDM14, WDR5, MLKL, or p53.

71. The protein conjugate of claim 70, wherein the monobody is selected from 12VC1, NS1,12D3, 12D4, 12D5, AS25, AS27, HA4, 7C12, Nsa1, NSa5, CS3, S14, S4, Mb1, Mb2, Mb3,Mb4, Mb5, Mb6, Mb19, Mb20, Mb24, Mb27, Mb32, Mb33, MS3-6, Mb13, R15, Fgr_1,Attorney Docket No: 243735.000431 Fgr_2, Hck_1, Hck_2, Lck_1, Lck_3, Lyn_2, Lyn_4, Src_2, Yes_1, Yes_3, MLKL_26, MLKL_27, MLKL_32, MLKL_33, and MLKL_37.

72. The protein conjugate of any one of claims 67-71, wherein the monobody comprises orconsists of an amino acid sequence set forth in any one of SEQ ID NOs:14-52 and 69-70 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:14-52 and 69-70.

73. The protein conjugate of claim 70, wherein the DARPin is K27, C10, or C10-H82R.

74. The protein conjugate of claim 67, wherein the antibody or antigen-binding fragment thereofis selected from a single chain antibody, a single domain antibody, a variable heavy homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, a monoclonal antibody, a bispecific antibody or antigen-binding fragment thereof, a bi-epitopic antibody or antigen-binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a VH domain, a monovalent antibody or a bivalent antibody.

75. The protein conjugate of claim 67, wherein the peptide is a stapled peptide or bicyclicpeptide.

76. The protein conjugate of any one of claims 1-75, further comprising one or more additionalmoieties.

77. The protein conjugate of claim 76, wherein the additional moiety is a His6-tag (SEQ ID NO:12).

78. The protein conjugate of claim 77, wherein the N-terminal polypeptide of the protein carriercomprises the His6-tag (SEQ ID NO: 12).

79. The protein conjugate of claim 76, wherein the additional moiety is a V5 tag, wherein the V5tag is at a C-terminus of the polypeptide cargo.Attorney Docket No: 243735.00043180. The protein conjugate of claim 76, wherein the additional moiety is a cell-targeting moiety,wherein the N-terminal polypeptide of the protein carrier comprises the cell-targeting moiety.

81. The protein conjugate of claim 80, wherein the cell-targeting moiety is a polypeptide thatbinds specifically to a cell-surface protein.

82. The protein conjugate of any one of claim 1-81, further comprising an albumin-bindingdomain.

83. The protein conjugate of claim 82, wherein the albumin-binding domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO:66 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of the SEQ ID NO:66.

84. The protein conjugate of any one of claims 1-18 and 21-83, wherein the contiguouspolypeptide comprises or consists of the sequence set forth in any one of SEQ ID Nos: 9, 11, 62-65, and 71 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, 62-65, and 71.

85. The protein conjugate of claim 84, wherein the contiguous polypeptide comprises or consistsof the sequence set forth in SEQ ID NO: 62 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62.

86. A nucleic acid molecule encoding the contiguous polypeptide in the protein conjugate of anyone of claims 1-18 and 21-85.

87. The nucleic acid molecule of claim 86, comprising (i) a nucleotide sequence encoding theprotein carrier; and (ii) a nucleotide sequence encoding the polypeptide cargo.Attorney Docket No: 243735.00043188. The nucleic acid molecule of claim 86 or claim 87, comprising or consisting of the nucleotidesequence set forth in SEQ ID NO: 10 or a sequence having at least 80% sequence identity to a nucleotide acid nucleotide sequence set forth in SEQ ID NO: 10.

89. A nucleic acid molecule comprising a nucleotide sequence encoding the protein carrier of theprotein conjugate of any one of claims 1-85.

90. The nucleic acid molecule of claim 89, wherein the nucleotide sequence encoding the proteincarrier comprises or consists of the sequence set forth in SEQ ID NO: 61 or a sequence having at least 80% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 61.

91. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide cargo ofthe protein conjugate of any one of claims 1-18 and 20-85.

92. The nucleic acid molecule of any one of claims 86-91, wherein the nucleic acid molecule is aDNA molecule.

93. The nucleic acid molecule of any one of claims 86-91, wherein the nucleic acid molecule isan RNA molecule.

94. A polypeptide encoded by the nucleic acid molecule of any one of claims 86-93.

95. A recombinant vector comprising the nucleic acid molecule of any one of claims 86-93.

96. An isolated host cell comprising the nucleic acid molecule of any one of claims 86-93 or thevector of claim 95.

97. The isolated host cell of claim 96, wherein the isolated host cell is a bacterial cell, a yeastcell, an insect cell, or a mammalian cell.

98. The isolated host cell of claim 97, wherein the bacterial cell is Escherichia coli (E. coli).Attorney Docket No: 243735.00043199. A method of producing a protein conjugate, comprisinga) generating a contiguous polypeptide comprising (i) a protein carrier and (ii) a polypeptide cargo; and b) conjugating a cationic polymer to the protein carrier to form a protein conjugate.

100. The method of claim 99, wherein the contiguous polypeptide is generated recombinantly.

101. The method of claim 100, wherein the contiguous polypeptide is generated by culturing ahost cell comprising the nucleic acid molecule of any one of claims 86-93 or the vector of claim 95 to express the contiguous polypeptide.

102. The method of claim 101, wherein the contiguous polypeptide is generated by attachingthe polypeptide cargo to the protein carrier chemically or enzymatically.

103. The method of any one of claims 99-102, further comprising purifying the contiguouspolypeptide from the host cell after step a).

104. A method of producing a protein conjugate, comprisinga) conjugating a cationic polymer to a protein carrier; and b) attaching a polypeptide cargo to the protein carrier chemically or enzymatically to form a contiguous polypeptide, thereby forming a protein conjugate.

105. The method of any one of claims 99-104, further comprising purifying the proteinconjugate after step b).

106. The method of any one of claims 99-105, wherein the protein carrier is linked to thepolypeptide cargo via a proteolytic cleavable linker.

107. The method of claim 106, wherein the proteolytic cleavable linker is recognized by anintracellular protease.Attorney Docket No: 243735.000431108. The method of any one of claims 99-107, wherein the protein carrier comprises an N-terminal polypeptide or a C-terminal polypeptide.

109. The method of claim 108, wherein the protein carrier comprises an N-terminalpolypeptide.

110. The method of any one of claims 99-109, wherein the protein carrier and / or thepolypeptide cargo do not contain native cysteine residue(s) or have been engineered to remove native cysteine residue(s).

111. The method of any one of claims 99-110, wherein the protein carrier is modified tointroduce one or more cysteine residues for site-specific conjugation with the cationic polymer.

112. The method of claim 111, wherein the protein carrier is modified to introduce one, two,three or four cysteine residues.

113. The method of claim 111 or claim 112, wherein the one or more cysteine residues areintroduced to the protein carrier outside the interface between the protein carrier and the intracellular protease.

114. The method of any one of claims 111-113, wherein the one or more cysteine residues areintroduced into the N-terminal polypeptide of the protein carrier.

115. The method of claim 114, wherein the one or more cysteine residues are introduced intothe N-terminal polypeptide as a serine-cysteine-serine (SCS), SCPCSG (SEQ ID NO: 67), or SCSG (SEQ ID NO: 68) peptide.

116. The method of any one of claims 111-115, wherein the protein carrier is conjugated toone or more cationic polymers at the one or more cysteine residues.Attorney Docket No: 243735.000431117. The method of any one of claims 99-116, wherein the protein carrier comprises aubiquitin-like protein (UBL), a functional fragment, or derivative thereof.

118. The method of any one of claims 111-113 and 117, wherein the one or more cysteineresidues are introduced to the UBL.

119. The method of claim 117 or claim 118, wherein the ubiquitin-like protein (UBL) isselected from ubiquitin, a small ubiquitin-like modifier (SUMO) protein, neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8), autophagy-related protein 8 (ATG8), autophagy-related 12 (ATG12), human leukocyte antigen (HLA)-F adjacent transcript 10 or Ubiquitin D (FAT10), interferon (IFN)-stimulated gene 15 (ISG15), ubiquitin related modifier 1 (URM1), or ubiquitin fold modifier 1 (UFM1).

120. The method of claim 119, wherein the UBL comprises or consists of an amino acidsequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72 or a sequence having at least 70 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8, 53-60, and 72.

121. The method of claim 119, wherein the SUMO protein is SUMO1, SUMO2, SUMO3, orSUMO4.

122. The method of claim 120, wherein the SUMO protein comprises or consists of an aminoacid sequence set forth in any one of SEQ ID NOs: 1-8 and 53-55 or at least 70 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-8 and 53-55.

123. The method of any one of claims 118-122, wherein the protein carrier comprisesSUMO1, a functional fragment, or derivative thereof.

124. The method of claim 122, wherein SUMO1 comprises or consists of an amino acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53 or a sequence having at least 70Attorney Docket No: 243735.000431 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 53.

125. The method of claim 123 or claim 124, wherein the cysteine residue at position 52 ofSUMO1 has been mutated to a non-cysteine residue.

126. The method of claim 125, wherein the cysteine residue at position 52 of SUMO1 hasbeen mutated to an alanine.

127. The method of any one of claims 123-126, wherein SUMO1 is modified to introduce oneor more cysteine residues at the amino acid position 33, amino acid position 46, amino acid position 53, amino acid position 85, and / or amino acid position 50.

128. The method of claim 127, wherein SUMO1 comprises or consists of an amino acidsequence set forth in SEQ ID NO: 8 or at least 70 % sequence identity to an amino acid sequence set forth in SEQ ID NO: 8.

129. The method of any one of claims 119-128, wherein the C-terminal or proximal to the C-terminal sequence of the SUMO protein comprises Gly-Gly.

130. The method of any one of claims 107-129, wherein the intracellular protease is a UBL-specific protease.

131. The method of claim 130, wherein the UBL-specific protease is a ubiquitin-specificprotease (USP) or SUMO-specific protease (SENP).

132. The method of claim 131, wherein the SENP is SENP1, SENP2, SENP3, SENP5,SENP6, and / or SENP7.

133. The method of any one of claims 99-132, wherein the cationic polymer is a polyamine.Attorney Docket No: 243735.000431134. The method of any one of claims 99-133, wherein the cationic polymer is selected frompolyethylenimine (PEI), poly(L-lysine) (PLL), poly(D-lysine) (PDL), polyamidoamine (PAA), Poly(amino-co-ester)s (PAEs), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), poly(propylene imine) (PPI), poly-L-arginine (PLA), poly-guanidine, poly-D- arginine, poly(D,L-lysine hydrobromide), poly-L-ornithine, PEI-Gua, PEI-Arg, PEI-His, PEI-Trp, or derivatives and combinations thereof.

135. The method of any one of claims 99-134, wherein the cationic polymer is PEI.

136. The method of claim 135, wherein the PEI is a linear PEI or branched PEI.

137. The method of claim 136, wherein the branched PEI is a short-chain PEI.

138. The method of any one of claims 134-137, wherein the PEI has a molecular weight ofless than 2000 Da.

139. The method of claim 138, wherein the PEI has a molecular weight ranging from about600 Da to about 1800Da.

140. The method of claim 138 or claim 139, wherein the PEI has a molecular weight of about600 Da (PEI600) or 1200 Da (PEI1200).

141. The method of claim 140, wherein the PEI has a molecular weight of about 600 Da(PEI600).

142. The method of claim 140, wherein the PEI is branched PEI600 (bPEI600) or branchedPEI1200 (bPEI1200).

143. The method of claim 142, wherein the PEI is branched PEI600 (bPEI600).Attorney Docket No: 243735.000431144. The method of any one of claims 99-143, wherein the protein carrier is conjugated to thecationic polymer via a linker.

145. The method of claim 144, wherein the linker is a heterobifunctional crosslinker.

146. The method of claim 145, wherein the linker comprises an electrophile selected fromhaloacetophenone, epoxide, haloacetamide, maleimide, isothiocyanate, vinyl sulfone, nitrile, iminonitrile, hydrazone, butynoate, acrylester, acrylonitrile, acrylamide, benzisothiazolone, and N-hydroxy(methyl)phthalimide.

147. The method of claim 145, wherein the linker comprises a N-hydroxysuccinimide (NHS)ester and a maleimide group.

148. The method of any one of claims 145-147, wherein the linker is selected from N-alpha-maleimidoacetoxyl succinimide ester (AMAS), N-beta-maleimidopropyl-oxysuccinimide ester (BMPS), N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS), m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-epsilon-malemidocaproyl- oxysuccinimide ester (EMCS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH), succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), N-(11- maleimidoundecanoyloxy)succinimide (KMUS), or derivatives and combinations thereof.

149. The method of any one of claims 144-148, wherein the linker is N-alpha-maleimidoacetoxyl succinimide ester (AMAS).

150. The method of any one of claims 99-149, wherein the cationic polymer is conjugated tothe protein carrier at a pH of 6-8.

151. The method of any one of claims 99-150, wherein the polypeptide cargo interacts with anintracellular target.Attorney Docket No: 243735.000431152. The method of claim 151, wherein the intracellular target is selected from rat sarcomavirus (RAS), Src homology 2 domain-containing phosphatase 2 (SHP2), Aurora A kinase, Src-family kinases, signal transducer and activator of transcription 3 (STAT3), PR domain containing 14 (PRDM14), WD repeat domain 5 (WDR5), mixed lineage kinase domain-like protein (MLKL), p53, and ABL kinase.

153. The method of claim 152, wherein RAS is Kirsten rat sarcoma viral oncogene homolog(KRAS), neuroblastoma RAS viral oncogene homolog (NRAS), or Harvey rat sarcoma viral oncogene homolog (HRAS).

154. The method of any one of claims 99-153, wherein the polypeptide cargo is selected froma monobody, a designed ankyrin repeat protein (DARPin), an Anticalin, an affilin, an affibody, a peptide, an antibody or antigen-binding fragment, an enzyme, or derivatives and combinations thereof.

155. The method of claim 154, wherein the polypeptide cargo is selected from a monobody, adesigned ankyrin repeat protein (DARPin), or derivatives thereof.

156. The method of claim 155, wherein the polypeptide cargo is tandem monobodiescomprising two or more monobody units.

157. The method of any one of claims 154-156, wherein the monobody or DARPin targetsKRAS, HRAS, NRAS, Aurora A kinase, ABL kinase, a Src-family kinase, SHP2, STAT3, PRDM14, WDR5, MLKL, or p53.

158. The method of claim 157, wherein the monobody is selected from 12VC1, NS1, 12D3,12D4, 12D5, AS25, AS27, HA4, 7C12, Nsa1, NSa5, CS3, S14, S4, Mb1, Mb2, Mb3, Mb4,Mb5, Mb6, Mb19, Mb20, Mb24, Mb27, Mb32, Mb33, MS3-6, Mb13, R15, Fgr_1, Fgr_2, Hck_1, Hck_2, Lck_1, Lck_3, Lyn_2, Lyn_4, Src_2, Yes_1, Yes_3, MLKL_26, MLKL_27, MLKL_32, MLKL_33, and MLKL_37.Attorney Docket No: 243735.000431159. The method of claim 157 or claim 158, wherein the monobody comprises or consists ofan amino acid sequence set forth in any one of SEQ ID NOs:14-52 and 69-70 or a sequence having at least 80 % sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:14-52 and 69-70.

160. The method of claim 157, wherein the DARPin is K27, C10, or C10-H82R.

161. The method of claim 154, wherein the antibody or antigen-binding fragment thereof isselected from a single chain antibody, a single domain antibody, a variable heavy homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, a monoclonal antibody, a bispecific antibody or antigen-binding fragment thereof, a bi-epitopic antibody or antigen- binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, an scFv, a VH domain, a monovalent antibody or a bivalent antibody.

162. The method of claim 154, wherein the peptide is a stapled peptide or bicyclic peptide.

163. The method of any one of claims 99-162, wherein the contiguous polypeptide comprisesor consists of the sequence set forth in any one of SEQ ID Nos: 9, 11, 62-65, and 71 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 9, 11, 62-65, and 71.

164. The method of claim 163, wherein the contiguous polypeptide comprises or consists ofthe sequence set forth in SEQ ID NO: 62 or a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62.

165. A protein conjugate produced by the method of any one of claims 99-164.

166. A pharmaceutical composition comprising the protein conjugate of any one of claims 1-85 and 165, the nucleic acid molecule of any one of claims 86-93, the vector of claim 95, orAttorney Docket No: 243735.000431 the host cell of any one of claims 96-98, and a pharmaceutically acceptable carrier and / or excipient.

167. A method of delivering a cargo into the cytoplasm of a cell, comprising a) producing aprotein conjugate comprising the cargo according to the method of any one of claims 99-164; and b) contacting the cell with the produced protein conjugate.

168. A method of targeting an intracellular target in a cell, comprising a) producing a proteinconjugate comprising a cargo that interacts with the intracellular target according the method of any one of claims 99-164; and b) contacting the cell with the produced protein conjugate.

169. The method of claim 167 or claim 168, wherein the cell is a mammalian cell.

170. The method of claim 169, wherein the mammalian cell is an adherent cell.

171. The method of claim 169, wherein the mammalian cell is a non-adherent cell.

172. The method of any one of claims 167-171, wherein the method occurs ex vivo.

173. The method of any one of claims 167-171, wherein the method occurs in vivo.

174. A method of preventing, treating, or diagnosing a disease in a subject in need thereof,comprising administering to the subject the protein conjugate of any one of claims 1-85 and 165, or the pharmaceutical composition of claim 166, wherein the cargo interacts with a target associated with disease.

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