WO2025101862A1 - Liants d'anticorps à domaine unique de myc - Google Patents

Liants d'anticorps à domaine unique de myc Download PDF

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WO2025101862A1
WO2025101862A1 PCT/US2024/055071 US2024055071W WO2025101862A1 WO 2025101862 A1 WO2025101862 A1 WO 2025101862A1 US 2024055071 W US2024055071 W US 2024055071W WO 2025101862 A1 WO2025101862 A1 WO 2025101862A1
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seq
polypeptide
optionally
sequence
myc
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Karl Dane Wittrup
Heng Li
Marius S. POP
Christopher M. WILFONG
Juwina WIJAYA
Kerry E. BENENATO
Bradley LUNDE
Alec DESAI
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Axelyf Ehf
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Axelyf Ehf
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/80—Immunoglobulins specific features remaining in the (producing) cell, i.e. intracellular antibodies or intrabodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal

Definitions

  • aspects of the present disclosure related to novel polypeptides comprising single domain antibody binders of Myc (e.g., c-Myc). Aspects of the present disclosure also relate generally to bifunctional polypeptides that promote ubiquitin-mediated proteasomal degradation of desired biological target(s), for example Myc, and uses such as for the treatment of a disease associated with the biological target(s). Aspects of the present disclosure also relate generally to dual-specific bifunctional polypeptides that promote ubiquitin-mediated proteasomal degradation of desired biological targets, for example Myc and another target, and uses such as for the treatment of a disease associated with the biological targets.
  • BACKGROUND Conventional therapeutics, such as small molecule and antibody inhibitors, operate by blocking or otherwise modulating the function of a therapeutic target (e.g., blocking an enzymatic or transcription promoting function). While many therapeutics belonging to this category have shown to be effective, confounding effects may arise as the therapeutic target is still present within the cellular milieu.
  • Molecular glues and proteolysis targeting chimeras are alternative classes of therapeutics that involve small molecule compounds that recruit E3 ubiquitin ligases to a therapeutic target, thereby inducing proteolysis of the therapeutic target through the endogenous proteasomal degradation machinery. The physical degradation of the target drives a therapeutic effect through the elimination of the dysfunctional and/or disease-associated target.
  • target binding moieties that can function in the intracellular space, and can be employed in compounds designed to utilize a proteasomal degradation tactic to degrade target proteins, for example in the intracellular space.
  • sdAb anti-Myc single domain antibody
  • HcAb heavy chain antibody
  • These anti-Myc sdAbs can be expressed intracellularly and can be employed as part of a designed to utilize a proteasomal degradation tactic to degrade Myc, and optionally one or more additional target proteins.
  • bifunctional polypeptides comprising the anti-Myc sdAb that promote proteasome-mediated degradation of Myc.
  • the bifunctional polypeptide comprises a) an anti-Myc sdAb; and b) a ubiquitin-proteasome system recruiting domain (URD).
  • URD ubiquitin-proteasome system recruiting domain
  • proximity of the bifunctional polypeptide to the target protein through binding of the targeting moiety induces ubiquitination of the target protein (Myc) via the URD, thereby promoting proteosome-mediated degradation of the target protein.
  • dual-specific bifunctional polypeptides that promote proteasome-mediated degradation of Myc and at least one second target protein.
  • the dual- specific bifunctional polypeptide comprises a) an anti-Myc sdAb (target binding domain) that is capable of binding to at least Myc; b) a second targeting moiety that is capable of binding to at least one second target protein; and c) a URD.
  • the polypeptides further comprise d) a first linker peptide; wherein the first linker peptide is positioned between the first targeting moiety and the URD; and/or optionally e) a second linker peptide, wherein the second linker peptide is positioned between the second targeting moiety and the URD.
  • dual-specific bifunctional polypeptide is in a “contralateral” configuration in which the first and second target binding domains are on either side of the URD, i.e., one is on the C-terminal side of the URD, and one is on the N-terminal side of the URD.
  • dual-specific bifunctional polypeptide is in a “tandem” configuration in which the first and second target binding domains are both on the same side of the URD, i.e., both are on the C-terminal side of the URD, or both are on the N-terminal side of the URD.
  • a linker peptide (a first or a second linker peptide) is positioned between the two target binding domains, and/or between a targeting moiety and the URD.
  • the bifunctional polypeptide or dual-specific bifunctional polypeptide comprises one or more localization sequences to direct the polypeptide to a preferred subcellular compartment (e.g., nucleus).
  • polynucleotides encoding for any of the polypeptides comprising or consisting of the anti-Myc sdAb, the bifunctional or dual-specific bifunctional polypeptides, or any portions thereof, provided herein.
  • pharmaceutical compositions comprising any of the polypeptides comprising or consisting of the anti-Myc sdAb, or the bifunctional or dual- specific bifunctional polypeptides provided herein and one or more pharmaceutically acceptable excipients, carriers, or diluents.
  • methods of treating a subject are also disclosed herein.
  • the methods comprise administering any of the polypeptides comprising or consisting of the anti- Myc sdAb, the bifunctional or dual-specific bifunctional polypeptides, polynucleotides, or pharmaceutical compositions provided herein to a subject in need thereof.
  • the methods may be for the treatment of a cancer.
  • Also disclosed herein are methods of reducing the amount of a target protein in a cell.
  • the methods comprise contacting the cell with any of polypeptides comprising or consisting of the anti-Myc sdAb, the bifunctional or dual-specific bifunctional polypeptides, polynucleotides, or pharmaceutical compositions provided herein.
  • the cell is in a subject, and the polypeptides comprising or consisting of the anti- Myc sdAb, the bifunctional or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition is administered to the subject.
  • the cell is contacted ex vivo, and after contacting the cell with the polypeptides comprising or consisting of the anti-Myc sdAb, the bifunctional or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition, the cell is administered to a subject, optionally wherein the subject is also the source of the cell (e.g., in an adoptive cell therapy).
  • sdAb anti-Myc single domain antibody
  • HcAb heavy chain antibody
  • the sdAb optionally a VHH domain, comprises a CDR3 comprising or consisting of the amino acid sequence AX1X2X3X4X5X6X7X8X9X10X11X12EX13DY (SEQ ID NO: 299), wherein X1 is A, H, Y, I, or T; X2 is L or I; X3 is G, D or P; X4 is D, Q, G or E; X5 is Y, E or I; X6 is G, L or S; X7 is D,T, S or G; X 8 is A, G, F, or no amino acid; X 9 is I, G, or no amino acid; X 10 is D, G, T or L; X 11 is Y, A, F or G; X12 is S, D or N; and X13 is A, H, G or Y; or optionally a conservative substitution of any of the foregoing; wherein the C
  • the polypeptide of embodiment 1 or 2, wherein the sdAb, optionally a V H H domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 177, 185, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273, 281, 289; a CDR2 comprising or consisting of any one of SEQ ID NOs: 178, 186, 194, 202, 210, 218, 226, 234, 242, 250, 266, 274, 282 or 290; and a CDR3 comprising or consisting of any one of SEQ ID Nos: 179, 195, 211, 219, 251, 267 or 283; optionally wherein the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: a.) SEQ ID NOs: 177, 178, and 179; b.) SEQ ID NOs: 185, 186, and 187; c.) SEQ ID
  • sdAb optionally a VHH domain, comprises: a CDR1 comprising or consisting of FX 1 FX 2 X 3 X 4 X 5 MX 6 (SEQ ID NO: 297), wherein X 1 is T or D; X 2 is A, S, E, G or D; X 3 is D, T, G, E, V or S; X 4 is T, M, Q, A, H, Y, V or N; X5 is D, P or A; and X6 is G or S; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of AIX 1 X 2 X 3 X 4 X 5 X 6 X 7 YYADSVX 8 G (SEQ ID NO: 298), wherein X1 is S or no amino acid; X2 is G, A, P, R or S; X3 is S, D, T or G; X4
  • sdAb optionally a VHH domain, comprises: a FR1 comprising or consisting of the amino acid sequence X 1 VX 2 LX 3 ESGGGLVQPGGSLRLSX 4 AASG (SEQ ID NO: 300), wherein X 1 is E or G; X2 is Q or R; X3 is L or V; and X4 is A or C; or optionally a conservative substitution of any of the foregoing; optionally wherein X1 is E, X2 is Q, X3 is L, and X4 is A or C; a FR2 comprising or consisting of the amino acid sequence SEQ ID NO: 301; a FR3 comprising or consisting of the amino acid sequence RFX1ISRDNSKNTLYLQMNX2LRAX3DTAVYYX4 (SEQ ID NO: 302), wherein X1 is T or S, X 2 is S or G, X 3 is E
  • polypeptide of any one of embodiments 1-7, wherein the sdAb, optionally a V H H domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288 or 296.
  • the polypeptide of embodiment 9, wherein the sdAb, optionally a V H H domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 177, 185, or 201; a CDR2 comprising or consisting of any one of SEQ ID NOs: 178, 186, 194 or 202; and a CDR3 comprising or consisting of any one of SEQ ID NOs: 179 or 195; optionally wherein the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: a.) SEQ ID NOs: 177, 178, and 179; b.) SEQ ID NOs: 185, 186, and 179; c.) SEQ ID NOs: 177, 194, and 195; and d.) SEQ ID NOs: 201, 202, and 179; optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more
  • sdAb optionally a VHH domain, comprises: a CDR1 comprising or consisting of the amino acid sequence FTFX 1 X 2 X 3 X 4 MX 5 (SEQ ID NO: 304), wherein X 1 is A, S or E; X 2 is D, T, or G; X 3 is T, M or Q; X4 is D or P; and X5 is G or S; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of the amino acid sequence AISX 1 X 2 X 3 X 4 X 5 TYYADSVX 6 G (SEQ ID NO: 305), wherein X 1 is G or A; X 2 is S or D; X3 is G or A; X4 is G, D, or E; X5 is N, V or A; and X6 is K or R; or optionally a conservative substitution of any of the foregoing
  • polypeptide of any one of embodiments 9-12, wherein the sdAb, optionally a V H H domain, comprises or consists of a sequence selected from any one of SEQ ID NOs: 184, 192, 200, 208, optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 177-179, 185-186, 194-195, or 201-202, and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%
  • polypeptide of any one of embodiments 9-13, wherein the sdAb, optionally a V H H domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 184, 192, 200, 208. 15.
  • the polypeptide of embodiment 15, wherein the sdAb, optionally a V H H domain, comprises: a CDR1 comprising or consisting of SEQ ID NO: 209, a CDR2 comprising or consisting of SEQ ID NO: 210, and a CDR3 comprising or consisting of SEQ ID NO: 211; optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 209-211. 17.
  • each V H framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 212-215.
  • polypeptide of any one of embodiments 15-19, wherein the sdAb, optionally a V H H domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 216. 21.
  • the polypeptide of embodiment 21, wherein the sdAb, optionally a VHH domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 217, 225, 233, 241, 249 or 257; a CDR2 comprising or consisting of any one of SEQ ID NOs: 218, 226, 234, 242 or 250, and a CDR3 comprising or consisting of any one of SEQ ID NOs: 219 or 251; optionally wherein the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: a.) SEQ ID NOs: 217, 218, and 219; b.) SEQ ID NOs: 225, 226, and 219; c.) SEQ ID NOs: 233, 234, and 219; d.) SEQ ID NOs: 241, 242, and 219; e.) SEQ ID NOs: 249, 250, and 251; and f.) SEQ ID NOs: 257,
  • sdAb optionally a V H H domain
  • a CDR1 comprising or consisting of the amino acid sequence FX1FX2X3X4X5MG (SEQ ID NO: 310), wherein X1 is T or D; X2 is A, E, D, S or G; X 3 is D, E or T; X 4 is T, H, Y, V or A; and X 5 is D, A or S; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of the amino acid sequence AISX1X2X3X4X5 X 6 YYADSVKG (SEQ ID NO: 311), wherein X 1 is G, P, R or A; X 2 is S or D, X 3 is G or A, X 4 is G, A, T or D; X 5 is S, T, A or V; and X 6 is T or A; or
  • polypeptide of any one of embodiments 21-23, wherein the sdAb, optionally a VHH domain, comprises a VH FR1 comprising or consisting of any one of SEQ ID NOs: 220 or 228, a V H FR2 comprising or consisting of SEQ ID NO: 221, a V H FR3 comprising or consisting of SEQ ID NO: 222, and a V H FR4 comprising or consisting of SEQ ID NO: 223, optionally wherein the combination of FR1, FR2, FR3, and FR4 is selected from one of the following combinations: a.) SEQ ID NOs: 220, 221, 222, and 223; and b.) SEQ ID NOs: 228, 221, 222, and 223; optionally wherein each VH framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and
  • polypeptide of any one of embodiments 21-24, wherein the sdAb, optionally a VHH domain, comprises or consists of a sequence selected from any one of SEQ ID NOs: 224, 232, 240, 248, 256 or 264, optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 217-219, 225-226, 233-234, 241-242, 249-251, or 257; and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%,
  • polypeptide of any one of embodiments 21-25, wherein the sdAb, optionally a V H H domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 224, 232, 240, 248, 256 or 264.
  • the polypeptide of any one of embodiments 1-8, wherein the sdAb, optionally a V H H domain comprises a CDR3 comprising or consisting of SEQ ID NO: 267. 28.
  • the polypeptide of embodiment 27, wherein the sdAb, optionally a VHH domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 265 or 273, a CDR2 comprising or consisting of any one of SEQ ID NOs: 266 or 274, and a CDR3 comprising or consisting of SEQ ID NO: 267; optionally wherein the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: a.) SEQ ID NOs: 265, 266, and 267; and b.) SEQ ID NOs: 273, 274, and 267; optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 265-267
  • the polypeptide of any one of embodiments 27-28, wherein the sdAb, optionally a V H H domain, comprises: a CDR1 comprising or consisting of the amino acid sequence FTFX 1 X 2 X 3 DMG (SEQ ID NO: 313), wherein X1 is D, G or E; X2 is V, S or G; and X3 is S, N, or Q; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of the amino acid sequence AIX1X2X3X4X5X6TYYADSVKG (SEQ ID NO: 314), wherein X1 is no amino acid or S; X2 is G or S; X3 is T, G, or S; X4 is E or G; X5 is G, D or E; and X6 is D, T or H; or optionally a conservative substitution of any of the foregoing; and a CDR3 comprising or consisting of the amino acid sequence SEQ ID
  • polypeptide of any one of embodiments 27-29, wherein the sdAb, optionally a VHH domain, comprises a VH FR1 comprising or consisting of SEQ ID NO: 268, a V H FR2 comprising or consisting of SEQ ID NO: 269, a V H FR3 comprising or consisting of any one of SEQ ID NOs: 270 or 278, and a V H FR4 comprising or consisting of SEQ ID NO: 271, optionally wherein the combination of FR1, FR2, FR3, and FR4 is selected from one of the following combinations: a.) SEQ ID NOs: 268, 269, 270, and 271; and b.) SEQ ID NOs: 268, 269, 278, and 271; optionally wherein each VH framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and
  • polypeptide of any one of embodiments 27-30, wherein the sdAb, optionally a V H H domain, comprises or consists of a sequence selected from any one of SEQ ID NOs: 272 or 280, optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 265-267, or 273-274, and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
  • polypeptide of any one of embodiments 27-31, wherein the sdAb, optionally a V H H domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 272 or 280.
  • the polypeptide of any one of embodiments 1-8, wherein the sdAb, optionally a V H H domain comprises a CDR3 comprising or consisting of SEQ ID NO: 283. 34.
  • the polypeptide of embodiment 33, wherein the sdAb, optionally a VHH domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 281 or 289, a CDR2 comprising or consisting of any one of SEQ ID NOs: 282 or 290, and a CDR3 comprising or consisting of SEQ ID NO: 283; optionally wherein the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: a.) SEQ ID NOs: 281, 282, and 283; and b.) SEQ ID NOs: 289, 290, and 283; optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NO
  • sdAb optionally a VHH domain
  • the sdAb comprises: a CDR1 comprising or consisting of the amino acid sequence FTFX 1 X 2 X 3 AMX 4 (SEQ ID NO: 316), wherein X 1 is S or D; X 2 is S, E or D; X 3 is Y or H, and X4 is S or G; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of the amino acid sequence AISX 1 X 2 X 3 X 4 X 5 TYYADSVKG (SEQ ID NO: 317), wherein X 1 G or A; X 2 is S or D; X3 is G or A; X4 is G or S; and X5 is S, V or T; or optionally a conservative substitution of any of the foregoing; and a CDR3 comprising or consisting of the amino acid sequence SEQ ID NO: 318; wherein
  • polypeptide of any one of embodiments 33-35, wherein the sdAb, optionally a VHH domain, comprises a VH FR1 comprising or consisting of SEQ ID NO: 284, a VH FR2 comprising or consisting of SEQ ID NO: 285, a VH FR3 comprising or consisting of SEQ ID NO: 286, and a V H FR4 comprising or consisting of SEQ ID NO: 287, optionally wherein the combination of FR1, FR2, FR3, and FR4 is 284, 285, 286, and 287; optionally wherein each VH framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NO
  • polypeptide of any one of embodiments 33-36, wherein the sdAb, optionally a VHH domain, comprises or consists of a sequence selected from any one of SEQ ID NOs: 288 or 296, optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 281-283, and 289-290, and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
  • polypeptide of any one of embodiments 33-37, wherein the sdAb, optionally a VHH domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 288 or 296. 39.
  • the polypeptide of any one of embodiments 9-38, wherein the sdAb, optionally a V H H domain, comprises: a FR1 comprising or consisting of the amino acid sequence X1VX2LX3ESGGGLVQPGGSLRLSX4AASG (SEQ ID NO: 300), wherein X1 is E or G; X2 is Q or R; X3 is L or V; and X4 is A or C; or optionally a conservative substitution of any of the foregoing; optionally wherein X 1 is E, X 2 is Q, X 3 is L, and X 4 is A or C; a FR2 comprising or consisting of the amino acid sequence SEQ ID NO: 301; a FR3 comprising or consisting of the amino acid sequence RFX 1 ISRDNSKNTLYLQMNX 2 LRAX 3 DTAVYYX 4 (SEQ ID NO: 302), wherein X 1 is T or S; X2 is S or G; X3
  • the sdAb comprises a VHH domain with a substitution at C22 of FR1 and/or C30 of FR3, optionally wherein the substitution is independently selected from a C to A and a C to V substitution.
  • PSR Poly Specificity Reagent
  • TM melting temperature
  • a bifunctional polypeptide that promotes proteasome-mediated degradation of at least one target protein comprising: a) a first targeting moiety comprising or consisting of the polypeptide any one of embodiments 1-48, wherein the polypeptide comprises or consists of the anti-Myc sdAb; b) a ubiquitin-proteasome system recruiting domain (URD), c) optionally a first linker peptide, optionally wherein the first linker peptide is positioned between the first targeting moiety and the URD, d) optionally one or more localization peptide sequences, wherein at least one target protein is MYC, optionally c-MYC, and wherein proximity of the bifunctional polypeptide to at the least one target protein through binding of the targeting moiety induces ubiquitination of the at least one target protein via the URD, thereby promoting proteasome-mediated degradation of the at least one target protein.
  • a first targeting moiety comprising or consisting of the polypeptide any one of embodiments
  • the URD is selected from: a) the URD derived from a protein which localizes to the cytoplasm, optionally selected from FBXW7 beta and Keap1; b) the URD derived from a protein which localizes to the nucleus, optionally selected from FBXW7 alpha, RNF165, and SPOP, optionally SPOP.2; and c) the URD derived from a protein which localizes to the cytoplasm and the nucleus, optionally selected from beta-TRCP, CHIP, RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL-b (Y363E), TRIM21, E6AP, DCAF1 VIF, RNF11, RNF111, RNF115, RNF12, RNF128, RNF149, RNF152, RNF165, RNF182, RNF20, RNF25, RNF4, RNF6, TRIM32, ZNRF1,
  • E3 ligase selected from the group consisting of DCAF1, beta-TRCP, FBXW7-alpha, FBXW7-beta, Keap1, and SPOP, optionally selected from the group consisting of beta-TRCP, FBXW7-alpha, and FBXW7-beta.
  • the URD is derived from an E3 ligase selected from the group consisting of NHLRC1, RNF125, RNF165, RNF4, and ZNRF1, optionally NHLRC1, RNF125, RNF165, and RNF4, optionally wherein the URD comprises or consists of NHLRC1.1, RNF125.2, RNF165.1, RNF4.1, or ZNRF1.1, optionally wherein the URD comprises or consists of NHLRC1.1, RNF125.2, RNF165.1, or RNF4.1. 64.
  • the one or more localization peptide sequences direct the bifunctional polypeptide to a subcellular compartment or compartments, optionally wherein the subcellular compartment is the nucleus and/or the cytoplasm. 75.
  • 76. The bifunctional polypeptide of any one of embodiments 73-75, wherein the one or more localization peptide sequences comprise or consist of a nuclear localization signal (NLS) peptide and/or a nuclear export signal (NES) peptide, optionally wherein the localization peptide comprises or consists of an amino acid sequence of any one of SEQ ID NO: 114-127. 77.
  • NLS nuclear localization signal
  • NES nuclear export signal
  • the bifunctional polypeptide of embodiment 78 wherein the endogenous localization peptide sequence is from a URD selected from: a URD derived from a protein which localizes to the cytoplasm, optionally selected from FBXW7 beta and Keap1; a URD derived from a protein which localizes to the nucleus, optionally selected from FBXW7 alpha, RNF165, and SPOP; and a URD derived from a protein which localizes to the cytoplasm and the nucleus, optionally selected from beta-TRCP, CHIP, RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL-b (Y363E), TRIM21, E6AP, DCAF1 VIF, RNF11, RNF111, RNF115, RNF12, RNF128, RNF149, RNF152, RNF165, RNF182, RNF20, RNF25, RNF4, RNF6, TRIM32, ZNRF1, ZNRF4.
  • the modification comprises or consists of a substitution, truncation or deletion of an endogenous localization peptide sequence.
  • the native endogenous localization peptide sequence directs the polypeptide to the nucleus, and the modified endogenous localization peptide sequence directs the polypeptide to the cytoplasm; b. the native endogenous localization peptide sequence directs the polypeptide to the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to the nucleus; c. the native endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to only the cytoplasm; d.
  • the native endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to only the nucleus; e. the native endogenous localization peptide sequence directs the polypeptide to the nucleus, and the modified endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm; or f. the native endogenous localization peptide sequence directs the polypeptide to the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm.
  • the bifunctional polypeptide of embodiment 73-82 wherein the one or more localization peptide sequences comprises or consists of an endogenous localization peptide sequence of the URD that is modified, wherein the native endogenous localization peptide sequence directs the polypeptide to the nucleus, and the modified endogenous localization peptide sequence directs the polypeptide to the cytoplasm.
  • bifunctional polypeptide of any one of embodiments 73-87 wherein the bifunctional polypeptide comprises or consists of two localization peptide sequences, optionally wherein the bifunctional polypeptide comprises or consists of 3, 4, or more localization peptide sequences, optionally wherein the bifunctional polypeptide comprises or consists of 2, 3, 4 or more copies of the same localization sequence in series. 89.
  • the bifunctional polypeptide of any one of embodiments 73-88 comprising one or more localization peptide sequences located at a location selected from the group consisting of: the N-terminus of the bifunctional polypeptide; the C-terminus of the bifunctional polypeptide; internally within the bifunctional polypeptide; as part of the URD, optionally an endogenous portion of the URD; as a part of the first targeting moiety, optionally an endogenous portion of the first targeting moiety; and a combination of any of the foregoing. 90.
  • the bifunctional polypeptide of embodiment 91 wherein a first NLS peptide is located at the N-terminus of the bifunctional polypeptide and a second NLS peptide is located at the C-terminus of the bifunctional polypeptide.
  • 93 The bifunctional polypeptide of any one of embodiments 50-92, wherein the orientation of the targeting moiety relative to the URD is the same orientation as compared to the naturally occurring substrate recognition domain of the protein from which the URD is derived.
  • 94 The bifunctional polypeptide of any one of embodiments 50-93, wherein the orientation of the first targeting moiety relative to the URD is the opposite orientation as compared to the naturally occurring substrate recognition domain of the protein from which the URD is derived. 95.
  • HA hemagglutinin
  • bifunctional polypeptide of any one of embodiments 50-96 wherein the bifunctional polypeptide does not comprise an epitope tag, optionally a hemagglutinin (HA) tag, optionally wherein the bifunctional polypeptide does not comprise an HA tag comprising or consisting of the amino acid sequence of SEQ ID NO: 384. 99.
  • HA hemagglutinin
  • bifunctional polypeptide of any one of embodiments 50-98 wherein the bifunctional polypeptide is less than or equal to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 amino acids long, or a range defined by any two of the preceding values, optionally 50-800, 50-650, 50-500, 50-400, 70-800, 70-650, 70-500, 70-400, 70-300, 70-200, 80-800, 80-650, 80- 500, 80-400, 80-300, or 80-200 amino acids long, optionally 90-550, 150-550, or 80-450 amino acids long.
  • 101. A polynucleotide encoding a bifunctional polypeptide comprising or consisting of the bifunctional polypeptide of any one of embodiments 50-100.
  • 102. The bifunctional polypeptide of any one of embodiments 50-100, wherein the at least one target protein comprises MYC, optionally c-MYC. 103.
  • the bifunctional polypeptide of embodiment 102 wherein the bifunctional polypeptide comprises a localization peptide sequence which directs the bifunctional polypeptide to the nucleus of a cell, optionally wherein the localization peptide sequence comprises or consists of the NLS sequence selected from the group of the NLS sequences of MYC, SPOP, Hrp1, SV40, 53BP1, and nucleoplasmin, optionally MYC, SPOP, Hrp1, optionally wherein the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of SPOP, optionally SPNLS.2, MYC, optionally mycNLS, and Hrp1, optionally hrpNLS. 104.
  • NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 114-127, optionally wherein the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 114, 122 and 116. 105.
  • the URD is the URD of NHLRC1, RNF125, RNF165, RNF4, or ZNRF1, or a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity, optionally wherein the URD comprises or consists of NHLRC1.1, RNF125.2, RNF165.1, RNF4.1, or ZNRF1.1, optionally wherein the URD comprises or consists of NHLRC1.1 RNF125.2, RNF165.1, or RNF4.1. 106.
  • the bifunctional polypeptide of any one of embodiments 102-105 wherein the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 76-80, 87- 93, 98, 104 and 111, optionally wherein the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 76, 88, 98, 104, and 111, or optionally SEQ ID NOs: 76, 88, 98, and 104. 107.
  • bifunctional polypeptide of any one of embodiments 102-109 wherein the bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 27, optionally excluding any one of the SEQ ID NOs listed as having a MYC degradation Grade of D, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). 111.
  • the bifunctional polypeptide of any one of embodiments 102-111, wherein the bifunctional polypeptide does not comprise or consist of an amino acid sequence of any one of the SEQ ID NOs in Table 27 listed as having a MYC degradation Grade of C and/or D, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). 113.
  • a second linker peptide wherein the second linker peptide is positioned between the second targeting moiety and the URD or between the first targeting moiety and the second targeting moiety, and wherein proximity of the dual-specific bifunctional polypeptide to at least one target protein through binding of the first targeting moiety and/or the second targeting moiety induces ubiquitination of the at least one target protein via the URD, thereby promoting proteasome- mediated degradation of the at least one target protein.
  • the dual-specific bifunctional polypeptide of embodiment 115 wherein the first targeting moiety binds to and/or is designed to bind a first target protein and the second targeting moiety binds to and/or is designed to bind a second target protein.
  • the dual-specific bifunctional polypeptide of embodiment 115 wherein the first targeting moiety and the second targeting moiety bind to and/or are designed to bind to the same target protein.
  • the dual-specific bifunctional polypeptide of embodiment 117 wherein the first targeting moiety and second targeting moiety bind to and/or are capable of binding to the same binding site of the target protein, wherein the dual-specific bifunctional polypeptide engages and/or is capable of engaging at least two molecules of the at least one target protein. 120.
  • sdAb single domain antibody
  • the dual-specific bifunctional polypeptide of embodiment 122 wherein the second targeting moiety is, or is less than, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70, 65, 60, 55, 54, 53, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids long, or a range defined by any two of the preceding values, optionally 10-100, 10-25, 14-100, 14-25, 30-100, 30-50, 30-45, or 80-100 amino acids long.
  • the second targeting moiety comprises or consists of an sdAb, optionally comprising or consisting of either a VH domain or a VL domain of an IgG antibody, or a VHH, VH, or VNAR domain of a heavy chain antibody (HcAb), optionally wherein the sdAb comprises or consists of a V H H or V L domain.
  • HcAb heavy chain antibody
  • the dual-specific bifunctional polypeptide of embodiment 124 wherein the second targeting moiety is, or is less than, 170, 165, 160, 155, 150, 145, 140, 135, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90, amino acids long, or a range defined by any two of the preceding values, optionally 90-150, 90-130, 110-150, 110-130, 110-125, or 114-124 amino acids long.
  • CNNB1 beta catenin 1
  • PCNA proliferating cell nuclear antigen
  • the dual-specific bifunctional polypeptide of embodiment 128, wherein the mutant form of the target protein is CTNNB1 (SEQ ID NO: 2) comprising one or more mutations selected from: D32Y; D32N; D32V; D32G; S33Y; S33L; S33F; S33C; S33P; G34V; G34E; I35S; H36P; S37C; S37P; S37P and D207G; T41A; T41I; T42_K49del; S45F; and Y670X wherein X indicates termination; S45C; S45F; S45P; S45Y; and S45del. 130.
  • the dual-specific bifunctional polypeptide of any one of embodiments 115-134 wherein the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 29 listed as having a degradation Grade of A for at least one of the target proteins, degradation Grade of B for at least one of the target proteins, degradation Grade of C for at least one of the target proteins, and/or degradation Grade of D for at least one of the target proteins, optionally excluding any one of the SEQ ID NOs listed as having a degradation Grade of D for at least one of the target proteins, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 29 listed as having degradation Grade of A for at least one of the target proteins, and/or degradation Grade of B for at least one of the target proteins, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • linker peptide is positioned between one or more of: the first targeting moiety and the URD; the second targeting moiety and the URD; the first targeting moiety and the second targeting moiety; a localization peptide and the first targeting moiety; a localization peptide and the second targeting moiety; a localization peptide and the URD; and/or a localization peptide and an epitope tag, optionally an
  • the dual-specific bifunctional polypeptide of any one of embodiments 115-143 comprising a first linker peptide positioned between the first targeting moiety and the URD or between the second targeting moiety and the URD, and a second linker peptide positioned between the second targeting moiety and the URD or between the first targeting moiety and the second targeting moiety, optionally wherein the first linker peptide is positioned between the first targeting moiety and the URD and the second linker peptide is positioned between the second targeting moiety and the URD. 145.
  • the dual-specific bifunctional polypeptide of embodiment 149 wherein the one or more localization peptide sequences direct the dual-specific bifunctional polypeptide to a subcellular compartment or compartments, optionally wherein the subcellular compartment is the nucleus and/or the cytoplasm.
  • NLS nuclear localization signal
  • NES nuclear export signal
  • the localization peptide comprises or consists of an amino acid sequence of any one of SEQ ID NO: 114-127.
  • the modification comprises or consists of a substitution, truncation or deletion of an endogenous localization peptide sequence.
  • the native endogenous localization peptide sequence directs the polypeptide to the nucleus, and the modified endogenous localization peptide sequence directs the polypeptide to the cytoplasm; b. the native endogenous localization peptide sequence directs the polypeptide to the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to the nucleus; c. the native endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to only the cytoplasm; d.
  • the native endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to only the nucleus; e. the native endogenous localization peptide sequence directs the polypeptide to the nucleus, and the modified endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm; or f. the native endogenous localization peptide sequence directs the polypeptide to the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm. 157.
  • the dual-specific bifunctional polypeptide of any one of embodiments 149-157 comprising one or more localization peptide sequences located at a location selected from the group consisting of: the N-terminus of the dual-specific bifunctional polypeptide; the C- terminus of the dual-specific bifunctional polypeptide; internally within the dual-specific bifunctional polypeptide; as part of the URD, optionally an endogenous portion of the URD; as a part of the first and/or second targeting moiety, optionally an endogenous portion of the first and/or second targeting moiety; and a combination of any of the foregoing. 159.
  • the dual-specific bifunctional polypeptide of embodiment 149-160 wherein a first NLS peptide is located at the N-terminus of the dual-specific bifunctional polypeptide and a second NLS peptide is located at the C-terminus of the dual-specific bifunctional polypeptide.
  • 162 The dual-specific bifunctional polypeptide of any one of embodiments 115-161, wherein the orientation of the first targeting moiety relative to the URD is the same orientation as compared to the naturally occurring substrate recognition domain of the protein from which the URD is derived. 163.
  • HA hemagglutinin
  • the dual-specific bifunctional polypeptide of any one of embodiments 115-172 wherein the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the peptide of any one of SEQ ID NO: 562-577. 174.
  • the dual-specific bifunctional polypeptide of embodiment 174 wherein the second targeting moiety binds, or binds at least in part, to at least a portion of: a.) the DNA binding domain of MYC; b.) amino acids 54-406 of MYC (SEQ ID NO: 1); c.) amino acids 354-406 of MYC (SEQ ID NO: 1); d.) amino acids 408-437 of MYC (SEQ ID NO: 1); and e.) amino acids 410-419 of MYC (SEQ ID NO: 1). 176.
  • the bifunctional polypeptide comprises a localization peptide sequence which directs the bifunctional polypeptide to the nucleus of a cell, optionally wherein the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequences of MYC, SPOP, Hrp1, SV40, 53BP1, and nucleoplasmin, optionally MYC, SPOP, Hrp1, optionally wherein the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of MYC, optionally mycNLS, SPOP, optionally spNLS.2, and Hrp1. 177.
  • the dual-specific bifunctional polypeptide of embodiment 176 wherein the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 114-127, optionally wherein the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 122, 114, and 116. 178.
  • a sdAb optionally comprising or consisting of either a V H domain or a V L domain of an IgG antibody, or a V H H, V H , or V NAR domain of a heavy chain antibody (HcAb)
  • HcAb heavy chain antibody
  • the dual-specific bifunctional polypeptide of embodiment 183, wherein the sdAb, optionally a VHH domain, of the second targeting moiety comprises a VH FR1 selected from any one of SEQ ID NOs: 138 and 170, a V H FR2 of SEQ ID NO: 140, a V H FR3 selected from any one of SEQ ID NOs: 142, 158, and 174, and a VH FR4 selected from any one of SEQ ID NOs: 144 and 152, optionally wherein the combination of FR1, FR2, FR3 and FR4 is SEQ ID NOs: 154, 156, 158, and 160; optionally wherein each VH framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • the dual-specific bifunctional polypeptide of any one of embodiments 174-188 wherein the bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 30, optionally excluding any one of the SEQ ID NOs listed as having a MYC degradation Grade of D, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 30, optionally excluding any one of the SEQ ID NOs listed as having a M
  • SEQ ID NO: 2 mutant form of CTNNB1
  • the bifunctional polypeptide comprises a localization peptide sequence which directs the bifunctional polypeptide to the nucleus of a cell, optionally wherein the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequences of MYC, SPOP, Hrp1, SV40, 53BP1, and nucleoplasmin, optionally wherein the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of MYC, optionally mycNLS, SPOP, optionally spNLS.2, and Hrp1.
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of MYC, optionally mycNLS, SPOP, optionally spNLS.2, and Hrp1.
  • the dual-specific bifunctional polypeptide of embodiment 195 wherein the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 114-127, optionally wherein the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 122, 114 and 116.
  • the second targeting moiety binds, or binds at least in part, to at least a portion of: a) the armadillo domain of CTNNB1, b) amino acids 150-663 of CTNNB1 (SEQ ID NO: 2), c) amino acids 15-29 of CTNNB1 (SEQ ID NO: 2), and d) amino acids 249-265, 2
  • the dual-specific bifunctional polypeptide of any one of embodiments 194-203 wherein the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 563, 564, 566, 568, and 569, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • SEQ ID NO: 563, 564, 566, 568, and 569 optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide of any one of embodiments 194-204 wherein the bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 31, optionally excluding any one of the SEQ ID NOs listed as having a degradation Grade of D for at least one of the target proteins, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 31, optionally excluding any one of the SEQ ID NOs listed
  • the dual-specific bifunctional polypeptide of any one of embodiments 194-205 wherein the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 31 listed as having a degradation Combined Grade of A/A or A, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide of any one of embodiments 194-205 wherein the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 31 listed as having a degradation Combined Grade of B/B or B, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the bifunctional polypeptide comprises a localization peptide sequence which directs the bifunctional polypeptide to the nucleus of a cell
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequences of MYC, SPOP, Hrp1, SV40, 53BP1, and nucleoplasmin
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of MYC, optionally mcyNLS and/or 3xmycNLS, SPOP, Hrp1, SV40
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of MYC, optionally mcyNLS, and SPOP, optionally spNLS.2.
  • the dual-specific bifunctional polypeptide of embodiment 115 wherein the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 562-577, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). 225.
  • a polypeptide comprising or consisting of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, and 385-577, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). 226.
  • polynucleotide of any one of embodiments 226-227 wherein the polynucleotide is packaged in a lipid nanoparticle, a polymeric nanoparticle, an extracellular vesicle, optionally an exosome, or a viral vector, optionally a replicating viral vector or a non- replicating viral vector, optionally an adenovirus, adeno-associated virus, lentivirus, or retrovirus vector. 229.
  • a pharmaceutical composition comprising the polypeptide comprising or consisting of the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, or polynucleotide of any one of embodiments 1-228 and one or more pharmaceutically acceptable excipients, carriers, or diluents.
  • polypeptide comprising or consisting of the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition of any one of embodiments 1-230 for use in the treatment of a cancer in a patient in need thereof.
  • a method of treating a subject comprising administering the polypeptide comprising or consisting of the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition of any one of embodiments 1-232 to a subject in need thereof, optionally wherein the subject has a cancer. 234.
  • a method of reducing the amount of a target protein in a cell comprising contacting the cell with the polypeptide comprising or consisting of the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition of any one of embodiments 1-232, optionally wherein the target protein is selected from MYC, (optionally c-MYC), CTNNB1, and/or PCNA. 235.
  • invention 233 or 234 wherein said cell is in a subject, and said contacting comprises administering the polypeptide comprising or consisting of the anti- Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition of any one of embodiments 1-232 to the subject, optionally wherein the subject has cancer.
  • the administering is intravenous, intraperitoneal, intra-arterial, subcutaneous, intramuscular, intrathecal, intratumoral, inhalation, or intracranial administration. 237.
  • any one of embodiments 234 or 235 wherein the cell is ex vivo, and said contacting comprises contacting the polypeptide comprising or consisting of the anti- Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition of any one of embodiments 1-232 to the cell ex vivo, optionally in an adoptive cell therapy.
  • the method of embodiment 237 further comprising administering the cell to a subject after the contacting step, optionally wherein the subject is also the source of the cell. 239.
  • polypeptide comprising or consisting of the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, pharmaceutical composition or method of any one of embodiments 1-238, wherein the polypeptide, bifunctional polypeptide, or dual-specific bifunctional polypeptide promotes proteasome-mediated degradation of the target protein when measured in one or more assays described in the Exemplary Assays for assessing proteasomal-mediated degradation, for example, by HiBiT tag assay and/or Western blot, optionally wherein the proteasome-mediated degradation is inhibited when the cells comprising the target protein and the dual-specific bifunctional polypeptide are treated with a proteasome inhibitor.
  • the polypeptide, bifunctional polypeptide, or dual-specific bifunctional polypeptide promotes proteasome-mediated degradation of the target protein when measured in one or more assays described in the Exemplary Assays for assessing proteasomal-mediated degradation, for example, by HiBiT
  • polypeptide comprising or consisting of the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, pharmaceutical composition or method of embodiment 239, wherein treatment of the cells comprising the target protein and the polypeptide, bifunctional polypeptide, or dual-specific bifunctional polypeptide with a proteasome inhibitor reduces the amount of degradation of the target protein by at least 50%, 60%, 70%, 80%, 90%, 95% or 100% as compared to cells not treated with the proteasome inhibitor. 241.
  • a compound comprising the polypeptide of any one of embodiments 1-48, the polypeptide comprising or consisting of the anti-Myc sdAb, and a therapeutic compound and/or drug and/or detectable label conjugated to the polypeptide. 243.
  • the conservative substitution is a conservative substitution from one of the following eight groups, wherein each group contains amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).
  • FIGS. 1A, 1B and 1C depict embodiments of a schematic describing proteasome- mediated degradation of a target protein using a bifunctional (FIG. 1A) or dual-specific bifunctional polypeptide (FIGS.
  • FIG. 1B depicts an embodiment of a “contralateral” configuration of a dual-specific bifunctional polypeptide in which the first and second targeting domains are located on either side of the URD.
  • FIG. 1C depicts and embodiment of a “tandem” configuration of a dual-specific functional polypeptide in which the first and second targeting domains are located on the same side (both on the N-terminal side or both on the C-terminal side of the URD).
  • FIGS. 2A, 2B and 2C depict embodiments of a schematic for different ordering combinations of the components that comprise the bifunctional or dual-specific bifunctional polypeptides of some embodiments disclosed herein.
  • FIG.2A depicts embodiments of a mono- specific bifunctional polypeptide comprising a single targeting moiety that is capable of binding to at least one target protein (e.g., Myc).
  • 2B depicts embodiments of a “contralateral” configuration in which the dual-specific bifunctional polypeptides comprise a first targeting moiety (target binding domain) (N-TBD or C-TBD) that is capable of binding to at least one target protein (e.g., Myc); a second targeting moiety (C-TBD or N-TBD) that is capable of binding to at least one target protein (e.g. Myc or a different target protein); and a URD.
  • first targeting moiety target binding domain
  • C-TBD or N-TBD second targeting moiety
  • 2C depicts embodiments of a “tandem” configuration in which the dual-specific bifunctional polypeptide is in a “tandem” configuration in which the first and second target binding domains are both on the same side of the URD, i.e., both are on the C-terminal side of the URD, or both are on the N-terminal side of the URD.
  • the bifunctional or dual-specific bifunctional polypeptide further comprises one or more linker peptides.
  • the dual-specific bifunctional polypeptide when in a “contralateral” configuration the dual-specific bifunctional polypeptide further comprises a first linker peptide (N-linker or C-linker), wherein the first linker peptide is positioned between the first targeting moiety and the URD; and/or a second linker peptide (C-linker or N-linker), wherein the second linker peptide is positioned between the second targeting moiety and the URD.
  • a “tandem” configuration is used (see, e.g., FIG.
  • a linker peptide (a first or a second linker peptide) is positioned between the two target binding domains, and/or a target binding domain and the URD.
  • one or both of the linkers is not present (not shown).
  • the linker(s), if present, may provide flexibility in positioning between the URD and target binding moiety.
  • the bifunctional or dual- specific bifunctional polypeptides may further comprise a localization sequence, (e.g., a nuclear localization signal (NLS)), which may be at the N-terminus, C-terminus, or both the N-terminus and C-terminus, or internally (not shown) within the bifunctional polypeptide (e.g., if the URD and/or target binder comprises an NLS within its sequence), if the target protein localizes to the nucleus of a cell.
  • the localization sequence(s) e.g., N- NLS, and/or C-NLS
  • FIG. 3 depicts an embodiment of subcellular localization of SPOP variant URD mono-specific bifunctional polypeptides to the nucleus and the cytoplasm.
  • FIG. 4 depicts an embodiment of subcellular localization of NHLRC1 containing mono-specific bifunctional polypeptides with and without an exogenous NLS sequence.
  • FIG.5 depicts an embodiment of subcellular localization of VL12.3 containing mono- specific bifunctional polypeptides with URDs containing endogenous NLS or NES signals.
  • FIG.6 is a table disclosing a summary of an embodiment of an experiment examining expression of various exemplary dual-specific bifunctional polypeptides.
  • FIGS. 7A-7C depict an embodiment of HA staining of the dual-specific bifunctional polypeptides disclosed in FIG. 6.
  • FIG. 8 depicts an embodiment of various exemplary bifunctional or dual-specific bifunctional polypeptides listing the target proteins (“Targets”), description of elements listed in a N- to C-terminal direction, and respective SEQ ID NOs.
  • Targets the target proteins
  • DETAILED DESCRIPTION OF THE DISCLOSURE Antibodies targeting Myc such as 9E10 are known in the art.
  • the anti-Myc sdAbs disclosed herein are designed to function in the intracellular environment. This allows these anti-Myc sdAbs to be expressed intracellularly and utilized as Myc-binding moieties on their own, or as portions of other compounds or polypeptides (e.g., antibody-drug conjugates, detectably labeled probes, diabodies, etc.), for example the bifunctional polypeptides disclosed herein.
  • the anti-Myc sdAbs disclosed herein are part of a bifunctional polypeptide that uses the ubiquitin-proteasome system to degrade Myc and optionally other target protein(s).
  • the ubiquitin-proteasome system is the major and essential mechanism by which eukaryotic cells regulate protein abundance and clear misfolded or damaged proteins through proteolytic degradation. This regulation is vital for many cellular functions, such as regulating the cell cycle and gene expression. Proteins are marked for degradation and recognized by the proteasomal complex when they are polyubiquitinated at lysine residues.
  • This polyubiquitination is mediated by recognition of the target proteins by an E3 ubiquitin ligase, which catalyzes the transfer of a ubiquitin subunit from an E2 ubiquitin-conjugating enzyme to the protein target.
  • an E3 ubiquitin ligase catalyzes the transfer of a ubiquitin subunit from an E2 ubiquitin-conjugating enzyme to the protein target.
  • the protein target can be polyubiquitinated, thereby marking it for degradation by the proteasome. It is estimated that there are over 600 unique E3 ubiquitin ligases encoded by the human genome, each having varying specificities to target proteins.
  • each E3 ubiquitin ligase generally comprises a substrate recognition domain specific for one or more targets and a ubiquitin-proteasome system recruiting domain (URD) that binds to an E2 ubiquitin-conjugating enzyme to enable ubiquitination of the target.
  • ULD ubiquitin-proteasome system recruiting domain
  • the ubiquitin-proteasome system may be exploited for the directed degradation of desired protein targets such a Myc.
  • desired protein targets such a Myc.
  • many diseases are associated with the abnormal function and/or expression of certain proteins (e.g., cancer caused by dysfunctional expression or localization of oncogenes). Degradation of said proteins can have a therapeutic effect in treating associated diseases, and the ubiquitin-proteasome system offers a naturally occurring process for effecting said degradation.
  • degradation of disease-associated proteins may be more effective than inhibition of the protein, for example, using a small molecule or antibody composition, as in these cases, the protein is still present within the cell and may perform various biological functions at interfaces other than the inhibited domain, for example scaffolding functions, or contribute to pathology as a result of accumulation, aggregation or mislocalization.
  • Several groups have reported on the structure and preclinical activity of various macromolecule degrader formats, including fusion proteins comprising a full-length or truncated E3 ligase and macromolecular targeting constructs.
  • a PROTAC peptide induces durable ⁇ -catenin degradation and suppresses Wnt-dependent intestinal cancer,” Cell Discov. (2020) 6:35; Liu et al. Targeted degradation of b-catenin by chimeric F-box fusion proteins. Biochem. and Biophys. Res. Comm. (2004) 313:1023-1029; Cong et al., “A protein knockdown strategy to study the function of ⁇ -catenin in tumorigenesis,” BMC Molecular Bio. (2003) 4:10; and Shu et al.
  • anti-Myc sdAbs and polypeptides comprising or consisting thereof.
  • polypeptides comprising or consisting thereof.
  • bifunctional, optionally dual-specific, polypeptides that promote proteasome-mediated degradation of a target protein, and uses thereof, such as for the treatment of a disease.
  • bifunctional polypeptides generally comprise a first component that is able to bind to a first target protein with specificity (e.g., an anti-Myc sdAb), a second component that is able to recruit a ubiquitination complex to mark the target protein for proteasomal degradation by ubiquitination, and optionally, a third component that is able to bind to a second target protein with specificity (e.g., Myc or a second target protein).
  • a first target protein with specificity e.g., an anti-Myc sdAb
  • a second target protein with specificity e.g., Myc or a second target protein
  • An exemplary schematic for the function of a mono-specific bifunctional polypeptide comprising a single targeting moiety as disclosed herein is depicted in FIG. 1A.
  • An exemplary schematic for the function of dual-specific bifunctional polypeptides disclosed herein may be seen in FIG.
  • FIG. 2A, 2B and 2C depict some non-limiting embodiments of mono-specific (FIG. 2A) dual-specific (FIG. 2B and 2C) bifunctional polypeptides with various combinations of targeting moieties and URD, and optionally localization sequences (e.g., NLS) and linkers (the depiction is in the conventional N-terminal to C-terminal (left to right) orientation).
  • the first and second targeting domains are on either side of the URD (see, e.g., FIG 2B) in a “contralateral” configuration.
  • the first and second target binding domains are on the same side of the URD (both on the N-terminal side, or both on the C-terminal side) (see, e.g., FIG. 2C) in a “tandem” configuration.
  • a localization sequence e.g., NLS
  • each of the localization signals (e.g., NLS) shown can represent one, two, three or more copies of the same or a different NLS sequence.
  • the localization sequence(s) may be separated from other components by a linker sequence (not shown).
  • a linker sequence not shown.
  • mono- or dual-specific bifunctional polypeptides which are engineered (e.g., by modification of an endogenous localization peptide sequence, by adding localization peptide sequences, and/or by selecting a component which has an endogenous localization sequence) to direct the bifunctional polypeptide to a particular subcellular compartment, e.g., the cytoplasm, the nucleus, or both.
  • polypeptides comprising or consisting of a portion derived from a an E3 ubiquitin ligase or viral homolog thereof.
  • the portion has ubiquitin-proteasome recruiting activity and may be designated as a ubiquitin recruiting domain (URD).
  • ULD ubiquitin recruiting domain
  • dual-specific bifunctional polypeptides comprising these portions.
  • Anti-Myc Single Domain Antibodies (sdAb) Disclosed herein are polypeptides comprising or consisting of an anti-Myc sdAb.
  • the anti-Myc sdAb comprises or consists of a V H domain of an IgG antibody, or a V H H, or V H domain of a heavy chain antibody (HcAb). Sequences of exemplary anti-Myc sdAbs are shown in Table 1A. Table 1A: Exemplary anti-Myc sdAb Sequences In the above table CDR refers to a complementarity-determining region of the sdAb, and FR refers to a framework region of the sdAb, while “full” is the entire anti-Myc sdAb sequence. Note that not every sequence in the above table is unique, as some exemplary anti- Myc sdAbs share one or more sequences.
  • FR2 sequences are identical even though a separate SEQ ID NO is provided for each FR2 sequence.
  • Table 1B below provides a list of SEQ ID NOs which have identical sequences.
  • Table 1B List of SEQ ID NOs having identical sequences.
  • the above exemplary anti-Myc sdAbs are affinity optimized antibodies of five parental anti-Myc sdAbs, the sequences of which are provided in Table 2 below.
  • Table 2 Parental anti-Myc sdAb sequences
  • the exemplary anti-Myc sdAbs disclosed in Table 1 have improved affinity for the target protein c-Myc as compared to the parental anti-Myc sdAb from which they are derived as evidenced by the Kd values calculated for each.
  • the Kd values for the exemplary anti-Myc sdAbs and the parental anti-Myc sdAbs are provided in Table 3.
  • “Octet sdAb Kd Human Myc Peptide Monovalent (M)” refers to the affinity of the anti-Myc sdAb against human c-Myc antigen peptide as described in more detail in Example 4.
  • “Octet sdAb Kd Human Myc-Fc Monovalent (M)” refers to the affinity of the anti-Myc sdAb against human c-Myc antigen-Fc fusion as described in more detail in Example 4.
  • Table 3 Kd Values of exemplary and parental anti-Myc sdAb
  • Tables 4-9 are alignments of each of CDR1, CDR2, CDR3, FR1, FR3, and FR4 of all of the exemplary and parental anti-Myc sdAbs listed in Tables 1A and 2 above, with the resulting consensus sequences for each.
  • the sequence of FR2 was identical across all anti-Myc sdAbs and therefore no table is provided for the FR2 consensus sequence.
  • Table 4 Consensus CDR1 Table 5: Consensus CDR2 with parental Table 6: Consensus CDR3 Table 7: Consensus FR1 Table 8: Consensus FR3 Table 9: Consensus FR4 Similar exemplary consensus sequences were prepared for each of CDR1-3 for each family of anti-Myc sdAbs (mycV.0300, mycV.1300, mycV.1500, mycV.3700, and mycV.6600) using the parental anti-Myc sdAb and exemplary variant anti-Myc sdAbs derived from the parental sequences.
  • a consensus sequence has variable residues having one of the amino acid specified, or optionally “a conservative substitution of any of the foregoing.”
  • variable residues X1X2X3X4 and X5 in SEQ ID NO: 317 as shown in Table 22 comprise the amino acid specified for the X X residues as shown in the table, or optionally a conservative substitution of any of the amino acids specified for that XX residue.
  • Table 22 specifies that X1 in SEQ ID NO: 317 may comprise G or A, and thus in some embodiments also contemplated is a conservative substitution of G or a conservative substitution for A.
  • X 2 may be S or D, and optionally in some embodiments a conservative substitution for S, or a conservative substitution for D.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, for example, Creighton, T.
  • sdAb Anti-Myc Single Domain Antibodies
  • the anti-Myc sdAb comprises or consists of a V H domain of an IgG antibody, or a V H H, or V H domain of a heavy chain antibody (HcAb), wherein the VH or VHH domain comprises a VH CDR3 comprising or consisting of any one of SEQ ID NOs: 179, 195, 211, 219, 251, 267 or 283.
  • the VH CDR3 sequence comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 179, 195, 211, 219, 251, 267 and 283.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises a CDR3 comprising or consisting of the amino acid sequence AX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 EX 13 DY (SEQ ID NO: 299), wherein X 1 is A, H, Y, I, or T; X 2 is L or I; X 3 is G, D or P; X 4 is D, Q, G or E; X 5 is Y, E or I; X 6 is G, L or S; X 7 is D,T, S or G; X8 is A, G, F, or no amino acid; X9 is I, G, or no amino acid; X10 is D, G, T or L; X11 is Y, A, F or G; X12 is S, D or N; and X13 is A, H, G or Y; or optionally a conservative substitution of any of the for
  • the sdAb optionally a VHH domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 177, 185, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273, 281, 289; a CDR2 comprising or consisting of any one of SEQ ID NOs: 178, 186, 194, 202, 210, 218, 226, 234, 242, 250, 266, 274, 282 or 290; and a CDR3 comprising or consisting of any one of SEQ ID Nos: 179, 195, 211, 219, 251, 267 or 283.
  • the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: a) SEQ ID NOs: 177, 178, and 179; b) SEQ ID NOs: 185, 186, and 187; c) SEQ ID NOs: 177, 194, and 195; d) SEQ ID NOs: 201, 202, and 179; e) SEQ ID NOs: 209, 210, and 211; f)SEQ ID NOs: 217, 218, and 219; g) SEQ ID NOs: 225, 226, and 219; h) SEQ ID NOs: 233, 234, and 219; i) SEQ ID NOs: 241, 242, and 219; j) SEQ ID NOs: 249, 250, and 251; k) SEQ ID NOs: 257, 218, and 219; l) SEQ ID NOs: 265, 266, and 267; m) SEQ ID NOs: 27
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 177-179, 185-186, 194-195, 201- 202, 209-211, 217-219, 225-226, 233-234, 241-242, 249-251, 257, 265-267, 273-274, 281- 283, or 289-290.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a V H H domain, comprises: a CDR1 comprising or consisting of FX1FX2X3X4X5MX6 (SEQ ID NO: 297), wherein X1 is T or D; X2 is A, S, E, G or D; X3 is D, T, G, E, V or S; X4 is T, M, Q, A, H, Y, V or N; X 5 is D, P or A; and X 6 is G or S; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of AIX1X2X3X4X5X6X7YYADSVX8G (SEQ ID NO: 298), wherein X1 is S or no amino acid; X2 is G, A, P, R or S; X3 is S, D, T or G; X 4 is G, A, S, or E; X
  • SEQ ID NO: 297 is blank, as sequence listing rules do not permit an amino acid sequence with fewer than four “specifically defined” amino acids, and SEQ ID NO: 297, (FX 1 FX 2 X 3 X 4 X 5 MX 6 ) has only three “specifically defined” amino acids at positions 1, 3 and 8.
  • X6 is either G or S (see Table 4 above).
  • SEQ ID NO: 297 is represented by two sequences, SEQ ID NO: 772 (FX 1 FX 2 X 3 X 4 X 5 MG) and SEQ ID NO: 773 (FX 1 FX 2 X 3 X 4 X 5 MS), which represent the sequences wherein X6 is specified as G or S, respectively, with X1, X2, X3, X4, and X5 defined as in SEQ ID NO: 297 (see Table 4 above).
  • SEQ ID NO: 297 is the same as the combined sequences of SEQ ID NOs 772 and 773, and the combination of SEQ ID NOs: 772 and 773 can replace SEQ ID NO: 297, optionally wherein the G or S at the position of X6 of SEQ ID NO: 772 and 772 is substituted with a conservative substitution of G or S.
  • the sdAb optionally a V H H domain, comprises a V H FR1 comprising or consisting of any one of SEQ ID NOs: 180, 188, 212, 228 or 284, a VH FR2 comprising or consisting of SEQ ID NO: 181, a VH FR3 comprising or consisting of any one of SEQ ID NOs: 182, 190, 278 or 286, and a V H FR4 comprising or consisting of any one of SEQ ID NOs: 183 or 215, optionally wherein the combination of FR1, FR2, FR3, and FR4 is selected from one of the following combinations: a.) SEQ ID NOs: 180, 181, 182, and 183; b.) SEQ ID NOs: 188, 181, 182, and 183; c.) SEQ ID NOs: 212, 181, 182, and 215; d.) SEQ ID NOs: 188, 181, 182, and 215
  • each VH framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 180-183, 188, 190, 212, 215, 228, 278, 284 or 286.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises: a FR1 comprising or consisting of the amino acid sequence X 1 VX 2 LX 3 ESGGGLVQPGGSLRLSX 4 AASG (SEQ ID NO: 300), wherein X 1 is E or G, X 2 is Q or R; X3 is L or V; and X4 is A or C; or optionally a conservative substitution of any of the foregoing; optionally wherein X1 is E; X2 is Q; X3 is L; and X4 is A or C; a FR2 comprising or consisting of the amino acid sequence SEQ ID NO: 301; a FR3 comprising or consisting of the amino acid sequence RFX1ISRDNSKNTLYLQMNX2LRAX3DTAVYYX4 (SEQ ID NO: 302), wherein X1 is T or S; X2 is S or G; X3 is E or A;
  • the sdAb optionally a V H H domain, comprises or consists of a sequence selected from any one of SEQ ID NOs: 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288 or 296.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 177-179, 185-186, 194-195, 201, 202, 209-211, 217-219, 225-226, 233-234, 241-242, 249-251, 257, 265-267, 273-274, 281-283, or 289-290, and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a V H H domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288 or 296.
  • SEQ ID NOs amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288
  • the sdAb optionally a V H H domain, comprises a CDR3 comprising or consisting of the amino acid sequence AALGDYGX1AIDYSEADY (SEQ ID NO: 306), wherein X1 is D or G; or optionally a conservative substitution of any of the foregoing.
  • the CDR3 comprises or consists of any one of SEQ ID NOs: 179 or 195.
  • the sdAb optionally a VHH domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 177, 185, or 201; a CDR2 comprising or consisting of any one of SEQ ID NOs: 178, 186, 194 or 202; and a CDR3 comprising or consisting of any one of SEQ ID NOs: 179 or 195.
  • the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: a.) SEQ ID NOs: 177, 178, and 179; b.) SEQ ID NOs: 185, 186, and 179; c.) SEQ ID NOs: 177, 194, and 195; and d.) SEQ ID NOs: 201, 202, and 179.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 177-179, 185-186, 194-195, or 201-202.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises: a CDR1 comprising or consisting of the amino acid sequence FTFX 1 X 2 X 3 X 4 MX 5 (SEQ ID NO: 304), wherein X1 is A, S or E; X2 is D, T, or G; X3 is T, M or Q; X4 is D or P; and X5 is G or S; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of the amino acid sequence AISX 1 X 2 X 3 X 4 X 5 TYYADSVX 6 G (SEQ ID NO: 305), wherein X 1 is G or A; X 2 is S or D; X 3 is G or A; X 4 is G, D, or E; X 5 is N, V or A; and X6 is K or R; or optionally a conservative substitution of any of the foregoing; and a CDR3 comprising or consisting or consisting of
  • the sdAb optionally a V H H domain, comprises a V H FR1 comprising or consisting of any one of SEQ ID NOs: 180 or 188, a VH FR2 comprising or consisting of SEQ ID NO: 181, a VH FR3 comprising or consisting of any one of SEQ ID NOs: 182 or 190, and a V H FR4 comprising or consisting of SEQ ID NO: 183, optionally wherein the combination of FR1, FR2, FR3, and FR4 is selected from one of the following combinations: a.) SEQ ID NOs: 180, 181, 182, and 183; b.) SEQ ID NOs: 188, 181, 190, and 183; and c.) SEQ ID NOs: 188, 181, 182, and 183.
  • each V H framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 180-183, 188 or 190.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises or consists of a sequence selected from any one of SEQ ID NOs: 184, 192, 200, 208, optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 177-179, 185-186, 194-195, or 201-202, and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 184, 192, 200, 208. B.
  • the sdAb optionally a VHH domain, comprises a CDR3 comprising or consisting of the amino acid sequence AX 1 LX 2 QELTGASEHDY (SEQ ID NO: 309), wherein X 1 is H or Y; and X 2 is D or G; or optionally a conservative substitution of any of the foregoing.
  • the CDR3 comprises or consists of SEQ ID NO: 211.
  • the sdAb optionally a VHH domain, comprises: a CDR1 comprising or consisting of SEQ ID NO: 209, a CDR2 comprising or consisting of SEQ ID NO: 210, and a CDR3 comprising or consisting of SEQ ID NO: 211.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 209-211.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a V H H domain, comprises: a CDR1 comprising or consisting of the amino acid sequence FTFX 1 X 2 X 3 X 4 MG (SEQ ID NO: 307), wherein X 1 is G or D; X2 is D or E; X3 is A or T; and X4 is D or A; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of the amino acid sequence AISASX 1 X 2 X 3 TYYADSVKG (SEQ ID NO: 308), wherein X 1 is G or S; X 2 is D or A; and X 3 is X or E; or optionally a conservative substitution of any of the foregoing; and a CDR3 comprising or consisting of the amino acid sequence AX1LX2QELTGASEHDY (SEQ ID NO: 309), wherein X 1 is H
  • the sdAb optionally a V H H domain, comprises a V H FR1 comprising or consisting of SEQ ID NO: 212, a V H FR2 comprising or consisting of SEQ ID NO: 213 a V H FR3 comprising or consisting of SEQ ID NO: 214, and a VH FR4 comprising or consisting of SEQ ID NO: 215.
  • each VH framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 212-215.
  • the sdAb optionally a V H H domain, comprises or consists of a sequence that is SEQ ID NO: 216.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 209-211, and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the framework region of any one of SEQ ID NOs: 212-215.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a V H H domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 216.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a V H H domain, comprises a CDR3 comprising or consisting of the amino acid sequence AIIDDILTX 1 GTFDEGDY (SEQ ID NO: 312), wherein X1 is G or D; or optionally a conservative substitution of any of the foregoing.
  • the CDR3 comprises or consists of any one of SEQ ID NOs: 219 or 251.
  • the sdAb optionally a V H H domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 217, 225, 233, 241, 249 or 257; a CDR2 comprising or consisting of any one of SEQ ID NOs: 218, 226, 234, 242 or 250, and a CDR3 comprising or consisting of any one of SEQ ID NOs: 219 or 251.
  • the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: a.) SEQ ID NOs: 217, 218, and 219; b.) SEQ ID NOs: 225, 226, and 219; c.) SEQ ID NOs: 233, 234, and 219; d.) SEQ ID NOs: 241, 242, and 219; e.) SEQ ID NOs: 249, 250, and 251; and f.) SEQ ID NOs: 257, 218, and 219.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 217-219, 225-226, 233-234, 241-242, 249-251, and 257.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a V H H domain, comprises: a CDR1 comprising or consisting of the amino acid sequence FX1FX2X3X4X5MG (SEQ ID NO: 310), wherein X1 is T or D; X2 is A, E, D, S or G; X3 is D, E or T; X4 is T, H, Y, V or A; and X5 is D, A or S; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of the amino acid sequence AISX 1 X 2 X 3 X 4 X 5 X 6 YYADSVKG (SEQ ID NO: 311), wherein X 1 is G, P, R or A; X 2 is S or D; X3 is G or A; X4 is G, A, T or D; X5 is S, T, A or V; and X6 is T or A; or optionally a conservative substitution of any
  • the sdAb optionally a V H H domain, comprises a VH FR1 comprising or consisting of any one of SEQ ID NOs: 220 or 228, a VH FR2 comprising or consisting of SEQ ID NO: 221, a VH FR3 comprising or consisting of SEQ ID NO: 222, and a V H FR4 comprising or consisting of SEQ ID NO: 223, optionally wherein the combination of FR1, FR2, FR3, and FR4 is selected from one of the following combinations: a) SEQ ID NOs: 220, 221, 222, and 223; and b) SEQ ID NOs: 228, 221, 222, and 223.
  • each V H framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 220-223, or 228.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises or consists of a sequence selected from any one of SEQ ID NOs: 224, 232, 240, 248, 256 or 264, optionally wherein each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 217-219, 225-226, 233-234, 241- 242, 249-251, or 257; and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 224, 232, 240, 248, 256 or 264. D.
  • the sdAb comprises a CDR3 comprising or consisting of SEQ ID NO: 267.
  • the sdAb optionally a V H H domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 265 or 273, a CDR2 comprising or consisting of any one of SEQ ID NOs: 266 or 274, and a CDR3 comprising or consisting of SEQ ID NO: 267.
  • the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: a) SEQ ID NOs: 265, 266, and 267; and b) SEQ ID NOs: 273, 274, and 267.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 265-267, or 273-274.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a V H H domain, comprises: a CDR1 comprising or consisting of the amino acid sequence FTFX 1 X 2 X 3 DMG (SEQ ID NO: 313), wherein X1 is D, G or E; X2 is V, S or G; and X3 is S, N, or Q; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of the amino acid sequence AIX 1 X 2 X 3 X 4 X 5 X 6 TYYADSVKG (SEQ ID NO: 314), wherein X 1 is no amino acid or S; X2 is G or S; X3 is T, G, or S; X4 is E or G; X5 is G, D or E; and X6 is D, T or H; or optionally a conservative substitution of any of the foregoing; and a CDR3 comprising or consisting of the amino acid sequence SEQ ID NO: 315; wherein X1 is
  • the sdAb optionally a VHH domain, comprises a V H FR1 comprising or consisting of SEQ ID NO: 268, a V H FR2 comprising or consisting of SEQ ID NO: 269, a V H FR3 comprising or consisting of any one of SEQ ID NOs: 270 or 278, and a VH FR4 comprising or consisting of SEQ ID NO: 271, optionally wherein the combination of FR1, FR2, FR3, and FR4 is selected from one of the following combinations: a) SEQ ID NOs: 268, 269, 270, and 271; and b) SEQ ID NOs: 268, 269, 278, and 271.
  • each VH framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 268-271, or 278.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a V H H domain, comprises or consists of a sequence selected from any one of SEQ ID NOs: 272 or 280.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 265-267, or 273-274, and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the framework region of any one of SEQ ID NOs: 268-271, or 278.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a V H H domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 272 or 280.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises a CDR3 comprising or consisting of SEQ ID NO: 283.
  • the sdAb optionally a V H H domain, comprises: a CDR1 comprising or consisting of any one of SEQ ID NOs: 281 or 289, a CDR2 comprising or consisting of any one of SEQ ID NOs: 282 or 290, and a CDR3 comprising or consisting of SEQ ID NO: 283.
  • the combination of CDR1, CDR2, and CDR3 is selected from one of the following combinations: SEQ ID NOs: 281, 282, and 283; and b) SEQ ID NOs: 289, 290, and 283.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 281-283, and 289-290.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises: a CDR1 comprising or consisting of the amino acid sequence FTFX 1 X 2 X 3 AMX 4 (SEQ ID NO: 316), wherein X1 is S or D, X2 is S, E or D; X3 is Y or H; and X4 is S or G; or optionally a conservative substitution of any of the foregoing; a CDR2 comprising or consisting of the amino acid sequence AISX 1 X 2 X 3 X 4 X 5 TYYADSVKG (SEQ ID NO: 317), wherein X 1 G or A; X 2 is S or D; X 3 is G or A; X 4 is G or S; and X 5 is S, V or T; or optionally a conservative substitution of any of the foregoing; and a CDR3 comprising or consisting of the amino acid sequence SEQ ID NO: 318; wherein the combination of CDR1, CDR2 and CDR3
  • the sdAb optionally a VHH domain, comprises a VH FR1 comprising or consisting of SEQ ID NO: 284, a VH FR2 comprising or consisting of SEQ ID NO: 285, a VH FR3 comprising or consisting of SEQ ID NO: 286, and a V H FR4 comprising or consisting of SEQ ID NO: 287.
  • the combination of FR1, FR2, FR3, and FR4 is 284, 285, 286, and 287.
  • each VH framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 284, 285, 286, and 287.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises or consists of a sequence selected from any one of SEQ ID NOs: 288 or 296.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 281-283, and 289-290, and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the framework region of any one of SEQ ID NOs: 284, 285, 286, and 287.
  • the substitution(s) are conservative substitution(s).
  • the sdAb optionally a VHH domain, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 288 or 296.
  • the substitution(s) are conservative substitution(s). F.
  • the sdAb optionally a VHH domain, comprises: a FR1 comprising or consisting of the amino acid sequence X 1 VX 2 LX 3 ESGGGLVQPGGSLRLSX 4 AASG (SEQ ID NO: 300), wherein X 1 is E or G, X 2 is Q or R; X3 is L or V; and X4 is A or C; or optionally a conservative substitution of any of the foregoing; optionally wherein X 1 is E; X 2 is Q; X 3 is L; and X 4 is A or C; a FR2 comprising or consisting of the amino acid sequence SEQ ID NO: 301; a FR3 comprising or consisting of the amino acid sequence RFX1ISRDNSKNTLYLQMNX2LRAX3
  • the sdAb comprises a VHH domain with a substitution at C22 of FR1 and/or C30 of FR3, optionally wherein the substitution is independently selected from a C to A and a C to V substitution.
  • the sdAb has a Kd value of less than 1x10 -7 and optionally at least 1x10- 10 , optionally wherein the Kd is 1.49x10 -7 to 5.56x10 -9 .
  • the Kd value is calculated using human c-Myc, optionally as described for values of Octet sdAb Kd Human Myc Peptide Monovalent (M) in Example 4.
  • the sdAb has a Poly Specificity Reagent (PSR) score of less than 1, optionally less than 0.43, optionally less than 0.26. In some embodiments, the sdAb has a PSR score of at least 0.01, optionally at least 0.26, optionally at least 0.43. In some embodiments, the sdAb has a PSR score of 1 to 0.01, 0.43 to 0.01, or 0.26 to 0.01. In some embodiments, the sdAb has a melting temperature (T M ) of at least 40°C, optionally at least 45.5°C, optionally at least 64°C.
  • T M melting temperature
  • the sdAb has a TM of less than 70°C, optionally wherein the TM is 45°C to 65°C. In some embodiments, any of the foregoing measures are calculated as detailed in Example 4. In some embodiments, the sdAb binds, or binds at least in part, to at least a portion of amino acids 410-419 of MYC (SEQ ID NO: 1). In some embodiments, the sdAb binds, or binds at least in part, to at least a portion of a peptide consisting of SEQ ID NO: 769.
  • the sdAb binds, or binds at least in part, to at least a portion of a peptide consisting of SEQ ID NO: 770. In some embodiments, the sdAb binds, or binds at least in part, to at least a portion of a peptide consisting of SEQ ID NO: 771. In some embodiments, the sdAb binds, or binds at least in part, to at least a portion of amino acids 410-419 of MYC (SEQ ID NO: 1), and/or one or more of SEQ ID NOs: 769-771. In some embodiments, the sdAb does not comprise or consist of any one of SEQ ID NO: 138-176 and 641-768.
  • the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • a polypeptide comprising or consisting of an anti-Myc sdAb disclosed above and elsewhere herein can be utilized as a myc-binding moiety in a larger polypeptide, compound or conjugate.
  • a polypeptide comprising or consisting of an anti-Myc sdAb disclosed herein is conjugated to a therapeutic compound such as a drug (e.g., a chemotherapeutic drug or agent).
  • a polypeptide comprising or consisting of an anti-Myc sdAb disclosed herein is conjugated to a detectable label (e.g., a radioisotope, fluorescent molecule, etc.).
  • detectable label e.g., a radioisotope, fluorescent molecule, etc.
  • Portions / URDs of E3 Ubiquitin Ligases and Viral Homologs Disclosed herein are polypeptides comprising or consisting of a portion derived from a protein selected from the group consisting of a RING family E3 ubiquitin ligase, a cullin family E3 ubiquitin ligase, a homologous to E6AP carboxyl terminus (HECT) family E3 ubiquitin ligase, and a viral homolog of an E3 ubiquitin ligase.
  • the viral homolog is VIF of HIV-1.
  • the portion comprises an amino acid substitution (optionally a conservative substitution), addition, and/or deletion to the protein.
  • the deletion is not an end terminal deletion or truncation of the protein.
  • the polypeptide and/or the portion is a non-natural polypeptide.
  • the portion has ubiquitin-proteasome recruiting activity.
  • the portion is a URD that has ubiquitin-proteasome recruiting activity.
  • the portion of E3 ligase or viral homolog is fused to a polypeptide.
  • the fusion polypeptide comprises a linker, localization sequence (e.g., NLS), first and/or second targeting moiety, and/or other polypeptide disclosed herein.
  • the portion is derived from an E3 ubiquitin ligase selected from the group consisting of CHIP, DCAF1, E6AP, FBXW7-alpha (also referred to herein as “FBXW7a”), FBXW7-beta (also referred to herein as “FBXW7b”), Keap1, NHLRC1, RNF4, RNF6, RNF11, RNF12, RNF20, RNF25, RNF111, RNF114, RNF115, RNF125, RNF128, RNF138, RNF149, RNF152, RNF165, RNF166, RNF182, SPOP, beta-TRCP, TRIM21, TRIM32, VIF, ZNRF1, ZNRF4, and CBL-b (Y363E).
  • E3 ubiquitin ligase selected from the group consisting of CHIP, DCAF1, E6AP, FBXW7-alpha (also referred to herein as “FBXW7a”), FBXW7
  • the portion is selected from the group consisting of CBLb.1, CHIP.1, DCAF1.1, E6AP.1, FBXW7a.1, FBXW7a.2, FBXW7a.3, FBXW7a.4, FBXW7a.5, FBXW7a.6, FBXW7a.7, FBXW7a.8, FBXW7a.9, FBXW7b.1, FBXW7b.2, FBXW7b.3, FBXW7b.4, Keap1.1, NHLRC1.1, NHLRC1.2, NHLRC1.3, NHLRC1.4, NHLRC1.5, RNF4.1, RNF6.1, RNF6.2, RNF11.1, RNF12.1, RNF12.2, RNF20.1, RNF25.1, RNF111.1, RNF114.1, RNF115.1, RNF125.1, RNF125.2, RNF125.3, RNF125.4, RNF125.5, RNF125.6
  • the portion is derived from a monomeric RING family E3 ligase.
  • the portion is derived from a monomeric RING family E3 ligase selected from the group consisting of NHLRC1, RNF11, RNF111, RNF114, RNF115, RNF12, RNF125, RNF128, RNF138, RNF149, RNF152, RNF165, RNF166, RNF182, RNF20, RNF25, RNF4, RNF6, ZNRF1, ZNRF4, and CBL-b (Y363E).
  • the portion is derived from a monomeric RING family E3 ligase selected from the group consisting of RNF125, NHL, RC1, RNF4, RNF6, RNF12, RNF138 and ZNRF1.
  • the portion is selected from the group consisting of NHLRC1.1, NHLRC1.2, NHLRC1.3, NHLRC1.4, NHLRC1.5, RNF11.1, RNF111.1, RNF114.1, RNF115.1, RNF12.1, RNF12.2, RNF125.1, RNF125.2, RNF125.3, RNF125.4, RNF125.5, RNF125.6, RNF125.7, RNF128.1, RNF138.1, RNF149.1, RNF152.1, RNF165.1, RNF166.1, RNF182.1, RNF182.2, RNF20.1, RNF25.1, RNF4.1, RNF6.1, RNF6.2, ZNRF1.1, ZNRF4.1, and CBLb.1.
  • the portion is selected from the group consisting of NHLRC1.1, NHLRC1.2, NHLRC1.3, NHLRC1.4, NHLRC1.5, RNF125.1, RNF125.2, RNF125.3, RNF125.4, RNF125.5, RNF125.6, RNF125.7, RNF4.1, RNF6.1, RNF6.2, RNF12.1, RNF12.2, RNF138.1, and ZNRF1.1.
  • the portion is derived from a cullin family E3 ligase.
  • the portion is derived from a cullin family E3 ligase selected from the group consisting of DCAF1, beta-TRCP, FBXW7-alpha, FBXW7-beta, Keap1, and SPOP.
  • the portion is derived from a cullin family E3 ligase selected from the group consisting of beta-TRCP, FBXW7-alpha, and FBXW7-beta. In some embodiments, the portion is selected from the group consisting of DCAF1.1, bTRCP.1, FBXW7a.1, FBXW7a.2, FBXW7a.3, FBXW7a.4, FBXW7a.5, FBXW7a.6, FBXW7a.7, FBXW7a.8, FBXW7a.9, FBXW7b.1, FBXW7b.2, FBXW7b.3, FBXW7b.4, Keap1.1, SPOP.1, SPOP.2, and SPOP.3.
  • the number of amino acids in the portion is, is about, or is less than, 80, 75, 70, 65, 60, 65, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%, of the number of amino acids in the wild-type full-length protein (e.g. sequences disclosed in Table 23), or a range defined by any two of the preceding values, optionally 15-80, 15-50, 25-80, 25-50, 30- 60, 1-80, 2-80, 1-70, 2-70, 2-65%. In some embodiments, the number of amino acids in the portion is, or is about 15-50% of the number of amino acids in the wild-type full-length protein.
  • the number of amino acids in the portion is, or is about, 2-61% of the number of amino acids in the wild-type full-length protein. In some embodiments, the portion is, is about, is less than or equal to 370, 369, 368, 367, 366, 365, 364, 363, 362, 361, 360, 300, 250, 200, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 129, 128, 127, 126, 125, 124, 123, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 25, or 20 amino acids long, or a range defined by any two of the preceding values.
  • the portion is, or is about, 20-370, 20-150, 20- 100, 20-80, 30-150, 30-100, 50-150 amino acids long. In some embodiments, the portion is, or is about, 50-100 or 20-100 amino acids long. In some embodiments, the portion is, or is about, 40-130 or 44-126 amino acids long.
  • the portion comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 39-113, optionally SEQ ID NOs: 54-113, optionally wherein the portion has ubiquitin-proteasome recruiting activity.
  • the portion comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 76-106 and 111-113, optionally wherein the portion has ubiquitin-proteasome recruiting activity.
  • the portion comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 76-80, 85-93, 95, 104-106, and 111, optionally wherein the portion has ubiquitin-proteasome recruiting activity.
  • the portion consists of an amino acid sequence selected from any one of SEQ ID NOs: 76-80, 85- 93, 95, 104-106, and 111.
  • the URD comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 39-113. In some embodiments, the URD comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of any one of SEQ ID NO: 54-113.
  • the URD comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 76, 88, 98, 104 and 111.
  • the URD comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 76, 88, 98, and 104.
  • the URD comprises or consists of a truncation or fragment of one of the forgoing that retains ubiquitin- proteasome recruiting activity.
  • the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the portion comprises an amino acid substitution, addition, and/or deletion to the protein.
  • the deletion is not an end terminal deletion or truncation of the protein.
  • the polypeptide and/or the portion is a non-natural polypeptide.
  • the portion is a URD that has ubiquitin- proteasome recruiting activity.
  • bifunctional polypeptides optionally dual-specific, comprising the polypeptide comprising or consisting of the portion derived from a protein selected from the group consisting of a RING family E3 ubiquitin ligase, a cullin family E3 ubiquitin ligase, a homologous to E6AP carboxyl terminus (HECT) family E3 ubiquitin ligase, and a viral homolog of an E3 ubiquitin ligase, as described in the paragraphs above and elsewhere herein, wherein the bifunctional polypeptide comprises: a) the portion of the protein and, b) a first and optionally a second targeting moiety (also referred to as a target “binder” or “binding moiety”) that is capable of binding to a
  • the first and/or second targeting moiety binds selectively or specifically to the target protein(s).
  • the first targeting moiety, and optionally the second targeting moiety comprises or consists of an anti-Myc sdAb disclosed herein.
  • the bifunctional polypeptides optionally dual-specific, promote proteasome-mediated degradation of a target protein or proteins.
  • proximity of the bifunctional polypeptide to the target protein(s) through binding of the targeting moiety induces ubiquitination of the target protein(s) via the URD, thereby promoting proteasome-mediated degradation of the target protein(s).
  • one or both of the target protein(s) is an intracellular target protein.
  • the bifunctional polypeptide that promotes proteasome-mediated degradation of a target protein(s) comprises or consisting of the polypeptide of comprising or consisting of the portion derived from a protein selected from the group consisting of a RING family E3 ubiquitin ligase, a cullin family E3 ubiquitin ligase, a homologous to E6AP carboxyl terminus (HECT) family E3 ubiquitin ligase, and a viral homolog of an E3 ubiquitin ligase, as described in the paragraphs above and elsewhere herein.
  • the URD consists of the URD derived from a protein selected from the group consisting of a RING family E3 ubiquitin ligase, a cullin family E3 ubiquitin ligase, a homologous to E6AP carboxyl terminus (HECT) family E3 ubiquitin ligase, and a viral homolog of an E3 ubiquitin ligase, as described in the paragraphs above and elsewhere herein.
  • the first and/or second targeting moiety binds selectively or specifically to the target protein(s).
  • the targeting moieties and the URD disclosed herein may be used in the construction of the bifunctional polypeptide.
  • Table 23 Representative E3 ligases and viral homologs for derivation of portions
  • Table 24 Exemplary Portions / URDs derived from E3 ligases and viral homologs
  • the component of the dual-specific bifunctional polypeptide that is capable of recruiting the ubiquitination complex is generally composed of a URD.
  • the URD may be derived from an E3 ubiquitin ligase, such as a human E3 ubiquitin ligase.
  • the URD does not strictly need to be derived from an E3 ubiquitin ligase, and may otherwise be derived from other sources, such as viral analogues of E3 ubiquitin ligases that have ubiquitin complex recruitment function (for example, the VIF protein of HIV).
  • the URD may be derived from any of the diverse family of E3 ubiquitin ligases based on factors such as size, localization in the cell (e.g., cytoplasm and/or nucleus), and orientation of the URD domain within the E3 ubiquitin ligase (i.e., if the URD appears N-terminally or C-terminally to the substrate recognition domain).
  • exemplary URD domains may include, but are not limited to, U-box, RING, HECT, F-box (Cul1-SKP1), BTB (Cul3), and H-box (Cul4-DDB1) domains.
  • exemplary URDs may be derived from E3 ubiquitin ligases including but not limited to CHIP (also termed STUB1), RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL- b (Y363E), TRIM21, E6AP, FBXW7 alpha, FBXW7 beta, beta-TRCP, Keap1, SPOP, or DCAF1.
  • the URD is derived from a RING domain (e.g., RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL-b (Y363E)) or an H-box (Cul4-DDB1) domain (e.g., DCAF1), or any truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • a RING domain e.g., RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL-b (Y363E)
  • Cul4-DDB1 domain e.g., DCAF1
  • the URD should be understood as a protein, or fragment or truncation thereof, that can function to recruit a ubiquitination complex (e.g., can recruit an E2 ubiquitin conjugating enzyme).
  • the URD is derived from an E3 ubiquitin ligase.
  • the URD is derived from CHIP, RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL-b (Y363E), TRIM21, E6AP, FBXW7 alpha, FBXW7 beta, beta-TRCP, Keap1, SPOP, or DCAF1, or a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the URD is derived from CHIP, RNF114, RNF125 (e.g., RNF125.2), RNF138, RNF166, NHLRC1, CBL-b (Y363E), TRIM21, E6AP, FBXW7 alpha, FBXW7 beta, beta-TRCP, Keap1, SPOP (e.g., SPOP.2 or SPOP.3), or DCAF1, or a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the URD is selected from a RING domain and an H-box (Cul4-DDB1) domain, or a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the RING domain is RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL-b (Y363E), or TRIM21.
  • the H-box domain is DCAF1.
  • the URD is selected from: the URD derived from a protein which localizes to the cytoplasm; the URD derived from a protein which localizes to the nucleus; and the URD derived from a protein which localizes to the cytoplasm and the nucleus; or a truncation or fragment of the URD that retains ubiquitin-proteasome recruiting activity.
  • the protein which localizes to the cytoplasm is FBXW7 beta.
  • the protein which localizes to the nucleus is selected from FBXW7 alpha and SPOP.
  • the protein which localizes to the cytoplasm and the nucleic is selected from CHIP, RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL-b (Y363E), TRIM21, E6AP, and DCAF1.
  • the URD is derived from an E3 ligase selected from the group consisting of NHLRC1, DCAF1, RNF125, RNF165, RNF4, and SPOP.
  • the URD is derived from an E3 ligase selected from the group consisting of optionally NHLRC1, RNF125, RNF165, RNF4 and SPOP.
  • the URD is derived from an E3 ligase selected from the group consisting of optionally NHLRC1, RNF125, RNF165, and RNF4.
  • the URD is selected from the group consisting of NHLRC1.1, DCAF1.1, RNF125.2, RNF125.3, RNF125.4, RNF125.5, RNF125.6, RNF165.1, RNF4.1, SPOP.2, and SPOP.3.
  • the URD is selected from the group consisting of NHLRC1.1, RNF125.2, RNF125.3, RNF125.4, RNF125.5, RNF125.6, RNF165.1, RNF4.1, SPOP.2, and SPOP.3.
  • the URD is selected from the group consisting of NHLRC1.1, RNF125.2, RNF165.1, and RNF4.1.
  • the protein is an E3 ubiquitin ligase.
  • the URD is, is about, is at least, is at least about, is not more than, or is not more than about, 370, 369, 368, 367, 366, 365, 364, 363, 362, 361, 360, 300, 250, 200, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 129, 128, 127, 126, 125, 124, 123, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 25, or 20 amino acids long, or a range defined by any two of the preceding values, for
  • the URD comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 39-53, or a truncation or fragment thereof that retains ubiquitin- proteasome recruiting activity.
  • the URD comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 54- 113, or a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the URD comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 73, 74, 76, 88-92, 98, and 104, or a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the URD comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 76, 88, 98, or a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the amino acid sequence differs from a recited reference sequence, the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the URD is selected from: a URD derived from a protein which localizes to the cytoplasm, optionally selected from FBXW7 beta and Keap1; a URD derived from a protein which localizes to the nucleus, optionally selected from FBXW7 alpha, RNF165, and SPOP; and a URD derived from a protein which localizes to the cytoplasm and the nucleus, optionally selected from beta-TRCP, CHIP, RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL-b (Y363E), TRIM21, E6AP, DCAF1 VIF, RNF11, RNF111, RNF115, RNF12, RNF128, RNF149, RNF152, RNF165, RNF182, RNF20, RNF25, RNF4, RNF6, TRIM32, ZNRF1, ZNRF4.
  • Table 24 depicts various E3 ubiquitin ligases and respective URDs embodied herein.
  • the portion comprises an exemplary URD described below and/or in Table 24.
  • the dual-specific bifunctional polypeptide comprises an exemplary URD described below and/or in Table 24. It is envisioned that alternative portions, fragments, or truncations of the E3 ubiquitin ligase proteins having ubiquitin-proteasome recruiting activity may also be used.
  • polypeptides, or bifunctional polypeptides optionally dual-specific, which are engineered (e.g., by modification of an endogenous localization peptide sequence, by adding localization peptide sequences, and/or by selecting a component which has an endogenous localization sequence) to direct the polypeptide or bifunctional polypeptide to a particular subcellular compartment, e.g., the cytoplasm, the nucleus, or both.
  • the polypeptide comprising a portion derived from an E3 ligase or viral homolog thereof comprises one or more localization peptide sequences.
  • the bifunctional polypeptide comprises one or more localization peptide sequences.
  • the polypeptide comprising a portion, or the bifunctional polypeptide comprises one or more localization peptide sequences that direct the polypeptide or bifunctional polypeptide to a subcellular compartment or compartments, optionally wherein the subcellular compartment is the nucleus and/or the cytoplasm.
  • the one or more localization peptide sequences direct the polypeptide or bifunctional polypeptide to a desired subcellular compartment or compartments, optionally wherein the one or more localization peptide sequences are selected or designed to direct the polypeptide or bifunctional polypeptide to a desired subcellular compartment or compartments.
  • the one or more localization peptide sequences comprise or consist of a nuclear localization signal (NLS) peptide and/or a nuclear export signal (NES) peptide.
  • the bifunctional polypeptide further comprises a nuclear localization signal (NLS) peptide.
  • the NLS peptide is located at the N-terminus of the bifunctional polypeptide, the C-terminus of the bifunctional polypeptide, or internally within the bifunctional polypeptide.
  • the NLS signal is an NLS that naturally occurs in one or more of the components of the bifunctional polypeptide, such as an NLS that is naturally found in the first and/or optional second targeting moiety and/or URD.
  • the first and/or optional second targeting moiety and/or URD of a bifunctional polypeptide may comprise an NLS within their sequence.
  • the NLS is an NLS that is engineered into the bifunctional polypeptide, for example, added N-terminally, C-terminally, or internally within a component of the bifunctional polypeptide, such as the first and/or optional second targeting moiety and/or URD.
  • the NLS peptide comprises or consists of the amino acid sequence of SEQ ID NO: 51.
  • suitable NLS peptides generally known in the art may also be used.
  • the localization sequence is a sequence from Table 25 or 26.
  • the one or more localization peptide sequences comprise or consist of an endogenous localization peptide sequence of the first and/or optional second targeting moiety and/or URD. In some embodiments, the one or more localization peptide sequences comprise or consist of an endogenous localization peptide sequence of the URD, for example the localization sequences disclosed in Table 26.
  • the URD is selected from: a URD derived from a protein which localizes to the cytoplasm, optionally selected from FBXW7 beta and Keap1; a URD derived from a protein which localizes to the nucleus, optionally selected from FBXW7 alpha, RNF165, and SPOP; and a URD derived from a protein which localizes to the cytoplasm and the nucleus, optionally selected from beta- TRCP, CHIP, RNF114, RNF125, RNF138, RNF166, NHLRC1, CBL-b (Y363E), TRIM21, E6AP, DCAF1 VIF, RNF11, RNF111, RNF115, RNF12, RNF128, RNF149, RNF152, RNF165, RNF182, RNF20, RNF25, RNF4, RNF6, TRIM32, ZNRF1, ZNRF4.
  • the one or more localization peptide sequences comprise or consist of an endogenous localization peptide sequence of the URD and/or first and/or optional second targeting moiety that is modified, wherein the modification alters the subcellular compartment or compartments to which the bifunctional polypeptide, optionally dual-specific, is directed as compared to the native endogenous localization peptide sequence.
  • the modification comprises or consists of a substitution, truncation or deletion of an endogenous localization peptide sequence.
  • the native endogenous localization peptide sequence directs the polypeptide to the nucleus
  • the modified endogenous localization peptide sequence directs the polypeptide to the cytoplasm
  • c the native endogenous localization peptide sequence directs the polypeptide to the nucleus
  • the native endogenous localization peptide sequence directs the polypeptide to the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to the nucleus; d. the native endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to only the cytoplasm; e. the native endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to only the nucleus; f.
  • the native endogenous localization peptide sequence directs the polypeptide to the nucleus, and the modified endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm; or g. the native endogenous localization peptide sequence directs the polypeptide to the cytoplasm, and the modified endogenous localization peptide sequence directs the polypeptide to the nucleus and the cytoplasm.
  • the one or more localization peptide sequences comprises or consists of an endogenous localization peptide sequence of the URD that is modified, wherein the native endogenous localization peptide sequence directs the polypeptide to the nucleus, and the modified endogenous localization peptide sequence directs the polypeptide to the cytoplasm.
  • the modified endogenous localization peptide sequence comprises or consists of a modified SPOP URD endogenous localization peptide sequence, optionally wherein the URD is SPOP.3.
  • the one or more localization peptide sequences comprise or consist of an exogenous localization peptide sequence.
  • the one or more localization peptide sequences comprise or consist of an NLS peptide that is or is a derivative of the NLS of MYC, simian virus 40 (SV40), SPOP, nucleoplasmin, 53BP1, Hrp1.
  • the one or more localization peptide sequences comprise or consists of an NLS sequence disclosed in Table 25.
  • the one or more localization peptide sequences comprise or consist of an NLS comprising or consisting of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 114-127, or SEQ ID NOs: 114-122.
  • the amino acid sequence differs from a recited reference sequence, the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the polypeptide comprising a portion derived from an E3 ligase or viral homolog thereof comprises 2, 3, 4, or more localization peptide sequences, optionally wherein the polypeptide comprises 2, 3, 4 or more copies of the same localization sequence in series.
  • the bifunctional polypeptide optionally dual- specific, comprises 2, 3, 4, or more localization peptide sequences, optionally wherein the bifunctional polypeptide comprises 2, 3, 4 or more copies of the same localization sequence in series.
  • the polypeptide or the bifunctional polypeptide optionally dual- specific, comprises one or more localization peptide sequences located at a location selected from the group consisting of: the N-terminus of the bifunctional polypeptide; the C-terminus of the bifunctional polypeptide; internally within the bifunctional polypeptide; as part of the URD, optionally an endogenous portion of the URD; as a part of the first and/or optional second targeting moiety, optionally an endogenous portion of the URD; and a combination of any of the foregoing.
  • a first localization peptide sequence is located at the N- terminus of the polypeptide or the bifunctional polypeptide and a second localization peptide sequence is located at the C-terminus of the polypeptide or the bifunctional polypeptide.
  • Table 25 Exemplary NLS sequences
  • Table 26 Exemplary URD endogenous NLS/NES sequences
  • the NLS peptide comprises or consists of the amino acid sequence of SEQ ID NOs: 51 or 1897-1903.
  • the NLS peptide is derived from MYC NLS, SV40 NLS, 3XMYC NLS, 3xSV40 NLS, SPOP NLS, nucleoplasmin NLS, 53BP1 NLS, or Hrp1 NLS (SEQ ID NOs: 51, 1897-1903.
  • other suitable NLS peptides generally known in the art are contemplated.
  • Some URDSs derived from E3 ubiquitin ligases contain an endogenous NLS, for example, SPOP and SPOP.2.
  • the dual-specific bifunctional polypeptide contains a URD with an endogenous NLS.
  • the dual-specific bifunctional polypeptide contains a URD with an endogenous NLS and an exogenous NLS. In some embodiments, the dual-specific bifunctional polypeptide contains a URD with a truncated or deleted endogenous NLS (e.g., SPOP.3) and an exogenous NLS. Additional localization sequences are disclosed in PCT/US2023/066619, filed May 4, 2023, and PCT/US2023/76886, filed October 13, 2023, each of which is herein incorporated by reference in its entirety. Exemplary Bifunctional Degraders Targeting Myc The following characteristics may apply to embodiments of the bifunctional polypeptides disclosed herein.
  • MYC proto- oncogene bHLH transcription factor
  • Myc bHLH transcription factor
  • Myc is a mutant form.
  • An example sequence for MYC is provided as SEQ ID NO: 1 (Uniprot #P01106).
  • Myc is a major oncogene involved in the pathology of many types of cancer, including solid tumors (including but not limited to hepatocellular carcinoma, liver metastases, and colorectal carcinoma) and hematological malignancies.
  • the bifunctional polypeptide comprises a targeting moiety (also referred to as a “binder” or “binding moiety”) that is capable of binding to Myc.
  • the bifunctional polypeptide promotes proteasome-mediated degradation of at least one target protein.
  • the at least one target protein is Myc, optionally c-Myc.
  • the bifunctional polypeptide comprises: a first targeting moiety comprising or consisting of an anti-Myc sdAb disclosed above under the heading “Anti-Myc Single Domain Antibodies (sdAb)” and elsewhere herein and a URD.
  • the bifunctional polypeptide further comprises a first linker peptide, optionally wherein the first linker peptide is positioned between the first targeting moiety and the URD.
  • the bifunctional polypeptide further comprises one or more localization peptide sequences.
  • the first targeting moiety binds selectively or specifically to the target protein Myc, preferably c-Myc.
  • the first targeting moiety comprises or consists of a VHH, VH, or VNAR domain of a heavy chain antibody (HcAb). In some embodiments, the first targeting moiety comprises or consists of an anti-Myc sdAb, comprising or consisting of a V H domain of an IgG antibody, or a V H H, or V H domain of a heavy chain antibody (HcAb). In some embodiments, the first targeting moiety comprises or consists of a VHH domain.
  • the first targeting moiety comprises or consists of a sdAb and the first targeting moiety is, or is less than, 170, 165, 160, 155, 150, 145, 140, 135, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90, amino acids long, or a range defined by any two of the preceding values, optionally 90-150, 90-130, 110-150, 110-130, 110-125, or 114-124 amino acids long.
  • the first targeting moiety that is capable of binding to Myc may be capable of binding, or binding at least in part, to at least a portion of Myc, such as the DNA binding domain of Myc. In some embodiments, the first targeting moiety that is capable of binding to Myc is capable of binding, or binding at least in part, to at least a portion of amino acids 54-406, amino acids 354-406 (the DNA binding domain of MYC that binds to Omomyc; as seen in PDB: 1NKP), or amino acids 408-437, of MYC (as represented as SEQ ID NO: 1).
  • the first targeting moiety that is capable of binding to Myc is capable of binding, or binding at least in part, to at least a portion of amino acids 410-419 of MYC (as represented as SEQ ID NO: 1), the binding epitope of antibody 9E10 (as seen in PDB: 2OR9).
  • the bifunctional polypeptides disclosed herein that are specific for Myc may be used for the treatment of a cancer, for example, a cancer exhibiting elevated levels of Myc, for example solid tumors (including but not limited to hepatocellular carcinoma, liver metastases, and colorectal carcinoma) and hematological malignancies.
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of SPOP, optionally SPNLS.2, MYC, optionally mycNLS, and Hrp1, optionally hrpNLS.
  • the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 114-127.
  • the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 114, 122 and 116.
  • the URD is the URD of NHLRC1, RNF125, RNF165, RNF4, or ZNRF1, or a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the URD comprises or consists of NHLRC1.1, RNF125.2, RNF165.1, RNF4.1, or ZNRF1.1.
  • the URD comprises or consists of NHLRC1.1 RNF125.2, RNF165.1, or RNF4.1.
  • the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 76-80, 87-93, 98, 104 and 111.
  • the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 76, 88, 98, 104, and 111, In some embodiments, the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 76, 88, 98, and 104.
  • FIG. 8 discloses embodiments of bifunctional polypeptides, with SEQ ID NOs 562- 577 being dual-specific bifunctional polypeptides. The description identifies the elements of the polypeptide in the N- to C-terminal direction.
  • the bifunctional polypeptide targeting Myc is one of the embodiments described in FIG. 8. In some embodiments, the bifunctional polypeptide comprises the embodiments in FIG.
  • the shared element or feature is the target. In some embodiments the shared element or feature is the URD name (e.g., those in Table 24). In some embodiments the shared element or feature is protein from which the URD is derived (e.g., those listed in Table 23). In some embodiments the shared element or feature is one or more of the following: the target; the protein from which the URD is derived; the URD name; the type of targeting moiety (e.g., sdAb); the targeting moiety name (e.g., those in Table 28; note that “full” is not included in the names in FIG. 8); the NLS name (e.g.
  • the first targeting moiety comprises or consists of an anti-Myc sdAb disclosed above under the heading “Anti-Myc Single Domain Antibodies (sdAb)” and elsewhere herein.
  • the first targeting moiety comprises or consists of a VHH domain.
  • the bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 428-561, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • Table 27 provided below lists the results of HiBiTTM degradation studies utilizing exemplary embodiments of bifunctional polypeptides disclosed herein. The tables list a “Grade” of A, B, C or D for the level of degradation of each of the targeted proteins. Details regarding the experimental protocol are provided in the Examples section below.
  • the bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 27, optionally excluding any one of the SEQ ID NOs listed as having a MYC degradation Grade of D, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 27 listed as having a MYC degradation Grade of A, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). In some embodiments, the bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 27 listed as having a MYC degradation Grade of A or B, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the bifunctional polypeptide does not comprise or consist of an amino acid sequence of any one of the SEQ ID NOs in Table 27 listed as having a MYC degradation Grade of C and/or D, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the URD is not derived from CHIP, optionally wherein the URD does not comprise or consist of CHIP.1, optionally wherein the URD does not comprise or consist of SEQ ID NO: 54.
  • a polynucleotide encoding any of the bifunctional polypeptides targeting Myc disclosed above, and elsewhere herein.
  • the dual-specific bifunctional polypeptide comprises the any one of the bifunctional polypeptides targeting Myc disclosed above and elsewhere herein, where the dual-specific bifunctional polypeptide further comprises a second targeting moiety that is capable of binding to at least one second target protein, optionally an intracellular target protein.
  • proximity of the dual-specific bifunctional polypeptide to at least one target protein through binding of the first targeting moiety and/or the second targeting moiety induces ubiquitination of the at least one target protein via the URD, thereby promoting proteasome-mediated degradation of the at least one target protein.
  • the first and the second target proteins can be or are bound by the dual-specific bifunctional polypeptide simultaneously, in some embodiments only one of the first and second target proteins can be or are bound by the dual-specific bifunctional polypeptide at a time.
  • dual-specific bifunctional polypeptide further comprises a second linker peptide, wherein the second linker peptide is positioned between the second targeting moiety and the URD or between the first targeting moiety and the second targeting moiety.
  • the first targeting moiety binds to and/or is designed to bind a first target protein and the second targeting moiety binds to and/or is designed to bind a second target protein.
  • the first targeting moiety and the second targeting moiety bind to and/or are designed to bind to the same target protein, Myc.
  • the first targeting moiety binds to and/or is capable of binding to a first binding site of the target protein and the second targeting moiety binds to and/or is capable of binding to a second binding site of the target protein Myc.
  • the first targeting moiety and second targeting moiety bind to and/or are capable of binding to the same binding site of the target protein, wherein the dual-specific bifunctional polypeptide engages and/or is capable of engaging at least two molecules of the at least one target protein Myc.
  • the first and second targeting moieties are two copies of the same class of targeting, an anti- Myc sdAb, or two copies of the same anti-Myc sdAb.
  • the first and second targeting moieties target the same protein (Myc), but are not the same class of targeting moieties (e.g., an anti-Myc sdAb and an endogenous binding partner of the target protein such as omomyc).
  • the first targeting moiety targets Myc and the second targeting moiety targets different target protein.
  • the first and/or second targeting moiety binds selectively or specifically to the target protein(s).
  • the second targeting moiety is, or is less than, 260, 259, 258, 257, 256, 255, 254, 253, 252, 251, 250, 245, 240, 235, 230, 225, 220, 215, 210, 205, 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70, 65, 60, 55, 54, 53
  • the second targeting moiety comprises or consists of an endogenous binding partner of the target protein, an antibody, Fab, F(ab’) 2 , Fab’, scFv, single domain antibody (sdAb), V H domain, V L domain, VHH, VNAR, diabody, intrabody, DARPin, monobody, affibody, avimer, or any binding fragment or derivative thereof.
  • the second targeting moiety comprises or consists of an endogenous protein binding partner of the target protein, a fragment and/or a derivative thereof.
  • the second targeting moiety is, or is less than, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70, 65, 60, 55, 54, 53, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids long, or a range defined by any two of the preceding values.
  • the second targeting moiety is 10-100, 10-25, 14-100, 14-25, 30-100, 30-50, 30-45, or 80-100 amino acids long.
  • the second targeting moiety comprises or consists of a sdAb.
  • the second targeting moiety comprises or consists of either a V H domain or a V L domain of an IgG antibody.
  • the second targeting moiety comprises or consists of a VHH, VH, or VNAR domain of a heavy chain antibody (HcAb).
  • the second targeting moiety comprises or consists of a VHH or VL domain.
  • the second targeting moiety comprises or consists of a sdAb and the second targeting moiety is, or is less than, 170, 165, 160, 155, 150, 145, 140, 135, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90, amino acids long, or a range defined by any two of the preceding values, optionally 90-150, 90-130, 110-150, 110-130, 110-125, or 114-124 amino acids long.
  • the second targeting moiety comprises or consists of an scFv. In some embodiments, the second targeting moiety comprises or consists of an scFv second targeting moiety is, or is less than, 260, 259, 258, 257, 256, 255, 254, 253, 252, 251, 250, 245, 240, 235, 230, 225, 220, 215, 210, 205, 200, 195, or 190 amino acids long, or a range defined by any two of the preceding values, optionally 190-260, 200-260, or 230-260 amino acids long.
  • the second target protein of the dual-specific bifunctional polypeptide is generally a protein that is involved in disease pathology, for example, cancer.
  • the dual-specific bifunctional polypeptide comprises a second targeting moiety that is capable of binding (optionally selectively or specifically) to target protein selected from Myc, (optionally c-Myc), beta catenin 1 (CTNNB1), and proliferating cell nuclear antigen (PCNA).
  • the target protein is a wild-type and/or a mutant form of the target protein, optionally wherein the targeting moiety preferentially or selectively binds the mutant form of the target protein relative to the wild-type form of the target protein.
  • the mutant form of the target protein is CTNNB1 (SEQ ID NO: 2) comprising one or more mutations selected from: D32Y; D32N; D32V; D32G; S33Y; S33L; S33F; S33C; S33P; G34V; G34E; I35S; H36P; S37C; S37P; S37P and D207G; T41A; T41I; T42_K49del; S45F and Y670X wherein X indicates termination; S45C; S45F; S45P; S45Y; and S45del.
  • the second targeting moiety binds, or binds at least in part, to at least a portion of: a) the DNA binding domain of MYC; b) the armadillo domain of CTNNB1, or c) the p21 binding domain of PCNA.
  • the second targeting moiety binds, or binds at least in part, to at least a portion of the target protein selected from the portion consisting of: a) amino acids 54-406 of MYC (SEQ ID NO: 1), b) amino acids 354-406 of MYC (SEQ ID NO: 1), c) amino acids 408-437 of MYC (SEQ ID NO: 1), d) amino acids 410-419 of MYC (SEQ ID NO: 1), e) amino acids 150-663 of CTNNB1 (SEQ ID NO: 2), f) amino acids 15-29 of CTNNB1 (SEQ ID NO: 2), g) amino acids 249-265, 292-306, 379-390, 415-429, 462-470, or 505-519, or any combination thereof, of CTNNB1 (SEQ ID NO: 2), and h) amino acids 38-48, 123-129, or 251-257 or any combination thereof, of PCNA (SEQ ID NO: 3).
  • the second targeting moiety is selected from Omomyc, TCF4, TCF4.1, TCF4.2, TCF4.3, TCF4.4, TCF4.5, TCF4.6, TCF4.7, TCF4.8, TCF4.9, TCF4.10, TCF4.11, TCF4.12, TCF4.13, TCF4.14, TCF4.15, TCF4.16, con1.1, con1.2, p21.1, p21.2, p21.3, p21.4, p21.5, p21.6, p21.7, p21.8, p21.9, mycV.0300, mycV.1300, mycV.1500, mycV.3700, mycV.6600, mycV.1516, mycV.1515, mycV.1514, mycV.1513, mycV.1512, mycV.1511, mycV.1510, mycV.1509, mycV.1508, mycV.1507, mycV.150
  • the second targeting moiety does not comprise full length or unmodified con1 or p21.
  • Exemplary sequences of second targeting moieties are found in Table 28.
  • the second targeting moiety comprises or consists comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 136, 137, 145, 153, 161, 169, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, 319-344, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, and 761.
  • the second targeting moiety comprises or consists comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, and 319-344.
  • the amino acid sequence differs from a recited reference sequence, the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • FIG. 8 discloses embodiments of dual-specific bifunctional polypeptides at SEQ ID NOs 562-577. The description identifies the elements of the polypeptide in the N- to C- terminal direction.
  • the dual-specific bifunctional polypeptide is or comprises one of the embodiments described in FIG. 8 at SEQ ID NOs: 562-577.
  • the dual-specific bifunctional polypeptide comprises one of the embodiments at SEQ ID NOs: 385-561 and further comprising a second targeting moiety.
  • the dual-specific bifunctional polypeptide comprises the embodiments in FIG.8 sharing one or more elements or features.
  • the shared element or feature is the target(s).
  • the shared element or feature is the URD name (e.g., those in Table 24).
  • the shared element or feature is protein from which the URD is derived (e.g., those listed in Table 23).
  • the shared element or feature is one or more of the following: the target; the protein from which the URD is derived; the URD name; the type of targeting moiety (e.g., sdAb, scFv, polypeptide binder); the targeting moiety name (e.g., those in Table 28; note that “full” is not included in the names in FIG.8); the NLS name (e.g. those in Tables 25 and 26); the linker name (e.g., those in Table 32); the position of the URD relative to the targeting moiety (upstream or downstream); and both the URD name and targeting moiety name.
  • the HA tag sequence (SEQ ID NO: 384) is excluded.
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 562-577. In some embodiments, the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 562-577.
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 562-577, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the amino acid sequence differs from a recited reference sequence, the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • Table 29 below lists the results of HiBiTTM degradation studies utilizing exemplary embodiments of dual-specific bifunctional polypeptides disclosed herein. The tables list a “Grade” of A, B, C or D for the level of degradation of each of the targeted proteins. For dual- specific bifunctional polypeptides where the first and the second targeting moieties target the same protein, only a single grade is provided, a grade for the second target is listed as “n.a.” signifying that it is not applicable. Details regarding the experimental protocol are provided in the Examples section below.
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 29 disclosing Grade A degradation of at least one of the target proteins, Grade B degradation of at least one of the target proteins, Grade C degradation of at least one of the target proteins, and/or Grade D degradation of at least one of the target proteins, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 29 disclosing Grade A degradation of at least one of the target proteins, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 29 disclosing Grade A degradation of at least one of the target proteins, and/or Grade B degradation of at least one of the target proteins, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 29 disclosing a Combined Grade A/A or A, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 29 disclosing a Combined Grade A/B or B/A, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). In some embodiments, the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 29 disclosing a Combined Grade B/B or B, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 29 disclosing Grade A degradation of both target proteins, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). In some embodiments, the dual-specific bifunctional polypeptide does not comprise or consist of an amino acid sequence of any one of the SEQ ID NOs in Table 29 disclosing Grade C and/or D degradation of at least one of the target proteins, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). In some embodiments, the dual-specific bifunctional polypeptide further shares one or more elements of features as described above and elsewhere herein.
  • the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • Table 28 Exemplary targeting moieties
  • Table 29 Exemplary dual-specific bifunctional polypeptides targeting MYC and CTNNB1 and corresponding exemplary target protein degradation grade Exemplary Dual-Specific Bifunctional Degraders Targeting Myc
  • the at least one second target protein is Myc (c-Myc).
  • the Myc is a mutant form.
  • the dual-specific bifunctional polypeptide comprises a second targeting moiety that is capable of binding to Myc.
  • second targeting moieties that target Myc include Omomyc (SEQ ID NO: 4), although other suitable targeting moieties that bind to Myc generally known in the art may also be used.
  • the second targeting moiety that is capable of binding to Myc may be capable of binding, or binding at least in part, to at least a portion of Myc, such as the DNA binding domain of Myc.
  • the second targeting moiety that is capable of binding to Myc is capable of binding, or binding at least in part, to at least a portion of amino acids 54-406, amino acids 354-406 (the DNA binding domain of MYC that binds to Omomyc; as seen in PDB: 1NKP), amino acids 408-437, or amino acids 410-419 (binding epitope of antibody 9E10; as seen in PDB: 2OR9) of MYC (as represented as SEQ ID NO: 1).
  • the dual- specific bifunctional polypeptides disclosed herein that are specific for Myc may be used for the treatment of a cancer, for example, a cancer exhibiting elevated levels of Myc, for example solid tumors (including but not limited to hepatocellular carcinoma, liver metastases, and colorectal carcinoma) and hematological malignancies.
  • the bifunction polypeptide targeting Myc comprises a localization peptide sequence which directs the dual-specific bifunctional polypeptide to the nucleus of a cell.
  • the localization peptide sequence comprises or consists of the NLS sequence selected from the group of the NLS sequences of MYC, SPOP, Hrp1, SV40, 53BP1, and nucleoplasmin.
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of SPOP, optionally SPNLS.2, MYC, optionally mycNLS, and Hrp1, optionally hrpNLS.
  • the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 114-127.
  • the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 122, 114 and 116.
  • the dual-specific bifunctional polypeptide targeting Myc comprises a URD derived from a URD selected from the group consisting of NHLRC1, RNF125, RNF165, and RNF4.
  • the URD is a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the URD comprises of consists of NHLRC1.1, RNF125.2, RNF165.1, or RNF4.1.
  • the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 76-80, 87-93, 98, and 104.
  • the URD comprises or consists of a sequence selected from any one of SEQ ID NO: 76, 88, 98, and 104.
  • the dual-specific bifunctional polypeptide targeting Myc comprises a second targeting moiety that comprises or consists of a targeting moiety disclosed in Table 28 as targeting Myc.
  • the second targeting moiety comprises or consists of an endogenous protein binding partner of the target protein, a fragment and/or a derivative thereof.
  • the second targeting moiety comprises or consists of Omomyc, or a fragment thereof that is capable of binding to the target protein.
  • the second targeting moiety comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 136.
  • the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the dual-specific bifunctional polypeptide targeting Myc comprises a second targeting moiety that comprises or consists of a targeting moiety disclosed in Table 28 as targeting Myc.
  • the second targeting moiety comprises or consists of a sdAb, optionally comprising or consisting of either a V H domain or a V L domain of an IgG antibody, or a VHH, VH, or VNAR domain of a heavy chain antibody (HcAb).
  • the sdAb comprises or consists of a VHH domain.
  • the second targeting moiety comprises or consists of a sdAb, optionally a V H H domain, comprising a CDR1 selected from any one of SEQ ID NOs: 139, 147, 163, and 171, a CDR2 selected from any one of SEQ ID NOs: 141, 149, 157, and 165, and a CDR3 selected from any one of SEQ ID NOs: 143, 151, 159, 167, and 175.
  • the combination of CDR1, CDR2, and CDR3 is 1936, 157 and 159.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions (optionally conservative substitutions) and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 139, 147, 163, 171, 141, 149, 157, 165, 143, 151, 159, 167, and 175.
  • the sdAb optionally a VHH domain, comprises a VH FR1 selected from any one of SEQ ID NOs: 138 and 170, a V H FR2 of SEQ ID NO: 140, a V H FR3 selected from any one of SEQ ID NOs: 142, 158, and 174, and a VH FR4 selected from any one of SEQ ID NOs: 144 and 152.
  • the combination of FR1, FR2, FR3 and FR4 is 154, 156, 158, and 160.
  • each V H framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions (optionally conservative substitutions) and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 138, 170, 140, 142, 158, 174, 144, and 152.
  • the second targeting moiety comprises or consists of a sdAb, optionally a VHH domain, sequence selected from any one of SEQ ID NOs: 137, 145, 153, 161, 169, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, and 761.
  • each CDR sequence independently comprises 1, 2, 3, 4, 5, 6, 7, or more substitutions (optionally conservative substitutions) and/or 1, 2, 3, 4, 5, 6, 7, or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CDR sequence of any one of SEQ ID NOs: 137, 145, 153, 161, 169, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, and 761, and/or wherein the framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more substitutions (optionally conservative substitutions) and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more deletions or additions, and/or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
  • the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the dual-specific bifunctional polypeptide targeting Myc comprises a second targeting moiety comprising or consisting of a sdAb, wherein the sdAb comprises or consists of a VHH domain.
  • the second targeting moiety comprises or consists of an anti-Myc sdAb described above under the heading “Anti-Myc Single Domain Antibodies (sdAb)”, and elsewhere herein.
  • the dual- specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 562, 565, 567, and 570-577, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprising a second targeting moiety targeting Myc comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 30, optionally excluding any one of the SEQ ID NOs having a Grade of D degradation of Myc and/or another target protein, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 30 having a Combined Grade A degradation of MYC, and/or a Combined Grade B degradation of MYC, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 30 having a Combined Grade of A, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 30 having a Combined Grade of B, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). In some embodiments, the dual-specific bifunctional polypeptide does not comprise or consist of an amino acid sequence of any one of the SEQ ID NOs in Table 30 having a combined of Grade C and/or D, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). In some embodiments where the amino acid sequence differs from a recited reference sequence, the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the dual-specific bifunctional polypeptide comprising a second targeting moiety targeting Myc comprises embodiments further sharing one or more elements or features.
  • the shared element or feature is the URD name.
  • the shared element or feature is protein from which the URD is derived.
  • the shared element or feature is one or more of the following: the protein from which the URD is derived; the URD name; the type of targeting moiety; the targeting moiety name; the NLS name; the linker name; the position of the URD relative to the targeting moiety (upstream or downstream); and both the URD name and targeting moiety name.
  • the HA tag sequence (SEQ ID NO: 384) is excluded.
  • the dual-specific bifunctional polypeptide comprising a second targeting moiety targeting Myc the URD is not derived from CHIP.
  • the URD does not comprise or consist of CHIP.1.
  • the URD does not comprise or consist of SEQ ID NO: 54.
  • a polynucleotide encoding any of the dual- specific bifunctional polypeptides having a first and second targeting moiety targeting Myc disclosed above, or elsewhere herein.
  • Table 30 Exemplary dual-specific bifunctional polypeptides comprising a first and second targeting moiety targeting MYC and corresponding exemplary target protein degradation grade Exemplary Dual-Specific Bifunctional Degraders Targeting Myc and CTNNB1
  • the at least one second target protein is beta catenin 1 (CTNNB1).
  • the CTNNB1 is a mutant form.
  • An example sequence for CTNNB1 is provided as SEQ ID NO: 2 (Uniprot #P35222).
  • the dual-specific bifunctional polypeptide comprises a second targeting moiety that is capable of binding to CTNNB1.
  • targeting moieties that target CTNNB1 include TCF4 (SEQ ID NO: 6; Uniprot #Q9NQB0), and amino acids 10- 54 of TCF4 (SEQ ID NO: 5), although other suitable CTNNB1 targeting moieties generally known in the art may also be used.
  • the second targeting moiety that is capable of binding to CTNNB1 may be capable of binding, or binding at least in part, to at least a portion of CTNNB1, such as the armadillo domain of CTNNB1.
  • the second targeting moiety that is capable of binding to CTNNB1 is capable of binding, or binding at least in part, to at least a portion of amino acids 150-663 (the armadillo domain of CTNNB1; as seen in PDB: 2GL7), amino acids 15-29 (binding epitope of antibody BC2), or amino acids 249-265, 292-306, 379-390, 415-429, 462-470, or 505-519, (which are residues in the armadillo domain that interact with TCF4, E-cadherin, Axin, APC, TCF/LEF family transcription factors and/or SOX9) or any combination thereof, of CTNNB1 (as represented as SEQ ID NO: 2).
  • the dual-specific bifunctional polypeptides disclosed herein comprising a second targeting moiety targeting CTNNB1 may be used for the treatment of a cancer, for example, a cancer involving nuclear expression of beta-catenin and/or harboring mutations or alterations in the Wnt/beta-catenin signaling pathway, for example, solid tumors (including but not limited to liver metastases, colorectal carcinoma, or other gastrointestinal cancers) and hematological malignancies.
  • the CTNNB1 target protein is a mutant form of CTNNB1 (SEQ ID NO: 2) comprising one or more mutations selected from: D32Y; D32N; D32V; D32G; S33Y; S33L; S33F; S33C; S33P; G34V; G34E; I35S; H36P; S37C; S37P; S37P and D207G; T41A; T41I; T42_K49del; S45F and Y670X wherein X indicates termination; S45C; S45F; S45P; S45Y; and S45del.
  • SEQ ID NO: 2 mutant form of CTNNB1
  • the dual-specific bifunctional polypeptide comprising a second targeting moiety targeting CTNNB1 comprises a localization peptide sequence which directs the dual-specific bifunctional polypeptide to the nucleus of a cell.
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequences of MYC, SPOP, Hrp1, SV40, 53BP1, and nucleoplasmin.
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of MYC, optionally mycNLS, SPOP, optionally spNLS.2, and Hrp1, optionally hrpNLS.
  • the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 114-127. In some embodiments, the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 122, 114 and 116.
  • the dual-specific bifunctional polypeptide comprising a second targeting moiety targeting CTNNB1 comprises a URD derived from a URD selected from the group consisting of NHLRC1, RNF125, RNF165, and RNF4, or a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity. In some embodiments, the URD comprises or consists of NHLRC1.1, RNF125.2, RNF165.1, or RNF4.1.
  • the URD is a truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 6-80, 87-93, 98, and 104.
  • the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 76, 88, 98, and 104.
  • the dual-specific bifunctional polypeptide comprising a second targeting moiety targeting CTNNB1 comprises a second targeting moiety wherein the second targeting moiety comprises or consists of a targeting moiety disclosed in Table 28 as targeting CTNNB1.
  • the second targeting moiety comprises or consists of an endogenous protein binding partner of the target protein, a fragment and/or a derivative thereof.
  • the second targeting moiety comprises or consists of TCF4.1, TCF4.2, TCF4.3, TCF4.4, TCF4.5, TCF4.6, TCF4.7, TCF4.8, TCF4.9, TCF4.10, TCF4.11, TCF4.12, TCF4.13, TCF4.14, TCF4.15, or TCF4.16, or a fragment thereof that is capable of binding to the target protein.
  • the second targeting moiety comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 319-333, optionally SEQ ID NO: 330.
  • the amino acid sequence differs from a recited reference sequence, the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the dual-specific bifunctional polypeptide targeting CTNNB1 comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 563, 564, 566, 568, and 569, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the SEQ ID NOs in Table 31, optionally excluding any one of the SEQ ID NOs having a Grade of D degradation of CTNNB1 and/or MYC, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 31 having a Grade A degradation of CTNNB1, and/or a Grade B degradation of CTNNB1, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 31 having a Combined Grade of A and/or A/A, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384).
  • the dual-specific bifunctional polypeptide comprises or consists of an amino acid sequence of any one of the SEQ ID NOs in Table 31 having a Combined Grade of A/B and/or B/A, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). In some embodiments, the dual-specific bifunctional polypeptide does not comprise or consist of an amino acid sequence of any one of the SEQ ID NOs in Table 31 having Grades C and/or D for either target protein, optionally wherein the sequence lacks the HA tag sequence (SEQ ID NO: 384). In some embodiments where the amino acid sequence differs from a recited reference sequence, the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the dual-specific bifunctional polypeptide targeting CTNNB1 comprises embodiments further sharing one or more elements or features.
  • the shared element or feature is the URD name.
  • the shared element or feature is protein from which the URD is derived.
  • the shared element or feature is one or more of the following: the protein from which the URD is derived; the URD name; the type of targeting moiety; the targeting moiety name; the NLS name; the linker name; the position of the URD relative to the targeting moiety (upstream or downstream); and both the URD name and targeting moiety name.
  • the HA tag sequence (SEQ ID NO: 384) is excluded.
  • the dual-specific bifunctional polypeptide targeting CTNNB1 the URD is not derived from beta-TRCP.
  • the URD does not comprise or consist of bTRCP.1.
  • the URD does not comprise or consist of SEQ ID NOs: 57.
  • the at least one second target protein is proliferating cell nuclear antigen (PCNA).
  • PCNA proliferating cell nuclear antigen
  • the PCNA is a mutant form.
  • An example sequence for PCNA is provided as SEQ ID NO: 3 (Uniprot # P12004).
  • PCNA is a co-factor for DNA polymerase delta and is involved in DNA synthesis and DNA repair.
  • the dual-specific bifunctional polypeptide comprises a second targeting moiety that is capable of binding to PCNA.
  • targeting moieties that target PCNA include those listed in Table 28 listed as targeting PCNA.
  • other suitable PCNA targeting moieties generally known in the art are used.
  • the dual-specific bifunctional polypeptides disclosed herein comprising a second targeting moiety targeting PCNA may be used for the treatment of a cancer, for example, solid tumor cancers (including but not limited to colorectal cancer, breast cancer, lung cancer, and liver cancer) and hematological malignancies.
  • the dual-specific bifunctional polypeptide comprising a second targeting moiety targeting PCNA comprises a localization peptide sequence which directs the dual-specific bifunctional polypeptide to the nucleus of a cell.
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequences of MYC, SPOP, Hrp1, SV40, 53BP1, and nucleoplasmin.
  • the localization peptide sequence comprises or consists of a NLS sequence selected from the group of the NLS sequence of MYC, optionally mcyNLS and/or 3xmycNLS, SPOP, Hrp1, SV40, optionally svNLS or 3xsvNLS, and nucleoplasmin.
  • the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 114-127.
  • the NLS comprises or consists of a sequence selected from any one of SEQ ID NOs: 115-116 and 118-122.
  • the dual-specific bifunctional polypeptide comprising a second targeting moiety targeting PCNA comprises a URD derived from a URD selected from the group consisting of DCAF1, NHLRC1, RNF125, or SPOP.
  • the URD comprises or consists of DCAF1.1, NHLRC1.1, RNF125.2, RNF125.3, RNF125.4, RNF125.5, RNF125.6, SPOP.2 or SPOP.3.
  • the URD comprises or consists of optionally NHLRC1.1 or RNF125.2.
  • the URD is truncation or fragment thereof that retains ubiquitin-proteasome recruiting activity.
  • the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 39-113. In some embodiments, the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 55, 73, 74, 76 and 88-92. In some embodiments, the URD comprises or consists of a sequence selected from any one of SEQ ID NOs: 76 and 88. In some embodiments, the dual-specific bifunctional polypeptide targeting PCNA comprises a second targeting moiety that comprises or consists of a targeting moiety disclosed in Table 28 as targeting PCNA.
  • the second targeting moiety binds, or binds at least in part, to at least a portion of amino acids 38-48, 123-129, or 251-257 or any combination thereof, of PCNA (SEQ ID NO: 3).
  • the second targeting moiety comprises or consists of an endogenous protein binding partner of the target protein, a fragment and/or a derivative thereof.
  • the second targeting moiety comprises or consists of con1, con1.1, con1.2, p21, p21.1, p21.2, p21.3, p21.4, p21.5, p21.6, p21.7, p21.8, p21.9, or a fragment thereof that is capable of binding to the target protein.
  • the second targeting moiety does not comprise full length or unmodified con1 or p21.
  • the second targeting moiety comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 334- 344.
  • the amino acid sequence differs from a recited reference sequence, the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the URD is not derived from SPOP.
  • the URD does not comprise or consist of SPOP.1, SPOP.2, or SPOP.3.
  • the URD does not comprise or consist of a sequence selected from any one of SEQ ID NOs: 72-74.
  • disclosed herein is a polynucleotide encoding any of the dual- specific bifunctional polypeptides targeting Myc and PCNA disclosed above, or elsewhere herein.
  • the bifunctional polypeptide or the dual-specific bifunctional polypeptide disclosed above and herein further comprise one or more linker peptides.
  • the linker peptide is a length that allows for ubiquitination of the target protein via the URD when the target protein is bound by the first and/or second targeting moiety. Any suitable linker peptide generally known in the art may be used in embodiments herein.
  • the linker peptide is positioned between one or more of: the first and the second targeting moieties; the first and/or second targeting moiety and the URD; the NLS and the first and/or second targeting moiety; the NLS and the URD; and/or the NLS and an epitope tag, optionally an HA tag.
  • the linker peptide is positioned between the first and/or second targeting moiety and the URD.
  • the linker peptide is 0 (i.e., there is no linker), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length, or a range defined by any two of the preceding values.
  • the linker peptide is 0-30, 1-30, 2-30, 0-20, 1-20, 2- 20, 0-15, 0-12, 0-10, 1-15, 1-12, 1-10, 2-15, 2-12, 2-10, 3-15, 3-12, or 3-10 amino acids in length, optionally 2-30 amino acids in length.
  • the linker peptide comprises or consists of glycine and serine.
  • the linker peptide comprises or consists of one or more alpha helixes, optionally flanked by one or more glycine and/or a serine residues.
  • the linker peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 345-383. In some embodiments, the linker peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 347-352. Note that in the sequence listing submitted herewith, SEQ ID NO: 352 is blank because the sequence listing rules do not permit an amino acid sequence of fewer than four specifically defined amino acids, and the sequence of SEQ ID NO: 352 is “GS,” which is only two amino acids long. Therefore, “SEQ ID NO: 352” can be replaced with “GS” herein.
  • SEQ ID NOs: 345-383 is the same as SEQ ID NOs: 345-351, GS, and 353-383.
  • SEQ ID NOs: 347-352 is the same as SEQ ID NOs: 347-351 and GS.
  • Exemplary linker sequences are disclosed in Table 32.
  • the bifunctional polypeptide or the dual-specific bifunctional polypeptide disclosed above and herein comprises a first targeting moiety, an optional second targeting moiety, and a URD.
  • the orientation of the URD and SRD of exemplary proteins from which URDs can be derived is provided in Table 23, with “URD-SRD” indicating the URD is upstream of the SRD, and “SRD-URD” indicating the SRD is upstream of the URD, when viewed in the standard N- to C-terminal direction.
  • the orientation of the first and/or second targeting moiety relative to the URD is the same orientation as compared to the naturally occurring substrate recognition domain (SRD) of the protein from which the URD is derived. In some embodiments, the orientation of the first and/or second targeting moiety relative to the URD is the opposite orientation as compared to the naturally occurring substrate recognition domain of the protein from which the URD is derived. In some embodiments, the first and/or second targeting moiety is N-terminal relative to the URD. In some embodiments, the first and/or second targeting moiety is C-terminal relative to the URD. In some embodiments of a dual-specific bifunctional polypeptide, both the first and the second targeting moiety are N-terminal relative to the URD.
  • a dual-specific bifunctional polypeptide both the first and the second targeting moiety are C-terminal relative to the URD.
  • Tag peptides In some embodiments, the bifunctional polypeptide or the dual-specific bifunctional polypeptide disclosed above and herein further comprises an epitope tag. In some embodiments, the epitope tag is a hemagglutinin (HA) tag. In some embodiments, the HA tag comprises or consists of the amino acid sequence of SEQ ID NO: 384. In some embodiments, the bifunctional polypeptide or dual-specific bifunctional polypeptide does not comprise an epitope tag.
  • HA hemagglutinin
  • the bifunctional polypeptide or dual-specific bifunctional polypeptide does not comprise a hemagglutinin (HA) tag. In some embodiments, the bifunctional polypeptide or dual-specific bifunctional polypeptide does not comprise an HA tag comprising or consisting of the amino acid sequence of SEQ ID NO: 384.
  • the epitope tag can be used to visualize the bifunctional polypeptide or dual- specific bifunctional polypeptide. Typically, bifunctional polypeptides or dual-specific bifunctional polypeptides comprising an epitope tag will be used for experimentation and visualization purposes. In some embodiments, the bifunctional polypeptide or dual-specific bifunctional polypeptides intended for treatment do not have an epitope tag.
  • the bifunctional polypeptide or dual-specific bifunctional polypeptides intended for treatment have an epitope tag.
  • Overall size of the bifunctional and dual-specific bifunctional polypeptides In some embodiments, the bifunctional polypeptide is less than or equal to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 amino acids long, or a range defined by any two of the preceding values, optionally 50-800, 50-650, 50-500, 50-400, 70-800, 70-650, 70-500, 70-400, 70-300, 70-200, 80-800, 80-650, 80-500, 80-400, 80-300, or 80-200 amino acids long, optionally 90-550, 150-550, or 80-450 amino acids long.
  • the dual- specific bifunctional polypeptide is, is about, is at least, is at least about, is not more than, or is not more than about, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950.
  • the first and/or second targeting moiety and/or dual-specific bifunctional polypeptide of the present disclosure have an equilibrium dissociation constant (Kd) of less (that is superior binding) than about 10 -7 or 10 -8 M, for example, less than about 10 -9 M or 10 -10 M, in some embodiments, less than about 10 -11 M, 10 -12 M, or 10 -13 M.
  • Kd equilibrium dissociation constant
  • the first and/or second targeting moiety comprises or consists of a sdAb that has a Kd value of less than 1x10 -7 and optionally at least 1x10 -10 .
  • the Kd is 1.22E -07 to 9.47E -09 .
  • the first and/or second targeting moiety and/or dual-specific bifunctional polypeptide of the present disclosure have an association rate constant of at least (that is superior binding rate) about 10 +2 or 10 +3 1/Ms, for example, at least about 10 +5 1/Ms or 10 +6 1/Ms, in some embodiments, at least about 10 +7 1/Ms, 10 +8 1/Ms, or 10 +9 1/Ms.
  • the first and/or second targeting moiety comprises or consists of a sdAb that has a kon value of at least 1x10 +4 1/Ms and optionally at least 1x10 +5 1/Ms, optionally wherein the k on is 6.79E +04 1/Ms to 4.04E +05 1/Ms.
  • the first and/or second targeting moiety and/or dual-specific bifunctional polypeptide of the present disclosure have a dissociation rate constant of less (that is superior binding) than about 10 -1 or 10 -2 1/s, for example, less than about 10 -3 1/s or 10 -5 1/s, in some embodiments, less than about 10 -6 1/s, 10 -7 1/s, or 10 -8 1/s.
  • the first and/or second targeting moiety comprises or consists of a sdAb that has a koff value of less than 1x10 -2 and optionally less than 1x10 -4 , optionally wherein the koff is 1.74E -02 to 8.67E -04 .
  • any of the preceding values is calculated using human c-Myc, optionally as described for values of Octet sdAb Kd Human Myc Peptide Monovalent (M) in Example 4.
  • first and/or second targeting moiety and/or dual-specific bifunctional polypeptide has Kd, k on , and/or k off value selected from a value in Table 36, or a range defined by any two of the values presented therein for a given parameter.
  • the first and/or second targeting moiety binds selectively to the target protein.
  • the first and/or second targeting moiety binds specifically to the target protein.
  • polypeptides comprising or consisting of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, and 385-577.
  • the polypeptide or dual-specific bifunctional polypeptide is a non-natural polypeptide.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the difference is the result of the inclusion of one or more modifications relative to the reference sequence.
  • the one or more modifications comprise a substitution, insertion and/or deletion.
  • the substitution is a conservative substitution.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • Exemplary Polynucleotides are polynucleotides encoding for a polypeptide having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of polypeptides, anti-Myc sdAbs, bifunctional polypeptides, or the dual-specific bifunctional polypeptides disclosed herein.
  • the polynucleotide is a DNA or an RNA.
  • the RNA is an mRNA or a circular RNA.
  • the polynucleotide is packaged in a lipid nanoparticle or a viral vector.
  • the viral vector is an adenovirus, adeno-associated virus, lentivirus, or retrovirus vector.
  • the % sequence identity is calculated over the entirety of the reference sequence.
  • the DNA the RNA, mRNA or circular RNA comprises one or more modified nucleosides. In some embodiments, up to 100% of the nucleotides comprising uracil are replaced with pseudouridine and/or N1-methylpseudouridine.
  • the polynucleotide is packaged in a lipid nanoparticle, a polymeric nanoparticle, an extracellular vesicle, optionally an exosome, or a viral vector, optionally a replicating viral vector or a non-replicating viral vector, optionally an adenovirus, adeno-associated virus, lentivirus, or retrovirus vector.
  • a viral vector optionally a replicating viral vector or a non-replicating viral vector
  • lipid nanoparticles for RNA delivery are known in the art.
  • the lipid nanoparticle is made from: an ionizable cationic lipid, a PEGylated lipid, a phospholipid, and cholesterol.
  • a neutral ionizable amino lipid is used.
  • Lipid nanoparticles for RNA delivery are disclosed in, for example: US11338044B2; US20180153822A1; US20220249699A1; WO2022251953A1; Mitchell et al., “Engineering precision nanoparticles for drug delivery,” Nature Rev. Drug Discovery (2021) 20:101-124; Kulkarni et al., “Lipid Nanoparticles Enabling Gene Therapies: From Concepts to Clinical Utility,” Nucleic Acid Therapeutics (2016) 28(3):146-157; Hou et al. “Lipid nanoparticles for mRNA delivery,” Nature Reviews Materials (2021) 6:1078-1094; each of which is incorporated herein by reference in its entirety.
  • compositions comprising any of the polypeptides, anti-Myc sdAbs, bifunctional polypeptides, dual-specific bifunctional polypeptides and/or the polynucleotides disclosed herein and one or more pharmaceutically acceptable excipients, carriers, or diluents.
  • pharmaceutical compositions comprising any of the polypeptides, anti-Myc sdAbs, bifunctional polypeptides, dual-specific bifunctional polypeptides and/or the polynucleotides disclosed herein and one or more pharmaceutically acceptable excipients, carriers, or diluents.
  • the composition is formulated for intravenous, intraperitoneal, intra- arterial, subcutaneous, intramuscular, intrathecal, intratumoral, inhalation, or intracranial administration.
  • the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition disclosed herein is for use in the treatment of a cancer in a patient in need thereof.
  • the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition disclosed herein is for use in the manufacture of a medicament.
  • the medicament is for the treatment of cancer.
  • Disclosed herein in some embodiments is a method of treating a subject comprising administering the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition disclosed herein to a subject in need thereof.
  • the subject has a cancer.
  • Disclosed herein in some embodiments is a method of reducing the amount of a target protein in a cell comprising contacting the cell with the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition disclosed herein.
  • the target protein is selected from Myc, (optionally c-Myc), CTNNB1, and/or PCNA.
  • the cell is in a subject, and the contacting comprises administering the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition to the subject.
  • the subject has cancer.
  • the administering is intravenous, intraperitoneal, intra-arterial, subcutaneous, intramuscular, intrathecal, intra-tumoral, or intracranial administration.
  • the cell is ex vivo, and said contacting comprises contacting the polypeptide comprising or consisting of the anti-Myc sdAb, bifunctional polypeptide, dual-specific bifunctional polypeptide, polypeptide, polynucleotide, or pharmaceutical composition to the cell ex vivo, optionally in an adoptive cell therapy.
  • the method further comprises administering the cell to a subject after the contacting step, optionally wherein the subject is also the source of the cell.
  • the bifunctional polypeptide, or dual-specific bifunctional polypeptide promotes proteasome-mediated degradation of a target protein when measured in one or more assays described in and Exemplary Assay for assessing proteasomal-mediated degradation of target protein disclosed herein.
  • the assay is a HiBiTTM tag assay.
  • the assay is a Western blot, optionally wherein the proteasome-mediated degradation is inhibited when the cells comprising the target protein and the bifunctional polypeptide or dual-specific bifunctional polypeptide are treated with a proteasome inhibitor.
  • the treatment with the proteasome inhibitor reduces the amount of degradation of the target protein by at least 50%, 60%, 70%, 80%, 90%, 95% or 100% as compared to cells not treated with the proteasome inhibitor.
  • Exemplary Assays for assessing proteasomal-mediated degradation of target protein(s) The following is an embodiment of exemplary assays for determining if a bifunctional polypeptide or dual-specific bifunctional polypeptide promotes proteasomal-mediated degradation of a target protein(s) and therefore that the URD retains ubiquitin-proteasome recruiting activity, and/or that the targeting moiety binds the target protein.
  • a URD is a domain derived from a ubiquitin-proteasome system component that has the physical property of recruiting the ubiquitin-proteasome system machinery and the functional property of enabling the polyubiquitination and degradation of the recruited target(s) of interest.
  • the target(s) of interest is engineered to harbor the HiBiTTM tag (Promega) in a relevant cell line.
  • the cell line is transfected with a plasmid encoding the bifunctional polypeptide, optionally dual-specific, designed to promote proteasome-mediated degradation of the target(s) of interest. 24 hours post transfection, doxycycline is added to induce expression of the bifunctional polypeptide.
  • the HiBiTTM assay is performed the next day according to manufacturer instructions.
  • Loss of HiBiTTM signal is indicative that the bifunctional polypeptide promotes proteasome-mediated degradation of the target(s), and that the URD retains ubiquitin-proteasome recruiting activity and/or that the targeting moiety binds the target.
  • a construct that leads to loss of HiBiTTM signal can be subjected to further confirmation in the assay described below. Additional information regarding the HiBiTTM system can be found in Dixon et al. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol. (2016) 11(2):400-8, which is hereby expressly incorporated by reference in its entirety.
  • An exemplary assay for confirmation of proteasome-mediated degradation activity of the bifunctional polypeptide, optionally dual-specific, is described. This assay can be used in addition to the assay described above, or independent of the above assay.
  • a suitable time to allow proteasome-mediated degradation of the target(s) of interest e.g., 24 to 72 hours
  • lysate of cells expressing the target(s) of interest and the bifunctional polypeptide of interest, and control cells that express the target(s) of interest but not the bifunctional polypeptide of interest are subjected to SDS-PAGE, transferred onto a suitable membrane, and probed with antibodies that are specific and selective for the target(s) of interest (Western Blot).
  • Loss of target protein is indicative that the bifunctional polypeptide promotes proteasome-mediated degradation of the target, and that the URD retains ubiquitin-proteasome recruiting activity.
  • a proteasome inhibitor such as bortezomib, MG-132, or lactacystin.
  • the cells expressing the target(s) and the bifunctional polypeptide of interest in the presence of the proteasome inhibitor will show a partial (e.g., at least 50%, 60%, 70%, 80%, 90%, or 95%) or total (100%) reduction in amount of degradation as compared to cells not treated with the proteasome inhibitor, which indicates that the loss of the target is proteasome-dependent, which is indicative that the bifunctional polypeptide promotes proteasome-mediated degradation of the target(s), and that the URD retains ubiquitin-proteasome recruiting activity.
  • Additional target proteins, target binding domains, linker sequences, and other disclosures related to components, features, uses, etc. of bispecific polypeptides are disclosed in PCT/US2023/066619, filed May 4, 2023, and PCT/US2023/76886, filed October 13, 2023, each of which is herein incorporated by reference in its entirety.
  • the terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and mean a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.
  • the term “mammal” is used in its usual biological sense.
  • the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man.
  • Isolated substances and/or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and/or spanning the aforementioned values).
  • isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and/or spanning the aforementioned values).
  • a substance that is “isolated” may be “pure” (e.g., substantially free of other components).
  • isolated cell may refer to a cell not contained in a multi-cellular organism or tissue.
  • in vivo is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.
  • ex vivo is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside a living organism with little alteration of natural conditions.
  • in vitro is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.
  • nucleic acid or “nucleic acid molecule” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • oligonucleotides those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, or phosphoramidate.
  • nucleic acid molecule also includes so-called “peptide nucleic acids,” which comprise naturally occurring or modified nucleic acid bases attached to a polyamide backbone.
  • Nucleic acids can be either single stranded or double stranded. “Oligonucleotide” can be used interchangeable with nucleic acid and can refer to either double stranded or single stranded DNA or RNA.
  • a nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g., plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)) that can be used for amplification and/or expression of the nucleic acid or nucleic acids in various biological systems.
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • HAC human artificial chromosome
  • the vector or construct will also contain elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.
  • a nucleic acid or nucleic acid molecule can comprise one or more sequences encoding different peptides, polypeptides, or proteins.
  • sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g. linkers, repeats or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths.
  • downstream on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the 3’-end of a previous sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded.
  • upstream on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the 5’-end of a subsequent sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded.
  • nucleic acid has its plain and ordinary meaning as understood in light of the specification and refers to two or more sequences that occur in proximity either directly or with extra nucleic acids in between, e.g. linkers, repeats, or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths, but generally not with a sequence in between that encodes for a functioning or catalytic polypeptide, protein, or protein domain.
  • nucleic acids described herein comprise nucleobases.
  • Primary, canonical, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil.
  • Other nucleobases include but are not limited to purines, pyrimidines, modified nucleobases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6- dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.
  • the uridine of the RNA, mRNA or circular RNA is pseudouridine. In some embodiments, the uridine of the RNA, mRNA or circular RNA is N1-methylpseudouridine. In some embodiments, the uracil of the RNA, mRNA or circular RNA is a mixture of pseudouridine and N1-methylpseudouridine. In some embodiments, up to 100% of the nucleotides comprising uracil are replaced with pseudouridine and/or N1-methylpseudouridine.
  • peptide “polypeptide”, and “protein” as used herein have their plain and ordinary meaning as understood in light of the specification and refer to macromolecules comprised of amino acids linked by peptide bonds.
  • the numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available.
  • nucleic acid template By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g.
  • the term “downstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the C-terminus of a previous sequence.
  • upstream on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the N-terminus of a subsequent sequence.
  • a polypeptide or amino acid sequence “derived from” a designated protein refers to the origin of the polypeptide.
  • the polypeptide has an amino acid sequence that is essentially identical to that of a polypeptide encoded in the sequence, or a portion thereof, or which is immunologically identifiable with a polypeptide encoded in the sequence. This terminology also includes a polypeptide expressed from a designated nucleic acid sequence.
  • Peptide sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of the peptide sequences disclosed herein and having the same or similar functional properties are envisioned.
  • the percent homology may be determined according to amino acid substitutions, deletions, or additions between two peptide sequences.
  • Peptide sequences having some percent homology to any one of the peptide sequences disclosed herein may be produced and tested by one skilled in the art through conventional methods.
  • non-natural denotes that the polypeptide or portion of a polypeptide in question has a sequence that is not present, in the noted state, in nature. In the present context the polypeptides have been altered from their native state, so that their sequences are no longer those found in wild-type proteins.
  • a portion derived from a naturally occurring protein e.g., a URD or NLS
  • a fragment of the naturally occurring protein which does not contain an altered sequence (other than being less than the complete sequence of the naturally occurring protein
  • a “non-natural” polypeptide comprising the derived portion does not encompass the naturally occurring protein from which the portion is derived.
  • a “non-natural” polypeptide comprising the portion must contain additional amino acids which are not found in the natural protein.
  • sequences having a % identity to any of the sequences disclosed herein are envisioned and may be used.
  • the terms “% identity” refer to the percentage of units (i.e., amino acids or nucleotides) that are the same between two or more sequences relative to the length of the sequence. When the two or more sequences being compared are the same length, the % identity will be respective to that length. When two or more sequences being compared are different lengths, deletions and/or insertions may be introduced to obtain the best alignment.
  • these sequences may include peptide sequences, nucleic acid sequences, CDR sequences, variable region sequences, or heavy or light chain sequences.
  • any sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the sequences disclosed herein may be used.
  • any sequence having at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 substitutions, deletions, or additions relative to any of the sequences disclosed herein may be used.
  • the changes in sequences may apply to, for example, single amino acids, single nucleic acid bases, or nucleic acid codons; however, differences in longer stretches of sequences are also envisioned.
  • sequences associated with antibodies or binding regions thereof may apply to antigen-binding regions (e.g., CDRs) or regions that do not bind to antigens or are only secondary to antigen binding (e.g., framework regions).
  • sequences having a % homology to any of the sequences disclosed herein are envisioned and may be used.
  • the term “% homology” refers to the degree of conservation between two sequences when considering their three-dimensional structure. For example, homology between two protein sequences may be dependent on structural motifs, such as beta strands, alpha helices, and other folds, as well as their distribution throughout the sequence. Homology may be determined through structural determination, either empirically or in silico.
  • any sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to any of the sequences disclosed herein may be used.
  • any sequence having at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 substitutions, deletions, or additions relative to any of the sequences disclosed herein, which may or may not affect the overall % homology, may be used.
  • sequences having a certain % similarity to any of the sequence disclosed herein are envisioned and may be used.
  • these sequences may include peptide sequences, nucleic acid sequences, CDR sequences, variable region sequences, or heavy or light chain sequences.
  • similarity refers to the comparison of amino acids based on their properties, including but not limited to size, polarity, charge, pK, aromaticity, hydrogen bonding properties, or presence of functional groups (e.g., hydroxyl, thiol, amine, carboxyl, and the like).
  • % similarity refers to the percentage of units (e.g., amino acids) that are the same between two or more sequences relative to the length of the sequence. When the two or more sequences being compared are the same length, the % similarity will be respective that length. When two or more sequences being compared are different lengths, deletions and/or insertions may be introduced to obtain the best alignment.
  • substitution matrices include BLOSUM45, BLOSUM62, BLOSUM80, PAM100, PAM120, PAM160, PAM200, PAM250, but other substitution matrices or approaches may be used as considered appropriate by the skilled person.
  • a certain substitution matrix may be preferential over the others when considering aspects such as stringency, conservation and/or divergence of related sequences (e.g., within the same species or broader), and length of the sequences in question.
  • a peptide sequence having a certain % similarity to another sequence will have up to that % of amino acids that are either identical or an acceptable substitution as governed by the method of similarity determination used.
  • a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to any of the sequences disclosed herein may be used.
  • any sequence having at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 similar substitutions relative to any of the sequences disclosed herein may be used.
  • these similar substitutions may apply to antigen-binding regions (i.e., CDRs) or regions that do not bind to antigens or are only secondary to antigen binding (i.e., framework regions).
  • the percent identity is over the CDR and/or FR regions noted herein.
  • the percent identity of the CDR or FR can be identified separately from the rest of the protein or nucleic acid sequence.
  • two CDRs or FRs can have a specified percentage of amino acid residues or nucleotides that are the same (for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), while allowing for the remainder of the protein to either stay 100% identical to the comparison protein, our while also allowing the remainder of the protein to also have variation by a specified percent identity.
  • consensus sequence may provide insight into the conserved regions of related sequences where the unit (e.g., amino acid or nucleotide) is the same in most or all of the sequences, and regions that exhibit divergence between sequences.
  • the consensus sequence of a CDR may indicate amino acids that are important or dispensable for antigen binding.
  • consensus sequences may be prepared with any of the sequences provided herein, and the resultant various sequences derived from the consensus sequence can be validated to have similar effects as the template sequences.
  • the term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual abundance of the substance, compound, or material relative to the expected abundance.
  • the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between.
  • Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof.
  • the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents.
  • Purity can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.
  • ELISA enzyme-linked immunosorbent assay
  • Yield of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual overall amount of the substance, compound, or material relative to the expected overall amount.
  • the yield of the substance, compound, or material is about, is at least, is at least about, is not more than, or is not more than about, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including all decimals in between. Yield may be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.
  • the terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to that amount of a recited composition or compound that results in an observable effect.
  • Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the desired response for a particular subject and/or application.
  • the selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated.
  • a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.
  • the terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to a biological, enzymatic, or therapeutic function.
  • the term “inhibit” as used herein has its plain and ordinary meaning as understood in light of the specification, and may refer to the reduction or prevention of a biological activity. The reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values.
  • the term “delay” has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected.
  • the delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values.
  • the terms inhibit and delay may not necessarily indicate a 100% inhibition or delay. A partial inhibition or delay may be realized.
  • treating or “treatment” have their plain and ordinary meaning as understood in light of the specification, and refer to an approach for obtaining beneficial or desired results in a subject's condition, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the recurrence of disease, and remission, whether partial or total and whether detectable or undetectable.
  • “Treating” and “treatment” as used herein also include prophylactic treatment.
  • Treatment methods comprise administering to a subject a therapeutically effective amount of an active agent.
  • the administering step may consist of a single administration or may comprise a series of administrations.
  • the compositions are administered to the subject in an amount and for a duration sufficient to treat the subject.
  • the length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the subject, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof.
  • the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.
  • Tumor as used herein, plain and ordinary meaning as understood in light of the specification, and refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cell proliferative disorder
  • proliferative disorder proliferative disorder
  • tumor tumor-derived cellular disorder
  • cancer examples include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include lung cancer including small-cell lung cancer, non-small cell lung cancer and lung adenocarcinomas with neuroendocrine features; neuroendocrine prostate cancer, melanoma, gliomas, low-grade gliomas and glioblastoma, medullary thyroid cancer, carcinoid tumors, neuroendocrine tumors in the pancreas, bladder cancer, testicular cancer squamous cell cancer (e.g.
  • neuroendocrine neoplasms such as neuroendocrine tumors of unknown primary, neuroendocrine neoplasms of the small bowel, carotid body, adrenal gland, colorectal gynecological organ, abdomen, esophagus, GI tract, bile duct, nervous system, appendix, liver, anal, thymus, ileocecal junction, head and neck, breast, peritoneum and retroperitoneum, kidney, thyroid, stomach, bone,; adenocarcinomas, such as adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, end
  • cancer includes adult and pediatric solid cancers.
  • the cancer can be a solid tumor.
  • the cancer is a highly fibrotic tumor or cancer.
  • the cancer is a desmoplasia.
  • therapeutic target refers to a gene or gene product that, upon modulation of its activity (e.g., by modulation of expression, biological activity, and the like), can provide for modulation of the disease phenotype.
  • modulation is meant to refer to an increase or a decrease in the indicated phenomenon (e.g., modulation of a biological activity refers to an increase in a biological activity or a decrease in a biological activity).
  • administering includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intra-tumoral, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
  • Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra- arteriole, intradermal, subcutaneous, intraperitoneal, intra-tumoral, intraventricular, and intracranial.
  • Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
  • co-administer it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein.
  • “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and/or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity.
  • a “pharmaceutically acceptable” “diluent,” “excipient,” and/or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts.
  • a pharmaceutically acceptable diluent, excipient, and/or carrier is a diluent, excipient, and/or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs.
  • the term diluent, excipient, and/or carrier can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical formulation is administered.
  • Such pharmaceutical diluent, excipient, and/or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin.
  • Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions.
  • suitable pharmaceutical diluents and/or excipients include sugars, starch, glucose, fructose, lactose, sucrose, maltose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, salts, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • a non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution.
  • the physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, isomalt, maltitol, or lactitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
  • antioxidants such as ascorbic acid,
  • the formulation can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These formulations can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.
  • Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, mannitol, cellulose, serum, amino acids, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline,
  • excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, ⁇ -propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof.
  • the amount of the excipient may be found in the formulation at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w/w or any percentage by weight in a range defined by any two of the aforementioned numbers.
  • pharmaceutically acceptable salts has its plain and ordinary meaning as understood in light of the specification and includes relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like.
  • pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like.
  • suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non- toxic and strong enough to form such salts.
  • the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N- methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl aminoethane.
  • ubiquitin-proteasome recruiting activity has its plain and ordinary meaning as understood in light of the specification and refers to the biological function of binding with proteins involved in the ubiquitination pathway to affect the ubiquitination of a target protein. More specifically in the embodiments disclosed herein, this generally applies to the ability of a peptide to recruit a ubiquitinated E2 ubiquitin-conjugating enzyme and catalyze the transfer of the ubiquitin from the E2 ubiquitin-conjugate enzyme to the target protein.
  • the peptide having this ubiquitin-proteasome recruiting activity will therefore generally be a “ubiquitin-proteasome system recruiting domain (URD)”, where the URD has the ability to recruit a ubiquitin E2 ubiquitin-conjugating enzyme and catalyze the transfer of the ubiquitin from the E2 ubiquitin-conjugate enzyme to the target protein.
  • URD ubiquitin-proteasome system recruiting domain
  • the URD is a portion, truncation, or fragment of a naturally occurring E3 ubiquitin ligase, where the portion, truncation, or fragment of the E3 ubiquitin ligase exhibits the ubiquitin-proteasome recruiting activity.
  • the E3 ubiquitin ligase can be obtained from any source (e.g., from any eukaryote or archaea) but, in some embodiments, is preferably a human E3 ubiquitin ligase. Nevertheless, the URD may also be derived from a protein that is not a E3 ubiquitin ligase but has a similar function, such as a viral analogue of an E3 ubiquitin ligase (e.g., VIF from HIV).
  • a viral analogue of an E3 ubiquitin ligase e.g., VIF from HIV
  • E3 ubiquitin ligase or analogue may exhibit a ubiquitin-proteasome recruiting activity, and it would be within the capabilities of a skilled person to determine a certain portion, truncation, or fragment of an E3 ubiquitin ligase or analogue that is preferable for their uses (e.g., exhibiting better E2 ubiquitin-conjugating enzyme binding activity, reducing off-target or off- mechanism protein-protein interactions, and/or more efficiently catalyzing ubiquitination of a target protein).
  • the URDs or the portions, truncations, or fragments of the URDs and/or E3 ubiquitin ligases disclosed herein can be reasonably modified while still retaining their ubiquitin-proteasome recruiting activity, for example, by including or excluding amino acids from the N-terminus or C-terminus of the URD or the portion, truncation, or fragment of the URD and/or E3 ubiquitin ligase or performing amino acid substitutions to enhance various aspects of the URD and/or the portion, truncation, or fragment of the URD and/or E3 ubiquitin ligase such as protein folding, expression, or catalytic activity, optionally within the context of the dual-specific bifunctional polypeptide.
  • the ubiquitin-proteasome recruiting activity of a certain URD and/or portion, truncation, or fragment of a URD and/or E3 ubiquitin ligase may be quantified or verified through methods generally known in the art. For example, activity may be observed by observing (a) loss of bioluminescent signal from a HiBiTTM-tagged intracellular target protein, (b) loss of target protein signature and/or presence of polyubiquitin chain in target protein by mass spectrometry, (c) ubiquitination of the target protein in cell or in recombinant in vitro assays, or (d) detecting ubiquitination of target protein by immunoprecipitation, upon introduction of a URD-containing polypeptide specific for a known target protein.
  • a URD that retains ubiquitin-proteasome recruiting activity has polyubiquitination activity against a target bound by its targeting moiety when measured in an assay disclosed in one of the preceding references.
  • a URD that retains ubiquitin-proteasome recruiting activity has polyubiquitination activity against a target bound by its targeting moiety when measured in an assay described in Exemplary Assays for assessing proteasomal-mediated degradation and/or Example 1.
  • a URD that retains ubiquitin-proteasome recruiting activity has polyubiquitination activity against a target bound by its targeting moiety when measured by Western blot (e.g., measuring the abundance of the target in cells).
  • a URD that retains ubiquitin-proteasome recruiting activity has polyubiquitination activity against a target bound by its targeting moiety when measured by Western blot, but where the target is partially or totally preserved when the cells comprising the URD and target are treated with a proteasome inhibitor.
  • the term “antibody” includes, but is not limited to, genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, scFv, and heteroconjugate antibodies (for example, bispecific antibodies, diabodies, triabodies, tetrabodies, and nanobodies, etc.).
  • the term “antibody” includes minibodies and diabodies.
  • antibody includes a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen.
  • An exemplary antibody structural unit comprises a tetramer.
  • a full length antibody can be composed of two identical pairs of polypeptide chains, each pair having one “light” and one “heavy” chain (connected through a disulfide bond).
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, hinge, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • variable light chain (V L ) and variable heavy chain (V H ) refer to these regions of light and heavy chains respectively.
  • an “antibody” encompasses all variations of antibody and fragments thereof.
  • the antibody binds specifically to a desired target.
  • complementarity-determining domains or “complementarity-determining regions (“CDRs") interchangeably refer to the hypervariable regions of VL and VH.
  • the CDRs are the target molecule-binding site of the antibody chains that harbors specificity for such target molecule.
  • CDRs there are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in each V L and/or V H , constituting about 15-20% of the variable domains.
  • the CDRs are structurally complementary to the epitope of the target molecule and are thus directly responsible for the binding specificity.
  • the remaining stretches of the V L or V H the so-called FRs, exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).
  • the positions of the CDRs and framework regions can be determined using various well known definitions in the art.
  • the polypeptide is numbered from the beginning of the polypeptide signal sequence.
  • the polypeptide is numbered according from the beginning of the polypeptide and not including the signal sequence.
  • An "antibody variable light chain” or an “antibody variable heavy chain” as used herein have their plain and ordinary meaning as understood in light of the specification, and refers to a polypeptide comprising the V L or V H , respectively.
  • the endogenous V L is encoded by the gene segments V (variable) and J (junctional), and the endogenous V H by V, D (diversity), and J.
  • Each of VL or VH includes the CDRs as well as the framework regions.
  • antibody variable light chains and/or antibody variable heavy chains may, from time to time, be collectively referred to as "antibody chains.” These terms encompass antibody chains containing mutations that do not disrupt the basic structure of VL or VH, as one skilled in the art will readily recognize. In some embodiments, full length heavy and/or light chains are contemplated. In some embodiments, only the variable region of the heavy and/or light chains are contemplated as being present. Antibodies can exist as intact immunoglobulins or as a number of fragments produced by digestion with various peptidases as is known in the art.
  • antibody has its plain and ordinary meaning as understood in light of the specification, and also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (for example, single chain Fv) or those identified using phage display libraries (see, for example, McCafferty, J. et al., “Phage antibodies: filamentous phage displaying antibody variable domains,” Nature, Vol.348, No.66301, pp.552-554, 1990).
  • any technique known in the art can be used. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of the present disclosure. Also, transgenic mice, or other organisms such as other mammals, may be used to express fully human monoclonal antibodies. Furthermore, E. Coli or yeast may be used to express and manufacture recombinant antibodies and antibody fragments. Alternatively, phage display and yeast display technologies can be used to identify high affinity binders to selected antigens.
  • antibodies can be produced through B-cell screening technologies from human hosts (Pedrioli A., Oxenius A., Single B cell technologies for monoclonal antibody discovery, Trends in Immunology, (2021) volume 42, issue 12, pp. 1143-1158). Furthermore, antibodies can be derived from immunization of camelid animals or screening of camelid phage libraries. Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. In some embodiments, the terms “donor” and “acceptor” sequences can be employed.
  • Humanization can be essentially performed following the method of Winter and co-workers by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some complementarity determining region ("CDR") residues and possibly some framework (“FR”) residues are substituted by residues from analogous sites in rodent antibodies.
  • antibody fragment includes but is not limited to one or more antigen binding fragments of antibodies alone or in combination with other molecules, including, but not limited to Fab', F(ab')2, Fab, Fv, rIgG (reduced IgG), scFv fragments, single domain fragments (nanobodies), peptibodies, nanobodies), minibodies, and diabodies.
  • scFv refers to a single chain Fv (“fragment variable”) antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain.
  • a “diabody” comprises a first polypeptide chain which comprises a heavy (VH) chain variable domain connected to a light chain variable domain (VL) on the first polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the first polypeptide chain and a second polypeptide chain comprising a light chain variable domain (VL) linked to a heavy chain variable domain VH on the second polypeptide chain (VL-VH) connected by a peptide linker that is too short to allow pairing between the two domains on the second polypeptide chain.
  • a peptide linker may be any suitable length that promotes such assembly, for example, between 5 and 20 amino acids in length.
  • a “cys-diabody” is a diabody with one or more than one N-terminal cysteines.
  • the targeting moiety includes all varieties of antibodies, including binding fragments thereof. Further included are constructs that include 1, 2, 3, 4, 5, and/or 6 CDRs. In some embodiments, these CDRs can be distributed between their appropriate framework regions in a traditional antibody.
  • the CDRs can be contained within a heavy and/or light chain variable region. In some embodiments, the CDRs can be within a heavy chain and/or a light chain. In some embodiments, the CDRs can be within a single peptide chain.
  • the targeting moieties e.g., protein binding partners, antibodies
  • the targeting moieties with a particular binding specificity bind to a particular target protein at least two times the background and do not substantially bind in a significant amount to other non-target molecules present in the sample.
  • Specific binding to a targeting moiety under such conditions may require the targeting moiety to have been selected for its specificity for a particular target protein.
  • a variety of assay formats may be used to select targeting moieties specifically reactive with a particular target protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, for example, Harlow, E.
  • Equilibrium dissociation constant refers to the dissociation rate constant (k d , time -1 ) divided by the association rate constant (k a , time -1 M -1 ). Equilibrium dissociation constants can be measured using any known method in the art.
  • the targeting moieties and/or bifunctional polypeptide of the present disclosure generally will have an equilibrium dissociation constant of less (that is superior binding) than about 10 -7 or 10 -8 M, for example, less than about 10 -9 M or 10 -10 M, in some embodiments, less than about 10 -11 M, 10 -12 M, or 10 -13 M.
  • the term “conservative” modification, substitution, or variant applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservative” modification, substitution or variant where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the present disclosure.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, for example, Creighton, T. E., “Proteins - Structures and Molecular Properties,” W. H.
  • % w/w or “% wt/wt” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100.
  • % v/v or “% vol/vol” as used herein has its plain and ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.
  • Example 1 HiBiTTM assay for proteasomal-mediated degradation of target protein(s)
  • HepG2 cells were transfected with plasmids for transiently expressing HiBiTTM-tagged target protein and for expressing the corresponding bifunctional polypeptide degrader at the time of plating.
  • Doxycycline was added 24 hours post- transfection to induce expression of the bifunctional polypeptide degrader from the doxycycline-inducible system.
  • HiBiTTM signal was measured the following day using Nano- Glo HiBiTTM lytic detection system according to manufacturer’s protocol.
  • Table 33 summarizes the assay conditions.
  • Table 33: Assay Conditions The results of the HiBiTTM assays are summarized in Tables 27, 29, 30, and 31 above. Average values of the percentage of target protein remaining relative to control was determined. A grade for the percent of degradation is provided based on the following cutoffs: A > 50% degradation ( ⁇ 50% remaining); B ⁇ 20% - ⁇ 50% degradation ( ⁇ 50% - ⁇ 80% remaining); C ⁇ 2% - ⁇ 20% degradation (> 80% - ⁇ 98 remaining); and D ⁇ 2% degradation (> 98% remaining) relative to control.
  • Example 2 Subcellular localization of mono-specific bifunctional polypeptide degraders Cells were seeded and transfected as described in Example 1. 48 hours post-plating, cells were fixed in 4% paraformaldehyde solution for 10 minutes, rinsed once in PBS pH 7.4, and permeabilized in 0.1% triton x-100 for 15 minutes at room temperature.
  • Table 34 Exemplary NLS sequences used in Example 2 The results are summarized in the following Table, where “Nuc” is predominantly nucleus, “Cyt” is predominantly cytoplasm, and “Mix” is both nucleus and cytoplasm. HA is the hemagglutinin epitope tag.
  • the URDs and linkers are described elsewhere herein.
  • Table 35 Subcellular localization of exemplary polypeptides The SPOP URD is derived from an E3 ubiquitin ligase. SPOP.2 and SPOP.3 variant URDs are truncated regions of the full-length SPOP URD. Compared to SPOP.2, SPOP.3 has a truncation of 14 residues at the C-terminus.
  • the additional 14 C-terminal residues of SPOP.2 function as an endogenous nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • Mono-specific bifunctional polypeptides comprising VL12.3 targeting moiety and URDs derived from either FBXW7a or FBW7b, E3 ligases with known nuclear and cytoplasmic localization (Yeh et al. “FBXW7: a critical tumor suppressor of human cancers,” Molecular Cancer (2018) 17:115), respectively directed the mono-specific bifunctional polypeptide exclusively the nucleus (SEQ ID NO: 2544) or to the cytoplasm (SEQ ID NO: 2592). This highlights the ability to design mono-specific bifunctional polypeptides to desired subcellular localization through URD selection.
  • Example 3 Subcellular localization of dual-specific bifunctional polypeptide degraders Cell assays were prepared as described in Example 2.
  • the dual-specific bifunctional polypeptide of SEQ ID NO: 2203 has an N-terminal mycNLS sequence, but no C-terminal NLS sequence.
  • Various NLS sequences were added to the C-terminus. The results show that adding additional NLS sequences to the C-terminus of can improve nuclear localization, The results of the experiment are summarized in FIG. 6, and exemplary photomicrographs are shown in FIG. 7A-7C.
  • Example 4 Development and Characterization of c-Myc sdABs Anti-c-Myc single domain antibodies were developed and characterized.
  • N-terminally and C-terminally biotinylated c-Myc peptide antigens were synthesized by Anaspec and c-Myc-Fc antigens including human, cynomolgus, and mouse were produced as N-terminal Fc fusions. All human c-Myc antigens were the portion of c-Myc with the sequence: AEEQKLISEEDLLRKRREQLKHKLEQLRNS (SEQ ID NO: 769). The cynomolgus c-Myc antigen was the portion of cynomolgus c-Myc with the sequence AEEQKLISEKDLLRKRREQLKHKLEQLRNS (SEQ ID NO: 770).
  • the mouse c-Myc antigen was the portion of murine c-Myc with the sequence ADEHKLTSEKDLLRKRREQLKHKLEQLRNS (SEQ ID NO: 771).
  • c-Myc-Fc antigens were biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kit from Thermo.
  • Goat F(ab’)2 anti-human IgG (HC-488), ExtrAvidin-PE (EA-PE) and Streptavidin-AF633 (SA-633) were obtained from Jackson Immuno Research, Sigma, and Molecular Probes, respectively. Streptavidin MicroBeads and MACS LC separation columns were purchased from Miltenyi Biotec.
  • c-Myc peptide antigens Prior to starting selections c-Myc peptide antigens were assessed for quality and suitability for selections. Quality assessments included Octet BLI kinetics to 9E10 control antibody, size exclusion chromatography, and non-specific binding checks to yeast heavy chain-only antibody libraries. Antigens were initially run on size exclusion chromatography to confirm that antigens are not aggregated and have suitable quality for selection (> 90% main peak). Antigens were run on an HPLC equipped with a UV detector and TSK mAb HTP, SW3000, or SWAggregate SEC column (Tosoh Bioscience, King of Prussia, PA, USA) along with a set of molecular weight standards.
  • Antigen binding to the benchmark antibody, 9E10 was assessed by Octet Biolayer Interferometry (BLI). All data were acquired at 25 °C with an Octet HTX instrument (Sartorius, Bohemia, NY, USA) at an orbital shaking speed of 1000 rpm. Reagents were formulated in a running buffer (PBSF) of phosphate buffered saline with 0.1% IgG-free bovine serum albumin.
  • PBSF running buffer
  • 9E10 For assessment of 9E10 binding to c-Myc-Fc antigens, 9E10 (100 nM) was captured to anti- human IgG Fc quantitation (AHQ) sensors (Sartorius, Bohemia, NY, USA) to a response level of 0.5-1.5 nm, the remaining unoccupied binding sites on the AHQ biosensors were blocked with an inert human antibody or inert biotinylated protein (for biotinylated antigens) (0.5 mg/mL for 10 min), and then allowed to stand in PBSF for a minimum of 15 minutes before proceeding to the kinetic measurements.
  • AHQ anti- human IgG Fc quantitation
  • the kinetic measurements began with a short (60 s) baseline dip into PBSF before exposing (180 s) the 9E10-loaded sensors to human, cyno or mouse c-Myc-Fc (100 nM). This was immediately followed by a dip (180 s) into PBSF to measure the rate of dissociation of the sample 9E10/c-Myc complex.
  • biotinylated c-Myc peptides were captured to streptavidin (SA) biosensors (Sartorius, Bohemia, NY, USA) to a response level of 0.5-1.5 nm, and then allowed to stand in PBSF for a minimum of 15 minutes before proceeding to the kinetic measurements.
  • SA streptavidin
  • the kinetic measurements began with a short (60 s) baseline dip into PBSF before exposing (180 s) the c-Myc peptide-loaded sensors to 9E10 (100 nM). This was immediately followed by a dip (180 s) into PBSF to measure the rate of dissociation of the 9E10/c-Myc peptide complex.
  • HCAb heavy-chain only antibody
  • yeast cells For incubations with biotinylated peptide antigens, yeast cells ( ⁇ 2 million cells/HCAb library) were incubated with either 0.1 mL of a 500 nM mixture of N- and C-terminally biotinylated peptides or a 100 nM mixture of N- and C-terminally biotinylated peptides preincubated with 25 nM Streptavidin Microbeads to create a tetrameric peptide antigen for 30 min at 30°C in wash buffer (phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA)).
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • yeast cells ⁇ 10 10 cells/HCAb library
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • yeast cells For incubations with biotinylated antigens, yeast cells ( ⁇ 10 10 cells/HCAb library) were incubated with either 3 mL of a 500 nM mixture of N- and C-terminally biotinylated peptides or a 100 nM mixture of N- and C-terminally biotinylated peptides preincubated with 25 nM Streptavidin Microbeads to create a tetrameric peptide antigen for 30 min at 30°C in wash buffer (phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA)).
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • the cell pellet was resuspended in 40 mL wash buffer, and Streptavidin MicroBeads (500 ⁇ L) were added to the yeast, except for the tetrameric peptide antigen, and incubated for 15 min at 4°C.
  • Streptavidin MicroBeads 500 ⁇ L were added to the yeast, except for the tetrameric peptide antigen, and incubated for 15 min at 4°C.
  • the yeast were pelleted, resuspended in 20 mL wash buffer, and loaded onto a Miltenyi LS column. The column was washed 3 times with 5 mL wash buffer, then the column was removed from the magnetic field and the yeast were eluted with 5 mL of growth media and grown overnight. The following rounds of selection were performed using flow cytometry.
  • Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sort gates were determined to select for antibodies with desired characteristics. Selection rounds were repeated and used decreasing c-Myc antigen concentrations until a population with all the desired characteristics was obtained. After the final round of sorting, yeast cells were plated and individual colonies were picked for characterization. CDRH1 and CDRH2 batch shuffle CDRH1 and CDRH2 batch shuffle diversification protocol was used during the primary discovery phase for further discovery and improvement of antibodies.
  • CDR H1 and CDR H2 batch diversification protocol Pools of CDR H3s from na ⁇ ve selection outputs were extracted from the yeast and transformed into a premade plasmid vector with CDR H1 and CDR H2 diversity of 2 x 10 5 . Additionally, this vector also contained limited diversities at positions C22 and C92 (Kabat) where the cysteines could be replaced by I, T, A, S, G, or V in order to remove the C22-C92 disulfide. Selections were performed with one round of MACS and two rounds of FACS as described in the na ⁇ ve discovery. In the different FACS rounds the libraries were assessed for c-Myc and PSR binding.
  • Affinity pressure was applied by antigen titration using either the biotinylated c-Myc peptides or the biotinylated c-Myc-Fc antigen. Selection rounds were repeated until a population with the desired characteristics was obtained.
  • Antibody Optimization Optimization of antibodies was performed by introducing diversities into the heavy chain variable regions as described below.
  • CDRH1 and CDRH2 selection The CDRH3 of a single antibody was recombined into a premade library with CDRH1 and CDRH2 variants including diversification at positions C22 and C92 (Kabat numbering; corresponding to residue 22 of FR1 and residue 30 of FR3 sequences presented herein) for a total library diversity of 2 x 10 7 and selections were performed with one round of MACS and two rounds of FACS as described in the na ⁇ ve discovery. During optimization, the libraries were assessed for PSR binding and affinity pressure in the different FACS rounds. Affinity pressure was generally applied by titrating the c-Myc biotinylated peptide antigens.
  • the final selection round included a parental HCAb pre-complexing pressure, this was performed by pre-complexing the c-Myc-Fc antigen with parental HCAb to select for antibodies with better affinity than the parent.
  • Antibody production and purification Yeast clones were grown to saturation and then induced for 48 h at 30°C with shaking. After induction, yeast cells were pelleted and the supernatants were harvested for purification. HCAbs were purified using a Protein A column and eluted with acetic acid, pH 2.0 into a neutralization buffer.
  • Single domain antibody (sdAb) fragments were generated by papain digestion and purified using FcXL resin to remove cleaved and uncleaved Fc fragments followed by purification of the sdAb using a Praesto AC resin and elution with acetic acid, pH 2.0 into a neutralization buffer.
  • Biolayer interferometry (BLI) binding and kinetic measurements of c-Myc BLI kinetic and epitope binning measurements were acquired at 25 °C with an Octet HTX instrument (Sartorius, Bohemia, NY, USA) at an orbital shaking speed of 1000 rpm.
  • HCAbs/IgGs were captured to anti-human IgG Fc capture (AHC) sensors (Sartorius, Bohemia, NY, USA) to a response level of 0.5-1.5 nm, and then allowed to stand in PBSF for a minimum of 15 minutes before proceeding to the kinetic measurements.
  • AHC anti-human IgG Fc capture
  • the kinetic measurements began with a short (60 s) baseline dip into PBSF before exposing (180 s) the IgG/HCAb-loaded sensors to human, cyno or mouse c-Myc-Fc (100 nM). This was immediately followed by a dip (180-600 s) into PBSF to measure the rate of dissociation of the sample HCAb/c-Myc complex.
  • c-Myc-Fc antigens To assess monovalent binding of sdAbs to human, cyno, or mouse, c-Myc-Fc antigens, c-Myc-Fc antigen (100 nM) was captured to anti-human IgG Fc capture (AHC) sensors (Sartorius, Bohemia, NY, USA) to a response level of 0.5-1.5 nm, and then allowed to stand in PBSF for a minimum of 15 minutes before proceeding to the kinetic measurements. The kinetic measurements began with a short (60 s) baseline dip into PBSF before exposing (180 s) the c-Myc-Fc-loaded sensors to sample sdAbs (100-300 nM).
  • AHC anti-human IgG Fc capture
  • Table 36 shows the results of the assessment of sdAb binding to human Myc-Fc.
  • Table 36 Human Myc-Fc on AHC Sensor, sdAb in Solution (300 nM) Monovalent
  • mycV variants bind human Myc-Fc with a Kd of between 1.22E -07 and 9.47E -09 , a kon of between 6.79E +04 and 4.04E +05 , and a Koff of between 1.74E -02 and 8.67E -04 .
  • Table 37 shows the results of the assessment of sdAb binding to Mouse Myc-Fc.
  • Table 37 Octet Image: Mouse Myc-Fc on AHC Sensor, sdAb in Solution (100 nM) Monovalent
  • mycV variants bind Mouse Myc-Fc with a Kd of between 1.09E -07 and 4.15E -10 , a k on of between 1.22E +05 and 4.18E +05 , and a K off of between 2.49E -02 and 7.27E -05 .
  • Table 38 shows the results of the assessment of sdAb binding to Cyno Myc-Fc. Table 38.
  • mycV variants bind Cyno Myc-Fc with a Kd of between 1.09E -07 and 4.15E -10 , a kon of between 1.22E +05 and 4.18E +05 , and a Koff of between 2.49E -02 and 7.27E -05 .
  • the C-terminally biotinylated human c-Myc antigen (SEQ ID NO: 769) was captured to streptavidin (SA) biosensors (Sartorius, Bohemia, NY, USA) to a response level of 0.5-1.5 nm, and then allowed to stand in PBSF for a minimum of 15 minutes before proceeding to the kinetic measurements.
  • SA streptavidin
  • the kinetic measurements began with a short (60 s) baseline dip into PBSF before exposing (180 s) the c- Myc-loaded sensors to sample sdAbs (300 nM).
  • Octet sdAb Kd Human Myc Peptide on Streptavidin Sensor, Monovalent
  • mycV variants bind human myc peptide with a Kd of between 1.09E -07 and 5.56E -09 , a k on of between 3.23E +04 and 1.31E +05 , and a K off of between 1.21E -02 and 6.06E -04 .
  • Epitope binning (sample IgG in solution)
  • the control IgGs or HCAbs (100 nM) were captured (0.5-1.5 nm) to AHC sensors, the remaining unoccupied binding sites on the AHC biosensors were blocked with an inert human antibody (0.5 mg/mL for 10 min), and then allowed to equilibrate in PBSF for a minimum of 30 min.
  • the loaded and blocked sensors were exposed (90-180 s) to the sample HCAbs (300 nM) prior to the binning analysis.
  • the loaded biosensor tips were exposed (180 s) to human c-Myc-Fc (100 nM) and then exposed (180 s) to the sample HCAbs (300 nM).
  • the data was y-axis aligned, and inter-step corrected using the ForteBio Data Analysis Software version 11.1.3.10. Additional binding by the sample HCAb indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).
  • PSR binding Poly-Specificity Reagent (PSR) binding assays were performed as described in, e.g., Xu et al.
  • the cells were washed twice with 200 ⁇ L of cold PBSF and the pellet was re- suspended in 50 ⁇ L of secondary labeling mix (Extravidin-R-PE, anti-human HC-488, and propidium iodide). The mix incubated on ice for 20 minutes and was then washed twice with 200 ⁇ l ice-cold PBSF. The cells were resuspended in 50 ⁇ L of ice-cold PBSF and analyzed on a FACSCanto (BD Biosciences) using an HTS sample injector. Flow cytometry data was analyzed for mean fluorescence intensity (MFI) in the R-PE channel and was normalized for background binding using appropriate controls.
  • MFI mean fluorescence intensity
  • a PSR score ⁇ 0.1 is considered clean, a PSR score > 0.10 and ⁇ 0.33 is considered low, > 0.33 and ⁇ 0.66 is considered medium, and > 0.66 and ⁇ 1.00 is considered high.
  • Table 40 shows the results of the PSR binding assay.
  • Table 40 Poly-Specificity Reagent (PSR) binding assay As can be seen in Table 40, mycV variants of between 0.01 and 0.43.
  • HIC retention time The method for measuring HIC retention time (RT) was described previously (Estep et al., MAbs.2015, 7(3): 553–561; Jain et al, Bioinformatics, 33(23), 2017, 3758-3766 ; which are hereby incorporated by reference herein in their entirety and for all purposes).
  • 5.0 ug of each sample 1.0 mg/ml was spiked into mobile phase A solution (1.8 M ammonium sulfate, 0.1 M sodium phosphate, pH 6.5) to achieve an ammonium sulfate concentration of about 1 M prior to analysis.
  • a Sepax Proteomix HIC butyl-NP5 column (Sigma Aldrich) was used with a linear gradient (of mobile phase A and mobile phase B solution (0.1 M sodium phosphate, pH 6.5) and protein was detected with UV absorbance (280 nm). The flow rate was 1.0 mL/min and the gradient time was 7.5 min. A standard IgG1 antibody sample corresponding to the variable regions of adalimumab was run periodically to assess assay reproducibility and used to normalize the measured RTs. A high throughput (HT) version of the experiment was adapted. The flow rate was increased to 2.0 mL/min and the gradient time shortened to 2.63 min.
  • HT high throughput
  • DSF measures protein unfolding by monitoring changes in fluorescence of a dye that binds preferentially to unfolded protein as a function of temperature.
  • 10 ⁇ L of 20 ⁇ Sypro Orange was added to 20 ⁇ L of 0.2- 1 mg/mL sdAb solution.
  • a RT-PCR instrument BioRad CFX96 RT PCR was used to ramp the sample plate temperature from 40 to 95° C at 0.5° C. increments, with 2 min allowed for equilibration at each temperature.
  • the negative of the first derivative for the raw data is used to extract Tm.
  • Table 42 shows the melting temperature of the tested mycV variants. Table 42.
  • mycV Variant Melting Temperature As can be seen in Table 42, the tested mycV variants had a melting temperature of between 45.5° and 64.5° C.
  • Example 5 Dual-specific bifunctional polypeptides that direct proteasomal-mediated degradation for treatment of cancer
  • the cancer may be a brain cancer, breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, pancreatic cancer, bladder cancer, stomach cancer, or a hematological malignancy, or another cancer not listed here.
  • the cancer may be non-responsive to standard of care treatments.
  • the cancer may be primary and/or metastatic.
  • a protein target associated with cancer e.g., Myc, CTNNB1, or PCNA
  • the bifunctional polypeptides, or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition is administered enterally, parenterally, intravenously, intratumorally, intramuscularly, intra-arterially, intradermally, subcutaneously, intraperitoneally, intraventricularly, intrathecally, or intracranially.
  • the bifunctional polypeptides, or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition is administered as at least one dose at an amount of 1, 10, 100, 1000 ng, or 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ⁇ g, or 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg, or any amount within a range defined by any two of the aforementioned amounts, or any other amount appropriate for optimal efficacy in humans.
  • the bifunctional polypeptides, or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition is administered in a number of doses sufficient to achieve an improvement in the disease pathology in the patient.
  • the bifunctional polypeptides, or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition is administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 doses, or any number of doses within a range defined by any two of the aforementioned number of doses, for example, 1-150 doses, 1-10 doses, 1-50 doses, 50-100 doses, or 50-150 doses.
  • the bifunctional polypeptides, or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition is administered in a single dose (for example, when using a gene therapy or neoadjuvant therapy approach).
  • the doses can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or weeks or any time within a range defined by any two of the aforementioned times, for example, 1 day to 48 weeks, 1-48 days, 1-48 weeks, or 10-30 days.
  • the patient After administration of the bifunctional polypeptides, or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition, the patient exhibits a prolongment of progression-free survival (PFS), a prolongment of overall survival (OS), a reduction in cancer burden (e.g., tumor size and number), or an amelioration of symptoms such as symptoms of pain, discomfort, or inflammation.
  • PFS progression-free survival
  • OS prolongment of overall survival
  • cancer burden e.g., tumor size and number
  • the bifunctional polypeptides, or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition is administered as a supplement to a standard of care therapy for a cancer.
  • the standard of care treatment may include but is not limited to surgery, radiation, chemotherapy, targeted therapy, or immunotherapy.
  • the bifunctional polypeptides, or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition may be administered in combination with anti-PD-1, anti-PD-L1, anti-CTLA4, anti-LAG3, or adoptive cell therapy.
  • the combination of the standard of care therapy and the bifunctional polypeptides, or dual-specific bifunctional polypeptide, polynucleotide, or pharmaceutical composition has a greater positive effect in reducing cancer burden or ameliorating symptoms compared to the standard of care therapy alone.
  • one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above.
  • a range includes each individual member.
  • a group having 1-3 articles refers to groups having 1, 2, or 3 articles.
  • a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth. While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art.

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Abstract

L'invention concerne des polypeptides comprenant ou constitués d'anticorps à domaine unique anti-Myc (sdAd), ainsi que des polypeptides bifonctionnels et bifonctionnels à double spécificité incorporant les sdAbs anti-Myc qui induisent la dégradation d'une première et éventuellement d'une seconde cible biologique à travers le mécanisme de protéasome cellulaire. Les polypeptides bifonctionnels selon l'invention comprennent généralement une première fraction de ciblage qui comprend l'anti-Myc sdAb, et un second composant qui a une fonction d'ubiquitination, et éventuellement une seconde fraction de ciblage ciblant au moins une seconde protéine cible. La liaison de Myc par la première fraction de ciblage favorise l'ubiquitination de Myc par le second composant, ce qui permet de marquer le Myc pour la dégradation protéasomale. Ces polypeptides bifonctionnels peuvent être utilisés pour le traitement d'une maladie chez un sujet, le premier et/ou le second composant se liant à une ou plusieurs cibles biologiques dont la fonction et/ou l'expression anormales est associée à la maladie.
PCT/US2024/055071 2023-11-08 2024-11-08 Liants d'anticorps à domaine unique de myc Pending WO2025101862A1 (fr)

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