De novo genetically-encodable polypeptides for extracellular protein degradation, and fusion proteins thereof

Abstract

Polypeptides that bind to asialoglycoprotein receptor (ASGPR), IGF-2R domain 11, IGF-2R, and fusion proteins thereof, that can promote extracellular protein degradation are provided, and methods for their use.

Description

[0001] De novo genetically-encodable polypeptides for extracellular protein degradation, and fusion proteins thereof Federal Funding Statement This invention was made with government support under Grant No. HR0011-21-2- 0012, awarded by the Defense Advanced Research Projects Agency and Grant Nos. 0001039633 and W81XWH2010230, awarded by the Department of Defense. The government has certain rights in the invention. Sequence Listing Statement A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on August 12, 2024 having the file name “23-0912-WO” and is 122,679 bytes in size. Background The endocytosis of many cell surface receptors is triggered by binding of their endogenous ligands which can shift the conformational or oligomerization state of the receptor4 and induce receptor clustering and adaptor protein recruitment. Native endocytosis inducing ligands have been utilized to enhance intracellular delivery and prompting extracellular protein degradation. Usage of native ligands could trigger off-targeting signaling, and their binding sites are potentially occupied by existing ligands. Additionally, many endogenous ligands are also hard for manufacturing due to instability to maintain physiological activity. Summary In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1, wherein the polypeptide binds to asialoglycoprotein receptor (ASGPR). In one embodiment, permissible amino acid substitutions relative to SEQ ID NO:1 are listed in Table 2 or 3. In another embodiment, no substitutions are permitted relative to SEQ ID NO:1 at residues 1, 16, 30, 39, 46-48, 50, and 53-54. In a further embodiment, permissible substitutions relative to SEQ ID NO:1 are shown in Table 3. In one embodiment, the polypeptides comprise an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1-10, wherein the polypeptide binds to asialoglycoprotein receptor (ASGPR). In another embodiment, the disclosure provides fusion polypeptide, comprising 2, 3, 4, 5, or more copies of the polypeptide of any embodiment of the first aspect of the disclosure. In one embodiment, the fusion polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:11 or 12, wherein the fusion protein binds to asialoglycoprotein receptor (ASGPR). In a second aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13-15 and 106, wherein the polypeptide binds to insulin-like growth factor 2 receptor (IGF-2R). In one embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13, wherein the polypeptide binds to IGF-2R domain 6. In one such embodiment, permissible amino acid substitutions relative to SEQ ID NO:13 are listed in Table 6 or 7. In another embodiment, no substitutions are permitted relative to SEQ ID NO:13 at residues 15, 23-24, 35, and 57. In a further embodiment, permissible substitutions relative to SEQ ID NO:13 are shown in Table 7. In another embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14, wherein the polypeptide binds to IGF-2R domain 11. In one embodiment, permissible amino acid substitutions relative to SEQ ID NO:14 are listed in Table 8 or 9. In another embodiment, no substitutions are permitted relative to SEQ ID NO:14 at residues 4, 8, 11, 13, 25, 31, 43, and 47. In a further embodiment, permissible substitutions relative to SEQ ID NO:14 are shown in Table 9. In one embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:15, wherein the polypeptide binds to IGF-2R domain 11. In another embodiment, permissible amino acid substitutions relative to SEQ ID NO:15 are listed in Table 10 or 11. In a further embodiment, no substitutions are permitted relative to SEQ ID NO:15 at residues 21, 32, and 36. In another embodiment, permissible substitutions relative to SEQ ID NO:14 are shown in Table 11. In one embodiment, the disclosure provides fusion polypeptides comprising (a) a first polypeptide according to any embodiment of the second aspect; and (b) a different second polypeptide according to any embodiment of the second aspect. In some embodiments, the fusion polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:16 or 17, wherein the fusion protein binds to IGF-2R. In a third aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18, wherein the polypeptide binds to IGF-2R. In one embodiment, X1 consists of an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:19-22. In another embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23-26, wherein optional residues may be present or deleted. In a fourth aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27, wherein the polypeptide binds to IGF-2R. In one embodiment, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or all 9 mutations relative to SEQ ID NO:27 selected from the group consisting of 13A, 17I, 18V, 19A, 27K, 35E, 46 R / Q, 54V, and 58K. In another embodiment, the polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27-30 wherein the polypeptide binds to IGF-2R. In one embodiment of any of the aspects, the disclosure provides fusion proteins, comprising (a) the polypeptide or fusion polypeptide of any aspect or embodiment; and (b) a protein that binds to a degradation target. In another aspect, the disclosure provides fusion protein, comprising: (a) a protein that binds to a degradation target; and (b) a transferrin receptor binding polypeptide selected from the group consisting of: (i) a polypeptide comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence selected from SEQ ID NO:32-37, wherein residues in parentheses are optional and may be present or absent; and (ii) a polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, or 98%, identical to the amino acid sequence of SEQ ID NO:31, wherein (A) the polypeptide is identical to SEQ ID NO:31 at residues corresponding to residues 20, 23, 24, 27, 28, 31, 32, 34, 35, 36, 37, 74, 77, 78, 81, 82, 84, 85, and 88 in SEQ ID NO:31; and (B) the polypeptide is substituted relative to SEQ ID NO:31 as shown in Table 12 at least at 5, 6, 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more residues relative to SEQ ID NO:31. In one embodiment, the polypeptide is identical to SEQ ID NO:31 at residues corresponding to residues 7, 9, 11, 13, 16, 18, 20-21, 23-24, 27-28, 30-32, 34-37, 41, 47, 49- 51, 56, 58, 60, 65-66, 68, 70-71, 74-75, 77-78, 80-82, 84-85, and 87-88; and optionally identical to SEQ ID NO:31 at residues corresponding to residues 3-5 in SEQ ID NO:31. In another embodiment, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 substitutions relative to SEQ ID NO:31: Q6K, A8F, E10K, R14E, E19K, R29K, A33S, R42E, S48A, R52K, R53K, A64E, L76E, and / or R79K. In a further embodiment, the polypeptide comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence selected from SEQ ID NO:32- 27, wherein residues in parentheses are optional and may be present or absent. In another embodiment, the disclosure provides fusion proteins comprising (a) the polypeptide or fusion polypeptide of any aspect or embodiment; and (b) a second polypeptide that binds to a therapeutic target of interest. In one embodiment, the second polypeptide comprises a monoclonal antibody heavy chain and / or light chain, or an antigen binding fragment thereof. In other embodiments, the second polypeptide comprises an amino acid sequence at least 90%, 95%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:38-97, or an antigen binding fragment thereof. In other embodiments, the disclosure provides antibodies, or antigen-binding fragments therein, comprising (a) the fusion protein of any embodiment herein wherein the second polypeptide comprises a monoclonal antibody heavy chain, or an antigen binding fragment thereof, and (b) a light chain for the monoclonal antibody, or an antigen binding fragment thereof, or (a) the fusion protein of any embodiment herein wherein the second polypeptide comprises a monoclonal antibody light chain, or an antigen binding fragment thereof, and (b) a heavy chain for the monoclonal antibody, or an antigen binding fragment thereof. The disclosure also provides nucleic acids encoding the polypeptide, polypeptide fusion, or or fusion protein of any aspect or embodiment; expression vectors comprising the nucleic acid operatively linked to a suitable control element, such as a promoter; host cells comprising the polypeptide, polypeptide fusion, fusion protein, nucleic acid, and / or expression vector of any aspect or embodiment, and pharmaceutical compositions and other compositions comprising the polypeptide, polypeptide fusion, fusion protein, nucleic acid, and / or expression vector of any aspect or embodiment. The disclosure also provides methods for degrading a target protein, comprising contacting a biological sample comprising the target with the fusion protein of any previous claim, under conditions to promote binding of the fusion protein to target protein present in the biological sample, thereby promoting degradation of the target protein. The disclosure further provides fusion proteins comprising (a) the polypeptide or fusion polypeptide of any aspect or embodiment; and (b) a therapeutic polypeptide; and methods for treating a disorder contacting a subject with the fusion protein, under conditions to promote binding of the fusion protein to ASGPR, transferrin receptor, or IGF-2R in cells of the subject, thus treating the disorder. In a further embodiment, the disclosure provides methods for treating a disease, comprising administering to a subject in need thereof an amount effective of the fusion protein or antibody or antigen binding fragment thereof of any aspect or embodiment, to degrade target protein present in the subject, thereby treating the disease. Description of the Figures Figure 1. Design strategies for endocytosis-triggering Tags. a,d,g, Schema of designed endocytosis mechanisms. (top) Design of binding to constitutively cycling receptors at sites that do not overlap with those of natural ligands to avoid competition. (middle) Design of binders that trigger endocytosis by eliciting a conformational change in the receptor. The Tag binds at two distinct epitopes on the target and actively triggers the conformational change. (bottom) Designed endocytosis via receptor clustering. The multivalent Tag clusters multiple copies of the target receptor and induces endocytosis. b, Design strategy for IGF_Tags. Starting from the structure of IGF-2 in complex with IGF-2R, de novo minibinders were generated and screened against the IGF-2 binding sites at IGF-2R domain 6 and domain 11, separately. Individual binders for each domain (D6mb and D11mb) were fused with flexible linkers or a rigid fusion interdomain connection. For flexible fusion, multiple linker lengths and fusion directions were sampled. For rigid fusion, the two major binding helices from D6mb and one major binding helix from D11mb were extracted as starting motifs. With protein Inpainting, geometries and fusion orders were sampled and ranked based on RosettaTMand AlphafoldTM2 metrics. c, Cellular uptake of IGF_Tags. Jurkat cells were treated with biotinylated 100 nM IGF_Tags or IGF-2, and 33 nM Strapavidin- AF647 for 24h. After washing 3 times, cellular uptake was measured by flow cytometry. The data were normalized with the control group treated with 33 nM Strapavidin-AF647 alone. e, Design strategy for TfR Tags. A designed binder to TfR binds at a site distant from that of transferrin46. f, Cellular uptake of TfR Tags. U-251MG cells were treated with 100nM AF647-labelled TfR-Tags for 2h. After washing 3 times, cellular uptake was measured by flow cytometry. The data were normalized with respect to a control group treated with 100nM AF647-labelled LHDB (supposed no endocytosis). h, Design strategy for ASGPR Tags. Based on the crystal structure of ASGPR47, binders were designed and then connected with flexible linkers. i, Cellular uptake of ASGPR Tags. Hep3B cells were treated with 100 nM AF-647 conjugated ASGPR Tags for 24h. After washing 3 times, the cellular uptake was measured by flow cytometry. The data were normalized with respect to a control group treated with 100nM AF647-labelled LHDB (supposed no endocytosis). Figure 2. Surface receptor degradation with tissue-specific pLYTACs. a, Schema of tissue-specific pLYTACs (lysosome-targeting chimeras (for receptor degradation. b, ASGPR. Left: Tissue expression of ASGPR. Right: Western blot analysis of total EGFR levels in Hep3B cells after treatment with 200 nM EGFRn or EGFRn-AS_Tag for 48 h. c, TfR. Left: Tissue expression of TfR. Right: Western blot analysis of total EGFR levels in Hela cells after treatment with 200 nM EGFRn or EGFRn-TfR_Tag for 48 h. e, IGF- 2R. Left: Tissue expression of IGF-2R. Right: Western blot analysis of total EGFR levels in H1975 cells after treatment with 200 nM of EGFRn / CTX with / without the fusion with Tag1 or M6Pn for 48 h. f, Western blot analysis of total EGFR levels in Hela cells after treatment with 200 nM EGFRn or EGFRn-IGF_Tags for 48 h. g, Quantitative proteomics analysis of protein abundance in H1975 cells after treatment with EGFRn or EGFRn-IGF_Tag2 for 48h relative to untreated cells. Data are the mean of three biological replicates. h, Western blot analysis of total PD-L1 levels in MDA-MB-231 cells after treatment with 200 nM of ATZ or ATZ-pLYTACs for 48 h. Figure 3. Clearance of soluble proteins by IGF-2R pLYTACs. a, Schema of soluble pLYTACs. b, Cellular uptake of LHDB-AF647 via LHDA-pLYTACs in Jurkat cells. Cells were incubated with 33nM LHDB-AF647 with / without 1uM LHDA-pLYTACs for 24h, washed twice with cold PBS and analyzed by flow cytometry. c, Remaining supernatant LHDB-AF647 levels in Jurkat cells. Jurkat cells were incubated with 100nM LHDB-AF647 with / without 500nM LHDA-pLYTACs. At timepoints 4h, 24h, 48h, the cells were pelleted down, and supernatant IgG levels were quantified by Neo2 plate reader. The percentage of IgG level was normalized with the IgG alone control group. d, Cellular uptake of IgG-AF647 via proteinG-pLYTACs in K562 cells. Cells were incubated with 33nM IgG-AF647 with / without 1uM proteinG-Tag3 for 24h, washed twice with cold PBS and analyzed by flow cytometry. The fold change in MFI (mean fluorescence intensity) was calculated by normalizing the IgG-AF647 alone group. e, Remaining supernatant IgG-AF647 levels in Jurkat cells. Jurkat cells were incubated with 33nM IgG-AF647 with / without 1uM proteinG- pLYTACs. At timepoints 4h, 24h, 48h, the cells were pelleted down, and supernatant IgG- AF647 levels were quantified by Neo2 plate reader. The percentage of IgG-AF647 level was normalized with the IgG-AF647 alone control group. f, Confocal imaging of lysosome co- localization of IgG-AF647 with lysosome in Hela cells. g, Confocal imaging of lysosome co- localization of IgG-AF647 with lysosome in Hela (IGF-2R KO) cells. For f,g, the cells were incubated with 200nM IgG-AF647 and 1uM of proteinG-Tag3 for 24h, washed and stained with LysotrackerTM. Figure 4. Enhanced signaling of de novo Sniper system. a, Schema of Signaling enhancing application of IGF-2R Tag. The SNIPR is a de novo designed SNIPR system including: 1) an extracellular LHDB protein that recognizes the LHDA ligand; 2) A cleavable membrane proximal domain; 3) an intracellular domain that releases transcription factor to activate the circuit to express blue fluorescent protein (BFP). Upon ligand binding of LHDB to LHDA SNIPR, the IGF-2R Tag will trigger the endocytosis of the complex. With decreased pH, the intracellular domain is cleaved and the released transcription factor is able to prompt the expression of BFP. Thus, the signaling activation level is quantified by the fluorescence intensity of BFP. b, Dose response curve of signaling activation of LHDA fusion with IGF-2R Tags. In Jurkat T-cells expressing LHDB SNIPR, the cells were incubated with IGF-2R Tags at titrated concentration. The corresponding mean fluorescence intensity (MFI) of BFP was recorded. The data was collected with replicates N=3. The error bar represents Standard Error of Mean (SEM). c, Live confocal imaging of lysosome co- localization with the Tag ligands and SNIPR receptors at 24 hours. d, Images were acquired at timepoints [0.25 hours, 3 hours, 6 hours, and 24 hours] for the experiment in (c) and analyzed for colocalization between the labeled Tag ligand and LysotrackerTMsignal. (N = 4 images per time point with at least 10 cells per image; + / - SEM) Figure 5. Target residue selection and Design model for IGF-2R minibinders. a, Selected target region for IGF-2R D6. b, Design model for IGF-2R D6mb. c, Selected target region for IGF-2R D11. d, Design model IGF-2R D11mb Figure 6. Binding affinity measurement for IGF-2R minibinders. a, BLI binding affinity measurement for D6mb against IGF-2R D6. b, BLI binding affinity measurement for D11mb against IGF-2R D11. c, BLI binding affinity measurement for Tag2 against IGF-2R D6 (left) and D11 (right). d, BLI binding affinity measurement for Tag3 against IGF-2R D6 (left) and D11 (right). e, BLI binding affinity measurement for Tag4 against IGF-2R D6 (left) and D11 (right). Figure 7. SSM validation for D6mb. a, Full SSM heatmap of D6mb. b, SSM heatmap for critical core and interface residues. The SC50 for each mutation collected from yeast display screening was estimated and normalized to the original D6mb sequence. Figure 8. SSM validation for D11mb. a, Full SSM heatmap of D11mb. b, SSM heatmap for critical core and interface residues. The SC50 for each mutation collected from yeast display screening was estimated and normalized to the original D6mb sequence. Figure 9. Computational design strategy to make rigid IGF-2R MDRE Figure 10. Cellular uptake evaluation of Linker length and homodimer fusion comparison for Tag1. a, Cellular uptake comparison between homodimer and heterodimer fusion of D6mb and D11mb. b, Cellular uptake comparison of linker length of D11mb-D6mb fusion with various lengths of GS linkers in the middle. For a and b, 200nM biotinylated fusion proteins were conjugated with 50 nM of Strapavidin-AF647 and incubated with Jurkat cells for 24h. After wash twice with cold PBS, the cellular uptake was measured by flow cytometry. Figure 11. Cellular uptake evaluation across cell lines. Biotinylated fusion proteins were conjugated with AlexaTM647-Strapavidin and incubated with different cell lines for 24h. After washing twice with cold PBS, the cellular uptake was measured by flow cytometry. The MFI for each group was calculated by normalizing to the group mean for the control group. Figure 12. Lysosome co-localization imaging. a, Fluorescence Microscopy imaging of IGF-2 co-localized with lysosomal. b, Fluorescence Microscopy imaging of Tag2 co- localized with lysosomal. c, Fluorescence Microscopy imaging of Tag4 co-localized with lysosomal. For a,b,c, HeLa cells were incubated with 100nM of biotinylated IGF-2, Tag2 or Tag4 for various time length. After cells were washed twice and fixed, they were stained with anti-LAMP2A antibody followed by goat secondary anti-IgG AlexaTMFluo 488 antibody and DAPI. Epifluorescence imaging was conducted on a Yokogawa CSU-X1 microscope. Figure 13. LHD clearance. a, LHDB-AF647 cellular uptake ability comparison between flexible and rigid designs in Jurkat cells. b, LHDB-AF647 cellular uptake ability comparison between flexible and rigid designs in K562 cells. c, Quantitative clearance of LHDB-AF647 in cell media with titrated LHDA-Tag3. For a,b, cells were incubated with 100nM LHDB-biotin + 33nM Strapavidin-AF647 with / without 500nM LHDB-pLYTACs for 48h, washed twice with cold PBS and analyzed by flow cytometry. The fold change in MFI (mean fluorescence intensity) was calculated by normalizing the LHDB-AF647 alone group. For c, cells were incubated with 100nM LHDB-AF647 with / without multiple concentrations of LHDA-Tag3. At timepoints 24h, 48h, the cells were pelleted down, and supernatant LHDB-AF647 levels were quantified by Neo2 plate reader. The percentage of LHDB-AF647 level was normalized with the LHDB-AF647 alone control group. Data shown above are collected with biological replicates with N=3. Figure 14. IgG clearance in Jurkat cells. a, IgG-AF647 cellular uptake ability comparison between flexible and rigid designs. b, Quantitative clearance of IgG-AF647 in cell media comparison between flexible and rigid designs. c, IgG-AF647 cellular uptake ability comparison between pLYTACs and M6Pn. d, Quantitative clearance of IgG-AF647 in cell media comparison between pLYTACs and M6Pn. e, Quantitative clearance of IgG- AF647 in cell media with titrated proteinG-Tag3. f, Quantitative clearance of IgG-AF647 in cell media with titrated proteinG-Tag4. For a,c, cells were incubated with 33nM IgG-AF647 with / without 1uM proteinG-pLYTACs for 24h, washed twice with cold PBS and analyzed by flow cytometry. The fold change in MFI (mean fluorescence intensity) was calculated by normalizing the IgG-AF647 alone group. For b,d,e,f, cells were incubated with 33nM IgG- AF647 with / without protein G-pLYTACs. At timepoints 4h, 24h, 48h, the cells were pelleted down, and supernatant IgG-AF647 levels were quantified by Neo2 plate reader. The percentage of IgG-AF647 level was normalized with the IgG-AF647 alone control group. Data shown are collected with biological replicates with N=3. Figure 15. Receptor degradation. a, Levels of EGFR after treatment with 10 nM CTX-M6P or titrated EGFRn-Tag1 in H1975 cells for 48 h. b, Surface EGFR level after treatment of EGFRn or EGFRn-Tag1 in Hela cells for 48h. N=3. c, Levels of EGFR after treatment with 10 nM or 100nM CTX-M6P or CTX-Tag1 in H1975 cells for 48 h. d, Levels of EGFR after treatment with 100nM EGFRn or EGFRn-pLYTACs in H1975 cells for 48 h. e, Levels of CTLA4 with 200nM of CTLA4mb or CTLA4mb-Tag1 in Jurkat-CTLA4 cells for 3h. Figure 16. Proteomic analysis. a, Fold change in abundance of EGFR with treatment of EGFRn compared with control. b, Fold change in abundance of EGFR with treatment of EGFRn-Tag1 compared with control. c, Fold change in abundance of EGFR with treatment of CTX compared with control. d, Fold change in abundance of EGFR with treatment of CTX-Tag1 compared with control. For a,b,c,d, the proteomic data was collected in H1975 cells with the treatment 100nM of corresponding reagents for 48h. All data was replicated with N=2. Figure 17. SSM validation for ASmb1. a, Full SSM heatmap of ASmb1. b, SSM heatmap for critical core and interface residues. The SC50 for each mutation collected from yeast display screening was estimated and normalized to the original D6mb sequence. Detailed Description All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol.185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R.I. Freshney.1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp.109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), Dang, B. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat. Methods 16, 319–322 (2019), and the Ambion 1998 Catalog (Ambion, Austin, TX). As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Any N-terminal methionine residue in any polypeptide of the disclosure may be present or may be deleted. In all embodiments of the polypeptides disclosed herein, 1, 2, 3, 4, or 5 residues may be deleted from the N-terminus and / or the C-terminus of the polypeptide while retaining activity. All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise. Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. In one aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1, wherein the polypeptide binds to asialoglycoprotein receptor (ASGPR). The polypeptides of this aspect of the invention can be used, for example, to treat lysosomal dysfunction by delivering therapeutic reagents to the lysosome; or to treat neurodegenerative diseases (including but not limited to Parkinson's disease. Alzheimer's disease. Huntington's disease), by degrading amyloid fibers; or to fuse to other proteins for targeted degradation of the fused protein, including but not limited to treating cancer by degrading proliferating-control receptors such as EGFR / HER2, immune checkpoints such as PD-L1 / CTLA4, or by degrading growth proliferating protein directly such as multiple growth factors; or treating autoimmune disease by degrading autoantibodies, cytokines or chemokines, or corresponding receptors. Table 1 The sequence of ASPGR to which the polypeptides of the disclosure bind is Shown in SEQ ID NO:100. MTKEYQDLQHLDNEESDHHQLRKGPPPPQPLLQRLCSGPRLLLLSLGLSLLLLVVVCVIGSQNSQLQEELRGLRE TFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSE RTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVD GTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL (Human ASGPR; SEQ ID NO:100) The binding interface for ASGPR in the polypeptide includes residues 158, 167, 171, 173, 181, 184, 185, 275 of SEQ ID NO:100. In one embodiment, permissible amino acid substitutions relative to SEQ ID NO:1 are listed in column 2 of Table 2 or 3. In another embodiment, permissible amino acid substitutions relative to SEQ ID NO:1 are listed in column 2 of Table 3. These permissible substitutions are based on site saturation mutagenesis studies, carried out by yeast surface display followed by next-generation sequencing to identify the single mutations that enhance binding. In another embodiment, no substitutions are permitted relative to SEQ ID NO:1 at residues 1, 16, 30, 39, 46-48, 50, and 53-54. Tables 2 and 3 list interface residues, present at the interface between the designed polypeptide and ASPGR. In one embodiment, only conservative substitutions, or no substitutions are permitted relative to SEQ ID NO:1 at interface residues identified in Table 2 or 3. Tables 2 and 3 list core residues, present in the core of the designed polypeptide. In one embodiment, only conservative substitutions, or no substitutions are permitted relative to SEQ ID NO:1 at core residues identified in Table 2 or 3. Table 2

[0002] Table 3

[0003] In other embodiments, the polypeptides of this aspect comprise an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1-10, wherein the polypeptide binds to asialoglycoprotein receptor (ASGPR). Table 4. Sequences of ASGPR binders In another embodiment, the disclosure provides fusion polypeptides comprising 2, 3, 4, 5, or more copies of the ASPGR-binding polypeptide of any embodiment disclosed above. The copies may be identical, or may be different. In some embodiments, an amino acid linker separates each copy of the polypeptide. In one such embodiment, the amino acid linker separating each copy of the first polypeptide comprises a GlySer-rich linker (e.g., GS / GGSx, wherein x is 1-20, or 1-15, or 1-10, or 1-5), or may comprise, but are not limited to (EAAAK)n (SEQ ID NO: 98) or (Gly)n where n is 1-20, 1-15, 1-10, 1-5, etc.; PAPA, or other chemical conjugation. In another embodiment, the fusion polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:11 or 12, wherein the fusion protein binds to asialoglycoprotein receptor (ASGPR). These fusion polypeptides trigger improved endocytosis and binding to ASGPR. >ASmb1-GSGGGGS-ASmb1 AAEEAIRRLKELNTPEAEDLIDFIETLLEKGVPEEEALRQAAALARRWGEPEAARVIEEALKSL GSGGGGSAAEEAIRRLKELNTPEAEDLIDFIETLLEKGVPEEEALRQAAALARRWGEPEAARV IEEALKSL (SEQ ID NO:11) >ASmb1-GSGGGGS-ASmb1-GSGGGGS-ASmb1 AAEEAIRRLKELNTPEAEDLIDFIETLLEKGVPEEEALRQAAALARRWGEPEAARVIEEALKSL GSGGGGSAAEEAIRRLKELNTPEAEDLIDFIETLLEKGVPEEEALRQAAALARRWGEPEAARV IEEALKSLGSGGGGSAAEEAIRRLKELNTPEAEDLIDFIETLLEKGVPEEEALRQAAALARRW GEPEAARVIEEALKSL (SEQ ID NO:12) In a second aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13-15 and 106, wherein the polypeptide binds to insulin-like growth factor 2 receptor (IGF-2R). The polypeptides of this aspect can be used, for example, in the methods described herein, or to fuse to a protein of interest for targeted degradation of the fused protein, as disclosed in the examples that follow. The amino acid sequences of SEQ ID NO:13-15 and 106 are provided in Table 5. Table 5 The sequence of IGF-2R to which the polypeptides of the disclosure bind is shown in SEQ ID NO:101. MGAAAGRSPHLGPAPARRPQRSLLLLQLLLLVAAPGSTQAQAAPFPELCSYTWEAVDTKNNVLYKINICGSVDIV QCGPSSAVCMHDLKTRTYHSVGDSVLRSATRSLLEFNTTVSCDQQGTNHRVQSSIAFLCGKTLGTPEFVTATECV HYFEWRTTAACKKDIFKANKEVPCYVFDEELRKHDLNPLIKLSGAYLVDDSDPDTSLFINVCRDIDTLRDPGSQL RACPPGTAACLVRGHQAFDVGQPRDGLKLVRKDRLVLSYVREEAGKLDFCDGHSPAVTITFVCPSERREGTIPKL TAKSNCRYEIEWITEYACHRDYLESKTCSLSGEQQDVSIDLTPLAQSGGSSYISDGKEYLFYLNVCGETEIQFCN KKQAAVCQVKKSDTSQVKAAGRYHNQTLRYSDGDLTLIYFGGDECSSGFQRMSVINFECNKTAGNDGKGTPVFTG EVDCTYFFTWDTEYACVKEKEDLLCGATDGKKRYDLSALVRHAEPEQNWEAVDGSQTETEKKHFFINICHRVLQE GKARGCPEDAAVCAVDKNGSKNLGKFISSPMKEKGNIQLSYSDGDDCGHGKKIKTNITLVCKPGDLESAPVLRTS GEGGCFYEFEWHTAAACVLSKTEGENCTVFDSQAGFSFDLSPLTKKNGAYKVETKKYDFYINVCGPVSVSPCQPD SGACQVAKSDEKTWNLGLSNAKLSYYDGMIQLNYRGGTPYNNERHTPRATLITFLCDRDAGVGFPEYQEEDNSTY NFRWYTSYACPEEPLECVVTDPSTLEQYDLSSLAKSEGGLGGNWYAMDNSGEHVTWRKYYINVCRPLNPVPGCNR YASACQMKYEKDQGSFTEVVSISNLGMAKTGPVVEDSGSLLLEYVNGSACTTSDGRQTTYTTRIHLVCSRGRLNS HPIFSLNWECVVSFLWNTEAACPIQTTTDTDQACSIRDPNSGFVFNLNPLNSSQGYNVSGIGKIFMFNVCGTMPV CGTILGKPASGCEAETQTEELKNWKPARPVGIEKSLQLSTEGFITLTYKGPLSAKGTADAFIVRFVCNDDVYSGP LKFLHQDIDSGQGIRNTYFEFETALACVPSPVDCQVTDLAGNEYDLTGLSTVRKPWTAVDTSVDGRKRTFYLSVC NPLPYIPGCQGSAVGSCLVSEGNSWNLGVVQMSPQAAANGSLSIMYVNGDKCGNQRFSTRITFECAQISGSPAFQ LQDGCEYVFIWRTVEACPVVRVEGDNCEVKDPRHGNLYDLKPLGLNDTIVSAGEYTYYFRVCGKLSSDVCPTSDK SKVVSSCQEKREPQGFHKVAGLLTQKLTYENGLLKMNFTGGDTCHKVYQRSTAIFFYCDRGTQRPVFLKETSDCS YLFEWRTQYACPPFDLTECSFKDGAGNSFDLSSLSRYSDNWEAITGTGDPEHYLINVCKSLAPQAGTEPCPPEAA ACLLGGSKPVNLGRVRDGPQWRDGIIVLKYVDGDLCPDGIRKKSTTIRFTCSESQVNSRPMFISAVEDCEYTFAW PTATACPMKSNEHDDCQVTNPSTGHLFDLSSLSGRAGFTAAYSEKGLVYMSICGENENCPPGVGACFGQTRISVG KANKRLRYVDQVLQLVYKDGSPCPSKSGLSYKSVISFVCRPEARPTNRPMLISLDKQTCTLFFSWHTPLACEQAT ECSVRNGSSIVDLSPLIHRTGGYEAYDESEDDASDTNPDFYINICQPLNPMHGVPCPAGAAVCKVPIDGPPIDIG RVAGPPILNPIANEIYLNFESSTPCLADKHFNYTSLIAFHCKRGVSMGTPKLLRTSECDFVFEWETPVVCPDEVR MDGCTLTDEQLLYSFNLSSLSTSTFKVTRDSRTYSVGVCTFAVGPEQGGCKDGGVCLLSGTKGASFGRLQSMKLD YRHQDEAVVLSYVNGDRCPPETDDGVPCVFPFIFNGKSYEECIIESRAKLWCSTTADYDRDHEWGFCRHSNSYRT SSIIFKCDEDEDIGRPQVFSEVRGCDVTFEWKTKVVCPPKKLECKFVQKHKTYDLRLLSSLTGSWSLVHNGVSYY INLCQKIYKGPLGCSERASICRRTTTGDVQVLGLVHTQKLGVIGDKVVVTYSKGYPCGGNKTASSVIELTCTKTV GRPAFKRFDIDSCTYYFSWDSRAACAVKPQEVQMVNGTITNPINGKSFSLGDIYFKLFRASGDMRTNGDNYLYEI QLSSITSSRNPACSGANICQVKPNDQHFSRKVGTSDKTKYYLQDGDLDVVFASSSKCGKDKTKSVSSTIFFHCDP LVEDGIPEFSHETADCQYLFSWYTSAVCPLGVGFDSENPGDDGQMHKGLSERSQAVGAVLSLLLVALTCCLLALL LYKKERRETVISKLTTCCRRSSNVSYKYSKVNKEEETDENETEWLMEEIQLPPPRQGKEGQENGHITTKSVKALS SLHGDDQDSEDEVLTIPEVKVHSGRGAGAESSHPVRNAQSNALQEREDDRVGLVRGEKARKGKSSSAQQKTVSST KLVSFHDDSDEDLLHI (Human IGF-2R; SEQ ID NO:101). In some embodiments, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13, wherein the polypeptide binds to IGF-2R domain 6. The binding interface for IGF-2R domain 6 (Residues 783L-917N of SEQ ID NO:101) in the polypeptides of the second aspect of present disclosure includes residues 866, 888 ,890, 892, 903, 905, 906, 911, 913, and 915. In one embodiment, permissible amino acid substitutions relative to SEQ ID NO:13 are listed in column 2 of Table 6 or 7. In another embodiment, permissible amino acid substitutions relative to SEQ ID NO:13 are listed in column 2 of Table 7. These permissible substitutions are based on site saturation mutagenesis studies, carried out by yeast surface display followed by next-generation sequencing to identify the single mutations that enhance binding. In another embodiment, no substitutions are permitted relative to SEQ ID NO:13 at residues 15, 23-24, 35, and 57. Tables 6 and 7 list interface residues, present at the interface between the designed polypeptide and IGF-2R domain 6. In one embodiment, only conservative substitutions, or no substitutions are permitted relative to SEQ ID NO:13 at interface residues identified in Table 6 or 7. Tables 6 and 7 list core residues, present in the core of the designed polypeptide.. In one embodiment, only conservative substitutions, or no substitutions are permitted relative to SEQ ID NO:13 at core residues identified in Table 6 or 7. Table 6

[0004] Table 7

[0005] In some embodiments, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14, wherein the polypeptide binds to IGF-2R domain 11. The binding interface on IGF-2R domain 11 (Residues 1503A-1647E of SEQ ID NO:101) for the polypeptides of the present disclosure includes residues 1542, 1567, 1570, 1572, 1599, 1606, 1629, and 1636. In one embodiment, permissible amino acid substitutions relative to SEQ ID NO:14 are listed in column 2 of Table 8 or 9. In another embodiment, permissible amino acid substitutions relative to SEQ ID NO:14 are listed in column 2 of Table 9. These permissible substitutions are based on site saturation mutagenesis studies, carried out by yeast surface display followed by next-generation sequencing to identify the single mutations that enhance binding. In another embodiment, no substitutions are permitted relative to SEQ ID NO:14 at residues 4, 8, 11, 13, 25, 31, 43, and 47. Tables 8 and 9 list interface residues, present at the interface between the designed polypeptide and IGF-2R domain 11. In one embodiment, only conservative substitutions, or no substitutions are permitted relative to SEQ ID NO:14 at interface residues identified in Table 8 or 9. Tables 8 and 9 list core residues, present in the core of the designed polypeptide.. In one embodiment, only conservative substitutions, or no substitutions are permitted relative to SEQ ID NO:14 at core residues identified in Table 8 or 9. Table 8

[0006] Table 9

[0007] In some embodiments, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:15, wherein the polypeptide binds to IGF-2R domain 11. The binding interface on IGF-2R domain 11 (Residues 1503A-1647E of SEQ ID NO:101) for the polypeptides of the present disclosure includes residues 1542, 1567, 1570, 1572, 1599, 1606, 1629, and 1636. In one embodiment, permissible amino acid substitutions relative to SEQ ID NO:15 are listed in column 2 of Table 10 or 11. In another embodiment, permissible amino acid substitutions relative to SEQ ID NO:15 are listed in column 2 of Table 11. These permissible substitutions are based on site saturation mutagenesis studies, carried out by yeast surface display followed by next-generation sequencing to identify the single mutations that enhance binding. In another embodiment, no substitutions are permitted relative to SEQ ID NO:15 at residues 21, 32, and 36. Tables 10 and 11 list interface residues, present at the interface between the designed polypeptide and IGF-2R domain 11. In one embodiment, only conservative substitutions, or no substitutions are permitted relative to SEQ ID NO:15 at interface residues identified in Table 10 or 11. Tables 10 and 11 list core residues, present in the core of the designed polypeptide.. In one embodiment, only conservative substitutions, or no substitutions are permitted relative to SEQ ID NO:15 at core residues identified in Table 10 or 11. Table 10

[0008] Table 11 In some embodiments of this second aspect, the disclosure provides fusion proteins comprising (a) a first polypeptide according to any embodiment disclosed above for polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14, wherein the polypeptide binds to IGF-2R domain 11; and (b) a second polypeptide according to any embodiment disclosed above for polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14, wherein the polypeptide binds to IGF-2R domain 11. These fusion proteins comprise binders against two different domains on IGF-2R domain 11, and thus provide improved endocytosis and lysosome targeting ability. In some embodiments, amino acid linker separates the first polypeptide and the second polypeptide. Any amino acid linkers can be used as appropriate for an intended use. In some embodiments, the amino acid linker separating the first polypeptide and the second polypeptide comprises a GlySer-rich linker (e.g., GS / GGSx, wherein x is 1-20, or 1-15, or 1- 10, or 1-5), or may comprise, but are not limited to (EAAAK)n (SEQ ID NO: 98) or (Gly)n where n is 1-20, 1-15, 1-10, 1-5, etc.; PAPA, or other chemical conjugation. In other embodiments, the fusion polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:16 or 17, wherein the fusion protein binds to IGF-2R. >D11mb-GGS-D6mb (IGF_Tag1) MVDARLLLLIWEAKELLERGNPEEARKVLEEAREIAERNNNEELLKELEVLLEKLEGGSTERRVIQVL EQILEDEDPEVVEKMLEILLEILEEAGDPARKLVEEILRVVRKNLEEARELVRRLS (SEQ ID NO:16) . >D11mb2-GGS-D6mb (IGF_Tag2) MVEASLWLLIWDAGELVERGNPEEARKVLEEAREIAERNNREEFLKELEVLLEKLEGGSTERRVIQVL EQILEDEDPEVVEKMLEILLEILEEAGDPARKLVEEILRVVRKNLEEARELVRRLS (SEQ ID NO:17) In a third aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18 wherein the polypeptide binds to IGF-2R. M(E / K)EAQRLL(L / E)EWE-X1-QI(L / I)QDPD(L / P)(D / E)V(R / K)(R / K) ML(D / E)IL(K / E)RIY(E / R)E(A / K)G, wherein X1 consists of 20 or 21 amino acids (SEQ ID NO:18) These IGF-2R binders are rigid and simplified fusion of the above IGF-2R binders, can achieve slightly better endocytosis / internalization functions, and have smaller size for manufacturing purposes. In various embodiments, X1 consists of an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:19-22. FQERVDELARKYGERVKEVGR (SEQ ID NO:19) AQEWADSQGEDAKRVKQVVQ (SEQ ID NO:20) AREWAKSQGEDAKRVLEVMK (SEQ ID NO:21) AREWAESQGEDSKRVKQVVE (SEQ ID NO:22) These IGF_Tags are designed to interact with IGF2R D6 and D11, respectively, using two separate binding regions. The different X1 domains differ in the angle connecting the two binding regions in the Tag that bind to D6 and D11 respectively, to control the relative orientation of D11 and D6 upon Tag binding to IGF2R. These structural differences result in different activity. In some embodiments, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23-26, wherein residues in parentheses are optional and may be present or deleted. >IGF_Tag3 (Rigid1) MEEAQRLLEEWEFQERVDELARKYGERVKEVGRQIIQDPDPEVRRKMLDILERIYREA(G) (SEQ ID NO:23) >IGF_Tag4 (Rigid4) MEEAQRLLLEWEAQEWADSQGEDAKRVKQVVQQILQDPDLEVQKKMLEILKRIYEEK(G) (SEQ ID NO:24) >IGF_Tag5 (Rigid2) MKEAQRLLLEWEAREWAKSQGEDAKRVLEVMKQILQDPDLDVQRKMLEILKRIYEEKG (SEQ ID NO:25) >IGF_Tag6 (Rigid3) MEEAQRLLLEWEAREWAESQGEDSKRVKQVVEQILQDPDLEVQRKMLEILKRIYEEKG (SEQ ID NO:26) In a fourth aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27 wherein the polypeptide binds to IGF-2R. The polypeptides of this aspect are variants of polypeptides of the third aspect, with structural differences result in increased internalization speed and amount. MDEARRLLLEWECRERVESLAKTEEERKRMLQVLSQILEDPDVEVVEKMVDILCRIYREAG (SEQ ID NO:27) In some embodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or all 9 mutations relative to SEQ ID NO:27 selected from the group consisting of 13A, 17I, 18V, 19A, 27K, 35E, 46 R / Q, 54V, and 58K. The mutations were identified by single mutant screening using yeast surface display. Single or combinations of these mutations result in improved binding affinity to IGF-2R. In other embodiments, the polypeptides comprise an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27-30 wherein the polypeptide binds to IGF-2R MDEAKRLLFEWEARERIESLAKTEEEKKRMLQVLSQILEDPDVEVREKMVDILVRIYKEAG (SEQ ID NO:28) >IGF_Tag8 (Rigid6) MEEAKRLLFEWEARERVVSLAKTEEERKRMLQVLSQILEDPDVEVREKMVDILCRIYREAG (SEQ ID NO:29) >IGF_Tag9 (Rigid7) MEEAKRLLFEWEARERVEALAKTEEERKRMLQVLEQILEDPDVEVQRKMVDILCRIYREAG (SEQ ID NO:30) >IGF_Tag10 (Rigid8) MDEARRLLLEWECRERVESLAKTEEERKRMLQVLSQILEDPDVEVVEKMVDILCRIYREAG (SEQ ID NO:27) In various further embodiments, the polypeptides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 mutations relative to the reference sequence selected from the group consisting of 2E, 5K, 9F, 13A, 17I, 18V, 19A, 27K, 35E, 46R / Q, 54V, and 56K. The mutations were identified by single mutant screening using yeast surface display. Single or combinations of these mutations result in improved binding affinity to IGF-2R. In one embodiment of each of the above aspects, amino acid substitutions relative to the reference polypeptide or fusion polypeptide are conservative amino acid substitutions. As used herein, “conservative amino acid substitution” means a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity is retained. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into H is; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Val, into Ile or into Leu. In one embodiment of all aspects, the disclosure provides fusion proteins comprising: (a) the polypeptide or fusion polypeptide of any preceding embodiment or combination of embodiments herein; and (b) a protein that binds to a degradation target. As discussed in the examples that follow, the polypeptides and fusion polypeptides of the disclosure can be linked to any protein that binds to a target to degrade, for administration to a cell, tissue, tumor, etc. that expresses the corresponding receptors that the polypeptides activate (either IGF-2R or ASGPR). In another aspect, the disclosure provides fusion proteins comprising: (a) a protein that binds to a degradation target; and (b) a transferrin receptor binding polypeptide selected from the group consisting of: (i) a polypeptide comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence selected from SEQ ID NO:32-37, wherein residues in parentheses are optional and may be present or absent; and (ii) a polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, or 98%, identical to the amino acid sequence of SEQ ID NO:31, wherein (A) the polypeptide is identical to SEQ ID NO:31 at residues corresponding to residues 20, 23, 24, 27, 28, 31, 32, 34, 35, 36, 37, 74, 77, 78, 81, 82, 84, 85, and 88 in SEQ ID NO:31; and (B) the polypeptide is substituted relative to SEQ ID NO:31 as shown in Table 12 at least at 5, 6, 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more residues relative to SEQ ID NO:31. >2DS25.5 DEEEIQKAIEELLRKGVSEEEAAIIIVQRFNVAVVVVVQDERQGKHISEYIRRYIPEADVILFANLVV IKVETHELSTRVWEAAQKAY (SEQ ID NO:31; 88 amino acids) >combo3_5_mpnn0 (NAEEI)KKFIKEKLEEGYSKEETAILVVQKFNVSVVVVVKNEEEGKRIAEYIKKHVPEAEVILYENL IVVKVDSHEESTKVWEAAQEAY (SEQ ID NO:32) > combo3_5_mpnn3 (SEEEI)KKFIKELLEQGISKEEAAILVVQKFNVSVVVVTENEEEAERIAEYIKKNVPSAKVIVHENL IVVKVESHEESTKVWEAAQEAY (EQ ID NO:33) > combo3_5_mpnn5 (DVEEI)KKFIKELLEQGYSKEETAIKVVQKFNVSVVVVTDDEEKAKEIAEYIKKNVPSATVVVYENL IVAKVESHEESTKVWELAQKAY (SEQ ID NO:34) >combo3_5_mpnn0 KKFIKEKLEEGYSKEETAILVVQKFNVSVVVVVKNEEEGKRIAEYIKKHVPEAEVILYENLIVVKVDS HEESTKVWEAAQEAY (SEQ ID NO:35) > combo3_5_mpnn3 KKFIKELLEQGISKEEAAILVVQKFNVSVVVVTENEEEAERIAEYIKKNVPSAKVIVHENLIVVKVES HEESTKVWEAAQEAY (SEQ ID NO:36) > combo3_5_mpnn5 KKFIKELLEQGYSKEETAIKVVQKFNVSVVVVTDDEEKAKEIAEYIKKNVPSATVVVYENLIVAKVES HEESTKVWELAQKAY (SEQ ID NO:37) The polypeptide of SEQ ID NO:31 (2DS25.5) is a previously described transferrin receptor binding protein (see PCT application publication WO / 2022 / 026555, incorporated by reference herein in its entirety). The polypeptides of SEQ ID NO:32-37 are modified transferrin receptor binding polypeptides. The polypeptides disclosed herein have substantially improved expression levels and solubility, and can thus be more efficiently coupled to therapeutic cargos for blood brain barrier (BBB) traversal. Residues 20, 23, 24, 27, 28, 31, 32, 34, 35, 36, 37, 74, 77, 78, 81, 82, 84, 85, and 88 in SEQ ID NO:31 are in the polypeptide-transferrin receptor protein binding interface, and were retained in polypeptides of the present disclosure. Table 12 shows the location of substitutions relative to 2DS25.5 residues in exemplary polypeptides of the disclosure, and does not show the residues that are maintained relative to SEQ ID NO:31. There are 3 columns in Table 12 of substitutions in an exemplary polypeptide of the disclosure relative to SEQ ID NO:31 (combo35_M0; combo35_M3; and combo35_M5). The amino acid position number relative to SEQ ID NO:31 is in the first column, and permissible substitutions at a given residue can be identified by looking across a given row in columns combo35_M0; combo35_M3; and combo35_M5. For example, amino acid position 1 in SEQ ID NO:31 is D, and it can be substituted with N or S. Those of skill in the art will understand other permissible substitutions based on Table 12. Table 12: Redesigned residues (“-“ indicates that residue is the same as in original):

[0009] ^ In some embodiments, the transferrin receptor binding polypeptide is identical to SEQ ID NO:31 at residues corresponding to residues 7, 9, 11, 13, 16, 18, 20-21, 23-24, 27-28, 30- 32, 34-37, 41, 47, 49-51, 56, 58, 60, 65-66, 68, 70-71, 74-75, 77-78, 80-82, 84-85, and 87-88; and optionally identical to SEQ ID NO:31 at residues corresponding to residues 3-5 in SEQ ID NO:31. In other embodiments, the transferrin receptor binding polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 substitutions relative to SEQ ID NO:31: Q6K, A8F, E10K, R14E, E19K, R29K, A33S, R42E, S48A, R52K, R53K, A64E, L76E, and / or R79K. In all of these fusion protein embodiments, any protein that binds to a degradation target may be used as suitable for an intended purpose. In non-limiting embodiments, the protein that binds to a degradation target may include cell surface proteins (for example, Cancer markers such as EGFR / HER2 / PD-L1, etc.); soluble proteins involved in disease (for example, autoantibodies, cytokines, amyloid fibers, albumin, neurotoxins / venom proteins, pathogens / viral proteins, etc.); proteins that have therapeutic effects in lysosomes (for example, for use in lysosomal dysfunction diseases), including but not limited to : glucosylceramide (in Gaucher disease, the native lysosome enzyme ȕ-GCase is defective, leading to accumulation of glucosylceramide), Į-L-iduronosidic (see nature.com article s41573-019-0036-1, table 1); or any other protein that plays a functional role in cells (the polypeptide will serve as a delivery reagent to transport the protein from membrane to inner side of the cell). In all embodiments, the protein that binds to a degradation target may be separated from the targeting polypeptide by an amino acid linker. Any amino acid linker may be used as suitable for an intended purpose. In some embodiments the amino acid linker comprises a GlySer-rich linker (e.g., GS / GGSx, wherein x is 1-20, or 1-15, or 1-10, or 1-5), or may comprise, but are not limited to (EAAAK)n (SEQ ID NO: 98) or (Gly)n where n is 1-20, 1- 15, 1-10, 1-5, etc.; PAPA, or other chemical conjugation. In a further embodiment, the disclosure provides fusion proteins comprising (a) the polypeptide or fusion polypeptide of any embodiment or combination of embodiments herein; and (b) a second polypeptide that binds to a therapeutic target of interest. The fusion proteins of the disclosure can be used, for example, to degrade a therapeutic target of interest that is bound by the second polypeptide, when administered to a cell / tissue / etc. that expresses the corresponding receptors that the first polypeptide activates (either IGF-2R or ASGPR). In all embodiments, the polypeptide or fusion polypeptide may be separated from the second polypeptide by an amino acid linker. Any amino acid linker may be used as suitable for an intended purpose. In some embodiments the amino acid linker comprises a GlySer- rich linker (e.g., GS / GGSx, wherein x is 1-20, or 1-15, or 1-10, or 1-5), or may comprise, but are not limited to (EAAAK)n (SEQ ID NO: 98) or (Gly)n where n is 1-20, 1-15, 1-10, 1-5, etc.; PAPA, or other chemical conjugation. The fusion proteins of these embodiments may be used, for example, to degrade a therapeutic target of interest that is bound by the second polypeptide, when administered to a cell / tissue / etc. that expresses the corresponding receptors that the first polypeptide activates (either IGF-2R or ASGPR). In non-limiting embodiments, the second polypeptide(s) may include cell surface proteins (for example, Cancer markers such as EGFR / HER2 / PD-L1, etc.); soluble proteins involved in disease (for example, autoantibodies, cytokines, amyloid fibers, albumin, neurotoxins / venom proteins, pathogens / viral proteins, etc.); proteins that have therapeutic effects in lysosomes (for example, for use in lysosomal dysfunction diseases), including but not limited to : glucosylceramide (in Gaucher disease, the native lysosome enzyme ȕ-GCase is defective, leading to accumulation of glucosylceramide), Į-L- iduronosidic; antibodies, and any other protein that plays a functional role in cells (the polypeptide(s) will serve as a delivery reagent to transport the protein from membrane to inner side of the cell). The fusion proteins can be used, for example, to treat lysosomal dysfunction by delivering therapeutic reagents to the lysosome; or to treat neurodegenerative diseases (including but not limited to Parkinson's disease, Alzheimer's disease, and Huntington's disease), by degrading amyloid fibers; or to fuse to other proteins for targeted degradation of the fused protein, including but not limited to treating cancer by degrading proliferating- control receptors such as EGFR / HER2, immune checkpoints such as PD-L1 / CTLA4, or by degrading growth proliferating protein directly such as multiple growth factors; or treating autoimmune disease by degrading autoantibodies, cytokines or chemokines, or corresponding receptors. In some embodiments, the second polypeptide comprises a monoclonal antibody heavy chain and / or light chain, or an antigen binding fragment thereof. The fusion proteins may include any monoclonal antibody heavy or light chain, or antigen binding fragment thereof, that binds to a therapeutic target of interest. In non-limiting embodiments, the second polypeptide comprises an amino acid sequence at least 90%, 95%, or 100% identical to the amino acid sequence of selected from the group consisting of SEQ ID NO:38-65, or an antigen binding fragment thereof. These sequences are shown in Table 13, and are monoclonal antibody heavy chain or light chains. The first SEQ ID NO shown in the left hand column is for the heacy chain, and the second SEQ ID NO shown in column 1 is the sequence of the light chain. In further embodiments, the second polypeptide comprises an amino acid sequence at least 90%, 95%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:40-41 (atezolizumab heavy or light chain) and 62-63 (cetuximab heavy or light chain), or an antigen binding fragment thereof. Table 13. Monoclonal antibodies In a further embodiment, the second polypeptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:66-70. Table 14 In some embodiments, the first polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1, wherein the first polypeptide binds to asialoglycoprotein receptor (ASGPR). The fusion proteins of this embodiment can be used, for example, to degrade a therapeutic target of interest that is bound by the second polypeptide, when administered to a cell / tissue / etc. that expresses ASGPR receptors that the first polypeptide activates. In one embodiment, the first polypeptide and the second polypeptide are separated by a separating amino acid linker. In other embodiments, the separating amino acid linker comprises a GlySer-rich linker (e.g., GS / GGSx, wherein x is 1-20, or 1-15, or 1-10, or 1-5), or may comprise, but are not limited to (EAAAK)n (SEQ ID NO: 98) or (Gly)n where n is 1- 20, 1-15, 1-10, 1-5, etc.; PAPA, or other chemical conjugation. In some embodiments, the first polypeptide is N-terminal to the second polypeptide. In other embodiments, the first polypeptide is C-terminal to the second polypeptide. In a further embodiment, fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:71-97. Table 15. Fusions

[0010] In another embodiment, the disclosure provides antibodies, or antigen binding fragment thereof, comprising: (a) the fusion protein of any embodiment or combination of embodiments herein, wherein the second polypeptide comprises a monoclonal antibody heavy chain, or an antigen binding fragment thereof; and (b) a light chain for the monoclonal antibody, or an antigen binding fragment thereof. In a further embodiment, the disclosure provides antibodies, or antigen binding fragment thereof, comprising: (a) the fusion protein of any embodiment or combination of embodiments herein, wherein the second polypeptide comprises a monoclonal antibody light chain, or an antigen binding fragment thereof; and (b) a heavy chain for the monoclonal antibody, or an antigen binding fragment thereof. In these embodiments, the amino acid sequence of heavy and light chains of exemplary monoclonal antibodies are shown in Table 13. In a further embodiment, the disclosure provides antibodies, or antigen binding fragment thereof, comprising: (a) a first fusion protein according to any embodiment or combination of embodiments herein, wherein the second polypeptide comprises a monoclonal antibody heavy chain, or an antigen binding fragment thereof; and (b) a second fusion protein according to any embodiment or combination of embodiments herein, wherein the second polypeptide comprises a monoclonal antibody light chain, or an antigen binding fragment thereof. In this embodiment, the heavy and light chains are fusion proteins according to any embodiment disclosed herein, wherein the first polypeptides in each of the first fusion protein and the second fusion protein bind a target. In one embodiment, the disclosure the polypeptides, fusion proteins, antibodies, or antigen-binding fragments thereof, of any preceding embodiment herein further comprise a detectable label, including but not limited to a fluorescent, luminescent, or radioactive label, or a detectable polypeptide (including but not limited to a fluorescent or bioluminescent polypeptide). In another aspect the disclosure provides nucleic acids encoding the polypeptide, polypeptide fusion, or fusion protein of any embodiment or combination of embodiments of the disclosure. The nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and / or purification of the encoded peptide or chimeric molecular construct, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptide, polypeptide fusion, or fusion protein of the disclosure. In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence. “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector. In another aspect, the disclosure provides host cells that comprise the polypeptide, fusion protein, nucleic acid or expression vector (i.e.: episomal or chromosomally integrated) disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate co- precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. In one embodiment, the disclosure provides pharmaceutical compositions, comprising: (a) the polypeptide, fusion protein, nucleic acid, expression vector, and / or host cell of any embodiment or combination of embodiments; and (b) a pharmaceutically acceptable carrier. The compositions may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and / or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition includes a bulking agent, like glycine. In yet other embodiments, the composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate- 85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the composition additionally includes a stabilizer, e.g., a molecule which substantially prevents or reduces chemical and / or physical instability of the nanostructure, in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride. The polypeptide, fusion protein, nucleic acid, expression vector, and / or host cell may be the sole active agent in the composition, or the composition may further comprise one or more other agents suitable for an intended use. In another aspect, the disclosure provides methods for degrading a target protein, comprising contacting a biological sample comprising the target with the fusion protein of any embodiment or combination of embodiments herein, under conditions to promote binding of the fusion protein to target protein present in the biological sample, thereby promoting degradation of the target protein. Exemplary such uses are described in the examples that follow. In another aspect, the disclosure provides methods for detecting an ASGPR, transferrin receptor, or IGF-2R receptor, comprising contacting a biological sample comprising the receptor with a detectable fusion protein of any embodiment herein, under conditions to promote binding of the fusion protein to receptor present in the sample, and detecting signal from the detectable polypeptide, thus detecting the receptor in the biological sample. For example, the fusion proteins can be used as biosensor to detect certain cells expressing the protein, and further label the lysosomes within a cell. In another embodiment, the disclosure provides fusion proteins, comprising (a) the polypeptide or fusion polypeptide of any preceding embodiment or combination of embodiments herein; and (b) a therapeutic polypeptide. The therapeutic polypeptide may be any polypeptide suitable for use in treating a disorder of interest. In another embodiment, the disclosure provides methods for treating a disorder, comprising contacting a subject with the fusion protein of the disclosure comprising a therapeutic polypeptide, under conditions to promote binding of the fusion protein to ASGPR, transferrin receptor, or IGF-2R in cells of the subject, thus treating the disorder. In some embodiments, the disorder is selected from the group consisting of lysosomal dysfunction, neurodegenerative diseases (including but not limited to Parkinson's disease. Alzheimer's disease. Huntington's disease), cancer, and autoimmune disease. Therapeutic antibodies (including but not limited to the antibodies listed in the Table13) bind to receptors that are relevant to certain diseases. For example, fusion of an IGF-2R Tag with Guselkumab (binds to IL-23) can help clear IL-23 in the cells to more potently relieve inflammation in subjects in need thereof. In another embodiment, the disclosure provides methods for treating a disease, comprising administering to a subject in need thereof an amount effective of the fusion protein or antibody or antigen binding fragment thereof of embodiment in which the second polypeptide comprises a binder for a therapeutic target of interest, to degrade target protein present in the subject, thereby treating the disease. In a further aspect, the disclosure provides compositions, comprising: (a) the polypeptide or fusion polypeptide of any embodiment or combination of embodiments herein; and (b) a non-protein functional moiety linked to the polypeptide or fusion polypeptide. In some embodiments, the non-protein functional moiety comprises a nucleic acid, including but not limited to an inhibitory nucleic acid. Exemplary such inhibitory nucleic acids include siRNA, antisense oligonucleotide, small molecule cytotoxic drug such as MMAE; and DNA aptamers that binding affinity to intracellular targets or in the lysosome). As used herein, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s). The subject may be any subject that has a relevant disorder. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc. Examples Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Current approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (Tags) that overcome these challenges. We present Tags for the IGF-2R, ASGPR, and Transferrin receptors, and show that fusing these to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different Tags could enable targeting of degradation to different tissues. The modularity and genetic encodability of Tags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. We find further that Tag fusion can amplify the signaling Emax more than 100 fold through a synthetic receptor system which could have considerable utility for cell therapy. The tunability and modularity of genetically- encodable Tags contributes to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates. Introduction The endocytosis of many cell surface receptors is triggered by binding of their endogenous ligands which can shift the conformational or oligomerization state of the receptor4and induce receptor clustering and adaptor protein recruitment5,6. Native endocytosis inducing ligands have been utilized to enhance intracellular delivery and prompting extracellular protein degradation1–3. While powerful, usage of native ligands could trigger off-targeting signaling3,7and their binding sites are potentially occupied by existing ligands. Additionally, many endogenous ligands are also hard for manufacturing due to instability to maintain physiological activity8. We reasoned that de novo protein design could enable the creation of bio-orthogonal endocytosis inducing proteins that avoid the above limitations using strategies customized for the target receptor. For receptors such as the transferrin receptor, that constitutively traffic between the cell surface and the endosome / lysosome, binding to a site on the receptor non- overlapping with the native ligands could be sufficient. For receptors such as the IGF-2R receptor, for which conformational change triggers endocytosis, binding must induce rearrangement of receptor extracellular domains, while for others, such as ASGPR, whose endocytosis is stimulated by clustering, binding should induce oligomerization. Fusion of designed proteins with these properties to a second target binding protein could promote endocytosis and lysosomal trafficking of the target. We set out to design such endocytosis targeting proteins, which we call Tags, for all four receptor systems, and to explore their utility for modulating protein degradation and cellular signaling. To enable tissue specific control over endocytosis for downstream applications, we selected target receptors with distinct tissue expression profiles. IGF-2R is expressed in most tissues, ASGPR is expressed primarily in the liver, and TfR is abundant in brain, where TfR has broader expression in liver and muscles (Fig 1). We developed customized design approaches to optimize endocytosis through each of these receptor systems, as described in the following sections. Design of Tags orthogonal to endogenous ligands Tfr constitutively cycles between the cell surface and intracellular compartments. Thus, for these receptors, the challenge is not to actively induce endocytosis, but to bind to the receptor at a site that does not compete for the natural ligand, which could have undesired side effects and reduce efficiency. De novo protein design has the advantage of being able to specifically create binders to sites of interest on a target, and thus is well suited to designing13, 16, protein binders that target receptor sites that do not overlap with those of native ligands. We applied a similar orthogonal binding strategy with TfR, a well-characterized trafficking receptor with enriched ligand Tf, where native iron-bound Tf is able to rapidly activate the intracellular trafficking pathway of TfR to transport iron inside the cells or brain26. We previously reported the design of TfR binders with high binding affinity at a binding site distant from transferrin Tf recognition spots17; here we explore the use of this binder as an Tag (TfR-Tag). We found the TfR-Tag was directed endocytosis in U-251MG glioblastoma cells and Jurkat cells [data pending]. These results with Tfr indicate that binding to bio-orthogonal sites with single high affinity binding domains is an effective strategy for hijacking constitutively endocytosing receptors for targeted degradation. Our bio-orthogonal binder design approach could be readily generalized to a wide range of other tissue specific targets. Design of Tags for triggering conformational change IGF-2R is a large cell surface receptor that rapidly transports its natural ligands IGF-2 and M6P to the lysosome for degradation11. IGF-2R has high abundance across tissues, and considerable overall lysosomal trafficking capacity which has made it an attractive target for protein degradation approaches. Structure investigation suggests that IGF-2 binding induces a conformational change in IGF-2R which brings We hypothesized that a designed binding protein that brings together domain 6 and 11 could similarly but exclusively trigger IGF-2R endocytosis and lysosomal targeting without triggering off-target signaling activation (Fig. 1a). We sought to use the RosettaTMRIFdock method14,15(Fig.1b) to design small proteins that bind to domain 11 and domain 6 of IGF-2R independently. Inverse rotamer fields were generated around residues interacting with IGF-2R in both domains (Fig.5a&c), and scaffolds were docked into these fields with RIFdock16. After sequence optimization and refinement with RosettaTMFastdesign, the best scoring binding motifs were identified and grafted back on to the scaffold library with RosettaTMMotifgraft17, followed by another round of RosettaTMFastdesign. Genes encoding thee top-ranked 15,000 designs based on RosettaTM interface metrics (ddg, contact_molecular_surface)14, were synthesized on an oligonucleotide array, transformed into a yeast display vector, and fluorescence-activated cell sorting (FACS) was carried out using biotinylated human IGF-2R domain 6 and domain 11. After next- generation sequencing, we identified one high-affinity candidate binder for domain 6 and two high-affinity candidate binders for domain 11; these were expressed and purified in E coli. The IGF-2R domain 6 binder (D6mb) affinity of 41nM KD was measured by using biolayer interferometry (BLI) (Fig.6a), and the best domain 11 minibinder (D11mb) bound IGF-2R domain 11 at an affinity of 190nM KD (Fig.6b). To validate the binder design models and identify potential substitutions that could increase affinity, yeast displayed Saturation Site Mutagenesis (SSM) libraries for D6mb and D11mb were generated, sorted using FACS for IGF-2R binding, and sequenced. For D6mb, the majority of the core and interface residues were conserved, consistent with the design model (Fig.7b). For D11mb, major core residues were conserved but several interface mutations favor binding (Fig.8b). A combinatorial library containing these substitutions was generated and subjected to FACS; the highest affinity variant (D11mb2) bound domain 11 at 6.5nM KD in BLI (Fig.6c). We next sought to develop IGF-2R Tags utilizing the distinct IGF-2R D6 and D11 minibinders (as shown in Fig.1c). Initially, we explored the use of flexible fusions between D11mb and D6mb, with different loop lengths and domain orders. We then expressed these fusions in E. coli and incorporated an avi-tag for biotinylation. Finally, we evaluated their cellular uptake in Jurkat cells by utilizing AlexaTM-647 (AF-647) and flow cytometry. A construct connecting D11mb and D6mb with a GGS linker (D11mb-GGS-D6mb, Tag1) was the most readily taken up, with increased cell associated fluorescence signal over native IGF- 2 or D6mb / D11mb alone in Jurkat cells (Fig.1f). This increased cellular uptake was observed across multiple human and mouse cell lines including K562, HEK293 and A20 (Fig.11). Co- localization imaging demonstrated that Tag1 is targeted to lysosomes (Fig.1g): this synthetic ligand thus recapitulates the trafficking of the endogenous IGF-2 ligands. Longer linkers decreased the uptake level, while a shorter GS linker abolished all uptake (Fig.10a), suggesting the orientation and distance of the two binding domains modulates the IGF-2R endocytosis process. Constructs with two copies of one mini-binder (D6mb-GGS-D6mb, D11mb-GGS-D11mb did not display any cellular uptake (Fig.10b); engagement of both domains (likely driving their reorientation within the receptor structure) appears to be necessary to trigger efficient cellular uptake. Substitution of D11mb with the higher affinity variant D11mb2 in Tag1 (generating Tag2) increased internalization to 2 fold compared to native IGF-2 in Jurkat cells (Fig 1f); Tag2 was clearly detectable in lysosomes at after a 30 minute incubation, unlike IGF-2 or Tag1 (Fig.12a&b). We reasoned that still more potent stimulation of endocytosis could be achieved by two domain constructs in which the individual domains are rigidly fused to each other to drive specific receptor conformational changes. We generated such fusions by using RFInpainting18to generate three-helix bundles combining the major interface helix from D11mb and two interface helices from D6mb (Fig.9). RFInpainting was implemented such that the interacting residues within 3 angstroms (A) of the receptor were maintained as the same identity as in the original minibinders. The D11mb helix was randomly perturbed by rigid body translations (up to 5 angstroms) and rotations (up to 10 degrees) relative to the D6mb helices, the two chains were connected by inpainting, and the sequence of the fusion designed in the context of IGF-2R using ProteinMPPN19keeping residues interacting with the receptor constant. Models with RosettaTMFold LDDT metrics (>0.5) for the inpainted region and AlphafoldTM220(AF2) structure predictions for the fusions matching the design models were experimentally screened for binding with yeast display against both IGF-2R domains. The eight most enriched candidates were selected for protein expression and BLI affinity evaluation. Different designs had distinct affinities for domains 6 and 11; for example, Tag3 possessed strong binding affinity to both domains (6nM for domain 6 and 190 nM for domain 11, Fig.6d), while Tag4 bound primarily domain 6 (15nM for domain 6 and 4.3uM for domain 11, Fig.6e). In the cellular uptake assay, Tag3 demonstrated internalization activity equivalent to IGF-2, and Tag4 showed 2 fold higher internalization than IGF-2 (Fig.1f) and co-localized with lysosome within 30 minutes as we observed in co-localization imaging (Fig.12c). Design of Tags for inducing receptor clustering The endocytosis of receptors such as ASGPR and EGFR is stimulated through dimerization or oligomerization21. ASGPR is a liver-specific ligand shuttling receptor to transport GalNAc labelled proteins into lysosome for clearance22. Multivalent GalNAc ligands have been used for multiple liver-specific degradation applications2,23and RNA delivery platforms24. However, these require chemical modification and hence are not genetically encodable and competition with native ligand raise the challenges. We sought to use a similar design strategy to generate orthogonal and synthetic ASGPR Tags (AS-Tag). Using a minibinder design approach25that integrated ProteinMPNN19and AlphafoldTM218We designed binders to ASGPR that do not overlap with the glycan binding sites using an augmented version of the RosettaTMdesign approach described above that uses ProteinMPNN19for sequence design and AlphafoldTM218for design evaluation.2689 designs passed AlphafoldTM2 pae_interaction cutoff were selected for experimental screening. After yeast display enrichment and NGS, we identified four candidates that bound to ASGPR on yeast surface. Following expression and purification from E coli, biolayer interferometry showed that the highest affinity of these, (ASmb1), bound to bind ASGPR with an affinity of 2.7 uM (Fig, 17). To stimulate ASGPR endocytosis through clustering, we connected two or three ASmb1 domains with GS linkers, which we refer to as ASGPR-Tags. Cellular uptake assay and confocal imaging in Hep3B cells indicated that ASGPR-Tags with two or three copies of the binding domain were endocytosed considerably more than monomeric ASmb1. Confocal imaging indicated lysosomal co-localization. We further optimized the binding affinity by SSM and then generated combo optimized ASGPR-Tag2, which increased cellular uptake by 2000 fold with trimer. Cell surface receptor degradation Targeted degradation of receptors and extracellular soluble proteins is a promising therapeutic strategy for cancer, autoimmune diseases and neurodegenerative diseases. Lysosome-targeting Chimeras (LYTACs) utilize mannose-6-phosphonate (M6Pn) ligands, which triggers lysosomal delivery and degradation of the targeted proteins through the IGF- 2R26, or N-acetylgalactosamine (GalNAc) to trigger the ASGPR lysosomal trafficking pathway2. While very promising, the LYTAC approach is hindered by the reliance on existing native ligands and on sophisticated chemistry required to generate multivalent modifications that increase endocytosis potency, limiting its manufacturing. Given their potent and rapid endocytosis and lysosomal targeting ability, we hypothesized that the fusion of Tags with POI-specific binders to generate protein-LYTACs (pLYTAC) could provide a genetically encoded approach for efficient extracellular protein degradation. (Fig.2a). With different tissue abundancy of the corresponding receptor, the four types of Tags could benefit the tissue specific degradation platform. To explore the potential of pLYTACs in promoting protein degradation, we investigated their ability to target and degrade cell surface receptors (Fig.2a). We first focused on the Epidermal Growth Factor Receptor (EGFR), a receptor that is often overexpressed in various cancer types and plays a crucial role in regulating cell proliferation27. To generate EGFR-Tags, we fused TfR-Tag and -Tag with a minibinder targeting EGFR N-terminal (EGFRn)14. Both construct demonstrated potent degradation in HeLa and H1975 cancer cells (Fig.2c-d). After fusing EGFRminibinder EGFRn with TfRmb, we observed efficient clearance of EGFR after 48h incubation in western blot (Fig. 2b), validating our hypothesis that the Tag could function as efficient degraders. With rich expression in brain, we believe that both TfR-Tag could function as brain-specific pLYTAC with promising therapeutic applications such as Amyloid degraders. Next, we assessed the degradation efficiency of liver specific ASGPR-Tag, we fused ASGPR-Tag with EGFRn but it did not significantly affect the EGFR level in Hep3B liver cells. To achieve a multi-domain effect for endocytosis induction, we then constructed the EGFRn fusion with daisy chain ASGPR-Tag-dimer and ASGPR-Tag-Trimer. When these constructs were introduced into HepB3 cells, the dimer and trimer of ASGPR-Tag in the daisy-chain configuration efficiently cleared EGFR, resulting in a 40% decrease in total EGFR levels (Fig.2f). This result correlated well with our cellular uptake experiment, validating that the clustering of ASGPR is critical to trigger efficient endocytosis and lysosomal targeting. It also validated that it is possible to direct ASGPR-mediated degradation using protein-based systems and generate liver-specific Tags. Next, we focused on a more general pLYTAC, IGF-2R that globally expressed across the tissues. Using similar approach, we fused IGF-2R Tags to EGFRn. In H1975 and HeLa cells, all EGFRn-Tags demonstrated effective clearance of EGFR in both the cell surface and whole cell lysate, as evidenced by Western blot analysis (Fig.2b&d, Fig.15b&d). EGFRn- Tag2 exhibited the highest capacity for degradation, leading to more than 80% clearance of EGFR, while EGFRn-Tag1 resulted in a 70% clearance. The flexible EGFRn-pLYTACs (Tag1&2) had higher degradation capacity than the rigid constructs (Tag3&4), perhaps due to better interactions with EGFR. MS proteomic analyses confirmed that EGFRn did not affect the EGFR itself (Fig.4c, Fig.15a), while EGFRn-Tag2 and EGFRn-Tag1 significantly reduced EGFR levels in both Hela and H1975 cells while maintaining IGF-2R levels (Fig.4c, Fig.15b). To compare with the original M6P-based LYTACs, we generated genetic fusions of Tag1 with Cetuximab (CTX), a clinically approved therapeutic antibody that targets EGFR with high affinity28. In H1975 cells, CTX-Tag1 led to more effective degradation of EGFR than the M6P-based LYTAC (Fig.4b)1. Proteomic analyses demonstrated that CTX-Tag1 caused a significantly greater reduction in EGFR levels compared to CTX alone (Fig. 15c&d), with little effect on IGF-2R levels (Fig.15d). CTX has a higher affinity for EGFR, and hence required a lower dose for effective degradation: CTX-Tag1 achieved 85% degradation at a concentration of 10nM (Fig.15a), whereas EGFRn-Tag1 did not induce any degradation at the same concentration (Fig.15a). We next investigated targeted degradation of Programmed Death-Ligand 1 (PD-L1), an immune checkpoint in cancer immunotherapy, and therefore an attractive target for this approach29. We genetically fused pLYTACs at the C-term of Atezolizumab (Atz), a potent PD-L1 inhibitor30, and then tested PD-L1 clearance ability. All of the Atz-Tags effectively degraded PD-L1 on the cell surface of MDA-MB-231 cells within 4 hours, with Tag2 achieving the highest clearance rate of 90% after 24 hours of incubation (Fig.4e). Western blot analysis of the whole cell lysis indicated that Atz-Tag2 molecules were able to deplete 77% of PD-L1 in the whole cell after 48h incubation (Fig.4f). Like PD-L1, cytotoxic T lymphocyte antigen 4 (CTLA4) is an immune checkpoint component for which inhibitors have shown promising anti-tumor effects18,19. To target CTLA4, we generated a genetic fusion between Tag1 and a minibinder against CTLA4 (CTLA4mb) which resulted in a 45% decrease of CTLA4 in Jurkat-CTLA4 cells after only 3 hours (Fig.15e). The anti-cancer ability of IGF_Tag fusion with ATZ was evaluated in a syngeneic tumor model, where BALB / c mice were inoculated subcutaneously with A20 cells to initiate tumor growth, and were treated intratumorally with Tag constructs when the tumor reached ~100 mm3. The proteins were administered for every 3 days, in a total of three injections, with a dosage of 5 mg / kg per injection. Results indicated that the ATZ-IGF_Tag3 and ATZ- IGF_Tag4 were considerably more effective than ATZ alone in reducing tumor size and tumor weight. ATZ-IGF_Tag4 dramatically increased overall survival compared to ATZ alone or control: after 55 days half the mice treated with the Tag fusion were still alive while all mice treated with ATZ alone had died. Western Blot analysis showed that ATZ-IGF_Tag3 and ATZ-IGF_Tag4 triggered significant degradation of PD-L1 compared with ATZ alone or control. Body weight assessment showed that the Tag treatments were well tolerated. Anti PD-L1 antibodies function by blocking the ability of PD-L1 expressed on tumor cells to activate the inhibitory receptor PD1 on T cells and dampen the host immune response. Our ATZ-Tag fusions likely have two mechanisms of action. Like ATZ, they block the PD-L1-PD1 interaction. In addition, they directly degrade PD-L1 in the tumor cells. The striking anti-tumor function observed with the Tag - ATZ fusions suggests that the efficacy of antagonistic antibodies may be generally increasable by fusion to Tags, which adds on the ability to not only antagonize a protein interaction but also degrade relevant target proteins for cancer or autoimmune diseases. Protein design is a powerful tool for the development of LYTACs against multiple receptors, including those that are tissue-specific. The example of ASGPR demonstrate the versatility of this approach and highlight the potential for developing LYTACs with cell- and tissue-specific activities. Furthermore, as more information becomes available about the structure and function of different receptors, it is readily doable to apply this approach to develop LYTACs against other new receptors. Clearance of soluble proteins In alternative to cell surface receptors, we further investigate the ability of Tags to degrade targeted soluble proteins (Fig.3a), we conducted a proof-of-concept study using the de novo designed proteins LHDA and LHDB, which exhibit nanomolar affinity for each other33. Firstly, we genetically fused LHDA with various IGF-2R Tags and expressed the resulting constructs in E. coli. Next, we performed a cellular uptake assay by incubating cells with LHDB-AF647, in the presence or absence of LHDA-pLYTACs. All Tags significantly enhanced the uptake of LHDB-AF647 in both Jurkat and K562 cells (Fig.3b, Fig.13a&b). Tag3 resulted in a remarkable 40-fold increase in mean fluorescence intensity (MFI) compared to the control, indicating the most significant uptake enhancement among all constructs evaluated. To quantify the clearance of LHDB, we measured the remaining fluorescence in the supernatant, revealing that Tag3 resulted in a 50% clearance of LHDB from the solution (Fig.3c). We next investigated the potential of the pLYTAC technology to eliminate disease- relevant soluble protein targets such as autoantibodies that recognize self-antigens, which have been linked to multiple autoimmune diseases34. We first tested whether the fusion of Tags with IgG binding protein G35could clear IgG in solution. ProteinG-Tags were generated by genetically fusing proteinG with various Tags and purified after expression in E coli. As a benchmark comparison to glycan-based LYTAC, ProteinG-M6Pn was generated by conjugating proteinG with azido-NHS ester followed by M6Pn-BCN peptide. All fusion proteins including Tag constructs together with proteinG-M6Pn were then incubated with IgG-AF647 and Jurkat cells. All candidates triggered considerable uptake of IgG in both Jurkat and K562 cells (Fig.14a&c). ProteinG-Tag3 elicited 2-fold higher cellular uptake of IgG than proteinG-M6Pn (Fig.3d), leading to an overall 80-fold increase in IgG in K562 cells and 360 fold increase in Jurkat cells (Fig.14c). To quantify the clearance of IgG in the solution, we further measured the remaining fluorescence intensity in the cell culture supernatant and normalized it with the control group treated by IgG-AF647 alone. Tag3 induced the highest clearance of IgG with a 72% drop in remaining fluorescence intensity of IgG-AF647 in the supernatant in K562 cells, while proteinG-M6Pn culminated in 50% decrease in fluorescence level (Fig.3e, Fig.14c). The flexible designs Tag1 and Tag2 only led to 20~30% reduction (Fig.14b). Overall, the decrease in fluorescence intensity in solution was consistent with the cellular uptake results, with Tag3 stimulating the highest cellular uptake and highest clearance in the solution. Tag3 clearance efficiency remained high at 100nM (Fig.14e), while that of Tag4 decreased considerably at 100nM (Fig.14f). Utilizing confocal microscopy in HeLa cells, we observed enhanced co-localization of IgG with lysosomes following treatment with proteinG-Tag3 for 24 hours, indicating that the Tags actively facilitated the transportation of IgG to the lysosomes for degradation (Fig.3f). Furthermore, we found that in HeLa cells lacking IGF-2R expression, this co-localization was not detectable, suggesting the essential role of IGF-2R in mediating the co-localization and subsequent degradation of targeted proteins by the Tags (Fig.3g). Modularity of Tags Many targets of interest are expressed not only in diseased tissue but also on healthy cells, and the ability to target protein degradation to specific cell sub-populations could reduce toxicity. For example, in the case of cancer, target degradation conditioned on the presence of a specific marker in the tumor microenvironment could help avoid undesired effects on healthy cells. Such logic gated targeted degradation has not been achieved with current extracellular protein degradation systems. To address this limitation, we utilized the colocalization-dependent protein switches (Co-LOCKR) system, which functions as an AND logic gate and will only expose a peptide binding motif when two target cell markers are present on the same cell. We utilized Co-LOCKR to selectively degrade EGFR and HER2 in response to their co-localization on the surface of cancer cells36. We expressed Tag fusions with Bcl2, which specifically binds to Bim peptide, and evaluated EGFR degradation level in both HER2+ and HER2- cells. In K562 cells overexpressing both EGFR and HER2, Bcl2- Tag2 resulted in 80% degradation of EGFR, while in K562 cells expressing only EGFR without HER2, the EGFR level remained unchanged (Fig.4i). Thus, the Tag system can be precisely targeted to specific cells based on combinations of surface markers. Another major challenge is the degradation of the degrader itself, upon binding cells that express IGF2R (or other endocytosing receptor) but lack the target protein, before reaching its target, leading to limited therapeutic applications as systemic delivery is required. In the context of adoptive cell therapies or mRNA delivery, the secretion of a degrader locally in response to stimuli could potentially overcome this challenge. Our system offers a solution as it is fully protein based and consequently can be locally secreted with high specificity and efficiency37,38. To investigate this possibility, we investigated the efficacy of Tag1 and Tag2 when secreted from cells by transiently transfecting IGF-2R KO HeLa cells with plasmids encoding EGFRn-Tag, collecting the cell supernatants, and incubating them with EGFR+ K562 cells while monitoring EGFR expression levels (Fig.4k). Secreted EGFRn-Tag1 led to similar EGFR clearance as the exogenously added EGFRn-Tag1 (Fig. 4l), while secreted EGFRn-Tag2 demonstrated greater EGFR clearance than EGFRn-Tag1, consistent with the results obtained with the purified proteins. Thus, Tags remain functional when secreted from cells, providing a potential avenue for their use in adoptive cell therapy control systems. We showcased the integration of modular Tags to logic-gate degrader and secretion degrader, further expanding the applications for the extracellular degradation strategies. Tag activated cell signaling Our understanding of the role of endocytosis in transmembrane signaling is still incomplete for most receptors. To address this, we sought to investigate the effect of potent enhancers of endocytosis on signaling pathways that involve endocytosis. For this purpose, we selected a minimalist Notch-derived synthetic receptor, SNIPR (described in the accompanying manuscript), which endocytosis inhibitor experiments suggested was enhanced by endocytosis. We fused the LHDA ligand for this synthetic system to our designed IGF-2R Tags and tested their effect on the SNIPR system, in which binding of the synthetic ligand LHDA to a SNIPR receptor with an LHDB extracellular domain results in cleavage and release of an intracellular transcription factor domain, which activates downstream BFP expression (Fig.4a). We found that all flexible and rigid inducers increased signal activation by at least 50-fold, while Tag2 led to a 100-fold increase which was the strongest signal we were able to achieve with any ligand designed for this receptor (Fig.4b). Confocal microscopy showed rapid lysosomal targeting of the LHDA-Tag2 construct (Fig. 4c). These results demonstrate that the Tags can significantly enhance signaling in the SNIPR system, making it a more useful tool for synthetic biology and adoptive cell therapy applications. Tags could also enhance the activity of endogenous pathways where endocytosis plays a critical role in signaling. Conversely, for pathways that are downregulated by endocytosis, Tags can be used to shorten the signaling half-life, which could have utility in applications such as TCR signaling where overstimulation can lead to pathway inactivation. Conclusions Targeted degradation as a therapeutic approach using native ligands or chemical modification to engage with rapidly trafficking cell surface receptors provides limited options for triggering endocytosis. Native ligands can trigger off-target signaling and may not stimulate endocytosis. We demonstrate here that computational design of orthogonal and synthetic all protein ligands provides a general approach to triggering endocytosis through both broadly distributed and more tissue specific transmembrane receptors. De novo protein design could enable the development of Tag through multiple endocytosis mechanisms for a given receptor. The small, stable and readily producible synthetic ligands that could be useful not only for therapeutic applications but as molecular tools for probing how receptor conformations modulate cellular trafficking. Moving forward, there are many avenues to pursue using our computational design approach to generating Tag stimulated enhancers of cell surface receptor endocytosis and trafficking. First, there are likely many more receptor targets which can undergo rapid endocytosis upon suitable triggering at the cell surface; our ability to design endocytosis stimulators without requiring native ligands or identification of chemical modifications should enable utilization of the full range of these receptors to achieve more tissue restricted targeting and modulatable intracellular trafficking (different receptors likely will have different intracellular compartment residence times and transition dynamics). In addition to the targeted degradation application pursued here, such designed endocytosis stimulators could be of great utility for enhancing uptake of nucleic acid (siRNA for example) and small molecule drug conjugates. Second, there are likely natural signaling pathways that, like the synthetic SNIPER system, can be more potently activated by promotion of receptor endocytosis. We achieve a 5-fold enhancement of signaling Emax by fusing the SNIPER ligand to Tags–if similar levels of signaling enhancement or modulation can be achieved by fusing natural signaling molecules to Tags there could be many applications for such increased potency molecules in therapeutics and biotechnology. Third, as illustrated by the use of the LOCKR system to make logic gated protein degradation systems and the secretion of Tag-degrader constructs from HeLa cells, the use of small designed all protein ligands opens the door to a wide range of applications where the capability for logic gated activation, cell based expression, and the overall robustness and modularity of de novo designed proteins can have therapeutic advantages. Methods Computational design of IGF-2R and ASGPR minibinders The minibinders against IGF-2R domain 6 and 11 were computationally designed via a RosettaTM-based approach previously described14. Briefly, the structures of IGF-2R domain 6 (PDB 6UM2) and IGF-2R domain 11 (PDB 1GP0) were refined using RosettaTMFastrelax with coordinate constraints. The residues at IGF-2 binding site for each domain were selected as “hotspot” residues. Helical protein scaffolds were docked against the hotspot residues via Patchdock followed by Rifdock protocol. After sequence optimization with RosettaTMFastDesign and filtering with RosettaTMinterface metrics including ddg and contact_molecular_surface, the top candidates were then resampled with RosettaTMMotifgraft and FastDesign. Candidates passing previous filters were then filtered again with exposed hydrophobicity (sap_score) and optimized with a net-charge of -7. The minibinders against ASGPR were designed with RosettaTM-based approach integrated with ProteinMPNN and AlphafoldTM2. The crystal structure of ASGPR (PDB 5JQ1) were refined and helical protein scaffolds were docked against the exposed hydrophobic residues via Patchdock followed by Rifdock. The sequences were optimized with protein-MPNN and interface scores were calculated with RosettaTMFastrelax. The models were then predicted by AlphafoldTM2 and scored after Fastrelax. Designs with pae_interaction<10 and relaxed_ddg<-40 were selected for resampling with another round of protein-MPNN prediction followed by RosettaTMFastrelax. After final round filtering with pae_interaction, relaxed_ddg and sap_score, the sequences were further optimized to have a net-charge of -7. Computational design of IGF-2R endocytosis enhancer To generate flexible IGF-2R agonists, all combinations of Gly-Ser (GS) linker with various lengths linking D6mb and D11mb were modeled with Alpahfold2. The designs with poor monomer plddt (plddt<85) were dropped. To generate rigid IGF-2R agonists, the major binding helix from D11mb or the native IGF-2 binding helix, and two interface helices from D6mb were extracted. Both crystal structures obtained for the domain 6 and 11 minibinders in complex with IGFII-M6PR were used as starting points for design. Domains 6 and 11 of the complex structures were aligned with the respective domains of CIM6P-IGFIIR in the putative receptor internalizing conformation available in the Protein Data Bank (PDB: 6UM2). In this orientation, the two interface helices from the domain 6 binder and the single interface helix from the domain 11 binder were extracted and used as motifs to scaffold by Protein Inpainting18. To increase the likelihood of design success, the D11 minibinder structure was adjusted to form an ideal three helical bundle with the two domain 6 helices. Protein Inpainting was implemented such that the interacting residues within 3 angstroms of the receptor maintained as the same identity as in the original minibinders. To increase design diversity, the d11 helix motif was randomly perturbed by rigid body translations (up to 5 angstroms) and rotations (up to 10 radians) for each design prior to inpainting a scaffold between the motifs. The best inpainting outputs were selected by RosettaTMFold LDDT metrics (> 0.5) for the inpainted region and used for sequence design with ProteinMPNN. ProteinMPNN sequence design was performed on the inpainted outputs in their desired complex orientation (with both domains 6 and 11 present) while fixing the original minibinder identities of interface residues (domain 6 minibinder: R4, V8, Q11, D15, V20, K24, M25, I27, I31, and E34, domain 11 minibinder: M1, A4, L7, L8, and W11). After 2000 sequences were generated for each ProteinMPNN input, designs were filtered by predicted RosettaTMddG. AlphaFoldTM2 structure predictions of the designed sequences were filtered by the pLDDT metric (keeping those with pLDDT > 90), and designs with a subangstrom backbone atom RMSD to the original design models realigned to the D6 and D11 minibinder crystal structures (in complex with the IGFII-M6PR target domains). Finally, the complexes were assessed by RosettaTMFastRelax. Designs with ddG metrics less than -40 and Spatial Aggregation Propensity scores less than 35 were selected for expression and experimental assays. Yeast surface display screening with FACS The yeast surface display screening was performed using the protocol as previously described14,17. Briefly, DNAs encoding the minibinder sequences were transformed into EBY-100 yeast strain. The yeast cells were grown in CTUG medium and induced in SGCAA medium. After washing with PBSF (PBS+1% BSA), the cells were incubated with 1uM biotinylated target proteins (IGF-2R, ASGPR) together with streptavidin–phycoerythrin (SAPE, ThermoFisher, 1:100) and anti-c-Myc fluorescein isothiocyanate (FITC, Miltenyi Biotech, 6.8:100) for 30min. After washing twice with PBSF, the yeast cells were then resuspended in PBSF and screened via FACS. Only cells with PE and FITC double-positive signals were sorted for next-round screening. After another round of enrichment, the cells were titrated with biotinylated target protein at different concentrations for 30min, washed, and further stained with both streptavidin–phycoerythrin (SAPE, ThermoFisher) and anti-c- Myc fluorescein isothiocyanate (FITC, Miltenyi Biotech) at 1:100 ratio for 30min. After washing twice with PBSF, the yeast cells at different concentrations were sorted individually via FACS and regrown for 2 days. Next the cells from each subpool were lysed and their sequences were determined next-generation sequencing or Miseq. Biolayer interferometry The binding affinity for the minibinders were determined by the OctetTMRED96 (ForteBio). To measure the binding affinity, streptavidin-coated biosensors (ForteBio) were first loaded with biotinylated target proteins at 50~100nM concentration, washed with OctetTMbuffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20 and 1% BSA), and incubated with titrated concentrations of corresponding binders. To measure the Koff, the biosensors were then dipped back into the OctetTMbuffer. The Kon, Koff and KD were further estimated with the OctetTMAnalysis software. Protein and purification Minibinders and minibinder fusions were expressed in E coli BL21 strain as previously described14. Briefly, the DNA fragments encoding the design sequences were assembled into pet-29 vectors via Gibson assembly and further transformed into BL21 strain with heat-shock. Protein expression was induced by the autoinduction system and protein were purified with Immobilized metal affinity chromatography (IMAC) approach. Next, the elutions were purified by FPLC SEC using SuperdexTM7510 / 300 GL column (GE Healthcare). Protein concentrations were determined by NanoDropTM(Thermo Scientific) and normalized by extinction coefficients. Antibody-pLYTAC fusions were produced with mammalian expression system. Light chain of CTX / ATZ antibody and heavy chain fused with pLYTAC at C-term constructs were ordered in CMVR from Genscript. Antibody-pLYTAC fusions were then expressed via transient co-transfection of the pLYTAC-heavy and light chains into Expi293F cells (Life Technologies) via PEI-MAX (Polyscience). In brief, 800mL cultures of Expi293F cells were transfected at a density of 3x10^6 cells per mL of culture using 1 ug of plasmid DNA and 3ug of PEI per mL of culture. These cultures were grown in Expi293F expression medium (Life Technologies) at 37^ in a humidified, 8% CO2 incubator rotating at 125rpm. After 6 days of expression, culture supernatants were harvested via 5 minutes of centrifugation at 4000 RCF, 5 minutes of incubation with PDADMAC solution (Sigma Aldrich) added to a final concentration of 0.0375%, followed by an additional 5 minutes of centrifugation at 4000 RCF. Supernatants were clarified via 0.22um vacuum filtration and then treated to a final concentration of 50mM Tris-HCl (pH 8) and 350mM NaCl for IMAC. Gravity IMAC was performed by batch binding the clarified supernatants with 10mL of Ni SepharoseTMExcel resin (GE Healthcare). After 20-30 minutes of incubation, the resin bed was washed with 10 CV of 20mM Tris-HCl (pH8), 300mM NaCl solution. The proteins were then eluted with three CV of 20mM Tris-HCl (pH 8), 300mM NaCl, 300mM imidazole solution. The batch bind process was then repeated with half the amount of resin (5mL) and the eluates from both batch binds were combined. SDS-PAGE was performed on the IMAC eluates to assess purity. The purified antibody-pLYTAC fusions were subsequently concentrated in a 10K MWCO AmiconTMUltra centrifugal filter unit (Millipore) and polished via SEC using a HiloadTM26 / 600 SuperoseTM200 column (GE Healthcare) in DPBS (Gibco). The SEC fractions were re-concentrated in the same manner as before to a final concentration of 5 mg / mL. Endotoxin levels were assayed via EndosafeTMLAL Endotoxin tests (Charles River) and analytical SEC was performed using a SuperdexTM200 Increase 5 / 150 column (GE Healthcare) to obtain a high-resolution size profile. Pre- and post-freeze stability was assessed via UV-Vis spectrophotometry as well as SDS-PAGE. Protein degradation via Western blot (Green) Cells were cultured in T75 flasks at 37 °C in a 5% CO2atmosphere. HEP3B (ATCC), HeLa (ATCC), and MDA-MB-231 were cultured in DMEM supplemented with 10% heat- inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin. Jurkat-CTLA4 and H1975 were cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin. Adherent cells were plated (100,000 cells / well in a 24-well plate) one day before the experiment, whereas suspension cells were plated on the day of the treatment. Cells were incubated with 250 μl of complete growth media with pLYTAC or controls for indicated time. Cells were then washed with PBS 3 times and lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche), 0.1% Benzonase (Millipore-Sigma), and phosphatase inhibitor cocktail (Roche) on ice for 30 minutes. The cells were scraped, transferred to Eppendorf tubes, and spun down at 21,000g for 15 minutes at 4 °C. The supernatant was collected and the protein concentration was determined by BCA assay (Pierce). Equal amounts of lysates were loaded onto 4-12% Bis-Tris gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the gel was transferred onto a nitrocellulose membrane and stained with REVERT Total Protein Stain (LI-COR), then blocked with Odyssey Blocking Buffer (TBS) (LI-COR) for 1 hour at rt. The membrane was incubated with primary antibodies (rabbit anti-EGFR D38B1 Cell Signaling Technologies , rabbit anti-HER22242 Cell Signaling Technologies, rabbit anti-PD- L1 E1L3N Cell Signaling Technologies, rabbit anti-CTAL4 E1V6T Cell Signaling Technologies, mouse anti- vinculin V284 Bio-Rad) overnight at 4 °C, washed 3 times with TBS-T. Subsequently, the membrane was incubated with secondary antibody (800CW goat- anti-mouse or goat-anti-rabbit LI-COR) for 1 hour at rt, and washed 3 times with TBS-T for visualization with an Odyssey CLx Imager (LI-COR). Image Studio (LI-COR) was used to quantify band intensities. Fluorescence Imaging WT HeLa (ATCC CCL-2) were cultured at 37ௗ°C with 5% CO2 in flasks with Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 1 mM L-glutamine (Gibco), 4.5 g / liter D-glucose (Gibco), 10% fetal bovine serum (FBS) (HycloneTM) and 1% penicillin-streptomycin (PenStrep) (Gibco). Cells were passaged twice per week. To passage, cells were dissociated using 0.05% trypsin EDTA (Gibco) and split 1:5 or 1:10 into a new tissue culture (TC)–treated T75 flask (Thermo Scientific ref 156499). 35 mm glass bottom dishes seeded at a density of 20k cells / dish. A final monomeric concentration of 100 nM of ligands were incubated with cultured cells. Cells were fixed 4% paraformaldehyde, permeabilized with 100% methanol, and blocked with PBS + 1% BSA. Cells were immunostained with Anti-LAMP2A antibody (Abcam ab18528) followed by goat anti-rabbit- IgG AlexaTMFluor 488 secondary antibody (Thermo Fisher A-11034) and 4ƍ,6- diamidino-2-phenylindole (DAPI) (Thermo Fisher D1306) and stored in the dark at 4°C until imaging. Cells were washed twice with HBSS and subsequently imaged in HBSS in the dark at 37°C. Right before imaging, cells were incubated with 25 μM DTZ. Epifluorescence imaging was conducted on a Yokogawa CSU-X1 microscope equipped with a Hamamatsu ORCA- Fusion scientific CMOS camera and Lumencor CelestaTMlight engine. Objectives used were: 10×, NA 0.45, WD 4.0 mm, 20×, NA 1.4, WD 0.13 mm, and 40×, NA 0.95, WD 0.17–0.25 mm with correction collar for cover glass thickness (0.11 mm to 0.23 mm) (Plan Apochromat Lambda). All epifluorescence experiments were subsequently analyzed using NIS Elements software. Confocal microscopy HeLa cells were seeded in 18 well glass bottom μ-Slides (Ibidi, Cat. No.81817) at a density of 15k / well. Fluorescently labeled ligands were incubated with the cultured cells for 23 hours and then LysotrackerTMwas added for 60 min. Fluorescently labeled anti-IGF-2R (Vendor, Cat. No.) was added for 30 min. Cells were washed 3× in PBS and immediately proceeded to imaging. Confocal laser scanning microscopy was performed on a Nikon A1R HD25 system equipped with a LU-N4 laser unit (Lasers used: 488 nm, 561 nm, 640 nm). Data was acquired using an 20×, NA 0.75, WD 1.00 mm air objective (Plan Apochromat Lambda) in combination with 1 multialkaline (EM 650 LP) and 2 GaAsP detectors (DM 560 LP EM 524 / 42 (503-545) and DM 652 EM 600 / 45 (578-623)). Acquisition was controlled via NIS Elements software and data was analyzed via Fiji and custom-written Python Scripts. Mass spectrometry and proteomic Cell pellets were thawed on ice and lysed in lysis buffer (400 ^L, 1 tablet of Pierce EDTA-free Protease Inhibitor Tablets dissolved in 50 mL of PBS) using a probe sonicator (3 × 3 pulses). Protein concentration was adjusted to 2.0 mg / mL and the samples (100 ^L, 200 ^g protein) were transferred to new Eppendorf tubes (1.5 mL) containing urea (48 mg / tube, final urea concentration: 8 M). DTT (five ^L, 200 mM fresh stock in H2O, final DTT concentration: 10 mM) was then added to the tubes and the samples were incubated at 65°C for 15 min. Following this incubation, iodoacetamide (5 ^L, 400 mM fresh stock in H2O, final IA concentration: 20 mM) was added and the samples were incubated in the dark at 37°C with shaking for 30 min. Ice-cold MeOH (600 ^L), CHCl3 (200 ^L), and H2O (500 ^L) were then added, and the mixture was vortexed and centrifuged (10,000 g, 10 min, 4°C) to afford a protein disc at the interface between CHCl3 and aqueous layers. The top layer was aspirated without perturbing the disk, additional MeOH (600 ^L) was added, and the proteins were pelleted (10,000 g, 10 min, 4°C) and used in the next step or stored at í80°C overnight. The resulting protein pellets were resuspended in EPPS buffer (160 ^L, 200 mM, pH 8) using probe sonicator (3 × 3 pulses). Trypsin (10 ^L, 0.5 ^g / ^L in trypsin reconstitute buffer) and CaCl2 (1.8 ^L, 100 mM in H2O) were added and the samples were incubated at 37°C with shaking overnight. Peptide concentration was determined using the microBCA assay (Thermo Scientific) according to the manufacturer’s instructions. For each sample, a volume corresponding to 25 ^g of peptides was transferred to a new Eppendorf tube and the total volume was brought up to 35 ^L with EPPS buffer (200 mM, pH 8). The samples were diluted with CH3CN (9 ^L) and incubated with the corresponding TMT tags (3 ^L / channel, 20 ^g / ^L) at room temperature for 30 min. An additional TMT tag (3 ^L / channel, 20 ^g / ^L, 30 min) was added and the samples were incubated for another 30 min. Labeling was quenched by the addition of hydroxylamine (6 ^L, 5% in H2O). Following a 15 min incubation at room temperature, formic acid was added (2.5 ^L, final FA concentration: 5%).20 ^L labeled peptides of each channel were combined into a 2.0 mL low-binding Eppendorf tube, and 25 ^L of 20% formic acid was added. The resulting mixture was lyophilized to remove the solvents before high pH fractionation. The spin columns from Pierce High pH Reversed-Phase Peptide Fractionation Kit were pre-equilibrated prior to use. Briefly, the columns were placed in Eppendorf tubes (2 mL), spun down to remove the storage solution (5,000 g, 2 min), and washed with CH3CN (2 × 300 ^L, 5,000 g, 2 min) and buffer A (2 × 300 ^L, 95% H2O, 5% CH3CN, 0.1% FA, 5,000 g, 2 min). TMT-labeled peptides were re-dissolved in buffer A (300 ^L, 95% H2O, 5% CH3CN, 0.1% FA) and loaded onto pre-equilibrated spin columns for high pH fractionation. The columns were spun down (2,000 g, 2 min) and the flow through was used to wash the original Eppendorf tube and passed through the spin column again (2,000 g, 2 min). The column was then washed with buffer A (300 ^L, 2,000 g, 2 min) and 10 mM aqueous NH4HCO3 containing 5% CH3CN (300 ^L, 2,000 g, 2 min), and the flow through was discarded. The peptides were eluted from the spin column into fresh Eppendorf tubes (2.0 mL) with a series of 10 mM NH4HCO3 / CH3CN buffers (2000 g, 2 min). The following buffers were used for peptide elution (% CH3CN): 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 80, and 95. Every 10th fraction was combined into a new clean Eppendorf tube (2 mL) and the solvent was removed using a benchtop lyophilizer and stored at -20 °C before analysis. The resulting 10 combined fractions were re-suspended in buffer A (25 ^L) and analyzed on the OrbitrapTMFusion mass-spectrometer (4 ^L injection volume) coupled to a Thermo Scientific EASY-nLC 1200 LC system and autosampler. The peptides were eluted onto a capillary column (75 ^m inner diameter fused silica, packed with C18 and separated at a flow rate of 0.3 ^L / min using the following gradient: 5% buffer B in buffer A from 0-10 min, 5%–35% buffer B from 10-129 min, 35%–100% buffer B from 129-130 min, 100% buffer B from 130-139 min, 100%–5% buffer B from 139-140 min, and 5% buffer B from 140-150 min (buffer A: 100% H2O, 0.1% FA; buffer B: 20% H2O, 80% CH3CN, 0.1% FA). Data were acquired using an MS3-based TMT method. Briefly, the scan sequence began with an MS1 master scan (OrbitrapTManalysis, resolution 120,000, 375í1600 m / z, cycle time 3s) with dynamic exclusion enabled (repeat count 1, duration 30 s). The top precursors were then selected for MS2 / MS3 analysis. MS2 analysis consisted of: quadrupole isolation (isolation window was set to 1.2 for charge state z = 2; 0.7 for charge state z = 3; 0.5 for charge states z = 4-6) of precursor ion followed by collision-induced dissociation (CID) in the ion trap (normalized collision energy 35%, maximum injection time 50 ms, MS2 resolution was set to turbo). Following the acquisition of each MS2 spectrum, synchronous precursor selection (SPS) enabled the selection of MS2 fragment ions for MS3 analysis (SPS isolation window was set to 1.3 for charge state z = 2; 0.7 for charge state z = 3; 0.5 for charge states z = 4-6). MS3 precursors were fragmented by HCD and analyzed using the OrbitrapTM(collision energy 65%, maximum injection time 120 ms). The raw files were converted to mzML files using the MSConvert tool from ProteoWizard (version 3.0.22088). A reverse concatenated, non-redundant variant of the Human UniProt database (2022-11-29) was searched using FragPipeTM(version 18.0) with the built-in ‘TMT10-MS3’ workflow. The virtual references were used for the data sets due to the lack of pooled sample. The quantified proteins were filtered with FDR < 1% with median centering normalization. Data are presented as the mean fold change to DMSO-treated controls. n= 3 per group. P values were calculated by a two- tailed unpaired t-test with Welch’s correction. IgG / LHDB supernatant clearance assay Jurkat / K562 cells seeded at 96 round bottom culture plate in 300uL media were incubated with AF647 conjugated IgG (Novusbio) or LHDB alone or together with pLYTACs reagents. At various time points, the cells were pelleted down and 30uL of supernatants were extracted and further diluted in 45uL DPBS. 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Brown, J., Jones, E. Y. & Forbes, B. E. Keeping IGF-II under control: Lessons from the IGF-II–IGF2R crystal structure. Trends Biochem. Sci.34, 612–619 (2009). 27. Sharma, S. V., Bell, D. W., Settleman, J. & Haber, D. A. Epidermal growth factor receptor mutations in lung cancer. Nat. Rev. Cancer 7, 169–181 (2007). 28. Steiner, P. et al. Tumor Growth Inhibition with Cetuximab and Chemotherapy in Non– Small Cell Lung Cancer Xenografts Expressing Wild-type and Mutated Epidermal Growth Factor Receptor. Clin. Cancer Res.13, 1540–1551 (2007). 29. Doroshow, D. B. et al. PD-L1 as a biomarker of response to immune-checkpoint inhibitors. Nat. Rev. Clin. Oncol.18, 345–362 (2021). 30. Ryu, R. & Ward, K. E. Atezolizumab for the First-Line Treatment of Non-small Cell Lung Cancer (NSCLC): Current Status and Future Prospects. Front. Oncol.8, 277 (2018). 31. Pardoll, D. M. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12, 252–264 (2012). 32. Walker, L. S. K. & Sansom, D. 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Direct demonstration of rapid insulin-like growth factor II Receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I- insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process. J. Biol. Chem.260, 9435–9442 (1985). Table 16. Binding affinity of IGF-2R binders / constructs Table 17. Binding affinity of ASGPR binders / constructs

[0011] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.

Claims

We claim:

1. A polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1, wherein the polypeptide binds to asialoglycoprotein receptor (ASGPR).

2. The polypeptide of claim 1, wherein permissible amino acid substitutions relative to SEQ ID NO:1 are listed in Table 2 or 3.

3. The polypeptide of claim 1 or 2, wherein no substitutions are permitted relative to SEQ ID NO:1 at residues 1, 16, 30, 39, 46-48, 50, and 53-54.

4. The polypeptide of any one of claims 1-3, wherein permissible substitutions relative to SEQ ID NO:1 are shown in Table 3.

5. The polypeptide of any one of claims 1-4, comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1-10, wherein the polypeptide binds to asialoglycoprotein receptor (ASGPR).

6. A fusion polypeptide, comprising 2, 3, 4, 5, or more copies of the polypeptide of any one of claims 1-5.

7. The fusion polypeptide of claim 6, wherein an amino acid linker separates each copy of the polypeptide.

8. The fusion polypeptide of claim 7 wherein the amino acid linker separating each copy of the first polypeptide comprises a GlySer-rich linker (e.g., GS / GGSx, wherein x is 1-20, or 1-15, or 1-10, or 1-5), or may comprise, but are not limited to (EAAAK)n (SEQ ID NO: 98) or (Gly)n where n is 1-20, 1-15, 1-10, 1-5, etc.; PAPA, or other chemical conjugation.

9. The fusion polypeptide of any one or claims 6-8, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:11 or 12, wherein the fusion protein binds to asialoglycoprotein receptor (ASGPR).

10. A polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13-15 and 106, wherein the polypeptide binds to insulin-like growth factor 2 receptor (IGF-2R).

11. The polypeptide of claim 10, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13, wherein the polypeptide binds to IGF-2R domain 6.

12. The polypeptide of claim 11, wherein permissible amino acid substitutions relative to SEQ ID NO:13 are listed in Table 6 or 7.

13. The polypeptide of claim 11 or 12, wherein no substitutions are permitted relative to SEQ ID NO:13 at residues 15, 23-24, 35, and 57.

14. The polypeptide of any one of claims 11-13, wherein permissible substitutions relative to SEQ ID NO:13 are shown in Table 7.

15. The polypeptide of claim 10, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14, wherein the polypeptide binds to IGF-2R domain 11.

16. The polypeptide of claim 15, wherein permissible amino acid substitutions relative to SEQ ID NO:14 are listed in Table 8 or 9.

17. The polypeptide of claim 15 or 16, wherein no substitutions are permitted relative to SEQ ID NO:14 at residues 4, 8, 11, 13, 25, 31, 43, and 47.

18. The polypeptide of any one of claims 15-17, wherein permissible substitutions relative to SEQ ID NO:14 are shown in Table 9.

19. The polypeptide of claim 10, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:15, wherein the polypeptide binds to IGF-2R domain 11.

20. The polypeptide of claim 19, wherein permissible amino acid substitutions relative to SEQ ID NO:15 are listed in Table 10 or 11.

21. The polypeptide of claim 19 or 20, wherein no substitutions are permitted relative to SEQ ID NO:15 at residues 21, 32, and 36.

22. The polypeptide of any one of claims 19-21, wherein permissible substitutions relative to SEQ ID NO:14 are shown in Table 11.

23. A fusion polypeptide, comprising: (a) a first polypeptide according to any one of claims 11-14; and (b) a second polypeptide according to any one of claims 15-22.

24. The fusion polypeptide of claim 23, wherein an amino acid linker separates the first polypeptide and the second polypeptide.

25. The fusion polypeptide of claim 24 wherein the amino acid linker separating the first polypeptide and the second polypeptide comprises a GlySer-rich linker (e.g., GS / GGSx, wherein x is 1-20, or 1-15, or 1-10, or 1-5), or may comprise, but are not limited to (EAAAK)n (SEQ ID NO: 98) or (Gly)n where n is 1-20, 1-15, 1-10, 1-5, etc.; PAPA, or other chemical conjugation.

26. The fusion polypeptide of any one or claims 23-25, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:16 or 17, wherein the fusion protein binds to IGF-2R.

27. A polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18, wherein the polypeptide binds to IGF-2R.

28. The polypeptide of claim 27, wherein X1 consists of an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:19-22.

29. The polypeptide of claim 27 or 28, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23-26, wherein optional residues may be present or deleted.

30. A polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27, wherein the polypeptide binds to IGF-2R.

31. The polypeptide of claim 30, comprising 1, 2, 3, 4, 5, 6, 7, 8, or all 9 mutations relative to SEQ ID NO:27 selected from the group consisting of 13A, 17I, 18V, 19A, 27K, 35E, 46 R / Q, 54V, and 58K.

32. The polypeptide of claim 30 or 31, comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27-30 wherein the polypeptide binds to IGF-2R.

33. The polypeptide of any one of claims 30-32, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 mutations relative to the reference sequence selected from the group consisting of 2E, 5K, 9F, 13A, 17I, 18V, 19A, 27K, 35E, 46R / Q, 54V, and 56K.

34. A fusion protein, comprising: (a) the polypeptide or fusion polypeptide of any preceding claim; and(b) a protein that binds to a degradation target.

35. A fusion protein, comprising: (a) a protein that binds to a degradation target; and (b) a transferrin receptor binding polypeptide selected from the group consisting of: (i) a polypeptide comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence selected from SEQ ID NO:32-37, wherein residues in parentheses are optional and may be present or absent; and (ii) a polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, or 98%, identical to the amino acid sequence of SEQ ID NO:31, wherein (A) the polypeptide is identical to SEQ ID NO:31 at residues corresponding to residues 20, 23, 24, 27, 28, 31, 32, 34, 35, 36, 37, 74, 77, 78, 81, 82, 84, 85, and 88 in SEQ ID NO:31; and (B) the polypeptide is substituted relative to SEQ ID NO:31 as shown in Table 12 at least at 5, 6, 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more residues relative to SEQ ID NO:

31.

36. The fusion protein claim 35, wherein the polypeptide is identical to SEQ ID NO:31 at residues corresponding to residues 7, 9, 11, 13, 16, 18, 20-21, 23-24, 27-28, 30-32, 34-37, 41, 47, 49-51, 56, 58, 60, 65-66, 68, 70-71, 74-75, 77-78, 80-82, 84-85, and 87-88; and optionally identical to SEQ ID NO:31 at residues corresponding to residues 3-5 in SEQ ID NO:

31.

37. The fusion protein of any one of claims 35 or 36, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 substitutions relative to SEQ ID NO:31: Q6K, A8F, E10K, R14E, E19K, R29K, A33S, R42E, S48A, R52K, R53K, A64E, L76E, and / or R79K.

38. The fusion protein of any one of claims 35-37, comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acidsequence selected from SEQ ID NO:32-27, wherein residues in parentheses are optional and may be present or absent.

39. The fusion protein of any one of claims 34-38, wherein component (a) is separated from component (b) by an amino acid linker.

40. The fusion protein of claim 39, wherein the amino acid linker comprises a GlySer-rich linker (e.g., GS / GGSx, wherein x is 1-20, or 1-15, or 1-10, or 1-5), or may comprise, but are not limited to (EAAAK)n (SEQ ID NO: 98) or (Gly)n where n is 1-20, 1-15, 1-10, 1-5, etc.; PAPA, or other chemical conjugation.

41. A fusion protein, comprising: (a) the polypeptide or fusion polypeptide of any preceding claim, or of any one of claims 1-32; and (b) a second polypeptide that binds to a therapeutic target of interest.

42. The fusion protein of claim41, wherein the second polypeptide comprises a monoclonal antibody heavy chain and / or light chain, or an antigen binding fragment thereof.

43. The fusion protein of claim 42, wherein the second polypeptide comprises an amino acid sequence at least 90%, 95%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:38-70, or an antigen binding fragment thereof.

44. The fusion protein of claim42, wherein the second polypeptide comprises an amino acid sequence at least 90%, 95%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:40, 41, 62, and 63, or an antigen binding fragment thereof.

45. The fusion protein of claim 41, wherein the second polypeptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of selected from the group consisting of SEQ ID NO:71-97.

46. The fusion protein of any one of claims 41-45, wherein the first polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1, wherein the first polypeptide binds to asialoglycoprotein receptor (ASGPR).

47. The fusion protein of any one of claims 41-46, wherein the first polypeptide and the second polypeptide are separated by a separating amino acid linker.

48. The fusion protein of claim 47, wherein the separating amino acid linker comprises a GlySer-rich linker (e.g., GS / GGSx, wherein x is 1-20, or 1-15, or 1-10, or 1-5), or may comprise, but are not limited to (EAAAK)n (SEQ ID NO: 98) or (Gly)n where n is 1-20, 1- 15, 1-10, 1-5, etc.; PAPA, or other chemical conjugation.

49. The fusion protein of any one of claims 41-48, wherein the first polypeptide is N- terminal to the second polypeptide.

50. The fusion protein of any one of claims 41-48, wherein the first polypeptide is C- terminal to the second polypeptide.

51. An antibody, or antigen binding fragment thereof, comprising: (a) the fusion protein of any one of claims 41-50, wherein the second polypeptide comprises a monoclonal antibody heavy chain, or an antigen binding fragment thereof; and (b) a light chain for the monoclonal antibody, or an antigen binding fragment thereof.

52. An antibody, or antigen binding fragment thereof, comprising: (a) the fusion protein of any one of claims 41-50, wherein the second polypeptide comprises a monoclonal antibody light chain, or an antigen binding fragment thereof; and (b) a heavy chain for the monoclonal antibody, or an antigen binding fragment thereof.

53. An antibody, or antigen binding fragment thereof, comprising: (a) a first fusion protein according to any one of claims 41-50, wherein the second polypeptide comprises a monoclonal antibody heavy chain, or an antigen binding fragment thereof; and(b) a second fusion protein according to any one of claims 41-50, wherein the second polypeptide comprises a monoclonal antibody light chain, or an antigen binding fragment thereof.

54. The polypeptide, fusion protein, antibody, or antigen-binding fragment thereof, of any preceding claim further comprising a detectable label, including but not limited to a fluorescent, luminescent, or radioactive label.

55. A nucleic acid encoding the polypeptide, polypeptide fusion, or fusion protein of any preceding claim.

56. An expression vector comprising the nucleic acid of claim 55 operatively linked to a suitable control element.

57. A host cell comprising the polypeptide, fusion protein, nucleic acid, and / or expression vector of any preceding claim.

58. A pharmaceutical composition, comprising: (a) the polypeptide, fusion protein, nucleic acid, expression vector, and / or host cell of any preceding claim; and (b) a pharmaceutically acceptable carrier.

59. A method for degrading a target protein, comprising contacting a biological sample comprising the target with the fusion protein of any previous claim, under conditions to promote binding of the fusion protein to target protein present in the biological sample, thereby promoting degradation of the target protein.

60. A fusion protein, comprising: (a) the polypeptide or fusion polypeptide of any preceding claim; and (b) a detectable polypeptide.

61. A method for detecting an ASGPR, transferrin receptor, or IGF-2R receptor, comprising contacting a biological sample comprising the receptor with the fusion protein of claim 60, under conditions to promote binding of the fusion protein to receptor present in thesample, and detecting signal from the detectable polypeptide, thus detecting the receptor in the biological sample.

62. A fusion protein, comprising: (a) the polypeptide or fusion polypeptide of any preceding claim; and (b) a therapeutic polypeptide.

63. A method for treating a disorder, comprising contacting a subject with the fusion protein of claim 62, under conditions to promote binding of the fusion protein to ASGPR, transferrin receptor, or IGF-2R in cells of the subject, thus treating the disorder.

64. The method of claim 63, wherein the disorder is selected from the group consisting of lysosomal dysfunction, neurodegenerative diseases (including but not limited to Parkinson's disease. Alzheimer's disease. Huntington's disease), cancer, and autoimmune disease.

65. A method for treating a disease, comprising administering to a subject in need thereof an amount effective of the fusion protein or antibody or antigen binding fragment thereof of any one of claims 41-54, to degrade target protein present in the subject, thereby treating the disease.

66. A composition comprising: (a) the polypeptide or fusion polypeptide of any preceding claim; and (b) a non-protein functional moiety linked to the polypeptide or fusion polypeptide.

67. The composition of claim 66, wherein the non-protein functional moiety comprises a nucleic acid, including but not limited to an inhibitor nucleic acid (siRNA, antisense oligonucleotide, small molecule cytotoxic drug such as MMAE; DNA aptamers that binding affinity to intracellular targets or in the lysosome; polysaccharide that binds to intracellular targets or in the lysosome etc.)

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