Sortilin-1 binders, constructs and uses thereof

Abstract

A polypeptide comprising a binder, the binder having a binding affinity to Sortilin-1 (Sort1) receptor and constructs thereof. The Sort1 binder capable of binding to the Sort1 receptor when the Sort1 receptor is in a protonated state, and wherein the polypeptide is further capable of being released from the Sort1 receptor when the Sort1 receptor is in a deprotonated state. The constructs a binder moiety and a cargo moiety, the cargo moiety linked to the binder moiety via a linker, the binder moiety being capable of binding to a Sortilin-1 (Sort1) receptor. The Sort1 binder and / or the construct comprising the Sort1 binder of use in promoting internalization a cargo, internalization of a therapeutic agent or extracellular protein degradation.

Description

[0001] SORTILIN-1 BINDERS, CONSTRUCTS AND USES THEREOF

[0002] TECHNICAL FIELD

[0003] Described herein are targeting moieties for use in molecules directed towards targeting membrane proteins, including molecules comprising Sortilin-1 (SORT1) binders.

[0004] BACKGROUND

[0005] Sortilin-1 (Sortl or SORT1) is a member of the vacuolar protein sorting 10 protein (VPSIOP)-domain receptor family (Zhao Z, Ukidve A, Kim J, Mitragotri S. Targeting Strategies for Tissue-Specific Drug Delivery. Cell. 2020;181(1):151-67). It is expressed in various tissues, including metabolically active tissues (e.g., liver, muscle, adipose tissue), immune cells (e.g., macrophages, lymphocytes), and the central nervous system.

[0006] It generally functions as a single-pass transmembrane protein involved in intracellular vesicle trafficking within the endocytic or exocytic pathways (Toth AE, Nielsen MS. Sortilins in the blood-brain barrier: impact on barrier integrity. Neural Regen Res. 2023;18(3):549-50), serving as a receptor that binds to ligands and internalizes them while further being capable of interacting with adaptor proteins to facilitate sorting thereafter.

[0007] More specifically, the luminal domain of Sortl interacts with its protein ligands within trafficking vesicles, while the cytoplasmic domain of Sortl facilitates the recruitment of adaptor proteins essential for vesicular trafficking.

[0008] This function of Sortl has been found to play a role in mediating the intracellular trafficking of proteins involved in diverse cellular pathways in different tissues and cell types (Hermey G. The Vps1 Op-domain receptor family. Cell Mol Life Sci. 2009;66(16):2677-89). For instance, Sortl receptors are believed to regulate cholesterol metabolism via low-density lipoprotein (LDL) uptake in the liver while also being involved in the uptake of neuropeptides and growth factors in the brain.

[0009] SUMMARY

[0010] The widespread availability of Sortl within different tissues and cells as well as its function of regulating uptake of ligands make of it a gateway for numerous applications. However, uses of this transmembrane protein generally require selective binding thereof which remains a challenge. There always remains room for improvement.

[0011] Described herein are Sortilin-1 ligands (generally referred to herein as Sortilin-1 binders) selective to the Sortilin-1 receptor. Also described herein are Sortilin-1 binders capable of taking advantage of pH sensitive conformations changes of Sortl receptor, and preferably to provide Sortilin-1 binders capable of being released after binding to Sortl . Also described herein are Sortilin-1 binders having a biologically relevant binding affinity, more preferably to provide Sortilin-1 binders having a binding affinity lower or equal to 2 uM. Also described herein are Sortilin-1 binders capable of binding at the surface of a cell. Also described herein are Sortilin-1 binders capable of internalizing via the Sortl biological mechanism.

[0012] Also described herein are constructs comprising a Sortilin-1 binder, such as those satisfying one or various of the above functions. Also described herein are constructs comprising a Sortilin-1 binder capable of binging to the Sortl receptor and leveraging the biological processes thereof. Also described herein are constructs comprising a Sortilin-1 binder capable of internalizing and carrying a cargo therewith into a cell. Also described herein are constructs comprising a Sortilin-1 binder having the capability to transcytose a cargo into a cell. Also described herein are constructs comprising a Sortilin-1 binder having the capability to promote protein degradation, such as within the use of protein lysosometargeting chimeras for instance. Also described herein are constructs comprising a Sortilin-1 binder and a cargo, where the cargo retains function.

[0013] As will be evident below, the inventors have surprisingly found that providing certain combinations of amino acid residues resulted in Sortl ligands, generally referred to herein as binders to Sortl or simply “binders”, which provide a biologically suitable binding affinity to Sortl, and which are capable of binding to Sortilin-1 receptor. Further, the inventors have identified that such binders may exploit the pH sensitive conformation changes of Sortl , as will be described in detail herein, to be released throughout the biological confirmational change of Sortl.

[0014] The inventors have further identified that the binders may form part of constructs with desired cargos for varied applications, leading to molecules that leverage the Sortl binder for use. For instance, in some embodiments, the constructs provide the capacity to internalize and carry a cargo into a cell and / or have the capacity for transcytosis of a cargo into other cell types, while retaining the function of the cargo. In another embodiment, the constructs comprising a Sortl binder forms a protein lysosome-targeting chimeras, promoting degradation of desired proteins.

[0015] In some embodiments, a combination of benefits is provided by constructs described herein. In some embodiments, a construct comprising a Sortl binder provides a benefit of being capable of internalizing and carrying a cargo therewith into a cell and also provides a benefit of taking advantage of pH sensitive conformations changes of Sortl. Sortilin- 1 binders described herein also solve further problems that will be apparent from the disclosure of the exemplary embodiments.

[0016] In accordance with one aspect, there is provided a polypeptide having a binding affinity to Sortilin-1 (SORT1) receptor.

[0017] In accordance with another aspect, there is provided a polypeptide comprising a binder, the binder having a binding affinity to Sortilin-1 (SORT1) receptor and an amino acid sequence at least 50%, 60%, 70%, 79%, 80%, 82%, 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 1-39, preferably at least 79%, 82%, 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1-4 or 6-39.

[0018] In accordance with another aspect, there is provided a polypeptide comprising the amino acid sequence according to Formula II (SEQ ID No. 92) :

[0019] Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu- Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu- Ile-Val-Glu-Glu-Leu-Xaa57-Xaa58, wherein

[0020] Xaa4 is Leu (L) or Phe (F);

[0021] Xaa6 is Gin (Q) or His (H) or Ser (S) or Thr (T);

[0022] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0023] XaalO is Vai (V) or lie (I);

[0024] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0025] Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);

[0026] Xaa25 is Glu (E) or Lys (K);

[0027] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0028] Xaa30 is Leu (L) or Thr (T) or lie (I);

[0029] Xaa37 is Asn (N) or Gin (Q) or His (H) or Lys (K);

[0030] Xaa57 is Absent or Leu (L); and

[0031] Xaa58 is Absent or Glu (E),

[0032] Preferably comprising the amino acid sequence as set out in any one of SEQ ID Nos. 5, 2, 6, 14, 24, 29.

[0033] In accordance with another aspect, there is provided a construct comprising a polypeptide as defined in any one of the above aspects, or as further discloses herein, linked to at least one cargo moiety, and preferably where the at least one cargo moiety is linked as a recombinant fusion, chemical conjugation, or combination thereof. In accordance with another aspect, there is provided a construct comprising a binding moiety and a cargo moiety, the binding moiety comprising a binder having a binding affinity to Sortilin-1 (SORT1) receptor and comprising an amino acid sequence as set out in any one of SEQ ID Nos. 5, 2, 6, 14, 24, 29.

[0034] In accordance with yet another embodiment, there is provided pharmaceutical compositions comprising any one of the polypeptides or constructs identified above or described herein and a pharmaceutical excipient. The pharmaceutical compositions further may comprise a pharmaceutically acceptable buffer. The pharmaceutical compositions may be suitable for parenteral administration such as subcutaneous, intramuscular, intraperitoneal, or intravenous administration. The pharmaceutical composition may be a liquid pharmaceutical formulation.

[0035] In accordance with another aspect, any one of the polypeptides, constructs, or pharmaceutical compositions identified above or described herein for use as a medicament, for use as therapeutic agent, for use in promoting internalization of a cargo, for use in promoting internalization of a therapeutic agent, for use in promoting clearance of a cargo, for use in promoting clearance of a therapeutic agent or for use in promoting extracellular protein degradation.

[0036] SEQUENCE LISTING

[0037] SEQ ID Nos. 1-39 represent the amino acid sequence of non-limitative examples of Sortl binders polypeptides CBO to CB39 in accordance with the invention;

[0038] SEQ ID No. 40 represents the amino acid sequence of huSORTI (78-756) with an extension;

[0039] SEQ ID No. 41 represents the amino acid sequence of the extension of SEQ ID No. 40;

[0040] SEQ ID Nos. 42 and 43 represent the amino acid sequence of huSORTI (78-756) and moSORTI (76-744), respectively, with an Avitag;

[0041] SEQ ID Nos. 44 and 45 represent the amino acid sequence of a TEV site and Myctag, respectively;

[0042] SEQ ID Nos. 46-81 represent the amino acid sequence of non-limitative example constructs comprising the exemplified Sortl binders disclosed herein;

[0043] SEQ ID Nos. 82 and 83 represent the amino acid sequence of huSORTI (78-756) with a point mutation at residue no. 500;

[0044] SEQ ID Nos. 84-86 represents the amino acid sequence of non-limitative example linkers provided within the constructs; SEQ ID No. 87 represents the amino acid sequence of a non-binding green fluorescent protein (GFP) construct;

[0045] SEQ ID Nos. 88 and 89 represent the amino acid sequence of non-binding GFP and mScarlet fusion constructs;

[0046] SEQ ID No. 90 represents the amino acid sequence of a hemaglutamin (HA) tagged moSORTI;

[0047] SEQ ID No. 91 represents the amino acid sequence of a minibinder targeting EGFR N-terminal (EGFRn); and

[0048] SEQ ID No. 92-95 represent the amino acid sequences according to Formulae I l-V respectively.

[0049] BRIEF DESCRIPTION OF DRAWINGS

[0050] Fig. 1A depicts an example construct comprising a Sortl binder and a cargo, linked to the Sortl binder at its C-terminus;

[0051] Fig. 1B depicts another example construct comprising a Sortl binder and a cargo, linked to the Sortl binder at its N-terminus;

[0052] Fig. 1C depicts yet another example construct comprising a Sortl binder, a cargo and tag;

[0053] Fig. 1 D depicts yet another example construct comprising a Sortl binder and more than one cargo;

[0054] Fig. 2 depicts another example construct comprising a Sortl and a cargo chemically conjugated to the Sortl binder;

[0055] Figs. 3 depicts a non-limitative flow chart of a computation methods used to design and identify potential Sortl binders;

[0056] Fig. 4A depicts ribbon diagrams of a Sortl binder of the present disclosure (CB1) docked onto a human Sortl receptor;

[0057] Figs. 4B and 4C depict a ribbon diagram of a Sortl binder of the present disclosure (CB1) with a surface representation of the human Sortl receptor at neutral pH (Fig. 4B) and acidic pH (4C) demonstrating complementarity and non-complementarity, respectively;

[0058] Figs. 5A-5D depict qualitative binding analysis results by SPR of example construct 3 against huSORTI, demonstrating pH dependence of binding;

[0059] Fig. 6 shows confocal imagery of a dose ranging study with Sortl binder construct 27 seen internalized (GFP stain, dark spots); Fig. 7 depicts a model for the blood brain barrier organoid used in the experimental section herein, identifying an example construct which goes through Sortl mediated transcytosis; and

[0060] Fig. 8 shows a western blot analysis of total EGFR level in HeLa cells after treatment with EGFRn and Construct 36 for 48h.

[0061] DESCRIPTION

[0062] As described herein, the disclosure provides Sortl binders. In some embodiments, the Sortl binders can be said to be ligands of the Sortl receptor. In some embodiments, the disclosure provides Sortl binders that are capable of binding to human and murine Sortl .

[0063] In some embodiments, the disclosure provides Sortl binders that are capable of binding to human and murine Sortl receptor and thus can be used in the context of constructs, ultimately playing the role of a Sortl ligand capable of interacting with the luminal domain of a Sortl receptor within such a construct.

[0064] In some embodiments, the constructs comprising a Sortl binder of the instant disclosure is used to internalize and / or carry a cargo into a cell, such a therapeutic agent, for instance. In some embodiments, the constructs comprising the Sortl binder of the instant disclosure is used as a protein lysosome-targeting chimera.

[0065] One of skill in the art understands that the Sortl binder, a fusion peptide comprising the Sortl binder or a construct comprising the Sortl binder of the instant disclosure can be used to leverage the biological mechanisms of the Sortl receptors and that applications are not to be limited to the explicit mechanisms exemplified herein, such as internalization of a cargo, delivery of cargo or promoting protein degradation

[0066] Sortl Binders

[0067] In some embodiments, the Sortl binder is a polypeptide of about 56 residues in length. In some embodiments, the Sortl binder is a polypeptide at least 57 residues in length. In some embodiments, the Sortl binder is a polypeptide of about 58 residues in length. In some embodiments, the Sortl binder is a polypeptide of no more than 58 residues in length. In some embodiments, the Sortl binder is a polypeptide of about 56-58 residues in length. In some embodiments, the Sortl binder is polypeptide of 56-58 residues in length.

[0068] In some embodiments, the Sortl binder is a polypeptide of 56-58 residues in length while further having a C-terminal, N-terminal, or both C-terminal and N-terminal extensions, preferably forming a construct. In some embodiments, the Sortl binder may be a polypeptide with an amino acid sequence at least 50%, such as at least 60%, at least 70%, at least 79%, at least 80%, at least 82%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 1-39.

[0069] In some embodiments, the Sortl binder may be a polypeptide with an amino acid sequence at least 79% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 1-39.

[0070] In some embodiments, the Sortl binder may be a polypeptide with an amino acid sequence at least 82% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 1-39.

[0071] In some embodiments, the Sortl binder may be a polypeptide with an amino acid sequence at least 87% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 1-39.

[0072] In some embodiments, the Sortl binder may have a polypeptide with no more than 12 modifications, such as 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modifications, relative to the full length of any one of SEQ ID Nos. 1-39. In some embodiments, the Sortl binder may have a polypeptide with no more than 12 modifications, such as 11, 10, 9 or 8 modifications, relative to the full length of any one of SEQ ID Nos. 1-39. In some embodiments, the Sortl binder may have a polypeptide with no more than 7 modifications, such as 6, 5, 4, 3 or 2 modifications, relative to the full length of any one of SEQ ID Nos. 1-39. In some embodiments, the Sortl binder may have a polypeptide with no more than 1 modification relative to the full length of any one of SEQ ID Nos. 1-39.

[0073] In some embodiments, the Sortl binder may be a polypeptide with an amino acid sequence at least 50%, such as at least 60%, at least 70%, at least 79%, at least 80%, at least 82%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1, 6, 14, 24, 29.

[0074] In some embodiments, the Sortl binder may be a polypeptide with an amino acid sequence at least 79% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1, 6, 14, 24, 29.

[0075] In some embodiments, the Sortl binder may be a polypeptide with an amino acid sequence at least 82% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1, 6, 14, 24, 29. In some embodiments, the Sortl binder may be a polypeptide with an amino acid sequence at least 87% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1, 6, 14, 24, 29.

[0076] In some embodiments, the Sortl binder may have a polypeptide with no more than 12 modifications, such as 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modifications, relative to the full length of any one of SEQ ID Nos. 5, 1, 6, 14, 24, 29. In some embodiments, the Sortl binder may have a polypeptide with no more than 12 modifications, such as 11, 10, 9 or 8 modifications, relative to the full length of any one of SEQ ID Nos. 5, 1, 6, 14, 24, 29. In some embodiments, the Sortl binder may have a polypeptide with no more than 7 modifications, such as 6, 5, 4, 3 or 2 modifications, relative to the full length of any one of SEQ ID Nos. 5, 1, 6, 14, 24, 29. In some embodiments, the Sortl binder may have a polypeptide with no more than 1 modification relative to the full length of any one of SEQ ID Nos. 5, 1 , 6, 14, 24, 29.

[0077] In some embodiments, the modifications, or variations relative to a reference sequence forming the difference in sequence identity are selected from those identified in Table 1.

[0078] In some embodiments, the Sortl binder disclosed herein may comprise a polypeptide with the amino acid (AA) residues identified in Table 1.

[0079] In some embodiments, the Sortl binder is a polypeptide of 56-58 residues numbered from N-terminus to C-terminus and comprising the residues identified in Table 1. The residues listed in a given row are alternatives to one another.

[0080] Table 1. Potential variation of Sortl binder residues.

[0081] In some embodiments, the Sortl binder disclosed herein may comprise a polypeptide of Formula I as follows:

[0082] Xaa1 -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-

[0083] Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Xaa22-Xaa23-Xaa24-

[0084] Xaa25-Trp-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36- Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45-Xaa46-Xaa47-

[0085] Xaa48-Xaa49-Xaa50-Xaa51-Xaa52-Xaa53-Xaa54-Xaa55-Xaa56-Xaa57-Xaa58 wherein

[0086] Xaa1 is Ser (S) or Gly (G) or Asn (N) or Ala (A) or Arg (R) or His (H);

[0087] Xaa2 is lie (I) or Tyr (Y) or Phe (F) or Leu (L) or Met (M) or Trp (W) or His (H) or Pro (P) or Vai (V);

[0088] Xaa3 is Leu (L) or lie (I) or Met (M);

[0089] Xaa4 is Tyr (Y) or Cys (C) or Leu (L) or Arg (R) or His (H) or Met (M) or Vai (V) or Gin (Q) or Pro (P) or Lys (K);

[0090] Xaa5 is Arg (R) or Lys (K) or Leu (L) or Vai (V) or Asn (N) or Ser (S) or Met (M) or Thr (T) or Gin (Q) or lie (I) or Ala (A) or Gly (G);

[0091] Xaa6 is Ser (S) or His (H) or Leu (L) or Thr (T) or Tyr (Y) or Gly (G) or Asn (N) or Gin (Q) or Phe (F) or Ala (A) or Pro (P) or Arg (R) or Met (M);

[0092] Xaa7 is Thr (T) or Ala (A) or Cys (C) or Ser (S);

[0093] Xaa8 is lie (I) or Vai (V) or Ala (A) or Met (M) or Thr (T);

[0094] Xaa9 is Arg (R) or Lys (K) or Vai (V);

[0095] Xaa10 is Vai (V) or lie (I) or Thr (T);

[0096] Xaa11 is lie (I) or Vai (V) or Cys (C) or Thr (T) or Ala (A) or Ser (S);

[0097] Xaa12 is Glu (E) or Asp (D) or Gin (Q) or Vai (V) or His (H) or Tyr (Y) or Met (M) or Gly (G) or lie (I) or Thr (T) or Ser (S) or Arg (R) or Lys (K) or Asn (N) or Ala (A);

[0098] Xaa13 is Arg (R) or Lys (K) or His (H) or Asn (N) or Ser (S); Xaa14 is Leu (L) or lie (I) or Arg (R) or Gin (Q) or Met (M);

[0099] Xaa15 is lie (I) or Leu (L) or Cys (C) or Vai (V) or Ala (A) or Met (M) or Thr (T);

[0100] Xaa16 is Arg (R) or Ala (A) or Gin (Q) or Gly (G) or Lys (K) or His (H) or Ser (S) or Asp (D) or Thr (T) or Tyr (Y) or Asn (N) or Met (M) or Leu (L) or Vai (V) or lie (I) or Glu (E) or Trp (W) or Phe (F) or Cys (C);

[0101] Xaa17 is Gin (Q) or Arg (R) or Ala (A) or Lys (K) or Asn (N) or Gly (G) or Glu (E) or Ser (S) or His (H) or Asp (D) or Met (M) or Leu (L) or Vai (V) or Phe (F) or Tyr (Y) or lie (I) or Thr (T) or Trp (W) or Cys (C);

[0102] Xaa18 is Lys (K) or Arg (R) or Ser (S) or Asp (D) or Gin (Q) or His (H) or Gly (G) or Asn (N) or Glu (E) or Ala (A) or Met (M) or Pro (P) or Phe (F) or Cys (C) or Thr (T) or Tyr (Y) or Vai (V) or Leu (L) or Trp (W);

[0103] Xaa19 is Arg (R) or Ser (S) or Lys (K) or Gin (Q) or Asn (N) or Glu (E) or Asp (D) or Ala (A) or His (H) or Thr (T) or Met (M) or Cys (C) or Phe (F) or Leu (L) or Vai (V) or Tyr (Y) or lie (I);

[0104] Xaa20 is Leu (L) or Thr (T) or Phe (F) or Cys (C) or Asn (N) or Ser (S) or His (H) or Met (M) or Vai (V) or Ala (A) or Trp (W) or Tyr (Y) or lie (I) or Lys (K) or Gly (G) or Gin (Q) or Arg (R) or Pro (P) or Asp (D);

[0105] Xaa21 is Arg (R) or Lys (K) or Asn (N) or Ser (S) or Thr (T) or Gly (G) or Leu (L) or Met (M) or His (H) or lie (I) or Phe (F) or Asp (D) or Ala (A) or Vai (V) or Pro (P) or Gin (Q) or Tyr (Y) or Glu (E) or Cys (C);

[0106] Xaa22 is Vai (V) or Thr (T) or Lys (K) or Arg (R) or Met (M) or lie (I) or Ala (A) or His (H) or Gin (Q) or Ser (S) or Cys (C) or Pro (P) or Asp (D) or Glu (E) or Leu (L) or Asn (N) or Trp (W) or Gly (G) or Phe (F);

[0107] Xaa23 is Asp (D) or Ala (A) or Gly (G) or Ser (S);

[0108] Xaa24 is Ser (S) or Met (M) or His (H) or Thr (T) or Ala (A) or Gly (G) or Phe (F) or Lys (K) or lie (I) or Asn (N) or Vai (V) or Arg (R) or Cys (C) or Leu (L) or Gin (Q) or Tyr (Y) or Glu (E) or Trp (W) or Asp (D);

[0109] Xaa25 is Arg (R) or Lys (K) or Gin (Q) or Ser (S) or Ala (A) or Thr (T) or Asp (D) or Asn (N) or His (H) or Glu (E) or Met (M) or Gly (G) or Pro (P) or Vai (V) or lie (I) or Leu (L);

[0110] Xaa27 is Leu (L) or Cys (C) or lie (I) or Vai (V) or Thr (T) or Asn (N);

[0111] Xaa28 is Pro (P) or Vai (V) or Tyr (Y) or Lys (K) or lie (I) or Ala (A) or Thr (T) or His (H) or Asn (N) or Gin (Q) or Leu (L) or Ser (S) or Trp (W) or Arg (R) or Met (M) or Gly (G) or Phe (F) or Cys (C) or Glu (E); Xaa29 is Cys (C) or Gin (Q) or Thr (T) or Vai (V) or His (H) or Ser (S) or Lys (K) or Ala (A) or Glu (E);

[0112] Xaa30 is Thr (T) or Vai (V) or Leu (L) or Cys (C) or lie (I);

[0113] Xaa31 is lie (I) or Lys (K) or Phe (F) or His (H) or Thr (T) or Met (M) or Arg (R) or Vai (V) or Leu (L) or Tyr (Y) or Gin (Q) or Trp (W) or Asn (N) or Ser (S) or Ala (A);

[0114] Xaa32 is Lys (K) or Asn (N) or Tyr (Y) or Trp (W) or His (H) or Arg (R) or Vai (V) or Gly (G) or Ser (S) or Phe (F) or Ala (A) or Gin (Q) or Met (M) or Leu (L) or Cys (C) or Thr (T) or lie (I);

[0115] Xaa33 is Leu (L) or lie (I) or Vai (V);

[0116] Xaa34 is Leu (L) or Ser (S) or Ala (A) or Gly (G) or Cys (C);

[0117] Xaa35 is Cys (C) or Tyr (Y) or lie (I) or Vai (V) or His (H) or Thr (T) or Lys (K) or Phe (F) or Arg (R) or Leu (L) or Met (M) or Trp (W) or Gin (Q) or Gly (G) or Ser (S) or Asn (N) or Glu (E);

[0118] Xaa36 is Leu (L) or Arg (R) or Lys (K) or Vai (V) or Ala (A) or Met (M) or lie (I) or Ser (S) or Gin (Q) or Thr (T) or Trp (W) or Tyr (Y) or Phe (F) or His (H) or Asn (N);

[0119] Xaa37 is Gin (Q) or Asn (N) or His (H) or Leu (L);

[0120] Xaa38 is Arg (R) or Ala (A) or His (H) or Gin (Q) or Lys (K) or Cys (C) or Met (M) or Thr (T) or Asp (D) or Asn (N) or Gly (G) or Phe (F) or Tyr (Y) or Vai (V) or Glu (E) or Ser (S) or lie (I) or Trp (W) or Leu (L);

[0121] Xaa39 is Asp (D) or Asn (N) or Gin (Q) or Ala (A) or Met (M) or Glu (E);

[0122] Xaa40 is Phe (F) or Trp (W) or Lys (K) or Arg (R) or Gin (Q) or Gly (G) or Tyr (Y) or Vai (V) or lie (I) or Asn (N) or His (H) or Ala (A) or Leu (L) or Met (M) or Pro (P) or Cys (C) or Ser (S) or Asp (D) or Thr (T);

[0123] Xaa41 is Trp (W) or Tyr (Y) or Gly (G) or His (H) or Phe (F) or Arg (R) or Leu (L) or Gin (Q) or Ser (S) or Asn (N) or Met (M) or Ala (A) or Vai (V) or Asp (D) or Glu (E) or Lys (K) or lie (I) or Thr (T) or Cys (C);

[0124] Xaa42 is Vai (V) or Ser (S) or Ala (A) or lie (I) or Cys (C) or Thr (T) or Met (M);

[0125] Xaa43 is Tyr (Y) or Trp (W) or Phe (F) or Met (M) or Leu (L) or Ala (A) or lie (I) or Gin (Q) or Vai (V) or Arg (R) or Ser (S) or Cys (C) or His (H) or Glu (E) or Thr (T) or Lys (K) or Gly (G) or Asn (N);

[0126] Xaa44 is Vai (V) or Leu (L) or His (H) or Arg (R) or Lys (K) or Ser (S) or Gin (Q) or Tyr (Y) or Phe (F) or Ala (A) or Met (M) or Cys (C) or lie (I) or Asn (N) or Glu (E) or Trp (W) or Gly (G) or Thr (T);

[0127] Xaa45 is Arg (R) or Lys (K) or Ala (A) or Cys (C) or His (H) or lie (I) or Vai (V) or Ser (S); Xaa46 is Ala (A) or Cys (C) or Ser (S) or Vai (V) or Gly (G);

[0128] Xaa47 is Vai (V) or Phe (F) or Tyr (Y) or Arg (R) or Leu (L) or lie (I) or Lys (K) or Gly

[0129] (G) or Ala (A) or Cys (C) or His (H) or T rp (W) or Glu (E) or Asn (N) or Gin (Q) or Thr

[0130] (T) or Met (M) or Asp (D) or Ser (S);

[0131] Xaa48 is Gin (Q) or Glu (E) or Ala (A) or Lys (K) or Asp (D) or Met (M) or Vai (V) or Arg (R) or Tyr (Y) or His (H) or Ser (S) or lie (I) or Asn (N) or Leu (L) or Cys (C);

[0132] Xaa49 is Leu (L) or Met (M);

[0133] Xaa50 is Gin (Q) or Arg (R) or Ser (S) or Met (M) or Trp (W) or Ala (A) or Tyr (Y) or His (H) or Lys (K) or Gly (G) or Thr (T) or Asn (N) or Phe (F) or Glu (E) or Asp (D) or Cys (C) or Leu (L);

[0134] Xaa51 is Lys (K) or Arg (R) or lie (I) or Phe (F) or Ala (A) or Cys (C) or Asn (N) or Gin (Q) or Asp (D) or Trp (W) or Gly (G) or Leu (L) or Ser (S) or Thr (T) or Vai (V) or Tyr (Y) or His (H) or Glu (E) or Met (M);

[0135] Xaa52 is Trp (W) or Tyr (Y) or Met (M) or Phe (F) or lie (I) or Leu (L) or Ala (A) or Lys (K) or Vai (V) or His (H) or Thr (T) or Ser (S) or Glu (E) or Arg (R) or Cys (C) or Gin (Q) or Asp (D) or Gly (G) or Asn (N);

[0136] Xaa53 is Ala (A) or Cys (C) or Arg (R) or Leu (L) or Vai (V) or Met (M) or lie (I) or Lys (K) or Thr (T) or Tyr (Y) or Gin (Q) or Trp (W) or Asn (N) or Ser (S) or His (H) or Phe (F) or Gly (G);

[0137] Xaa54 is Gly (G) or Gin (Q) or Thr (T) or Leu (L) or Arg (R) or Asp (D) or lie (I) or Tyr (Y) or Asn (N) or Lys (K) or Ala (A) or Met (M) or Phe (F) or His (H) or Glu (E) or T rp (W) or Ser (S) or Cys (C);

[0138] Xaa55 is Gly (G) or Lys (K) or Vai (V) or Ser (S) or His (H) or Met (M) or Arg (R) or Leu (L) or Asp (D) or Asn (N) or Gin (Q) or Phe (F) or Ala (A) or Thr (T) or Glu (E) or Tyr (Y) or Cys (C) or lie (I) or Trp (W);

[0139] Xaa56 is Met (M) or lie (I) or Leu (L) or Phe (F) or Arg (R) or Lys (K) or Vai (V) or Asn (N) or Trp (W) or Ala (A) or Tyr (Y) or Cys (C) or Thr (T) or Gin (Q) or His (H) or Ser (S) or Gly (G);

[0140] Xaa57 is Absent or Leu (L); and

[0141] Xaa58 is Absent or Glu (E).

[0142] In some embodiments, the Sortl binder disclosed herein may comprise a polypeptide of Formula II, identified in the sequence listing SEQ ID No. 92 as follows:

[0143] Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu-

[0144] Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu-

[0145] Ile-Val-Glu-Glu-Leu-Xaa57-Xaa58, wherein

[0146] Xaa4 is Leu (L) or Phe (F);

[0147] Xaa6 is Gin (Q) or His (H) or Ser (S) or Thr (T);

[0148] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0149] XaalO is Vai (V) or lie (I);

[0150] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0151] Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);

[0152] Xaa25 is Glu (E) or Lys (K);

[0153] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0154] Xaa30 is Leu (L) or Thr (T) or lie (I);

[0155] Xaa37 is Asn (N) or Gin (Q) or His (H) or Lys (K);

[0156] Xaa57 is Absent or Leu (L); and

[0157] Xaa58 is Absent or Glu (E).

[0158] In some embodiments, the Sortl binder disclosed herein may comprise a polypeptide of Formula III, identified in the sequence listing SEQ ID No. 93 as follows:

[0159] Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu-

[0160] Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu- lle-Val-Glu-Glu-Leu-Leu-Glu, wherein

[0161] Xaa4 is Leu (L) or Phe (F);

[0162] Xaa6 is His (H) or Ser (S) or Thr (T) or Gin (Q);

[0163] Xaa8 is Vai (V) or lie (I) or Thr (T) or Met (M);

[0164] XaalO is Vai (V) or lie (I);

[0165] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0166] Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);

[0167] Xaa25 is Lys (K) or Glu (E);

[0168] Xaa27 is Vai (V) or lie (I) or Leu (L);

[0169] Xaa30 is Leu (L) or Thr (T) or lie (I); and

[0170] Xaa37 is Gin (Q) or Asn (N) or His (H) or Lys (K). In some embodiments, the Sortl binder disclosed herein may comprise a polypeptide of Formula IV, identified in the sequence listing SEQ ID No. 94 as follows:

[0171] Ser-Phe-Leu-Xaa4-Lys-His-Ala-Xaa8-Arg-Val-Xaa11-Glu-Arg-Leu-Leu-Glu-Glu-Gly- Asn-Val-Glu-Val-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-lle-Lys-Arg-Leu-Ala-His-Ala- Gln-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu-lle-Val-Glu-Glu- Leu-Xaa57-Xaa58, wherein

[0172] Xaa4 is Leu (L) or Phe (F);

[0173] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0174] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0175] Xaa25 is Glu (E) or Lys (K);

[0176] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0177] Xaa57 is Absent or Leu (L); and

[0178] Xaa58 is Absent or Glu (E).

[0179] In some embodiments, the Sortl binder disclosed herein may comprise a polypeptide of Formula V, identified in the sequence listing SEQ ID No. 95 as follows:

[0180] Ser-Phe-Leu-Leu-Lys-Xaa6-Ala-Xaa8-Arg-Val-Xaa11-Glu-Arg-Leu-Leu-Glu-Glu-Gly- Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg-Leu-Ala-His- Ala-GIn-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu-lle-Val-Glu-Glu- Leu-Leu-Glu, wherein

[0181] Xaa6 is His (H) or Ser (S);

[0182] Xaa8 is Vai (V) or lie (I) or Met (M);

[0183] Xaa11 is lie (I) or Vai (V);

[0184] Xaa22 is Leu (L) or Vai (V) or lie (I) or Thr (T);

[0185] Xaa25 is Lys (K) or Glu (E);

[0186] Xaa27 is Vai (V) or lie (I); and

[0187] Xaa30 is Leu (L) or lie (I) or Thr (T).

[0188] In some embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 or all 18 of the following residues of a Sortl binder, with reference to the numbering of any one of Formulas l-V or in any one of SEQ ID No. 1-39, are invariant: Seri , Phe2, Leu3, Lys5, His6, Ala7, Arg9, Val10, Arg13, Leu14, Val22, Glu25, Trp26, Gln29, He30, Leu33, Ala36, Gln37. In some embodiments, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11 , 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 of the following residues of a Sortl binder, with reference to the numbering of any one of Formulas l-V or in any one of SEQ ID No. 1-39, are invariant: Seri , Phe2, Leu3, Lys5, His6, Ala7, Arg9, Val10, Arg13, Leu14, Val22, Glu25, Trp26, Gln29, lle30, Leu33, Ala36, Gln37. In some embodiments, no more than 18, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11 , no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3 or no more than 2 of the following residues of a Sortl binder, with reference to the numbering of any one of Formulas l-V or in any one of SEQ ID No. 1-39, are invariant: Seri, Phe2, Leu3, Lys5, His6, Ala7, Arg9, Val10, Arg13, Leu14, Val22, Glu25, Trp26, Gln29, He30, Leu33, Ala36, Gln37. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 at least 13, at least 14, at least 15, at least 16 or at least 17 of the following residues of a Sortl binder, with reference to the numbering of any one of Formulas l-V or in any one of SEQ ID No. 1-39, are invariant: Seri, Phe2, Leu3, Lys5, His6, Ala7, Arg9, Val10, Arg13, Leu14, Val22, Glu25, Trp26, Gln29, lle30, Leu33, Ala36, Gln37. In some embodiments, the following residues of a Sortl binder, with reference to the numbering of any one of Formulas l-V or in any one of SEQ ID No. 1-39, are invariant: Seri , Phe2, Leu3, Lys5, His6, Ala7, Arg9, Val10, Arg13, Leu14, Val22, Glu25, Trp26, Gln29, He30, Leu33, Ala36, Gln37.

[0189] In some embodiments, the Sortl binder has a binding affinity to Sortl receptor. In some embodiments, the Sortl binder is capable of binding to human or murine Sortl , preferably at the Sortl receptor interface. In some embodiments, the Sortl binder is capable of binding to human and murine Sortl , preferably at the Sortl receptor interface. In some embodiments, the Sortl binder is capable of binding to human Sortl , preferably at the Sortl receptor interface. In some embodiments, the Sortl binder is capable of binding to murine Sortl, preferably at the Sortl receptor interface.

[0190] In some embodiments, the Sortl binder disclosed herein binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or all 17 of the following residues of the Sortl receptor: Thr396, Leu419, Asp422, Asn423, Ile425, Ala450, Thr451 , Ala452, Tyr466, Gln470, Lys471, Leu472, Ala497, Ile498, Ser499, Val500, Met501, where the residue positions refers to the native positions of human Sortilin-1 (Uniprot Q99523). In some embodiments, the Sortl binder disclosed herein binds between 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to

[0191] 11 , 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 of the following residues of the Sortl receptor: Thr396, Leu419, Asp422, Asn423, Ile425, Ala450, Thr451, Ala452, Tyr466, Gln470, Lys471 , Leu472, Ala497, Ile498, Ser499, Val500, Met501 , where the residue positions refers to the native positions of human Sortilin-1 (Uniprot Q99523). In some embodiments, the Sortl binder disclosed herein binds to at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16 or all 17 of the following residues of the Sortl receptor: Thr396, Leu419, Asp422, Asn423, Ile425, Ala450, Thr451, Ala452, Tyr466, Gln470, Lys471, Leu472, Ala497, Ile498, Ser499, Val500, Met501, where the residue positions refers to the native positions of human Sortilin-1 (Uniprot Q99523).

[0192] In some embodiments, the Sortl binder is capable of binding to human or murine Sortl with a Kdof about 2uM or less. In some embodiments, the Sortl binder is capable of binding to human and murine Sortl with a Kdof about 2uM or less. In some embodiments, the Sortl binder is capable of binding to human or murine Sortl with a Kd of about 1uM or less. In some embodiments, the Sortl binder is capable of binding to human and murine Sortl with a Kdof about 1uM or less. In some embodiments, the Sortl binder is capable of binding to human or murine Sortl with a Kdof about 100nM or less. In some embodiments, the Sortl binder is capable of binding to human and murine Sortl with a Kdof about 100nM or less. In some embodiments, the Sortl binder is capable of binding to human or murine Sortl with a Kdof about 75nM or less. In some embodiments, the Sortl binder is capable of binding to human and murine Sortl with a Kdof about 75nM or less. In some embodiments, the Sortl binder is capable of binding to human or murine Sortl with a Kdof about 64nM or less. In some embodiments, the Sortl binder is capable of binding to human and murine Sortl with a Kdof about 64nM or less. In some embodiments, the Sortl binder is capable of binding to human or murine Sortl with a Kdof about 59nM or less. In some embodiments, the Sortl binder is capable of binding to human and murine Sortl with a Kdof about 59nM or less. In some embodiments, the Sortl binder is capable of binding to human or murine Sortl with a Kdof about 50nM or less. In some embodiments, the Sortl binder is capable of binding to human and murine Sortl with a Kdof about 50nM or less.

[0193] In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kd of about 2uM or less. In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kdof about 1uM or less. In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kdof about 100nM or less. In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kdof about 75nM or less. In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kdof about 64nM or less. In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kdof about 59nM or less. In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kdof about 50nM or less. In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kd of about 34nM or less. In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kd of about 30nM or less. In some embodiments, the Sortl binder is capable of binding to human Sortl with a Kd of about 25nM or less.

[0194] In some embodiments, the Sortl binder is capable of binding to murine Sortl with a Kd of about 2uM or less. In some embodiments, the Sortl binder is capable of binding to murine Sortl with a Kdof about 1uM or less. In some embodiments, the Sortl binder is capable of binding to murine Sortl with a Kdof about 100nM or less. In some embodiments, the Sortl binder is capable of binding to murine Sortl with a Kdof about 75nM or less. In some embodiments, the Sortl binder is capable of binding to murine Sortl with a Kd of about 64nM or less. In some embodiments, the Sortl binder is capable of binding to murine Sortl with a Kd of about 59nM or less. In some embodiments, the Sortl binder is capable of binding to murine Sortl with a Kd of about 50nM or less. In some embodiments, the Sortl binder is capable of binding to murine Sortl with a Kd of about 35nM or less. In some embodiments, the Sortl binder is capable of binding to murine Sortl with a Kd of about 27nM or less.

[0195] One of skill in the art understands that, in some embodiments, the binding of the Sortl binder to Sortl refers to the binding of the Sortl binder to the Sortl receptor or Sortl receptor interface.

[0196] As will become clear below, one of skill understands that such binding capacity may be determined in suitable conditions for binding, such as in pH conditions where the Sortl receptor is considered to have complementarity with the Sortl binders of the instant application. In some embodiments, the binding capacity is determined when the Sortl receptor is in a monomeric state. In some embodiments, the binding capacity is determined when the Sortl receptor is in neutral or alkaline pH conditions. In some embodiments, the binding capacity is determined when the Sortl receptor is an environment with a pH of 6.5 or more. In some embodiments, the binding capacity is determined when the Sortl receptor is an environment with a pH of 7 or more. In some embodiments, the binding capacity is determined when the Sortl receptor is an environment with a pH of 7.4 or more. pH Dependent Binding of Sortl Binders

[0197] Sortl undergoes a pH-mediated conformational change. At neutral or alkaline pH, the Sortl receptors exist in a monomeric state and are capable of binding and regulating the cellular distribution of its ligands. However, under acidic pH conditions, such as may be the case within the endosome or lysosome for instance, protonation of histidine residues may induce a conformational change in Sortl. This conformational change may enable the formation of a homodimer.

[0198] In some embodiments, the Sortl binder of the instant disclosure takes advantage of the conformational changes of the Sortl receptors, providing a binder capable of binding to the Sortl receptor in certain environmental conditions while further capable of being released in alternate environmental conditions. In some embodiments, the Sortl binder of the instant disclosure is capable of binding to the Sortl receptor when the Sortl receptor is in a first protonation state while further capable of being released when the Sortl receptor is in a different protonation state.

[0199] In some embodiments, the Sortl binder’s capacity to bind to the Sortl is dependent on the state of Sortl . In some embodiments, the Sortl binder’s capacity to bind to Sortl is dependent on the protonation state of Sortl . In some embodiments, the Sortl binder is capable to bind to the Sortl receptor when in a deprotonated state. In some embodiments, the Sortl binder is incapable to bind to the Sortl receptor when in a protonated state. In some embodiments, the Sortl binder is capable of binding to the Sortl receptor in neutral or alkaline pH conditions. In some embodiments, the Sortl binder is incapable of binding to the Sortl receptor in acidic pH conditions.

[0200] In some embodiments, the Sortl binder is capable of binding to the Sortl receptor in an environmental pH above 6, above 6.1, above 6.2, above 6.3, above 6.4, above 6.5, above 6.6, above 6.7, above 6.8, above 6.9, above 7.0, above 7.1 , above 7.2, above 7.3, above 7.4, or above 7.5. In some embodiments, the Sortl binder is capable of binding to the Sortl receptor in an environmental pH of 6.1 or more, 6.2 or more, 6.3 or more, 6.4 or more, 6.5 or more, 6.6 or more, 6.7 or more, 6.8 or more, 6.9 or more, 7.0 or more, 7.1 or more, 7.2 or more, 7.3 or more, 7.4 or more, or 7.5 or more. In some embodiments, the Sortl binder is capable of binding to the Sortl receptor in an environmental pH between about 6.1-14, about 6.1-12, about 6.1-10, about 6.1-9. In some embodiments, the Sortl binder is capable of binding to the Sortl receptor in an environmental pH between about 6.4-14, about 6.4-12, about 6.4-10, about 6.4-9, between 6.4-8.5 or between 6.4-8. In some embodiments, the Sortl binder is capable of binding to the Sortl receptor in an environmental pH between about 6.5-14, about 6.5-12, about 6.5-10, about 6.5-9, between 6.5-8.5 or between 6.5-8. In some embodiments, the Sortl binder is capable of binding to the Sortl receptor in an environmental pH between about 6.6-14, about 6.6-12, about 6.6-10, about 6.6-9, between 6.6-8.5 or between 6.6-8. In some embodiments, the Sortl binder is capable of binding to the Sortl receptor in an environmental pH between about 7-14, about 7-12, about 7-10, about 7- 9, between 7-8.5 or between 7-8. In some embodiments, the Sortl binder is capable of binding to the Sortl receptor in an environmental pH between about 7.4-14, about 7.4-12, about 7.4-10, about 7.4-9, between 7.4-8.5 or between 7.4-8.

[0201] In some embodiments, the Sortl binder is incapable of binding to the Sortl receptor in an environmental pH below or equal to 6. In some embodiments, the Sortl binder is incapable of binding to the Sortl receptor in an environmental pH of 6 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, or 5.5 or less. In some embodiments, the Sortl binder is incapable of binding to the Sortl receptor in an environmental pH of less than 6, less than 5.9, less than 5.8, less than 5.7, less than 5.6 or less than 5.5.

[0202] In some embodiments, the Sortl binder is capable of binding to the Sortl receptor when the Sortl receptor is in a deprotonated state and is further be capable of being released from the Sortl receptor when the Sortl receptor is in a protonated state.

[0203] In some embodiments, the Sortl binder may be capable of binding to the Sortl receptor when the Sortl receptor is in neutral or alkaline conditions, such as in an environmental pH above 6, above 6.1 , above 6.2, above 6.3, above 6.4 or above 6.5 or in an environmental pH between about 6.1-14, about 6.1-12, about 6.1-10, about 6.1-9, between about 6.5-14, about 6.5-12, about 6.5-10, about 6.5-9, between 6.5-8.5 or between 6.5-8, and may further be capable of being released from the Sortl receptor when the Sortl receptor is in acidic conditions, such as in an environmental pH below or equal to 6 or in an environmental pH of less than 6, less than 5.9, less than 5.8, less than 5.7, less than 5.6 or less than 5.5.

[0204] In some embodiments, the Sortl binder may be capable of binding to the Sortl receptor when the Sortl receptor is in an environmental pH above 6 and may further be capable of being released from the Sortl receptor when the Sortl receptor is in an environmental pH below or equal to 6.

[0205] It is understood that any of the pH conditions referred to above with reference to the capacity of the Sortl binder to bind to the Sortl receptor may be used in combination to provide a binding / releasing mechanism.

[0206] One of skill in the art understands that, while the above passage refers to the binding and release capacity of the Sortl binders disclosed herein, the binding and releasing capacity further applies to constructs comprising the Sortl binder, discussed hereinafter without departing from the present disclosure.

[0207] Constructs comprising a Sortl Binder

[0208] In some embodiments, the Sortl binders of the instant application is combined with at least one functional or non-functional entity to form a construct comprising the Sortl binder. The entity combined to the Sortl binder may generally be referred to as a cargo. As will be discussed in detail below the cargo may be linked to the Sortl binder as a recombinant fusion (as shown in Figs. 1A-1D for instance), as chemical conjugation (as shown in Fig. 2 for instance), or a combination thereof.

[0209] As will be made clear below, it is understood that a cargo as discussed herein may generally be a cargo moiety within the context of a construct, which may comprise at least 1 functional or non-functional entity. For instance, a cargo moiety may be attached to the Sortl binder and may comprise 1 or more entities (or cargos) linked to one another. Likewise, the Sortl binder may be referred to as a binder moiety in the context of a construct.

[0210] One of skill in the art understands that various constructs comprising a combination of at least one Sortl binder and at least one cargo may be established for a given purpose without departing from the instant disclosure. One of skill in the art further understands that, using Sortl binder, linker and cargo “building blocks”, various constructs may be established for a given purpose without departing from the instant disclosure. Further, it is understood that a construct of the Sortl binder may attach one or more cargos, independently from one another via methods that may be the same or different for each one of the cargos. For instance, in some embodiments, one or more cargos are attached to the Sortl binder independently from one another via a method selected from recombinant fusion and chemical conjugation. In some embodiments, at least one cargo is attached to the Sortl binder as a recombinant fusion. In some embodiments, a construct comprising a Sortl binder and a cargo is provided as a recombinant fusion. In some embodiments, at least one cargo is attached to the Sortl binder as a chemical conjugation. In some embodiments, a construct comprises a cargo chemically conjugated to the Sortl binder.

[0211] Recombinant Fusion

[0212] In some embodiments, a functional or non-functional entity, which can generally be referred to as a cargo, may be linked to the Sortl binder as a recombinant fusion at either the N-terminus or C-terminus. In some embodiments, the cargo may be linked to the Sortl binder via a linker. One of skill in the art understands that various linkers may be used without departing from the instant disclosure.

[0213] This fusion strategy allows for the direct attachment of the cargo to the Sortl binder, which may enable its simultaneous presentation and binding. In some embodiments, the linker sequence can be optimized to maintain the structural integrity and functionality of both the binder and the attached cargo entity. As shown in Fig. 1A, in some embodiments, a construct comprising the Sortl binder may have a linker attached at the C-terminus of the Sortl binder, linking it to a given cargo. Similarly, as shown in Fig. 1 B, in some embodiments, a construct comprising the Sortl binder may have a linker attached at the N-terminus of the Sortl binder, linking it to a given cargo. One of skill in the art understands that various combinations of the Sortl binder, linker and cargo (as “building blocks”) may be envisioned to form recombinant fusion constructs for a desired purpose.

[0214] For instance, in a non-limitative example shown in Fig. 1 C, one may provide a recombinant fusion with the cargo provided as an extension of the N-terminus of the Sortl binder, while further having an entity attached to the Sortl binder at the C-terminus. In the example shown in Fig. 1 C, and in contrast to the example construct shown in Fig. 1B, the additional entity provided is a tag. It is understood that the additional entities provided need not be limited to a tag and may be any other functional or non-functional entity without departing from the instant disclosure.

[0215] For instance, in another embodiment and as shown in Fig. 1D, the construct may comprise more than 1 cargo attached thereto. In the example construct of Fig. 1 D, the Sortl binder may be linked to a first cargo (Cargol) via a first linker (Linkerl) attaching the first cargo (Cargol) to the N-terminus of the Sortl binder and may further be linked to a second cargo (Cargo2) via a second linker (Linker2) attaching the second cargo (Cargo2) to the C- terminus of the Sortl binder. In such an embodiment, the construct may be said to comprise two cargo moieties for instance, each comprising a single cargo. For abundance of clarity, in the embodiment shown in Fig. 1D, the first cargo (Cargol) and first linker (linkerl) may be referred to as the first cargo moiety, while the second cargo (Cargo2) and second linker (Iinker2) may be referred to as the second cargo moiety. It is understood that the first cargo (Cargol) and second cargo (Cargo2), may be the same or different, and that the first linker (Linkerl) and second linker (Linker2) may also, and independently from the first and second cargos, be the same of different. In some embodiments, the first cargo and second cargo linked to the Sortl binder are different cargos. In some embodiments, the first cargo and second cargo linked to the Sortl binder as the same cargo. In some embodiments, the first linker and second linker attached to the Sortl binder are different linkers. In some embodiments, the first linker and second linker attached to the Sortl binder are the same linker.

[0216] It is understood that additional cargo entities may be provided consecutively, one after the other, without departing from the instant disclosure. For instance, in some embodiments, there may be provided a construct similar to Fig. 1A, where an additional cargo is linked to the first cargo (said first cargo being the cargo shown in Fig. 1A, for instance), said first cargo linked to the Sortl binder, resulting in two cargos linked to C- terminus of the Sortl binder. One may refer to the two consecutive cargos as a single cargo moiety, comprising two cargos, linked to the Sortl binder.

[0217] Chemical Conjugation

[0218] In some embodiments, a functional or non-functional entity, which can generally be referred to as a cargo, may be chemically conjugated to the Sortl binder. This may involve the covalent attachment of the cargo to one or more specific amino acid residues or reactive groups on the Sortl binder. For instance, these amino acid residues or reactive groups may include, but are not limited to, Cysteine or Lysine residues, or non-natural amino acids that contain reactive chemistries allowing for conjugation between two elements. It is understood that the chemical conjugation may take place at the N-terminus or C-terminus of the Sortl binder or between the N-terminus and the C-terminus of the Sortl binder.

[0219] In some embodiments, the attachment of a cargo may take place between the N- terminus and the C-terminus of the Sortl binder, leaving these termini of the Sortl binder free. This chemical conjugation is exemplified in Fig. 2, which is depicted via a dark star symbol, identifying the conjugation between the Sortl binder and the Cargo at a location between the N-terminus and C-terminus of the Sortl binder.

[0220] In some embodiments, the Sortl binder comprises an introduced Cysteine with surface accessibility for the purpose of chemically conjugating a cargo.

[0221] One of skill in the art understands that, while Fig. 2 shows the N-terminus and the C- terminus of the Sortl binder as being free, that a construct may comprise additional entities attached to the Sortl binder, at the N-terminus, C-terminus or elsewhere without departing from the instant disclosure. Such entities may be provided as a recombinant fusion or via another chemical conjugation.

[0222] In some embodiments, more than 1 cargo may be chemically conjugated to the Sortl binder. For instance, in some embodiments, a construct may comprise 2 or more cargos chemically conjugated to the Sort 1 binder.

[0223] It is understood that additional cargo entities may be provided consecutively, one after the other, without departing from the instant disclosure. For instance, in some embodiments, there may be provided a construct similar to Fig. 2, where an additional cargo is linked to the first cargo (said first cargo being the cargo currently shown in Fig. 2), said first cargo chemically conjugated to the Sortl binder, thus resulting in two cargos attached to the Sortl binder. One may refer to the two consecutive cargos as a single cargo moiety, comprising two cargos, attached to the Sortl binder.

[0224] Other

[0225] One of skill in the art understands that, in some embodiments, the Sortl binder as identified in any one of Formulas l-V or in any one of SEQ ID No. 1-39 may further having a N-terminus or C-terminus extension, which may generally be referred to as a tag to instance, without departing form the instant disclosure. For instance, and purely for the purpose of exemplifying, the Sortl binder of the instant disclosure may comprise a polypeptide in accordance with any one of SEQ ID No. 1-39 or Formulas l-V and further have a “GGG” tag as an N- or C- terminus extension to the Sortl binder sequence. Such a structure of the Sortl binder with the tag may be referred to, in the general sense, as a construct. In some embodiments, the constructs may comprise the Sortl binder combined with non-functional entity, such as a tag for instance. It is understood that such a cargo or extension may be provided via a recombinant fusion, chemical conjugation or combination thereof as described above.

[0226] It is understood that by utilizing recombinant fusions, chemical conjugation, or combinations thereof, a wide range of entities may be attached to the Sortl binders of the instant disclosure.

[0227] It is understood that, no matter how a construct is provided, the linker selected to be used with a given binder will be clear to one of skill in the art for a given construct desired. Any suitable linker may be used to provide a construct as described herein without departing from the instant disclosure. For instance, one of skill in the art may decide to select a linker based on one or more desired parameters such as, for example, length variation of the linker and / or flexibility / rigidity, hydrophilicity / hydrophobicity, acid / base, etc. and thus select a linker length and composition to provide suitable parameters for a given construct. In some embodiments, the linker is selected such as to permit the construct to comprise a correctly folded cargo and / or binder moiety. In some embodiments, the linker in a construct may be a Gly-rich linker.

[0228] Pharmaceutical Compositions

[0229] In some embodiments, there is provided pharmaceutical compositions comprising a Sortl binder as disclosed in the instant application and according to the embodiments of the invention identified above, and at least one pharmaceutically acceptable excipient. In some embodiments, there is provided pharmaceutical compositions comprising a Sortl binder within a construct as disclosed in the instant application and according to the embodiments of the invention identified above, and at least one pharmaceutically acceptable excipient. Such pharmaceutical compositions may be prepared as is known in the art. The excipient may serve various purposes, e.g. as a carrier, vehicle, diluent, and / or to improve administration, and / or absorption of the active substance.

[0230] The formulation of pharmaceutically active ingredients with various excipients is known in the art, see e.g. Remington: The Science and Practice of Pharmacy, 19th edition (1995), and any later editions.

[0231] Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity regulating agents, chelating agents, and stabilisers. Examples of formulations include liquid formulations and solid formulations.

[0232] Liquid Pharmaceutical Formulations

[0233] Examples of a liquid formulation are aqueous formulations, i.e. formulations that comprise water. A liquid formulation may be a solution. An aqueous formulation typically comprises at least 50% (w / w) water, or at least 60%, 70%, 80% or even at least 90% (w / w) water.

[0234] In some embodiment, the formulation may comprise a buffer. In a further embodiment of the invention the formulation further comprises a pharmaceutically acceptable preservative. In a further embodiment of the invention the formulation further comprises an isotonic agent. In some embodiments, the pharmaceutical composition may comprise a chelating agent. In some embodiments, the pharmaceutical composition may comprise a stabiliser.

[0235] Route of administration

[0236] A Sortl binder and / or constructs comprising a Sortl binder of the present invention may be administered in the form of a pharmaceutical composition.

[0237] Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal, or intravenous injection. In some embodiments, the administration may be performed by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump.

[0238] Preparation of Sortl Binder and constructs comprising a Sortl Binder

[0239] Further disclosed herein are methods of making a Sortl binder and constructs comprising a Sortl binder according to the invention The Sortl binders or constructs comprising the Sortl binders according to the present disclosure may for instance be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques, see e.g. Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1999; Florencio Zaragoza Ddrwald, “Organic Synthesis on Solid Phase”, Wiley-VCH Verlag GmbH, 2000; and “Fmoc Solid Phase Peptide Synthesis”, Edited by W.C. Chan and P.D. White, Oxford University Press, 2000.

[0240] Alternatively, the Sortl binders or constructs comprising the Sortl binder according to the present disclosure may be produced by recombinant methods, e.g. by culturing a host cell containing a DNA sequence encoding the necessary peptide sequence and capable of expressing the peptide, in a suitable nutrient medium under conditions permitting the expression of the peptide. Non-limiting examples of host cells suitable for expression of these peptides are Escherichia coli, Saccharomyces cerevisiae and mammalian BHK or CHO cell lines.

[0241] Specific examples of methods of preparing the Sortl binders and constructs comprising a Sortl binder in accordance with the present disclosure are included in the experimental section.

[0242] Methods of Use

[0243] The instant application enables one of skill in the art to provide constructs which leverage the Sortl binder’s binding capacity, enabling specific applications tailored to a variety of needs in different areas such as targeted therapy, imaging, or diagnostics, to name a few non-limitative examples. It is understood that the exact application will vary depending in the Sortl construct provided.

[0244] For instance, in a non-limitative example, there is provided constructs comprising the Sortl binder and which are further linked to a pharmaceutical composition, wherein the construct provides an increased uptake to the target cell and where the pharmaceutical composition may provide its expected medical treatment. In some embodiments, Sortl binder constructs of the present invention provided with a pharmaceutical composition provide for superior pharmacodynamic properties in comparison to the pharmaceutical composition on itself.

[0245] In yet another non-limitative example, there is provided constructs comprising the Sortl binder and which are further linked to another cell surface receptor binder, where the construct provides for protein lysosome-targeting chimeras, promoting targeted protein degradation. In some embodiments, there is provided a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting internalization of a cargo. In some embodiments, there is provided a pharmaceutical composition comprising a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting internalization of a cargo. In some embodiments, there is provided a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting clearance of a cargo. In some embodiments, there is provided a pharmaceutical composition comprising a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting clearance of a cargo. In some embodiments, there is provided a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting internalization of a therapeutic agent. In some embodiments, there is provided a pharmaceutical composition comprising a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting internalization of a therapeutic agent.

[0246] In some embodiments, there is provided a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting protein degradation. In some embodiments, there is provided a pharmaceutical composition comprising a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting protein degradation. In some embodiments, there is provided a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting extracellular protein degradation. In some embodiments, there is provided a pharmaceutical composition comprising a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use in promoting extracellular protein degradation.

[0247] In some embodiments, there is provided a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use as a medicament. In some embodiments, there is provided a pharmaceutical composition comprising a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use as a medicament. In some embodiments, there is provided a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use as a therapeutic agent. In some embodiments, there is provided a pharmaceutical composition comprising a Sortl binder or a construct comprising a Sortl binder in accordance with the instant disclosure for use as a therapeutic agent.

[0248] In some embodiments, there is provided a construct comprising a Sortl binder and a therapeutic agent in accordance with the instant disclosure for use in the treatment of a given condition via the therapeutic agent. In some embodiments, there is provided a pharmaceutical composition comprising a construct comprising a Sortl binder and a therapeutic agent in accordance with the instant disclosure for use in the treatment of a given condition via the therapeutic agent.

[0249] The term “treatment”, as used herein, refers to the medical treatment of any human subject in need thereof. The treatment may be preventive, prophylactic, palliative, symptomatic and / or curative. The timing and purpose of said treatment may vary from one individual to another, according to the status of the subject’s health.

[0250] In some embodiments, the methods comprise administering the peptide or the pharmaceutical composition as described herein to a patient.

[0251] Definitions

[0252] Unless otherwise specified, “Sortilin 1”, “Sortilin-1 ”, “Sortl”, “Sort 1”, “Sort-1”, “SORT1" are synonymous and refer to the Sortilin-1 transmembrane protein. When referring to an entity binding to Sortl, it may be understood that said entity is binding to a Sortilin-1 receptor.

[0253] As used herein, the terms "subject", "individual," and "patient" are used interchangeably to refer to a vertebrate, for example, a mammal. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. None of the terms entail supervision of a medical professional.

[0254] "Amino acid" refers to both canonical and non-canonical amino acids. Non-canonical amino acids, or non-natural amino acid include, but are not limited to, 2-aminoisobutyric acid (Aib) and pyroglutamate.

[0255] A "conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For purposes of the present disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another:

[0256] 1) Alanine (A) and Glycine (G);

[0257] 2) Aspartic acid (D) and Glutamic acid (E);

[0258] 3) Asparagine (N) and Glutamine (Q);

[0259] 4) Arginine (R) and Lysine (K);

[0260] 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V);

[0261] 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W);

[0262] 7) Serine (S) and Threonine (T); and 8) Cysteine (C) and Methionine (M).

[0263] Naturally occurring residues can be divided into classes based on common side group properties, for example: polar positive (histidine (H), lysine (K), and arginine (R)); polar negative (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)), non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)), proline (P) and glycine (G); and cysteine (C). A "semi-conservative" amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.

[0264] “Pharmaceutically acceptable” indicates that the substance or composition must be chemically and / or toxicologically suitable for the treatment of mammals.

[0265] "Pharmaceutical composition” refers to the combination of an active agent (e.g., a construct with a Sortl binder) with a carrier, inert or active, making the composition especially suitable for therapeutic or diagnostic use.

[0266] Unless otherwise specified, “modification” refers to amino acid modifications selected from elongations, truncations, substitutions (e.g. substitution of one amino acid for another), and chemical modifications, or a combination thereof.

[0267] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For illustration purposes, a numerical range of "about 1 to about 5" should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc., as well as 1 , 2, 3, 4, and 5, individually. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0268] “Functional Entity” refers to any type of entity that may carry out a function or chemical or biological process. A functional entity may be imaging reporter, diagnostic reporter, a naturally occurring peptide or a therapeutic agent, to name a few examples. A functional entity may be a therapeutic agent. “Therapeutic Agent” refers to a drug, protein, peptide, gene, compound, or any other pharmaceutically active ingredient. More generally referring to a known or unknown biologically active compound capable of treating at least one disease state or condition.

[0269] All embodiments of any aspect of the disclosure can be used in combination unless context clearly dictates otherwise.

[0270] Unless otherwise indicated in the specification, terms presented in singular form also include the plural situation

[0271] The term “linked” in the context of linking entities refers to the general linkage or attachment between said two entities via any means and is not to be construed as being limited to the attachment of the two entities via a linker.

[0272] “huSORTI” refers to native human Sortilin-1 receptor identifiable with Uniprot number Q99523.

[0273] “moSORTT’ refers to native mouse Sortilin-1 receptor identifiable with Uniprot number Q6PHU5.

[0274] LIST OF FURTHER EMBODIMENTS OF THE INVENTION

[0275] The invention is further described by the following non-limiting embodiments:

[0276] 1. A polypeptide comprising an amino acid sequence at least 50%, 60%, 70%, 79%, 80%, 82%, 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 1-39.

[0277] 2. A polypeptide comprising an amino acid sequence at least 50%, 60%, 70%, 79%, 80%, 82%, 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1-4 or 6-39.

[0278] 3. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide comprises a binder.

[0279] 4. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide has a binding affinity to Sortilin-1 (SORT1) receptor.

[0280] 5. A polypeptide comprising a binder, the binder having a binding affinity to Sortilin-1 (SORT1) receptor and comprising an amino acid sequence at least 50%, 60%, 70%, 79%, 80%, 82%, 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 1-39.

[0281] 6. A polypeptide comprising a binder, the binder having a binding affinity to Sortilin- 1 (SORT1) receptor and comprising an amino acid sequence at least 50%, 60%, 70%, 79%, 80%, 82%, 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1-4 or 6-39.

[0282] 7. A polypeptide comprising a binder, the binder having a binding affinity to Sortilin- 1 (SORT1) receptor and comprising an amino acid sequence at least 50%, 60%, 70%, 79%, 80%, 82%, 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1 , 6, 14, 24, 29.

[0283] 8. The polypeptide according to any one of the preceding embodiments, wherein amino acid substitutions relative to a reference polypeptide are selected from those listed in Table 1.

[0284] 9. The polypeptide according to any one of the preceding embodiments, comprising the amino acid sequence according to Formula I :

[0285] Xaa1 -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13- Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Xaa22-Xaa23-Xaa24- Xaa25-Trp-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36- Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45-Xaa46-Xaa47- Xaa48-Xaa49-Xaa50-Xaa51-Xaa52-Xaa53-Xaa54-Xaa55-Xaa56-Xaa57-Xaa58 wherein

[0286] Xaa1 is Ser (S) or Gly (G) or Asn (N) or Ala (A) or Arg (R) or His (H);

[0287] Xaa2 is lie (I) or Tyr (Y) or Phe (F) or Leu (L) or Met (M) or Trp (W) or His (H) or Pro (P) or Vai (V);

[0288] Xaa3 is Leu (L) or lie (I) or Met (M);

[0289] Xaa4 is Tyr (Y) or Cys (C) or Leu (L) or Arg (R) or His (H) or Met (M) or Vai (V) or Gin (Q) or Pro (P) or Lys (K);

[0290] Xaa5 is Arg (R) or Lys (K) or Leu (L) or Vai (V) or Asn (N) or Ser (S) or Met (M) or Thr (T) or Gin (Q) or lie (I) or Ala (A) or Gly (G);

[0291] Xaa6 is Ser (S) or His (H) or Leu (L) or Thr (T) or Tyr (Y) or Gly (G) or Asn (N) or Gin (Q) or Phe (F) or Ala (A) or Pro (P) or Arg (R) or Met (M);

[0292] Xaa7 is Thr (T) or Ala (A) or Cys (C) or Ser (S); Xaa8 is lie (I) or Vai (V) or Ala (A) or Met (M) or Thr (T);

[0293] Xaa9 is Arg (R) or Lys (K) or Vai (V);

[0294] Xaa10 is Vai (V) or lie (I) or Thr (T);

[0295] Xaa11 is lie (I) or Vai (V) or Cys (C) or Thr (T) or Ala (A) or Ser (S);

[0296] Xaa12 is Glu (E) or Asp (D) or Gin (Q) or Vai (V) or His (H) or Tyr (Y) or Met (M) or Gly (G) or lie (I) or Thr (T) or Ser (S) or Arg (R) or Lys (K) or Asn (N) or Ala (A);

[0297] Xaa13 is Arg (R) or Lys (K) or His (H) or Asn (N) or Ser (S);

[0298] Xaa14 is Leu (L) or lie (I) or Arg (R) or Gin (Q) or Met (M);

[0299] Xaa15 is lie (I) or Leu (L) or Cys (C) or Vai (V) or Ala (A) or Met (M) or Thr (T);

[0300] Xaa16 is Arg (R) or Ala (A) or Gin (Q) or Gly (G) or Lys (K) or His (H) or Ser (S) or Asp (D) or Thr (T) or Tyr (Y) or Asn (N) or Met (M) or Leu (L) or Vai (V) or lie (I) or Glu (E) or Trp (W) or Phe (F) or Cys (C);

[0301] Xaa17 is Gin (Q) or Arg (R) or Ala (A) or Lys (K) or Asn (N) or Gly (G) or Glu (E) or Ser (S) or His (H) or Asp (D) or Met (M) or Leu (L) or Vai (V) or Phe (F) or Tyr (Y) or lie (I) or Thr (T) or Trp (W) or Cys (C);

[0302] Xaa18 is Lys (K) or Arg (R) or Ser (S) or Asp (D) or Gin (Q) or His (H) or Gly (G) or Asn (N) or Glu (E) or Ala (A) or Met (M) or Pro (P) or Phe (F) or Cys (C) or Thr (T) or Tyr (Y) or Vai (V) or Leu (L) or Trp (W);

[0303] Xaa19 is Arg (R) or Ser (S) or Lys (K) or Gin (Q) or Asn (N) or Glu (E) or Asp (D) or Ala (A) or His (H) or Thr (T) or Met (M) or Cys (C) or Phe (F) or Leu (L) or Vai (V) or Tyr (Y) or lie (I);

[0304] Xaa20 is Leu (L) or Thr (T) or Phe (F) or Cys (C) or Asn (N) or Ser (S) or His (H) or Met (M) or Vai (V) or Ala (A) or Trp (W) or Tyr (Y) or lie (I) or Lys (K) or Gly (G) or Gin (Q) or Arg (R) or Pro (P) or Asp (D);

[0305] Xaa21 is Arg (R) or Lys (K) or Asn (N) or Ser (S) or Thr (T) or Gly (G) or Leu (L) or Met (M) or His (H) or lie (I) or Phe (F) or Asp (D) or Ala (A) or Vai (V) or Pro (P) or Gin (Q) or Tyr (Y) or Glu (E) or Cys (C);

[0306] Xaa22 is Vai (V) or Thr (T) or Lys (K) or Arg (R) or Met (M) or lie (I) or Ala (A) or His (H) or Gin (Q) or Ser (S) or Cys (C) or Pro (P) or Asp (D) or Glu (E) or Leu (L) or Asn (N) or Trp (W) or Gly (G) or Phe (F);

[0307] Xaa23 is Asp (D) or Ala (A) or Gly (G) or Ser (S);

[0308] Xaa24 is Ser (S) or Met (M) or His (H) or Thr (T) or Ala (A) or Gly (G) or Phe (F) or Lys (K) or lie (I) or Asn (N) or Vai (V) or Arg (R) or Cys (C) or Leu (L) or Gin (Q) or Tyr (Y) or Glu (E) or Trp (W) or Asp (D); Xaa25 is Arg (R) or Lys (K) or Gin (Q) or Ser (S) or Ala (A) or Thr (T) or Asp (D) or Asn (N) or His (H) or Glu (E) or Met (M) or Gly (G) or Pro (P) or Vai (V) or lie (I) or Leu (L);

[0309] Xaa27 is Leu (L) or Cys (C) or lie (I) or Vai (V) or Thr (T) or Asn (N);

[0310] Xaa28 is Pro (P) or Vai (V) or Tyr (Y) or Lys (K) or lie (I) or Ala (A) or Thr (T) or His (H) or Asn (N) or Gin (Q) or Leu (L) or Ser (S) or T rp (W) or Arg (R) or Met (M) or Gly (G) or Phe (F) or Cys (C) or Glu (E);

[0311] Xaa29 is Cys (C) or Gin (Q) or Thr (T) or Vai (V) or His (H) or Ser (S) or Lys (K) or Ala (A) or Glu (E);

[0312] Xaa30 is Thr (T) or Vai (V) or Leu (L) or Cys (C) or lie (I);

[0313] Xaa31 is lie (I) or Lys (K) or Phe (F) or His (H) or Thr (T) or Met (M) or Arg (R) or Vai (V) or Leu (L) or Tyr (Y) or Gin (Q) or Trp (W) or Asn (N) or Ser (S) or Ala (A);

[0314] Xaa32 is Lys (K) or Asn (N) or Tyr (Y) or Trp (W) or His (H) or Arg (R) or Vai (V) or Gly (G) or Ser (S) or Phe (F) or Ala (A) or Gin (Q) or Met (M) or Leu (L) or Cys (C) or Thr (T) or lie (I);

[0315] Xaa33 is Leu (L) or lie (I) or Vai (V);

[0316] Xaa34 is Leu (L) or Ser (S) or Ala (A) or Gly (G) or Cys (C);

[0317] Xaa35 is Cys (C) or Tyr (Y) or lie (I) or Vai (V) or His (H) or Thr (T) or Lys (K) or Phe (F) or Arg (R) or Leu (L) or Met (M) or T rp (W) or Gin (Q) or Gly (G) or Ser (S) or Asn (N) or Glu (E);

[0318] Xaa36 is Leu (L) or Arg (R) or Lys (K) or Vai (V) or Ala (A) or Met (M) or lie (I) or Ser (S) or Gin (Q) or Thr (T) or Trp (W) or Tyr (Y) or Phe (F) or His (H) or Asn (N);

[0319] Xaa37 is Gin (Q) or Asn (N) or His (H) or Leu (L);

[0320] Xaa38 is Arg (R) or Ala (A) or His (H) or Gin (Q) or Lys (K) or Cys (C) or Met (M) or Thr (T) or Asp (D) or Asn (N) or Gly (G) or Phe (F) or Tyr (Y) or Vai (V) or Glu (E) or Ser (S) or lie (I) or Trp (W) or Leu (L);

[0321] Xaa39 is Asp (D) or Asn (N) or Gin (Q) or Ala (A) or Met (M) or Glu (E);

[0322] Xaa40 is Phe (F) or Trp (W) or Lys (K) or Arg (R) or Gin (Q) or Gly (G) or Tyr (Y) or Vai (V) or lie (I) or Asn (N) or His (H) or Ala (A) or Leu (L) or Met (M) or Pro (P) or Cys (C) or Ser (S) or Asp (D) or Thr (T);

[0323] Xaa41 is Trp (W) or Tyr (Y) or Gly (G) or His (H) or Phe (F) or Arg (R) or Leu (L) or Gin (Q) or Ser (S) or Asn (N) or Met (M) or Ala (A) or Vai (V) or Asp (D) or Glu (E) or Lys (K) or lie (I) or Thr (T) or Cys (C);

[0324] Xaa42 is Vai (V) or Ser (S) or Ala (A) or lie (I) or Cys (C) or Thr (T) or Met (M); Xaa43 is Tyr (Y) or Trp (W) or Phe (F) or Met (M) or Leu (L) or Ala (A) or lie (I) or Gin (Q) or Vai (V) or Arg (R) or Ser (S) or Cys (C) or His (H) or Glu (E) or Thr (T) or Lys (K) or Gly (G) or Asn (N);

[0325] Xaa44 is Vai (V) or Leu (L) or His (H) or Arg (R) or Lys (K) or Ser (S) or Gin (Q) or Tyr (Y) or Phe (F) or Ala (A) or Met (M) or Cys (C) or lie (I) or Asn (N) or Glu (E) or Trp (W) or Gly (G) or Thr (T);

[0326] Xaa45 is Arg (R) or Lys (K) or Ala (A) or Cys (C) or His (H) or lie (I) or Vai (V) or Ser

[0327] (S);

[0328] Xaa46 is Ala (A) or Cys (C) or Ser (S) or Vai (V) or Gly (G);

[0329] Xaa47 is Vai (V) or Phe (F) or Tyr (Y) or Arg (R) or Leu (L) or lie (I) or Lys (K) or Gly

[0330] (G) or Ala (A) or Cys (C) or His (H) or T rp (W) or Glu (E) or Asn (N) or Gin (Q) or Thr

[0331] (T) or Met (M) or Asp (D) or Ser (S);

[0332] Xaa48 is Gin (Q) or Glu (E) or Ala (A) or Lys (K) or Asp (D) or Met (M) or Vai (V) or Arg (R) or Tyr (Y) or His (H) or Ser (S) or lie (I) or Asn (N) or Leu (L) or Cys (C); Xaa49 is Leu (L) or Met (M);

[0333] Xaa50 is Gin (Q) or Arg (R) or Ser (S) or Met (M) or Trp (W) or Ala (A) or Tyr (Y) or His (H) or Lys (K) or Gly (G) or Thr (T) or Asn (N) or Phe (F) or Glu (E) or Asp (D) or Cys (C) or Leu (L);

[0334] Xaa51 is Lys (K) or Arg (R) or lie (I) or Phe (F) or Ala (A) or Cys (C) or Asn (N) or Gin (Q) or Asp (D) or T rp (W) or Gly (G) or Leu (L) or Ser (S) or Thr (T) or Vai (V) or Tyr (Y) or His (H) or Glu (E) or Met (M);

[0335] Xaa52 is Trp (W) or Tyr (Y) or Met (M) or Phe (F) or lie (I) or Leu (L) or Ala (A) or Lys (K) or Vai (V) or His (H) or Thr (T) or Ser (S) or Glu (E) or Arg (R) or Cys (C) or Gin (Q) or Asp (D) or Gly (G) or Asn (N);

[0336] Xaa53 is Ala (A) or Cys (C) or Arg (R) or Leu (L) or Vai (V) or Met (M) or lie (I) or Lys (K) or Thr (T) or Tyr (Y) or Gin (Q) or Trp (W) or Asn (N) or Ser (S) or His (H) or Phe (F) or Gly (G);

[0337] Xaa54 is Gly (G) or Gin (Q) or Thr (T) or Leu (L) or Arg (R) or Asp (D) or lie (I) or Tyr (Y) or Asn (N) or Lys (K) or Ala (A) or Met (M) or Phe (F) or His (H) or Glu (E) or T rp (W) or Ser (S) or Cys (C);

[0338] Xaa55 is Gly (G) or Lys (K) or Vai (V) or Ser (S) or His (H) or Met (M) or Arg (R) or Leu (L) or Asp (D) or Asn (N) or Gin (Q) or Phe (F) or Ala (A) or Thr (T) or Glu (E) or Tyr (Y) or Cys (C) or lie (I) or Trp (W); Xaa56 is Met (M) or lie (I) or Leu (L) or Phe (F) or Arg (R) or Lys (K) or Vai (V) or Asn (N) or Trp (W) or Ala (A) or Tyr (Y) or Cys (C) or Thr (T) or Gin (Q) or His (H) or Ser (S) or Gly (G);

[0339] Xaa57 is Absent or Leu (L); and

[0340] Xaa58 is Absent or Glu (E).

[0341] 10. The polypeptide according to any one of the preceding embodiments, wherein the binder comprises the amino acid sequence according to Formula I :

[0342] Xaa1 -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-

[0343] Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Xaa22-Xaa23-Xaa24-

[0344] Xaa25-Trp-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36- Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45-Xaa46-Xaa47- Xaa48-Xaa49-Xaa50-Xaa51-Xaa52-Xaa53-Xaa54-Xaa55-Xaa56-Xaa57-Xaa58 wherein

[0345] Xaa1 is Ser (S) or Gly (G) or Asn (N) or Ala (A) or Arg (R) or His (H);

[0346] Xaa2 is lie (I) or Tyr (Y) or Phe (F) or Leu (L) or Met (M) or Trp (W) or His (H) or Pro (P) or Vai (V);

[0347] Xaa3 is Leu (L) or lie (I) or Met (M);

[0348] Xaa4 is Tyr (Y) or Cys (C) or Leu (L) or Arg (R) or His (H) or Met (M) or Vai (V) or Gin (Q) or Pro (P) or Lys (K);

[0349] Xaa5 is Arg (R) or Lys (K) or Leu (L) or Vai (V) or Asn (N) or Ser (S) or Met (M) or Thr (T) or Gin (Q) or lie (I) or Ala (A) or Gly (G);

[0350] Xaa6 is Ser (S) or His (H) or Leu (L) or Thr (T) or Tyr (Y) or Gly (G) or Asn (N) or Gin (Q) or Phe (F) or Ala (A) or Pro (P) or Arg (R) or Met (M);

[0351] Xaa7 is Thr (T) or Ala (A) or Cys (C) or Ser (S);

[0352] Xaa8 is lie (I) or Vai (V) or Ala (A) or Met (M) or Thr (T);

[0353] Xaa9 is Arg (R) or Lys (K) or Vai (V);

[0354] Xaa10 is Vai (V) or lie (I) or Thr (T);

[0355] Xaa11 is lie (I) or Vai (V) or Cys (C) or Thr (T) or Ala (A) or Ser (S);

[0356] Xaa12 is Glu (E) or Asp (D) or Gin (Q) or Vai (V) or His (H) or Tyr (Y) or Met (M) or Gly (G) or lie (I) or Thr (T) or Ser (S) or Arg (R) or Lys (K) or Asn (N) or Ala (A);

[0357] Xaa13 is Arg (R) or Lys (K) or His (H) or Asn (N) or Ser (S);

[0358] Xaa14 is Leu (L) or lie (I) or Arg (R) or Gin (Q) or Met (M);

[0359] Xaa15 is lie (I) or Leu (L) or Cys (C) or Vai (V) or Ala (A) or Met (M) or Thr (T); Xaa16 is Arg (R) or Ala (A) or Gin (Q) or Gly (G) or Lys (K) or His (H) or Ser (S) or Asp (D) or Thr (T) or Tyr (Y) or Asn (N) or Met (M) or Leu (L) or Vai (V) or lie (I) or Glu (E) or Trp (W) or Phe (F) or Cys (C);

[0360] Xaa17 is Gin (Q) or Arg (R) or Ala (A) or Lys (K) or Asn (N) or Gly (G) or Glu (E) or Ser (S) or His (H) or Asp (D) or Met (M) or Leu (L) or Vai (V) or Phe (F) or Tyr (Y) or lie (I) or Thr (T) or Trp (W) or Cys (C);

[0361] Xaa18 is Lys (K) or Arg (R) or Ser (S) or Asp (D) or Gin (Q) or His (H) or Gly (G) or Asn (N) or Glu (E) or Ala (A) or Met (M) or Pro (P) or Phe (F) or Cys (C) or Thr (T) or Tyr (Y) or Vai (V) or Leu (L) or Trp (W);

[0362] Xaa19 is Arg (R) or Ser (S) or Lys (K) or Gin (Q) or Asn (N) or Glu (E) or Asp (D) or Ala (A) or His (H) or Thr (T) or Met (M) or Cys (C) or Phe (F) or Leu (L) or Vai (V) or Tyr (Y) or lie (I);

[0363] Xaa20 is Leu (L) or Thr (T) or Phe (F) or Cys (C) or Asn (N) or Ser (S) or His (H) or Met (M) or Vai (V) or Ala (A) or Trp (W) or Tyr (Y) or lie (I) or Lys (K) or Gly (G) or Gin (Q) or Arg (R) or Pro (P) or Asp (D);

[0364] Xaa21 is Arg (R) or Lys (K) or Asn (N) or Ser (S) or Thr (T) or Gly (G) or Leu (L) or Met (M) or His (H) or lie (I) or Phe (F) or Asp (D) or Ala (A) or Vai (V) or Pro (P) or Gin (Q) or Tyr (Y) or Glu (E) or Cys (C);

[0365] Xaa22 is Vai (V) or Thr (T) or Lys (K) or Arg (R) or Met (M) or lie (I) or Ala (A) or His (H) or Gin (Q) or Ser (S) or Cys (C) or Pro (P) or Asp (D) or Glu (E) or Leu (L) or Asn (N) or Trp (W) or Gly (G) or Phe (F);

[0366] Xaa23 is Asp (D) or Ala (A) or Gly (G) or Ser (S);

[0367] Xaa24 is Ser (S) or Met (M) or His (H) or Thr (T) or Ala (A) or Gly (G) or Phe (F) or Lys (K) or lie (I) or Asn (N) or Vai (V) or Arg (R) or Cys (C) or Leu (L) or Gin (Q) or Tyr (Y) or Glu (E) or Trp (W) or Asp (D);

[0368] Xaa25 is Arg (R) or Lys (K) or Gin (Q) or Ser (S) or Ala (A) or Thr (T) or Asp (D) or Asn (N) or His (H) or Glu (E) or Met (M) or Gly (G) or Pro (P) or Vai (V) or lie (I) or Leu (L);

[0369] Xaa27 is Leu (L) or Cys (C) or lie (I) or Vai (V) or Thr (T) or Asn (N);

[0370] Xaa28 is Pro (P) or Vai (V) or Tyr (Y) or Lys (K) or lie (I) or Ala (A) or Thr (T) or His (H) or Asn (N) or Gin (Q) or Leu (L) or Ser (S) or T rp (W) or Arg (R) or Met (M) or Gly (G) or Phe (F) or Cys (C) or Glu (E);

[0371] Xaa29 is Cys (C) or Gin (Q) or Thr (T) or Vai (V) or His (H) or Ser (S) or Lys (K) or Ala (A) or Glu (E);

[0372] Xaa30 is Thr (T) or Vai (V) or Leu (L) or Cys (C) or lie (I); Xaa31 is lie (I) or Lys (K) or Phe (F) or His (H) or Thr (T) or Met (M) or Arg (R) or Vai (V) or Leu (L) or Tyr (Y) or Gin (Q) or Trp (W) or Asn (N) or Ser (S) or Ala (A);

[0373] Xaa32 is Lys (K) or Asn (N) or Tyr (Y) or Trp (W) or His (H) or Arg (R) or Vai (V) or Gly (G) or Ser (S) or Phe (F) or Ala (A) or Gin (Q) or Met (M) or Leu (L) or Cys (C) or Thr (T) or lie (I);

[0374] Xaa33 is Leu (L) or lie (I) or Vai (V);

[0375] Xaa34 is Leu (L) or Ser (S) or Ala (A) or Gly (G) or Cys (C);

[0376] Xaa35 is Cys (C) or Tyr (Y) or lie (I) or Vai (V) or His (H) or Thr (T) or Lys (K) or Phe (F) or Arg (R) or Leu (L) or Met (M) or T rp (W) or Gin (Q) or Gly (G) or Ser (S) or Asn (N) or Glu (E);

[0377] Xaa36 is Leu (L) or Arg (R) or Lys (K) or Vai (V) or Ala (A) or Met (M) or lie (I) or Ser (S) or Gin (Q) or Thr (T) or Trp (W) or Tyr (Y) or Phe (F) or His (H) or Asn (N);

[0378] Xaa37 is Gin (Q) or Asn (N) or His (H) or Leu (L);

[0379] Xaa38 is Arg (R) or Ala (A) or His (H) or Gin (Q) or Lys (K) or Cys (C) or Met (M) or Thr (T) or Asp (D) or Asn (N) or Gly (G) or Phe (F) or Tyr (Y) or Vai (V) or Glu (E) or Ser (S) or lie (I) or Trp (W) or Leu (L);

[0380] Xaa39 is Asp (D) or Asn (N) or Gin (Q) or Ala (A) or Met (M) or Glu (E);

[0381] Xaa40 is Phe (F) or Trp (W) or Lys (K) or Arg (R) or Gin (Q) or Gly (G) or Tyr (Y) or Vai (V) or lie (I) or Asn (N) or His (H) or Ala (A) or Leu (L) or Met (M) or Pro (P) or Cys (C) or Ser (S) or Asp (D) or Thr (T);

[0382] Xaa41 is Trp (W) or Tyr (Y) or Gly (G) or His (H) or Phe (F) or Arg (R) or Leu (L) or Gin (Q) or Ser (S) or Asn (N) or Met (M) or Ala (A) or Vai (V) or Asp (D) or Glu (E) or Lys (K) or lie (I) or Thr (T) or Cys (C);

[0383] Xaa42 is Vai (V) or Ser (S) or Ala (A) or lie (I) or Cys (C) or Thr (T) or Met (M);

[0384] Xaa43 is Tyr (Y) or Trp (W) or Phe (F) or Met (M) or Leu (L) or Ala (A) or lie (I) or Gin (Q) or Vai (V) or Arg (R) or Ser (S) or Cys (C) or His (H) or Glu (E) or Thr (T) or Lys (K) or Gly (G) or Asn (N);

[0385] Xaa44 is Vai (V) or Leu (L) or His (H) or Arg (R) or Lys (K) or Ser (S) or Gin (Q) or Tyr (Y) or Phe (F) or Ala (A) or Met (M) or Cys (C) or lie (I) or Asn (N) or Glu (E) or Trp (W) or Gly (G) or Thr (T);

[0386] Xaa45 is Arg (R) or Lys (K) or Ala (A) or Cys (C) or His (H) or lie (I) or Vai (V) or Ser (S);

[0387] Xaa46 is Ala (A) or Cys (C) or Ser (S) or Vai (V) or Gly (G); Xaa47 is Vai (V) or Phe (F) or Tyr (Y) or Arg (R) or Leu (L) or lie (I) or Lys (K) or Gly (G) or Ala (A) or Cys (C) or His (H) or T rp (W) or Glu (E) or Asn (N) or Gin (Q) or Thr (T) or Met (M) or Asp (D) or Ser (S);

[0388] Xaa48 is Gin (Q) or Glu (E) or Ala (A) or Lys (K) or Asp (D) or Met (M) or Vai (V) or Arg (R) or Tyr (Y) or His (H) or Ser (S) or lie (I) or Asn (N) or Leu (L) or Cys (C); Xaa49 is Leu (L) or Met (M);

[0389] Xaa50 is Gin (Q) or Arg (R) or Ser (S) or Met (M) or Trp (W) or Ala (A) or Tyr (Y) or His (H) or Lys (K) or Gly (G) or Thr (T) or Asn (N) or Phe (F) or Glu (E) or Asp (D) or Cys (C) or Leu (L);

[0390] Xaa51 is Lys (K) or Arg (R) or lie (I) or Phe (F) or Ala (A) or Cys (C) or Asn (N) or Gin (Q) or Asp (D) or T rp (W) or Gly (G) or Leu (L) or Ser (S) or Thr (T) or Vai (V) or Tyr (Y) or His (H) or Glu (E) or Met (M);

[0391] Xaa52 is Trp (W) or Tyr (Y) or Met (M) or Phe (F) or lie (I) or Leu (L) or Ala (A) or Lys (K) or Vai (V) or His (H) or Thr (T) or Ser (S) or Glu (E) or Arg (R) or Cys (C) or Gin (Q) or Asp (D) or Gly (G) or Asn (N);

[0392] Xaa53 is Ala (A) or Cys (C) or Arg (R) or Leu (L) or Vai (V) or Met (M) or lie (I) or Lys (K) or Thr (T) or Tyr (Y) or Gin (Q) or Trp (W) or Asn (N) or Ser (S) or His (H) or Phe (F) or Gly (G);

[0393] Xaa54 is Gly (G) or Gin (Q) or Thr (T) or Leu (L) or Arg (R) or Asp (D) or lie (I) or Tyr (Y) or Asn (N) or Lys (K) or Ala (A) or Met (M) or Phe (F) or His (H) or Glu (E) or T rp (W) or Ser (S) or Cys (C);

[0394] Xaa55 is Gly (G) or Lys (K) or Vai (V) or Ser (S) or His (H) or Met (M) or Arg (R) or Leu (L) or Asp (D) or Asn (N) or Gin (Q) or Phe (F) or Ala (A) or Thr (T) or Glu (E) or Tyr (Y) or Cys (C) or lie (I) or Trp (W);

[0395] Xaa56 is Met (M) or lie (I) or Leu (L) or Phe (F) or Arg (R) or Lys (K) or Vai (V) or Asn (N) or Trp (W) or Ala (A) or Tyr (Y) or Cys (C) or Thr (T) or Gin (Q) or His (H) or Ser (S) or Gly (G);

[0396] Xaa57 is Absent or Leu (L); and

[0397] Xaa58 is Absent or Glu (E). The polypeptide according to any one of the preceding embodiments, wherein the binder comprises an amino acid sequence at least 79%, 82%, 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1-4 or 6-39. 12. The polypeptide according to any one of the preceding embodiments, comprising the amino acid sequence according to Formula II (SEQ ID No. 92) :

[0398] Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu-

[0399] Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu- Ile-Val-Glu-Glu-Leu-Xaa57-Xaa58, wherein

[0400] Xaa4 is Leu (L) or Phe (F);

[0401] Xaa6 is Gin (Q) or His (H) or Ser (S) or Thr (T);

[0402] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0403] XaalO is Vai (V) or lie (I);

[0404] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0405] Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);

[0406] Xaa25 is Glu (E) or Lys (K);

[0407] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0408] Xaa30 is Leu (L) or Thr (T) or lie (I);

[0409] Xaa37 is Asn (N) or Gin (Q) or His (H) or Lys (K);

[0410] Xaa57 is Absent or Leu (L); and

[0411] Xaa58 is Absent or Glu (E).

[0412] 13. The polypeptide according to any one of the preceding embodiments, comprising the amino acid sequence according to Formula III (SEQ ID No. 93) :

[0413] Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu-

[0414] Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu- lle-Val-Glu-Glu-Leu-Leu-Glu, wherein

[0415] Xaa4 is Leu (L) or Phe (F);

[0416] Xaa6 is His (H) or Ser (S) or Thr (T) or Gin (Q);

[0417] Xaa8 is Vai (V) or lie (I) or Thr (T) or Met (M);

[0418] XaalO is Vai (V) or lie (I);

[0419] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0420] Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);

[0421] Xaa25 is Lys (K) or Glu (E);

[0422] Xaa27 is Vai (V) or lie (I) or Leu (L); Xaa30 is Leu (L) or Thr (T) or lie (I); and

[0423] Xaa37 is Gin (Q) or Asn (N) or His (H) or Lys (K).

[0424] 14. The polypeptide according to any one of the preceding embodiments, comprising the amino acid sequence according to Formula IV (SEQ ID No. 94):

[0425] Ser-Phe-Leu-Xaa4-Lys-His-Ala-Xaa8-Arg-Val-Xaa11-Glu-Arg-Leu-Leu-Glu-Glu-Gly-

[0426] Asn-Val-Glu-Val-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-lle-Lys-Arg-Leu-Ala-His-Ala- Gln-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu-lle-Val-Glu-Glu- Leu-Xaa57-Xaa58, wherein

[0427] Xaa4 is Leu (L) or Phe (F);

[0428] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0429] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0430] Xaa25 is Glu (E) or Lys (K);

[0431] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0432] Xaa57 is Absent or Leu (L); and

[0433] Xaa58 is Absent or Glu (E).

[0434] 15. The polypeptide according to any one of the preceding embodiments, wherein the binder comprises the amino acid sequence according to Formula II (SEQ ID No. 92) :

[0435] Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu-

[0436] Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu- Ile-Val-Glu-Glu-Leu-Xaa57-Xaa58, wherein

[0437] Xaa4 is Leu (L) or Phe (F);

[0438] Xaa6 is Gin (Q) or His (H) or Ser (S) or Thr (T);

[0439] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0440] XaalO is Vai (V) or lie (I);

[0441] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0442] Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);

[0443] Xaa25 is Glu (E) or Lys (K);

[0444] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0445] Xaa30 is Leu (L) or Thr (T) or lie (I);

[0446] Xaa37 is Asn (N) or Gin (Q) or His (H) or Lys (K); Xaa57 is Absent or Leu (L); and Xaa58 is Absent or Glu (E).

[0447] 16. The polypeptide according to any one of the preceding embodiments, wherein the binder comprises the amino acid sequence according to Formula IV (SEQ ID No. 94):

[0448] Ser-Phe-Leu-Xaa4-Lys-His-Ala-Xaa8-Arg-Val-Xaa11-Glu-Arg-Leu-Leu-Glu-Glu-Gly- Asn-Val-Glu-Val-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-lle-Lys-Arg-Leu-Ala-His-Ala- Gln-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu-lle-Val-Glu-Glu- Leu-Xaa57-Xaa58, wherein

[0449] Xaa4 is Leu (L) or Phe (F);

[0450] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0451] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0452] Xaa25 is Glu (E) or Lys (K);

[0453] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0454] Xaa57 is Absent or Leu (L); and

[0455] Xaa58 is Absent or Glu (E).

[0456] 17. The polypeptide according to any one of the preceding embodiments, wherein the binder comprises an amino acid sequence at least 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 2, 6, 14, 24, 29.

[0457] 18. The polypeptide according to any one of the preceding embodiments, wherein the binder comprises the amino acid sequence according to Formula V (SEQ ID No. 95):

[0458] Ser-Phe-Leu-Leu-Lys-Xaa6-Ala-Xaa8-Arg-Val-Xaa11-Glu-Arg-Leu-Leu-Glu-Glu-Gly- Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg-Leu-Ala-His- Ala-GIn-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu-lle-Val-Glu-Glu- Leu-Leu-Glu, wherein

[0459] Xaa6 is His (H) or Ser (S);

[0460] Xaa8 is Vai (V) or lie (I) or Met (M);

[0461] Xaa11 is lie (I) or Vai (V);

[0462] Xaa22 is Leu (L) or Vai (V) or lie (I) or Thr (T);

[0463] Xaa25 is Lys (K) or Glu (E); Xaa27 is Vai (V) or lie (I); and

[0464] Xaa30 is Leu (L) or lie (I) or Thr (T).

[0465] 19. The polypeptide according to any one of the preceding embodiments, wherein 1-18 of the following residues are invariant: Seri , Phe2, Leu3, Lys5, His6, Ala7, Arg9, Val10, Arg13, Leu14, Val22, Glu25, Trp26, Gln29, He30, Leu33, Ala36, Gln37.

[0466] 20. The polypeptide according to any one of the preceding embodiments, wherein no more than 18 of the following residues are invariant: Seri , Phe2, Leu3, Lys5, His6, Ala7, Arg9, Val10, Arg13, Leu14, Val22, Glu25, Trp26, Gln29, He30, Leu33, Ala36, Gln37, optionally wherein no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11 , no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3 or no more than 2 of the residues are invariant.

[0467] 21. The polypeptide according to any one of the preceding embodiments, wherein the following residues are invariant: Seri , Phe2, Leu3, Lys5, His6, Ala7, Arg9, Val10, Arg13, Leu14, Val22, Glu25, Trp26, Gln29, He30, Leu33, Ala36, Gln37.

[0468] 22. The polypeptide according to any one of the preceding embodiments, comprising the amino acid sequence as set out in any one of SEQ ID Nos. 5, 2, 6, 14, 24, 29.

[0469] 23. The polypeptide according to any one of the preceding embodiments, wherein the binder comprises the amino acid sequence as set out in any one of SEQ ID Nos. 5, 2, 6, 14, 24, 29.

[0470] 24. The polypeptide according to any one of the preceding embodiments, wherein the binder comprises the amino acid sequence as set out in SEQ ID No. 5.

[0471] 25. The polypeptide according to any one of embodiments 1-23, wherein the binder comprises the amino acid sequence as set out in SEQ ID No. 2.

[0472] 26. The polypeptide according to any one of embodiments 1-23, wherein the binder comprises the amino acid sequence as set out in SEQ ID No. 6. 27. The polypeptide according to any one of embodiments 1-23, wherein the binder comprises the amino acid sequence as set out in SEQ ID No. 14.

[0473] 28. The polypeptide according to any one of embodiments 1-23, wherein the binder comprises the amino acid sequence as set out in SEQ ID No. 24.

[0474] 29. The polypeptide according to any one of embodiments 1-23, wherein the binder comprises the amino acid sequence as set out in SEQ ID No. 29.

[0475] 30. The polypeptide according to any one of the preceding embodiments, wherein the binder comprises a N-terminal or C-terminal amino acid extension.

[0476] 31 . The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to a SORT 1 receptor.

[0477] 32. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface and / or murine SORT 1 at the SORT 1 receptor interface.

[0478] 33. The polypeptide according to any one of the preceding embodiments, wherein the binder is capable of binding to a SORT 1 receptor.

[0479] 34. The polypeptide according to any one of the preceding embodiments, wherein the binder is capable of binding to human SORT 1 at the SORT 1 receptor interface and / or murine SORT 1 at the SORT 1 receptor interface.

[0480] 35. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 2uM or less, and / or murine SORT 1 at the SORT 1 receptor interface with a Kd of 2uM or less.

[0481] 36. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 1uM or less, and / or murine SORT 1 at the SORT 1 receptor interface with a Kd of 1uM or less. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 100nM or less, and / or murine SORT 1 at the SORT 1 receptor interface with a Kd of 100nM or less. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 75nM or less, and / or murine SORT 1 at the SORT 1 receptor interface with a Kd of 75nM or less. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 64nM or less, and / or murine SORT 1 at the SORT 1 receptor interface with a Kd of 64nM or less. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 59nM or less, and / or murine SORT 1 at the SORT 1 receptor interface with a Kd of 59nM or less. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 50nM or less, and / or murine SORT 1 at the SORT 1 receptor interface with a Kd of 50nM or less. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 2uM or less. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 1uM or less. 44. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 100nM or less.

[0482] 45. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 75nM or less.

[0483] 46. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 64nM or less.

[0484] 47. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 59nM or less.

[0485] 48. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 50nM or less.

[0486] 49. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 34nM or less.

[0487] 50. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 30nM or less.

[0488] 51 . The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to human SORT 1 at the SORT 1 receptor interface with a Kd of 25nM or less.

[0489] 52. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide binds between 1 to 17 of the following residues of human Sortl : Thr396, Leu419, Asp422, Asn423, Ile425, Ala450, Thr451 , Ala452, Tyr466, Gln470, Lys471 , Leu472, Ala497, Ile498, Ser499, Val500, Met501.

[0490] 53. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide binds between at least 1 of the following residues of human Sortl : Thr396, Leu419, Asp422, Asn423, Ile425, Ala450, Thr451 , Ala452, Tyr466, Gln470, Lys471 , Leu472, Ala497, Ile498, Ser499, Val500, Met501 , optionally wherein the polypeptide binds at least at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15 or at least 16 of the residues.

[0491] 54. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide binds the following residues of human Sortl : Thr396, Leu419, Asp422, Asn423, Ile425, Ala450, Thr451 , Ala452, Tyr466, Gln470, Lys471 , Leu472, Ala497, Ile498, Ser499, Val500, Met501.

[0492] 55. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to murine SORT 1 at the SORT 1 receptor interface with a Kd of 2uM or less.

[0493] 56. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to murine SORT 1 at the SORT 1 receptor interface with a Kd of 1uM or less.

[0494] 57. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to murine SORT 1 at the SORT 1 receptor interface with a Kd of 100nM or less.

[0495] 58. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to murine SORT 1 at the SORT 1 receptor interface with a Kd of 75nM or less.

[0496] 59. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to murine SORT 1 at the SORT 1 receptor interface with a Kd of 64nM or less. 60. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to murine SORT 1 at the SORT 1 receptor interface with a Kd of 59nM or less.

[0497] 61 . The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to murine SORT 1 at the SORT 1 receptor interface with a Kd of 50nM or less.

[0498] 62. The polypeptide according to any one of the preceding embodiments, wherein the capacity of the polypeptide to bind to the SORT 1 receptor is dependent on the protonation state of the SORT 1 receptor.

[0499] 63. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of being released from the SORT1 receptor under acidic conditions.

[0500] 64. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of being released from the SORT1 receptor when the SORT1 receptor is in an environmental pH below or equal to 6.

[0501] 65. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is incapable of binding to the SORT 1 receptor when the SORT 1 receptor is in a protonated state.

[0502] 66. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is incapable of binding to the SORT 1 receptor when the SORT 1 receptor is in an acidic environment.

[0503] 67. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is incapable of binding to the SORT 1 receptor when the SORT 1 receptor is in an environment below or equal to pH 6. 68. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH above 6.

[0504] 69. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH above 6.1.

[0505] 70. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH above 6.2.

[0506] 71. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH above 6.3.

[0507] 72. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH above 6.4.

[0508] 73. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH above 6.5.

[0509] 74. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH between 6.1-9.

[0510] 75. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH between 6.5-9.

[0511] 76. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH between 6.5-8.5. 77. The polypeptide according to any one of the preceding embodiments, wherein the binding of the polypeptide to the SORT1 receptor is dependent on an environmental pH between 6.5-8.

[0512] 78. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to the SORT 1 receptor when the SORT 1 receptor is in a deprotonated state, and wherein the polypeptide is further capable of being released from the SORT1 receptor when the SORT1 receptor is in a protonated state.

[0513] 79. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to the SORT 1 receptor when the SORT 1 receptor is in neutral or alkaline conditions, and wherein the polypeptide is further capable of being released from the SORT 1 receptor when the SORT 1 receptor is in acidic conditions.

[0514] 80. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is capable of binding to the SORT 1 receptor when the SORT 1 receptor is in an environmental pH above 6, and wherein the polypeptide is further capable of being released from the SORT1 receptor when the SORT1 receptor is in an environmental pH below or equal to 6.

[0515] 81. The polypeptide according to any one of the preceding embodiments, wherein the binder forms part of a binder moiety.

[0516] 82. The polypeptide according to embodiment 81 , further comprising at least one cargo moiety.

[0517] 83. The polypeptide according to any one of embodiments 82, wherein the cargo moiety is a therapeutic agent, optionally wherein the cargo moiety is a polypeptide.

[0518] 84. The polypeptide according to any one of embodiments 82-83, wherein the cargo moiety is linked to the binder moiety.

[0519] 85. The polypeptide according to any one of embodiments 81-84, wherein the cargo moiety is linked to the binder moiety with a linker. 86. The polypeptide according to any one of embodiments 81-85, wherein the cargo moiety is linked to the N-terminus of the binder moiety.

[0520] 87. The polypeptide according to any one of embodiments 81-85, wherein the cargo moiety is linked to the C-terminus of the binder moiety.

[0521] 88. The polypeptide according to any one of embodiments 81-85, wherein the cargo moiety is linked to the C-terminus or the N-terminus of the binder moiety.

[0522] 89. The polypeptide according to any one of embodiments 81-85, wherein the at least one cargo moiety is 2 cargo moieties and each one of the cargo moieties is linked to the C- terminus and the N-terminus of the binder moiety.

[0523] 90. The polypeptide according to any one of embodiments 81-85, wherein the cargo moiety is linked to an amino acid residue found between the N-terminus and C-terminus of the binder moiety.

[0524] 91. The polypeptide according to any one of embodiments 81-85, wherein the binder moiety further comprises a reactive group, and wherein the cargo moiety is linked to the reactive group.

[0525] 92. The polypeptide according to any one of embodiments 81-91, wherein the cargo moiety is linked to the binder moiety as a recombinant fusion, chemical conjugation, or combination thereof.

[0526] 93. The polypeptide according to any one of embodiments 81-92, wherein the cargo moiety is linked as a chemical conjugation to the binder moiety.

[0527] 94. The polypeptide according to any one of embodiments 81-87, wherein the cargo moiety is linked as a recombinant fusion to the binder moiety.

[0528] 95. A construct comprising the polypeptide according to any one of embodiments 1-81, the polypeptide or binder forming part of a binder moiety linked to at least one cargo moiety. 96. The construct according to embodiment 95, wherein the at least one cargo moiety is a therapeutic agent.

[0529] 97. The construct according to embodiment 96, wherein the therapeutic agent is a polypeptide.

[0530] 98. The construct according to embodiment 95, wherein the construct comprises a fusion protein.

[0531] 99. The construct according to any one of embodiments 95-98, wherein the cargo moiety is linked to the polypeptide with a linker.

[0532] 100. The construct according to any one of embodiments 95-99, wherein the cargo moiety is linked to the N-terminus of the polypeptide.

[0533] 101. The construct according to any one of embodiments 95-99, wherein the cargo moiety is linked to the C-terminus of the polypeptide.

[0534] 102. The construct according to any one of embodiments 95-99, wherein the cargo moiety is linked to the C-terminus or the N-terminus of the polypeptide.

[0535] 103. The construct according to any one of embodiments 95-99, wherein the cargo moiety is linked to an amino acid residue found between the N-terminus and C-terminus of the binder moiety.

[0536] 104. The construct according to any one of embodiments 95-99, wherein the polypeptide further comprises a reactive group, and wherein the cargo moiety is linked to the reactive group.

[0537] 105. The construct according to any one of embodiments 95-104, wherein the cargo moiety is linked as a recombinant fusion, chemical conjugation, or combination thereof.

[0538] 106. The construct according to any one of embodiments 95-104, wherein the cargo moiety is linked as a chemical conjugation to the polypeptide. 107. The construct according to any one of embodiments 95-101 , wherein the cargo moiety is linked as a recombinant fusion to the binder moiety.

[0539] 108. A construct comprising a binding moiety and a cargo moiety, the binding moiety comprising a binder having a binding affinity to Sortilin-1 (SORT1) receptor and comprising an amino acid sequence as set out in any one of SEQ ID Nos. 1-39.

[0540] 109. A polypeptide comprising a binding moiety and a cargo moiety, the binding moiety comprising a binder having a binding affinity to Sortilin-1 (SORT1) receptor and comprising an amino acid sequence as set out in any one of SEQ ID Nos. 1-39.

[0541] 110. A construct comprising a binding moiety and a cargo moiety, the binding moiety comprising a binder having a binding affinity to Sortilin-1 (SORT1) receptor and comprising an amino acid sequence as set out in any one of SEQ ID Nos. 5, 2, 6, 14, 24, 29.

[0542] 111. A polypeptide comprising a binding moiety and a cargo moiety, the binding moiety comprising a binder having a binding affinity to Sortilin-1 (SORT1) receptor and comprising an amino acid sequence as set out in any one of SEQ ID Nos. 5, 2, 6, 14, 24, 29.

[0543] 112. A polypeptide comprising a binder, the binder having a binding affinity to Sortilin-1 (Sortl) receptor and comprising an amino acid sequence according to Formula II (SEQ ID No. 92):

[0544] Ser- Phe- Leu-Xaa4- Lys-Xaa6-AI a-Xaa8-Arg-Xaa 10-Xaa 11 -G I u- Arg- Leu- Leu-GI u- Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu- Ile-Val-Glu-Glu-Leu-Xaa57-Xaa58, wherein

[0545] Xaa4 is Leu (L) or Phe (F);

[0546] Xaa6 is Gin (Q) or His (H) or Ser (S) or Thr (T);

[0547] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0548] XaalO is Vai (V) or lie (I);

[0549] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0550] Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P); Xaa25 is Glu (E) or Lys (K);

[0551] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0552] Xaa30 is Leu (L) or Thr (T) or lie (I);

[0553] Xaa37 is Asn (N) or Gin (Q) or His (H) or Lys (K);

[0554] Xaa57 is Absent or Leu (L); and

[0555] Xaa58 is Absent or Glu (E).

[0556] 113. A polypeptide comprising a binder, the binder having a binding affinity to Sortilin-1 (Sortl) receptor and comprising an amino acid sequence according to Formula V (SEQ ID No. 95)

[0557] Ser-Phe-Leu-Leu-Lys-Xaa6-Ala-Xaa8-Arg-Val-Xaa11-Glu-Arg-Leu-Leu-Glu-Glu-Gly- Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg-Leu-Ala-His- Ala-GIn-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu-lle-Val-Glu-Glu- Leu-Leu-Glu, wherein

[0558] Xaa6 is His (H) or Ser (S);

[0559] Xaa8 is Vai (V) or lie (I) or Met (M);

[0560] Xaa11 is lie (I) or Vai (V);

[0561] Xaa22 is Leu (L) or Vai (V) or lie (I) or Thr (T);

[0562] Xaa25 is Lys (K) or Glu (E);

[0563] Xaa27 is Vai (V) or lie (I); and

[0564] Xaa30 is Leu (L) or lie (I) or Thr (T).

[0565] 114. A construct comprising a binder moiety and a cargo moiety, the binder moiety being capable of binding to a Sortilin-1 (Sortl) receptor, and the binder moiety comprising an amino acid sequence according to Formula II (SEQ ID No. 92) :

[0566] Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu- Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu-

[0567] Ile-Val-Glu-Glu-Leu-Xaa57-Xaa58, wherein

[0568] Xaa4 is Leu (L) or Phe (F);

[0569] Xaa6 is Gin (Q) or His (H) or Ser (S) or Thr (T);

[0570] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0571] XaalO is Vai (V) or lie (I); Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0572] Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);

[0573] Xaa25 is Glu (E) or Lys (K);

[0574] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0575] Xaa30 is Leu (L) or Thr (T) or lie (I);

[0576] Xaa37 is Asn (N) or Gin (Q) or His (H) or Lys (K);

[0577] Xaa57 is Absent or Leu (L); and

[0578] Xaa58 is Absent or Glu (E).

[0579] 115. A construct comprising a binder moiety and a cargo moiety, the binder moiety being capable of binding to a Sortilin-1 (Sortl) receptor, and the binder moiety comprising an amino acid sequence according to Formula V (SEQ ID No. 95) :

[0580] Ser-Phe-Leu-Leu-Lys-Xaa6-Ala-Xaa8-Arg-Val-Xaa11-Glu-Arg-Leu-Leu-Glu-Glu-Gly- Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg-Leu-Ala-His- Ala-GIn-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu-lle-Val-Glu-Glu- Leu-Leu-Glu, wherein

[0581] Xaa6 is His (H) or Ser (S);

[0582] Xaa8 is Vai (V) or lie (I) or Met (M);

[0583] Xaa11 is lie (I) or Vai (V);

[0584] Xaa22 is Leu (L) or Vai (V) or lie (I) or Thr (T);

[0585] Xaa25 is Lys (K) or Glu (E);

[0586] Xaa27 is Vai (V) or lie (I); and

[0587] Xaa30 is Leu (L) or lie (I) or Thr (T).

[0588] 116. A construct comprising a binder moiety and a cargo moiety, the cargo moiety linked to the binder moiety via a linker, the binder moiety being capable of binding to a Sortilin-1 (SORT1) receptor, and the binder moiety comprising an amino acid sequence according to Formula II (SEQ ID No. 92) :

[0589] Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu- Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu- Ile-Val-Glu-Glu-Leu-Xaa57-Xaa58, wherein

[0590] Xaa4 is Leu (L) or Phe (F); Xaa6 is Gin (Q) or His (H) or Ser (S) or Thr (T);

[0591] Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);

[0592] XaalO is Vai (V) or lie (I);

[0593] Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);

[0594] Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);

[0595] Xaa25 is Glu (E) or Lys (K);

[0596] Xaa27 is lie (I) or Vai (V) or Leu (L);

[0597] Xaa30 is Leu (L) or Thr (T) or lie (I);

[0598] Xaa37 is Asn (N) or Gin (Q) or His (H) or Lys (K);

[0599] Xaa57 is Absent or Leu (L); and

[0600] Xaa58 is Absent or Glu (E). . A pharmaceutical composition comprising the polypeptide or construct according to any one of the preceding embodiments and a pharmaceutical excipient. . A pharmaceutical composition comprising the polypeptide or construct according to any one of the preceding embodiments and a pharmaceutically acceptable buffer. . The pharmaceutical composition according to any one of embodiments 117-118, wherein the pharmaceutical composition is suitable for parenteral administration such as subcutaneous, intramuscular, intraperitoneal, or intravenous administration. . The pharmaceutical composition according to any one of embodiments 117-119, wherein the pharmaceutical composition, wherein the pharmaceutical composition is a liquid pharmaceutical formulation. . The pharmaceutical composition according to any one of embodiments 117-120, wherein the pharmaceutical composition, wherein the pharmaceutical composition is an aqueous pharmaceutical formulation. . The polypeptide or construct according to any one of embodiments 1-116 or the pharmaceutical composition according to any one of embodiments 117-121 for use as a medicament. 123. The polypeptide or construct according to any one of embodiments 1-116 or the pharmaceutical composition according to any one of embodiments 117-121 for use as use as a therapeutic agent.

[0601] 124. The polypeptide or construct according to any one of embodiments 1-116 or the pharmaceutical composition according to any one of embodiments 117-121 for use in promoting internalization of a cargo.

[0602] 125. The polypeptide or construct according to any one of embodiments 1-116 or the pharmaceutical composition according to any one of embodiments 117-121 for use in promoting internalization of a therapeutic agent.

[0603] 126. The polypeptide or construct according to any one of embodiments 1-116 or the pharmaceutical composition according to any one of embodiments 117-121 for use in promoting clearance of a cargo.

[0604] 127. The polypeptide or construct according to any one of embodiments 1-116 or the pharmaceutical composition according to any one of embodiments 117-121 for use as use in promoting clearance of a therapeutic agent.

[0605] 128. The polypeptide or construct according to any one of embodiments 1-116 or the pharmaceutical composition according to any one of embodiments 117-121 for use in promoting extracellular protein degradation.

[0606] 129. The polypeptide or construct according to any one of embodiments 1-116 or the pharmaceutical composition according to any one of embodiments 117-121 for use as use in promoting internalization a cargo, internalization of a therapeutic agent or extracellular protein degradation.

[0607] 130. The polypeptide or construct according to any one of embodiments 1-116 or the pharmaceutical composition according to any one of embodiments 117-121 for use as use in promoting clearance of a cargo, clearance of a therapeutic agent or extracellular protein degradation. EXAMPLES

[0608] This experimental part starts with a list of abbreviations and is followed by a section including the general computational assays, the list of binders forming par the invention herein as well as constructs synthesized and used, experimental assays relating to the activity and properties of the Sortl binders and constructs and finally the experimental methods. The examples serve to illustrate the invention and are not to be construed as limiting in any way.

[0609] Abbreviations

[0610] PDB Protein Data Bank

[0611] CBx Combiatorial Binder (x= any number)

[0612] ECD Extraellular Domain

[0613] BINDER A computationally derived protein that binds to a Target, herein described as SORT1. This refers to the CBx series

[0614] Sortl or SORT1 Sortilin-1

[0615] BBB Blood Brain Barrier huSORTI(ECD) huSORTI (78-756), uniprot Q99523-1

[0616] SPR Surface Plasmon Resonance

[0617] SSM single site mutagenesis

[0618] FACS fluorescence-activated cell sorting hCMEC / D3 cells human cerebral microvascular endothelial cells

[0619] GFP Green fluorescent protein

[0620] IPTG Isopropyl p-d-1-thiogalactopyranoside

[0621] IMAC I mobilized Metal Affinity Chromatography

[0622] SEC Size Exclusion Chromatography

[0623] EGFR Epidermal Growth Factor Receptor pLYTACs Protein lysosomal-targeting chimeras

[0624] FBS fetal bovine serum

[0625] PBS Phosphate-buffered saline

[0626] Computational Assays

[0627] Computational Binders design method and variants Following the protocol from Cao et al. (Cao L, Goreshnik I, Coventry B, Case JB, Miller L, Kozodoy L, et al. De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science. 2020;370(6515):426-31), 21,000 binders were generated to each of two sites on the sortilin- 1 receptor. The epitope of Sitel is comprised of five amino acids (Uniprot numbering: F92, V93, T546, T559, and T561) was chosen since it provided a modest patch of exposed hydrophobicity while avoiding any sites of known interactions. Site2 is comprised of six amino acids residues (Uniprot: L394, L419, I425, Y466, I498, and V500) was chosen. For sitel , the selected epitope has the added feature that a binder to this location would be pH dependent since this region undergoes significant structural change at low pH (Leloup N, Lossl P, Meijer DH, Brennich M, Heck AJR, Thies-Weesie DME, et al. Low pH-induced conformational change and dimerization of sortilin triggers endocytosed ligand release. Nat Commun. 2017;8(1):1708). As described in Cao et al. (see reference above), we used a set of scaffold libraries to generate 44M docks using PatchDock which were subsequently refined with Rifdock to bring the total to around 6M docks to each site of the sites. From here, the standard predict / FastDesign process from Cao et. al. (see reference above) was used to first to quickly down sample the docks to the most promising 500,000 and then run those through Rosetta FastDesign to produce finalized sequences. Interface helices were extracted from those 500,000, clustered, and ranked by binding energy to produce 3,000 de novo motifs. The original 22,000 scaffolds were then docked into each of the motifs to produce roughly 6M docks once more. The docks were once again down sampled with the predictor to 500K and FastDesigned. The final pool of 1M FastDesign outputs was filtered by Rosetta ddG, Contact Molecular Surface, and Contact Molecular Surface to hotspot residues (Contact Patch). This computational protocol (Figure 3) generated the best 21 ,000 per site for a total of 42,000 tested against SORT 1.

[0628] SORT 1 Site Selection

[0629] Selection of the target site in SORT1 for designing a computational binder is aimed at specifically targeting a region that undergoes conformational changes in response to a shift from neutral pH to acidic pH, such as within endosome or lysosomal compartments. Notably, the transition from neutral to acidic pH triggers the conversion of SORT1 from a monomeric to a dimeric state. The computational methods described herein have yielded a set of SORT1 binders to this region. As exemplified in Figures 4A-4C modeled interactions illustrate the successful binding of a binder (in this case comprising ligand sequence 2) to the surface of human SORT1 at physiological pH, particularly within the extracellular region at the cell surface. However, at acidic pH, where SORT1 undergoes a conformational change, clashes occur between SORT1 and the binder, thereby inhibiting their interaction. This mechanism provides a means to regulate binding, where the native states of SORT1 are modulated by pH, ultimately affecting the apparent compatibility of the binder with the epitope.

[0630] With reference to Figs. 4A-4C, human SORT1 models were generated based on structures obtained at neutral pH (PDB: 4PO7) and acidic pH (PDB: 6EHO). The surfaces of these models differ due to variations in histitdine protonation states, resulting in changes in the protein's tertiary fold. In Fig. 4A, at neutral pH, the computationally derived model CB1 is docked onto SORT1. Histidine residues are depicted as dark spheres. In Fig. 4B, the surface representation of the neutral pH human SORT1 structure is shown, while CB1 is represented as a ribbon diagram. Complementary interactions between SORT1 and CB1 are observed. In Fig. 4C, the surface representation of at acidic pH human SORT1 structure is shown, along with CB1 represented as a ribbon diagram. Here non-complementary interactions between SORT1 and CB1 are evident. These non-complementary interactions at acidic pH represent a mechanism through which the Sortl receptor can release the Sortl binder, and facilitating the Sortl protein dimer formation.

[0631] Single Site Mutagenesis Screen (SSM)

[0632] A specific parent design (CBO, 1) was selected for single site mutagenesis (SSM), where each position was changed to all other native amino acids (saturation of each position) to observe impacts on protein's core, and interface to SORT1, and solvent exposed residues. The saturated site-directed mutagenesis utilized Yeast Display and FACS to determine that shifts in binding to SORT1 relative to CBO were beneficial or tolerated. The data indicates strong sequence preferences at positions relevant to the modeled interface to SORT1. Conversely, residues exposed on the protein's surface or with high surface accessibility showed higher variability. Table 2 illustrates positions categorized as best or tolerant and based on their association with the interface, core, or surface-exposed residues. It is understood that both best and tolerable are acceptable single site mutations of the parent CBO designed sequence.

[0633] Table 2. demonstrates both tolerant and intolerant positions related to interface, core, and surface exposed residues by yeast display shifts.

[0634] A library of SSM sequences were derived to generate a combinatorial (CB) library based on the parent (CBO) that included both single site and by combining two or more changes suggested as beneficial by SSM and Rosetta output, identified herein as CB1-CB14 and CB16-CB39. Without preference, a sub-set of ‘CB’ binders that demonstrated significant binding in Yeast Display and Rosetta output were selected, and were considered for conversion to constructs amenable for E.coli or Mammalian cell production (HEK) for functional and specificity assays, among other things.

[0635] Binders and Constructs

[0636] Binders

[0637] Reference to a binder of the invention via the name CBx, where x refers to any integer, refers to a binder from the list of binders identified below. The below list are examples only and are not to be construed as limitative in any way.

[0638] It is understood that, where a construct refers to having a structure comprising a CBx binder, it refers to the incorporation of the sequence associated to said CBx binder within the construct.

[0639] CBO

[0640] SFLLKQAIRVIERLLEEGNVELAEEWIEQLKRLAHANRDPEALRRAEELEEIVEEL

[0641] SEQ ID No. 1

[0642] CB1

[0643] SFLLKHAVRVIERLLEEGNVELAEKWVEQLKRLAHAQRDPEALRRAEELEEIVEELLE SEQ ID No. 2

[0644] CB2

[0645] SFLLKHAIRVIERLLEEGNVEIAEKWVEQTKRLAHAQRDPEALRRAEELEEIVEELLE SEQ ID No. 3

[0646] CB3

[0647] SFLLKSAVRVI ERLLEEGN VEVAEKWI EQLKRLAHAQRDPEALRRAEELEEI VEELLE

[0648] SEQ ID No. 4

[0649] CB4

[0650] SFLLKHAIRVVERLLEEGNVEVAEEWVEQIKRLAHAQRDPEALRRAEELEEI VEELLE

[0651] SEQ ID No. 5

[0652] CB5

[0653] SFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0654] SEQ ID No. 6

[0655] CB6

[0656] SFLLKTAIRVIERLLEEGNVETAEKWIEQLKRLAHAQRDPEALRRAEELEEIVEELLE

[0657] SEQ ID No. 7

[0658] CB7

[0659] SFLLKSAVRVVERLLEEGNVEVAEKWIEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0660] SEQ ID No. 8

[0661] CB8

[0662] SFLLKSAVRWERLLEEGNVEIAEKWVEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0663] SEQ ID No. 9

[0664] CB9

[0665] SFLLKSAVRWERLLEEGNVEIAEKWVEQIKRLAHANRDPEALRRAEELEEIVEELLE

[0666] SEQ ID No. 10

[0667] CB10

[0668] SFLLKSAVRVIERLLEEGNVEIAEKWIEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0669] SEQ ID No. 11

[0670] CB11 SFLLKSAIRVTERLLEEGNVETAEKWIEQLKRLAHAQRDPEALRRAEELEEIVEELLE

[0671] SEQ ID No. 12

[0672] CB12

[0673] SFLLKSAIRVIERLLEEGNVEVAEKWIEQLKRLAHAQRDPEALRRAEELEEIVEELLE

[0674] SEQ ID No. 13

[0675] CB13

[0676] SFLLKSAIRVIERLLEEGNVETAEKWVEQTKRLAHAQRDPEALRRAEELEEIVEELLE

[0677] SEQ ID No. 14

[0678] CB14

[0679] SFLLKSAIRVAERLLEEGNVEVAEKWVEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0680] SEQ ID No. 15

[0681] CB16

[0682] SFLLKHAVRVVERLLEEGNVETAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0683] SEQ ID No. 16

[0684] CB17

[0685] SFLLKHAVRVVERLLEEGNVEIAEEWIEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0686] SEQ ID No. 17

[0687] CB18

[0688] SFLLKHAVRVIERLLEEGNVEVAEKWLEQLKRLAHANRDPEALRRAEELEEIVEELLE

[0689] SEQ ID No. 18

[0690] CB19

[0691] SFLLKHAVRVIERLLEEGNVETAEKWLEQLKRLAHAQRDPEALRRAEELEEIVEELLE

[0692] SEQ ID No. 19

[0693] CB20

[0694] SFLLKHAVRIIERLLEEGNVELAEKWLEQLKRLAHAQRDPEALRRAEELEEIVEELLE

[0695] SEQ ID No. 20 CB21

[0696] SFLLKHATRVVERLLEEGNVELAEKWLEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0697] SEQ ID No. 21

[0698] CB22

[0699] SFLLKHATRVVERLLEEGNVEIAEKWIEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0700] SEQ ID No. 22

[0701] CB23

[0702] SFLLKHAM RVI ERLLEEGN VEVAEKWVEQTKRLAHAQRDPEALRRAEELEEI VEELLE

[0703] SEQ ID No. 23

[0704] CB24

[0705] SFLLKHAM RVI ERLLEEGN VEVAEKWI EQTKRLAHAQRDPEALRRAEELEEI VEELLE

[0706] SEQ ID No. 24

[0707] CB25

[0708] SFLLKHAM RVI ERLLEEGN VEIAEKWVEQI KRLAHAQRDPEALRRAEELEEI VEELLE

[0709] SEQ ID No. 25

[0710] CB26

[0711] SFLLKHAIRVVERLLEEGNVETAEKWVEQIKRLAHAHRDPEALRRAEELEEIVEELLE

[0712] SEQ ID No. 26

[0713] CB27

[0714] SFLLKHAIRVVERLLEEGNVELAEEWIEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0715] SEQ ID No. 27

[0716] CB28

[0717] SFLLKHAIRVVERLLEEGNVEIAEKWVEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0718] SEQ ID No. 28

[0719] CB29

[0720] SFLLKHAIRVIERLLEEGNVETAEKWVEQTKRLAHAQRDPEALRRAEELEEIVEELLE

[0721] SEQ ID No. 29 CB30

[0722] SFLLKHAIRVIERLLEEGNVETAEEWIEQTKRLAHAQRDPEALRRAEELEEIVEELLE

[0723] SEQ ID No. 30

[0724] CB31

[0725] SFLLKHAIRVIERLLEEGNVETAEEWIEQTKRLAHANRDPEALRRAEELEEIVEELLE

[0726] SEQ ID No. 31

[0727] CB32

[0728] SFLLKHAIRVIERLLEEGNVEPAEKWIEQLKRLAHAQRDPEALRRAEELEEIVEELLE

[0729] SEQ ID No. 32

[0730] CB33

[0731] SFLLKHAIRVIERLLEEGNVEIAEKWIEQLKRLAHAHRDPEALRRAEELEEIVEELLE

[0732] SEQ ID No. 33

[0733] CB34

[0734] SFLLKHAIRVIERLLEEGNVEIAEKWIEQIKRLAHAQRDPEALRRAEELEEIVEELLE

[0735] SEQ ID No. 34

[0736] CB35

[0737] SFLLKHAIRVAERLLEEGNVEIAEKWVEQIKRLAHANRDPEALRRAEELEEIVEELLE

[0738] SEQ ID No. 35

[0739] CB36

[0740] SFLLKHAIRIIERLLEEGNVEVAEKWVEQLKRLAHAQRDPEALRRAEELEEIVEELLE

[0741] SEQ ID No. 36

[0742] CB37

[0743] SFLLKHAIRIIERLLEEGNVEVAEKWIEQLKRLAHAQRDPEALRRAEELEEIVEELLE

[0744] SEQ ID No. 37

[0745] CB38

[0746] SFLLKHAIRIIERLLEEGNVELAEKWVEQTKRLAHAQRDPEALRRAEELEEIVEELLE SEQ ID No. 38

[0747] CB39

[0748] SFLFKQAVRVVERLLEEGNVEIAEEWIEQTKRLAHAKRDPEALRRAEELEEIVEELLE

[0749] SEQ ID No. 39

[0750] Example Constructs

[0751] Construct 1

[0752] 6xHis-[TEV]-CB1-GGG-MycTag

[0753] MSHHHHHHHHSENLYFQSMSFLLKHAVRVIERLLEEGNVELAEKWVEQLKRLAHAQRDPE

[0754] ALRRAEELEEIVEELLEGGGSEQKLISEEDL

[0755] SEQ ID No. 46

[0756] Construct comprising CB1 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV (SENLYFQS, SEQ ID No. 44) site, the CB binding sequence, and a Myc-tag (EQKLISEEDL, SEQ ID No. 45).

[0757] Construct 2

[0758] 6xHis-[TEV]-CB4-GGG-MycTag

[0759] MSHHHHHHHHSENLYFQSMSFLLKHAIRVVERLLEEGNVEVAEEWVEQIKRLAHAQRDPEA LRRAEELEEIVEELLEGGGSEQKLISEEDL

[0760] SEQ ID No. 47

[0761] Construct comprising CB4 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV (SENLYFQS, SEQ ID No. 44) site, the CB binding sequence, and a Myc-tag (EQKLISEEDL, SEQ ID No. 45).

[0762] Construct 3

[0763] 6xHis-[TEV]-CB5-GGG-MycTag

[0764] MSHHHHHHHHSENLYFQSMSFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRDPEA LRRAEELEEIVEELLEGGGSEQKLISEEDL

[0765] SEQ ID No. 48

[0766] Construct comprising CB5 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV (SENLYFQS, SEQ ID No. 44) site, the CB binding sequence, and a Myc-tag (EQKLISEEDL, SEQ ID No. 45). Construct 4

[0767] 6xHis-[TEV]-CB13-GGG-MycTag

[0768] MSHHHHHHHHSENLYFQSMSFLLKSAIRVIERLLEEGNVETAEKWVEQTKRLAHAQRDPEA

[0769] LRRAEELEEIVEELLEGGGSEQKLISEEDL

[0770] SEQ ID No. 49

[0771] Construct comprising CB13 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV (SENLYFQS, SEQ ID No. 44) site, the CB binding sequence, and a Myc-tag (EQKLISEEDL, SEQ ID No. 45).

[0772] Construct 5

[0773] 6xHis-[TEV]-CB24-GGG-MycTag

[0774] MSHHHHHHHHSENLYFQSMSFLLKHAMRVIERLLEEGNVEVAEKWIEQTKRLAHAQRDPEA LRRAEELEEIVEELLEGGGSEQKLISEEDL

[0775] SEQ ID No. 50

[0776] Construct comprising CB24 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV (SENLYFQS, SEQ ID No. 44) site, the CB binding sequence, and a Myc-tag (EQKLISEEDL, SEQ ID No. 45).

[0777] Construct 6

[0778] 6xHis-[TEV]-CB29-GGG-MycTag

[0779] MSHHHHHHHHSENLYFQSMSFLLKHAIRVIERLLEEGNVETAEKWVEQTKRLAHAQRDPEA LRRAEELEEIVEELLEGGGSEQKLISEEDL

[0780] SEQ ID No. 51

[0781] Construct comprising CB29 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV (SENLYFQS, SEQ ID No. 44) site, the CB binding sequence, and a Myc-tag (EQKLISEEDL, SEQ ID No. 45).

[0782] Construct 7

[0783] GP-GGG-CB4

[0784] GPGGGSFLLKHAIRVVERLLEEGNVEVAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELL E

[0785] SEQ ID No. 52

[0786] Construct comprising a GP and GGG tag and the CB4 binding sequence.

[0787] Construct 8 6xHis-[HRV3C]-CB5

[0788] MSQSSHHHHHHGGSEVLFQGPGSFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRD

[0789] PEALRRAEELEEIVEELLE

[0790] SEQ ID No. 53

[0791] Construct comprising CB5 binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0792] Construct 9

[0793] 6xHis-[H RV3C]-CB5(S1 A)

[0794] MSQSSHHHHHHGGSEVLFQGPGAFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRD

[0795] PEALRRAEELEEIVEELLE

[0796] SEQ ID No. 54

[0797] Construct comprising a modified CB5(S1 A) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0798] Construct 10

[0799] 6xHis-[HRV3C]-CB5(F2A)

[0800] MSQSSHHHHHHGGSEVLFQGPGSALLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRD

[0801] PEALRRAEELEEIVEELLE

[0802] SEQ ID No. 55

[0803] Construct comprising a modified CB5(F2A) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0804] Construct 11

[0805] 6xHis-[HRV3C]-CB5(F2E)

[0806] MSQSSHHHHHHGGSEVLFQGPGSELLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRD

[0807] PEALRRAEELEEIVEELLE

[0808] SEQ ID No. 56

[0809] Construct comprising a modified CB5(F2E) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0810] Construct 12

[0811] 6xHis-[HRV3C]-CB5(L3A)

[0812] MSQSSHHHHHHGGSEVLFQGPGSFALKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRD

[0813] PEALRRAEELEEIVEELLE SEQ ID No. 57

[0814] Construct comprising a modified CB5(L3A) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0815] Construct 13

[0816] 6xHis-[HRV3C]-CB5(L3E)

[0817] MSQSSHHHHHHGGSEVLFQGPGSFELKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRD

[0818] PEALRRAEELEEIVEELLE

[0819] SEQ ID No. 58

[0820] Construct comprising a modified CB5(L3E) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0821] Construct 14

[0822] 6xHis-[HRV3C]-CB5(R9A)

[0823] MSQSSHHHHHHGGSEVLFQGPGSFLLKHAIAVVERLLEEGNVEIAEEWVEQIKRLAHAQRD

[0824] PEALRRAEELEEIVEELLE

[0825] SEQ ID No. 59

[0826] Construct comprising a modified CB5(R9A) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0827] Construct 15

[0828] 6xHis-[HRV3C]-CB5(V10A)

[0829] MSQSSHHHHHHGGSEVLFQGPGSFLLKHAIRAVERLLEEGNVEIAEEWVEQIKRLAHAQRD

[0830] PEALRRAEELEEIVEELLE

[0831] SEQ ID No. 60

[0832] Construct comprising a modified CB5(V10A) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0833] Construct 16

[0834] 6xHis-[HRV3C]-CB5(R13A)

[0835] MSQSSHHHHHHGGSEVLFQGPGSFLLKHAIRVVEALLEEGNVEIAEEWVEQIKRLAHAQRD

[0836] PEALRRAEELEEIVEELLE

[0837] SEQ ID No. 61

[0838] Construct comprising a modified CB5(R13A) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence. Construct 17

[0839] 6xH is-[H RV3C]-CB5(W26A)

[0840] MSQSSHHHHHHGGSEVLFQGPGSFLLKHAIRVVERLLEEGNVEIAEEAVEQIKRLAHAQRD

[0841] PEALRRAEELEEIVEELLE

[0842] SEQ ID No. 62

[0843] Construct comprising a modified CB5(W26A) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0844] Construct 18

[0845] 6xHis-[HRV3C]-CB5(W26L)

[0846] MSQSSHHHHHHGGSEVLFQGPGSFLLKHAIRVVERLLEEGNVEIAEELVEQIKRLAHAQRDP

[0847] EALRRAEELEEIVEELLE

[0848] SEQ ID No. 63

[0849] Construct comprising a modified CB5(W26L) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0850] Construct 19

[0851] 6xHis-[HRV3C]-CB5(W26E)

[0852] MSQSSHHHHHHGGSEVLFQGPGSFLLKHAIRVVERLLEEGNVEIAEEEVEQIKRLAHAQRD

[0853] PEALRRAEELEEIVEELLE

[0854] SEQ ID No. 64

[0855] Construct comprising a modified CB5(W26E) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0856] Construct 20

[0857] 6xHis-[HRV3C]-CB5(L33A)

[0858] MSQSSHHHHHHGGSEVLFQGPGSFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRAAHAQRD

[0859] PEALRRAEELEEIVEELLE

[0860] SEQ ID No. 65

[0861] Construct comprising a modified CB5(L33A) binder therein: a 6-repeat histidine and HRV3C tag, followed by the CB binding sequence.

[0862] Construct 21

[0863] 6xHis-[TEV]-CB1-[G4SG4]-mScarlet MHHHHHHGGSSENLYFQSFLLKHAVRVIERLLEEGNVELAEKWVEQLKRLAHAQRDPEALR RAEELEEIVEELLEGGGGSGGGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRP YEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMN FEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLK GDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTWEQYERSEG RHSTGGMDELYK SEQ ID No. 66

[0864] Construct comprising CB1 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV site, the CB binding sequence, and the fluorescent protein mScarlet.

[0865] Construct 22

[0866] 6xHis-[TEV]-CB4-[G4SG4]-mScarlet

[0867] MHHHHHHGGSSENLYFQSFLLKHAIRVVERLLEEGNVEVAEEWVEQIKRLAHAQRDPEALR RAEELEEIVEELLEGGGGSGGGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRP YEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMN FEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLK GDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTWEQYERSEG RHSTGGMDELYK SEQ ID No. 67

[0868] Construct comprising CB4 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV site, the CB binding sequence, and the fluorescent protein mScarlet.

[0869] Construct 23

[0870] 6xHis-[TEV]-CB5-[G4SG4]-mScarlet

[0871] MHHHHHHGGSSENLYFQSFLLKHAIRWERLLEEGNVEIAEEWVEQIKRLAHAQRDPEALR RAEELEEIVEELLEGGGGSGGGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRP YEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMN FEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLK GDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTWEQYERSEG RHSTGGMDELYK SEQ ID No. 68 Construct comprising CB5 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV site, the CB binding sequence, and the fluorescent protein mScarlet.

[0872] Construct 24

[0873] 6xHis-[TEV]-mScarlet-[G4SG4]-CB1

[0874] MHHHHHHGGSSENLYFQSGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYE GTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFE DGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDI KMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH STGGMDELYKGGGGSGGGGSFLLKHAVRVIERLLEEGNVELAEKWVEQLKRLAHAQRDPE ALRRAEELEEIVEELLE

[0875] SEQ ID No. 69

[0876] Construct comprising CB1 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV site, the fluorescent protein mScarlet and the CB binding sequence.

[0877] Construct 25

[0878] 6xHis-[TEV]-mScarlet-[G4SG4]-CB4

[0879] MHHHHHHGGSSENLYFQSGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYE GTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFE DGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDI KMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH STGGM DELYKGGGGSGGGGSFLLKHAI RVVERLLEEGN VEVAEEWVEQI KRLAHAQRDPE ALRRAEELEEIVEELLE SEQ ID No. 70

[0880] Construct comprising CB4 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV site, the fluorescent protein mScarlet and the CB binding sequence.

[0881] Construct 26

[0882] 6xHis-[TEV]-mScarlet-[G4SG4]-CB5

[0883] MHHHHHHGGSSENLYFQSGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYE GTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFE DGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDI KMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH STGGMDELYKGGGGSGGGGSFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRDPEA LRRAEELEEIVEELLE

[0884] SEQ ID No. 71

[0885] Construct comprising CB5 binder therein: a purification affinity tag (6-repeat histidine tag), followed by a protease sensitive TEV site, the fluorescent protein mScarlet and the CB binding sequence.

[0886] Construct 27

[0887] GP-msGFP2-[G4SG4]-CB5

[0888] GPGGGDSTESLFTGWPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPW

[0889] PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDGTYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADH YQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITGGGGSGGGGSFL LKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELLE SEQ ID No. 72

[0890] Construct comprising CB5 binder therein: the monomeric superfolder green fluorescent protein (msGFP2), followed by a glycine rich linker G4SG4 (GGGGSGGGG, SEQ ID No. 84) and the CB binding sequence.

[0891] Construct 28

[0892] GP-msGFP2-[TEV]-CB5

[0893] GPGGDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWP TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHY QQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITGGEGGENLYFQGS FLLKHAIRWERLLEEGNVEIAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELLE SEQ ID No. 73

[0894] Construct comprising CB5 binder therein, the monomeric superfolder green fluorescent protein (msGFP2), followed by a TEV site and the CB binding sequence.

[0895] Construct 29

[0896] GP-msGFP2-[TEV]-CB24

[0897] GPGGDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWP TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHY QQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITGGEGGENLYFQGS FLLKHAMRVIERLLEEGNVEVAEKWIEQTKRLAHAQRDPEALRRAEELEEIVEELLE SEQ ID No. 74

[0898] Construct comprising CB24 binder therein: the monomeric superfolder green fluorescent protein (msGFP2), followed by a TEV site and the CB binding sequence.

[0899] Construct 30

[0900] GP-msGFP2-[Lnk10]-CB5-[Lnk15]-Neurotensin

[0901] GPGGDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWP TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHY QQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITGGEGGENLYFQGS FLLKHAIRWERLLEEGNVEIAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELLEGGEGAQ GEAGQGEGGQLYENKPRRPYIL SEQ ID No. 75

[0902] Construct comprising CB5 binder therein: the monomeric superfolder green fluorescent protein (msGFP2), the CB binding sequence via a first linker and the Neurotensin peptide via a second linker.

[0903] Construct 31

[0904] CB5-[G4SG4]-mScarlet

[0905] SFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELLEGGGGS GGGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF SWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDG TLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADF KTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK SEQ ID No. 76

[0906] Construct comprising CB5 binder therein: the CB binding sequence followed by the fluorescent protein mScarlet via a glycine rich linker G4SG4 (GGGGSGGGG, SEQ ID No. 84).

[0907] Construct 32 mScarlet-[G4SG4]-CB5 SGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFS WDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGT LIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFK TTYKAKKPVQMPGAYNVDRKLDITSHNEDYTWEQYERSEGRHSTGGMDELYKGGGGSG GGGSFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELLE SEQ ID No. 77

[0908] Construct comprising CB5 binder therein: the fluorescent protein mScarlet followed by the CB binding sequence via a glycine rich linker G4SG4 (GGGGSGGGG, SEQ ID No. 84).

[0909] Construct 33 mScarlet-GGGS-[G4Sx3]-G-CB5

[0910] GPGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF SWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDG TLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADF KTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYKGGGSG GGGSGGGGSGGGGSGSFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRDPEALRR AEELEEIVEELLE

[0911] SEQ ID No. 78

[0912] Construct comprising CB5 binder therein: the fluorescent protein mScarlet followed by the CB binding sequence via a glycine rich linker GGGSGGGGSGGGGSGGGGSG (SEQ ID No. 86).

[0913] Construct 34

[0914] CB4-[G4SG4]-mScarlet

[0915] SGSFLLKHAIRVVERLLEEGNVEVAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELLEGG GGSGGGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGG PLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSL EDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYL ADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTWEQYERSEGRHSTGGMDELYK SEQ ID No. 79

[0916] Construct comprising CB4 binder therein: the CB binding sequence followed by the fluorescent protein mScarlet via a glycine rich linker G4SG4 (GGGGSGGGG, SEQ ID No. 84).

[0917] Construct 35 CB5-[G4SG4]-mScarlet

[0918] SGSFLLKHAIRVVERLLEEGNVEIAEEWVEQIKRLAHAQRDPEALRRAEELEEIVEELLEGGG GSGGGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPL PFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLE DGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLA DFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK SEQ ID No. 80

[0919] Construct comprising CB5 binder therein: the CB binding sequence followed by the fluorescent protein mScarlet via a glycine rich linker G4SG4 (GGGGSGGGG, SEQ ID No. 84).

[0920] Construct 36

[0921] CB1-[GGSGGGS]-EGFRn

[0922] SFLLKHAVRVIERLLEEGNVELAEKWVEQLKRLAHAQRDPEALRRAEELEEIVEELGGSGGG SDHWEEVFRNALEHLQEATQQNDPQKAKKILEEAHKKLRRELSEEEARSVVRWLKQLVDR EKS

[0923] Construct comprising CB1 binder therein: the CB binding sequence followed by the minibinder targeting EGFR N-terminal (SEQ ID No. 91) via a glycine rick linker GGSGGGS (SEQ ID No. 85).

[0924] Other

[0925] Sortl Binding Site huSORTI (78-756)-GGGGSGEVLFQ

[0926] SAPGEDEECGRVRDFVAKLANNTHQHVFDDLRGSVSLSWVGDSTGVILVLTTFHVPLVIMT FGQSKLYRSEDYGKNFKDITDLINNTFIRTEFGMAIGPENSGKVVLTAEVSGGSRGGRIFRSS DFAKNFVQTDLPFHPLTQMMYSPQNSDYLLALSTENGLWVSKNFGGKWEEIHKAVCLAKW GSDNTIFFTTYANGSCKADLGALELWRTSDLGKSFKTIGVKIYSFGLGGRFLFASVMADKDT TRRIHVSTDQGDTWSMAQLPSVGQEQFYSILAANDDMVFMHVDEPGDTGFGTIFTSDDRGI VYSKSLDRHLYTTTGGETDFTNVTSLRGVYITSVLSEDNSIQTMITFDQGGRWTHLRKPENS ECDATAKNKNECSLHIHASYSISQKLNVPMAPLSEPNAVGIVIAHGSVGDAISVMVPDVYISD DGGYSWTKMLEGPHYYTILDSGGIIVAIEHSSRPINVIKFSTDEGQCWQTYTFTRDPIYFTGL ASEPGARSMNISIWGFTESFLTSQWVSYTIDFKDILERNCEEKDYTIWLAHSTDPEDYEDGCI LGYKEQFLRLRKSSVCQNGRDYVVTKQPSICLCSLEDFLCDFGYYRPENDSKCVEQPELKG HDLEFCLYGREEHLTTNGYRKIPGDKCQGGVNPVREVKDLKKKCTSNFLSPEKQNSKSNSG

[0927] GGGSGEVLFQ

[0928] SEQ ID No. 40

[0929] Composed of native huSORTI (Uniprot Q99523) residues 78-756, commonly referred to as huSORTI (78-756), with a GGGGSGEVLFQ (SEQ ID No. 41) tag.

[0930] Extracellular domain (ECD) of huSORTI huSORTI (78-756)-[HRV3C]-8xHis-Avitag

[0931] SAPGEDEECGRVRDFVAKLANNTHQHVFDDLRGSVSLSWVGDSTGVILVLTTFHVPLVIMT

[0932] FGQSKLYRSEDYGKNFKDITDLINNTFIRTEFGMAIGPENSGKVVLTAEVSGGSRGGRIFRSS DFAKNFVQTDLPFHPLTQMMYSPQNSDYLLALSTENGLWVSKNFGGKWEEIHKAVCLAKW GSDNTIFFTTYANGSCKADLGALELWRTSDLGKSFKTIGVKIYSFGLGGRFLFASVMADKDT TRRIHVSTDQGDTWSMAQLPSVGQEQFYSILAANDDMVFMHVDEPGDTGFGTIFTSDDRGI VYSKSLDRHLYTTTGGETDFTNVTSLRGVYITSVLSEDNSIQTMITFDQGGRWTHLRKPENS ECDATAKNKNECSLHIHASYSISQKLNVPMAPLSEPNAVGIVIAHGSVGDAISVMVPDVYISD DGGYSWTKMLEGPHYYTILDSGGIIVAIEHSSRPINVIKFSTDEGQCWQTYTFTRDPIYFTGL ASEPGARSMNISIWGFTESFLTSQWVSYTIDFKDILERNCEEKDYTIWLAHSTDPEDYEDGCI

[0933] LGYKEQFLRLRKSSVCQNGRDYWTKQPSICLCSLEDFLCDFGYYRPENDSKCVEQPELKG

[0934] HDLEFCLYGREEHLTTNGYRKIPGDKCQGGVNPVREVKDLKKKCTSNFLSPEKQNSKSNSG

[0935] GGGSGEVLFQGPGGGHHHHHHHHGGGGSGLNDIFEAQKIEWHE

[0936] SEQ ID No. 42

[0937] Composed of native huSORTI (Uniprot Q99523) residues 78-756, commonly referred to as huSORTI (78-756), with an HRV3C, 8x Histidine and Avi tag ([HRV3C]-8xHis-Avitag).

[0938] Extracellular domain (ECD) of moSORTI moSORT1(76-744)-[HRV3C]-8xHis-Avitag

[0939] GAPAEDQDCGRLPDFIAKLTNNTHQHVFDDLSGSVSLSWVGDSTGVILVLTTFQVPLVIVSF

[0940] GQSKLYRSEDYGKNFKDITNLINNTFIRTEFGMAIGPENSGKVILTAEVSGGSRGGRVFRSS DFAKNFVQTDLPFHPLTQMMYSPQNSDYLLALSTENGLWVSKNFGEKWEEIHKAVCLAKW

[0941] GPNNIIFFTTHVNGSCKADLGALELWRTSDLGKTFKTIGVKIYSFGLGGRFLFASVMADKDTT RRIHVSTDQGDTWSMAQLPSVGQEQFYSILAANEDMVFMHVDEPGDTGFGTIFTSDDRGIV YSKSLDRHLYTTTGGETDFTNVTSLRGVYITSTLSEDNSIQSMITFDQGGRWEHLRKPENSK CDATAKNKNECSLHIHASYSISQKLNVPMAPLSEPNAVGIVIAHGSVGDAISVMVPDVYISDD GGYSWAKMLEGPHYYTILDSGGIIVAIEHSNRPINVIKFSTDEGQCWQSYVFTQEPIYFTGLA SEPGARSMNISIWGFTESFITRQWVSYTVDFKDILERNCEEDDYTTWLAHSTDPGDYKDGCI LGYKEQFLRLRKSSVCQNGRDYWAKQPSVCPCSLEDFLCDFGYFRPENASECVEQPELK

[0942] GHELEFCLYGKEEHLTTNGYRKIPGDKCQGGMNPAREVKDLKKKCTSNFLNGGGGSGEVL

[0943] FQGPGGGHHHHHHHHGGGGSGLNDIFEAQKIEWHE

[0944] SEQ ID No. 43

[0945] Composed of native moSORTI (Uniprot Q6PHLI5) residues 76-744, commonly referred to as moSORTI (76-744), with an HRV3C, 8x Histidine and Avi tag ([HRV3C]-8xHis-Avitag). huSORTI Epitope Modification 1 huSORTI (78-756, V500A)-[HRV3C]-8xHis-Avitag

[0946] SAPGEDEECGRVRDFVAKLANNTHQHVFDDLRGSVSLSWVGDSTGVILVLTTFHVPLVIMT

[0947] FGQSKLYRSEDYGKNFKDITDLINNTFIRTEFGMAIGPENSGKVVLTAEVSGGSRGGRIFRSS DFAKNFVQTDLPFHPLTQMMYSPQNSDYLLALSTENGLWVSKNFGGKWEEIHKAVCLAKW GSDNTIFFTTYANGSCKADLGALELWRTSDLGKSFKTIGVKIYSFGLGGRFLFASVMADKDT TRRIHVSTDQGDTWSMAQLPSVGQEQFYSILAANDDMVFMHVDEPGDTGFGTIFTSDDRGI VYSKSLDRHLYTTTGGETDFTNVTSLRGVYITSVLSEDNSIQTMITFDQGGRWTHLRKPENS ECDATAKNKNECSLHIHASYSISQKLNVPMAPLSEPNAVGIVIAHGSVGDAISAMVPDVYISD DGGYSWTKMLEGPHYYTILDSGGIIVAIEHSSRPINVIKFSTDEGQCWQTYTFTRDPIYFTGL ASEPGARSMNISIWGFTESFLTSQWVSYTIDFKDILERNCEEKDYTIWLAHSTDPEDYEDGCI

[0948] LGYKEQFLRLRKSSVCQNGRDYWTKQPSICLCSLEDFLCDFGYYRPENDSKCVEQPELKG

[0949] HDLEFCLYGREEHLTTNGYRKIPGDKCQGGVNPVREVKDLKKKCTSNFLSPEKQNSKSNSG

[0950] GGGSGEVLFQGPGGGHHHHHHHHGGGGSGLNDIFEAQKIEWHE

[0951] SEQ ID No. 82

[0952] Composed of huSORTI residues 78-756, commonly referred to as huSORTI (78-756), with V500A modification, an HRV3C, 8x Histidine and Avi tag ([HRV3C]-8xHis-Avitag). huSORTI Epitope Modification 2 huSORTI (78-756, V500K)-[HRV3C]-8xHis-Avitag

[0953] SAPGEDEECGRVRDFVAKLANNTHQHVFDDLRGSVSLSWVGDSTGVILVLTTFHVPLVIMT

[0954] FGQSKLYRSEDYGKNFKDITDLINNTFIRTEFGMAIGPENSGKVVLTAEVSGGSRGGRIFRSS DFAKNFVQTDLPFHPLTQMMYSPQNSDYLLALSTENGLWVSKNFGGKWEEIHKAVCLAKW GSDNTIFFTTYANGSCKADLGALELWRTSDLGKSFKTIGVKIYSFGLGGRFLFASVMADKDT TRRIHVSTDQGDTWSMAQLPSVGQEQFYSILAANDDMVFMHVDEPGDTGFGTIFTSDDRGI VYSKSLDRHLYTTTGGETDFTNVTSLRGVYITSVLSEDNSIQTMITFDQGGRWTHLRKPENS ECDATAKNKNECSLHIHASYSISQKLNVPMAPLSEPNAVGIVIAHGSVGDAISKMVPDVYISD DGGYSWTKMLEGPHYYTILDSGGIIVAIEHSSRPINVIKFSTDEGQCWQTYTFTRDPIYFTGL ASEPGARSMNISIWGFTESFLTSQWVSYTIDFKDILERNCEEKDYTIWLAHSTDPEDYEDGCI

[0955] LGYKEQFLRLRKSSVCQNGRDYWTKQPSICLCSLEDFLCDFGYYRPENDSKCVEQPELKG

[0956] HDLEFCLYGREEHLTTNGYRKIPGDKCQGGVNPVREVKDLKKKCTSNFLSPEKQNSKSNSG

[0957] GGGSGEVLFQGPGGGHHHHHHHHGGGGSGLNDIFEAQKIEWHE

[0958] SEQ ID No. 83

[0959] Composed of huSORTI residues 78-756, commonly referred to as huSORTI (78-756), with

[0960] V500K modification, an HRV3C, 8x Histidine and Avi tag ([HRV3C]-8xHis-Avitag).

[0961] Green Fluorescent Protein, msGFP2

[0962] GP-msGFP2

[0963] GPGGGDSTESLFTGWPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPW

[0964] PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDGTYKTRAEVKFEGDT

[0965] LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADH

[0966] YQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITG

[0967] SEQ ID No. 87

[0968] Non-Binding GFP control fusion with similar molecular molecular weight to fluorescent constructs

[0969] GP-msGFP-[G4SG4]-[Control]

[0970] GPGGGDSTESLFTGWPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPW

[0971] PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDGTYKTRAEVKFEGDT

[0972] LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADH

[0973] YQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITGGGGSGGGGEEE

[0974] VREELRKLIPDPRLRKIVEEALELAKRLGISPEEVLRLLKELIEKLGDPEEAAKVLLELVR

[0975] SEQ ID No. 88

[0976] Non-Binding mScarlet control fusion with similar molecular weight to fluorescent constructs

[0977] GP-mScarlet-Control

[0978] GPGGVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF

[0979] SWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDG

[0980] TLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADF

[0981] KTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYKGGGPEE

[0982] EVREELRKLIPDPRLRKIVEEALELAKRLGISPEEVLRLLKELIEKLGDPEEAAKVLLELVR

[0983] SEQ ID No. 89 C-terminus Hemaglutamin (HA) tagged moSORTI protein (Uniprot Q6PHU5)

[0984] MERPRGAADGLLRWPLGLLLLLQLLPPAAVGQDRLDAPPPPAPPLLRWAGPVGVSWGLRA

[0985] AAPGGPVPRAGRWRRGAPAEDQDCGRLPDFIAKLTNNTHQHVFDDLSGSVSLSWVGDST

[0986] GVILVLTTFQVPLVIVSFGQSKLYRSEDYGKNFKDITNLINNTFIRTEFGMAIGPENSGKVILTA

[0987] EVSGGSRGGRVFRSSDFAKNFVQTDLPFHPLTQMMYSPQNSDYLLALSTENGLWVSKNFG

[0988] EKWEEIHKAVCLAKWGPNNIIFFTTHVNGSCKADLGALELWRTSDLGKTFKTIGVKIYSFGLG

[0989] GRFLFASVMADKDTTRRIHVSTDQGDTWSMAQLPSVGQEQFYSILAANEDMVFMHVDEPG

[0990] DTGFGTIFTSDDRGIVYSKSLDRHLYTTTGGETDFTNVTSLRGVYITSTLSEDNSIQSMITFDQ

[0991] GGRWEHLRKPENSKCDATAKNKNECSLHIHASYSISQKLNVPMAPLSEPNAVGIVIAHGSVG

[0992] DAISVMVPDVYISDDGGYSWAKMLEGPHYYTILDSGGIIVAIEHSNRPINVIKFSTDEGQCWQ

[0993] SYVFTQEPIYFTGLASEPGARSMNISIWGFTESFITRQWVSYTVDFKDILERNCEEDDYTTWL

[0994] AHSTDPGDYKDGCILGYKEQFLRLRKSSVCQNGRDYVVAKQPSVCPCSLEDFLCDFGYFR

[0995] PENASECVEQPELKGHELEFCLYGKEEHLTTNGYRKIPGDKCQGGMNPAREVKDLKKKCT

[0996] SNFLNPTKQNSKSNSVPIILAIVGLMLVTVVAGVLIVKKYVCGGRFLVHRYSVLQQHAEADGV

[0997] EALDSTSHAKSGYHDDSDEDLLETRTRPLYPYDVPDYAV

[0998] SEQ ID No. 90

[0999] Mini binder targeting EGFR N -termin al (EGFRn)

[1000] DHWEEVFRNALEHLQEATQQNDPQKAKKILEEAHKKLRRELSEEEARSVVRWLKQLVDRE

[1001] KS

[1002] SEQ ID No. 91

[1003] Experimental Assays

[1004] Example 1. Direct Binding Validation

[1005] A selection of the computation designs discussed above were recombinantly produced and confirmed my mass spectrometry analysis. The binding affinity of these computational designs were validated as binding to recombinant SORT1 using surface plasmon resonance (SPR). Here the extracellular domain (ECD) of huSORTI (SEQ ID No. 42) and moSORTI (SEQ ID No. 43) are utilized. Binders CB1 , CB4, and CB5 exhibited binding affinities in the double-digit nanomolar range (25-64 nM) for both human and mouse SORT1 ECD proteins (Table 3). To investigate the impact of temperature on binding affinity, CB5, CB13, CB24, and CB29 were evaluated at both 25°C and 37°C. It was observed that all four binder constructs demonstrated adequate affinity at 37°C and 25°C. Table 3. Kd (nM) for the binders against human / mouse SORT 1 ECD at 25°C and 37°C.

[1006] The testing at 37°C was conducted to aid in the demonstration relative stability of these binders under conditions closer to physiological conditions.

[1007] Example 2. pH Dependency

[1008] To verify the binding dependency of these designs on the protonation state of the SORT1 receptor, the binding affinity of the computational designs was evaluated using surface plasmon resonance (SPR) at 37°C, in neutral and acidic pH, such as to correspond to the lower pH environment where the SORT 1 receptor undergoes conformational changes due to the protonation of histidine residues. As can be seen from Figs. 5A-5D, robust binding of CB5 (Construct 3) was maintained at pH 7.4 and 6.5, affirming the affinity under physiologically relevant conditions. However, complete abolition of binding was observed at pH 5.5, indicating a strong dependency on the protonation state of histidines and conformational epitope. These findings strongly support the notion that binding can be regulated by altering the pH.

[1009] Figs. 5A-5D provide qualitative binding analysis results by SPR of CB5 (Construct 3) against human Sortl (SEQ ID No. 42) which demonstrated pH dependence of binding. This supported modelled data that the conformation changes from neutral to acidic pH induced by protonation of Histidines on Sortl . Fig. 5A shows the results at pH 7.4, which retained binding. Fig. 5B shows the results at pH 6.5, which retained binding. Figs. 5C and 5D shows the results at pH 6.0, where binding is reduced, and pH 5.5, where binding capacity is lost.

[1010] Example 3. Verification of Epitope and Paratope from crystal structure of complex

[1011] To validate the interaction between the epitope and paratope at a high resolution, a crystal structure of the complex between the extracellular domain (ECD) of human SORT-1 (SEQ ID No. 40) and the computational designed binder CB4 (Construct 7) was successfully determined. The epitope and paratope regions were defined based on a distance cutoff of 4.5 A for all heavy atoms, providing a detailed understanding of their spatial arrangement and interaction (see Table 4).

[1012] Comparing the computational model of the binder CB4 with the crystal structure, a significant agreement was observed, with a root mean square deviation (RMSD) of approximately 1.1 A for the alpha-carbon atoms. This close correspondence between the computational model and the experimental crystal structure serves as a validation of the binding specificity and highlights the accuracy of the designed binder in capturing the desired interaction.

[1013] Table 4. Residues of binder CB4 involved in Epitope and Paratope contacts from crystal structure (4.5 A distance cut-off as determined in Molecular Operating Environment (MOE) program). Example 4. Verification of Epitope and Paratope through site-directed mutations

[1014] To gain further insights into the interactions between the epitope and paratope, point mutations were introduced and constructed. These mutated variants were subsequently tested for their binding affinity using surface plasmon resonance (SPR). This analysis is crucial for elucidating the key contributions and specific residues involved in the epitopeparatope interaction. By comparing the binding affinities of these mutated variants to the wild-type complex, we can better understand the individual amino acid residues and their impact on the binding affinity and overall interaction between the epitope and paratope. This comprehensive approach combines both computational modelling and experimental structural analysis to provide a comprehensive understanding of the epitope-paratope interface.

[1015] Paratope

[1016] To further validate the computational and crystallographic structures of the interaction, twelve variants were designed with the specific aim of impacting binding by introducing steric hindrance or removing interface contacts within the paratope region of CB5. Binding experiments were conducted using surface plasmon resonance (SPR) at 37°C to assess the binding affinity of these variants (Table 5). All twelve variants demonstrated a reduction in affinity compared to the parent construct with CB5 (Construct 8). These consistent reductions in affinity support the observed structural interactions and align closely with the computational design and predicted epitope. Note that the construct identifies the modification to the CB5 and refers to the residue number of the binder sequence alone. Written otherwise, the residue number being modified refers to the numbers found in parent CB5 SEQ ID No. 6. For instance, construct 9 identifies CB5(S1A) which refers to a modification the CB5 binder found therein, where residue no. 1 of the parent CB5 SEQ ID No. 6 is modified from a Serine (S) residue to an Alanine (A) residue.

[1017] Table 5. SPR measurements of impact on affinity of CB5 paratope mutations have to huSORTI (SEQ ID No. 42). Interface changes result in a reduction in affinity.

[1018] Epitope

[1019] To investigate the interaction between the epitope of huSORTI and the CB5 binder (Construct 8), a specific residue in the epitope region, V500, was targeted. This residue is a central hydrophobic interface residue that plays a crucial role in organizing the hydrophobic interface of the paratope. Two mutations were introduced at position V500: V500A (ie. replacement of residue V with residue A at position 500), which aimed to remove the interaction; and V500K (ie. replacement of residue V with residue K at position 500), which aimed to introduce steric hindrance. The goal was to assess the impact of these mutations on the interaction between the epitope and paratope of CB5. While V500A reduced affinity modestly, the V500K mutation resulted in the complete abolition of binding between the epitope and the CB5 binder (See Table 6). This finding provided strong evidence for the essential role of residue V500 in the interaction. Specifically, it confirmed that residue V500, through central hydrophobic-driven interactions, plays a pivotal role in mediating the binding between the paratope and epitope.

[1020] The V500A modification results in loss of a central epitope contacts and results in minor loss of affinity. However, V500K removes hydrophobic contacts and likely introduces a steric hinder thus largely abolishing the central hydrophobic interaction made by the binder CB5 paratope. Characterization using SPR at 25°C. This demonstrates the impact of a central SORT1 (SEQ ID No. 42) epitope residue interaction to a SORT1 binder, such as CB5 comprised in Construct 8 for instance, when modified.

[1021] Table 6. Affinity modulation of V500 epitope modification By systematically mutating key residues within the interface, these experiments provided valuable insights into the specific amino acid residues involved in the epitopeparatope interaction and further elucidate the essential components of the binding interface between the huSORTI epitope and the CB5 binder (Construct 8).

[1022] Example 5. Impact of binding when a binder is combined with a cargo

[1023] The binder to SORT1 holds utility in various applications, including targeting binding to SORT1 -expressing cells or tissues and delivering functional cargo to specific target cells and tissues. To investigate the binding of the binder to the full-length SORT1 receptor in a cellular context, the binder molecules were recombinantly produced as fusions with well- known fluorescent proteins, such as green fluorescent protein (GFP) or mScarlet. This fusion strategy aids in the visualization of the binder molecules within cells and more complex 3- dimensional organoid targets.

[1024] In Table 7, the binding characteristics of the binder fused to either the amino or carboxy termini of SORT1 (CB5) are demonstrated using surface plasmon resonance (SPR). The results showed that adeguate binding affinity is retained after the fusion of the binder to either the amino or carboxy termini of the SORT1 receptor. This indicates that the fusion of the binder with a cargo, such as fluorescent proteins, does not significantly impair its ability to bind to SORT1, ensuring its suitability for visualization and targeting purposes.

[1025] Table 7. SPR results demonstrating minimal impact of a standard G4S Linker and Fluorescent Protein fusion construct.

[1026] Example 6. SORT 1 BINDER-Cargo interaction in the context of cell uptake Binder-Cargo Cell Internalization

[1027] It was desirable to investigate whether the biological role of Sortl could be leveraged with Sortl binder constructs to leverage it for shuttling entities form the cell surface to intracellular compartments. We sought to determine whether we could utilize this mechanism to facilitate the internalization of a SORT1 Binder and its cargo fusion construct from a binding event at the cell surface to an intracellular compartment.

[1028] To verify the presence of SORT1 on the hCMEC / D3 cell line, which can serve as a model for the human blood-brain barrier, we utilized a commercially available monoclonal antibody (mAb) specific for huSORTI , FAB31541G conjugated with alexafluor-488 (R&D Systems, Catalog #FAB110G). High Content Imaging was used to validate that the hCMEC / D3 endothelial cell line expresses SORT1 at levels suitable for the studies.

[1029] Next, a green fluorescent protein (GFP) recombinant fusion constructs to SORT1 binder formats were generated to determine if they have the ability to bind and internalize to cells (CMEC / D3). Here we utilized the CB5 in fusion with GFP (Construct 27), which has a Kd of 64nM (SPR) to the human SORT-1 (SEQ ID No. 42). Internalization was compared against controls of GFP alone (SEQ ID No. 87) and a non-binding control fusion with GFP of similar molecular weight as CB5 (SEQ ID No. 88).

[1030] Here we found construct 27 can bind and then internalize to the hCMEC / D3 cell line. Figure 6 shows a set of confocal images (as inverse greyscale) of a dose ranging study with Construct 27, where punctate GFP stain (dark spots) are observered intracellularly. Cells were counterstained with a membrane marker, WGA, and DAPI for a nuclear maker (both light grey).

[1031] This demonstrated that SORT1 (Construct 27) is internalized to a cell (dark punctate staining in Fig. 6). In Table 8 this imagery is quantified in relative units (duplicate experiments) demonstrating a dose dependent uptake. The non-binding GFP control fusion (SEQ ID No. 88) did not show uptake nor punctate staining pattern (data not shown). Here the experiment was run in duplicate.

[1032] Table 8. Relative quantification (AU / Cell) with GFP-CB5 (Construct 27) internalized in a confocal imagery field. | 1000 nM I 47 I 49 I

[1033] Confocal imagery utilizing a lysotracker demonstrated that a proportion is in the endosome or lysozome, and a significant portion is in other non-lysozome compartments.

[1034] To assess the potential of constructs comprising the Sortl Binder as structures which may provide a therapeutic effect, such as a drug, various cargos were added to the binders, while different linker compositions and lengths were incorporated to observe their impact on their ability to internalize. Here, we employed high content imaging to visualize and quantitatively measure their uptake in hCMEC / D3 cells (human cerebral microvascular endothelial cells) as well as human adipocytes. Similar internalization capability was observed for hCMEC / D3 cells when comparing different recombinant fusion constructs of SORT1 binder CB5 with various linkers (Construct 27 and 28), the fusion construct of GFP with the CB24 binder (Construct 29), and the fusion construct of GFP with the CB5 binder further fused with a Neurotensin peptide at the C-terminus (Construct 30). In this last construct, construct 30, it was determined that neurotensin was not only internalized similarly to other constructs (see Table 9 below), but further retained its function within the construct. This was ascertained via a cell-based readout, where corresponding potency was identified in comparison to the standalone a peptide.

[1035] These results indicates that variations in the Binder-Linker-Cargo combinations, in any order understood by one of skill in the art, only have a modest impact on the binding to SORT1 and its subsequent internalization despite the differences in linkers and cargo. The ability of the binders to undergo internalization into hCMEC / D3 cells remains comparable with previous examples.

[1036] These findings are important as they indicate that modifications in the fusion design do not significantly alter the SORT1 binders' internalization capability, supporting their potential utility as versatile tools for targeted delivery and intracellular trafficking applications. This adaptability can facilitate the development of customized SORT1 binders for specific applications and therapeutic interventions.

[1037] Table 9. A comparison of SORT1 binders in fusion with GFP demonstrating internalization to CMEC / D3 cells. Data is describing internalized fluorescent intensity as characterized by High Content Imaging, and expressed in relative units.

[1038] Binder-Cargo Orientation on cell binding

[1039] To understand if orientation of a cargo (such as mScarlet, for instance) at the N- termini or C-termini impacts binding and internalizing to hCMEC / D3 cells, constructs of CB5- mScarlet (Construct 31) and mScarlet-CB5 (Construct 32) were utilized in a confocal experiment. In Table 10 we show that cargo orientation (in this case mScarlet), does not have significant impact on internalization efficiency. Additionally in Table 10, we ran a competition study against the CMEC / D3 endogenous SORT1 receptor to demonstrate specificity to SORT1 on hCMEC / D3 cells and to preclude a non-specific uptake of the CB5 binder by the cell (such as, for instance via pinocytosis or other known mechanisms). Here extracellular domain (ECD) SORT1 (SEQ ID No. 42) was added to the cell culture at 50x molar excess relative to construct 31 and construct 32 to demonstrate a potent reduction in uptake and demonstrated that the binding interaction with native SORT1 is critical for uptake.

[1040] Table 10. Orientation of Fluorescent protein has minimal impact on uptake in hCMEC-D3.

[1041] SORTI(ECD) competes with native SORT1 on the cell surface.

[1042] Additional cell types

[1043] Published reports indicate that SORT1 is broadly expressed, and so it was desirable to investigate if binding and internalization was also possible with other cell types. Shown to express SORT1 include primary differentiated Adipocytes (Li, Jibiao, et al., Biochimica et Biophysica Acta (BBA) (2017): 2924-2933) and a Hepatic cancer cell line, HEPG2 (Gao, Yan, et al. "Sortilin 1 promotes hepatocellular carcinoma cell proliferation and migration by regulating immune cell infiltration." Journal of Oncology 2022 (2022)). Here the computational design SORT1 binder CB5 in fusion constructs with GFP were screened against human adipocytes and the human liver cell line along with prior noted controls, as well as a HEPG2 at 50nM, 500nM and 5000nM. As with CMEC / D3, a clear signal was observed for the SORT1 binder construct, GFP-CB5 (construct 27), in adipocytes (Table 11) and HEPG2 (Table 12).

[1044] Table 11. High content screen of CB5 constructs with different cargos and linkers (Adipocytes). Units correspond to regional intensity of cell body.

[1045] Table 12. High content screen of CB5 constructs with different cargos and linkers (HepG2 (liver cell line)). Units correspond to regional intensity of cell body.

[1046] Example 7. Brain Organoid Uptake - A complex tissue proxy

[1047] Moving from monolayers of cells to a proxy for complex tissues, the blood-brain organoid model represents a powerful tool to elucidate the utility of SORT1 binders in targeting the endothelium (CMEC / D3) and facilitating transcytosis to a neuronal layer. Bloodbrain organoids are three-dimensional in vitro models that recapitulate the intricate cellular and structural complexity of the human brain's neurovascular unit. They are comprised of the vascular barrier endothelium, and internal pericytes and astrocytes, which together form the blood-brain barrier (BBB) (Nature Communications, 8:15623, DOI: 10.1038 / ncomms15623). A process of transcytosis involves a binder to an endothelium target being internalized into the endothelial cells and subsequently transported through the cellular machinery before being released at the basolateral side of the endothelium. From there, an endothelium cell targeting binder can transcytose to the underlying neuronal layer, reaching and interacting with specific neurons. Figure 7 shows a model for the blood brain barrier organoid, which is comprised of an endothelium barrier (CMED / D3), which forms into barrier sphere around an inner neuronal space (Pericytes and Astrocytes). Sortl Binder first interacts with SORT1 protein on the endothelium in the periphery, that then transcytosises it to the neuronal space. As described herein, the cargo could be any functional or non-functional entity, including a fluorophore or fluorescent protein such as to aid tracking of transcytosis events (Bergmann, S. et al. Nature Protocols 2018).

[1048] In Table 13, we utilized the following Sortl binders fused with the fluorescent protein mScarlet to investigate their transcytosis capabilities: construct 33 (comprising Sortl binder CB5 proximal the C-terminal), construct 34 (comprising Sortl binder CB4), and construct 35 (comprising Sortl binder CB5 proximal the N-terminal). All SORT1 binders demonstrated efficient transcytosis, showcasing their ability to traverse cellular barriers and reach the neuronal space. Further the orientation of mScarlet had only a modest effect on transcytosis efficiency as both N-terminal and C-terminal fusion configurations among the Sortl Binders exhibited significant transcytosis capabilities. This data further indicated that neither the cargo (fluorescent protein) nor the cargo’s position hindered the binders' internalization and transport to the neuronal layer.

[1049] Table 13. Blood brain barrier (BBB) organoid. SORT1 construct 33 (comprising binder CB5), construct 34 (comprising binder CB4), and construct 35 (comprising binder CB5) demonstrate transcytosis. Relative units are utilized related to fluorescence intensity of mScarlet in analysis.

[1050] In Table 14, we refer to the same constructs from Table 9 that internalized to CM EC as monoculture, and here demonstrates a transcytosis signal for GFP fusions as well. Similar BBB organoid results were seen with CB5 when utilizing a GlySer linker (construct 27) or a TEV protease linker (construct 28), or with appendage of the functional Neurotensin peptide at the C-termini (construct 30). Further CB24 and CB5 (Construct 29 and 28, respectively), which have similar Kd to SORT1 , demonstrated similar uptake.

[1051] Table 14. Blood brain barier (BBB) organoid. Relative units are utilized related to fluorescence intensity of mScarlet in analysis.

[1052] The blood-brain organoid model allows researchers to study the precise dynamics of this transcytosis process and the factors influencing its efficiency. By introducing different Binder-Linder-Cargo combinations, researchers can enable enhanced targeting and delivery to specific neuronal subtypes. Further the blood-brain organoid model offers a valuable platform to investigate the potential of SORT1 binders in selectively targeting the BBB and mediating transcytosis to reach neurons and investigate these SORT1 binders utility can extend into therapeutic applications, such as targeted drug delivery and neurological disorder treatments.

[1053] Example 8: Construct for Promotion of Protein Degradation

[1054] To explore the potential of protein lysosome-targeting chimeras (pLYTACs) in promoting protein degradation, we investigated their ability to target and degrade cell surface receptors via targeting with Sortilin binders. We focused on the Epidermal Growth Factor Receptor (EGFR). To generate EGFR-pLYTACs targeting to brain via Sortilin, we fused the Sortilin binder CB1 with a minibinder targeting EGFR N-terminal, which may be commonly referred to as EGFRn (SEQ ID NO. 91) (Cao, L. et al. Nature 1-1 (2022). This fusion is described herein as Construct 36.

[1055] HeLa cells were treated with a PBS buffer control, 200nM EGFRn (SEQ ID No. 91) and 200nM of Construct 36 (SEQ ID No. 81) for 48h and a western blot analysis was taken to ascertain the total EGFR levels in the HeLa cells after treatment. The results of the western blot are shown in Fig. 8. As can be seen, efficient clearance of EGFR in HeLa cancer cells after 48h incubation was observed for Construct 36 by western blot, demonstrating a 77% reduction of EGFR in comparison to the PBS buffer control. Further, it can be seen that no reduction in EGFR levels was obtained by only providing EGFRn (SEQ ID NO. 91), which shows the role played by the Sortl binder in Construct 36.

[1056] With rich expression of the corresponding receptor in the brain, a construct such as that provided in example Construct 36 demonstrates that the Sortilin-1 binders can function as pLYTACs for neurodegenerative disease applications, or other adequate applications. for cell surface bi

[1057] To assess the binding of a Sortl binder to mouse Sortl receptors, knock-in experiments were performed in human HEK293 cells. The mouse Sortl receptor (Uniprot Q6PHLI5) was introduced into the human HEK293 cells as a knock-in, allowing for the investigation of binding interactions on the cell surface. To ensure the presence of the mouse Sortl knock-in, a Hemaglutamin (HA) tag was introduced at the C-terminus of the full-length Sortl protein (Uniprot Q6PHU5), see SEQ ID No. 90. The presence of mouse SORT1 knock- in HEK293 cells was confirmed via an anti-HA-FITC and mouse cross-reactive SORT1 polyclonal (BAF3145) antibody, as shown in table 15 below. The background signal was determined against parental HEK293 cells and using two negative controls: standalone GFP, identified below as GP-msGFP2 (SEQ ID No. 87), and a non-Binding GFP control fusion with similar molecular weight to fluorescent constructs, identified as GP-msGFP-[G4SG4]- [Control] (SEQ ID No. 88).

[1058] For the SORT1 binder, construct 27 (comprising CB5) was utilized. Here an increase in the binding signal was observed, indicating that both the human and mouse SORT1 receptors share the same native epitope. Thus, the CB binders of the present application successfully targeted a conserved epitope between mouse and human SORT 1 receptors.

[1059] By confirming the binding of construct 27 to the mouse SORT1 knock-in HEK293 cells, it provided further evidence of the cross-reactivity and shared epitope between the human and mouse SORT1 receptors. This information is valuable for the development and application of SORT1 binders in studying and manipulating SORT1 -mediated processes in both mouse models and in human systems.

[1060] Table 15. FACS binding evaluation of HEK293 parental cell line versus a mouse SORT1 knock-in on HEK293 cells (relative counts). Anti-HA-FITC and BAF3145 antibodies were used to verify the presence of mouse SORT1 Knock-In (KI). The designed binder CB5 to S0RT1 , shown via construct 27 (GP-msGFP2-[G4SG4]-CB5), was also verified to bind to the mouse SORT1 relative to controls GP-msGFP2 (SEQ ID No. 87), and GP-msGFP- [G4SG4]-[Control] (SEQ ID No. 88).

[1061] Experimental Methods

[1062] Generation of SORT1 extracellular domain (ECD)

[1063] SORT1 extracellular domain (ECD), and at its Carboxy-terminal sequences for purification (His-tag) and an Avi-tag, which can be biotinylated. Here standard construct generation of a vector that allows for production in Expi293™ (Thermo, Catalog #A14635). Purification was through a standard immobilized metal affinity chromatography binding and elution methods. Biotynilation by BIRA (Avidity, Catalog #BirA500).

[1064] Synthesis of Ligands

[1065] Transfections of BL21(DE3) E.coli strain with standard E.coli growth conditions followed with IPTG inductions was carried out. Post growth and cell lysis, homogeneous proteins were isolated through standard IMAC and SEC purification methods. Standard methods of mass spec and analytical size exclusion methods (Superdex 200 5 / 30, PBS) were utilized to confirm mass and quality.

[1066] SPR Binding Assay

[1067] All experiments were performed using Biacore T200 system. Biotinylated human and mouse SORT 1 were captured on the experimental flow cells of a Series S CAP sensor chip (Cytiva, Product ID 28920234). For single cycle kinetics analysis at 25 °C and 37°C at neutral pH, five concentrations of the de novo design protein binders were injected over the flow cells at 30 pl / min using HBS-EPB (0.01 M HEPES pH 7.4, 0.5 M NaCI, 3 mM EDTA, 0.005% (v / v) Surfactant P20 (Cytiva, Catalog #50-105-5279), 0.1% BSA) as the running buffer. For qualitative binding analysis pH shift experiment by SPR binding of construct 3, utilizing flowing buffer of 12.5mM Citrate and 12.5mM NaPhosphate 500mM NaCI, 3 mM EDTA, 0.005% (v / v) Surfactant P20, 0.1% BSA at pH 7.4, pH 7.0, pH 6.5, pH 6.0, pH 5.5. Surface regeneration was achieved by injecting 0.25N NaOH, 6 M guanidine twice after each binding cycle. Curve fitting was done using a 1 :1 binding interaction model. All sensorgrams were processed by double referencing.

[1068] Human Endothelial Microvascular Cell Line Cell Culture

[1069] For experiments involving the human SORT1 protein, we used the cell line hCMEC / D3 (SigmaAldrich, Catalog #SCC066). The medium kit for culture of these cells is EndoGRO-MV Complete Culture Kit (EMD Millipore, Catalog #SCME004). Cells were grown in T75 flasks coated with 1% bovine collagen in 20mM acetic acid. Cells were split every 2-3 days, and then harvested as needed. Typical yield from a T75 flask is approx. 4.5E6 - 7.5E6 cells.

[1070] Cell Line

[1071] The primary human cell line used is hCMEC / D3, which is a human-derived immortalized cell line comprised of endothelial cells (SigmaAldrich, Catalog #SCC066). The cells require a special media, which is available from EMD Millipore (Product ID SCME004). Cells are propagated 2X / week, 1 :4 split (~1 E6 cells / mL density). Cells are seeded onto flasks pre-coated with bovine collagen. These cells are slow to come out of thaw, but after ~2 passages, grow robustly.

[1072] Several mouse models of BBB have been investigated. Two immortalized cell lines have been tested: bEnd.3 (ATCC, Catalog #CRL-2299) and cEND ((ABM, Catalog #T0290). Both are endothelial cell lines derived from murine tissue and express von Willebrand factor. The media for both these cell lines is standard: DMEM with 10% FBS and 1% Pen-Strep. Cells can be passaged 2X / week, 1 :4 split.

[1073] Cell binding evaluation by flow cytometry

[1074] The binding of different binders to human and mouse cells were evaluated by flow cytometry. The cells were first washed twice with 1xPBS 0.5% HSA. Subsequently, 1x10A5 cells were mixed with 0.2-2 pM purified compounds in a total 30 pL system under 37 °C for 30 min, followed by wash with 1xPBS twice. Then the cells were loaded to the NovoCyte flow cytometer (Agilent) for fluorescence intensity quantitation, in which msGFP2 and mScarlet fluorescent intensities were detected with the FITC channel, 530 / 30 nm band pass filter and PE channel, 600 / 30 nm band pass filter, respectively. The binding efficiencies were evaluated by Mean Fluorescence Intensity (M.F.I.) of msGFP2 or mScarlet, and the results were normalized by control compounds. • 1x10A5 cell in 30uL HEK or HEK-mSORT1 KI

[1075] • anti-HA-FITC (GenScript, 40nM) : 4 °C 15 min followed by 20 °C 15 min

[1076] • Sortilin Antibody [Biotin] (BAF3154): Novus Biologicals (40nM): 37 °C 30 min, Avidin-APC will be 20 °C 5 min

[1077] • Sortl binders (construct 27) : 1uM 37 °C 30 min, Avidin-APC will be 20 °C 5 min phy methods

[1078] Complex preparation: huSORTI and CB4 were mixed in a 1 :2 molar ratio and subjected to size exclusion chromatography on a Superdex75 (Cytiva, Product ID 29148721) column. The eluted peak fractions were pooled and concentrated to a concentration of 6.5 mg / ml.

[1079] Deglycosylation

[1080] 5 mg HuSORTI was incubated with 250 pl PNgaseF (New England Biolabs, Catalog #P0704L) and incubated at room temperature for 16 hours. Deglycosylated huSORT 1 was isolated using a standard Ni Sepharose column, and a complex with CB4 was prepared as described above. This complex was concentrated to 8.5 mg / ml and used for crystallization trials.

[1081] Crystallisation

[1082] A solution of either 6.5 mg / ml huSORT1 / CB4 complex or 8.5 mg / ml deglycosylated hu / SORT1 / CB4 complex was crystallized with the sitting drop method using commercially available crystallization screens (Hampton research). Crystals readily grew within a few days in several conditions, and crystals from different pH values were used for structure determination. Conditions, unit cell and symmetry data are shown in Table 1.

[1083] Crystal structure determination

[1084] X-ray diffraction data were collected at beamline X10SA of the Swiss Light Source (SLS; Villigen, Switzerland). Structures were solved by molecular replacement with PHASER (McCoy, A. J., R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storoni and R. J. Read (2007). "Phaser crystallographic software." J Appl Crystallogr 40(Pt 4): 658-674.) as implemented in the Phenix program suite (Adams, P. D., P. V. Afonine, G. Bunkoczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L. W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger and P. H. Zwart (2010). "PHENIX: a comprehensive Python-based system for macromolecular structure solution." Acta Crystallogr D Biol Crystallogr 66(Pt 2): 213-221.). Alphafold2 generated coordinates of huSORTI and CB4 were used as search model. Subsequent rounds of refinement with Phenix and model building in COOT (Emsley, P., B. Lohkamp, W. G. Scott and K. Cowtan (2010). "Features and development of Coot." Acta Crystallogr D Biol Crystallogr 66(Pt 4): 486-501.) yielded the final coordinates. content imaging screen hCMEC / D3 were maintained in EndoGRO-MV Complete culture media (Millipore Sigma, SCME004). For binder internalization experiments cells were seeded in a 384-well plate and incubated with binders at 250nM, 500nM or 1uM in EndoGRO-MV media (MillPore Sigma, Catalog # SCME004) at 37C 5% CO2 for 3hr. Following incubation, cells were washed 3x with PBS then fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Catalog #15710) for 15 minutes at room temperature. Following fixation, cells were stained with 1.5ug / ml Wheat Germ Agglutinin Alexa Fluor 555 conjugate (Thermo Fisher, Catalog #W32464) and 1ug / ml Hoechst 33342 (Life Technologies, Catalog #H3570) in PBS for 30 minutes at room temperature, then washed 3x with PBS. Images were acquired at 20x using the Perkin Elmer Operetta CLS high content imager and GFP intensity was measured exclusively in the cytoplasmic region.

[1085] Human

[1086] Generation of human blood brain-barrier (BBB) organoids and measurement of compounds across the BBB was based on (Bergmann, S. et al. Nature Protocols 2018) with optimization. Briefly, human brain endothelial cells (hCMEC / d3), primary human astrocytes and primary human pericytes were co-cultured in AggreWell™ Microwell Plates at a ratio of 1:1:1. After 48 hours when the BBB organoids were formed, each 50 organoids were pooled and treated with fluorescently labelled compounds at the concentrations of 0.2pM, 1 pM or 5pM for 3 hours at 37°C. Confocal images at Z-stack 50 pm away from the surface of the organoids were then taken by a high content imaging system (Operetta®, PerkinElmer). The fluorescence signal inside each organoid was quantified by the Perkin Elmer Harmony analysis software.

[1087] Degradation Assay

[1088] Western blot analysis of total EGFR levels in Hela cells after treatment with 200 nM

[1089] EGFRn or Construct 36 for 48 h. Cells were cultured in T75 flasks at 37 °C in a 5% CO2 atmosphere. HeLa (ATCC, CCL-2) cells were cultured in DMEM supplemented with 10% heat-inactivated 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 pl of complete growth media with Construct 36, EGFRn or controls for indicated time. Cells were then washed with PBS 3 times and lysed with RIPA buffer supplemented with 0.1% Benzonase (Millipore- Sigma) and phosphatase inhibitor cocktail (Roche, 4693132001) 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, Catalog #23225). Equal amounts of lysates were loaded onto 4-12% Bis-Tris gel and separated by sodium dodecyl sulfate-polacrylamide gel electrophoresis. Then, the gel was transferred onto a nitrocellulose membrane and stained with REVERT Total Protein Stain (LI-COR, 926-11011) and then blocked with Odyssey Blocking Buffer (TBS) (LI-COR, 927-50100) for 1 hour at rt. The membrane was incubated with primary antibodies (rabbit anti-EGFR D38B1 Cell Signaling Technologies, 4267T) overnight at 4 °C, washed 3 times with TBS. Subsequently, the membrane was incubated with secondary antibody (800CWgoat-anti-rabbit LI-COR, 926-32211) for 1 hour at rt, and washed 3 times with TBS for visualization with an Odyssey CLx Imager (LI-COR).

[1090] Murine Sortl Knock-In Cell Line

[1091] A murine Sortl knock-in cell line was generated using a custom mammalian baculovirus (BacMam), generated by Montana Molecular (Bozeman, MT). The mammalian baculovirus was engineered to contain both mouse Sortl (Accession ID: NM_019972) and an HA tag (to identify transfected cells).

[1092] The BacMam particles were used to perform a transient transduction into an adherent HEK293 parental cell line (ATCC, Catalog #CRL-1573). HEK293 cells were thawed and grown in growth medium (DMEM supplemented with 10% FBS and 1% Pen / Strep) in T75 flasks to -75% confluence in a humidified incubator maintained at 37°C and 5% CO2. Once -75% confluence was achieved, the growth medium was removed and replaced with growth medium containing 5% virus suspension (e.g. for a T75 flask, 750 pL added to 14.25 mL growth medium). The original lot of virus contained 6.85 x 1010VG / mL, corresponding to 5.14 x 1010viral genomes in one T75 flask. Following virus addition, cells were incubated for another 24 h in a humidified incubator maintained at 37°C and 5% CO2, to near-full confluence. Cells were then harvested using Assay Complete Cell Detachment Reagent (DiscoverX, Catalog #92-0009). Cell suspension was centrifuged at 1400 RPM for 3 minutes, the trypsin-containing media was removed, and cells were re-suspended in assay running buffer. Transduction was verified using an anti-HA-FITC antibody (GenScript, Catalog #A01621) and the mouse cross-reactive SORT1 polyclonal antibody (Human Sortilin Biotinylated Antibody, Novus Biologies, Catalog #BAF3145).

Claims

CLAIMS1. A polypeptide comprising a binder, the binder having a binding affinity to Sortilin-1 (Sortl) receptor and comprising an amino acid sequence at least 60%, 70%, 79%, 80%, 82%, 87%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1-4 or 6-39.

2. The polypeptide according to claim 1 , wherein the binder comprises an amino acid sequence at least 79% identical to the full length of the amino acid sequence as set out in any one of SEQ ID Nos. 5, 1-4 or 6-39.

3. The polypeptide according to any one of the preceding claims, wherein amino acid substitutions relative to a reference polypeptide are selected from those listed in Table 1.

4. The polypeptide according to any one of the preceding claims, wherein the binder comprises the amino acid sequence according to Formula II (SEQ ID No. 92) :Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu- Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu- Ile-Val-Glu-Glu-Leu-Xaa57-Xaa58, whereinXaa4 is Leu (L) or Phe (F);Xaa6 is Gin (Q) or His (H) or Ser (S) or Thr (T);Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);XaalO is Vai (V) or lie (I);Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);Xaa25 is Glu (E) or Lys (K);Xaa27 is lie (I) or Vai (V) or Leu (L);Xaa30 is Leu (L) or Thr (T) or lie (I);Xaa37 is Asn (N) or Gin (Q) or His (H) or Lys (K);Xaa57 is Absent or Leu (L); andXaa58 is Absent or Glu (E).

5. The polypeptide according to any one of the preceding claims, wherein the binder comprises the amino acid sequence as set out in any one of SEQ ID Nos. 5, 2, 6, 14, 24, 29.

6. The polypeptide according to any one of the preceding claims, wherein the polypeptide is capable of binding to a Sortl receptor.

7. The polypeptide according to any one of the preceding claims, wherein the polypeptide is capable of binding to the Sortl receptor when the Sortl receptor is in a protonated state, and wherein the polypeptide is further capable of being released from the Sortl receptor when the Sortl receptor is in a deprotonated state.

8. A construct comprising the polypeptide according to any one of the preceding claims, the binder forming part of a binder moiety linked to at least one cargo moiety.

9. The construct according to claim 8, wherein the cargo moiety is linked to the C-terminus or the N-terminus of the binder moiety.

10. The construct according to claim 8, wherein the cargo moiety is linked to an amino acid residue found between the N-terminus and C-terminus of the binder moiety.

11. The construct according to claim according to any one of claims 8-10, wherein the construct comprises a fusion protein.

12. The construct according to any one of claims 8-11, wherein the at least one cargo moiety is a therapeutic agent.

13. A construct comprising a binder moiety and a cargo moiety, the cargo moiety linked to the binder moiety via a linker, the binder moiety being capable of binding to a Sortilin-1 (Sortl) receptor, and the binder moiety comprising an amino acid sequence according to Formula II (SEQ ID No. 92) :Ser-Phe-Leu-Xaa4-Lys-Xaa6-Ala-Xaa8-Arg-Xaa10-Xaa11-Glu-Arg-Leu-Leu-Glu- Glu-Gly-Asn-Val-Glu-Xaa22-Ala-Glu-Xaa25-Trp-Xaa27-Glu-Gln-Xaa30-Lys-Arg- Leu-Ala-His-Ala-Xaa37-Arg-Asp-Pro-Glu-Ala-Leu-Arg-Arg-Ala-Glu-Glu-Leu-Glu-Glu- Ile-Val-Glu-Glu-Leu-Xaa57-Xaa58,whereinXaa4 is Leu (L) or Phe (F);Xaa6 is Gin (Q) or His (H) or Ser (S) or Thr (T);Xaa8 is lie (I) or Vai (V) or Thr (T) or Met (M);XaalO is Vai (V) or lie (I);Xaa11 is lie (I) or Vai (V) or Thr (T) or Ala (A);Xaa22 is Leu (L) or lie (I) or Vai (V) or Thr (T) or Pro (P);Xaa25 is Glu (E) or Lys (K);Xaa27 is lie (I) or Vai (V) or Leu (L);Xaa30 is Leu (L) or Thr (T) or lie (I);Xaa37 is Asn (N) or Gin (Q) or His (H) or Lys (K);Xaa57 is Absent or Leu (L); andXaa58 is Absent or Glu (E).

14. A pharmaceutical composition comprising the polypeptide or construct according to any one of the preceding claims and a pharmaceutical excipient.

15. The polypeptide or construct according to any one of claims 1-12 or the pharmaceutical composition according to claim 13 for use in promoting internalization a cargo, internalization of a therapeutic agent or extracellular protein degradation.

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