Targeting sortilin

Abstract

Disclosed herein are sortilin binding agents, compositions comprising the same, and methods of making and using the same, including conjugated agents comprising a sortilin-binding moiety conjugated directly or indirectly to a payload moiety. The disclosure provides sortilin targeting techniques, e.g., for delivering a payload to a cell that expresses sortilin (e.g., has sortilin on its surface). In particular, the disclosure provides sortilin targeting agents and conjugates thereof (e.g., in which a payload is conjugated to the sortilin targeting agent).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 430,643, filed December 6, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Sortilin, also known as neurotensin receptor 3 (NTSR3), has transmembrane and soluble forms and has been reported to play a role in a variety of physiological processes and conditions, including certain cancers, cardiovascular disease, Alzheimer's disease, diabetes, depression, etc. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides sortilin targeting techniques, e.g., for delivering a payload to a cell that expresses sortilin (e.g., has sortilin on its surface). Among other things, the present disclosure provides sortilin targeting agents and conjugates thereof (e.g., in which a payload is conjugated to the sortilin targeting agent).

[0004] In some embodiments, the provided sortilin binding agents (and / or conjugates thereof) have been shown to bind to sortilin with high affinity and / or have useful stability properties, including, for example, in serum.

[0005] In some embodiments, provided sortilin binding agents (and / or conjugates thereof) have been shown to efficiently deliver a payload (e.g., a therapeutic payload, which in some embodiments may be a toxic payload) to cells (e.g., sortilin-expressing cells), and in some embodiments have shown a correlation between affinity for sortilin and efficient cell killing (e.g., by delivery of a toxic payload).

[0006] In some embodiments, provided sortilin binding agents (and / or conjugates thereof) have been shown to efficiently deliver payloads (e.g., toxic payloads) to cells within a dose range between the minimum active dose and the maximum tolerated dose, and in some embodiments, such range has been shown to span at least three-fold in mice.

[0007] In some embodiments, provided sortilin binding agents (and / or conjugates thereof) are characterized by one or more of the following: (i) affinity (Kd) for human sortilin of up to 1 μM as examined by fluorescence polarization; (ii) IC in competitive binding assays 50 is less than about 12 μM; (iii) exhibiting stability of greater than 3.16 minutes when maintained in mouse serum;

[0008] Among other things, the present disclosure provides insight into the sources of challenges in previous efforts to develop sortilin binding agents, and in particular, previous attempts to develop conjugates that bind to sortilin on cells that express sortilin and deliver payloads to such cells. For example, the present disclosure observes that certain previous efforts to target sortilin have focused on developing agents structurally related to sortilin ligand(s) other than progranulin (e.g., certain bacterial cell-permeable proteins and / or neurotensin). Furthermore, these efforts that have focused on developing agents structurally related to progranulin have followed different paths.

[0009] For example, in certain embodiments, the present disclosure provides and / or utilizes sortilin-binding peptides having amino acid sequences substantially corresponding to fragments of human Progranulin, but including one or more amino acid substitutions at positions P3, P4, and / or P17, as described herein. Alternatively, or in addition, in certain embodiments, the present disclosure provides and / or utilizes sortilin-binding peptides having amino acid sequences substantially corresponding to fragments of human Progranulin, but including one or more substitutions with unnatural amino acids (in some embodiments, specifically at one or more positions corresponding to, and including, one or more of P15, P16, and P17, e.g., in one or more of the last three C-terminal residues). Still further alternatively, or in addition, in certain embodiments, the present disclosure provides and / or utilizes cyclic sortilin-binding peptides, including cyclic peptides having certain amino acid sequence feature(s) found in human Progranulin, such as certain C-terminal sequences.

[0010] The present disclosure recognizes that certain other approaches to developing sortilin binding agents suggest deleting residues corresponding to P1-P6 found in human Progranulin, and the present disclosure takes a different approach in some embodiments, including these residues. That is, the C-terminal 24 amino acids of wild-type human Progranulin are as follows: [Table 14]

[0011] Certain previous efforts to develop sortilin-binding peptides based on progranulin (e.g., as described in one or more of WO2017 / 088058; WO2018 / 213928; and / or WO2020 / 037434, among others) have failed to recognize that residues P1-P6 (APRWDA) can beneficially contribute to sortilin binding, and further that removal or substitution of certain of these residues may also be recommended. Such teachings lead away from the development of peptides containing such residues (e.g., APRWDA elements), such as certain of the residues provided.

[0012] Among other things, the present disclosure provides the insight that the C-terminal portion of certain provided peptides, e.g., the sequence R(D / F / X)PALR(Q / X)(L / X)(L / X), can be responsible for sortilin binding and, e.g., contain the primary driver for binding affinity, and further that the N-terminal portion of the sequence, APRWDAPL, can bind to a secondary site on the target, which contributes to binding affinity, albeit to a lesser extent. Thus, the present disclosure identifies a source of challenges with certain methods proposed by others, including, e.g., methods that propose peptides of different lengths and / or sequences and / or peptides that may not include part or all of the APRWDAPL sequence.

[0013] In some embodiments, the peptide according to the present disclosure comprises X n RDPALRXLL sequence, where X is any standard or non-standard amino acid according to the present disclosure, and n is 0, 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, for example, a peptide according to the present disclosure has an amino acid sequence that is or includes the sequence RDPALRQLL. In some embodiments, for example, a peptide according to the present disclosure has an amino acid sequence that is or includes RDPALR(B43)LL.

[0014] As noted above, among other things, the present disclosure demonstrates that, in some embodiments, the inclusion of an APRWDAPL sequence can improve one or more aspects or characteristics of sortilin binding (e.g., in some embodiments, affinity, association rate, and / or dissociation rate, etc.) by provided peptides (e.g., provided sortilin-binding moieties). In some particular embodiments, for example, the present disclosure demonstrates that the inclusion of APRWDAPL can improve the sortilin affinity of an RDPALR(B43)LL peptide. That is, among other things, the present disclosure demonstrates that a peptide having APRWDAPLRDPALR(B43)LL can exhibit improved sortilin affinity compared to a peptide having the amino acid sequence RDPALR(B43)LL.

[0015] In some embodiments, the present disclosure provides a peptide whose amino acid sequence includes an APRWDA element. In some such embodiments, such peptides are linear peptides. Alternatively, or in addition, in some embodiments, such peptides include one or more of: (i) a PLR(D / F)P element; (ii) an ALRQLL element, or a variant of ALRQLL, where one or more Qs, or one or both Ls, are substituted with a non-natural amino acid.

[0016] Furthermore, certain previous efforts to develop sortilin-binding peptides based on Progranulin (e.g., as described in one or more of WO2017 / 088058; WO2018 / 213928; and / or WO2020 / 037434, among others) have recommended the inclusion of amino acids corresponding to residues at positions P-6 through P-0 of human Sortilin, and in particular the inclusion of a YKXLRRX (e.g., YKSLRRK) element. The present disclosure provides alternative approaches to sortilin-binding peptides. In some embodiments, such approaches include peptides that do not include residues corresponding to amino acids 18 or more residues N-terminal to the human Progranulin C-terminus and / or that lack the YKXLRRX (e.g., YKSLRRK) sequence element. In some embodiments, provided peptides lack a YKX (e.g., YKS), KXL (e.g., KSL), XLR (e.g., SLR), LRR, and / or LRX (e.g., LRK) element.

[0017] Furthermore, it is worth noting that WO2020 / 037434 (and the referenced prior publications WO2017 / 088058 and WO2018 / 213928), which themselves describe providing peptides that target sortilin, identifies three specific types of such peptides: (1) those derived from bacterial cell-penetrating proteins, (2) those based on an optimized primary sequence derived from progranulin; and (3) those based on an optimized primary sequence derived from neurotensin. As already mentioned above, the progranulin sequence identified by WO2020 / 037434 as the basis for the desired sortilin-binding peptides is different from that utilized herein. Furthermore, the peptides and / or conjugates described herein have a different structure than that taught or suggested in WO2020 / 037434.

[0018] In particular, in this context, the contribution of the present disclosure surprisingly demonstrates the particular utility of the sortilin-binding peptides described herein, including, for example, peptides that (1) include residues corresponding to a sequence found in a C-terminal fragment of human Progranulin, (b) do not include residues corresponding to 18 or more amino acids N-terminal to the human Progranulin C-terminus, (c) do not include a sequence element that includes or consists of a YKSLRR, (d) do not include a sequence element that includes or consists of a YKS, KSL, SLR, or LRR, (e) include an ALRQLL element, or (f) include a variant of the ALRQLL sequence element (in which one or more of (i) Q, or (ii) one or both of L, are substituted with a non-natural amino acid), (2) are cyclic, (3) are less than 20 amino acids in length, e.g., are about 17 amino acids in length, or are exactly 17 amino acids in length, and / or otherwise have the structural and / or functional characteristics described herein.

[0019] In particular, in some particular embodiments, the present disclosure provides: (a) a polypeptide; (b) a payload; (c) optionally a linker; The polypeptide is (a) a sortilin-binding moiety having a length in the range of about 12 to about 20 amino acids and comprising a characteristic sequence represented by: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO:2); wherein each X is independently any standard or non-standard amino acid.

[0020] In some embodiments, the provided binding moieties of Sortilin correspond to a C-terminal fragment of Progranulin, or a variant thereof, and comprise no more than about 20 consecutive residues corresponding to consecutive Progranulin residues.

[0021] In some embodiments, the binding moieties of provided Sortilin are or comprise cyclic peptides, hi some embodiments, the binding moieties of provided Sortilin comprise a characteristic sequence represented by: (R / N) X 2-3 CX 0-1 R (Q / E) (SEQ ID NO: 3).

[0022] In some embodiments, the binding moieties of provided Sortilin are or comprise linear peptides. In some embodiments, the binding moieties of provided Sortilin are or comprise linear peptides. In some embodiments, the binding moieties of provided Sortilin comprise the characteristic sequence represented by SEQ ID NO:2. In some embodiments, the binding moieties of provided Sortilin comprise the characteristic sequence represented by: (R / N) X 2-3 LX 0-1 R (Q / B43 / B50) (SEQ ID NO: 4).

[0023] In some embodiments, provided binding agents of Sortilin (and / or conjugates thereof), or Sortilin binding moieties, are or comprise the amino acid sequence APRWDAPLRDPALRQLL. In some embodiments, provided binding agents of Sortilin (and / or conjugates thereof), or Sortilin binding moieties, are or comprise the amino acid sequence APRWDAPLRDPALRQ(B13)(G48).

[0024] In some embodiments, the provided nucleic acid comprises a nucleotide sequence comprising a coding region encoding a peptide comprising the sequence of APRWDAPLRDPALRQLL. In some embodiments, the provided nucleic acid comprises a nucleotide sequence comprising a coding region encoding a peptide comprising the sequence of APRWDAPLRDPALRQ.

[0025] In some embodiments, the provided linker is a VCPAB linker.

[0026] In some embodiments, the payload provided is MMAE.

[0027] These and other aspects encompassed by the present disclosure are described in more detail below and in the claims. [Brief explanation of the drawings]

[0028] [Figure 1] The amino acid sequence of human progranulin (SEQ ID NO: 1) is shown. [Figure 2] 1 shows exemplary conjugation agents provided by the present disclosure. [Figure 3] (a) Binding affinity of PRGN-based homing peptides measured by direct titration. (b) Binding affinity of PRGN-based homing peptides measured by competitive activity in the FP assay. [Figure 4] (a) shows the binding affinity of PRGN_WT (also referred to as PRGN_C18 or PRGN_WT_C18) measured by grating-binding interferometry in a protein-loaded format. (b) shows the binding affinity of PRGN_WT (also referred to as PRGN_C18 or PRGN_WT_C18) measured by grating-binding interferometry in a peptide-loaded format. [Figure 5] (a) shows the binding affinity of PRGN_RL measured by grating binding interferometry in a protein-loaded format. (b) shows the binding affinity of PRGN_RL measured by grating binding interferometry in a peptide-loaded format. [Figure 6] (a) Biophysical characterization of the homing peptide by dynamic light scattering, showing that the homing peptide does not aggregate. (b) Biophysical characterization of the homing peptide by circular dichroism, showing that the homing peptide is an unstructured random coil in buffer solution. [Figure 7](a) Biophysical characterization of peptide drug conjugates (PDC) by dynamic light scattering, showing no aggregation. (b) Biophysical characterization of peptide drug conjugates (PDC) by circular dichroism, showing that PDC is an unstructured random coil in buffer solution. [Figure 8] Binding of PDC to SORT1 in an FP binding assay is shown. [Figure 9-1] The chemical structures of PRGN_PDC_WT and PRGN_PDC_RL are shown. [Figure 9-2] Same as above [Figure 10A] Figure 1 shows the characterization of PRGN_WT and PRGN_PDC_WT by LC-MS. The readout is free of contaminants and the fragments correspond to the masses of the parent compounds. (a) LC-MS trace for PRGN_WT. (b) MS / MS spectrum for PRGN_PDC_WT. (c) HPLC chromatogram for PRGN_PDC_WT. [Figure 10B] Same as above [Figure 10C] Same as above [Figure 11] (a) The potency of PGRN-based PDC in a 72-hour cell killing assay using HCC70 cells. (b) The potency of PGRN-based PDC in a 72-hour cell killing assay using MDA-MB-231 cells. [Figure 12] (a) Characterization of PRGN-based PDC at the in vivo maximum tolerated dose (MTD) in WT_PDC. n=3. (b) Characterization of PRGN-based PDC at the in vivo maximum tolerated dose (MTD) in RL_PDC. n=3. [Figure 13-1](a) PQ-designed peptides that bind to SORT1, as identified by peptide microarray, are shown for linear peptides. His-tag positive controls were printed at positions A1-A5. (b) PQ-designed peptides that bind to SORT1, as identified by peptide microarray, are shown for cyclic peptides. His-tag positive controls were printed at positions A7-A11, T1-T2, and T30. [Figure 13-2] Same as above [Figure 14-1] (a) Dynamic light scattering biophysical characterization of a linear peptide designed by PQ. The aggregating peptide (colored bar) was removed from the workflow. (b) Representative circular dichroism traces of a linear peptide designed by PQ. [Figure 14-2] Same as above [Figure 15-1] (a) Dynamic light scattering and (b) representative circular dichroism traces show biophysical characterization of PQ-designed cyclic peptides. [Figure 15-2] Same as above [Figure 16] Figure 1 shows the affinity of PQ-designed linear peptides for SORT1 in a fluorescence polarization assay. PRGN_WT and the natural ligand neurotensin are highlighted in red and blue, respectively. The computer-designed linear peptides show improved affinity for SORT1 compared to the control PRGN_WT. [Figure 17] 1 shows the affinity of PQ-designed cyclic peptides to SORT1 in a fluorescence polarization assay. [Figure 18] The affinity and mouse serum stability of the complete list of SORT1-binding peptides are shown. PRGN_WT and the natural ligand neurotensin are highlighted in green and red, respectively. [Figure 19](a) Shown is the sequence logo of a cyclic peptide of cyclic group 1. The following amino acids are shown as natural variants: F02, B13, and Nle, as L, L, and M, respectively. (b) Shown is the sequence logo of a cyclic peptide of cyclic group 2. The following amino acids are shown as natural variants: F02, B13, and Nle, as L, L, and M, respectively. [Figure 20] Figure 1 shows the internalization and lysosomal colocalization of the PRGN_WT homing peptide conjugated to fluorescently labeled AF488 in MDA-MB-231 TNBC cells. Two different exemplary fields of MDA-MB-231 cells treated with Lysotracker (red), AF488-PRGN_WT (green), and Hoechst nuclear stain (blue) are shown. The merged channel is depicted on the right, where yellow pixels indicate colocalization of AF488-PRGN_WT with lysosomes. [Figure 21] Figure 1 shows the in vivo efficacy of PRGN_WT_PDC and free monomethylauristatin E (MMAE) in the MDA-MB-231 mouse model, with n=6 for each treatment group. (a) Tumor growth curves after treatment with three different doses of PRGN_WT_PDC and free MMAE administered at an amount equivalent to 3 mg / kg WT_PDC. (b) Changes in body weight as a result of treatment with PRGN_WT_PDC and free MMAE (P<0.0001; two-way ANOVA with Sidek's multiple comparison test). [Figure 22] In vivo efficacy of PRGN_WT_PDC, Arb-SAR-Q15-PDC, Arb-cyclic-G23-PDC, control PRGN_scr_PDC, and free MMAE in an MDA-MB-231 mouse xenograft model, n=8 for each treatment group. (a) Tumor growth curves after treatment with PRGN_WT_PDC, Arb-SAR-Q15-PDC, Arb-cyclic-G23-PDC, control PRGN_scr_PDC, and free MMAE administered at a molar concentration equivalent to 3 mg / kg PDC. (b) Body weight change as a function of treatment group (**P≦0.01; two-way ANOVA with Sidek's multiple comparison test). [Figure 23] Figure 1 shows the characterization of peptide drug conjugates PRGN_WT_PDC, Arb-SAR-Q15-PDC, and Arb_cyclic_G23-PDC together with vehicle and MMAE in an in vivo hematotoxicity assay. n=3. (*P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001). DETAILED DESCRIPTION OF THE INVENTION

[0029] definition In order to further define this invention, the following terms and definitions are provided herein.

[0030] In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," (iii) the terms "comprising" and "including" may be understood to encompass the itemized components or steps, whether presented by themselves or with one or more additional components or steps, (iv) the terms "about" and "approximately" may be understood to allow for standard variations that would be understood by one of ordinary skill in the art, and (v) when ranges are specified, both endpoints are included.

[0031] About: When used herein in connection with a value, the term "about" refers to a value that is similar to the reference value in the context. Generally, a person skilled in the art who is familiar with the context will fully understand the relative degree of difference encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

[0032] Administration: As used herein, the term "administration" means administering a composition (e.g., a compound described herein [e.g., a conjugate], or a preparation containing or otherwise delivering such a compound) to a subject or system, or to cells or tissues thereof. Administration to an animal subject (e.g., a human) can be by any suitable route, such as those described herein. In some embodiments, administration can be topical. In some embodiments, administration can be systemic. In some embodiments, administration can be enteral. In many embodiments, administration can be parenteral. In some embodiments, parenteral administration can be intradermal, intramuscular, intrathecal, intravenous, subcutaneous, etc.

[0033] Affinity: As known in the art, "affinity" is a measure of the tightness with which two or more binding partners bind to one another. Those skilled in the art will be knowledgeable of various assays that can be used to assess affinity and will also be aware of appropriate controls for such assays. In some embodiments, affinity is assessed in a quantitative assay. In some embodiments, affinity (e.g., of one binding partner at a time) is assessed across multiple concentrations. In some embodiments, affinity is assessed in the presence of one or more potential competitors (e.g., that may be present in a relevant physiological situation). In some embodiments, affinity is compared to a reference (e.g., a known affinity above a certain threshold [see "positive control"] or with a known affinity below a certain threshold [see "negative control"]). In some embodiments, affinity may be assessed relative to a concurrent reference, and in some embodiments, affinity may be assessed relative to a background reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.

[0034] Agent / Drug / Agent: As used herein, the term "agent / drug / agent" may refer to a physical entity or phenomenon. In some embodiments, an agent / drug / agent may be characterized by a particular property and / or effect. In some embodiments, an agent / drug / agent may be a compound, molecule, or entity of any chemical class, including, for example, a small molecule, polypeptide, nucleic acid, monosaccharide, lipid, metal, or combination or complex thereof. In some embodiments, the term "agent / drug / agent" may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or element that comprises one or more polymer moieties. In some embodiments, the term "agent / drug / agent" may refer to a compound, molecule, or element that is substantially free of a particular polymer or polymer moiety. In some embodiments, the term may refer to a compound, molecule, or entity that is devoid of or substantially free of any polymer or polymer moiety.

[0035] Amino acid: As used herein in its broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through one or more peptide bond formats. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is an unnatural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" or "canonical amino acid" amino acid refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "nonstandard amino acid" or "non-canonical amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. Certain non-standard amino acids that may be particularly useful in accordance with certain embodiments of the present disclosure are provided herein (see, e.g., Tables 2-7), and in some embodiments, the particular positions where certain such non-standard amino acids may be most useful are named.

[0036] Antibody: As used herein, the term "antibody" refers to a polypeptide containing standard immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As known in the art, intact antibodies, as produced in nature, are approximately 150 kD tetrameric chemical entities composed of two identical heavy chain polypeptides (approximately 50 kD each) and two identical light chain polypeptides (approximately 25 kD each) associated with each other in a commonly referred to "Y" structure. Each heavy chain is composed of at least four domains, each approximately 110 amino acids long: an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 domain (located at the base of the Y stem). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in intact antibodies. Each light chain is composed of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch." An intact antibody tetramer is composed of two heavy-light chain dimers in which the heavy and light chains are linked to each other by one disulfide bond and two other disulfide bonds connect the heavy chain hinge regions to form a tetramer. Each variable domain contains three hypervariable loops (CDR1, CDR2, and CDR3) known as "complement-determining regions" and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the CDR loop regions of both the heavy and light chains join in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure, with the FR regions forming a beta sheet to provide the structural framework for the domain. The Fc region of a naturally occurring antibody binds to components of the complement system and also to receptors on effector cells, such as effector cells that mediate cytotoxicity.As is known in the art, the affinity and / or other binding properties of the Fc region for an Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present disclosure comprise one or more modifications on the Fc domain. For purposes of the present disclosure, in certain embodiments, any polypeptide or polypeptide complex comprising a sufficient immunoglobulin domain sequence as found in a native antibody may be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., generated by an organism in response to an antigen) or by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of a canine, feline, murine, rabbit, primate, or human antibody. In some embodiments, the antibody sequence elements are human, humanized, primatized, chimeric, etc., as known in the art. In some embodiments, the term "antibody" (or "antibody portion") can refer, in appropriate embodiments (unless otherwise specified or clear from the context), to any construct or format known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations. For example, in some embodiments, "antibodies" (or antigen-binding elements thereof) can be utilized in accordance with the present invention in a single-chain format or in another format that is smaller than a complete antibody or is otherwise particularly amenable to inclusion in the conjugates described herein. In some embodiments, antibodies can contain covalent modifications (e.g., attachment of glycans, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0037] Approximately: As used herein, as applied to one or more subject values, the term "approximately" or "about" refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction (above or below) of the stated reference value, unless otherwise specified or a different meaning is apparent from the context (except where such number would exceed 100% of the possible values).

[0038] Associated: As used herein, two events or entities are "associated" with one another when the presence, level, degree, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, etc.) is considered to be associated with a particular cell type or a particular disease, disorder, or condition if its presence, level, and / or form correlates (e.g., across a relevant population) with the identity of such cell type or the incidence of, susceptibility to, severity of, stage of, etc., of the disease, disorder, or condition. In some embodiments, two or more entities are physically "associated" with one another if they are in physical proximity to one another and / or interact, directly or indirectly, to remain in physical proximity. In some embodiments, two or more entities that are physically associated with one another are covalently bonded to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently bonded to one another but are non-covalently associated, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0039] Binding: Those skilled in the art will understand that the term "binding," as used herein, typically refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between the entities or moieties, while indirect binding involves a physical interaction due to physical contact through one or more intermediate entities. Generally, binding between two or more entities can be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied alone or in the context of more complex systems (e.g., while covalently or otherwise bound to a carrier entity and / or within a biological system or cell). Binding between two entities can be considered "specific" if, under the conditions being assessed, the associated entities are more likely to associate with each other than with other available binding partners. In some embodiments, an agent (e.g., a peptide described herein) that specifically binds to a particular agent (e.g., sortilin) ​​is said to be "homed" to such target or to be a "homing agent" for such agent.

[0040] Carrier: As used herein, refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, the carrier can comprise a sterile liquid, such as water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or comprises one or more solid ingredients.

[0041] Cell Surface Factors. As used herein, the term "cell surface factor" refers to a factor (e.g., a polypeptide or including a polypeptide) that is present on the surface of a cell(s) of interest (e.g., a target cell(s) described herein, which in many embodiments may be cells that express Sortilin, such as cancer cells that express Sortilin). In some embodiments, the cell surface factor is present primarily on the surface of the target cell(s) (e.g., cancer cells) compared to cells of one or more other tissues. In some embodiments, the cell surface factor is present on certain non-target cells in addition to the target cells. In some embodiments, the cell surface factor is not present primarily or specifically on the relevant target cells of interest. In some embodiments, the cell surface factor is or includes a receptor. In some embodiments, the cell surface factor is internalized when bound by one or more specific ligands (e.g., those having a Sortilin-binding moiety described herein). In some embodiments, a cell surface factor can interact with (e.g., bind to, form a complex with, etc.) one or more other components of the cell on whose surface the cell surface factor is found (e.g., one or more cell membrane components, and / or one or more cell surface components, and / or one or more internal cell components). In some embodiments, a cell surface factor, and / or a particular form or variant thereof, and / or any of the foregoing cell surface factors may be associated with a particular cellular state or condition (e.g., developmental stage, disease state, etc.).

[0042] Characteristic sequence element: As used herein, the phrase "characteristic sequence element" refers to a sequence element found in a polymer (e.g., a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., sequentially linked monomers). In some embodiments, a characteristic sequence element comprises at least first and second stretches of consecutive monomers separated by one or more spacer regions that may or may not vary in length between polymers sharing the sequence element.

[0043] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, the two or more regimens may be administered simultaneously. In some embodiments, the regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before any doses of a second regimen are administered). In some embodiments, the agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may include administering one or more agent(s) or modality to a subject who is also receiving the other agent(s) or modality. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily simultaneously), although in some embodiments, two or more agents, or active portions thereof, may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0044] Equivalent / Comparable: As used herein, the term "equivalent / comparable" refers to two or more agents, entities, circumstances, settings, etc. that may not be identical to one another, but that are sufficiently similar to permit a comparison between them, where one of skill in the art would understand that conclusions can reasonably be drawn based on observed differences or similarities. In some embodiments, equivalent settings, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few altered characteristics. Those of skill in the art will understand what degree of identity is required in any given situation for two or more such agents, entities, circumstances, settings, etc. to be considered comparable, depending on the context. For example, one skilled in the art will understand that situational settings, individuals, or populations are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or observed phenomena under or with different situational settings, individuals, or populations are caused by or exhibit variation in the diverse characteristics.

[0045] Conservative: As used herein, the term "conservative" refers to cases that represent conservative amino acid substitutions, including the replacement of an amino acid residue with another amino acid residue having a side chain R group with similar structural, chemical (e.g., charge or hydrophobicity), and / or functional properties. Generally, conservative amino acid substitutions do not substantially alter the desired functional properties of a protein, such as, for example, the ability of a receptor to bind a ligand. Examples of groups of amino acids having side chains with similar chemical properties include aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (Ile, I); aliphatic hydroxyl side chains such as serine (Ser, S) and threonine (Thr, T); amide-containing side chains such as asparagine (Asn, N) and glutamine (Gln, Q); aromatic side chains such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfate-containing side chains such as cysteine ​​(Cys, C) and methionine (Met, M). Conservative amino acid substitutions include, for example, valine / leucine / isoleucine (Val / Leu / Ile, V / L / I), phenylalanine / tyrosine (Phe / Tyr, F / Y), lysine / arginine (Lys / Arg, K / R), alanine / valine (Ala / Val, A / V), glutamic acid / aspartic acid (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, a conservative amino acid substitution may be the substitution of any naturally occurring residue in a protein with alanine, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet, et al., Science 256:1443, 1992, which is incorporated herein by reference in its entirety. In some embodiments, the substitution is a moderately conservative substitution, and the substitution has a non-negative value in the PAM250 log-likelihood matrix.Those skilled in the art will understand that changes (e.g., substitutions, additions, deletions, etc.) of amino acids that are not conserved between the same proteins from different species are unlikely to affect the function of the protein, and therefore, these amino acids should be selected for mutation. Amino acids that are conserved between the same proteins from different species should not be altered (e.g., deleted, added, substituted, etc.) because these mutations are likely to result in changes in protein function. In some embodiments, "conservative" substitutions are considered "homologous" residues for purposes of calculating the percentage homology between amino acid sequences.

[0046] Conjugated Agents. As used herein, the term "conjugated agent" refers to an agent that has been modified to bind to different moieties. According to the present disclosure, conjugated agents typically comprise at least one sortilin binding moiety conjugated directly or indirectly to at least one payload moiety. In some embodiments, the sortilin binding moiety is or comprises a peptide. In some embodiments, the conjugated agent comprises at least one sortilin binding moiety, at least a first payload moiety, and at least one other moiety, which may be, for example, a second sortilin binding moiety, a second payload moiety, or another moiety, for example, a binding moiety for a target other than sortilin. In some embodiments, the conjugated agent comprises at least two binding moieties, at least one of which is a sortilin binding moiety. Alternatively, or in addition, in some embodiments, the conjugated agent comprises at least two payload moieties (which may in some embodiments be two or more of the same payload moieties or, in some embodiments, two or more different payload moieties). In some embodiments, the conjugated agent comprises a plurality of sortilin binding moieties, in some such embodiments, all of the plurality are the same sortilin binding moieties, in some such embodiments, the plurality comprises at least two different sortilin binding moieties, and in some such embodiments, each sortilin binding moiety of the plurality is different from each other of the plurality of sortilin binding moieties. In some embodiments, the conjugated agent comprises a plurality of payload moieties, in some such embodiments, all of the plurality are the same payload moiety, in some such embodiments, the plurality comprises at least two different payload moieties, and in some such embodiments, each payload moiety of the plurality is different from each other of the plurality of payload moieties. In some particular embodiments, a single sortilin binding moiety may be conjugated to multiple payload moieties, and in some such embodiments, all such payload moieties are conjugated at the same position on the sortilin binding moiety.In other such embodiments, at least two such payload moieties are conjugated at different sites on the Sortilin binding moiety, and in some such embodiments, each payload moiety is conjugated at a different site on the Sortilin binding moiety. Alternatively, or in addition, for each such embodiment, multiple payload moieties are conjugated to a single Sortilin binding moiety, and in some embodiments, all of the multiple are the same payload moiety, and in some such embodiments, the multiple comprises at least two different payload moieties, and in some such embodiments, each payload moiety of the multiple is different from each other of the multiple payload moieties. In some particular embodiments, a single payload moiety is conjugated to multiple Sortilin binding moieties. In some such embodiments, all such Sortilin binding moieties are conjugated at the same site on the payload moiety. In other such embodiments, at least two such Sortilin binding moieties are conjugated at different sites on the payload moiety, and in some such embodiments, each Sortilin binding moiety is conjugated at a different site on the payload moiety. Alternatively, or additionally, for each such embodiment, a plurality of Sortilin binding moieties are conjugated to a single Sortilin binding moiety, in some embodiments all of the plurality are the same Sortilin binding moiety, in some such embodiments the plurality comprises at least two different Sortilin binding moieties, and in some such embodiments each Sortilin binding moiety of the plurality is different from each other of the plurality of Sortilin binding moieties.

[0047] Corresponding to: As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to indicate the location / identity of a structural element in a compound or composition compared to another compound or composition (e.g., an appropriate reference compound or composition). For example, in some embodiments, the binding portion of Sortilin corresponds to a C-terminal fragment of Progranulin, or a variant thereof, and includes no more than about 20 consecutive residues corresponding to consecutive Progranulin residues.

[0048] Designed / Made: As used herein, the term "designed / made" refers to (i) an agent whose structure is selected or chosen by the human hand, (ii) an agent produced by a process requiring human intervention, and / or (iii) an agent that is distinct from natural substances and other known agents.

[0049] Domain: As used herein, the term "domain" refers to a section or portion of an entity. In some embodiments, a "domain" relates to a particular structural and / or functional characteristic of an entity such that when the domain is physically separated from the remainder of its parent entity, it substantially or completely retains the particular structural and / or functional characteristic. Alternatively, or in addition, a domain may be or comprise a portion of an entity that, when separated from its (parent) entity and associated with a different (recipient) entity, substantially retains and / or confers to the recipient entity one or more structural and / or functional characteristics that characterized the domain in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide, and in some such embodiments, a domain is characterized by particular structural elements (e.g., particular amino acid sequences or sequence motifs, alpha-helical properties, beta-sheet properties, coiled-coil properties, random coil properties, etc.) and / or particular functional properties (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0050] Engineered: In general, the term "engineered" refers to aspects that have been manipulated by human hands. For example, a polynucleotide is considered "engineered" when the hand of man manipulates two or more sequences that are not naturally linked together in that order so that they are directly linked to one another in the engineered polynucleotide, and / or when certain residues within the polynucleotide are caused through the action of man to be linked to entities or moieties that are not naturally occurring and / or not naturally linked. For example, in some embodiments described and / or utilized herein, a recombinant polynucleotide comprises a regulatory sequence that is found in nature in operable association with a first coding sequence but not with a second coding sequence, and which has been joined by the hand of man into operable association with the second coding sequence. Similarly, a cell or organism is considered "engineered" when, after having been subjected to manipulation, its genetic, epigenetic, and / or phenotypic identity has been altered compared to an appropriate reference cell, such as an otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or includes a genetic operation that changes the genetic information (e.g., new genetic material not previously present is introduced, e.g., by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is altered or removed, e.g., by substitution or deletion mutations, or mating protocols). In some embodiments, an engineered cell is one that has been engineered to contain and / or express a particular agent of interest (e.g., a protein, nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. As is common practice and understood by those skilled in the art, the progeny of an engineered polynucleotide or cell are typically still referred to as "engineered," despite the actual manipulation performed on the earlier entity.

[0051] Excipient: As used herein means a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect.

[0052] Fragment: A "fragment" of a material or entity described herein comprises a discrete portion of the whole, but has a structure that lacks one or more portions found in the whole. In some embodiments, the fragment consists of such a discrete portion. In some embodiments, the fragment consists of or comprises a characteristic structural element or portion found in the whole. In some embodiments, a fragment of a polymer comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., residues) present in the entire polymer, e.g., wholly intact and consecutively connected. In some embodiments, a polymeric fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer. The whole substance or entity may, in some embodiments, be referred to as the "parent" of the fragment.

[0053] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., polypeptide molecules. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymer molecules are considered to be "homologous" to one another when their sequences are at least 80%, 85%, 90%, 95%, or 99% identical, e.g., 80%, 85%, 90%, 95%, or 99% identical when substitutions with "similar" residues are not counted as differences.

[0054] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. For example, calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. Residues at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be achieved using a mathematical algorithm. For example, the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0) can be used to determine the percent identity between two nucleotide sequences. In some exemplary embodiments, comparison of nucleic acid sequences using the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.The percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix.

[0055] "Improve," "Increase," "Inhibit," or "Decrease": As used herein, the terms "improve," "increase," "inhibit," "decrease," or their grammatical equivalents refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of an appropriate comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in an equivalent system known or expected to respond in a particular way in the presence of the relevant agent or treatment.

[0056] Peptide: As used herein, the term "peptide" refers to a polypeptide that is typically relatively short, e.g., less than about 100 amino acids, less than about 50 amino acids, less than about 40 amino acids, less than about 30 amino acids, less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, or less than 10 amino acids in length. In certain embodiments, the peptide has a length in the range of about 12 to about 20 amino acids. In some embodiments, the peptide has a length of about 15 to about 20 amino acids, or about 15 to about 18 amino acids. In some embodiments, the peptide has a length of about 17 amino acids, e.g., 17 amino acids.

[0057] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in an amount of a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in a particular form (e.g., a solid or liquid form) and / or may be specifically adapted for, for example, oral administration (e.g., specially formulated for buccal, sublingual, or systemic absorption, e.g., as a drench [aqueous or non-aqueous solution or suspension], tablet, capsule, bolus, powder, granules, paste, etc.); parenteral administration (e.g., subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation); topical application (e.g., as a cream, ointment, patch, or spray applied, e.g., to the skin, lungs, or buccal cavity); vaginal or rectal administration (e.g., as a pessary, suppository, cream, or foam); intraocular administration; nasal or pulmonary administration, etc.

[0058] Polypeptide: As used herein, refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an amino acid sequence that is modified, in that it is artificially designed and / or created. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.

[0059] Prevent or prophylaxis: As used herein, when used in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete when the onset of the disease, disorder, or condition has been delayed for a predetermined period of time.

[0060] Reference: As used herein, refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated. Those of skill in the art will understand when sufficient similarity exists to justify reliance on and / or comparison to a particular potential reference or control.

[0061] Sortilin-binding moiety: As used herein, the term "Sortilin-binding moiety" refers to a moiety that, when contacted with a system containing Sortilin, or a related moiety thereof, can be measured or concluded to specifically bind to such Sortilin. In some embodiments, the system is an in vitro system. In some embodiments, the system can include cells (e.g., cells measured or known to express Sortilin). In some embodiments, such cells (which can be considered "target cells of interest") can be present in culture, tissue, and / or organism. In many embodiments, the Sortilin-binding moiety has been characterized (e.g., measured or concluded to specifically bind to Sortilin) ​​as binding to a cell surface factor (e.g., a factor that is found primarily or specifically on the surface(s) known to express Sortilin). In some embodiments, binding of the Sortilin-binding moiety to the cell surface factor results in internalization of the Sortilin-binding moiety. Typically, Sortilin-binding moieties useful according to the present disclosure maintain their specific binding properties when included in the conjugated agents described herein. In some embodiments, binding of such conjugated agents to relevant cell surface factors results in internalization of the conjugated agent, hi some embodiments, the sortilin binding moiety specifically binds to factors on the surface of cancer cells that express sortilin.

[0062] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in the environment in which the binding occurs. A binding agent that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to that interacting target. In some embodiments, specific binding is assessed by detecting or measuring the degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or measuring the degree of dissociation of the binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or measuring the ability of a binding agent to compete with an alternative interaction of its partner with another entity. In some embodiments, specific binding is assessed by performing such detection or measurement over a range of concentrations.

[0063] Subject: As used herein, the term "subject" refers to an organism, e.g., a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, the human subject is an adult, an adolescent, or a pediatric subject. In some embodiments, the subject is suffering from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, the subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or exhibits an increased risk (compared to the average risk observed in a reference subject or a reference population) of developing the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit certain symptoms (e.g., clinical symptoms of the disease) or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom diagnosis and / or therapy is being administered.

[0064] Substantially: As used herein, the term "substantially" refers to the quantitative condition of exhibiting a total or near-total degree or degree of a desired characteristic or attribute. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0065] Substantial sequence identity: As used herein, refers to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN for nucleotide sequences, BLASTP for amino acid sequences, gapped BLAST, and PSI-BLAST. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above programs typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant stretch of residues, which in some embodiments is the complete sequence.In some embodiments, the relevant extension is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0066] Treat: As used herein, the terms "treat," "treated," and "treating" refer to the administration of a regimen or therapy shown or reasonably expected to delay the onset and / or reduce the severity and / or frequency of one or more undesirable physiological events or conditions (e.g., which may be indicative of a particular condition, disease, or disorder), and / or achieve certain beneficial or desired physiological or outcome(s), and / or to effect such delay, reduction, or achievement. In some embodiments, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms; reduction in the severity of the condition, disease, or disorder; stabilization of the state of the condition, disease, or disorder (e.g., not worsening); delaying the onset or slowing the rate of progression of the condition, disease, or disorder; remission (whether partial or total), whether detectable or undetectable, of the condition, disease, or disorder; recovery of at least one measurable physical parameter, not necessarily discernible by the patient; improvement or amelioration of the condition, disease, or disorder, and the like. In some embodiments, treatment may involve inducing a clinically significant response without undue side effects, hi some embodiments, treatment may be or may include prolonging survival as compared to expected survival if not receiving treatment.

[0067] Variant: As used herein, the term "variant" refers to a molecule or entity (e.g., a nucleic acid, protein, or small molecule, e.g., that exhibits significant structural identity with a reference molecule or entity, but that structurally differs from the reference molecule or entity, e.g., in the presence or absence, or level, of one or more chemical moieties compared to the reference molecule or entity. In some embodiments, a "variant" can be referred to as a "derivative." In some embodiments, a variant is functionally different from its reference molecule or entity. In many embodiments, whether a particular molecule or entity is properly considered a "variant" of a reference is based on the degree of structural identity with the reference molecule. As will be understood by those skilled in the art, biological or chemical reference molecules are typically characterized by certain characteristic structural elements. A variant, by definition, is a distinct molecule or entity that shares one or more such characteristic structural elements but differs from the reference molecule or entity in at least one aspect. To give a few examples, a polypeptide may have characteristic sequence elements composed of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or that contribute to a particular structural motif and / or biological function, and a nucleic acid may have characteristic sequence elements composed of multiple nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., to which the polypeptide or nucleic acid backbone is attached). In some embodiments, a variant polypeptide or nucleic acid exhibits an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some embodiments, a subject polypeptide or nucleic acid is considered a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to that of the reference, but with minor sequence modifications at specific positions. Typically, less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to the reference. Often, a variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues responsible for a particular biological activity) compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, a variant polypeptide or nucleic acid contains fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically fewer than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the reference polypeptide or nucleic acid is one found in nature.In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0068] Disclosed herein, among other things, are sortilin binding agents (e.g., peptides) and various compositions comprising them. Among other things, the disclosure provides conjugates in which a sortilin binding moiety is linked directly or indirectly to a payload moiety. In some embodiments, the sortilin binding moiety specifically binds to a surface factor on a target cell of interest (e.g., a sortilin-expressing cell, such as a sortilin-expressing cancer cell). In some embodiments, the payload moiety is or includes a therapeutic agent (e.g., an anti-cancer agent) or a detectable agent (e.g., a diagnostic agent).

[0069] Among other things, the present disclosure provides the insight that the sortilin binding agents described herein can be particularly useful or effective for delivering therapeutic agents to cancer cells and / or other cells that express or otherwise contain a surface factor that is specifically bound by a sortilin binding moiety described herein. For example, the present disclosure provides techniques to achieve delivery of a payload to a sortilin-expressing cell (e.g., that displays sortilin on its surface) by contacting the cell with a conjugate described herein.

[0070] Sortilin Sortilin (SORT1) belongs to the Vps10p domain sorting receptor family, which is composed of heterogeneous type 1 receptors that are widely expressed in mammalian tissues, particularly the brain (see, e.g., Petersen et al., 1999). For example, sortilin has been described to be highly expressed in the brain, heart, skeletal muscle, and spinal cord, and is also expressed, albeit at lower levels, in the kidney, liver, pancreas, small intestine, and spleen (see, e.g., Petersen et al., J Biol Chem 6:3599, 1997). Sortilin is also expressed in immune cells (see, e.g., Patel et al., Circ Res 116:789, 2015; Mortensen et al., J Clin Invest 124:5317, 2014). In the brain, sortilin has been reported to be expressed primarily in neurons, with some cell type and regional variability (see, e.g., Xu et al., Front Neuroanat. 13:31, 2021).

[0071] Sortilin has been reported to function as a sorting and transport receptor, shuttling proteins between the cell surface and various intracellular compartments (see, e.g., Ouyang, et al. J. Cell Physiol. 235:8958, 2020). Dysregulation of sortilin has been reported to contribute to the development of various diseases, including cancer, certain neurological diseases (e.g., Alzheimer's disease, Parkinson's disease), cardiovascular disease, and certain metabolic diseases (e.g., type 2 diabetes) (reviewed, e.g., in Al-Yozbaki et al., Rev. on Cancer 1874:188429, 2020).

[0072] For example, sortilin has been reported to mediate beta-secretase transport, thereby increasing the cleavage of beta-amyloid precursor protein, which is associated with the development of Alzheimer's disease (see, e.g., Walter et al., Curr Opin Neurobiol 11:585, 2001). Also, sortilin levels have been reported to correlate with the level of circulating low-density lipoprotein, a hallmark of cardiovascular disease (see, e.g., Gustafsen et al., Cell Metab. 19:310, 2014). Furthermore, there have been many notable reports of sortilin overexpression in cancer cells. Its clinicopathological importance in oncology has been reported in various types of human cancers, including neuroendocrine (see, e.g., Kim et al., 2018; Rhost et al., 2018; Roselli et al., 2015), breast (see, e.g., Rhost, S., Breast Cancer Res, 2018; Berger, BMC Cancer, 2021), pancreatic (see, e.g., Gao, Am J Path, 2020), colon (see, e.g., Akil et al., 2011), ovarian (see, e.g., Ghaemimanesh et al., 2014; Hemmati et al., 2009), and hematological malignancies (e.g., chronic lymphocytic leukemia [CLL]; see, e.g., Lia Farahi et al., 2019).

[0073] Sortilin has a large extracellular domain and a short intracellular domain, sometimes referred to as its cytoplasmic tail (see, e.g., Petersen et al., J Biol Chem 272:3599, 1997). The extracellular domain contains two smaller domains, 10CC-a and 10CC-b (representing 10 conserved cysteines (10CC)) N-terminal to the 10-bladed β-propeller domain, which interacts with the β-propeller domain (see, Quistgaard & Thirup BMC Struct Biol 9:46, 2009).

[0074] Sortilin is predominantly monomeric at neutral pH and predominantly dimeric at acidic pH. Many ligands exhibit high affinity for (monomeric) sortilin at neutral pH and reduced affinity for (dimeric) sortilin at acidic pH. Dimerization has been proposed to disrupt certain ligand-binding site(s). It has further been proposed that dimerization and / or pH effects may help sortilin achieve differential transport of different ligands (see, e.g., Mitok et al., J Lipid Res 63:100243, and references cited therein).

[0075] Over 50 different drugs have been reported to bind to sortilin and / or be transported by mechanisms affected by sortilin activity (see Table 1 in Mitok et al., J Lipid Res 63:100243, 2022). Certain competitive binding studies suggest that different sortilin ligands can bind to unique, yet potentially partially overlapping, sites on sortilin (see, e.g., Quistgaard, et al., Nat. Struct. Mol. Biol. 16:96, 2009; Trabjerg, et al., Structure. 27:1103, 2019; Serup et al., J. Biol. Chem. 285: 12210, 2010). Furthermore, it is established that different ligands can have different effects on sortilin, including different structural changes (see Trajberg et al., Structure 27:1103, 2019).

[0076] Progranulin One sortilin ligand that is particularly relevant to the present disclosure is progranulin.

[0077] Progranulin is a highly conserved, secreted protein expressed in multiple cell types, including both CNS and peripheral tissues. It is initially produced and secreted as a glycosylated protein, which is then cleaved into 6-kDa peptides known as granulins A–G (reviewed in Townley et al., Neurology 90:118, 2018). Progranulin and / or the granulins it produces have been reported to be involved in various biological pathways, including cell proliferation, survival, repair, and inflammation. Specifically, progranulin and various granulins have been shown to activate or repress genes involved in transcription, splicing, stress response, endosomal sorting, cytoskeletal maintenance, and proteostasis (see Rollinson et al., Eur J Neurosci 44:2214, 2016). Progranulin has also been suggested to promote tumorigenesis and / or metastasis (see, e.g., Berge et al., BMC Cancer 21:185, 2021).

[0078] Studies have shown that sortilin regulates progranulin transport and is the primary determinant of progranulin levels in the brain.

[0079] The amino acid sequence of human Progranulin is shown in Figure 1. This is the C-terminal portion of Progranulin that binds to Sortilin (and specifically, the β-propeller region of Sortilin). In fact, the C-terminal Progranulin fragment is completely sufficient for Sortilin binding and will displace full-length Progranulin bound to Sortilin.

[0080] Specifically, Zheng et al. reported that the last 24 aa of Progranulin (C24, aa 570–593) are fully sufficient for binding to Sortilin. The last six residues (C6, ALRQLL) can mediate the Progranulin-Sortilin interaction, although not to the same extent as the 24 residues. Furthermore, Zheng et al. discovered that adding residues to the C-terminus (+7 aa) of Progranulin prevented Progranulin from binding to Sortilin, further confirming the important role of the C-terminal leucine residues of Progranulin in mediating Progranulin binding to Sortilin (Zheng et al., 2011). Deleting the last three residues (QLL) of Progranulin prevented Progranulin from binding to Sortilin and also prevented Sortilin-dependent regulation of Progranulin trafficking. Although Zheng et al. report certain sequences that are sufficient for binding to sortilin, Zheng et al. do not mention sortilin binding agents, as described herein, (e.g., sortilin-binding moieties (e.g., sortilin-binding peptides described herein) that may be conjugated to or otherwise associated with one or more additional moieties).

[0081] Progranulin has been described as a "key mediator involved in breast cancer progression" (see Berger et al., BMC Cancer 21:185, 2021), and sortilin-mediated progranulin endocytosis has been reported to contribute to, and indeed be required for, progranulin-induced metastasis of breast cancer cells (Rhost et al., Breast Cancer Res. 20:137, 2018). Sortilin is highly expressed in breast cancer cell lines compared with non-tumorigenic breast epithelial cells (Berger BMC Cancer 2021). Researchers suggest that co-expression of progranulin and sortilin may be an effective biomarker for identifying highly aggressive subtypes of breast cancer, which may be responsive to therapy using drugs targeting the sortilin-progranulin interaction.

[0082] Sortilin binders The present disclosure provides certain sortilin binding agents, and compositions (e.g., conjugates) comprising same.

[0083] The provided sortilin binding agents are or comprise a sortilin binding moiety described herein, and optionally comprise one or more other moieties (e.g., one or more linker moieties and / or one or more payload moieties). In some embodiments, a sortilin binding agent comprises a sortilin binding moiety (e.g., a sortilin-binding peptide described herein) conjugated to or otherwise associated with one or more additional moieties (e.g., one or more linker moieties and / or one or more payload moieties), although some embodiments may utilize an unconjugated sortilin binding agent (e.g., a sortilin-binding peptide).

[0084] In some embodiments, provided sortilin binding agents are characterized by one or more functional or performance attributes, e.g., as described below. In some such embodiments, such functional or performance attributes are evaluated for a sortilin binding moiety or sortilin binding agent absent the payload, and in some such embodiments, such functional or performance attributes are evaluated for a conjugate comprising a sortilin binding moiety, a payload, and optionally a linker.

[0085] In some embodiments, provided sortilin binding agents (or sortilin binding portions thereof) are characterized by their ability to bind to sortilin.

[0086] In some embodiments, provided sortilin binding agents (e.g., sortilin binding moieties) bind to sortilin with an affinity of 1 μM or less, and / or with an affinity that is reasonably comparable to or greater than that of Progranulin and / or the affinity of a C-terminal fragment of Progranulin (e.g., a peptide that is or includes about 15-20, or specifically 17, C-terminal residues of Progranulin).

[0087] In some embodiments, affinity can be assessed, for example, by fluorescence polarization. In some such embodiments, provided sortilin binding agents (e.g., sortilin binding moieties) exhibit an affinity for sortilin of about 500 nM or less, about 450 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 40 nM or less, about 35 nM or less, or less, when assessed by fluorescence polarization.

[0088] In some embodiments, affinity can be examined, for example, by grating coupling interferometry (GCI). In some such embodiments, provided sortilin binding agents (e.g., sortilin binding moieties) exhibit an affinity for sortilin of 1 μM or less, e.g., about 500 nM or less, about 450 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, or less, when examined by GCI.

[0089] In some embodiments, provided sortilin binding agents (e.g., sortilin binding moieties) are characterized by their ability to compete with Progranulin and / or a C-terminal fragment of Progranulin (e.g., a peptide that is or includes about 15-20, or specifically 17, C-terminal residues of Progranulin) for binding to Sortilin. For example, in some embodiments, provided sortilin binding agents (e.g., sortilin binding moieties) have a binding affinity of less than about 12 μM, less than about 11 μM, less than about 10 μM, less than about 9 μM, less than about 8 μM, less than about 7 μM, less than about 6 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 750 nM, less than about 700 nM, less than about 650 nM, or less than about 12 μM. The compound exhibits an IC50 of less than about 600 nM, less than about 550 nM, less than about 500 nM, less than about 450 nM, less than about 400 nM, less than about 350 nM, less than about 300 nM, or less than this, for example, less than about 300 to 350 nM, or less than about 300 to 400 nM, or less than 400 to 600 nM, or less than 600 to 800 nM, or less than 600 to 700 nM, or less than about 600 to 650 nM. In some particular embodiments, provided Sortilin binding agents that are or comprise cyclic peptide Sortilin binding agents are characterized in such assays by an IC50 that is within the range of about 500 nM or less to about 12 μM, or about 500 μM or less to about 5 μM, or less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, or less (e.g., less than about 600-650 nM). In some particular embodiments, provided Sortilin binding agents that are, or comprise, linear peptide Sortilin binding agents are characterized in such assays by an IC50 in the range of about 300 nM or less to about 10 μM, or about 300 nM or less to about 4 μM, or about 300 nM or less to about 3 μM, or about 300 nM or less to about 2 μM, or about 300 nM or less to about 1 μM, or about 300 nM or less to about 700 nM, or about 300 nM or less to about 600 nM, or about 300 nM or less to about 500 nM, or about 300 nM or less to about 400 nM, or about 300 nM or less to about 350 nM, e.g., less than about 300-400 nM, or less than about 300-350 nM.

[0090] In some embodiments, provided sortilin binding agents (e.g., sortilin binding moieties) are characterized, for example, by their stability in a particular environment and / or under certain conditions (e.g., in serum, during storage, etc.). In some embodiments, the disclosure provides compositions or preparations of sortilin binding agents in, and / or exposed to, and / or stored (for a particular period of time) in such environments or under such conditions.

[0091] In some embodiments, provided sortilin binding agents (e.g., sortilin binding moieties) are characterized by stability in a particular environment and / or under certain conditions that is comparable to or better than the stability of Progranulin or a fragment thereof (e.g., a C-terminal fragment of Progranulin, such as a peptide that is or includes about 15-20, or specifically 17, C-terminal residues of Progranulin). In some particular embodiments, such stability is in serum (e.g., mammalian serum, such as in mouse serum or human serum).

[0092] In some embodiments, provided sortilin binding agents (e.g., sortilin binding moieties) are characterized by a half-life in serum (e.g., mammalian serum, such as in mouse serum and / or human serum) of at least about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes or more. Among other things, the present disclosure teaches that cyclic peptides, e.g., as provided herein, can often have longer half-lives.

[0093] In some embodiments, provided sortilin binding agents (e.g., sortilin binding moieties) are characterized by a half-life of less than or equal to a particular period. That is, among other things, the present disclosure recognizes that in some contexts (e.g., as recognized by those of skill in the art upon reading this disclosure), it may be desirable to utilize agents with shorter half-lives, e.g., less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, or even less.

[0094] In some embodiments, it may be desirable to utilize provided sortilin binding agents that have a serum half-life that is reasonably comparable to the half-life of one or more endogenous ligands of sortilin (e.g., progranulin).

[0095] In some embodiments, provided sortilin binding agents comprise a sortilin binding moiety, wherein at least one amino acid is conjugated to a payload moiety. In some embodiments, provided sortilin binding agents comprise a sortilin binding moiety, wherein two or more amino acids are conjugated to a payload moiety. In some embodiments, at least one amino acid within the sequence outside P15, P16, and P17 of the sortilin binding moiety is conjugated to a payload moiety. In some embodiments, two or more amino acids within the sequence outside P15, P16, and P17 of the sortilin binding moiety are conjugated to a payload moiety. In some embodiments, provided sortilin binding agents comprise a linear sortilin binding moiety comprising at least one lysine residue conjugated to a payload moiety. In some embodiments, provided sortilin binding agents comprise a linear sortilin binding moiety, wherein any lysine residue within the sequence is conjugated to a payload moiety. In some embodiments, the sortilin binding moiety is conjugated to the payload moiety through a linker moiety.

[0096] In some embodiments, provided sortilin binding agents comprise a sortilin binding moiety, wherein at least one amino acid is conjugated to a linker and payload moiety at a particular site. In some embodiments, provided sortilin binding moieties can be characterized by a sequence pattern for cyclic group 1 disclosed in the present disclosure, wherein the amino acid residue at the P3 site is conjugated to a linker and payload moiety. In some embodiments, provided sortilin binding moieties can be characterized by a sequence pattern for cyclic group 1 disclosed in the present disclosure, wherein the amino acid residue at the P8 site is conjugated to a linker and payload moiety. In some embodiments, provided sortilin binding moieties can be characterized by a sequence pattern for cyclic group 2 disclosed in the present disclosure, wherein the amino acid residue at the P10 site is conjugated to a linker and payload moiety. In some embodiments, provided sortilin binding moieties can be characterized by a sequence pattern for either cyclic group 1 or cyclic group 2 disclosed in the present disclosure, wherein the amino acid residue at the P4 site is conjugated to a linker and payload moiety. In some embodiments, a provided sortilin binding moiety can be conjugated to a linker and a payload at two or more sites.

[0097] Sortilin binding site Among other things, the present disclosure provides the insight that peptide agents corresponding to fragments of Progranulin, or variants thereof, can be particularly useful as Sortilin binding agents or moieties. In particular, the present disclosure provides the insight that peptide agents corresponding to C-terminal fragments of Progranulin, or variants thereof, are particularly useful.

[0098] In some embodiments, the C-terminal fragment of Progranulin comprises the Progranulin C-terminus. In some embodiments, the C-terminal fragment of Progranulin comprises about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 C-terminal residues of Progranulin. In some embodiments, the C-terminal fragment of Progranulin comprises a C-terminal "QLL" motif.

[0099] In some embodiments, a peptide variant of Progranulin, or a peptide variant of a fragment thereof (e.g., a C-terminal fragment), is a peptide whose amino acid sequence substantially corresponds to the amino acid sequence of Progranulin (or a related fragment thereof, such as a C-terminal fragment), but which contains at least an amino acid substitution, addition, or deletion compared to the amino acid sequence of Progranulin (or a related fragment thereof). In many embodiments, a peptide variant of Progranulin has an amino acid sequence that substantially corresponds to the amino acid sequence of a relevant portion of Progranulin, except for one or more amino acid substitutions. One of skill in the art will understand that a peptide variant of Progranulin, or a peptide variant of a fragment thereof (e.g., a C-terminal fragment), contains sufficient amino acid identity or similarity (e.g., substitution of homologous residues) to be recognizable as related to Progranulin. For example, in many embodiments, a peptide variant of Progranulin has an amino acid sequence corresponding to a contiguous stretch of about 10 or more (e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 consecutive residues of Progranulin) and contains one, two, three, four or more amino acid substitutions compared to such contiguous stretch of Progranulin.

[0100] The present disclosure provides insight that unnatural amino acids can be particularly useful for inclusion in the sortilin-binding peptides described herein.

[0101] The present disclosure further provides insight that linear peptides exhibiting at least about 60%, 65%, 70%, 75% or more sequence identity to fragments of Progranulin (e.g., to its C-terminal fragment), e.g., linear peptides having lengths in the range of about 12 to about 20 amino acids, can be particularly useful in certain embodiments.

[0102] The present disclosure further provides the insight that, in many embodiments, certain cyclic peptides described herein can be particularly useful, which include at least one, and often multiple, unnatural amino acids, and alternatively or in addition, include at least one sequence feature found in fragments of Progranulin (e.g., C-terminal fragments, such as those comprising at least about 12, about 13, about 14, about 15, about 16, or about 17 C-terminal residues).

[0103] The present disclosure provides that, in some embodiments, the peptide sortilin binding moiety contains: (i) a basic residue (e.g., specific such residues, such as L-arginine or an analog thereof, as exemplified in Table 3) at a position corresponding to position P3 and / or P4 of the 17-mer C-terminal fragment of Progranulin; (ii) a hydrophobic residue (e.g., specific such residues, such as L-tryptophan or an analog thereof, as exemplified in Table 4) at a position corresponding to position P4 of the 17-mer C-terminal fragment of Progranulin; (iii) a long-chain hydrophobic residue (e.g., specific such residues, such as L-tryptophan or an analog thereof, as exemplified in Table 5) at a position corresponding to position P10 of the 17-mer C-terminal fragment of Progranulin. , specific such residues such as L-tryptophan or analogs thereof; (iv) a covalent bond (e.g., a disulfide bond) between residues (e.g., between cysteine ​​residues or analogs thereof) between positions corresponding to positions P5 and P13, or P8 and P12, of the 17-mer C-terminal fragment of Progranulin; and / or (v) a hydrophobic residue (e.g., specific such residues such as leucine or a leucine analog, as exemplified in Table 7) at positions corresponding to positions P16 and / or P17 of the 17-mer C-terminal fragment of Progranulin.

[0104] In some embodiments, the provided sortilin binding agents or moieties have a length in the range of about 12 to about 20 amino acids and are: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO:2); wherein X is any standard or non-standard amino acid.

[0105] As described in various embodiments with reference to the signature sequences described herein, each instance of "X" is independently any standard or non-standard amino acid. In some embodiments, each X is independently selected from the standard or non-standard amino acids in Tables 2-7 herein. As also described herein, "Xn" (e.g., X4, X5, X6, etc.) refers to the amino acid "X" at position "n" in the signature sequence. For example, X4 refers to the amino acid that is the fourth residue in the signature sequence, X5 refers to the amino acid that is the fifth residue in the signature sequence, etc. In a given sequence, X m-n A reference to "X" is intended to mean m to n instances of X, where each instance of X is independently selected from any standard or non-standard amino acid. For example, "X" 2-3 " is intended to mean two or three instances of X in a characteristic sequence, where each instance of X is independently selected from any standard or non-standard amino acid. X 0 is intended to indicate the absence of an amino acid. For example, CX 0 R is intended to mean a sequence that is simply C, directly attached to R.

[0106] Additionally, as described in various embodiments herein, two or more amino acids within parentheses and separated by a slash " / " are intended to refer to the amino acid at a given position in the alternative, e.g., (R / N) is intended to mean either the amino acid R or the amino acid N at that position.

[0107] In some embodiments, the provided sortilin binding agents or moieties correspond to a C-terminal fragment of Progranulin, or a variant thereof, and comprise no more than about 20 consecutive residues corresponding to consecutive Progranulin residues.

[0108] In some embodiments, the binding moieties of provided Sortilin are or comprise cyclic peptides, hi some embodiments, the binding moieties of provided Sortilin comprise a characteristic sequence represented by: (R / N) X 2-3 CX 0-1 R (Q / E) (SEQ ID NO: 3).

[0109] In some embodiments, the binding moieties of provided Sortilin are or comprise linear peptides. In some embodiments, the binding moieties of provided Sortilin are or comprise linear peptides. In some embodiments, the binding moieties of provided Sortilin comprise the characteristic sequence represented by SEQ ID NO:2. In some embodiments, the binding moieties of provided Sortilin comprise the characteristic sequence represented by: (R / N) X 2-3 LX 0-1 R (Q / B43 / B50) (SEQ ID NO: 4).

[0110] In some embodiments, the provided binding moieties of Sortilin comprise a characteristic sequence represented by: (R / N) X 2-3 C / LX 0-1 RQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 5).

[0111] In some embodiments, the provided binding moieties of Sortilin comprise a characteristic sequence represented by: (R / N) X 2-3 CX 0-1 RQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 6).

[0112] In some embodiments, the sortilin binding moiety comprises a characteristic sequence according to any one of SEQ ID NOs: 1, 3, 5, or 6, wherein the characteristic sequence comprises at least a second cysteine ​​residue, and wherein the polypeptide comprises at least one disulfide bond between the cysteine ​​residues.

[0113] In some embodiments, the provided binding moieties of Sortilin comprise a characteristic sequence represented by: (R / N) X10 X11 X12 CRQ (SEQ ID NO: 7).

[0114] In some embodiments, the provided binding moieties of Sortilin comprise a characteristic sequence represented by: X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (Sequence number 8) wherein at least one of X, X, and X is a cysteine ​​residue. In some embodiments, provided sortilin-binding polypeptides comprise at least one disulfide bond between cysteine ​​residues.

[0115] In some embodiments, the provided binding moieties of Sortilin comprise a characteristic sequence represented by: (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (Sequence number 9) wherein at least one of X, X, and X is a cysteine ​​residue. In some embodiments, provided sortilin binding polypeptides comprise at least one disulfide bond between cysteine ​​residues.

[0116] In some embodiments, the provided binding moieties of Sortilin comprise a characteristic sequence represented by: (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (L / B13 / F02) (L / B13 / F02) (Sequence number 10) wherein at least one of X4, X5, and X6 is a cysteine ​​residue. In some embodiments, the sortilin binding polypeptide comprises at least one disulfide bond between the cysteine ​​residues.

[0117] In some embodiments, the provided binding moieties of Sortilin comprise a characteristic sequence represented by: CR X10 X11 C X13 RQ (SEQ ID NO: 11) wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0118] In some embodiments, the provided binding moieties of Sortilin comprise a characteristic sequence represented by: (R / H) N X6 X7 CR X10 X11 C X13 RQ (SEQ ID NO: 12) wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0119] In some embodiments, the provided binding moieties of Sortilin comprise a characteristic sequence represented by: (R / H) N X6 X7 CR X10 X11 C X13 RQ (L / B13 / F02) L / B13 / F02) (SEQ ID NO: 13) wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0120] In some embodiments, the sortilin binding moiety is X nIt has the sequence RDPALRXLL (SEQ ID NO: 14), where X is any standard or non-standard amino acid according to the present disclosure, and n is 0, 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, for example, a peptide compound according to the present disclosure has the sequence RDPALRQLL. In some embodiments, for example, a peptide compound according to the present disclosure has the sequence RDPALR(B43)LL. In some embodiments, RDPALR(B43)LL exhibits reduced affinity compared to APRWDAPLRDPALR(B43)LL.

[0121] In some embodiments, provided sortilin binding moieties comprise one or more of the following: (i) a basic residue, such as L-arginine or an analog thereof, at a position corresponding to position P3 and / or P4 of the 17-mer C-terminal fragment of Progranulin; (ii) a hydrophobic residue, such as L-tryptophan or an analogue thereof, at a position corresponding to position P4 of the 17-mer C-terminal fragment of Progranulin; (iii) a long-chain hydrophobic residue at a position corresponding to position P10 of the 17-mer C-terminal fragment of Progranulin; (iv) a covalent bond, such as a disulfide bond, between residues at positions corresponding to positions P5 and P13, or P8 and P12, of the 17-mer C-terminal fragment of Progranulin; (v) a hydrophobic residue, such as leucine or a leucine analog, at a position corresponding to position P16 and / or P17 of the 17-mer C-terminal fragment of Progranulin.

[0122] In some embodiments, X represents an amino acid included in one of Tables 2-7.

[0123] In some embodiments, X represents a standard amino acid. In some embodiments, X is a non-standard amino acid. In some embodiments, the non-standard amino acid is selected from those in one or more of Tables 2-7.

[0124] In some embodiments, provided sortilin binding agents or moieties have a length in the range of about 12 to about 20 amino acids and are selected from the group consisting of: (R / N) X 2-3 (C / L) X 0-1 RZ (SEQ ID NO: 15) wherein each X is independently any standard or non-standard amino acid. In some embodiments, X represents a standard amino acid. In some embodiments, X is a non-standard amino acid. In some embodiments, the non-standard amino acid is selected from one or more of Tables 2-7. In some embodiments, Z represents a standard amino acid. In some embodiments, Z represents a polar amino acid. In some embodiments, Z represents an amino acid comprising a side chain having an acidic group. In some embodiments, the side chain of Z comprises -COOH. In some embodiments, Z is Q, E, or an analog thereof. In some such embodiments, an analog of Q can be, for example, but is not limited to, theanine, 2,4-diaminopentanedioic acid, D-glutamine, Nα-acetyl-L-glutamine, 5-methylglutamine, 2-methyl-L-glutamine, etc. In some embodiments, Z is L or an analog thereof. In some such embodiments, an analog of L is selected from Table 7. In some embodiments, Z represents a non-standard amino acid selected from those in one or more of Tables 2 through 7. In some embodiments, Z can be, for example (but is not limited to), D-leucine, L-3-cyclopropyl-alanine, L-4-methyl-leucine, L-homoleucine, 3-cyclobutyl-alanine, L-norleucine, L-3-methyl-valine, (2S,4R)-2-amino-4-hydroxypentanoic acid, (2R,4S)-2-amino-4-hydroxypentanoic acid, (4S)-4-hydroxy-L-norvaline, (4R)-4-hydroxy-L-norvaline, 5-methyl-D-norleucine, L-homoserine, or β-alanine.

[0125] In some embodiments, provided Sortilin binding agents or moieties are or comprise peptides having the amino acid sequence APRWDAPLRDPALRQLL. In some embodiments, provided Sortilin binding agents or moieties are or comprise peptides having the amino acid sequence APRWDAPLRDPALRQ(B13)(G48).

[0126] In some embodiments, provided sortilin binding agents or moieties are characterized by: (i) exhibit an affinity (Kd) for human sortilin 1 of less than about 11 μM, as examined by fluorescence polarization; (ii) an IC of less than about 12 μM in a competitive binding assay with a reference C-terminal Progranulin fragment 50 Showing; (iii) exhibits greater stability than that of a reference C-terminal Progranulin fragment when maintained in mouse serum;

[0127] In some embodiments, provided sortilin binding agents or moieties are characterized by: (i) exhibit an affinity (Kd) for human sortilin 1 of less than about 11 μM, as examined by fluorescence polarization; (ii) an IC of less than about 12 μM in a competitive binding assay with a reference C-terminal Progranulin fragment 50 Showing; (iii) exhibits lower stability than that of a reference C-terminal Progranulin fragment when maintained in mouse serum;

[0128] In some embodiments, the reference C-terminal Progranulin fragment has an amino acid sequence that is or includes: APRWDAPLRDPALRQLL. In some embodiments, a provided Sortilin binding agent or moiety has an amino acid sequence that is or includes: DDPRAPWPALQRLALRL.

[0129] In some embodiments, provided sortilin binding agents or moieties are characterized by a stability half-life in mouse serum of greater than about 3 minutes. In some embodiments, the stability half-life in mouse serum is about 5 minutes. In some embodiments, the stability half-life in mouse serum is about 10 minutes. In some embodiments, the stability half-life in mouse serum is about 14 minutes.

[0130] In some embodiments, provided sortilin binding agents or moieties are characterized by an IC50 in a competitive binding assay that is less than about 5000 nM. In some embodiments, the IC50 is less than about 4000 nM. In some embodiments, the IC50 is less than about 3000 nM.

[0131] In some embodiments, the IC50 is less than 2000 nM. In some embodiments, the IC50 is less than about 1000 nM. In some embodiments, the IC50 is less than about 750 nM. In some embodiments, the IC50 is about 600-650 nM. In some embodiments, the IC50 is less than about 600 nM. In some embodiments, the IC50 is less than about 500 nM. In some embodiments, the IC50 is less than about 400 nM. In some embodiments, the IC50 is less than about 300-400 nM. In some embodiments, the IC50 is less than about 300-350 nM.

[0132] Linker As noted above, the present disclosure provides conjugates comprising (a) a polypeptide, and (b) a payload, and (c) optionally a linker, wherein the polypeptide comprises a sortilin binding moiety.

[0133] In some embodiments, the conjugation agent has the structure of Formula I shown in FIG. 2, where each of the homing peptide, linker, and payload is as described above and herein.

[0134] Those skilled in the art will recognize the various types of linkers and their chemical compositions available for associating one or more payloads with one or more sortilin binding moieties described herein.

[0135] Linkers are generally designated as "cleavable" or "non-cleavable." Cleavable linkers are typically used when it is desirable for the conjugated payload and binding moiety to be released (e.g., when the conjugate is exposed to a particular environment or condition, such as a particular pH, or to a particular enzyme, such as a particular protease).

[0136] In some embodiments, the cleavable linker is cleaved chemically, e.g., by hydrolysis, pH change, reduction, or oxidation. In some embodiments, the cleavable linker is cleaved enzymatically, e.g., by the action of a protease, esterase, glucosidase, glucuronidase, galactosidase, phosphatase, phosphodiesterase, nuclease, lipase, or any enzyme capable of cleaving the relevant linker.

[0137] In some embodiments, the cleavable linker is or comprises a disulfide bond, an ester, a phosphodiester, a sugar, or a lipid.

[0138] In some embodiments, a non-cleavable linker is chemically, enzymatically, or otherwise biochemically and physiologically stable, and thus typically lacks a chemically, biochemically, enzymatically cleavable, or otherwise physiologically unstable linkage.

[0139] In some embodiments, the linker is multivalent (e.g., so that multiple payload moieties can be or are capable of being attached to the sortilin binding moiety, or multiple sortilin binding moieties to the payload moiety, or both). In some such embodiments, the linker is bivalent, trivalent, etc.

[0140] In some embodiments, the linker is a divalent straight or branched C 1-40 is or comprises an aliphatic chain, wherein one or more methylene units of the aliphatic chain are -CH(R)-, -C(R)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N( and substituted by a group selected from a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; During the ceremony, R is hydrogen or optionally substituted C 1-6 It is selected from an aliphatic, 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0141] In some embodiments, the linker is a divalent straight or branched C 1-35is or comprises an aliphatic chain, wherein one or more methylene units of the aliphatic chain are -CH(R)-, -C(R)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R) substituted by a group selected from -, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; During the ceremony, R is hydrogen or optionally substituted C 1-6 It is selected from an aliphatic, 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0142] In some embodiments, the linker is a divalent straight or branched C 1-30is or comprises an aliphatic chain, wherein one or more methylene units of the aliphatic chain are -CH(R)-, -C(R)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R) substituted by a group selected from -, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; During the ceremony, R is hydrogen or optionally substituted C 1-6 It is selected from an aliphatic, 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0143] In some embodiments, the linker is a divalent straight or branched C 1-25is or comprises an aliphatic chain, wherein one or more methylene units of the aliphatic chain are -CH(R)-, -C(R)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R) substituted by a group selected from -, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; During the ceremony, R is hydrogen or optionally substituted C 1-6 It is selected from an aliphatic, 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0144] In some embodiments, the linker is a divalent straight or branched C 1-20is or comprises an aliphatic chain, wherein one or more methylene units of the aliphatic chain are -CH(R)-, -C(R)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R) substituted by a group selected from -, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; During the ceremony, R is hydrogen or optionally substituted C 1-6 It is selected from an aliphatic, 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0145] In some embodiments, the linker is a divalent straight or branched C 1-15is or comprises an aliphatic chain, wherein one or more methylene units of the aliphatic chain are -CH(R)-, -C(R)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R) substituted by a group selected from -, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; During the ceremony, R is hydrogen or optionally substituted C 1-6 It is selected from an aliphatic, 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0146] In some embodiments, the linker is a divalent straight or branched C 1-10is or comprises an aliphatic chain, wherein one or more methylene units of the aliphatic chain are -CH(R)-, -C(R)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R) substituted by a group selected from -, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; During the ceremony, R is hydrogen or optionally substituted C 1-6 It is selected from an aliphatic, 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0147] In some embodiments, the linker is a divalent straight or branched C 1-5is or comprises an aliphatic chain, wherein one or more methylene units of the aliphatic chain are -CH(R)-, -C(R)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R) substituted by a group selected from -, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; During the ceremony, R is hydrogen or optionally substituted C 1-6 It is selected from an aliphatic, 3- to 6-membered saturated or partially unsaturated carbocyclic ring, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0148] In some embodiments, the linker is or includes a structure selected from: [ka] wherein X is NH or O.

[0149] In some embodiments, the cleavable linker is a cathepsin-cleavable linker. In some such embodiments, the linker is or includes a valine-citrulline (Val-Cit) motif, [ka] where R is hydrogen or C 1-6 It is aliphatic.

[0150] In some embodiments, the valine-citrulline linker is or comprises: [ka]

[0151] In some embodiments, the valine-citrulline linker is or comprises: [ka] where R is hydrogen or C 1-6 It is aliphatic.

[0152] In some embodiments, the valine-citrulline linker is or comprises: [ka]

[0153] In some embodiments, the valine-citrulline linker is or comprises: [ka]

[0154] In some embodiments, the linker comprises a disulfide bond. In some embodiments, the linker comprises a poly(ethylene glycol) moiety (e.g., -(CH2CH2O) b -), wherein b is 1 to 50.

[0155] In some embodiments, the linker is or includes a group selected from: [ka] [ka] [ka] wherein each of k, m, n, p, q, r, s, t, u, v, w, x, y, and z is 1 to 20; and R is hydrogen or C 1-10 It is aliphatic.

[0156] In some embodiments, k is 3.

[0157] In some embodiments, m is 3.

[0158] In some embodiments, n is 2. In some embodiments, n is 12.

[0159] In some embodiments, p is 3.

[0160] In some embodiments, each of m and p is 3.

[0161] In some embodiments, q is 1.

[0162] In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 6.

[0163] In some embodiments, s is 3. In some embodiments, s is 4. In some embodiments, s is 6.

[0164] In some embodiments, each of r and s is 3. In some embodiments, each of r and s is 4. In some embodiments, each of r and s is 6.

[0165] In some embodiments, t is 3. In some embodiments, t is 5.

[0166] In some embodiments, u is 3. In some embodiments, u is 5.

[0167] In some embodiments, each of t and u is 3. In some embodiments, each of t and u is 5.

[0168] In some embodiments, v is 3.

[0169] In some embodiments, w is 4.

[0170] In some embodiments, x is 8.

[0171] In some embodiments, y is 2.

[0172] In some embodiments, z is 1.

[0173] payload Those of skill in the art reading this disclosure will understand that any of a variety of payloads can be conveniently conjugated to the sortilin binding moieties described herein. Those of skill in the art reading this disclosure will further understand that the present teachings are not limited to any particular specific payload.

[0174] In many embodiments, useful payload moieties are capable of achieving (e.g., correlating with) a particular biological or physiological effect on sortilin or a sortilin-expressing system, such as a sortilin-expressing cell or tissue. In some embodiments, useful payloads are capable of conferring detectability to the complex (and / or sortilin or a sortilin-expressing system, such as a sortilin-expressing cell or tissue). Those of skill in the art are familiar with categories of therapeutic agents based on their biological effect (see, e.g., www.fda.gov / regulatory-information / fdaaa-implementation-chart / usp-therapeutic-categories-model-guidelines, incorporated herein by reference).

[0175] In many embodiments, the effect of the payload moiety is a change in one or more parameters of one or more targets of interest (e.g., expression parameters and / or activity of the targets of interest). In some embodiments, the targets of interest can be specific genes or gene products, or forms thereof (e.g., disease-associated forms, splice variant forms, etc.).

[0176] In some embodiments, the payload can be or include a cytotoxic or cytostatic agent, particularly when provided sortilin binding agents are utilized to treat cancer. In some embodiments, the cytotoxic or cytostatic agent can be or include a toxin (e.g., a bacterial toxin such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A, cholera toxin, etc.). In some embodiments, the cytotoxic or cytostatic agent can be or include a maytansinoid, a calicheamicin, an amitoxin, an amanitin, or a combination thereof. In some embodiments, the cytotoxic or cytostatic agent can be or include an alkaloid, an alkylating agent, an antimetabolite (e.g., hydroxyurea), an anthracycline, a cytotoxic antibiotic (e.g., actinomycin D, doxorubicin, daunorubicin, epirubicin, bleomycin, mitomycin C), an antimetabolite, an auristatin, a camptothecin, an enzyme (e.g., L-asparaginase), a folic acid derivative, a metal complex, a microtubule damaging agent (e.g., vincristine, vinblastine, vinorelbine, cabazitaxel, paclitaxel, and docetaxel), a nucleoside analog, a taxane, a topoisomerase-2 inhibitor (e.g., etoposide), a topoisomerase-1 inhibitor (e.g., topotecan, irinotecan), a vinca alkaloid analog, or a combination thereof.

[0177] In some embodiments, the payload can be or include an anti-cancer drug. In some embodiments, the payload is selected from the group consisting of alkylating agents, antimetabolites, antitumor antibiotics, boron neutron capture therapeutics, cell cycle inhibitors, kinesin spindle protein inhibitors, microtubule binding agents, topoisomerase inhibitors, and combinations thereof. In some embodiments, the payload can be or include an anti-tumor peptide, including D-peptides A, B, C, D, gomesin, hepcidin, and PTP7. In some embodiments, the payload can be or include a targeting agent. In some embodiments, the targeting agent can be or include an angiogenesis inhibitor (e.g., bevacizunab, thalidomide, endostatin, angiostatin, angiopoietin, cannabinoid), EGF receptor inhibitor (e.g., gefitinib and erlotinib), monoclonal antibody (e.g., rituximab and trastuzumab), proteasome inhibitor (e.g., bortezomib and tilidomide), tyrosine protein kinase inhibitor (e.g., imatinib and dasatinib), or combinations thereof.

[0178] In some embodiments, the payload can be or include an immunotherapeutic agent, hi some embodiments, the payload is selected from the group consisting of a checkpoint inhibitor, a CAR-T cell therapy, an antibody, an oncolytic virus, or a combination thereof.

[0179] In some embodiments, the payload can be or can include an anti-inflammatory agent (e.g., a phytochemical, a nonsteroidal anti-inflammatory drug (NSAID), a steroidal anti-inflammatory drug, an anti-leukotriene agent, a biologic agent, or an immunoselective anti-inflammatory derivative (ImSAID)).

[0180] In some embodiments, the payload can be or include a phytochemical. In some embodiments, the payload can be an alkaloid (e.g., chlorogenic acid, theobromine, theophylline), anthocyanins / anthocyanidins (e.g., cyanidin, malvidin), carotenoids (e.g., beta-carotene, lutein, lycopene), capsaicin, catechin, coumestans, flavan-3-ols, flavonoids (e.g., epicatechin, hesperidin, isorhamnetin, campherol, myricetin, naringin, nobiletin, proanthocyanidins, quercetin, rutin, tangeretin), hydroxycinnamic acids (e.g., thiamin ... The hydroxybenzoates are selected from the group consisting of hydroxybenzoates, hydroxybenzoates (e.g., hydroxybenzoates, hydroxybenzoates), ...

[0181] In some embodiments, the payload moiety is or comprises a nucleic acid. In some embodiments, the payload moiety is or comprises a single-stranded nucleic acid. In some embodiments, the payload moiety is or comprises a double-stranded nucleic acid. In some embodiments, the payload moiety is or comprises an oligonucleotide.

[0182] In some embodiments, the nucleic acid is about 10 to 50 nucleotides, about 10 to 49 nucleotides, about 10 to 48 nucleotides, about 10 to 47 nucleotides, about 10 to 46 nucleotides, about 10 to 45 nucleotides, about 10 to 44 nucleotides, about 10 to 43 nucleotides, about 10 to 42 nucleotides, about 10 to 41 nucleotides, about 10 to 40 nucleotides, about 10 to 39 nucleotides, about 10 to 38 nucleotides, about 10 to 37 nucleotides, about 10 to 36 nucleotides, about 10 to 35 nucleotides, about 10 to 34 nucleotides, about 10 to 33 nucleotides, about 10 to 32 nucleotides, about 10 to 31 nucleotides. The length ranges from about 10 to 30 nucleotides, about 10 to 29 nucleotides, about 10 to 28 nucleotides, about 10 to 27 nucleotides, about 10 to 26 nucleotides, about 10 to 25 nucleotides, about 10 to 24 nucleotides, about 10 to 23 nucleotides, about 10 to 22 nucleotides, about 10 to 21 nucleotides, about 10 to 20 nucleotides, about 10 to 19 nucleotides, about 10 to 18 nucleotides, about 10 to 17 nucleotides, about 10 to 16 nucleotides, about 10 to 15 nucleotides, about 10 to 14 nucleotides, about 10 to 13 nucleotides, about 10 to 12 nucleotides, and about 10 to 11 nucleotides.In some embodiments, the nucleic acid is from about 11 to 50 nucleotides, from about 12 to 50 nucleotides, from about 13 to 50 nucleotides, from about 14 to 50 nucleotides, from about 15 to 50 nucleotides, from about 16 to 50 nucleotides, from about 17 to 50 nucleotides, from about 18 to 50 nucleotides, from about 19 to 50 nucleotides, from about 20 to 50 nucleotides, from about 21 to 50 nucleotides, from about 22 to 50 nucleotides, from about 23 to 50 nucleotides, from about 24 to 50 nucleotides, from about 25 to 50 nucleotides, from about 26 to 50 nucleotides, from about 27 to 50 nucleotides, from about 28 to 50 nucleotides, from about 29 to 50 nucleotides, from about 30 The length ranges from about 50 nucleotides, about 31 to 50 nucleotides, about 32 to 50 nucleotides, about 33 to 50 nucleotides, about 34 to 50 nucleotides, about 35 to 50 nucleotides, about 36 to 50 nucleotides, about 37 to 50 nucleotides, about 38 to 50 nucleotides, about 39 to 50 nucleotides, about 40 to 50 nucleotides, about 41 to 50 nucleotides, about 42 to 50 nucleotides, about 43 to 50 nucleotides, about 44 to 50 nucleotides, about 45 to 50 nucleotides, about 46 to 50 nucleotides, about 47 to 50 nucleotides, about 48 to 50 nucleotides, and about 49 to 50 nucleotides.

[0183] In some embodiments, the nucleic acid is about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides, about 45 nucleotides, about 46 nucleotides, about 47 nucleotides, about 48 nucleotides, about 49 nucleotides, or about 50 nucleotides in length.

[0184] In some embodiments, particularly when a nucleic acid encoding a polypeptide is used as the payload, the nucleic acid may be about 10 to 20,000 nucleotides, about 10 to 19,000 nucleotides, about 10 to 18,000 nucleotides, about 10 to 17,000 nucleotides, about 10 to 16,000 nucleotides, about 10 to 15,000 nucleotides, about 10 to 14,000 nucleotides, about 10 to 13,000 nucleotides, about 10 to 12,000 nucleotides, about 10 to 11,000 nucleotides, about 10 to 10 ,000 nucleotides, about 10 to 9,500 nucleotides, about 10 to 9,000 nucleotides, about 10 to 8,500 nucleotides, about 10 to 8,000 nucleotides, about 10 to 7,500 nucleotides, about 10 to 7,000 nucleotides, about 10 to 6,500 nucleotides, about 10 to 6,000 nucleotides, about 10 to 5,500 nucleotides, about 10 to 5,000 nucleotides, about 10 to 4,500 nucleotides, about 10 to 4,000 nucleotides, about 10 to 3,500 nucleotides, about 10 to 3,0 00 nucleotides, about 10 to 2,500 nucleotides, about 10 to 2,000 nucleotides, about 10 to 1,500 nucleotides, about 10 to 1,000 nucleotides, about 10 to 950 nucleotides, about 10 to 900 nucleotides, about 10 to 850 nucleotides, about 10 to 800 nucleotides, about 10 to 750 nucleotides, about 10 to 700 nucleotides, about 10 to 650 nucleotides, about 10 to 600 nucleotides, about 10 to 550 nucleotides, about 10 to 500 nucleotides, about 10 to 450 nucleotides , about 10 to 400 nucleotides, about 10 to 350 nucleotides, about 10 to 300 nucleotides, about 10 to 250 nucleotides, about 10 to 200 nucleotides, about 10 to 150 nucleotides, about 10 to 100 nucleotides, about 10 to 95 nucleotides, about 10 to 90 nucleotides, about 10 to 85 nucleotides, about 10 to 80 nucleotides, about 10 to 75 nucleotides, about 10 to 70 nucleotides, about 10 to 65 nucleotides, about 10 to 60 nucleotides, or about 10 to 50 nucleotides in length.In some embodiments, the nucleic acid is from about 100 to 20,000 nucleotides, from about 200 to 20,000 nucleotides, from about 300 to 20,000 nucleotides, from about 400 to 20,000 nucleotides, from about 500 to 20,000 nucleotides, from about 600 to 20,000 nucleotides, from about 700 to 20,000 nucleotides, from about 800 to 20,000 nucleotides, from about 900 to 20,000 nucleotides, from about 1,000 to 20,000 nucleotides, from about 1,100 to 20,000 nucleotides, from about 1,200 to 20,000 nucleotides, from about 1,300 to 20,000 nucleotides, from about 1,400 to 20,000 nucleotides, from about 1,500 to 20,000 nucleotides, from about 1,600 to 20,000 nucleotides, or from about 1,700 to 20,000 nucleotides. 000 nucleotides, about 1,800 to 20,000 nucleotides, about 1,900 to 20,000 nucleotides, about 2,000 to 20,000 nucleotides, about 3,000 to 20,000 nucleotides, about 4,000 to 20,000 nucleotides, about 5,000 to 20,000 nucleotides, about 6,000 to 20,000 nucleotides, about 7,000 to 20,000 nucleotides The nucleotides have lengths ranging from about 8,000 to 20,000 nucleotides, about 9,000 to 20,000 nucleotides, about 10,000 to 20,000 nucleotides, about 11,000 to 20,000 nucleotides, about 12,000 to 20,000 nucleotides, about 13,000 to 20,000 nucleotides, about 14,000 to 20,000 nucleotides, about 15,000 to 20,000 nucleotides, about 16,000 to 20,000 nucleotides, about 17,000 to 20,000 nucleotides, about 18,000 to 20,000 nucleotides, and about 19,000 to 20,000 nucleotides.

[0185] In some embodiments, a nucleic acid agent, e.g., an oligonucleotide agent or a polypeptide-encoding agent, for use in accordance with the present disclosure can comprise a single strand. In some embodiments, a nucleic acid can comprise two or more strands. In some embodiments, a nucleic acid can comprise one or more double-stranded portions. In some such embodiments, some or all of such portion(s) can be formed by self-hybridization of a single-stranded sequence, and in some embodiments, some or all of such portion(s) can be formed by hybridization of separate strands. In some embodiments, a nucleic acid comprising one or more double-stranded portions can comprise one or more nicks or gaps and / or one or more bulges or loops.

[0186] In some embodiments, nucleic acid agents, e.g., oligonucleotide agents, for use in accordance with the present disclosure can include one or more structural features or characteristics related to their mechanism of action. For example, those skilled in the art will recognize an extensive literature on structural features such as, for example, oligonucleotides that trigger target degradation (e.g., by recruiting RNase H) (such oligonucleotides are often referred to as "antisense" agents or "ASOs"), and / or oligonucleotides that trigger degradation of Dicer and / or other elements of the RNA-induced silencing complex (RISC) (such oligonucleotides are often referred to as "siRNA" agents), and / or oligonucleotides that modulate splicing of target transcripts (e.g., to generate one splice form in preference to others), and / or oligonucleotides that function as guide RNAs to recruit other mechanisms (e.g., nucleases such as CRISPR / Cas or dsRNA-binding proteins, or conjugates thereof) to specific nucleic acid sequences or as aptamers that bind to specific targets.

[0187] In some embodiments, the nucleic acid agent is directed to (e.g., hybridizes with) a target nucleic acid (e.g., DNA or, more commonly, RNA, such as mRNA, that may be present in, e.g., expressed in, a cell to which the administered agent is delivered). In some embodiments, delivery of the nucleic acid agent inhibits (e.g., reduces the level and / or activity of) the target nucleic acid. In some embodiments, delivery of the nucleic acid alters (e.g., cleaves, edits, alters splicing of, the target nucleic acid, etc.) the target nucleic acid. In some embodiments, delivery of the nucleic acid agent enhances or protects (e.g., reduces degradation of, activates expression of, the target nucleic acid) the target nucleic acid.

[0188] In some embodiments, the target nucleic acid is a target nucleic acid whose activity or level (e.g., of a particular form or variant thereof) can be associated with a particular disease, disorder, or condition. In some such embodiments, delivery of the nucleic acid agent modulates such activity or level toward a non-disease state (e.g., activity or level). In some embodiments, the target nucleic acid is a target nucleic acid whose activity or level (e.g., of a particular form or variant thereof) is associated with cell viability or lack thereof. In some such embodiments, delivery of the nucleic acid agent reduces cell viability (e.g., induces cell death or apoptosis, etc.).

[0189] In some embodiments, a target is a particular allele at which alteration in expression and / or activity of one or more products (e.g., RNA and / or protein products) is intended. In many embodiments, the target allele is a target allele whose presence and / or expression is associated with (e.g., correlated with) the presence, occurrence, and / or severity of one or more diseases and / or conditions. Alternatively, or in addition, in some embodiments, the target allele is a target allele at which altered levels and / or activity of one or more gene products correlate with improvement (e.g., delayed onset, reduced severity, responsiveness to other therapies, etc.) in one or more aspects of a disease and / or condition.

[0190] In some embodiments, in which the presence and / or activity of a particular allele (disease-associated allele) is associated (e.g., correlated) with the presence, occurrence, and / or severity of one or more disorders, diseases, and / or conditions, different alleles of the same gene are present and are unassociated or less associated (e.g., exhibit a less pronounced or less statistically significant correlation). In some such embodiments, the oligonucleotides and methods described herein can preferentially or specifically target the associated allele relative to one or more less associated / unassociated allele(s), thereby mediating allele-specific suppression.

[0191] In some embodiments, the target sequence is a sequence to which an oligonucleotide agent described herein binds. In many embodiments, the target sequence is identical to or is the perfect complement of the sequence of a provided oligonucleotide, or of consecutive residues therein (e.g., a provided oligonucleotide comprises a target binding sequence that is identical to or is the perfect complement of the target sequence). In some embodiments, the target binding sequence is the perfect complement of the target sequence of a transcript (e.g., pre-mRNA, mRNA, etc.). The target binding sequence / target sequence can have various lengths to provide an oligonucleotide with desired activity and / or properties. In some embodiments, the target binding sequence / target sequence comprises 5-50 (e.g., 10-40, 15-30, 15-25, 16-25, 17-25, 18-25, 19-25, 20-25, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) bases. In some embodiments, a small number of differences / mismatches are tolerated between the oligonucleotide and (relevant portions of) its target sequence, including, but not limited to, the 5' and / or 3' terminal regions of the target and / or oligonucleotide sequence. In many embodiments, the target sequence is present within a target gene. In some embodiments, the target sequence is present within a transcript (e.g., mRNA and / or pre-mRNA) produced from the target gene. In some embodiments, the target sequence comprises one or more allelic sites (i.e., locations within the target gene where an allelic variation occurs). In some embodiments, the allelic sites are mutations. In some embodiments, the allele site is a SNP, where SNP refers to a single nucleotide polymorphism (a single nucleotide is substituted compared to the original sequence). In some such embodiments, the oligonucleotide agent preferentially or specifically binds to one allele compared to one or more other alleles. In some embodiments, the oligonucleotide agent preferentially binds to a disease-associated allele.For example, in some embodiments, an oligonucleotide agent (or its target binding sequence portion) has a sequence identical to a particular allelic version of a target sequence, or a perfect complement of a particular allelic version of a target sequence. In some embodiments, the target sequence is the sequence of a particular allele. In some embodiments, an oligonucleotide agent (or its target binding sequence portion) has a sequence identical to a disease-associated allele, or a perfect complement of a target sequence that includes an allelic site of a disease-associated allele, or an allelic site of a disease-associated allele. In some embodiments, an oligonucleotide agent has a target binding sequence that is a perfect complement of a target sequence that includes an allelic site of a transcript of an allele (in many embodiments, a disease-associated allele), where the allelic site is a mutation. In some embodiments, an oligonucleotide agent has a target binding sequence that is a perfect complement of a target sequence that includes an allelic site of a transcript of an allele (in many embodiments, a disease-associated allele), where the allelic site is a SNP.

[0192] In some embodiments, the nucleic acid is or comprises an interfering RNA (RNAi) agent. In some embodiments, the RNA is or comprises a single-stranded interfering RNA (ssRNAi) agent. In some embodiments, the RNA is or comprises a double-stranded interfering RNA (dsRNAi) agent. In some embodiments, the RNA is or comprises a short interfering RNA (siRNA) agent. In some embodiments, the RNA is or comprises a microRNA (miRNA) agent. In some embodiments, the nucleic acid is or comprises a guide RNA (gRNA) agent.

[0193] In some embodiments, the nucleic acid is or comprises a short interfering RNA (siRNA) agent. In some embodiments, the nucleic acid comprising the siRNA agent can be bound (e.g., directly or indirectly) to the sortilin binding moiety on the sense strand. In some embodiments, the nucleic acid comprising the siRNA agent can be bound (e.g., directly or indirectly) to the sortilin binding moiety on the antisense strand. In some embodiments, the nucleic acid comprising the siRNA agent can be bound (e.g., directly or indirectly) to the sortilin binding moiety at the 5' end of the siRNA agent. In some embodiments, the nucleic acid comprising the siRNA agent can be bound (e.g., directly or indirectly) to the sortilin binding moiety at the 3' end of the siRNA agent.

[0194] In some embodiments, the nucleic acid is or comprises an exon skipping agent, exon inclusion agent, or other splicing modulator.

[0195] In some embodiments, the nucleic acid is or comprises an aptamer agent.

[0196] In some embodiments, the nucleic acid agent is or comprises an antisense oligo (ASO). In some embodiments, the ASO regulates gene expression by an RNase H-mediated mechanism. In some embodiments, the ASO regulates gene expression by steric hindrance.

[0197] In some embodiments, the nucleic acid agent is or comprises a phosphoramidate morpholino oligonucleotide (PMO).

[0198] In some embodiments, the nucleic acid agent is or comprises a peptide-nucleic acid (PNA).

[0199] In some embodiments, the nucleic acid agent is or includes a nucleic acid analog, such as an RNA analog or a DNA analog, or a combination thereof.

[0200] In some embodiments, the nucleic acid agent is or includes the sense or antisense strand (or both) of a sequence encoding a polypeptide. In some embodiments, delivery of such a nucleic acid results in expression of such encoded polypeptide. In some embodiments, the encoded polypeptide has an activity that improves the condition of a cell (e.g., tends to move the condition away from a condition characterized by a disease, disorder, or pathology). In some embodiments, the encoded polypeptide corrects a defect in the cell (e.g., replaces a missing activity). In some embodiments, the encoded polypeptide is toxic to the cell or otherwise reduces cell viability and / or proliferation. In some embodiments, the encoded polypeptide is or includes an antigen-binding sequence of an immunoglobulin (e.g., of an antibody agent such as an scFv, camelid antibody, antibody heavy or light chain, etc.). In some embodiments, the encoded polypeptide suppresses an undesired biological event or response (e.g., associated with disease and / or an undesired immune response, etc.). In some embodiments, the encoded polypeptide supports or activates a desirable biological event or response (e.g., an immune response).

[0201] In some embodiments, the nucleic acid can be bound (e.g., directly or indirectly) to the sortilin binding moiety on the sense strand. In some embodiments, the nucleic acid can be bound (e.g., directly or indirectly) to the sortilin binding moiety on the antisense strand. In some embodiments, the nucleic acid can be bound (e.g., directly or indirectly) to the sortilin binding moiety at the 5' end of the nucleic acid. In some embodiments, the nucleic acid can be bound (e.g., directly or indirectly) to the sortilin binding moiety at the 3' end of the nucleic acid.

[0202] For example, in some embodiments, nucleic acid analogs comprise one or more modified nucleotides (compared to standard DNA and / or RNA). In some embodiments, the modified nucleotides comprise one or more of a modified backbone, a modified nucleobase, a modified sugar (e.g., a modified ribose or a modified deoxyribose), or a combination thereof. In some embodiments, the modified nucleotides can be or include one or more naturally occurring modifications. In some embodiments, the modified nucleotides can be or include one or more non-naturally occurring modifications.

[0203] In some embodiments, nucleic acid analogs include one or more linkages that are not phosphodiester linkages (eg, that are or include phosphorothioate or phosphorodiamidate linkages).

[0204] In some embodiments, the nucleic acid agent has a negative charge.

[0205] In some embodiments, the nucleic acid agent is substantially uncharged, eg, has a neutral charge.

[0206] Those of skill in the art reading this disclosure will understand that in some embodiments, nucleic acid agents for use in accordance with the present disclosure can include one or more DNA residues or analogs thereof, one or more RNA residues or analogs thereof, and / or combinations thereof. Furthermore, those of skill in the art will understand that in some embodiments, nucleic acid agents can include one or more, or entirely, phosphodiester, phosphorothioate, or other suitable linkages.

[0207] In some embodiments, nucleic acid agents include naturally occurring residues, eg, DNA residues and / or RNA residues.

[0208] In some embodiments, a nucleic acid agent comprises one or more analogs, eg, a DNA analog and / or an RNA analog.

[0209] In some embodiments, nucleic acid agents include DNA and / or RNA residues, eg, naturally occurring residues or analogs.

[0210] In some embodiments, nucleic acids contain one or more chiral centers (e.g., as may be present in a phosphorothioate linkage). In some embodiments, a preparation of nucleic acids having a chiral center is stereopure with respect to that center, in that the nucleic acid contains only one stereoisomer of that center. In some embodiments, both stereoisomers are present. In some embodiments, the preparation represents a racemic mixture of stereoisomers at that position. In some embodiments, a preparation of nucleic acids having two or more chiral linkages is stereopure with respect to one or more centers and mixed with respect to one or more other centers (e.g., racemic). In some embodiments, the preparation can be stereopure at all chiral centers. In some embodiments, the preparation can be racemic (e.g., at all or entirely at all chiral centers).

[0211] In some embodiments, the nucleic acid comprises one or more modified nucleotides, in some embodiments, the modified nucleotides comprise one or more of a modified backbone, a modified nucleobase, a modified ribose, a modified deoxyribose, or a combination thereof.

[0212] In some embodiments, the modified nucleotide is selected from 2-O'-methyl modified nucleotides, 5-methylcytidine, 5-methyluridine, nucleotides containing a 5'-phosphorothioate group, morpholino nucleotides (e.g., PMO), terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides (e.g., PMO), phosphoramidates, phosphorylguanidine (PN)-based backbones, or non-natural base containing nucleotides, or combinations thereof.

[0213] In some embodiments, the modified nucleobase comprises a C7-modified deaza-adenine, a C7-modified deaza-guanosine, a C5-modified cytosine, a C5-modified uridine, N1-methyl-pseudouridine (m1Ψ), 1-ethyl-pseudouridine (e1Ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (Ψ), 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, 5-methoxycytidine, or a combination thereof.

[0214] In some embodiments, the modified sugar (e.g., a modified ribose or a modified deoxyribose) comprises a 2' fluoro modification, a 2'-O-methyl (2'OMe) modification, a locked nucleic acid (LNA), a 2'-fluoroarabinose nucleic acid (FANA), a hexitol nucleic acid (HNA), a 2'O-methoxyethyl (2'MOE) modification, or a combination thereof.

[0215] In some embodiments, the modified backbone comprises a phosphorothioate (PS) modification, a phosphorylguanidine (PN) modification, a borano-phosphate modification, an alkylphosphonate nucleic acid (phNA), a peptide nucleic acid (PNA), or a combination thereof.

[0216] In some embodiments, the nucleic acid comprises one or more modifications, for example, to the 5' end of an oligonucleotide, hi some embodiments, the nucleic acid comprises a 5' amino modification.

[0217] In some embodiments, the nucleic acid is partially (eg, at least 5%) modified for a particular modification, eg, over the entire length of the sequence.

[0218] In some embodiments, the nucleic acid is fully modified for a particular modification throughout the length of the sequence.

[0219] In some embodiments, at least 5% of a particular nucleotide (eg, A, G, C, T, or U) is modified in the oligonucleotide.

[0220] In some embodiments, all (eg, 100%) of a particular nucleotide (eg, A, G, C, T, or U) are modified in the oligonucleotide.

[0221] In some embodiments, the nucleic acid agent comprises a structure comprising a first wing sequence, a gap sequence, and a second wing sequence. Nucleic acids comprising such wing-gap-wing sequences are commonly referred to as gapmers. In some embodiments, the gap sequence flanks the first wing sequence and the second wing sequence. In some embodiments, the gap sequence comprises about 6-10 nucleotides. In some embodiments, the wing sequence comprises one or more nucleotides. In some embodiments, the wing sequence comprises one or more modified nucleotides, for example, as disclosed herein. In some embodiments, gapmers act by recruiting RNase H.

[0222] In some embodiments, the nucleic acid comprises an overhang. In some embodiments, the overhang is a 3' overhang or a 5' overhang. In some embodiments, the overhang is a 3' overhang. In some embodiments, the overhang comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the nucleic acid is double-stranded and comprises an overhang.

[0223] In some embodiments, the nucleic acid, e.g., oligonucleotide, is characterized in that when delivered to a cell, tissue, or organism that expresses the target, the expression and / or activity of the target is modulated, e.g., decreased, compared to a cell, tissue, or organism to which the oligonucleotide has not been delivered.

[0224] Without wishing to be bound by theory, it is believed that in some embodiments, a sortilin binding moiety, e.g., a peptide disclosed herein, can be conjugated to a payload moiety comprising a nucleic acid, e.g., an oligonucleotide.

[0225] In some embodiments, the payload can be or include particles (e.g., microparticles or nanoparticles), such as, for example, metal particles, crystalline particles, polymer particles, lipid-containing particles, etc. Those skilled in the art will be familiar with various particulate systems useful in delivering payload agents. For example, various polymer systems (e.g., polyacrylamide, polyacrylate, or polysaccharide-containing systems, including PLGA-based systems, among others) have been described. Inorganic particle (e.g., nanoparticle) systems have also been reported, such as those utilizing metals (e.g., gold, titanium) to detect and / or associate with active agents. In some embodiments, the payload can be or include quantum dots.

[0226] A variety of lipid-based and / or viral-based particle systems have been described as useful for delivery of nucleic acids and can be utilized in or as payloads in accordance with the present disclosure, hi some embodiments, liposomes or lipid nanoparticles are utilized.

[0227] Preparations and Compositions The present disclosure provides, inter alia, certain preparations of sortilin binding agents (eg, sortilin binding moieties or conjugates).

[0228] For example, in some embodiments, the disclosure provides liquid preparations. In some embodiments, the disclosure provides solid preparations (e.g., powder preparations such as lyophilized compositions, aerosolized preparations, or tablet or capsule preparations, etc.).

[0229] In some embodiments, provided compositions are pharmaceutical compositions that comprise or deliver a sortilin binding agent (e.g., a sortilin binding moiety or conjugate agent); typically, such pharmaceutical compositions comprise an active agent (e.g., a sortilin binding agent, which in some embodiments can be a sortilin binding moiety, or can include a sortilin binding moiety, or can be or include a conjugate agent, or can be or include a composition that includes a conjugate agent), and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0230] In some embodiments, the pharmaceutical compositions described herein can include a buffer such as neutral buffered saline or phosphate buffered saline (PBS), a carbohydrate such as glucose, mannose, sucrose, dextran, or mannitol, a protein, polypeptide, or amino acid (e.g., glycine), an antioxidant, a chelating agent such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide), and / or a preservative. In some embodiments, the pharmaceutical composition is substantially free of one or more specific contaminants. For example, in some embodiments, a specific contaminant or set of contaminants is absent from the composition above a certain threshold level. In some such embodiments, the relevant contaminant can be or include endotoxin.

[0231] In some embodiments, the pharmaceutical compositions described herein can be administered in a manner appropriate for the disease, disorder, or condition being treated or prevented. In some embodiments, the amount and / or frequency of administration can be determined by factors such as the condition of the patient and / or the type and / or severity of the patient's disease, disorder, or condition, although appropriate dosages can be determined through clinical trials.

[0232] In some embodiments, pharmaceutical compositions provided by the present disclosure may be in the form of liquid, semi-solid, and solid dosage forms, such as, for example, liquid solutions (e.g., injectable and insoluble solutions), dispersions or suspensions, liposomes, and suppositories. Typically, pharmaceutical compositions containing or delivering antibody agents are injectable or infusible solutions, and in some such embodiments, such compositions can be formulated for intravenous, subcutaneous, intradermal, intranasal, intratumoral, intramedullary, intramuscular, intranodal, intraperitoneal, intrathecal, sublingual, topical, or intraarterial administration. In some embodiments, provided pharmaceutical compositions are formulated for intravenous administration. In some embodiments, provided pharmaceutical compositions are formulated for subcutaneous administration.

[0233] The pharmaceutical compositions described herein can be formulated for administration using injection techniques commonly known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, which is incorporated herein by reference in its entirety).

[0234] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously with, or after) other treatments for a particular disease, disorder, or condition, or symptom thereof, such as the standard of care ("SOC") for such disease, disorder, or condition, or symptom thereof. In some embodiments, the pharmaceutical compositions described herein may be administered before or after surgery.

[0235] In some embodiments, provided sortilin binding agents are useful in the treatment of cancer and can be administered in combination with other cancer therapies, such as, for example, one or more of surgery, radiation, immunotherapy, checkpoint inhibitor therapy, and the like.

[0236] produce Sortilin-binding peptides Those skilled in the art who read this disclosure will understand that a variety of techniques are available for preparing sortilin binding moieties. In some embodiments, sortilin binding moieties are prepared according to the methods described herein. In some embodiments, sortilin binding moieties are prepared by peptide solid-phase synthesis protocols. Many reviews of peptide solid-phase synthesis methods can be found, for example, in Coin et al., Nat. Protoc. 2007, 2, 3247-3256.

[0237] In some embodiments, one or more peptides provided and / or utilized in accordance with the present disclosure can be cyclic peptides. Those skilled in the art will recognize various techniques for achieving peptide cyclization. In some embodiments, cyclization can involve only side chain residues. In some embodiments, cyclization can involve only backbone residues. In some embodiments, cyclization can involve both side chain and backbone residues.

[0238] In some embodiments, cyclization can involve disulfide bond formation, amide bond formation, ester bond formation, stapling, and the like.

[0239] The present disclosure illustrates that certain cyclization methods can be utilized to form the provided Sortilin binding moieties, including cyclic peptides. In some embodiments, the provided Sortilin binding agents or moieties contain at least two cysteine ​​residues that form a disulfide bond. Those skilled in the art will understand, upon reading this disclosure, that a variety of alternative cyclization methods are available. In some embodiments, the two cysteines that form an intramolecular disulfide are replaced with standard and non-standard amino acids capable of forming covalent bonds, including amides, disulfides, thioethers, diselenides, triazoles, and azo bridges. In some embodiments, a cyclic Sortilin binding moiety is formed by conjugation of two cysteine ​​residues in a peptide sequence with a molecular scaffold containing a thiol-reactive group (e.g., hexafluorobenzene or di(bromomethyl)benzene). In some embodiments, a bicyclic Sortilin binding moiety is formed by conjugation of three cysteine ​​residues in a peptide sequence with a molecular scaffold containing a thiol-reactive group (e.g., 1,3,5-tri(bromomethyl)benzene). In some embodiments, the molecular scaffold comprising thiol reactive groups is [ka] is selected from.

[0240] In some embodiments, the cyclic sortilin binding moiety is formed by the conjugation of an acidic side chain of certain amino acids (e.g., aspartic acid, glutamic acid) with a basic side chain of certain amino acids (e.g., lysine) within the peptide sequence. In some embodiments, the acidic and basic side chains of certain amino acids within the peptide sequence can form an amide bond.

[0241] In some embodiments, cyclic sortilin binding moieties are formed by conjugation of the peptide N-terminus with a reactive side chain of certain amino acids in the peptide sequence. In some embodiments, such a method is referred to as "head to side chain" cyclization. In some embodiments, the N-terminus and reactive side chain of certain amino acids in the peptide sequence (e.g., aspartic acid, glutamic acid) can form an amide bond. In some embodiments, the N-terminus and reactive side chain of certain amino acids in the peptide sequence can form a thioether bond.

[0242] In some embodiments, a cyclic sortilin binding moiety is formed by introducing a staple between certain side chains of certain amino acids within a peptide sequence. A staple is a linker that can link one amino acid residue to another by joining two peptide backbone atoms of the amino acid residue; as will be understood by those of skill in the art, the resulting bond does not extend through the peptide backbone between the linked amino acid residues. In some embodiments, the staple is attached to the peptide backbone by replacing one or more hydrogens and / or substituents (e.g., side chains, O, S, etc.) on a peptide backbone atom (e.g., C, N, etc.). Those skilled in the art, upon reading this disclosure, will understand that a variety of peptide stapling techniques are available, including hydrocarbon stapling techniques and non-hydrocarbon stapling techniques. In some embodiments, the staple is a hydrocarbon staple. In some embodiments, the staple is a non-hydrocarbon staple. In some embodiments, the non-hydrocarbon staple contains one or more chain heteroatoms, and the chain of the staple is the shortest covalent connection within the staple, from one end of the staple to the other end of the staple. In some embodiments, a non-hydrocarbon staple is or comprises at least one sulfur atom derived from an amino acid residue of a polypeptide. In some embodiments, a non-hydrocarbon staple comprises two sulfur atoms derived from two different amino acid residues of a polypeptide. In some embodiments, a non-hydrocarbon staple comprises two sulfur atoms derived from two different cysteine ​​residues of a polypeptide. In some embodiments, a staple is a cysteine ​​staple. In some embodiments, a staple is a non-cysteine ​​staple. In some embodiments, staples can be constructed utilizing amino acid residues having double or triple bonds and, optionally, side chains containing various heteroatoms.

[0243] Conjugates In some embodiments, the conjugate agent is prepared by conjugating or covalently attaching a payload moiety to a sortilin binding moiety. In some embodiments, the payload moiety can be attached to the sortilin binding moiety by, for example, reaction of the payload moiety with the sortilin binding moiety in solution.

[0244] In some embodiments, the conjugate can have two or more payloads. In some embodiments, two identical payload moieties are attached to the sortilin binding moiety. In some embodiments, two different payload moieties are attached to the sortilin binding moiety. In some embodiments, a payload moiety can be attached to two or more sortilin binding moieties. In some embodiments, a payload moiety can be attached to two or more different binding moieties. In some embodiments, a payload moiety can be attached to two or more binding moieties, and one of the binding moieties is a sortilin binding moiety.

[0245] In some embodiments, a linker, whether cleavable or non-cleavable, can be introduced by a chemical conjugation reaction between the payload and the binding moiety to which the payload is conjugated. The payload and binding moiety may or may not first be modified to increase or promote their reactivity with each other. Such modifications can increase or improve the specificity of the conjugation reaction and, if desired, the degree of conjugation. In some embodiments, the linker can be introduced in a single reaction or by stepwise reactions until the desired conjugate is prepared.

[0246] Non-limiting examples of chemical coupling reactions to form conjugate agents include the reaction of carboxylic acids with amines, thiols, or alcohols (i.e., nucleophiles) to form amides, the reaction of various thiols to form disulfides, the reaction of thiols with alkyl halides or maleimides to form thioethers, the reaction of alkynes with azides to form triazoles (the "click reaction"), and the reaction of aldehydes with hydrazides or amines, or aminooxy compounds to form hydrazones, imines, and oximines, thioesters, and esters. Carboxylic acids can be activated in situ in the presence of amines, thiols, or alcohols to become reactive, or they can be preactivated before the addition of the nucleophile, for example, by conversion to an activated ester of the nucleophile N-hydroxysuccinimide (NHS) or sulfonated NHS. A comprehensive review of chemical bonding reactions can be found, for example, in Spicer et al. (2018) Chem. Rev. 2018, 118, 16, 7702-7743.

[0247] use Those of skill in the art reading this disclosure will appreciate various uses of the provided sortilin binding agents (e.g., sortilin binding moieties and / or conjugates comprising a sortilin binding moiety and at least a payload moiety). In some embodiments, provided sortilin binding agents are useful for binding to sortilin. In some embodiments, provided sortilin binding agents are usefully internalized into sortilin-expressing cells. In some embodiments, provided sortilin binding agents are useful for delivering a payload to (or, in some embodiments, within) a sortilin-expressing cell.

[0248] In some embodiments, provided sortilin binding agents are contacted with a system that includes sortilin, e.g., is or includes one or more cells that express (on their surface) sortilin. In some embodiments, the system is an in vitro system. In some embodiments, the system includes cultured cells. In some embodiments, the system includes a cell line. In some embodiments, the system is or includes a living organism.

[0249] In some embodiments, provided sortilin binding agents are administered to a subject (e.g., a human or animal subject) to achieve binding, e.g., to one or more sortilin-expressing cells or tissues in such a subject. In some such embodiments, such binding is detectable (e.g., when the bound agent comprises a payload whose presence can be detected), e.g., by imaging (e.g., color, or fluorescence, or structures such as nanoparticles) or other means (e.g., detection of radioactivity).

[0250] In some embodiments, the sortilin-expressing cell is or comprises a cell (e.g., of a tissue) selected from: an immune cell (e.g., a bone marrow cell, a lymph node cell, a thymocyte, a peripheral blood mononuclear cell [e.g., a myeloid and / or lymphoid cell], an erythrocyte, an eosinophil, a neutrophil, and / or a platelet); a nervous system cell (e.g., brain tissue, cortex, cerebellum, retinal cell, spinal cord cell, nerve cell, neuron, and / or support cell); an endothelial cell; a muscle (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle); a small intestinal cell; a colon cell; an adipocyte; a kidney cell; a liver cell; a lung cell; a spleen cell; a stomach cell; an esophageal cell; a bladder cell; a pancreatic cell; a thyroid cell; a salivary gland cell; an adrenal gland cell; a pituitary cell; a breast cell; a skin cell; an ovarian cell; a uterine cell; a placental cell; a prostate cell; or a testicular cell; or a combination thereof.

[0251] In some embodiments, the sortilin-expressing cell is or comprises, e.g., a cancer cell described herein, hi some embodiments, the sortilin-expressing cancer cell is selected from the group consisting of bladder cancer cells, breast cancer cells, colorectal cancer cells, endometrial cancer cells, glioblastoma cells, kidney cancer cells, liver cancer cells, lung cancer cells, ovarian cancer cells, pancreatic cancer cells, prostate cancer cells, skin cancer cells, small intestine cancer cells, thymus cancer cells, gastric cancer cells, thyroid cancer cells, and combinations thereof.

[0252] In some embodiments, a provided sortilin binding agent is contacted with a cell in a system comprising both sortilin and a target of the payload moiety. In some such embodiments, the relevant system is or comprises a cell that expresses both sortilin and such target.

[0253] In some embodiments, provided sortilin binding agents are administered to a subject having, for example, a disease, disorder, or condition disclosed herein. In some embodiments, the disease, disorder, or condition is associated with sortilin and / or cells in which the target of the payload moiety is present. In some embodiments, the subject to whom a provided agent is administered is first determined to express sortilin (e.g., in one or more disease-associated cells or tissues). In some such embodiments, the same sortilin binding moiety can be used to detect such sortilin (e.g., by association with a detectable payload moiety) and deliver a therapeutic payload.

[0254] The sortilin binding agents described herein (eg, which are or include sortilin binding polypeptides) are useful in the treatment of a variety of diseases, disorders, and conditions.

[0255] In some embodiments, provided sortilin binding agents are administered to a system (e.g., a subject) that expresses sortilin, and binding reduces the associated level (e.g., cell surface level) and / or activity of sortilin. Alternatively, or in addition, in some embodiments, the sortilin binding agent delivers a payload to the sortilin-expressing system. In some embodiments, the sortilin binding agent is administered to a system comprising cells that express sortilin on their surface, and the sortilin binding agent is internalized by such cells. In some such embodiments, such internalization achieves delivery of a payload (which may be associated with the internalized sortilin binding agent, e.g., by being conjugated to the internalized sortilin binding agent) into the cell.

[0256] In some embodiments, the relevant disease, disorder, or condition for which the conjugates disclosed herein are provided is associated with increased or otherwise abnormal expression (e.g., surface expression) and / or activity of Sortilin. In some embodiments, the relevant disease, disorder, or condition is not associated with increased or otherwise abnormal expression and / or activity of Sortilin, but can nevertheless be effected by binding to Sortilin and / or by such binding to effect payload delivery.

[0257] Sortilin may be a desirable target for the treatment of a variety of diseases, disorders, and conditions. Sortilin expression and / or activity has been described to be associated with numerous physiological events, conditions, etc., and its role(s) in human disease have been the subject of extensive research and review (see, e.g., Mazella Int J Molec Sci 23:11888, 2022; Mitok et al, J Lipid Res 63:8, 2022; Ayodele et al., Curr Neurol Neurosci Rept 21:1, 2021; Ghaemimanesh et al., J Cell Physiol 236:6271, 2021; Al-Yozbaki et al., BBA- Rev on Cancer 1874:188429, 2020; Blondeau et al., Front Pharmacol 9:1561, 2019; Talbot, Front Pharmacol 9:1507, 2019; etc.).

[0258] In some embodiments, the associated disease, disorder, or condition is a nervous system disease, disorder, or condition, a metabolic disease, disorder, or condition, a cardiovascular disease, disorder, or condition, a proliferative disease, a lysosomal storage disease, an inflammatory disease, disorder, or condition, or cancer.

[0259] In some embodiments, the associated disease, disorder, or condition is Alzheimer's disease, inflammatory disease, Parkinson's disease, diabetes mellitus (e.g., type II diabetes mellitus), cardiovascular disease, α-1 antitrypsin deficiency, lysosomal storage disease, or cancer. In some embodiments, the associated disease, disorder, or condition is cancer. In some embodiments, the associated disease, disorder, or condition is cancer, and the cancer is a specific type of cancer (e.g., blood cancer, tumor, non-tumor cancer, brain tumor, lung cancer, bone cancer, etc.). In some embodiments, the associated disease, disorder, or condition is a cancer selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, bladder cancer, osteosarcoma, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, glioblastoma, glioma, head cancer, Hodgkin's lymphoma, kidney cancer, liver cancer, lung cancer, melanoma, neuroblastoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small intestine cancer, soft tissue cancer, spleen cancer, thymus cancer, stomach cancer, testicular cancer, thyroid cancer, transitional bladder cancer, urothelial carcinoma, Wilms' tumor, and combinations thereof.

[0260] In some embodiments, the present invention provides methods for diagnosing a disease, disorder, or condition in a subject suspected of suffering from such a disease, disorder, or condition, the method comprising: a) contacting a sample from the subject with a conjugate described herein under conditions that result in binding of the conjugate to sortilin-expressing cells; and b) measuring binding of the conjugate to sortilin-expressing cells. In some embodiments, the associated disease, disorder, or condition is a nervous system disease, disorder, or condition, a metabolic disease, disorder, or condition, a cardiovascular disease, disorder, or condition, a proliferative disease, lysosomal storage disease, or cancer.

[0261] Dosing regimen Those skilled in the art will recognize that certain drugs, such as abaloparatide, afamelanotide, angiotensin II, atosiban, aviptadil, bremelanotide, carbetocin, carfilzomib, cosyntropin, degarelix, dulaglutide, enfuvirtide, epifibatide, etelcalcetide, exenatide, glatimelamer, gramicidin D, icatibant, lepirudin, leuprolide, linac, lotide, liraglutide, lixisenatide, lucinactate, mifamurtide, nesiritide, oxytocin, pasireotide, peginesatide, plecanatide, pramlintide, romiplostim, semaglutide, sermorelin, setomelanotide, taltirelin, teduglutide, teriparatide, tesamorelin, thymalfasin, ziconotide, and melflufen and 177 They will be familiar with administration regimens utilizing approved therapeutic agents, including specifically approved peptide therapeutic agents, such as approved peptide drug conjugate therapeutic agents, such as Lu-dotatate.

[0262] In many embodiments, provided sortilin binding agents are administered parenterally. In some embodiments, provided sortilin binding agents can be administered intravenously, intramuscularly, subcutaneously, etc. In some embodiments, provided sortilin binding agents can be administered by injection. In some embodiments, provided sortilin binding agents can be administered by intrathecal injection. In some embodiments, provided sortilin binding agents can be administered by inhalation. In some embodiments, provided sortilin binding agents can be administered by nebulization.

[0263] In some embodiments, the conjugated agent is administered at a fixed dose, ie, regardless of body weight.

[0264] In some embodiments, the conjugated agent is administered based on body weight, for example, in mg / kg doses.

[0265] In some embodiments, the conjugate agent is administered in an initial dose. In some embodiments, the initial dose can be followed by one or more subsequent doses. In some embodiments, the one or more subsequent doses can be administered daily, weekly, monthly, or at other intervals therebetween. In some embodiments, the administration regimen disclosed herein can be repeated one or more times.

[0266] In some embodiments, provided methods include administering to a patient (e.g., a human patient) in need thereof a conjugate agent, or a composition comprising or delivering the conjugate agent, in an amount equivalent to (e.g., corresponding to, equal to) about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 30 mg / kg in mice. In some such embodiments, the amount of conjugate agent administered to the subject is about 15 mg, about 25 mg, about 50 mg, about 75 mg, about 150 mg, or about 500 mg.

[0267] The present specification is deemed sufficient to enable those skilled in the art to practice the invention. The examples are intended to be merely illustrative of one aspect of the invention, and the present disclosure should not be limited in scope by the examples shown, as other, functionally equivalent embodiments are intended to be within the scope of the invention. In addition to the modifications shown and described herein, various modifications of the invention will become apparent to those skilled in the art from the foregoing description and are included in the accompanying embodiments. Certain advantages and thematic characteristics of some embodiments of the invention may not necessarily be characteristics of all embodiments of the invention.

[0268] Embodiment Embodiment 1. A conjugate comprising: (a) a polypeptide; (b) a payload; (c) optionally a linker; The polypeptide is a sortilin binding portion having a length in the range of about 12 to about 20 amino acids and comprising a characteristic sequence represented by: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO:2); wherein each X is independently any standard or non-standard amino acid.

[0269] Embodiment 2. The sortilin binding moiety is 2. The conjugate of embodiment 1, which corresponds to a C-terminal fragment of Progranulin, or a variant thereof, and comprises no more than about 20 consecutive residues corresponding to consecutive Progranulin residues.

[0270] Embodiment 3. The conjugate of embodiment 1, wherein the sortilin binding moiety is or comprises a cyclic peptide.

[0271] Embodiment 4. The characteristic sequence is: (R / N) X 2-3 CX 0-1 2. The conjugate of embodiment 1, represented by R (Q / E) (SEQ ID NO: 3).

[0272] Embodiment 5. The characteristic sequence is: (R / N) X 2-3 LX 0-1 2. The conjugate of embodiment 1, represented by R (Q / B43 / B50) (SEQ ID NO: 4).

[0273] Embodiment 6. The conjugate of embodiment 1, wherein the sortilin binding moiety is or comprises a linear peptide, and the characteristic sequence is represented by SEQ ID NO:2.

[0274] Embodiment 7. The conjugate of embodiment 5, wherein the sortilin binding moiety is or comprises a linear peptide.

[0275] Embodiment 8. The conjugate of any one of the preceding embodiments, wherein X is an amino acid included in any one of Tables 2-7.

[0276] Embodiment 9. The characteristic sequence is: (R / N) X 2-3 C / LX 0-1 2. The conjugate of embodiment 1, represented by RQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 5).

[0277] Embodiment 10. The characteristic sequence is: (R / N) X 2-3 CX 0-1 2. The conjugate of embodiment 1, represented by RQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 6).

[0278] Embodiment 11. The conjugate of embodiment 1, wherein the characteristic sequence comprises at least a second cysteine ​​residue and the polypeptide comprises at least one disulfide bond between the cysteine ​​residues.

[0279] Embodiment 12. The characteristic sequence is: The conjugate of embodiment 1, represented by (R / N) X10 X11 X12 CRQ (SEQ ID NO: 7).

[0280] Embodiment 13. The characteristic sequence is: X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (SEQ ID NO: 8), 2. The conjugate of embodiment 1, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

[0281] Embodiment 14. The conjugate of embodiment 13, wherein the polypeptide comprises at least one disulfide bond between cysteine ​​residues.

[0282] Embodiment 15. The characteristic sequence is: (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (SEQ ID NO: 9), 2. The conjugate of embodiment 1, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

[0283] Embodiment 16. The conjugate of embodiment 15, wherein the polypeptide comprises at least one disulfide bond between cysteine ​​residues.

[0284] Embodiment 17. The characteristic sequence is: (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 10), 2. The conjugate of embodiment 1, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

[0285] Embodiment 18. The conjugate of embodiment 17, wherein the polypeptide comprises at least one disulfide bond between cysteine ​​residues.

[0286] Embodiment 19. The characteristic sequence is: CR X10 X11 C X13 RQ (SEQ ID NO: 11), 2. The conjugate of embodiment 1, wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0287] Embodiment 20. The characteristic sequence is: (R / H) N X6 X7 CR X10 X11 C X13 RQ (SEQ ID NO: 12), 2. The conjugate of embodiment 1, wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0288] Embodiment 21. The characteristic sequence is: (R / H) N X6 X7 CR X10 X11 C X13 RQ (L / B13 / F02) L / B13 / F02) (SEQ ID NO: 13), 2. The conjugate of embodiment 1, wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0289] Embodiment 22. The sortilin binding moiety is (i) a basic residue, such as L-arginine or an analog thereof, at a position corresponding to position P3 and / or P4 of the 17-mer C-terminal fragment of Progranulin; (ii) a hydrophobic residue, such as L-tryptophan or an analogue thereof, at a position corresponding to position P4 of the 17-mer C-terminal fragment of Progranulin; (iii) a long-chain hydrophobic residue at a position corresponding to position P10 of the 17-mer C-terminal fragment of Progranulin; (iv) a covalent bond, such as a disulfide bond, between residues at positions corresponding to positions P5 and P13, or P8 and P12, of the 17-mer C-terminal fragment of Progranulin; and (v) a hydrophobic residue such as leucine or leucine at a position corresponding to position P16 and / or P17 of the 17-mer C-terminal fragment of Progranulin. 10. The conjugate of any one of the preceding embodiments, comprising one or more of:

[0290] Embodiment 23. A conjugate according to any one of the preceding embodiments, wherein X represents a standard amino acid.

[0291] Embodiment 24. A conjugate according to any one of the preceding embodiments, wherein X is a non-standard amino acid.

[0292] Embodiment 25. The conjugate of embodiment 24, wherein the non-standard amino acid is selected from those in one or more of Tables 2-7.

[0293] Embodiment 26. The conjugate of any one of the preceding embodiments, wherein the payload is or comprises a therapeutic payload.

[0294] Embodiment 27. The sortilin binding moiety is (i) exhibit an affinity (Kd) for human sortilin 1 of less than about 1 μM, as examined by fluorescence polarization; (ii) an IC of less than about 12 μM in a competitive binding assay with a reference C-terminal Progranulin fragment 50 Showing; 2. The conjugate of embodiment 1, characterized in that

[0295] Embodiment 28. The conjugate of embodiment 27, further characterized in that the sortilin binding moiety exhibits greater stability when maintained in mouse serum than the stability of a reference C-terminal Progranulin fragment.

[0296] Embodiment 29. The conjugate of embodiment 27, further characterized in that the sortilin binding moiety exhibits a stability when maintained in mouse serum that is less than the stability of a reference C-terminal Progranulin fragment.

[0297] Embodiment 30. A conjugate according to any one of embodiments 27 to 29, wherein the reference C-terminal Progranulin fragment has an amino acid sequence that is or includes: APRWDAPLRDPALRQLL.

[0298] Embodiment 31. The conjugate of any one of embodiments 27 to 29, wherein the sortilin binding moiety is characterized by a stability half-life in mouse serum of greater than about 3 minutes.

[0299] Embodiment 32. The conjugate of embodiment 31, wherein the stability half-life in mouse serum is about 5 minutes.

[0300] Embodiment 33 The conjugate of embodiment 31, wherein the stability half-life in mouse serum is about 10 minutes.

[0301] Embodiment 34 The conjugate of embodiment 31, wherein the stability half-life in mouse serum is about 14 minutes.

[0302] Embodiment 35. The conjugate of embodiment 30, characterized by an IC50 in said competitive binding assay of less than about 5000 nM.

[0303] Embodiment 36. The conjugate of embodiment 35, wherein the IC50 is less than about 4000 nM.

[0304] Embodiment 37. The conjugate of embodiment 35, wherein the IC50 is less than about 3000 nM.

[0305] Embodiment 38. The conjugate of embodiment 35, wherein the IC50 is less than about 2000 nM.

[0306] Embodiment 39. The conjugate of embodiment 35, wherein the IC50 is less than about 1000 nM.

[0307] Embodiment 40. The conjugate of embodiment 35, wherein the IC50 is less than about 750 nM.

[0308] Embodiment 41. The conjugate of embodiment 35, wherein the IC50 is about 600 to 650 nM.

[0309] Embodiment 42. A conjugate according to any one of embodiments 35 to 41, wherein the sortilin binding moiety is or comprises a cyclic peptide.

[0310] Embodiment 43. The conjugate of embodiment 35, wherein the IC50 is less than about 600 nM.

[0311] Embodiment 44. The conjugate of embodiment 35, wherein the IC50 is less than about 500 nM.

[0312] Embodiment 45. The conjugate of embodiment 35, wherein the IC50 is less than about 400 nM.

[0313] Embodiment 46. The conjugate of embodiment 35, wherein the IC50 is less than about 300-400 nM.

[0314] Embodiment 47. The conjugate of embodiment 35, wherein the IC50 is less than about 300-350 nM.

[0315] Embodiment 48. A conjugate according to any one of embodiments 43, wherein the sortilin binding moiety is or comprises a linear peptide.

[0316] Embodiment 49. The conjugate of embodiment 1, wherein the conjugate selectively or specifically targets sortilin-expressing cells.

[0317] Embodiment 50. The conjugate of embodiment 1, wherein the conjugate selectively or specifically targets cancer cells over normal cells.

[0318] Embodiment 51. The conjugate of embodiment 50, wherein the payload is or comprises a cytotoxic moiety, and the conjugate kills the sortilin-expressing cells.

[0319] Embodiment 52. The conjugate of embodiment 51, wherein the conjugate kills said sortilin-expressing cells with a potency that is greater than the potency observed for an otherwise identical conjugate in which the sortilin-binding moiety is or comprises a reference peptide that is a C-terminal fragment of Progranulin.

[0320] Embodiment 53. The conjugate of embodiment 52, wherein the reference peptide has the amino acid sequence APRWDAPLRDPALRQLL.

[0321] Embodiment 54. The conjugate of embodiment 53, wherein the potency is at least about 5 times greater.

[0322] Embodiment 55. The conjugate of embodiment 50, wherein the cancer cells express sortilin at levels equal to or greater than those expressed by otherwise comparable non-cancer cells.

[0323] Embodiment 56 The conjugate of embodiment 1, wherein the conjugate undergoes cellular internalization.

[0324] Embodiment 57. The conjugate of embodiment 1, wherein the conjugate exhibits improved affinity for sortilin compared to that observed with the unconjugated sortilin binding moiety.

[0325] Embodiment 58. The conjugate of embodiment 57, wherein such improved affinity is at least about two-fold greater.

[0326] Embodiment 59. The conjugate of embodiment 1, wherein the payload is or comprises a therapeutic or diagnostic moiety.

[0327] Embodiment 60. The conjugate of embodiment 1, wherein the payload is or comprises a therapeutic moiety.

[0328] Embodiment 61. The conjugate of embodiment 1, wherein the payload is or comprises a cytostatic or cytotoxic moiety.

[0329] Embodiment 62. The conjugate of embodiment 61, wherein the payload is or comprises a cytotoxic moiety.

[0330] Embodiment 63. The conjugate of embodiment 62, wherein the cytotoxic moiety is characterized by a subnanomolar IC50 against relevant cells.

[0331] Embodiment 64. The conjugate of embodiment 61, wherein the cytotoxic moiety is or comprises a bacterial toxin.

[0332] Embodiment 65. The conjugate of embodiment 1, wherein the payload is or comprises an anticancer drug.

[0333] Embodiment 66. The conjugate of embodiment 1, wherein the payload is selected from the group consisting of alkylating agents, antimetabolites, antitumor antibiotics, boron neutron capture therapeutics, cell cycle inhibitors, kinesin spindle protein inhibitors, microtubule binding agents, topoisomerase inhibitors, and combinations thereof.

[0334] Embodiment 67. The conjugate of embodiment 1, wherein the payload is or comprises an alkaloid, an anthracycline, an auristatin, a camptothecin, a folic acid derivative, a metal complex, a nucleoside analog, a taxane, a vinca alkaloid analog, or a combination thereof.

[0335] Embodiment 68. The conjugate of embodiment 1, wherein the payload is or comprises a phytochemical.

[0336] Embodiment 69. The conjugate of embodiment 1, wherein the payload is monomethyl auristatin E (MMAE).

[0337] Embodiment 70. The conjugate of embodiment 1, wherein the payload is a detectable entity.

[0338] Embodiment 71. The conjugate of embodiment 1, wherein the payload is a small molecule.

[0339] Embodiment 72. The conjugate of embodiment 1, wherein the payload is a polypeptide.

[0340] Embodiment 73. The conjugate of embodiment 1, wherein the payload is an oligonucleotide.

[0341] Embodiment 74. The conjugate of embodiment 73, wherein the polypeptide is mRNA.

[0342] Embodiment 75. The conjugate of embodiment 1, wherein the payload is a particle.

[0343] Embodiment 76. The conjugate of embodiment 75, wherein the payload is a lipid nanoparticle.

[0344] Embodiment 77. The conjugate of embodiment 1, wherein the payload is a viral capsid.

[0345] Embodiment 78. The conjugate of embodiment 1, wherein the payload is a lipid vesicle such as an exosome or a liposome.

[0346] Embodiment 79. The conjugate of embodiment 1, wherein the payload is or comprises a radioisotope.

[0347] Embodiment 80. The conjugate of embodiment 1, wherein the payload is covalently attached to the linker or the peptide through a hydroxyl group, a carboxyl group, or an amine group.

[0348] Embodiment 81. The conjugate of embodiment 1, wherein the payload is or comprises a protein degradation modulator, such as a proteasome inhibitor or PROTAC agent.

[0349] Embodiment 82. The conjugate of embodiment 1, wherein the payload is or comprises a nucleic acid editing system such as CRISPR / Cas, TALEN, ADAR, etc.

[0350] Embodiment 83. A conjugate according to any one of embodiments 12 to 21, wherein the payload is attached to an amino acid residue at position P4.

[0351] Embodiment 84. A conjugate according to any one of embodiments 12 to 18, wherein the payload is attached to an amino acid residue at position P3.

[0352] Embodiment 85. A conjugate according to any one of embodiments 12 to 18, wherein the payload is attached to an amino acid residue at position P8.

[0353] Embodiment 86. A conjugate according to any one of embodiments 19 to 21, wherein the payload is attached to an amino acid residue at position P10.

[0354] Embodiment 87. A conjugate according to any one of embodiments 1 to 21, wherein at least one amino acid residue within said sequence of the sortilin binding moiety is conjugated to a payload moiety.

[0355] Embodiment 88. A conjugate according to any one of embodiments 1 to 21, wherein at least one amino acid within said sequence outside of P15, P16, and P17 of the sortilin binding moiety is conjugated to a payload moiety.

[0356] Embodiment 89. A conjugate according to any one of embodiments 1 to 21, wherein two or more amino acids within said sequence of the sortilin binding moiety are conjugated to a payload moiety.

[0357] Embodiment 90. A conjugate according to any one of embodiments 1 to 21, wherein two or more amino acids within said sequence outside of P15, P16, and P17 of the sortilin binding moiety are conjugated to a payload moiety.

[0358] Embodiment 91. The conjugate of embodiment 1, wherein the linker is cleavable.

[0359] Embodiment 92. The conjugate of embodiment 91, wherein the linker is an acid-cleavable linker.

[0360] Embodiment 93. The conjugate of embodiment 91, wherein the linker is an enzyme-cleavable linker.

[0361] Embodiment 94. The conjugate of embodiment 1, wherein the linker is a VCPAB linker.

[0362] Embodiment 95. The conjugate of embodiment 1, wherein the linker is or comprises an ester, amide, hydrozone, carbonate, reducible disulfide, and combinations thereof.

[0363] Embodiment 96. The conjugate of embodiment 1, wherein the linker is or comprises a thioether, an oxime, a triazole, and combinations thereof.

[0364] Embodiment 97. The conjugate of embodiment 1, wherein the linker is redox-sensitive.

[0365] Embodiment 98. An engineered sortilin-binding peptide, comprising: (a) a C-terminal fragment of Progranulin or a variant thereof, wherein the fragment comprises about 20 or fewer residues corresponding to consecutive C-terminal Progranulin residues; (b) a characteristic sequence having a length ranging from about 12 to about 20 amino acids and represented by: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO: 2) Including, wherein X is any standard or non-standard amino acid.

[0366] Embodiment 99. An amino acid sequence having a length ranging from about 12 to about 20 amino acids and comprising a characteristic sequence represented by: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO: 2) and wherein each X is independently any standard or non-standard amino acid; An engineered sortilin-binding peptide, wherein at least one X residue is an unnatural amino acid.

[0367] Embodiment 100. The peptide of embodiment 98 or 99, wherein the unnatural amino acid is selected from those in one or more of Tables 2-7.

[0368] Embodiment 101. A peptide according to embodiment 98 or 99, comprising two cysteine ​​residues within said sequence that form a disulfide bond.

[0369] Embodiment 102. The characteristic sequence is (R / N) X 2-3 CX 0-1 100. The peptide of any one of embodiments 98 or 99, represented by R (Q / E) (SEQ ID NO: 3).

[0370] Embodiment 103. The characteristic sequence is (R / N) X 2-3 LX 0-1 100. The peptide of any one of embodiments 98 or 99, represented by R (Q / B43 / B50) (SEQ ID NO: 4).

[0371] Embodiment 104. The characteristic sequence is (R / N) X 2-3 C / LX 0-1100. The peptide of any one of embodiments 98 or 99, represented by RQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 5).

[0372] Embodiment 105. The characteristic sequence is (R / N) X 2-3 CX 0-1 100. The peptide of any one of embodiments 98 or 99, represented by RQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 6).

[0373] Embodiment 106. The characteristic sequence is 100. The peptide of any one of embodiments 98 or 99, represented by (R / N) X10 X11 X12 CRQ (SEQ ID NO: 7).

[0374] Embodiment 107. The characteristic sequence is X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (SEQ ID NO: 8), 100. The peptide of any one of embodiments 98 or 99, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

[0375] Embodiment 108. The peptide of embodiment 107, wherein the two cysteine ​​residues in the sequence form a disulfide bond.

[0376] Embodiment 109. The characteristic sequence is (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (SEQ ID NO: 9), 100. The peptide of any one of embodiments 98 or 99, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

[0377] Embodiment 110. The peptide of embodiment 109, wherein the two cysteine ​​residues in the sequence form a disulfide bond.

[0378] Embodiment 111. The characteristic sequence is: (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 10), 100. The peptide of any one of embodiments 98 or 99, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

[0379] Embodiment 112. The peptide of embodiment 111, wherein the two cysteine ​​residues in the sequence form a disulfide bond.

[0380] Embodiment 113. The characteristic sequence is: CR X10 X11 C X13 RQ (SEQ ID NO: 11), 100. The peptide of any one of embodiments 98 or 99, wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0381] Embodiment 114. The characteristic sequence is: (R / H) N X6 X7 CR X10 X11 C X13 RQ (SEQ ID NO: 12), 100. The peptide of any one of embodiments 98 or 99, wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0382] Embodiment 115. The characteristic sequence is: (R / H) N X6 X7 CR X10 X11 C X13 RQ (L / B13 / F02) L / B13 / F02) (SEQ ID NO: 13), 100. The peptide of any one of embodiments 98 or 99, wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0383] Embodiment 116 (i) a basic residue, such as L-arginine or an analog thereof, at a position corresponding to position P3 and / or P4 of the 17-mer C-terminal fragment of Progranulin; (ii) a hydrophobic residue, such as L-tryptophan or an analogue thereof, at a position corresponding to position P4 of the 17-mer C-terminal fragment of Progranulin; (iii) a long-chain hydrophobic residue at a position corresponding to position P10 of the 17-mer C-terminal fragment of Progranulin; (iv) a covalent bond, such as a disulfide bond, between residues at positions corresponding to positions P5 and P13, or P8 and P12, of the 17-mer C-terminal fragment of Progranulin; (v) The peptide of the preceding embodiment, comprising one or more hydrophobic residues, such as leucine or leucine, at positions corresponding to positions P16 and / or P17 of the 17-mer C-terminal fragment of Progranulin.

[0384] Embodiment 117.X n An engineered sortilin-binding peptide comprising the sequence RDPALRXLL (SEQ ID NO: 14).

[0385] Embodiment 118. A peptide according to embodiment 117, wherein n is 0.

[0386] Embodiment 119. The peptide of embodiment 118, wherein X corresponds to an unnatural amino acid selected from those in one or more of Tables 2-7.

[0387] Embodiment 120. The peptide of embodiment 118, wherein X is Q.

[0388] Embodiment 121. An engineered sortilin-binding peptide comprising the sequence DDPRAPWPALQRLALRL.

[0389] Embodiment 122. A nucleic acid, wherein the nucleotide sequence of the nucleic acid has a length in the range of about 12 to about 20 amino acids; (R / N) X 2-3 (C / L) X 0-1R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO: 2) a coding region encoding a peptide having an amino acid sequence comprising a characteristic sequence represented by wherein each X is independently any standard or non-standard amino acid.

[0390] Embodiment 123. The nucleic acid of embodiment 122, wherein the nucleic acid is DNA.

[0391] Embodiment 124. The nucleic acid of embodiment 122, wherein the nucleic acid is RNA.

[0392] Embodiment 125. The nucleic acid of embodiment 122, wherein the nucleic acid is mRNA.

[0393] Embodiment 126. The nucleic acid of any one of embodiments 122 to 125, wherein the nucleic acid is part of a viral vector.

[0394] Embodiment 127. A host cell comprising the nucleic acid of embodiment 122.

[0395] Embodiment 128. A pharmaceutical composition comprising or delivering a conjugate according to any one of embodiments 1 to 21, or an engineered peptide according to embodiment 98 or 99.

[0396] Embodiment 129. An active agent which is or comprises a conjugate according to any one of embodiments 1 to 21 or an engineered peptide according to embodiment 98 or 99, at least one pharmaceutically acceptable carrier; 127. The pharmaceutical composition of embodiment 126, comprising:

[0397] Embodiment 130. The pharmaceutical composition of embodiment 129, wherein the payload is an anticancer drug.

[0398] Embodiment 131. The pharmaceutical composition of embodiment 130, wherein the active agent further comprises an additional anticancer agent.

[0399] Embodiment 132. A conjugate according to embodiment 1 or an engineered peptide according to embodiment 98 or 99, and at least one additional therapeutic agent.

[0400] Embodiment 133. The combination of embodiment 132, wherein the conjugate and the at least one additional therapeutic agent are contained in the same pharmaceutical composition.

[0401] Embodiment 134. A pharmaceutical composition according to embodiment 128, at least one additional therapeutic agent; Including, combinations.

[0402] Embodiment 135. The combination of embodiment 132 or 134, wherein one or both of the payload and the additional therapeutic agent is or comprises an anti-cancer agent.

[0403] Embodiment 136. The combination of embodiment 135, wherein the anticancer agent is or comprises a small molecule.

[0404] Embodiment 137. The combination of embodiment 135, wherein the anti-cancer agent is or comprises an antibody.

[0405] Embodiment 138. A method of binding to sortilin, said method comprising: The method comprising contacting a system comprising sortilin with a conjugate or engineered peptide according to any one of the preceding embodiments.

[0406] Embodiment 139. A method for inhibiting sortilin, said method comprising: The method comprising contacting a system in which sortilin is active with the conjugate or engineered peptide of any one of the preceding embodiments.

[0407] Embodiment 140. A method of reducing sortilin on a cell surface, the method comprising: 10. The method of claim 1, wherein the Sortilin is internalized by the cells, the method comprising contacting a system comprising cells having Sortilin on their surface with the conjugate or engineered peptide of any one of the preceding embodiments, wherein said contacting is performed under conditions and for a time sufficient to allow said Sortilin to be internalized by said cells.

[0408] Embodiment 141. A method for increasing the specificity of a therapeutic or diagnostic moiety for a target cell, said method comprising: providing said therapeutic moiety with a length in the range of about 12 to about 20 amino acids; (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or to a sortilin-binding peptide having an amino acid sequence including the characteristic sequence represented by APRWDAPLRXPALR (SEQ ID NO: 2), wherein each X is independently any standard or non-standard amino acid.

[0409] Embodiment 142. A method for delivering a payload to a sortilin-expressing cell, the method comprising: 2. The method comprising contacting the cell with the conjugate of embodiment 1.

[0410] Embodiment 143. A method for increasing the specificity of a payload for sortilin-expressing cells, the method comprising: associating the payload with a polypeptide, the polypeptide comprising: comprising a sortilin binding moiety having a length in the range of about 12 to about 20 amino acids and having a characteristic sequence represented by: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO:) or APRWDAPLRXPALR (SEQ ID NO:2); wherein each X is independently any standard or non-standard amino acid.

[0411] Embodiment 144. A method for increasing cellular uptake of a payload by a target cell, the method comprising: associating the payload with a polypeptide, the polypeptide comprising: (a) a fragment of Progranulin, or a variant thereof, comprising no more than about 20 consecutive residues corresponding to consecutive C-terminal Progranulin residues; (b) a sortilin-binding moiety having a length in the range of about 12 to about 20 amino acids and having a characteristic sequence represented by: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO:2); wherein each X is independently any standard or non-standard amino acid.

[0412] Embodiment 145. The method of any one of embodiments 141 to 144, wherein the associating step is or includes covalent bonding.

[0413] Embodiment 146. The method of any one of embodiments 141-144, wherein the sortilin binding moiety and the therapeutic moiety are covalently associated through a linker.

[0414] Embodiment 147. The method of any one of embodiments 141 to 144, wherein the sortilin-expressing cell is a cancer cell.

[0415] Embodiment 148. The method of any one of embodiments 141 to 144, wherein the sortilin-expressing cell is a mammalian cell.

[0416] Embodiment 149. The method of any one of embodiments 141 to 144, wherein the sortilin-expressing cell is a human cell.

[0417] Embodiment 150. A method of treating a subject suffering from a disease, disorder, or condition associated with sortilin-expressing cells, said method comprising the step of delivering to said subject a conjugate of embodiment 1.

[0418] Embodiment 151. The method of embodiment 150, wherein the subject has cancer and cancer cells in the subject express elevated levels of sortilin compared to reference non-cancerous cells.

[0419] Embodiment 152. The method of embodiment 150 or 151, wherein the subject is undergoing or has undergone other cancer therapy.

[0420] Embodiment 153. The method of embodiment 152, wherein the other cancer therapy is or includes chemotherapy, cryotherapy, hormone therapy, immunotherapy, radiation therapy, surgery, and combinations thereof.

[0421] Embodiment 154. The method of embodiment 150, wherein the administering step comprises parenterally administering the conjugate.

[0422] Embodiment 155. The method of embodiment 150, wherein the patient has a cancer that is a member of the group consisting of breast cancer, colorectal cancer, glioblastoma, lung cancer, ovarian cancer, pancreatic cancer, small intestine cancer, thymus cancer, thyroid cancer, bladder cancer, prostate cancer, kidney cancer, liver cancer, endometrial cancer, skin cancer, gastric cancer, and combinations thereof.

[0423] Embodiment 156. A method for preparing a conjugate according to embodiment 1 or a peptide according to embodiment 98 or embodiment 99, said method comprising: synthesizing said conjugate or peptide using standard solid phase peptide synthesis (SPPS) with Fmoc chemistry; deprotecting the conjugate or peptide and cleaning the resin; purifying the conjugate or peptide using reverse-phase high performance liquid chromatography; The method comprising:

[0424] Embodiment 157. A method for producing a conjugate, the method comprising: covalently associating a peptide corresponding to a C-terminal fragment of Progranulin, or a variant thereof, with a cytotoxic payload, wherein the peptide has an amino acid sequence comprising characteristic sequence elements represented by: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO:2); wherein each X is independently any standard or non-standard amino acid.

[0425] Embodiment 158. A method for producing a peptide having a length ranging from about 12 to about 20 amino acids and an amino acid sequence comprising characteristic sequence elements represented by: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO:2); wherein X is any standard or non-standard amino acid. The method comprises: synthesizing said peptide using standard solid phase peptide synthesis (SPPS) with Fmoc chemistry; deprotecting the peptide and cleaning the resin; purifying the peptide using reverse-phase high performance liquid chromatography.

[0426] Embodiment 159. A method for producing a pharmaceutical composition, the method comprising: an active agent which is or comprises a conjugate or engineered peptide according to any one of the preceding embodiments, The method further comprising the step of bringing into association with at least one pharmaceutically acceptable carrier.

[0427] Embodiment 160. A conjugate according to any one of embodiments 1 to 97, wherein the sortilin binding moiety is or comprises the amino acid sequence APRWDAPLRDPALRQLL.

[0428] Embodiment 161. A conjugate according to any one of embodiments 1 to 97, wherein the sortilin binding moiety is or comprises the amino acid sequence APRWDAPLRDPALRQ(B13)(G48).

[0429] Embodiment 162. A conjugate according to any one of embodiments 160 or 161, wherein the linker is a VCPAB linker.

[0430] Embodiment 163. A conjugate according to any one of embodiments 160 to 162, wherein the payload is MMAE.

[0431] Embodiment 164. A peptide according to any one of embodiments 98 to 120, comprising the sequence APRWDAPLRDPALRQLL.

[0432] Embodiment 165. A peptide according to any one of embodiments 98 to 120, comprising the sequence APRWDAPLRDPALRQ(B13)(G48).

[0433] Embodiment 166. The nucleic acid of any one of embodiments 122 to 126, wherein the nucleotide sequence comprises a coding region encoding a peptide comprising the sequence APRWDAPLRDPALRQLL.

[0434] Embodiment 167. The nucleic acid of any one of embodiments 122 to 126, wherein the nucleotide sequence comprises a coding region encoding a peptide comprising the sequence APRWDAPLRDPALRQ. [Example]

[0435] Example 1: Materials and Methods Peptide microarray Synthesis. Peptide microarrays were generated using SPOT synthesis technology on a MutiPep synthesizer (Intavis). SPOT synthesis of two peptide arrays was performed on commercially available amino-PEG-functionalized cellulose membranes. One array contained linear peptides, cyclized by two cysteines. During standard coupling cycles, peptides were synthesized using solutions of preactivated amino acids. Unnatural amino acids were activated in situ using HOBt and DIC. To efficiently couple the first amino acid to the linker group required for orientation change, the first coupling cycle was performed using amino acids activated in situ with DIC. All peptides were membrane-bound via their N-terminus, with the C-terminus free.

[0436] Membranes were prepared to ensure free peptide termini. The membranes bearing the arrays were modified with glutamic acid. After denaturation, the membranes were modified with additional linker molecules to release the assembled peptide chains. After the initial peptide synthesis, all peptides were modified with β-alanine to achieve more uniform coupling behavior. After assembly of the protected peptide chains, the membranes were treated with diluted TFA, which made the glutamic acid side chain(s) available for coupling to the membrane. The peptides on the arrays were cyclized by forming an amide bond between the free glutamic acid side chain and the free N-terminus. Cleavage of the peptide protecting group(s) on both arrays and release of the free C-terminus on the linear array were performed using a TFA solution (water, triisopropanol, thioanisole) containing scavengers. After cleavage, the membranes were washed, dried, and stored at -20 °C until probing. To cyclize arrays containing two cysteine ​​residues, the arrays were treated with a DMSO-water mixture to achieve the formation of disulfide bridge(s) within the peptide. After treatment, the cyclic peptide arrays were also dried and stored at -20°C until probing.

[0437] Probing. As a negative control, the array was probed with a secondary antibody (HRP-conjugated rabbit anti-His6 antibody from ICL, diluted 1:20,000). T-TBS was used during the corresponding incubation time to mimic the preceding primary probing step. Detection of bound secondary antibody was performed using enhanced chemiluminescence (ECL). ECL scanning was performed with a scan time of 50 seconds. Eight images of the scanned array were generated during the scan time. Immediately after probing, a two-step regeneration of the peptide array was performed (without adding β-mercaptoethanol (BME)), and the membrane was stored at -20°C until the next probing. As a next step, the array was probed with SORT1 (Acrobiosystems, SON-H52H5) at a final protein concentration of 30 μg / mL. Detection of bound protein was performed as described above.

[0438] Peptide synthesis: SPPS Peptides were synthesized on Rink amide resin with Fmoc chemistry. The protecting groups used for the amino acids were t-butyl for Ser, Thr, Tyr, Asp, and Glu, Trt for Asn, Cyc, His, and Gln, Pbf for Arg, and Boc for Lys and Boc.

[0439] Each peptide chain was assembled on the resin by repeatedly removing the Fmoc protecting group and coupling protected amino acids. DIC and HOBt were used as coupling reagents, and NMM was used as the base. 20% piperidine in DMF was used as the de-Fmoc reagent. After each coupling, a ninhydrin test was performed to confirm the coupling efficiency.

[0440] After removal of the final Fmoc protecting group, the resin was treated with a TFA cocktail for cleavage and removal of side-chain protecting groups. The crude peptide was purified by RP-HPLC. Peptide fractions with the desired purity were lyophilized to produce a powder preparation of the purified peptide.

[0441] Dynamic Light Scattering (DLS) Unless otherwise specified, peptide samples were prepared in 1x PBS (pH 7.4) to final concentrations of 250, 125, or 62.5 μM. Samples were spun down in a tabletop centrifuge for 15 minutes at 15,000 rpm at room temperature to pellet any aggregated peptides / dust before measurements were taken. Tubes were visually inspected to identify whether any large pellets, indicative of large aggregates, had formed. The clear solution was then loaded onto a DLS plate (Aurora 384-well, black, clear-bottom plate (P8806-38403), 20 μL per well), and the plate was spun down at 4000 rpm for 1 minute to collect the solution at the bottom of the well. The plate was then transferred and read on a DLS instrument (Wyatt Technology DynaPro Plate Reader III) at 25°C.

[0442] Circular dichroism (CD) Unless otherwise specified, peptide samples were diluted in 1x PBS (pH 7.4) to a final concentration of 50 μM. Samples were allowed to equilibrate to room temperature for 30 min before reading and then spun down in a tabletop centrifuge for 15 min at 15,000 rpm at room temperature to pellet any aggregated peptides / dust before measurements were taken. Tubes were visually inspected to identify whether any large pellets, indicative of large aggregates, had formed. A 220 μL aliquot of peptide sample or buffer blank was added to a CD quartz cuvette (Jasco J / 0556). Samples were then read on a Jasco J-1500 CD machine at 25 °C with a wavelength scan from 250 to 190 nM, and the raw mdeg values ​​were converted to mean residue ellipticity (MRE).

[0443] Fluorescence Polarization (FP) Related peptides (e.g., reference peptide PRGN_WT, negative control peptide PRGN_scr, and peptide PRGN_RL) were synthesized with an N-terminal FAM tag and a free end. Peptides were dissolved in stock (aqueous) solution and then brought to a final concentration in 1x PBS (pH 7.4) and tested by DLS and CD (methods described above) to ensure peptide solubility. Peptide concentrations were determined by quantifying the dye using Nanodrop and dye-specific extinction coefficients at specific wavelengths. For FAM-labeled dyes, the extinction coefficient was 83000 cm. -1 M -1 The damping coefficient was used.

[0444] Direct binding in FP The FAM-labeled peptide was diluted to 1 μM in FP buffer (1x PBS, pH 7.4, 0.1% Tween®-20) and the concentration was determined by serially diluting 2-fold in a 12-point series, at which point the fluorescence intensity was 10,000 RFU and the peptide polarization was uniform. Therefore, the 10x working solution was a 10x concentration of FAM-labeled peptide that met the above criteria.

[0445] A stock solution of the target protein, sortilin (Acrobiosystems, SON-H52H5), was prepared in FP buffer (1x PBS, pH 7.4, 0.1% Tween-20) to a maximum SORT1 concentration of 3.85 μM. A 12-point, 2-fold dilution series of the target protein was prepared in a V-bottom 96-well plate in a total volume of 25 μL.

[0446] A 10x working solution of the FAM-labeled peptide was prepared in FP buffer (1x PBS, pH 7.4, 0.1% Tween-20). A 2.5 μL aliquot of the 10x working solution of the FAM-labeled peptide was then added to 22.5 μL of the SORT1 dilution series, pipetted, and mixed in a V-bottom 96-well plate. A negative control without target protein was also prepared to investigate the baseline polarization of the FAM-labeled peptide. The plate was then spun down to pellet the solution, and 10 μL of the mixture was then transferred in technical duplicate to an opaque black 384-well non-binding FP plate (Corning® Low Volume 384-well Black Flat Bottom Polystyrene NBS Microplate, #3820). The 384-well plate was then centrifuged at 4000 rpm for 1 minute, and the fluorescence and polarization of the dyes were measured on a Biotek Synergy Neo2 plate reader using the following settings: Excitation 485 / 20, Emission 528 / 20, Top Read, Grain: 75, Light Source: Xenon Flash, Lamp Energy: Low, Standard Dynamic Range, Read Speed: Normal, Delay: 0 ms, Measurements / Data Points: 10, Read Height: 9 mm. The data were then fitted to a single site-specific binding curve in GraphPad to obtain the K D was calculated.

[0447] Competitive binding in FP. Competitive binding studies were performed in a manner similar to the direct binding protocol. Unlabeled peptides were prepared in a 12-point, 2-fold dilution series in a V-bottom 96-well plate in a total volume of 25 μL at a starting concentration of 50 μM. 10x working stock solutions of FAM-labeled peptides were prepared as described above for the direct binding experiments.

[0448] A 500 nM working solution (EC80) of the target protein was prepared in FP buffer (1x PBS, pH 7.4, 0.1% Tween-20). An aliquot (20 μL) of the 500 nM working solution was then added to 2.5 μL of FAM peptide, followed by 2.5 μL of the unlabeled peptide dilution series, and mixed by pipetting into a V-bottom 96-well plate. After allowing the samples to incubate for 15 minutes, 10 μL of the mixture was transferred in technical duplicate to an opaque black 384-well non-binding FP plate (Corning® Low Volume 384-well Black Flat Bottom Polystyrene NBS Microplate, #3820). The 384-well plate was then centrifuged at 4000 rpm for 1 minute, and the fluorescence and polarization of the dye were measured on a Biotek Synergy Neo2 plate reader using the same settings as in the direct binding experiments. The data were then fitted to a single site-fit logIC50 curve in GraphPad to obtain IC 50 was calculated.

[0449] Grating Coupling Interferometry (GCI) The direct binding affinity of sortilin-binding peptides was measured using grating coupling interferometry (GCI) assays in two different formats: (a) immobilized protein as ligand and detection of peptide as analyte in solution, and (b) immobilized peptide as ligand and detection of protein as analyte in solution. Immobilization of protein / peptide as ligand was achieved by capturing biotinylated ligand on the surface of a streptavidin sensor chip, and upon ligand immobilization, the interaction between the immobilized ligand and analyte was measured in a multiple-cycle kinetic assay setup.

[0450] Immobilization. For protein immobilization, SORT1 (Acrobiosystems, SON-H52H5) was biotinylated at a 1:1 ratio using NHS-PEG4 biotin (Thermo Fisher, 21330). The protein was diluted to 0.5 mg / mL in 1x PBS (pH 7.4), 10.3% trehalose, and the biotinylation reaction was carried out at 25°C for 30 min. After biotinylation, unbound biotin was removed by a desalting step (Zeba Spin Desalting Columns (Thermo Fisher)), and the ligand protein was loaded into a final buffer of 1x PBS (pH 7.4), 0.005% Tween-20. The total protein concentration was then determined by measuring absorbance at 280 nm using an Implen spectrometer. The biotinylated SORT1 target protein was immobilized on the surface of a streptavidin sensor chip (PCH-STA) as a ligand. The chip was first conditioned using a solution of 1 M NaCl, 0.1 M Na-borate (pH 9.0) with a 180 s injection time and a flow rate of 2.5 μL / min. Ligand capture and immobilization of biotinylated SORT1 (20 μg / mL in running buffer, 1× PBS (pH 7.4), 0.005% Tween-20) was performed by streptavidin capture with 2× 1200 s injection times and a flow rate of 10 μL / min. To avoid nonspecific binding to remaining accessible streptavidin-binding pockets, the surface was blocked by a biotin injection (10 μg / mL in running buffer) (60 s injection time and a flow rate of 10 μL / min). Ligand capture stability was then investigated by rinsing the surface with buffer at a high flow rate (100 μL / min). For peptide immobilization, sortilin-binding peptides were synthesized with an N-terminal biotinylated group (e.g., PRGN_WT-biotin, PRGN_RL-biotin). The immobilization process on the streptavidin sensor chip was repeated as described above.

[0451] Multi-cycle reaction kinetics. Binding of analytes (free peptide or unlabeled SORT1) was performed in multiple-cycle kinetic experiments. Peptide analytes or SORT1 protein were diluted in running buffer in a 1:2 serial dilution with a maximum concentration of 5000 nM and injected at increasing concentrations over the ligand and reference surfaces. Raw sensorgrams were examined for nonspecific and ligand-specific analyte binding, and data were double-referenced, solvent-corrected, and fitted to a 1:1 kinetic model.

[0452] Heat-induced unfolding To verify the integrity of the SORT1 protein after biotinylation, a comparative thermal unfolding experiment was performed. For this, the thermal melting temperatures of the non-biotinylated and biotinylated target proteins were measured using a Tycho NT.6 nanoDSF instrument with a thermal gradient of 30°C / min. For the non-biotinylated reference protein, a single unfolding peak was observed at 66.7°C. For the labeled target protein, a similar unfolding peak at 66.3°C was observed. Therefore, the structural integrity of the target protein was not negatively affected by the biotinylation procedure.

[0453] Mouse serum / plasma stability assay serum Serum was collected from male CD-1 mice by Pharmidex. Lyophilized peptides were solubilized in DMSO and then further diluted in male mouse (CD-1) serum in duplicate at 37°C. Reactions were incubated at 37°C, and samples were extracted at six time points: 0, 0.16, 0.5, 1, 3, and 6 hours. The incubation was stopped by the addition of the internal standard (tolbutamide) in acetonitrile (ACN). Samples were centrifuged, and the supernatants were analyzed by HPLC-MS / MS for the parent compound.

[0454] plasma Frozen plasma was thawed at 37°C, centrifuged at 4000 rpm to remove any clots, and transferred to a 96-well reaction plate by WuXi AppTec. Test compounds were added to the reaction plate at a final concentration of 2 μM and either stopped immediately or incubated at 37°C for 5, 10, 30, 60, or 120 minutes. At the end of the incubation, an ACN stop solution consisting of 200 ng / mL tolbutamide and 200 ng / mL labetalol was added to precipitate proteins and mixed thoroughly. Samples were then transferred to a bioanalysis plate and shaken for 10 minutes before HPLC-MS / MS analysis of the percentage of compound remaining.

[0455] Cell killing assay The antiproliferative effects of peptides were investigated in a cell viability assay. HCC70 and MDA-MB-231 cells were grown according to the manufacturer's specifications: HCC70 in RPMI 1640 + 10% FBS + 1X PS / AA, and MDA-MB-231 in L-15 + 10% FBS + 1X PS / AA. Cells were counted using a hemocytometer using trypan blue staining, and the cell concentration was adjusted to the desired cell density. 90 μL of cell suspension was added to the assay plate, and 90 μL of assay medium was added to the blank well. Cells were then incubated overnight at 37°C, 5% CO2, 95% air, and 100% relative humidity for 16–24 hours. The peptide under investigation was dissolved in DMSO, and a 400-fold compound stock plate was prepared, with serial dilutions of the stock solution. A 10x compound plate was prepared by further diluting the 400x compound stock plate in cell culture medium and added directly to the cells to reach the final test concentrations. Cells were returned to the incubator and viability measurements were performed 72 hours later. Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) according to the manufacturer's procedure. The inhibition rate (IR) of the test compound was determined using the following formula: IR(%)=(1-(RLU compound-RLU blank) / (RLU control-RLU blank))*100%

[0456] Cellular internalization assay MDA-MB-231 cells were grown on glass coverslips in Petri dishes in L-15 + 10% FBS + 1X PS / AA. When cells reached the desired confluence (approximately 80%), growth medium was removed and prewarmed (37°C) medium containing 100 nM LysoTraker was added. Cells were incubated for 30 minutes to 2 hours under growth conditions appropriate for the particular cell type. LysoTraker-containing medium was removed, and cells were incubated with 1 μM Alexa488-tagged PRGN-based peptide for 2, 4, and 8 hours. At the indicated time points, cells were analyzed using a confocal fluorescence microscope fitted with compensation filter sets for the indicated time points.

[0457] CMC and formulation In this example, an exemplary conjugated agent (e.g., a sortilin binding agent), referred to as a "peptide drug conjugate," or "PDC," was investigated in a formulation screen to determine an appropriate vehicle for in vivo studies and to investigate precipitation risk. PDC solutions were made at 0.5 mg / mL or less in the following buffers: 25 mM histidine, 10% sucrose (pH 7), 50 mM acetate / acetic acid, 10% sucrose (pH 5), 1×PBS (pH 7.4), saline, 50 mM HEPES, 10% sucrose (pH 7). Solubility was investigated using the shake-flask method, and test compound concentrations in the filtrates were confirmed using HPLC-UV.

[0458] Maximum tolerated dose (MTD) study Balb / c nude mice (female, 6-8 weeks, approximately 18-22 g body weight) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The MTD study was conducted at WuXi AppTec, and animal care and use was carried out in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) regulations. Two MTD studies were conducted: (a) a low-dose study with doses ranging from 0.1, 0.5, 1, and 3 mg / kg, and (b) a high-dose study with doses ranging from 3, 5, and 10 mg / kg. Dose volume was determined based on body weight (10 μL / g), and PDC was dissolved in 25 mM histidine 10% sucrose (pH 7) and administered intravenously as a single dose on day 0. Treatment and observation were performed for 7 days or less. A weight loss of 20% or more was defined as the MTD. Body weights were measured daily, and deaths and observed clinical signs were recorded.

[0459] Mouse xenografts Mouse xenograft experiments were performed at WuXi AppTec and / or Charles River Laboratories, and animal care and use was carried out in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) regulations. MDA-MB-231 tumor cells (ATCC, Manassas, VA, catalog no. HTB-26) were grown and maintained in L15 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% antibiotic-antimycotic, and L-glutamine (2 mM) at 37°C in a 5% CO2, 95% air atmosphere. Balb / c nude mice (female, 6-8 weeks, weighing approximately 18-22 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. For tumor growth, inoculate the right flank of the mice with MDA-MB-231 tumor cells (e.g., triple-negative breast cancer cells) (10 × 10) in 0.2 mL of PBS containing Matrigel (1:1). 6) were inoculated subcutaneously. Animals were randomized and in the efficacy study, the mean tumor volume was approximately 150-200 mm 3 Treatment was initiated when tumor size reached 100 mg / kg. Dosage was adjusted based on body weight and administered at 10 mL / kg. PRGN_WT_PDC, PRGN_RL_PDC, Arb-SAR-Q15-PDC, Arb_cyclic_G23-PDC, and PRGN_scr_PDC were dissolved in 25 mM histidine 10% sucrose (pH 7) and administered i.v. to mice at one of the following doses: 3, 1, or 0.3 mg / kg on a QW x 4 schedule. Free MMAE payload was also tested and similarly dissolved in 25 mM histidine 10% sucrose pH 7 and administered i.v. to mice at one of the following doses: 0.6 or 0.06 mg / kg on a QA x 4 schedule. Tumor size was measured twice weekly in two dimensions using calipers and calculated as volume (mm 3 ) was calculated using the following formula: V = 0.5a × b 2 where a and b are the long and short sides of the tumor, respectively. TGI was calculated for each group using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] x 100; where Ti is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on the first day of treatment, Vi is the mean tumor volume of the vehicle control group on the same day as Ti, and V0 is the mean tumor volume of the vehicle group on the first day of treatment.

[0460] Surface Plasmon Resonance (SPR) SPR analysis was performed on a Series S CM5 chip (Cytiva) using a Biacore 8K instrument (Cytiva). Sensorgrams were double-referenced by subtracting the response from both the reference flow cell and the blank sample. Human SORT1(78-755)-His (Acrobiosystems) was diluted to 60 μg / mL in 10 mM sodium acetate (pH 4.5) buffer and immobilized to a Series S CM5 chip using amine coupling for 45 seconds at a flow rate of 10 μL / min. The final immobilization level of SORT1 was 2200-2600 RU.

[0461] For multiple-cycle kinetic analysis runs, a running buffer of 1x PBS (pH 7.4), 0.005% Tween-20 was used. Five start-up cycles and one blank cycle were performed before each analytical run. The run was performed by sequential injection of analyte (0-25 nM) at a flow rate of 30 μL / min with 150 s(es) of association and 300 s of dissociation, followed by 200 s of regeneration with 2 M MgCl2 at a flow rate of 10 μL / min. The kinetic constant (k a and k d ), and the equilibrium binding constant (K d ) was calculated using a 1:1 kinetic binding model with Biacore Insight Evaluation Software (Cytiva).

[0462] Mouse hematotoxicity assay Mouse hematotoxicity studies were performed at Charles River Laboratories, and animal care and use was in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) regulations. Wild-type Balb / c mice (female, 7-8 weeks old, weighing approximately 18-22 g) were randomized and administered control or test article intravenously on a QW x 4 schedule. Dose was adjusted based on body weight and administered at 10 mL / kg. PDC was dissolved in 25 mM histidine, 10% sucrose, pH 7, and administered at 3 mg / kg. Free MMAE payload was also tested; it was dissolved in 25 mM histidine, 10% sucrose, pH 7, and administered to mice via intravenous injection at a dose of 0.6 mg / kg on a QW x 4 schedule. Three days after each dose, 150 μL of whole blood was collected submandibularly and placed in a K2 EDTA tube stored on ice. Blood neutrophil counts were performed by IDEXX laboratories on the day of collection. Body weights were recorded twice weekly until the end of the study.

[0463] Example 2 - Homing peptides for SORT1 This example describes the development of certain sortilin-binding peptides (referred to in this example as "homing peptides") that are useful in accordance with the present disclosure.

[0464] Specifically, a specific sortilin-binding peptide was synthesized based on the C-terminal region of progranulin (Zheng, Y., PlosOne, 2011; Hu, F., Neuron, 2010).

[0465] A reference peptide, designated "PRGN_WT," was synthesized with the amino acid sequence APRWDAPLRDPALRQLL-COOH, which corresponds to the 17 C-terminal residues of human progranulin. Direct binding experiments confirmed that PRGN_WT bound to SORT1-Ag with high affinity. Specifically, PRGN_WT bound to SORT1-Ag with affinities of 36.6 nM for FP and 51 nM for GCI (Figures 3a and 4b).

[0466] To better understand the effect of affinity (e.g., low nM affinity vs. high nM affinity) on downstream efficacy and toxicity studies, we developed a derivative peptide, which we call "PRGN_RL," with the amino acid sequence DDPRAPWPALQRLALRL. We measured that PRGN_RL bound to SORT1-Ag with approximately 7-fold weaker affinity than the WT sequence (i.e., PRGN_WT). Specifically, we measured that PRGN_RL bound to SORT1-Ag with affinities of 132.9 nM for FP and 407 nM for GCI (Figures 3a and 5b).

[0467] As a negative control, we synthesized a reference peptide, which we called "PRGN_scr." This amino acid sequence was a scrambled version of the PRGN_WT sequence. Specifically, the amino acid sequence of PRGN_scr was QRLARLPRDLLAWPPAD. This peptide was shown to not bind in the FP assay.

[0468] Detailed biophysical characterization by DLS and CD further demonstrated that all three synthetic peptides (i.e., PRGN_WT, PRGN_RL, and PRGN_scr) exhibited good solubility and random coil secondary structure at the working concentration (250 nM, PBS, pH 7.4) (Figure 6b).

[0469] The stability of these PRGN-based peptides was further investigated in a mouse serum stability assay. PRGN_WT and PRGN_RL, respectively, showed T 1 / 2 = 3.16 and 27.7 minutes. In particular, additional peptides described herein were designed and developed to have improved stability characteristics (e.g., in a mouse serum stability assay) compared to one or more of PRGN_WT, PRGN_RL, and PRGN_scr, with a particular focus on such improved stability compared to PRGN_WT.

[0470] Example 3 - Biophysical characterization of PRGN-based PDCs This example describes certain conjugated agents (e.g., peptide drug conjugates (PDCs)) that use the provided sortilin-binding peptides, and certain biophysical characterization thereof.

[0471] Certain homing peptides (e.g., certain sortilin-binding peptides and / or certain reference peptides) were converted into peptide-drug conjugates (PDCs). Specifically, the cytotoxic drug MMAE was conjugated to the N-terminus of the homing peptide via a valine-citrulline VCPAB linker to generate PDCs. The structure of PRGN_WT_PDC is shown in Figure 9. Additional homing peptides PRGN_RL and PRGN_scr were chemically functionalized to PDCs using the same method. PDCs were synthesized to 95% purity for downstream experiments, as monitored by MS-MS, LC-MS, and HPLC (Figure 10).

[0472] PDC was recharacterized in certain biophysical assays (specifically, DLS and CD in this example) and demonstrated that it remained soluble and behaved well in DLS and in a random coil secondary structure ( FIG. 7 b). Characterization in FP competitive binding assays demonstrated an approximately 2.5-fold increase in affinity for both PRGN_WT_PDC and PRGN_WT_PDC compared to the parent homing peptide ( FIG. 8 ), while the negative control PRGN_scr_PDC continued to fail to bind. Additional functional assays were then performed with these PDCs.

[0473] Example 4 - PRGN-based PDC kills SORT1-expressing cancer cells This example demonstrates the performance of certain provided PRGN-based PDCs in a cancer cell killing assay using cancer cells that overexpress sortilin.

[0474] The breast cancer cell lines HCC70 and MDA-MB-231 were selected based on high SORT1 gene expression levels.

[0475] Both PRGN_WT_PDC and PRGN_WT_RL potently killed sortilin-overexpressing cancer cells (IC50 = 20.8 nM and 111.9 nM, respectively, in MDA-MB-231 cells), with PRGN_WT_PDC exhibiting approximately 5-fold greater potency than PRGN_RL_PDC (Figure 11). The difference in potency between PRGN_WT_PDC and PRGN_RL_PDC correlated well with the difference in affinity for SORT1, supporting a SORT1-specific mechanism.

[0476] Example 5 - PRGN-based PDC is well tolerated in a mouse MTD study CMC Survey This example demonstrates, among other things, the investigation of optimal vehicles and precipitation risk for in vivo studies by testing certain provided PDC in a formulation buffer screen. The following buffers were screened: 25 mM histidine, 10% sucrose (pH 7), 50 mM acetate / acetic acid 10% sucrose (pH 5), 1× PBS (pH 7.4), saline, 50 mM HEPES, 10% sucrose (pH 7). PDC was soluble in all buffers tested (Table 1), and we proceeded with 25 mM histidine, 10% sucrose (pH 7) as the optimal buffer for in vivo studies. [Table 1]

[0477] Mouse MTD study This example specifically demonstrates the identification of the maximum tolerated dose (MTD) of a specific PRGN-based PDC provided as a single agent in naive female BALB / c nude mice. Doses of 3, 5, and 10 mg / kg were administered. A weight loss of 20% or greater was defined as the MTD, and body weights were measured daily.

[0478] It was observed that high doses of PRGN_WT_PDC showed significant toxicity (Fig. 12c, P<0.0001 by two-way anova). Upon treatment with 10 mg / kg PRGN_WT_PDC, mice showed significant toxicity and a weight loss of over 20% 48 hours after treatment and were euthanized 96 hours after treatment. Upon treatment with 5 mg / kg PRGN_WT_PDC, mice showed toxicity and a sustained weight loss of approximately 15% 48 hours after treatment (P=0.029), which recovered by day 7.

[0479] PRGN_RL_PDC showed mild toxicity at the highest dose of 10 mg / kg. Without wishing to be bound by any particular theory, we propose that the relatively mild toxicity observed may be attributed to the significantly lower affinity of the homing peptide for SORT1.

[0480] Example 6 - PRGN-based PDC tested in mouse xenograft studies This example demonstrates the efficacy of certain provided PRGN-based PDCs in an MDA-MB-231 xenograft efficacy model. PRGN_WT_PDC was dissolved in 25 mm histidine 10% sucrose (pH 7) and administered intravenously to mice at 3, 1, or 0.3 mg / kg on a QW x 4 schedule.

[0481] To characterize the effects of the MMAE payload alone, free MMAE was included at molar equivalents to PDC and tested as a separate group at 0.6 and 0.06 mg / kg.

[0482] In a xenograft efficacy model, dose-dependent tumor growth inhibition by PRGN_WT_PDC was observed (Fig. 21a, P<0.0001 by two-way Anova). Treatment with 3 mg / kg PRGN_WT_PDC inhibited tumor growth by 63.45% compared to vehicle at day 27 (Fig. 21a).

[0483] The toxicity of PRGN_WT_PDC compared to free MMAE was assessed by comparing body weight loss in an MDA-MB-231 xenograft model. At each administration of PRGN_WT-PDC, body weight fluctuations mirrored those observed with vehicle treatment (FIG. 21b). Free MMAE, administered at an equivalent dose to 3 mg / kg PRGN_WT_PDC, resulted in body weight fluctuations that corresponded to the duration of treatment.

[0484] Without wishing to be bound by theory, we propose that the low toxicity observed with PRGN_WT_PDC may result from specific targeting of SORT1-expressing MDA-MB-231 in the xenograft model, whereas the weight fluctuations caused by an equivalent dose of MMAE may result from nonspecific targeting of the free drug.

[0485] Example 7 - Exemplary peptides provided This example describes certain sortilin-binding peptides provided by the present disclosure. Among other things, the present disclosure provides insight that unnatural amino acids can be particularly useful in sortilin-binding peptides (especially in sortilin-binding polypeptides corresponding to C-terminal Progranulin fragments). The peptides exemplified in this example include such unnatural amino acids.

[0486] Two different scaffolds (linear and cyclic) are utilized in these exemplary peptides. Approximately 600 linear peptides and approximately 600 cyclic peptides were designed, each of which incorporates one or more unnatural amino acids.

[0487] Peptides were investigated for binding to SORT1-Ag in a peptide microarray screen (Figures 13 and 14). As a negative control, peptide arrays were first probed with a secondary antibody (HRP-conjugated rabbit anti-His6 antibody from ICL, diluted 1:20,000). Peptide arrays containing linear designs showed low levels of nonspecific binding when probed with the negative control (Figure 13a). In arrays containing cyclic designs, some sequences showed nonspecific binding to the negative control secondary antibody, particularly at positions G8, B29, and T23 (Figure 13b). Peptides that showed nonspecific binding were excluded from further investigation and were not scored as positive hits.

[0488] After testing for nonspecific binding, peptides that specifically bound to sortilin were identified by probing the arrays with 30 μg / mL SORT1 antigen. Sortilin-specific hits were identified on both arrays. Specifically, 19 linear peptides and 15 cyclic peptides listed in Table 2 were measured and showed sortilin-specific binding. [Table 2-1] [Table 2-2]

[0489] As can be seen, these peptides feature consensus sequence elements: (R / N) X 2-3 (C / L) X 0-1 R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or APRWDAPLRXPALR (SEQ ID NO:2); wherein each X is independently any standard or non-standard amino acid.

[0490] Some of these peptides feature a consensus sequence: (R / N) X 2-3 CX 0-1 RQ (L / B13 / F02) (L / B13 / F02)

[0491] Some of these peptides feature a consensus sequence: (R / N) X10 X11 X12 CRQ

[0492] Some of these peptides feature a consensus sequence: X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

[0493] Some of these peptides feature a consensus sequence: (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

[0494] Some of these peptides feature a consensus sequence: (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (L / B13 / F02) (L / B13 / F02) wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

[0495] Some of these peptides feature a consensus sequence: CR X10 X11 C X13 RQ wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0496] Some of these peptides feature a consensus sequence: (R / H) N X6 X7 CR X10 X11 C X13 RQ wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0497] Some of these peptides feature a consensus sequence: (R / H) N X6 X7 CR X10 X11 C X13 RQ (L / B13 / F02) L / B13 / F02) wherein the two cysteine ​​residues form a disulfide bond and the polypeptide is a cyclic polypeptide.

[0498] Alternatively, or in addition, the cyclic peptides of Table 2 can be characterized by the following sequence patterns: Cyclic group 1: has a length of 14 or 15 amino acids and has a cysteine ​​residue at P13 which is disulfide bonded to a cysteine ​​residue at P4, P5, or P6. The sequence patterns are as follows: (R / E / X)(X / C)(C / X)(X / C)XX(R / N)XXXCRQ(L / X)(L / X) Cyclic group 2: 14 amino acids in length, with a cysteine ​​residue at P12, which is disulfide bonded to a cysteine ​​residue at P8. The sequence pattern is as follows: (R / H / X)NXXCRXXCXRQ(L / X)(L / X) wherein each X is independently any standard or non-standard amino acid.

[0499] A specific exemplary linear peptide has the amino acid sequence: APRWDAPLR(D / F / X)PALR(Q / X)(L / X)(L / X). Thus, no more than four positions are altered in these exemplary 17-mer linear sequences compared to that of Progranulin. These exemplary specific peptides therefore have amino acid sequences that are at least 76% identical to the amino acid sequence of the corresponding fragment of PGRN (or PGRN-SAR-Q15).

[0500] Without wishing to be bound by any particular theory, we propose that the C-terminal portion of the sequence, R(D / F / X)PALR(Q / X)(L / X)(L / X), is responsible for sortilin binding and, for example, contains the major driver of binding affinity. We further propose that the N-terminal portion of the sequence, APRWDAPL, can bind to a secondary site on the target, which contributes to binding affinity, albeit to a lesser extent.

[0501] Taking into account the 3D structural data, additional peptides were designed that may have further affinity and improved properties. Specifically, peptides (e.g., linear peptides) were designed with the following sequences: XP(R / X)(W / X)(D / X)APL(R / X)(D / F / X)PAL(R / X)(Q / X)(L / X)(L / X) wherein X represents any one or more of the naturally occurring amino acids specified in Residue Substitution Tables 3-5 below. Certain exemplified preferred linear peptides have a sequence at least about 70% identical to that of a corresponding fragment of Progranulin, or more preferably at least about 75% identical to such a corresponding fragment, as described above (e.g., having a length in the range of about 4-20, preferably about 15-20, and often 17 amino acids). In certain exemplified preferred peptides (and specifically in certain exemplified preferred linear peptides), one or more of positions P3, P4, P10, and P17 are modified (e.g., substituted with a natural or non-natural amino acid compared to the residue at those positions in human Progranulin). [Table 15] [Table 3] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 6] [Table 7]

[0502] Conjugation site At least the following sites are identified as suitable for conjugation with a linker and payload: P3 relative to cyclic group 1 P4 for cyclic groups 1 and 2 P8 for cyclic group 1 P10 for cyclic group 2

[0503] FIG. 19 depicts exemplary sequence logos for these two cyclic groups.

[0504] Peptides showing the greatest binding (highest i density on the array) were selected and further synthesized by SPPS.

[0505] A total of 19 linear and 15 cyclic hits were characterized for solubility by DLS and secondary structure by CD (Table 2). 95% were found to exhibit good solubility and random coil secondary structure at the working concentration (250 nM, PBS, pH 7.4). Three of the linear designs had DLS results suggesting aggregation and were therefore excluded from the remainder of the workflow (Figures 14-15).

[0506] Using the FP competition assay, 16 soluble linear sequence hits were profiled. Of these, 10 sequences showed improved affinity for SORT1 compared to PRGN_WT (Figure 16, Table 8). In particular, Arb-SAR-Q15 showed a 2-fold higher affinity (IC50 = 233.35 nM) compared to PRGN_WT. [Table 8]

[0507] Of the 15 cyclic array hits, Arb_cyclic_G23 showed comparable SORT1 affinity compared to PRGN_WT (Figure 17, Table 9, IC50 = 620 nM). The SORT1 affinities of the remaining cyclic designs range from 1621 nM to 4592 nM.

[0508] The stability of the computationally designed peptides was further investigated in a mouse serum stability assay (Tables 10-11). Of the 17 profiled linear sequences, 15 exhibited half-lives that were more than three-fold longer than PRGN_WT. In particular, Arb-SAR-Q15 had a T1 / 2 of 16.92 min. The cyclic peptides, overall, exhibited significantly longer half-lives. A total of 15 profiled cyclic sequences exhibited half-lives that were more than three-fold longer than PRGN_WT, with 10 of these exhibiting a greater than 10-fold increase. Arb_cyclic_F3 and Arb_cyclic_G10 exhibited particularly long half-lives of 172.58 min and 112.02 min, respectively. [Table 9] [Table 10] [Table 11]

[0509] Example 8 - PRGN-based non-native and cyclic PDC tested in mouse xenograft studies This example demonstrates the efficacy of certain provided PRGN-based PDCs in an MDA-MB-231 xenograft efficacy model. PRGN_WT_PDC, Arb-SAR-Q15-PDC, Arb_cyclic_G23-PDC, and control PRGN_scr_PDC were each dissolved in 25 mM histidine 10% sucrose (pH 7) and administered intravenously to mice at 3 mg / kg QW x 4.

[0510] To characterize the effect of the MMAE payload alone, free MMAE was included at molar equivalent to PDC and tested at 0.6 mg / kg.

[0511] In a xenograft efficacy model, tumor growth inhibition was observed with PRGN_WT_PDC, Arb-SAR-Q15-PDC, Arb_cyclic_G23-PDC, and PRGN_scr_PDC (Fig. 22a, P < 0.01 by two-way Anova). Upon treatment with 3 mg / kg of PRGN_WT_PDC, Arb-SAR-Q15-PDC, Arb_cyclic_G23-PDC, and PRGN_scr_PDC, tumor growth was inhibited by 96%, 98%, 68%, and 73%, respectively, compared to vehicle at day 22 (Fig. 22a). It is hypothesized that PRGN_scr_PDC has a lower affinity for the target and is therefore likely to exhibit lower tumor inhibition.

[0512] The toxicity of PRGN_WT_PDC, Arb-SAR-Q15-PDC, Arb_cyclic_G23-PDC, and PRGN_scr_PDC compared to free MMAE was evaluated by comparing body weight loss in an MDA-MB-231 xenograft model. Treatment with PRGN_WT_PDC, Arb-SAR-Q15-PDC, Arb_cyclic_G23-PDC, and PRGN_scr_PDC tracked weight loss relative to each other (Figure 22b). Free MMAE administered at molar equivalents to PDC resulted in body weight fluctuations that corresponded to the treatment time, while treatment with vehicle resulted in the least weight loss.

[0513] Without wishing to be bound by theory, we propose that the low toxicity observed with PRGN_WT_PDC, Arb-SAR-Q15-PDC, and Arb_cyclic_G23-PDC may result from specific targeting of SORT1-expressing MDA-MB-231 in the xenograft model. On the other hand, the body weight fluctuations caused by an equivalent dose of MMAE may result from nonspecific targeting of the free drug. Furthermore, we propose that the low toxicity observed with PRGN_scr_PDC may result from conjugating the scrambled (scr) peptide with various components to form PDC.

[0514] Pharmacokinetic evaluation The reaction rate constants (k a and k d ), and the equilibrium binding constant (K d ) was assessed using SPR as detailed in Example 1. Table 12 lists the binding constants for each of the three PDCs tested. [Table 12]

[0515] Mouse plasma stability assay The plasma stability for each of PRGN_WT_PDC, Arb-SAR-Q15-PDC, and Arb_cyclic_G23-PDC was assessed using the mouse plasma stability assay detailed in Example 1. Table 13 tabulates the plasma stability for each of the three PDCs tested. [Table 13]

[0516] As shown in Table 13, T for PGRN_WT_PDC 1 / 2 was measured to be 17.1 min, which is comparable to the other two PDCs tested, i.e., Arb-SAR-Q15-PDC (T 1 / 2 =49.7 minutes), and Arb_cyclic_G23-PDC(T 1 / 2 = 57.4 min). Despite the lower serum half-life of PRGN_WT_PDC compared to the other PDCs tested, PRGN_WT_PDC treatment resulted in significant tumor growth inhibition. For example, both PRGN_WT_PDC and Arb-SAR-Q15-PDC treatment surprisingly resulted in similar tumor growth inhibition of 96% and 98%, respectively, in a mouse xenograft efficacy model. Furthermore, PRGN_WT_PDC treatment surprisingly resulted in a significantly increased 96% tumor growth inhibition compared to the 68% inhibition with Arb_cyclic_G23-PDC treatment (see Figure 22a).

[0517] That is, the inventors of the present application have surprisingly discovered that although PGRN_WT_PDC may not appear to be effective in targeting and / or reducing tumor growth in vitro due to its lower half-life compared to other PDCs such as Arb-SAR-Q15-PDC and Arb_cyclic_G23-PDC, in an in vivo mouse model PGRN_WT_PDC performs surprisingly well, if not significantly better than PDCs with longer half-lives.

[0518] That is, in some embodiments, PDCs with less in vitro stability can be used in the therapeutic methods or compositions described herein to achieve comparable or better therapeutic results (e.g., tumor growth inhibition).

[0519] Mouse hematotoxicity assay As outlined in the Materials and Methods section of Example 1, wild-type Balb / c mice (female, 7-8 weeks old, weighing approximately 18-22 g) were randomized and administered control or test article intravenously on a QA x 4 schedule. Dose was adjusted based on body weight and administered at 10 mL / kg. PRGN_WT_PDC, Arb-SAR-Q15-PDC, and Arb_cyclic_G23-PDC were dissolved in 25 mM histidine, 10% sucrose, pH 7, and administered at 3 mg / kg. Free MMAE payload was also tested; similarly, it was dissolved in 25 mM histidine, 10% sucrose, pH 7, and administered to mice via intravenous injection at a dose of 0.6 mg / kg on a QW x 4 schedule. Three days after each dose, 150 μL of whole blood was collected submandibularly and placed in a K2 EDTA tube stored on ice. Blood neutrophil counts were performed by IDEXX laboratories on the day of collection. Body weights were recorded twice weekly until the end of the study.

[0520] The results of this study are shown in Figure 23. As shown, PRGN_WT_PDC, Arb-SAR-Q15-PDC, and Arb_cyclic_G23-PDC show a reduction in neutrophil counts compared to vehicle or MMAE treatment.

[0521] References 1. Petersen, C. M., Nielsen, M. S., Jacobsen, C., Tauris, J., Jacobsen, L., Gliemann, J., Moestrup, S. K. & Madsen, P. (1999). Propeptide cleavage conditions sortilin / neurotensin receptor-3 for ligand binding. The EMBO Journal, 18(3), 595-604. https: / / doi.org / 10.1093 / emboj / 18.3.595 2. Bartkowska, K., Turlejski, K. & Djavadian, R. L. (2010). Neurotrophins and their receptors in early development of the mammalian nervous system. Acta Neurobiologiae Experimentalis, 70(4), 454-467. 3. Zhou, X., Paushter, D. H., Feng, T., Sun, L., Reinheckel, T. & Hu, F. (2017). Lysosomal processing of progranulin. Molecular Neurodegeneration, 12(1), 62. https: / / doi.org / 10.1186 / s13024-017-0205-9 4. Nielsen, M. S., Jacobsen, C., Olivecrona, G., Gliemann, J. & Petersen, C. M. (1999). Sortilin / Neurotensin Receptor-3 Binds and Mediates Degradation of Lipoprotein Lipase*. Journal of Biological Chemistry, 274(13), 8832-8836. https: / / doi.org / 10.1074 / jbc.274.13.8832 5. Kim, J. T., Napier, D. L., Weiss, H. L., Lee, E. Y., Townsend, C. M. & Evers, B. M. (2017). Neurotensin Receptor 3 / Sortilin Contributes to Tumorigenesis of Neuroendocrine Tumors Through Augmentation of Cell Adhesion and Migration1. Neoplasia (New York, N.Y.), 20(2), 175-181. https: / / doi.org / 10.1016 / j.neo.2017.11.012 6. Rhost, S., Hughes, E., Harrison, H., Rafnsdottir, S., Jacobsson, H., Gregersson, P., Magnusson, Y., Fitzpatrick, P., Andersson, D., Berger, K., Stahlberg, A. & Landberg, G. (2018). Sortilin inhibition limits secretion-induced progranulin-dependent breast cancer progression and cancer stem cell expansion. Breast Cancer Research: BCR, 20(1), 137. https: / / doi.org / 10.1186 / s13058-018-1060-5 7. Roselli, S., Pundavela, J., Demont, Y., Faulkner, S., Keene, S., Attia, J., Jiang, C. C., Zhang, X. D., Walker, M. M. & Hondermarck, H. (2015). Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion. Oncotarget, 6(12), 10473-10486. https: / / doi.org / 10.18632 / oncotarget.3401 8. Berger, K., Rhost, S., Rafnsdottir, S., Hughes, E., Magnusson, Y., Ekholm, M., Stal, O., Ryden, L. & Landberg, G. (2021). Tumor co-expression of progranulin and sortilin as a prognostic biomarker in breast cancer. BMC Cancer, 21(1), 185. https: / / doi.org / 10.1186 / s12885-021-07854-0 9. Gao, F., Griffin, N., Faulkner, S., Li, X., King, S. J., Jobling, P., Denham, J. W., Jiang, C. C. & Hondermarck, H. (2020). The Membrane Protein Sortilin Can Be Targeted to Inhibit Pancreatic Cancer Cell Invasion. The American Journal of Pathology, 190(9), 1931-1942. https: / / doi.org / 10.1016 / j.ajpath.2020.05.018 10. Akil, H., Perraud, A., Melin, C., Jauberteau, M.-O. & Mathonnet, M. (2011). Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival. PloS ONE, 6(9), e25097. https: / / doi.org / 10.1371 / journal.pone.0025097 11. Ghaemimanesh, F. (n.d.). The Effect of Sortilin Silencing on Ovarian Carcinoma Cells. 12. Hemmati, S., Zarnani, A. H., Mahmoudi, A. R., Sadeghi, M.-R., Soltanghoraee, H., Akhondi, M. M., Tarahomi, M., Jeddi-Tehrani, M. & Rabbani, H. (2009). Ectopic Expression of Sortilin 1 (NTR-3) in Patients with Ovarian Carcinoma. Avicenna Journal of Medical Biotechnology, 1(2), 125-131. 13. Farahi, L., Ghaemimanesh, F., Milani, S., Razavi, S. M., Akhondi, M. M. & Rabbani, H. (2019). Sortilin as a Novel Diagnostic and Therapeutic Biomarker in Chronic Lymphocytic Leukemia. Avicenna Journal of Medical Biotechnology, 11(4), 270-276. 14. Zheng, Y., Brady, OA, Meng, PS, Mao, Y. & Hu, F. (2011). C-Terminus of Progranulin Interacts with the Beta-Propeller Region of Sortilin to Regulate Progranulin Trafficking. PloS ONE, 6(6), e21023. https: / / doi.org / 10.1371 / journal.pone.0021023 15. Hu, F., Padukkavidana, T., Vaegter, CB, Brady, OA, Zheng, Y., Mackenzie, IR, Feldman, HH, Nykjaer, A. & Strittmatter, SM (2010). Sortilin-Mediated Endocytosis Determines Levels of the Frontotemporal Dementia Protein, Progranulin. Neuron, 68(4), 654-667. https: / / doi.org / 10.1016 / j.neuron.2010.09.034

[0522] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

1. A conjugate comprising: (a) a polypeptide; (b) a payload; and (c) optionally a linker; The polypeptide is comprising a sortilin binding moiety having a length in the range of about 12 to about 20 amino acids and having a characteristic sequence represented by: (R / N) X ２－３ (C / L) X ０－１ R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or A P R W D A P L R X P A L R (SEQ ID NO: 2); wherein X is any standard or non-standard amino acid.

2. The sortilin binding moiety is 2. The conjugate of claim 1, which corresponds to a C-terminal fragment of Progranulin, or a variant thereof, and comprises no more than about 20 consecutive residues corresponding to consecutive Progranulin residues.

3. 2. The conjugate of claim 1, wherein the sortilin binding moiety is or comprises a cyclic peptide.

4. The characteristic sequence is (R / N) X ２－３ C X ０－１ The conjugate of claim 1 , represented by R (Q / E) (SEQ ID NO: 3).

5. The characteristic sequence is (R / N) X ２－３ L X ０－１ The conjugate of claim 1 , represented by R (Q / B43 / B50) (SEQ ID NO: 4).

6. 2. The conjugate of claim 1, wherein the sortilin binding moiety is or comprises a linear peptide and the characteristic sequence is represented by SEQ ID NO:

2.

7. 6. The conjugate of claim 5, wherein the sortilin binding moiety is or comprises a linear peptide.

8. 10. A conjugate according to any one of the preceding claims, wherein X is an amino acid contained in one or more of Tables 2 to 7.

9. The characteristic sequence is (R / N) X ２－３ C / L X ０－１ 2. The conjugate of claim 1, represented by RQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 5).

10. The characteristic sequence is (R / N) X ２－３ C X ０－１ 2. The conjugate of claim 1, represented by R Q (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 6).

11. The conjugate of claim 1 , wherein the characteristic sequence comprises at least a second cysteine ​​residue and the polypeptide comprises at least one disulfide bond between the cysteine ​​residues.

12. The characteristic sequence is 2. The conjugate of claim 1, represented by (R / N) X10 X11 X12 C R Q (SEQ ID NO: 7).

13. The characteristic sequence is X4 X5 X6 X7 X8 (R / N) X10 X11 X12 C R Q (SEQ ID NO: 8), 2. The conjugate of claim 1, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

14. The conjugate of claim 13 , wherein the polypeptide comprises at least one disulfide bond between cysteine ​​residues.

15. The characteristic sequence is (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 C R Q (SEQ ID NO: 9), 2. The conjugate of claim 1, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

16. 16. The conjugate of claim 15, wherein the polypeptide comprises at least one disulfide bond between cysteine ​​residues.

17. The characteristic sequence is (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 C R Q (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 10), 2. The conjugate of claim 1, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

18. 18. The conjugate of claim 17, wherein the polypeptide comprises at least one disulfide bond between cysteine ​​residues.

19. The characteristic sequence is C R X10 X11 C X13 R Q (SEQ ID NO: 11), 2. The conjugate of claim 1, wherein the two cysteine ​​residues form a disulfide bond and the polypeptide is a cyclic polypeptide.

20. The characteristic sequence is (R / H) N X6 X7 C R X10 X11 C X13 R Q (SEQ ID NO: 12), 2. The conjugate of claim 1, wherein the two cysteine ​​residues form a disulfide bond and the polypeptide is a cyclic polypeptide.

21. The characteristic sequence is (R / H) N X6 X7 C R X10 X11 C X13 R Q (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 13), 2. The conjugate of claim 1, wherein the two cysteine ​​residues form a disulfide bond and the polypeptide is a cyclic polypeptide.

22. The sortilin binding moiety is (i) a basic residue, such as L-arginine or an analog thereof, at a position corresponding to position P3 and / or P4 of the 17-mer C-terminal fragment of Progranulin; (ii) a hydrophobic residue, such as L-tryptophan or an analog thereof, at a position corresponding to position P4 of the 17-mer C-terminal fragment of Progranulin; (iii) a long-chain hydrophobic residue at a position corresponding to position P10 of the 17-mer C-terminal fragment of Progranulin; (iv) a covalent bond, such as a disulfide bond, between residues at positions corresponding to positions P5 and P13, or P8 and P12, of the 17-mer C-terminal fragment of Progranulin; and (v) A conjugate according to any one of the preceding claims, comprising one or more hydrophobic residues such as leucine or leucine at positions corresponding to positions P16 and / or P17 of the 17-mer C-terminal fragment of Progranulin.

23. 10. A conjugate according to any one of the preceding claims, wherein X represents a standard amino acid.

24. 10. The conjugate of any one of the preceding claims, wherein X is a non-standard amino acid.

25. 25. The conjugate of claim 24, wherein the non-standard amino acid is selected from those in one or more of Tables 2-7.

26. 10. The conjugate of any one of the preceding claims, wherein the payload is or comprises a therapeutic payload.

27. The sortilin binding moiety is (i) exhibit an affinity (Kd) for human sortilin 1 of less than about 1 μM, as determined by fluorescence polarization; (ii) an IC of less than about 12 μM in a competitive binding assay with a reference C-terminal Progranulin fragment ５０ 2. The conjugate of claim 1, wherein:

28. 28. The conjugate of claim 27, further characterized in that the sortilin binding moiety exhibits greater stability when maintained in mouse serum than the stability of the reference C-terminal Progranulin fragment.

29. 28. The conjugate of claim 27, further characterized in that the sortilin binding moiety exhibits lower stability when maintained in mouse serum than the stability of the reference C-terminal Progranulin fragment.

30. The conjugate of any one of claims 27 to 29, wherein the reference C-terminal Progranulin fragment has an amino acid sequence that is or includes APRWDAPLRDPALRQLL.

31. 30. The conjugate of any one of claims 27 to 29, wherein the sortilin binding moiety is characterized by a stability half-life in mouse serum of greater than about 3 minutes.

32. 32. The conjugate of claim 31, wherein the stability half-life in mouse serum is about 5 minutes.

33. 32. The conjugate of claim 31, wherein the stability half-life in mouse serum is about 10 minutes.

34. 32. The conjugate of claim 31, wherein the stability half-life in mouse serum is about 14 minutes.

35. 31. The conjugate of claim 30, characterized by an IC50 in the competitive binding assay of less than about 5000 nM.

36. 36. The conjugate of claim 35, wherein the IC50 is less than about 4000 nM.

37. 36. The conjugate of claim 35, wherein the IC50 is less than about 3000 nM.

38. 36. The conjugate of claim 35, wherein the IC50 is less than about 2000 nM.

39. 36. The conjugate of claim 35, wherein the IC50 is less than about 1000 nM.

40. 36. The conjugate of claim 35, wherein the IC50 is less than about 750 nM.

41. The conjugate of claim 35, wherein the IC50 is about 600-650 nM.

42. 42. The conjugate of any one of claims 35 to 41, wherein the sortilin binding moiety is or comprises a cyclic peptide.

43. 36. The conjugate of claim 35, wherein the IC50 is less than about 600 nM.

44. 36. The conjugate of claim 35, wherein the IC50 is less than about 500 nM.

45. 36. The conjugate of claim 35, wherein the IC50 is less than about 400 nM.

46. 36. The conjugate of claim 35, wherein the IC50 is less than about 300-400 nM.

47. 36. The conjugate of claim 35, wherein the IC50 is less than about 300-350 nM.

48. 44. The conjugate of any one of claims 43, wherein the sortilin binding moiety is or comprises a linear peptide.

49. 10. The conjugate of claim 1, wherein the conjugate selectively or specifically targets sortilin-expressing cells.

50. 10. The conjugate of claim 1, wherein the conjugate selectively or specifically targets cancer cells over normal cells.

51. 51. The conjugate of claim 50, wherein the payload is or comprises a cytotoxic moiety, and the conjugate kills the sortilin-expressing cells.

52. 52. The conjugate of claim 51, wherein the conjugate kills the sortilin-expressing cells with a potency greater than that observed for an otherwise identical conjugate in which the sortilin binding moiety is or comprises a reference peptide that is a C-terminal fragment of Progranulin.

53. 53. The conjugate of claim 52, wherein the reference peptide has the amino acid sequence APRWDAPLRDPALRQLL.

54. 54. The conjugate of claim 53, wherein the potency is at least about 5 times greater.

55. 51. The conjugate of claim 50, wherein the cancer cells express sortilin at levels equal to or greater than those expressed by otherwise comparable non-cancer cells.

56. The conjugate of claim 1, wherein the conjugate undergoes cellular internalization.

57. 2. The conjugate of claim 1, wherein the conjugate exhibits improved affinity for sortilin compared to that observed with the unconjugated sortilin binding moiety.

58. 58. The conjugate of claim 57, wherein such improved affinity is at least about two-fold greater.

59. The conjugate of claim 1 , wherein the payload is or comprises a therapeutic or diagnostic moiety.

60. The conjugate of claim 1 , wherein the payload is or comprises a therapeutic moiety.

61. The conjugate of claim 1 , wherein the payload is or comprises a cytostatic or cytotoxic moiety.

62. 62. The conjugate of claim 61, wherein the payload is or comprises a cytotoxic moiety.

63. 63. The conjugate of claim 62, wherein the cytotoxic moiety is characterized by a sub-nanomolar IC50 against relevant cells.

64. 62. The conjugate of claim 61, wherein the cytotoxic moiety is or comprises a bacterial toxin.

65. 10. The conjugate of claim 1, wherein the payload is or comprises an anti-cancer drug.

66. 2. The conjugate of claim 1, wherein the payload is selected from the group consisting of alkylating agents, antimetabolites, antitumor antibiotics, boron neutron capture therapeutics, cell cycle inhibitors, kinesin spindle protein inhibitors, microtubule binding agents, topoisomerase inhibitors, and combinations thereof.

67. 2. The conjugate of claim 1, wherein the payload is or comprises an alkaloid, an anthracycline, an auristatin, a camptothecin, a folic acid derivative, a metal complex, a nucleoside analog, a taxane, a vinca alkaloid analog, or a combination thereof.

68. 10. The conjugate of claim 1, wherein the payload is or comprises a phytochemical.

69. 2. The conjugate of claim 1, wherein the payload is monomethyl auristatin E (MMAE).

70. The conjugate of claim 1 , wherein the payload is a detectable entity.

71. The conjugate of claim 1 , wherein the payload is a small molecule.

72. The conjugate of claim 1 , wherein the payload is a polypeptide.

73. The conjugate of claim 1 , wherein the payload is an oligonucleotide.

74. 74. The conjugate of claim 73, wherein the polypeptide is mRNA.

75. The conjugate of claim 1 , wherein the payload is a particle.

76. 76. The conjugate of claim 75, wherein the payload is a lipid nanoparticle.

77. The conjugate of claim 1 , wherein the payload is a viral capsid.

78. The conjugate of claim 1 , wherein the payload is a lipid vesicle such as an exosome or a liposome.

79. The conjugate of claim 1 , wherein the payload is or comprises a radioisotope.

80. 2. The conjugate of claim 1, wherein the payload is covalently attached to the linker or the peptide through a hydroxyl group, a carboxyl group, or an amine group.

81. 2. The conjugate of claim 1, wherein the payload is or comprises a protein degradation modulator, such as a proteasome inhibitor or a PROTAC agent.

82. The conjugate of claim 1, wherein the payload is or comprises a nucleic acid editing system such as CRISPR / Cas, TALEN, ADAR, etc.

83. The conjugate of any one of claims 12 to 21, wherein the payload is attached to an amino acid residue at position P4.

84. The conjugate according to any one of claims 12 to 18, wherein the payload is attached to an amino acid residue at position P3.

85. The conjugate of any one of claims 12 to 18, wherein the payload is attached to an amino acid residue at position P8.

86. The conjugate according to any one of claims 19 to 21, wherein the payload is attached to an amino acid residue at position P10.

87. 22. The conjugate of any one of claims 1 to 21, wherein at least one amino acid residue within the sequence of the sortilin binding moiety is conjugated to a payload moiety.

88. 22. The conjugate of any one of claims 1 to 21, wherein at least one amino acid within the sequence outside of P15, P16, and P17 of the sortilin binding moiety is conjugated to a payload moiety.

89. 22. The conjugate of any one of claims 1 to 21, wherein two or more amino acids within the sequence of the sortilin binding moiety are conjugated to a payload moiety.

90. 22. The conjugate of any one of claims 1 to 21, wherein two or more amino acids within the sequence outside P15, P16, and P17 of the sortilin binding moiety are conjugated to a payload moiety.

91. The conjugate of claim 1 , wherein the linker is cleavable.

92. 92. The conjugate of claim 91, wherein the linker is an acid-cleavable linker.

93. 92. The conjugate of claim 91, wherein the linker is an enzyme-cleavable linker.

94. The conjugate of claim 1 , wherein the linker is a VCPAB linker.

95. 10. The conjugate of claim 1, wherein the linker is or comprises an ester, an amide, a hydrozone, a carbonate, a reducible disulfide, and combinations thereof.

96. 2. The conjugate of claim 1, wherein the linker is or comprises a thioether, an oxime, a triazole, and combinations thereof.

97. The conjugate of claim 1 , wherein the linker is redox sensitive.

98. 1. An engineered sortilin-binding peptide, comprising: (a) a C-terminal fragment of Progranulin or a variant thereof, wherein the fragment comprises about 20 or fewer residues corresponding to consecutive C-terminal Progranulin residues; (b) a characteristic sequence having a length in the range of about 12 to about 20 amino acids and represented by: (R / N) X ２－３ (C / L) X ０－１ R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or A P R W D A P L R X P A L R (SEQ ID NO: 2), wherein X is any standard or non-standard amino acid.

99. an amino acid sequence ranging in length from about 12 to about 20 amino acids and comprising a characteristic sequence represented by: (R / N) X ２－３ (C / L) X ０－１ R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or A P R W D A P L R X P A L R (SEQ ID NO: 2), where X is any standard or non-standard amino acid; An engineered sortilin-binding peptide, wherein at least one X residue is an unnatural amino acid.

100. 100. The peptide of claim 98 or 99, wherein the unnatural amino acid is selected from those in one or more of Tables 2-7.

101. 100. The peptide of claim 98 or 99, comprising two cysteine ​​residues within said sequence that form a disulfide bond.

102. The characteristic sequence is (R / N) X ２－３ C X ０－１ 100. The peptide of any one of claims 98 or 99, represented by R(Q / E) (SEQ ID NO: 3).

103. The characteristic sequence is (R / N) X ２－３ L X ０－１ 100. The peptide of any one of claims 98 or 99, represented by R(Q / B43 / B50) (SEQ ID NO: 4).

104. The characteristic sequence is (R / N) X ２－３ C / LX ０－１ 100. The peptide of any one of claims 98 or 99, represented by RQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 5).

105. The characteristic sequence is (R / N) X ２－３ C X ０－１ 100. The peptide of any one of claims 98 or 99, represented by RQ(L / B13 / F02)(L / B13 / F02)(SEQ ID NO:6).

106. The characteristic sequence is 100. The peptide of any one of claims 98 or 99, represented by (R / N) X10 X11 X12 CRQ (SEQ ID NO: 7).

107. The characteristic sequence is X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (SEQ ID NO: 8) 100. The peptide of any one of claims 98 or 99, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

108. 108. The peptide of claim 107, wherein the two cysteine ​​residues in the sequence form a disulfide bond.

109. The characteristic sequence is (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 C R Q (SEQ ID NO: 9), 100. The peptide of any one of claims 98 or 99, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

110. 110. The peptide of claim 109, wherein the two cysteine ​​residues in the sequence form a disulfide bond.

111. The characteristic sequence is (R / E) X4 X5 X6 X7 X8 (R / N) X10 X11 X12 CRQ (L / B13 / F02) (L / B13 / F02) (SEQ ID NO: 10), 100. The peptide of any one of claims 98 or 99, wherein at least one of X4, X5, and X6 is a cysteine ​​residue.

112. 112. The peptide of claim 111, wherein the two cysteine ​​residues in the sequence form a disulfide bond.

113. The characteristic sequence is C R X10 X11 C X13 R Q (SEQ ID NO: 11), 100. The peptide of any one of claims 98 or 99, wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

114. The characteristic sequence is (R / H) N X6 X7 CR X10 X11 C X13 RQ (SEQ ID NO: 12), 100. The peptide of any one of claims 98 or 99, wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

115. The characteristic sequence is (R / H) N X6 X7 CR X10 X11 C X13 RQ (L / B13 / F02) L / B13 / F02) (SEQ ID NO: 13), 100. The peptide of any one of claims 98 or 99, wherein the two cysteines form a disulfide bond and the polypeptide is a cyclic polypeptide.

116. (i) a basic residue, such as L-arginine or an analog thereof, at a position corresponding to position P3 and / or P4 of the 17-mer C-terminal fragment of Progranulin; (ii) a hydrophobic residue, such as L-tryptophan or an analog thereof, at a position corresponding to position P4 of the 17-mer C-terminal fragment of Progranulin; (iii) a long-chain hydrophobic residue at a position corresponding to position P10 of the 17-mer C-terminal fragment of Progranulin; (iv) a covalent bond, such as a disulfide bond, between residues at positions corresponding to positions P5 and P13, or P8 and P12, of the 17-mer C-terminal fragment of Progranulin; (v) a hydrophobic residue such as leucine or leucine at a position corresponding to position P16 and / or P17 of the 17-mer C-terminal fragment of Progranulin.

10. A peptide according to the preceding claims, comprising one or more of:

117. X ｎ An engineered sortilin-binding peptide comprising the sequence RDPALRXLL (SEQ ID NO: 14).

118. 118. The peptide of claim 117, wherein n is 0.

119. 119. The peptide of claim 118, wherein X corresponds to an unnatural amino acid selected from those in one or more of Tables 2-7.

120. 119. The peptide of claim 118, wherein X is Q.

121. An engineered sortilin-binding peptide containing the sequence DDPRAPWPALQRLALRL.

122. a nucleic acid, the nucleotide sequence of which has a length in the range of about 12 to about 20 amino acids; (R / N) X ２－３ (C / L) X ０－１ R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or A P R W D A P L R X P A L R (SEQ ID NO: 2) a coding region encoding a peptide having an amino acid sequence comprising a characteristic sequence represented by wherein X is any standard or non-standard amino acid.

123. 123. The nucleic acid of claim 122, wherein the nucleic acid is DNA.

124. 123. The nucleic acid of claim 122, wherein the nucleic acid is RNA.

125. 123. The nucleic acid of claim 122, wherein the nucleic acid is mRNA.

126. The nucleic acid of any one of claims 122 to 125, wherein the nucleic acid is part of a viral vector.

127. A host cell comprising the nucleic acid of claim 122.

128. A pharmaceutical composition comprising or delivering a conjugate according to any one of claims 1 to 21 or an engineered peptide according to claim 98 or 99.

129. an active agent which is or comprises a conjugate according to any one of claims 1 to 21 or an engineered peptide according to claim 98 or 99; at least one pharmaceutically acceptable carrier; 129. The pharmaceutical composition of claim 128, comprising:

130. 130. The pharmaceutical composition of claim 129, wherein the payload is an anti-cancer drug.

131. 131. The pharmaceutical composition of claim 130, wherein the active agent further comprises an additional anti-cancer agent.

132. A conjugate according to claim 1 or an engineered peptide according to claim 98 or 99; at least one additional therapeutic agent; Including, combinations.

133. 133. The combination of claim 132, wherein the conjugate and the at least one additional therapeutic agent are comprised in the same pharmaceutical composition.

134. A pharmaceutical composition according to claim 128; at least one additional therapeutic agent; Including, combinations.

135. 135. The combination of claim 132 or 134, wherein one or both of the payload and the additional therapeutic agent is or comprises an anti-cancer agent.

136. 136. The combination of claim 135, wherein the anti-cancer agent is or comprises a small molecule.

137. 136. The combination of claim 135, wherein the anti-cancer agent is or comprises an antibody.

138. 1. A method of binding to sortilin, said method comprising:

10. The method comprising the step of contacting a system comprising sortilin with a conjugate or engineered peptide according to any one of the preceding claims.

139. 1. A method for inhibiting sortilin, said method comprising:

10. The method comprising the step of contacting a system in which sortilin is active with a conjugate or engineered peptide according to any one of the preceding claims.

140. 1. A method for reducing sortilin on a cell surface, the method comprising:

10. A method for treating a cell-containing cell line comprising contacting a cell line comprising: contacting a cell line comprising a cell surface comprising a Sortilin molecule with a conjugate or engineered peptide according to any one of the preceding claims, said contacting being carried out under conditions and for a time sufficient to allow said Sortilin to be internalised by said cell line. The method.

141. 1. A method for increasing the specificity of a therapeutic or diagnostic moiety for a target cell, said method comprising: providing said therapeutic moiety having a length in the range of about 12 to about 20 amino acids; (R / N) X ２－３ (C / L) X ０－１ R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or A P R W D A P L R X P A L R (SEQ ID NO: 2) to a sortilin-binding peptide having an amino acid sequence comprising a characteristic sequence represented by wherein X is any standard or non-standard amino acid.

142. 1. A method for delivering a payload to a sortilin-expressing cell, the method comprising: contacting the cells with the conjugate of claim 1, The method.

143. 1. A method for increasing the specificity of a payload for a sortilin-expressing cell, the method comprising: associating the payload with a polypeptide, the polypeptide comprising: comprising a sortilin binding moiety having a length in the range of about 12 to about 20 amino acids and having a characteristic sequence represented by: (R / N) X ２－３ (C / L) X ０－１ R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or A P R W D A P L R X P A L R (SEQ ID NO: 2); wherein X is any standard or non-standard amino acid.

144. 1. A method for increasing cellular uptake of a payload by a target cell, the method comprising: associating the payload with a polypeptide, the polypeptide comprising: (a) corresponds to a fragment of Progranulin, or a variant thereof, and comprises no more than about 20 consecutive residues corresponding to consecutive C-terminal Progranulin residues; (b) a characteristic sequence having a length in the range of about 12 to about 20 amino acids and represented by: (R / N) X ２－３ (C / L) X ０－１ R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or A P R W D A P L R X P A L R (SEQ ID NO: 2), wherein X is any standard or non-standard amino acid.

145. 145. The method of any one of claims 141 to 144, wherein the associating step is or comprises covalent bonding.

146. 145. The method of any one of claims 141-144, wherein the sortilin binding moiety and the therapeutic moiety are covalently associated through a linker.

147. The method of any one of claims 141 to 144, wherein the sortilin-expressing cell is a cancer cell.

148. 145. The method of any one of claims 141 to 144, wherein the sortilin-expressing cell is a mammalian cell.

149. 145. The method of any one of claims 141 to 144, wherein the sortilin-expressing cell is a human cell.

150. 10. A method of treating a subject suffering from a disease, disorder, or condition associated with sortilin-expressing cells, the method comprising delivering to the subject the conjugate of claim 1.

151. 151. The method of claim 150, wherein the subject has cancer and cancer cells in the subject express elevated levels of sortilin compared to reference non-cancerous cells.

152. 152. The method of claim 150 or claim 151, wherein the subject is undergoing or has undergone other cancer therapies.

153. 153. The method of claim 152, wherein the other cancer therapy is or comprises chemotherapy, cryotherapy, hormone therapy, immunotherapy, radiation therapy, surgery, and combinations thereof.

154. 151. The method of claim 150, wherein the administering step comprises parenterally administering the conjugate.

155. 151. The method of claim 150, wherein the patient has a cancer that is a member of the group consisting of breast cancer, colorectal cancer, glioblastoma, lung cancer, ovarian cancer, pancreatic cancer, small intestine cancer, thymus cancer, thyroid cancer, bladder cancer, prostate cancer, kidney cancer, liver cancer, endometrial cancer, skin cancer, gastric cancer, and combinations thereof.

156. 100. A method for preparing a conjugate according to claim 1 or a peptide according to claim 98 or claim 99, said method comprising: synthesizing said conjugate or peptide using standard solid phase peptide synthesis (SPPS) with Fmoc chemistry; deprotecting the conjugate or the peptide and cleaning the resin; purifying the conjugate or peptide using reverse-phase high performance liquid chromatography; The method comprising:

157. 1. A method for producing a conjugate, the method comprising: covalently associating a peptide corresponding to a C-terminal fragment of Progranulin, or a variant thereof, with a cytotoxic payload, wherein the peptide has an amino acid sequence comprising characteristic sequence elements represented by: (R / N) X ２－３ (C / L) X ０－１ R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or A P R W D A P L R X P A L R (SEQ ID NO: 2) wherein each X is independently any standard or non-standard amino acid.

158. 1. A method for producing a peptide having a length in the range of about 12 to about 20 amino acids and an amino acid sequence containing characteristic sequence elements represented by: (R / N) X ２－３ (C / L) X ０－１ R (Q / E / L / B43 / B50) (SEQ ID NO: 1) or A P R W D A P L R X P A L R (SEQ ID NO: 2); where X is any standard or non-standard amino acid. The method comprises: synthesizing said peptide using standard solid phase peptide synthesis (SPPS) with Fmoc chemistry; deprotecting the conjugate or the peptide and cleaning the resin; purifying the conjugate or peptide using reverse-phase high performance liquid chromatography; The method comprising:

159. 1. A method for producing a pharmaceutical composition, said method comprising: An active agent which is or comprises a conjugate or engineered peptide according to any one of the preceding claims, The method further comprising the step of contacting with at least one pharmaceutically acceptable carrier.

160. 98. The conjugate of any one of claims 1 to 97, wherein the sortilin binding moiety is or comprises the amino acid sequence APRWDAPLRDPALRQLL.

161. 98. The conjugate of any one of claims 1 to 97, wherein the sortilin binding moiety is or comprises the amino acid sequence APRWDAPLRDPALRQ(B13)(G48).

162. 162. The conjugate of any one of claims 160 or 161, wherein the linker is a VCPAB linker.

163. The conjugate of any one of claims 160 to 162, wherein the payload is MMAE.

164. 121. The peptide of any one of claims 98 to 120, comprising the sequence APRWDAPLRDPALRQLL.

165. A peptide according to any one of claims 98 to 120, comprising the sequence APRWDAPLRDPALRQ (B13) (G48).

166. 127. The nucleic acid of any one of claims 122 to 126, wherein the nucleotide sequence comprises a coding region encoding a peptide comprising the sequence APRWDAPLRDPALRQLL.

167. 127. The nucleic acid of any one of claims 122 to 126, wherein the nucleotide sequence comprises a coding region encoding a peptide comprising the sequence APRWDAPLRDPALRQ.

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