Transferrin receptor targeting peptides

Abstract

To provide a method delivering a drug into a target in a central nervous system (CNS), and a composition for treating and diagnosing CNS.SOLUTION: Provided are peptides capable of binding to transferrin receptor (TfR) and variants thereof as well as peptide constructs comprising peptides conjugated to, linked to, or fused to medicines. Therein the peptides and peptide constructs are capable of binding to the TfR. Binding of the peptide or peptide construct as described herein to the TfR can enable transcytosis across an endothelial layer, e.g., a blood brain barrier, or crossing of a cell membrane. Pharmaceutical compositions and uses of peptides and peptide constructs, as well as methods of designing and manufacturing such peptides and peptide constructs, to treat a disease or condition are also described herein.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 779,885, filed December 14, 2018, and U.S. Provisional Patent Application No. 62 / 836,520, filed April 19, 2019, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains an electronically submitted Sequence Listing in ASCII format, which is incorporated herein by reference in its entirety. This ASCII copy was created on December 12, 2019, is named 44189-723_601_SL.txt, and is 552,571 bytes in size. [Background technology]

[0003] Drug delivery to targets in the central nervous system (CNS) is hindered by the blood-brain barrier (BBB), a term referring to the endothelial cells of CNS capillaries, which are extremely difficult to cross. The BBB exists to prevent harmful metabolites and pathogens from entering the brain, but it also makes treating CNS diseases with conventional drugs particularly difficult. For example, primary CNS tumors (e.g., gliomas) and neuroinflammatory and neurodegenerative diseases (e.g., multiple sclerosis and Alzheimer's disease) respond particularly poorly to treatments that are highly effective for similar diseases affecting peripheral tissues. Therefore, improved approaches to delivering therapeutic and / or diagnostic molecules to the CNS and other organs and tissues are needed. Summary of the Invention

[0004] In various aspects, the present disclosure provides compositions comprising engineered transferrin receptor (TfR) binding peptides, or variants, homologs, fragments, or analogs thereof.

[0005] In various aspects, the present disclosure provides compositions comprising a peptide construct, wherein the peptide construct comprises: a) a transferrin receptor (TfR)-binding peptide, or a variant, homolog, fragment, or analog thereof; and b) an active agent, wherein the peptide is conjugated, linked, or fused to the active agent.

[0006] In various aspects, the present disclosure provides compositions comprising an engineered peptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs:1-32, SEQ ID NOs:36-67, SEQ ID NOs:136-167, SEQ ID NOs:171-202, or SEQ ID NOs:356-358, or a variant or functional fragment thereof.

[0007] In some embodiments, the peptide penetrates a cell layer, including the blood-brain barrier (BBB). In some embodiments, the peptide penetrates a membrane, wherein the membrane is a cell membrane. In some embodiments, the cell expresses TfR. In some embodiments, the peptide penetrates a cell membrane via TfR-mediated endocytosis.

[0008] In some embodiments, the peptide is delivered across the blood-brain barrier to the CNS by binding to and then dissociating from TfR. In some embodiments, the peptide is delivered across the blood-brain barrier by receptor-mediated transcytosis. In some embodiments, the cell is a tumor cell. In some embodiments, the cell or organ to which the peptide is localized overexpresses TfR. In some embodiments, the peptide comprises SEQ ID NOs: 206-224 and 334-344.

[0009] In some embodiments, the peptide comprises surface interface residues corresponding to G5, A7, S8, N14, L17, E18, E21, L38, L42, L45, D46, H47, S50, and Q51, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the peptide comprises hydrophilic surface distal residues corresponding to any one of positions 3, 4, 9, 11, 15, 16, 19, 23, 26, 28, 29, 30, 31, 32, 33, 35, 36, 37, 39, and 40, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the hydrophilic surface distal residues are selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises any one of the hydrophilic surface-distal residues corresponding to R3, E4, R9, K12, D15, E16, K19, R23, S26, S28, N29, T30, E31, E32, D33, E35, Q36, E37, E39, and D40, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the peptide comprises a hydrophilic residue at a position corresponding to any one of 15, 35, 39, or 49, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the hydrophilic residue is selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises a hydrophilic residue corresponding to D15, E35, E39, and H49, or any combination thereof, relative to SEQ ID NO: 32.

[0010] In some embodiments, the peptide comprises a hydrophobic residue at a position corresponding to 11, 25, or 27, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the hydrophobic residue is selected from amino acids A, M, I, L, V, F, W, or Y, or any combination thereof. In some embodiments, the peptide comprises any one of the hydrophobic residues corresponding to M11, M25, M27, or any combination thereof, relative to SEQ ID NO: 32.

[0011] In some embodiments, the peptide comprises an aliphatic residue corresponding to position 45, relative to SEQ ID NO: 32. In some embodiments, the aliphatic residue is selected from amino acids A, M, I, L, or V. In some embodiments, the peptide comprises an aliphatic residue corresponding to L45, relative to SEQ ID NO: 32.

[0012] In some embodiments, the peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least five disulfide bonds. In some embodiments, the peptide comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-32, SEQ ID NOs: 36-67, SEQ ID NOs: 136-167, SEQ ID NOs: 171-202, SEQ ID NOs: 356-358, or a variant or fragment thereof. In some embodiments, the peptide comprises any one of SEQ ID NOs: 1-32, SEQ ID NOs: 36-67, SEQ ID NOs: 136-167, SEQ ID NOs: 171-202, SEQ ID NOs: 356-358, or a variant or fragment thereof. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 2. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 4. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 30. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 32.

[0013] In some embodiments, the peptide is further formulated, fused, or conjugated with a cell-penetrating moiety, such as a polycation, a polyorganic acid, an endosome-releasing polymer, poly(2-propylacrylic acid), poly(2-ethylacrylic acid), Tat peptide, Arg patch, knot peptide, CysTAT, S19-TAT, R8 (SEQ ID NO: 73), pAntp, Pas-TAT, Pas-R8 (SEQ ID NO: 76), Pas-FHV, Pas-pAntP, F2R4 (SEQ ID NO: 79), B55, aurein, IMT-P8, BR2, OMOTAG1, OMOTAG2, p In some embodiments, the cell-penetrating moiety is any one of SEQ ID NOs:71-96, SEQ ID NOs:98-101, SEQ ID NOs:116-126, and SEQ ID NOs:403-455.

[0014] In some embodiments, the peptide sequence is at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58 residues, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, or at least 81 amino acid residues.

[0015] In some embodiments, an active sequence is grafted onto the peptide, or the peptide is conjugated, linked, fused, or grafted to an active agent to provide a peptide construct. In some embodiments, the peptide construct has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs:33-35, SEQ ID NOs:68-70, SEQ ID NOs:168-170, SEQ ID NOs:203-205, SEQ ID NOs:225-332, SEQ ID NOs:359-361, SEQ ID NOs:373, SEQ ID NO:375, SEQ ID NO:381, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:393, SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:400, or SEQ ID NO:401.

[0016] In some embodiments, the active agent is linked via a linker, wherein the linker is selected from any one of Table 7 or SEQ ID NO:103-SEQ ID NO:115, or SEQ ID NO:125. In some embodiments, the active agent is an immunotherapeutic agent, a CTLA-4 targeted agent, a PD-1 targeted agent, a PDL-1 targeted agent, an IL15 agent, a fused IL-15 / IL-15Ra combination agent, an IFN gamma agent, an anti-CD3 agent, an ion channel modulator, a Kv1.3 inhibitor, an auristatin, MMAE, a maytansinoid, DM1, DM4, doxorubicin, a calicheamicin, a platinum compound, cisplatin, a taxane, paclitaxel, SN-38, a BACE inhibitor, a Bcl-xL inhibitor, WEHI-539, venetoclax, ABT-199, navitoclax, AT-101, obatoclax, a pyrrolobenzodiazepine or pyrrolobenzodiazepine dimer, a dolastatin, or a neurotransmitter. In some embodiments, the Kv1.3 inhibitor is Vm24, ShK-170, ShK-186, or ShK-192. In some embodiments, Vm24 has the sequence of SEQ ID NO:378 or SEQ ID NO:391. In some embodiments, Shk-185 has the sequence of SEQ ID NO:379, SEQ ID NO:390, or SEQ ID NO:402. In some embodiments, the neurotransmitter is neurotensin, a neurotensin peptide variant, or a functional fragment thereof. In some embodiments, the neurotensin peptide variant has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:350 or SEQ ID NOs:365-369.

[0017] In some embodiments, the composition further comprises a half-life modifying agent conjugated to the peptide. In some embodiments, the half-life modifying agent comprises a polymer, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, or a molecule that binds to albumin. In some embodiments, the half-life modifying agent comprises SA21. In some embodiments, SA21 has the sequence of SEQ ID NO: 376 or SEQ ID NO: 377.

[0018] In some embodiments, the composition further comprises a detectable agent attached to the peptide by a linker. In some embodiments, the detectable agent is linked via a linker, wherein the linker is selected from any one of Table 7 or SEQ ID NOs: 103-115, or SEQ ID NO: 125. In some embodiments, the detectable agent is a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an x-ray contrast agent, a PET agent, a radionuclide, or a radionuclide chelator.

[0019] In various aspects, the present disclosure provides a pharmaceutical composition comprising any of the compositions described herein, or a salt thereof, and a pharmaceutically acceptable carrier.

[0020] In various aspects, the present disclosure provides methods for transporting a composition across a cell layer, the method comprising: a) contacting the cell layer with a composition comprising any of the peptides described herein or any of the pharmaceutical compositions described herein; and b) transporting the composition across the cell layer.

[0021] In various aspects, the present disclosure provides methods for treating a condition in a subject in need thereof, comprising: a) administering to the subject a composition comprising any of the peptides described herein or any of the pharmaceutical compositions described herein; and b) transporting the composition across a cell layer.

[0022] In various aspects, the present disclosure provides methods of treating a condition in a subject in need thereof, comprising administering to the subject an amount of a composition comprising any of the peptides described herein or any of the pharmaceutical compositions described herein based on expression of TfR in a tissue of the subject.

[0023] In some embodiments, the peptide construct is capable of binding to and activating a neurotensin receptor. In some embodiments, the method further comprises reducing the level of pain by administering the peptide to the subject. In some embodiments, the administration is by inhalation, intranasal administration, oral administration, topical administration, intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intratumoral administration, intrathecal administration, or a combination thereof. In some embodiments, the composition is administered intravenously as a bolus, injection, or continuous infusion.

[0024] In some embodiments, the method further comprises administering the peptide to a patient suffering from schizophrenia, substance abuse, Parkinson's disease, hypertension, eating disorders, stroke, Alzheimer's disease, cancer, inflammation, pain, epilepsy, mood disorders, or endocrine disorders. In some embodiments, the subject has cancer. In some embodiments, the subject has a CNS disease. In some embodiments, the subject has neuropathic pain, migraine, anxiety, or epilepsy.

[0025] In various aspects, the present disclosure provides a method for transporting a composition according to any one of claims 1-53 or a pharmaceutical composition according to claim 54 across a cell layer, the method comprising contacting the composition with a cell layer and transporting the composition across the cell layer.

[0026] In various aspects, the present disclosure provides a method of treating a condition in a subject in need thereof, comprising administering to the subject a composition of any one of claims 1-53 or a pharmaceutical composition of claim 54.

[0027] In some embodiments, transporting comprises binding of the composition to TfR. In some embodiments, transporting further comprises transcytosis. In some embodiments, the transcytosis is vesicular transcytosis. In some embodiments, the transcytosis is TfR-mediated. In some embodiments, transporting further comprises dissociating the composition from TfR. In some embodiments, transporting further comprises releasing the active agent from the composition.

[0028] In various aspects, the disclosure provides methods for designing TfR-binding peptides, the method comprising: a) using a computer program to identify stability- and folding-optimized peptides from a library of peptides, wherein each peptide in the library of peptides contains at least three intramolecular disulfide bonds; b) using a computer program to superimpose amino acid residues of binding patches of a peptide:TfR complex and determine stable peptides that bind to TfR after grafting the binding patches onto the peptides; and c) grafting the binding patches onto the peptides to generate stable peptides.

[0029] In some embodiments, the method further comprises performing site-saturation mutagenesis to obtain mutant peptides that have a higher binding affinity for TfR than the stable peptide. In some embodiments, the peptides in the library of peptides are 20 to 75 amino acid residues in length.

[0030] In various aspects, the present disclosure provides compositions comprising a non-natural peptide capable of binding to the transferrin receptor (TfR), or a variant, homolog, fragment, or analog thereof.

[0031] In various aspects, the disclosure provides compositions comprising a peptide construct, wherein the peptide construct comprises a peptide capable of binding to transferrin receptor (TfR), or a variant, homolog, fragment, or analog thereof, and an active agent, wherein the peptide is conjugated, linked, or fused to the active agent.

[0032] In some embodiments, the transferrin receptor (TfR) is a human transferrin receptor, or a mouse transferrin receptor, or a human transferrin receptor and a mouse transferrin receptor.

[0033] In various aspects, the disclosure provides a composition comprising a peptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs:1-32, SEQ ID NOs:36-67, SEQ ID NOs:136-167, SEQ ID NOs:171-202, or SEQ ID NOs:356-358, or a variant or functional fragment thereof.

[0034] In some embodiments, the peptide penetrates a cell layer comprising endothelial cells. In some embodiments, the peptide penetrates a cell layer comprising the blood-brain barrier (BBB). In some embodiments, the peptide penetrates a cell layer comprising endothelial cells via TfR-mediated transcytosis. In some embodiments, the peptide penetrates a membrane, and the membrane is the membrane of a cell. In some embodiments, the cell expresses TfR. In some embodiments, the peptide penetrates the membrane of a cell via TfR-mediated endocytosis. In some embodiments, the peptide can localize within or with any cell that expresses TfR. In some embodiments, the peptide remains bound to the cell surface membrane that expresses TfR. In some embodiments, the peptide is internalized into the cell by binding to TfR. In some embodiments, the peptide is delivered across the blood-brain barrier to the CNS by binding to TfR and then dissociating. In some embodiments, the peptide is delivered across the blood-brain barrier by receptor-mediated transcytosis.

[0035] In some embodiments, the cell is a CNS cell, an erythrocyte, an erythroid progenitor cell, an immune cell, a stem cell, a muscle cell, a brain cell, a thyroid cell, a parathyroid cell, an adrenal cell, a bone marrow cell, an appendix cell, a lymph node cell, a tonsil cell, a splenocyte, a muscle cell, a liver cell, a gallbladder cell, a pancreatic cell, a cell of the gastrointestinal tract, a glandular cell, a kidney cell, a bladder cell, an endothelial cell, an epithelial cell, a choroid plexus epithelial cell, a glial cell, an astrocyte, or a cell associated with the nervous system.

[0036] In some embodiments, the cell is a tumor cell. In some embodiments, the tumor cell is an ovarian cancer cell, a colon cancer cell, a lung cancer cell, a cancer cell located in bone or bone marrow, a glioblastoma cell, an astrocytoma cell, a glioma cell, a medulloblastoma cell, an ependymoma cell, a choroid plexus carcinoma cell, a midline glioma cell, a diffuse intrinsic pontine glioma (DIPG) cell, a breast cancer cell, a liver cancer cell, a colon cancer cell, a brain cancer cell, a spleen cancer cell, a salivary gland cancer cell, a kidney cancer cell, a muscle cancer cell, a bone marrow cell cancer cell, a skin cancer cell, a genitourinary cancer cell, an osteosarcoma cell, a muscle-derived sarcoma cell, a melanoma cell, a head and neck cancer cell, a neuroblastoma cell, a prostate cancer cell, a bladder cancer cell, an acute lymphocytic leukemia cell, an acute myeloid leukemia cell, a chronic lymphocytic leukemia cell, a chronic myeloid leukemia cell, a Hodgkin's lymphoma cell, a non-Hodgkin's lymphoma cell, or a CMYC-overexpressing cancer cell. In some embodiments, the peptide is localized to an organ or tissue.

[0037] In some embodiments, the organ or tissue is the brain, thyroid, parathyroid, adrenal gland, bone marrow, appendix, lymph node, tonsil, spleen, muscle, liver, gallbladder, pancreas, gastrointestinal tract, kidney, bladder, fallopian tube, breast, vagina, cervix, endometrium, ovary, placenta, and skin. In some embodiments, the cell or organ to which the peptide is localized overexpresses TfR. In some embodiments, the peptide is localized in the organ parenchyma.

[0038] In some embodiments, the peptide binds to at least one amino acid residue selected from the group consisting of residues 506-510, 523-531, or 611-662, which correspond to SEQ ID NO: 363() of human TfR1 (UniProtKB-P02786). In some embodiments, the peptide comprises SEQ ID NOs: 206-224 and SEQ ID NOs: 334-344.In some embodiments, the peptide comprises surface interface residues corresponding to G5, A7, S8, N14, L17, E18, E21, L38, L42, L45, D46, H47, S50, and Q51, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the peptide comprises hydrophilic surface distal residues corresponding to any one of positions 3, 4, 9, 11, 15, 16, 19, 23, 26, 28, 29, 30, 31, 32, 33, 35, 36, 37, 39, and 40, relative to SEQ ID NO: 32, or any combination thereof.

[0039] In some embodiments, the hydrophilic surface-distal residue is selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises any one of the hydrophilic surface-distal residues corresponding to R3, E4, R9, K12, D15, E16, K19, R23, S26, S28, N29, T30, E31, E32, D33, E35, Q36, E37, E39, and D40, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the peptide comprises a hydrophilic residue at a position corresponding to any one of 15, 35, 39, or 49, or any combination thereof, relative to SEQ ID NO: 32.

[0040] In some embodiments, the hydrophilic residue is selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises hydrophilic residues corresponding to D15, E35, E39, and H49, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the peptide comprises a hydrophobic residue at a position corresponding to 11, 25, or 27, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the hydrophobic residue is selected from amino acids A, M, I, L, V, F, W, or Y, or any combination thereof. In some embodiments, the peptide comprises any one of hydrophobic residues corresponding to M11, M25, M27, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the peptide comprises an aliphatic residue corresponding to position 45, relative to SEQ ID NO: 32. In some embodiments, the aliphatic residue is selected from amino acids A, M, I, L, or V.

[0041] In some embodiments, the peptide comprises an aliphatic residue corresponding to L45 in SEQ ID NO: 32. In some embodiments, the peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least five disulfide bonds. In some embodiments, the peptide comprises three disulfide bonds. In some embodiments, the peptide comprises at least two, at least four, or at least six cysteine ​​residues. In some embodiments, the peptide comprises six cysteine ​​residues.

[0042] In some embodiments, the peptide comprises a cysteine ​​residue at a position corresponding to C6, C10, C20, C34, C44, and C48, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, at least one cysteine ​​residue of the peptide participates in a disulfide bond. In some embodiments, six cysteine ​​residues participate in a disulfide bond. In some embodiments, the disulfide bond increases the stability of the peptide.

[0043] In some embodiments, the peptide comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-32, 36-67, 136-167, 171-202, or 356-358, or a variant or fragment thereof. In some embodiments, the peptide comprises a sequence of any one of SEQ ID NOs: 1-32, 36-67, 136-167, 171-202, or 356-358, or a variant or fragment thereof.

[0044] In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 2. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 4. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 30. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 32.

[0045] In some embodiments, the peptide comprising three disulfide bonds is derived from a cysteine-dense peptide (CDP). In some embodiments, the peptide comprising three disulfide bonds is a CDP. In some embodiments, the peptide competes with transferrin, or a homolog, variant, or fragment thereof, for binding to TfR. In some embodiments, the peptide sequesteres TfR or prevents another protein from binding to TfR. In some embodiments, the peptide has equal or greater affinity or specificity for TfR compared to transferrin, or an endogenous derivative thereof. In some embodiments, the peptide has equal or smaller dissociation constant for TfR compared to transferrin, or an endogenous derivative thereof. In further embodiments, the peptide has a K of less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 1 nM, or less than 0.1 nM. D In some embodiments, the peptide penetrates or localizes within the target cell.

[0046] In some embodiments, the peptide is further formulated, fused, or conjugated with a cell-penetrating moiety, such as a polycation, a polyorganic acid, an endosome-releasing polymer, poly(2-propylacrylic acid), poly(2-ethylacrylic acid), Tat peptide, Arg patch, knot peptide, CysTAT, S19-TAT, R8 (SEQ ID NO: 73), pAntp, Pas-TAT, Pas-R8 (SEQ ID NO: 76), Pas-FHV, Pas-pAntP, F2R4 (SEQ ID NO: 79), B55, aurin, IMT-P8, BR2, OMOTAG1, OMO In some embodiments, the cell-penetrating moiety is maurocalcine, imperatoxin, haddrucalcin, hemicalcin, opicalcin-1, opicalcin-2, midkine(62-104), MCoTI-II, chlorotoxin, DRI-TAT, cFΦR4 (SEQ ID NO:96), myristate, yBBR, or a fragment or variant thereof, or any combination thereof. In some embodiments, the cell-penetrating moiety is maurocalcine, imperatoxin, haddrucalcin, hemicalcin, opicalcin-1, opicalcin-2, midkine(62-104), MCoTI-II, or chlorotoxin.

[0047] In some embodiments, the peptide targets, homes to, accumulates in, or selectively penetrates tissues or cells. In some embodiments, the peptide is localized in the nucleus of the target cell. In some embodiments, the peptide is localized in the nucleus of the target cell by formulation, fusion, or conjugation with a signal peptide or moiety. In some embodiments, the peptide is further conjugated, linked, or fused to a moiety or second peptide that targets, homes to, accumulates in, or selectively penetrates cancerous tissues or cancer cells. In some embodiments, the moiety or second peptide is a knot peptide or a variant or fragment thereof. In some embodiments, the target cell is a cancerous cell or tumor cell. In some embodiments, the target cell is a CNS cell, an erythrocyte, an erythroid progenitor cell, an immune cell, a stem cell, a muscle cell, a brain cell, a thyroid cell, a parathyroid cell, an adrenal gland cell, a bone marrow cell, an appendix cell, a lymph node cell, a tonsil cell, a spleen cell, a muscle cell, a liver cell, a gallbladder cell, a pancreatic cell, a cell of the gastrointestinal tract, a glandular cell, a kidney cell, a bladder cell, an endothelial cell, an epithelial cell, a choroid plexus epithelial cell, a glial cell, an astrocyte, or a cell associated with the nervous system.

[0048] In some embodiments, the peptide contains disulfides via disulfide knots. In some embodiments, the peptide contains multiple disulfide bridges formed between cysteine ​​residues. In some embodiments, the peptide contains three or more disulfide bridges formed between cysteine ​​residues, and one of the disulfide bridges passes through a loop formed by the other two disulfide bridges. In some embodiments, the peptide does not contain a disulfide bond or a disulfide knot. In some embodiments, at least one amino acid residue of the peptide is in the L-configuration or at least one amino acid residue is in the D-configuration.

[0049] In some embodiments, the peptide contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged residues. In some embodiments, the peptide contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive positively charged residues. In some embodiments, the positively charged residues are Arg, Lys, or any combination thereof. In some embodiments, the peptide contains an Arg patch. In some embodiments, the peptide contains two or more consecutive Arg residues at the N-terminus.

[0050] In some embodiments, the peptide comprises a Tat peptide, or a fragment thereof. In some embodiments, the Tat peptide comprises the sequence YGRKKRRQRRR (SEQ ID NO: 99), GRKKRRQRRR (SEQ ID NO: 100), or a fragment or variant thereof. In some embodiments, the Tat peptide comprises the sequence (GS) x (SEQ ID NO: 105) or G x S y (SEQ ID NO: 104), where x and y are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the Tat peptide is attached to any residue of the peptide.

[0051] In some embodiments, the peptide comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 cysteine ​​residues. In some embodiments, the peptide comprises one or more regions of negatively charged residues. In some embodiments, the peptide comprises one or more regions of positively charged residues.

[0052] In some embodiments, the peptide sequence is at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58 residues, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, or at least 81 amino acid residues.

[0053] In some embodiments, the peptide comprises or is derived from a human protein or peptide. In some embodiments, the peptide is arranged in a multimeric structure with at least one other peptide. In some embodiments, the multimeric structure comprises a dimer, trimer, tetramer, pentamer, hexamer, or heptamer. In some embodiments, the peptide has an isoelectric point in the range of about 3.0 to about 10.0. In some embodiments, the peptide has an isoelectric point in the range of about 4.5 to about 8.9. In some embodiments, the peptide has a heterogeneous charge distribution. In some embodiments, the peptide is stable at physiological or intracellular pH values.

[0054] In some embodiments, the peptide is stable at a pH of about 7 or 7.5. In some embodiments, the peptide is stable at a pH of about 6.5 to 7.5. In some embodiments, the peptide is stable at a pH value of about 5.0 or less, about 3.0 or less, or within the range of about 3.0 to about 5.0. In some embodiments, the peptide is stable at a pH value of about 5.0 to about 7.0. In some embodiments, the peptide comprises a hydrophobic core.

[0055] In some embodiments, the peptide is resistant to protease degradation. In some embodiments, the protease is any one of pepsin, trypsin, chymotrypsin, serum proteases, intracellular proteases, or any combination thereof. In some embodiments, the peptide is resistant to reduction or stable in a reducing environment. In some embodiments, the reducing or reducing environment includes DTT or GSH. In some embodiments, the peptide is stable at high temperatures. In some embodiments, the peptide exhibits anti-cancer activity. In some embodiments, the peptide comprises one or more chemical modifications. In some embodiments, the chemical modifications alter one or more pharmacokinetic properties of the peptide. In some embodiments, the chemical modifications extend the plasma half-life and / or biological half-life of the peptide.

[0056] In some embodiments, the chemical modification is blocking the N-terminus of the peptide. In some embodiments, the chemical modification is methylation, acetylation, or acylation. In some embodiments, the chemical modification is methylation of one or more lysine residues or analogs thereof; methylation of the N-terminus; or methylation of one or more lysine residues or analogs thereof and methylation of the N-terminus. In some embodiments, the peptide is linked to an acyl adduct. In some embodiments, an active sequence is grafted onto the peptide, or the peptide is conjugated, linked, fused, or grafted to an active agent to produce a peptide construct.

[0057] In some embodiments, the peptide construct has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 33-35, 68-70, 168-170, 203-205, 225-332, and 359-361. In some embodiments, the peptide construct is SEQ ID NO: 359, SEQ ID NO: 360, or SEQ ID NO: 361. In some embodiments, an active agent is conjugated, linked, or fused to the peptide at the N-terminus or C-terminus of the peptide. In some embodiments, the active agent is an antibody, antibody fragment, Fc, single-chain Fv, polypeptide, peptide, small molecule, or nucleic acid.

[0058] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 active agents are linked to the peptide by a linker. In some embodiments, the peptide is linked to the active agent via a cleavable linker or a pH-sensitive linker. In some embodiments, the peptide is linked to the active agent by a linker at the N-terminus, the epsilon amine of an internal lysine residue, the carboxylic acid of an aspartic acid or glutamic acid residue, or the C-terminus of the peptide. In some embodiments, the composition further comprises an unnatural amino acid, wherein the unnatural amino acid is inserted, added, or substituted with another amino acid. In some embodiments, the peptide is linked to the active agent at the unnatural amino acid by a linker. In some embodiments, the linker comprises an amide bond, an ester bond, an ether bond, a triazole, a macrocyclic polymer, an oxime bond, a carbamate bond, a carbonate bond, a hydrazone bond, an azo bond, an oxime bond, a disulfide bond, a thioester bond, a thioether bond, a carbon-carbon single, double, or triple bond, a disulfide bond, a two-carbon bridge between two cysteines, a three-carbon bridge between two cysteines, or a carbon-nitrogen bond.

[0059] In some embodiments, the cleavable linker comprises a cleavage site for a protease. In some embodiments, the protease is a matrix metalloprotease, thrombin, cathepsin, or beta-glucuronidase. In some embodiments, the linker is a hydrolytically unstable linker. In some embodiments, the peptide is linked to the active agent via a non-cleavable linker. In some embodiments, the active agent is linked via a linker, wherein the linker is selected from Table 4. In some embodiments, the active agent is an anti-cancer agent. In some embodiments, the active agent is an immunotherapeutic agent, a CTLA-4 targeted agent, a PD-1 targeted agent, a PDL-1 targeted agent, an IL15 agent, a fused IL-15 / IL-15Ra combination agent, an IFN gamma agent, an anti-CD3 agent, an ion channel modulator, a Kv1.3 inhibitor, an auristatin, MMAE, a maytansinoid, DM1, DM4, doxorubicin, a calicheamicin, a platinum compound, cisplatin, a taxane, paclitaxel, SN-38, a BACE inhibitor, a Bcl-xL inhibitor, WEHI-539, venetoclax, ABT-199, navitoclax, AT-101, obatoclax, a pyrrolobenzodiazepine or pyrrolobenzodiazepine dimer, a dolastatin, or a neurotransmitter.

[0060] In some embodiments, the neurotransmitter is neurotensin, a neurotensin peptide variant, or a functional fragment thereof. In some embodiments, the functional fragment is at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, or at least 12 amino acid residues.

[0061] In some embodiments, the neurotensin peptide variant has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 365-369. In some embodiments, the neurotensin has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 350. In some embodiments, the peptide construct is capable of binding to and activating a neurotensin receptor. In some embodiments, the peptide is capable of reducing pain levels in a subject. In some embodiments, the peptide is administered to patients with schizophrenia, substance abuse, Parkinson's disease, hypertension, eating disorders, stroke, Alzheimer's disease, cancer, inflammation, or endocrine disorders. In some embodiments, the peptide interacts with dopamine signaling neurons. In some embodiments, the peptide increases dopamine release in neurons. In some embodiments, the composition further comprises a half-life modifying agent attached to the peptide. In some embodiments, the half-life modifying agent comprises a polymer, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, or a molecule that binds to albumin.

[0062] In some embodiments, the composition further comprises a detectable agent attached to the peptide by a linker. In some embodiments, the detectable agent is conjugated, linked, or fused to the peptide at the N-terminus or C-terminus of the peptide. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 detectable agents are linked to the peptide. In some embodiments, the peptide is linked to the detectable agent via a cleavable linker. In some embodiments, the peptide is linked to the detectable agent at the N-terminus, the epsilon amine of an internal lysine residue, or the C-terminus of the peptide by a linker. In some embodiments, the composition further comprises an unnatural amino acid, wherein the unnatural amino acid is an insertion, addition, or substitution with another amino acid. In some embodiments, the peptide is linked to the detectable agent at the unnatural amino acid by a linker. In some embodiments, the linker comprises an amide bond, an ester bond, an ether bond, a triazole, a macrocyclic polymer, an oxime bond, a carbamate bond, a carbonate bond, a hydrazone bond, an azo bond, an oxime bond, a disulfide bond, a thioester bond, a thioether bond, a carbon-carbon single bond, a double bond, or a triple bond, a disulfide bond, a two-carbon bridge between two cysteines, a three-carbon bridge between two cysteines, or a carbon-nitrogen bond. In some embodiments, the cleavable linker comprises a cleavage site for a matrix metalloproteinase, thrombin, cathepsin, or beta-glucuronidase. In some embodiments, the peptide is linked to the detectable agent via a stable linker. In some embodiments, the detectable agent is linked via a linker, wherein the linker is selected from Table 4.

[0063] In some embodiments, the detectable agent is a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radionuclide, or a radionuclide chelator. In some embodiments, the detectable agent is a fluorescent dye.

[0064] In various aspects, the present disclosure provides a pharmaceutical composition comprising any of the compositions disclosed herein, or a salt thereof, and a pharmaceutically acceptable carrier.

[0065] In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the pharmaceutical composition is formulated for oral administration, intravenous administration, subcutaneous administration, intramuscular administration, or a combination thereof.

[0066] In various aspects, the present disclosure provides methods for transporting a composition across a cell layer, the method comprising contacting the composition with the cell layer and transporting the composition across the cell layer.

[0067] In some embodiments, the composition comprises a peptide. In some embodiments, the peptide comprises an amino acid sequence comprising any one of SEQ ID NOs:206-224 and SEQ ID NOs:334-344, or a functional fragment thereof. In some embodiments, the peptide is capable of binding to the transferrin receptor (TfR). In some embodiments, the peptide comprises surface-interface residues corresponding to G5, A7, S8, N14, L17, E18, E21, L38, L42, L45, D46, H47, S50, and Q51, relative to SEQ ID NO:32, or any combination thereof. In some embodiments, the peptide comprises a hydrophilic surface-distal residue at one position corresponding to 3, 4, 9, 11, 15, 16, 19, 23, 26, 28, 29, 30, 31, 32, 33, 35, 36, 37, 39, and 40, relative to SEQ ID NO:32, or any combination thereof. In some embodiments, the hydrophilic surface-distal residue is selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises any one of the hydrophilic surface-distal residues corresponding to R3, E4, R9, K12, D15, E16, K19, R23, S26, S28, N29, T30, E31, E32, D33, E35, Q36, E37, E39, and D40, relative to SEQ ID NO: 32, or any combination thereof. In some embodiments, the peptide comprises a hydrophilic residue at any one of positions corresponding to 15, 35, 39, or 49, relative to SEQ ID NO: 32, or any combination thereof.

[0068] In some embodiments, the hydrophilic residue is selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises any one of the hydrophilic residues corresponding to D15, E35, E39, and H49, or any combination thereof, based on SEQ ID NO: 32. In some embodiments, the peptide comprises a hydrophobic residue at any of positions corresponding to 11, 25, or 27, or any combination thereof, based on SEQ ID NO: 32. In some embodiments, the hydrophobic residue is selected from amino acids A, M, I, L, V, F, W, or Y, or any combination thereof. In some embodiments, the peptide comprises any one of the hydrophobic residues corresponding to M11, M25, M27, or any combination thereof, based on SEQ ID NO: 32. In some embodiments, the peptide comprises an aliphatic residue corresponding to position 45, based on SEQ ID NO: 32. In some embodiments, the aliphatic residue is selected from amino acids A, M, I, L, or V. In some embodiments, the peptide comprises an aliphatic residue corresponding to L45 relative to SEQ ID NO:32.

[0069] In some embodiments, the peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least five disulfide bonds. In some embodiments, the peptide comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-32, SEQ ID NOs: 36-67, SEQ ID NOs: 136-167, SEQ ID NOs: 171-202, or SEQ ID NOs: 356-358, or a variant or fragment thereof. In some embodiments, the peptide comprises a sequence of any one of SEQ ID NOs: 1-32, SEQ ID NOs: 36-67, SEQ ID NOs: 136-167, SEQ ID NOs: 171-202, or SEQ ID NOs: 356-358, or a variant or fragment thereof. In some embodiments, the peptide penetrates or localizes in or on the target cell. In some embodiments, an active sequence is grafted onto the peptide, or the peptide is conjugated, linked, fused, or grafted to an active agent to provide a peptide construct.

[0070] In some embodiments, the peptide is conjugated, linked, or fused to an active agent. In some embodiments, the active agent is linked via a linker, wherein the linker is selected from Table 4. In some embodiments, the active agent is an antibody, antibody fragment, Fc, single-chain Fv, polypeptide, peptide, small molecule, or nucleic acid. In some embodiments, the peptide construct has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs:33-35, SEQ ID NOs:68-70, SEQ ID NOs:168-170, SEQ ID NOs:203-205, SEQ ID NOs:225-332, and SEQ ID NOs:359-361. In some embodiments, the peptide construct is SEQ ID NO:359, SEQ ID NO:360, or SEQ ID NO:361.

[0071] In some embodiments, the active agent is an immunotherapeutic agent, a CTLA-4 targeting agent, a PD-1 targeting agent, a PDL-1 targeting agent, an IL15 agent, a fused IL-15 / IL-15Ra combination agent, an IFN gamma agent, an anti-CD3 agent, an ion channel modulator, a Kv1.3 inhibitor, an auristatin, MMAE, a maytansinoid, DM1, DM4, doxorubicin, a calicheamicin, a platinum compound, cisplatin, a taxane, paclitaxel, SN-38, a BACE inhibitor, a Bcl-xL inhibitor, WEHI-539, venetoclax, ABT-199, navitoclax, AT-101, obatoclax, a pyrrolobenzodiazepine or pyrrolobenzodiazepine dimer, a dolastatin, or a neurotransmitter. In some embodiments, the neurotransmitter is neurotensin, a neurotensin peptide variant, or a functional fragment thereof.

[0072] In some embodiments, a functional fragment is at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, or at least 12 amino acid residues. In some embodiments, a neurotensin peptide variant has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 365-369. In some embodiments, a neurotensin has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 350.

[0073] In some embodiments, the peptide construct is capable of binding to and activating a neurotensin receptor. In some embodiments, the peptide is capable of reducing pain levels upon administration to a subject. In some embodiments, the peptide is administered to a patient with schizophrenia, substance abuse, Parkinson's disease, hypertension, eating disorders, stroke, Alzheimer's disease, cancer, inflammation, or an endocrine disorder. In some embodiments, the peptide interacts with dopamine signaling neurons. In some embodiments, the peptide increases dopamine release in neurons. In some embodiments, the peptide is further formulated, fused, or conjugated with a cell-penetrating moiety. In some embodiments, the cell layer is an endothelial cell layer or an epithelial cell layer. In some embodiments, the endothelial cell layer is the blood-brain barrier. In some embodiments, the endothelial cell layer is an intestinal endothelial cell layer. In some embodiments, the epithelial cell layer is an intestinal epithelial cell layer. In some embodiments, the transporting comprises binding of the composition to TfR. In some embodiments, the transporting further comprises transcytosis. In some embodiments, the transcytosis is vesicular transcytosis. In some embodiments, the transcytosis is TfR-mediated. In some embodiments, transporting further comprises dissociating the composition from the TfR.

[0074] In various aspects, the present disclosure provides methods for treating a condition in a subject in need thereof, comprising administering a composition to the subject and transporting the composition across a cell layer.

[0075] In some embodiments, the composition comprises a peptide. In some embodiments, the peptide comprises the amino acid sequence of any one of SEQ ID NOs:206-224 and 334-344. In some embodiments, the peptide is capable of binding to the transferrin receptor (TfR). In some embodiments, the peptide comprises surface-interface residues corresponding to G5, A7, S8, N14, L17, E18, E21, L38, L42, L45, D46, H47, S50, and Q51, or any combination thereof, relative to SEQ ID NO:32. In some embodiments, the peptide comprises a hydrophilic surface-distal residue at any one of positions corresponding to 3, 4, 9, 11, 15, 16, 19, 23, 26, 28, 29, 30, 31, 32, 33, 35, 36, 37, 39, and 40, or any combination thereof, relative to SEQ ID NO:32. In some embodiments, the hydrophilic surface distal residues are selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises any one, or any combination of, hydrophilic surface distal residues corresponding to R3, E4, R9, K12, D15, E16, K19, R23, S26, S28, N29, T30, E31, E32, D33, E35, Q36, E37, E39, and D40, based on SEQ ID NO:32.

[0076] In some embodiments, the peptide comprises a hydrophilic residue at any one of positions corresponding to 15, 35, 39, or 49, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the hydrophilic residue is selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises a hydrophilic residue at any one of positions corresponding to D15, E35, E39, and H49, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the peptide comprises a hydrophobic residue at any one of positions corresponding to 11, 25, or 27, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, the hydrophobic residue is selected from amino acids A, M, I, L, V, F, W, or Y, or any combination thereof. In some embodiments, the peptide comprises a hydrophobic residue at any one of positions corresponding to M11, M25, M27, or any combination thereof, relative to SEQ ID NO: 32.

[0077] In some embodiments, the peptide comprises an aliphatic residue corresponding to position 45 relative to SEQ ID NO: 32. In some embodiments, the aliphatic residue is selected from amino acids A, M, I, L, or V. In some embodiments, the peptide comprises an aliphatic residue corresponding to L45 relative to SEQ ID NO: 32. In some embodiments, the peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least five disulfide bonds.

[0078] In some embodiments, the peptide comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-32, 36-67, 136-167, 171-202, or 356-358, or a variant or fragment thereof. In some embodiments, the peptide comprises a sequence of any one of SEQ ID NOs: 1-32, 36-67, 136-167, 171-202, or 356-358, or a variant or fragment thereof.

[0079] In some embodiments, the peptide penetrates target cells or localizes in target cells.In some embodiments, the peptide is grafted with an active sequence, or the peptide is conjugated, linked, fused, or grafted with an active agent to produce a peptide construct.In some embodiments, the peptide is conjugated, linked, or fused with an active agent.In some embodiments, the active agent is linked via a linker, wherein the linker is selected from Table 4.

[0080] In some embodiments, the active agent is an antibody, antibody fragment, Fc, single-chain Fv, polypeptide, peptide, small molecule, or nucleic acid. In some embodiments, the peptide construct has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs:33-35, 68-70, 168-170, 203-205, 225-332, and 359-361. In some embodiments, the peptide construct is SEQ ID NO:359, SEQ ID NO:360, or SEQ ID NO:361.

[0081] In some embodiments, the active agent is an immunotherapeutic agent, a CTLA-4 targeted agent, a PD-1 targeted agent, a PDL-1 targeted agent, an IL15 agent, a fused IL-15 / IL-15Ra combination agent, an IFN gamma agent, an anti-CD3 agent, an ion channel modulator, a Kv1.3 inhibitor, an auristatin, MMAE, a maytansinoid, DM1, DM4, doxorubicin, a calicheamicin, a platinum compound, cisplatin, a taxane, paclitaxel, SN-38, a BACE inhibitor, a Bcl-xL inhibitor, WEHI-539, venetoclax, ABT-199, navitoclax, AT-101, obatoclax, a pyrrolobenzodiazepine or pyrrolobenzodiazepine dimer, a dolastatin, or a neurotransmitter.

[0082] In some embodiments, the neurotransmitter is neurotensin, a neurotensin peptide variant, or a functional fragment thereof. In some embodiments, the functional fragment is at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, or at least 12 amino acid residues. In some embodiments, the neurotensin peptide variant has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 365-369. In some embodiments, the neurotensin has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 350.

[0083] In some embodiments, the peptide construct is capable of binding to and activating a neurotensin receptor. In some embodiments, the peptide is capable of reducing pain levels upon administration to a subject. In some embodiments, the peptide is administered to a patient with schizophrenia, substance abuse, Parkinson's disease, hypertension, eating disorders, stroke, Alzheimer's disease, cancer, inflammation, or an endocrine disorder. In some embodiments, the peptide interacts with dopamine signaling neurons. In some embodiments, the peptide increases dopamine release in neurons. In some embodiments, the peptide is further formulated, fused, or conjugated with a cell-penetrating moiety. In some embodiments, the cell layer is an endothelial cell layer or an epithelial cell layer. In some embodiments, the endothelial cell layer is the blood-brain barrier.

[0084] In some embodiments, the endothelial cell layer is an intestinal endothelial cell layer. In some embodiments, the epithelial cell layer is an intestinal epithelial cell layer. In some embodiments, transporting comprises binding of the composition to TfR. In some embodiments, transporting further comprises transcytosis. In some embodiments, transcytosis is vesicular transcytosis. In some embodiments, transcytosis is TfR-mediated. In some embodiments, transporting further comprises dissociation of the composition from TfR. In some embodiments, transporting further comprises releasing the active agent from the composition.

[0085] In various aspects, the present disclosure provides methods of treating a condition in a subject in need thereof, comprising administering to the subject an amount of a composition based on expression of TfR in the subject.

[0086] In some embodiments, the composition comprises a peptide. In some embodiments, the peptide comprises the amino acid sequence of any one of SEQ ID NOs:206-224 and 334-344. In some embodiments, the peptide is capable of binding to the transferrin receptor (TfR). In some embodiments, the peptide comprises surface-interface residues corresponding to G5, A7, S8, N14, L17, E18, E21, L38, L42, L45, D46, H47, S50, and Q51, or any combination thereof, relative to SEQ ID NO:32. In some embodiments, the peptide comprises a hydrophilic surface-distal residue at any one of positions corresponding to 3, 4, 9, 11, 15, 16, 19, 23, 26, 28, 29, 30, 31, 32, 33, 35, 36, 37, 39, and 40, or any combination thereof, relative to SEQ ID NO:32. In some embodiments, the hydrophilic surface-distal residue is selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises any one of the hydrophilic surface-distal residues corresponding to R3, E4, R9, K12, D15, E16, K19, R23, S26, S28, N29, T30, E31, E32, D33, E35, Q36, E37, E39, and D40, relative to SEQ ID NO: 32, or any combination thereof. In some embodiments, the peptide comprises a hydrophilic residue at any one of positions corresponding to 15, 35, 39, or 49, relative to SEQ ID NO: 32, or any combination thereof. In some embodiments, the hydrophilic residue is selected from amino acids D, E, H, K, R, N, Q, S, or T. In some embodiments, the peptide comprises any one of, or any combination of, hydrophilic residues corresponding to D15, E35, E39, and H49, relative to SEQ ID NO: 32. In some embodiments, the peptide comprises a hydrophobic residue at any of, or any combination of, positions corresponding to 11, 25, or 27, relative to SEQ ID NO: 32.

[0087] In some embodiments, the hydrophobic residue is selected from the amino acids A, M, I, L, V, F, W, or Y, or any combination thereof. In some embodiments, the peptide comprises any one of the hydrophobic residues corresponding to M11, M25, M27, or any combination thereof, based on SEQ ID NO: 32. In some embodiments, the peptide comprises an aliphatic residue corresponding to position 45 based on SEQ ID NO: 32. In some embodiments, the aliphatic residue is selected from the amino acids A, M, I, L, or V. In some embodiments, the peptide comprises an aliphatic residue corresponding to L45 based on SEQ ID NO: 32. In some embodiments, the peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least five disulfide bonds.

[0088] In some embodiments, the peptide comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-32, 36-67, 136-167, 171-202, or 356-358, or a variant or fragment thereof. In some embodiments, the peptide comprises a sequence of any one of SEQ ID NOs: 1-32, 36-67, 136-167, 171-202, or 356-358, or a variant or fragment thereof.

[0089] In some embodiments, the peptide penetrates target cells or localizes in target cells.In some embodiments, the peptide is grafted with an active sequence, or the peptide is conjugated, linked, fused, or grafted with an active agent to produce a peptide construct.In some embodiments, the peptide is conjugated, linked, or fused with an active agent.In some embodiments, the active agent is linked via a linker, wherein the linker is selected from Table 4.

[0090] In some embodiments, the active agent is an antibody, antibody fragment, Fc, single-chain Fv, polypeptide, peptide, small molecule, or nucleic acid. In some embodiments, the peptide construct has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs:33-35, 68-70, 168-170, 203-205, 225-332, and 359-361. In some embodiments, the peptide construct is SEQ ID NO:359, SEQ ID NO:360, or SEQ ID NO:361.

[0091] In some embodiments, the active agent is an immunotherapeutic agent, a CTLA-4 targeted agent, a PD-1 targeted agent, a PDL-1 targeted agent, an IL15 agent, a fused IL-15 / IL-15Ra combination agent, an IFN gamma agent, an anti-CD3 agent, an ion channel modulator, a Kv1.3 inhibitor, an auristatin, MMAE, a maytansinoid, DM1, DM4, doxorubicin, a calicheamicin, a platinum compound, cisplatin, a taxane, paclitaxel, SN-38, a BACE inhibitor, a Bcl-xL inhibitor, WEHI-539, venetoclax, ABT-199, navitoclax, AT-101, obatoclax, a pyrrolobenzodiazepine or pyrrolobenzodiazepine dimer, a dolastatin, or a neurotransmitter.

[0092] In some embodiments, the neurotransmitter is neurotensin, a neurotensin peptide variant, or a functional fragment thereof. In some embodiments, the functional fragment is at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, or at least 12 amino acid residues. In some embodiments, the neurotensin peptide variant has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 365-369. In some embodiments, the neurotensin has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 350.

[0093] In some embodiments, the peptide construct is capable of binding to and activating a neurotensin receptor. In some embodiments, the peptide is capable of reducing pain levels upon administration to a subject. In some embodiments, the peptide is administered to a patient with schizophrenia, substance abuse, Parkinson's disease, hypertension, eating disorders, stroke, Alzheimer's disease, cancer, inflammation, or an endocrine disorder. In some embodiments, the peptide interacts with dopamine signaling neurons. In some embodiments, the peptide increases dopamine release in neurons. In some embodiments, the peptide is further formulated, fused, or conjugated with a cell-penetrating moiety. In some embodiments, the composition is administered by inhalation, intranasal, oral, topical, intravenous, subcutaneous, intramuscular, intraperitoneal, intratumoral, intrathecal, or a combination thereof.

[0094] In some embodiments, the composition is administered intravenously as a bolus, injection, or continuous infusion.In some embodiments, the composition or pharmaceutical composition is administered multiple times a day, twice a day, once a day, twice a week, three times a week, once a week, once every two weeks, or once a month or once every three months.In some embodiments, the composition is administered subcutaneously once a day, once a week, or once a month.In some embodiments, the condition is cancer.

[0095] In some embodiments, the cancer is ovarian cancer, colon cancer, lung cancer, cancer located in bone or bone marrow, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma (DIPG), breast cancer, liver cancer, colon cancer, brain cancer, spleen cancer, salivary gland cancer, renal cancer, muscle cancer, myeloid cell carcinoma, skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, neuroblastoma, prostate cancer, bladder cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or CMYC-overexpressing cancer. In some embodiments, the condition is brain cancer. In some embodiments, the brain cancer is glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, or diffuse intrinsic pontine glioma.

[0096] In some embodiments, the condition is a CNS disease. In some embodiments, the CNS disease is a neuroinflammatory or neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, motor neuron disease, Lyme disease, ataxia-telangiectasia, autosomal dominant cerebellar ataxia, Batten disease, corticobasal syndrome, Creutzfeldt-Jakob disease, paraneoplastic tremor / ataxia syndrome, Kufour-Rakeb syndrome, Machado-Joseph disease, multiple sclerosis, chronic traumatic encephalopathy, or frontotemporal dementia. In some embodiments, the condition is neuropathic pain, migraine, anxiety, or epilepsy. In some embodiments, the condition is chronic pain, acute pain, nociceptive pain, neuropathic pain, allodynia, phantom pain, visceral pain, or breakthrough pain.

[0097] In some embodiments, the condition is chronic pain, acute pain, injury, mechanical pain, heat pain, cold pain, ischemic pain, and chemical-induced pain, inflammatory pain, migraine-related pain, headache-related pain, irritable bowel syndrome-related pain, fibromyalgia-related pain, arthritis pain, skeletal pain, joint pain, gastrointestinal pain, myalgia, angina pain, facial pain, pelvic pain, claudication, postoperative pain, posttraumatic pain, tension headache, obstetric pain, gynecological pain, or chemotherapy-induced pain, nociceptive pain, neuropathic pain, allodynia, phantom pain, visceral pain, breakthrough pain, familial episodic pain syndrome, paroxysmal excruciating pain, congenital analgesia, or opioid withdrawal-related pain. In some embodiments, the condition is muscular dystrophy or spinal-bulbar muscular dystrophy (SBMA). In some embodiments, the condition is inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn's disease.

[0098] In various aspects, the present disclosure provides methods for transporting any of the compositions disclosed herein across a cell layer, the method comprising contacting the composition with the cell layer and transporting the composition across the cell layer.

[0099] In some embodiments, the cell layer is an endothelial cell layer or an epithelial cell layer. In some embodiments, the endothelial cell layer is the blood-brain barrier. In some embodiments, the endothelial cell layer is an intestinal endothelial cell layer. In some embodiments, the epithelial cell layer is an intestinal epithelial cell layer. In some embodiments, the transporting comprises binding of the composition to TfR. In some embodiments, the transporting further comprises transcytosis. In some embodiments, the transcytosis is vesicular transcytosis. In some embodiments, the transcytosis is TfR-mediated. In some embodiments, the transporting further comprises dissociation of the composition from TfR. In some embodiments, the transporting further comprises releasing the active agent from the composition.

[0100] In various aspects, the present disclosure provides methods for treating a condition in a subject in need thereof, comprising administering to the subject any of the compositions disclosed herein or any of the pharmaceutical compositions disclosed herein.

[0101] In some embodiments, the composition is administered by inhalation, intranasal, oral, topical, intravenous, subcutaneous, intramuscular, intraperitoneal, intratumoral, intrathecal, or a combination thereof. In some embodiments, the composition is administered intravenously as a bolus, injection, or continuous infusion. In some embodiments, the composition or pharmaceutical composition is administered multiple times a day, twice a day, once a day, twice a week, three times a week, once a week, once every two weeks, or once a month or once every three months. In some embodiments, the composition is administered subcutaneously once a day, once a week, or once a month. In some embodiments, the condition is cancer. In some embodiments, the cancer is ovarian cancer, colon cancer, lung cancer, cancer located in bone or bone marrow, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma (DIPG), breast cancer, liver cancer, colon cancer, brain cancer, spleen cancer, salivary gland cancer, renal cancer, muscle cancer, myeloid cell carcinoma, skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, neuroblastoma, prostate cancer, bladder cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or CMYC-overexpressing cancer. In some embodiments, the condition is brain cancer.

[0102] In some embodiments, the brain cancer is glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, or diffuse intrinsic pontine glioma. In some embodiments, the condition is a CNS disease. In some embodiments, the CNS condition is a neuroinflammatory or neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, motor neuron disease, Lyme disease, ataxia-telangiectasia, autosomal dominant cerebellar ataxia, Batten disease, corticobasal syndrome, Creutzfeldt-Jakob disease, parafragile X tremor / ataxia syndrome, Kufour-Rakeb syndrome, Machado-Joseph disease, multiple sclerosis, chronic traumatic encephalopathy, or frontotemporal dementia. In some embodiments, the condition is neuropathic pain, migraine, anxiety, or epilepsy.

[0103] In some embodiments, the condition is chronic pain, acute pain, injury, mechanical pain, heat pain, cold pain, ischemic pain, and chemical-induced pain, inflammatory pain, migraine-related pain, headache-related pain, irritable bowel syndrome-related pain, fibromyalgia-related pain, arthritis pain, skeletal pain, joint pain, gastrointestinal pain, myalgia, angina pain, facial pain, pelvic pain, claudication, post-operative pain, post-traumatic pain, tension headache, obstetric pain, gynecological pain, or chemotherapy-induced pain, nociceptive pain, neuropathic pain, allodynia, phantom pain, visceral pain, breakthrough pain, familial episodic pain syndrome, paroxysmal excruciating pain, congenital analgesia, opioid withdrawal-related pain. In some embodiments, the condition is muscular dystrophy or spinal-bulbar muscular dystrophy (SBMA).

[0104] In some embodiments, the condition is inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn's disease. In some embodiments, the method further comprises transporting the composition across a cell layer. In some embodiments, the cell layer is an endothelial cell layer or an epithelial cell layer. In some embodiments, the endothelial cell layer is the blood-brain barrier. In some embodiments, the endothelial cell layer is an intestinal endothelial cell layer.

[0105] In some embodiments, the epithelial cell layer is an intestinal epithelial cell layer. In some embodiments, the transporting comprises binding of the composition to TfR. In some embodiments, the transporting further comprises transcytosis. In some embodiments, the transcytosis is vesicular transcytosis. In some embodiments, the transcytosis is TfR-mediated. In some embodiments, the transporting further comprises dissociating the composition from TfR. In some embodiments, the transporting further comprises releasing the active agent from the composition.

[0106] In various aspects, the present disclosure provides methods for treating a condition in a subject in need thereof, comprising administering to the subject an amount of any of the compositions disclosed herein or any of the pharmaceutical compositions disclosed herein that is dependent on the expression of TfR in the subject.

[0107] In some embodiments, the composition is administered by inhalation, intranasal, oral, topical, intravenous, subcutaneous, intramuscular, intraperitoneal, intratumoral, intrathecal, or a combination thereof. In some embodiments, the composition is administered intravenously as a bolus, injection, or continuous infusion. In some embodiments, the composition or pharmaceutical composition is administered multiple times a day, twice a day, once a day, twice a week, three times a week, once a week, once every two weeks, or once a month or once every three months. In some embodiments, the composition is administered subcutaneously once a day, once a week, or once a month. In some embodiments, the condition is cancer.

[0108] In some embodiments, the cancer is ovarian cancer, colon cancer, lung cancer, cancer located in bone or bone marrow, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma (DIPG), breast cancer, liver cancer, colon cancer, brain cancer, spleen cancer, salivary gland cancer, renal cancer, muscle cancer, myeloid cell carcinoma, skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, neuroblastoma, prostate cancer, bladder cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or CMYC-overexpressing cancer. In some embodiments, the condition is brain cancer. In some embodiments, the brain cancer is glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, or diffuse intrinsic pontine glioma.

[0109] In some embodiments, the condition is a CNS disease. In some embodiments, the CNS disease is a neuroinflammatory or neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, motor neuron disease, Lyme disease, ataxia-telangiectasia, autosomal dominant cerebellar ataxia, Batten disease, corticobasal syndrome, Creutzfeldt-Jakob disease, para-fragile X tremor / ataxia syndrome, Kufour-Rakeb syndrome, Machado-Joseph disease, multiple sclerosis, chronic traumatic encephalopathy, or frontotemporal dementia.

[0110] In some embodiments, the condition is neuropathic pain, migraine, anxiety, or epilepsy. In some embodiments, the condition is chronic pain, acute pain, injury, mechanical pain, heat pain, cold pain, ischemic pain, and chemical-induced pain, inflammatory pain, migraine-related pain, headache-related pain, irritable bowel syndrome-related pain, fibromyalgia-related pain, arthritis pain, skeletal pain, joint pain, gastrointestinal pain, muscle pain, angina pain, facial pain, pelvic pain, claudication, postoperative pain, posttraumatic pain, tension headache, obstetric pain, gynecological pain, or chemotherapy-induced pain, nociceptive pain, neuropathic pain, allodynia, phantom pain, visceral pain, breakthrough pain, familial episodic pain syndrome, paroxysmal excruciating pain syndrome, congenital analgesia, or opioid withdrawal-related pain. In some embodiments, the condition is muscular dystrophy or spinal-bulbar muscular dystrophy (SBMA).

[0111] In some embodiments, the condition is inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn's disease. In some embodiments, the method further comprises transporting the composition across a cell layer. In some embodiments, the cell layer is an endothelial cell layer or an epithelial cell layer. In some embodiments, the endothelial cell layer is the blood-brain barrier. In some embodiments, the endothelial cell layer is an intestinal endothelial cell layer. In some embodiments, the epithelial cell layer is an intestinal epithelial cell layer.

[0112] In some embodiments, transporting comprises binding of the composition to TfR. In some embodiments, transporting further comprises transcytosis. In some embodiments, the transcytosis is vesicular transcytosis. In some embodiments, the transcytosis is TfR-mediated. In some embodiments, transporting further comprises dissociating the composition from TfR. In some embodiments, transporting further comprises releasing the active agent from the composition.

[0113] In various aspects, the disclosure provides methods for designing peptides capable of binding to TfR, the method comprising: using a computer program to identify stability- and folding-optimized peptides from a library of peptides, wherein each peptide in the library of peptides contains at least three intramolecular disulfide bonds; using a computer program to superimpose amino acid residues of binding patches of a peptide:TfR complex and determine stable peptides that bind to TfR after grafting the binding patches onto the peptides; and grafting the binding patches onto the peptides to generate stable peptides.

[0114] In some embodiments, the method further comprises performing site-saturation mutagenesis to obtain mutant peptides that have a higher binding affinity for TfR than the stable peptide. In some embodiments, the peptides in the library of peptides are 20 to 75 amino acid residues in length.

[0115] In various aspects, the present disclosure provides any of the compositions disclosed herein, wherein the neurotensin comprises any one of SEQ ID NO: 350, SEQ ID NO: 365-SEQ ID NO: 371, or a functional fragment thereof.

[0116] In various aspects, the present disclosure provides any of the methods disclosed herein, wherein the neurotensin comprises any one of SEQ ID NO: 350, SEQ ID NO: 365-SEQ ID NO: 371, or a functional fragment thereof.

[0117] In various aspects, the present disclosure provides any of the methods disclosed herein, wherein the neurotensin comprises any one of SEQ ID NO: 350, SEQ ID NO: 365-SEQ ID NO: 371, or a functional fragment thereof.

[0118] In various aspects, the present disclosure provides any of the methods disclosed herein, wherein the neurotensin comprises any one of SEQ ID NO: 350, SEQ ID NO: 365-SEQ ID NO: 371, or a functional fragment thereof.

[0119] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0120] This patent and application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, in which: [Brief explanation of the drawings]

[0121] [Figure 1A] Figure 1 shows a Coomassie stained gel of human soluble transferrin receptor (hTfR) ectodomain protein and flow cytometry plots demonstrating the sequential enrichment of cells that bind to the hTfR ectodomain from a pooled high diversity peptide library. [Figure 1B] Figure 1 shows a Coomassie-stained gel of human soluble transferrin receptor (hTfR) ectodomain protein and flow cytometry plots demonstrating the sequential enrichment of cells that bind to the hTfR ectodomain from a pooled high-diversity peptide library. Figure 2 shows flow cytometry plots demonstrating cells that bind to the TfR after a single flow sorting run. [Figure 1C]Figure 1 shows a Coomassie-stained gel of human soluble transferrin receptor (hTfR) ectodomain protein and flow cytometry plots demonstrating the sequential enrichment of cells that bind to the hTfR ectodomain from a pooled high-diversity peptide library. Figure 2 shows a flow cytometry plot of control-stained cells after a single flow sort. [Figure 1D] Figure 1 shows a Coomassie-stained gel of human soluble transferrin receptor (hTfR) ectodomain protein and flow cytometry plots demonstrating the sequential enrichment of cells that bind to the hTfR ectodomain from a pooled high-diversity peptide library. Figure 2 shows flow cytometry plots demonstrating cells that bind to the TfR after two rounds of flow sorting. [Figure 1E] Figure 1 shows a Coomassie-stained gel of human soluble transferrin receptor (hTfR) ectodomain protein and flow cytometry plots demonstrating the sequential enrichment of cells that bind to the hTfR ectodomain from a pooled high-diversity peptide library. Figure 2 shows a flow cytometry plot of control-stained cells after two rounds of flow sorting. [Figure 1F] Figure 1 shows a Coomassie-stained gel of human soluble transferrin receptor (hTfR) ectodomain protein and flow cytometry plots demonstrating the sequential enrichment of cells that bind to the hTfR ectodomain from a pooled high-diversity peptide library. Figure 2 shows flow cytometry plots demonstrating cells that bind to the TfR after three rounds of flow sorting. [Figure 1G] Figure 1 shows a Coomassie-stained gel of human soluble transferrin receptor (hTfR) ectodomain protein and flow cytometry plots demonstrating the sequential enrichment of cells that bind to the hTfR ectodomain from a pooled high-diversity peptide library. Figure 2 shows a flow cytometry plot of control-stained cells after three rounds of flow sorting. [Figure 2A]Figure 1 shows cells presenting TfR and a single cloned peptide. The control protein used in this experiment has the amino acid sequence shown in SEQ ID NO: 333(). Flow cytometry plots of cells expressing the peptide of SEQ ID NO: 1 (x-axis, GFP) and the control protein (y-axis, stained with fluorescent anti-His antibody) are shown. [Figure 2B]Figure 1 shows cells presenting TfR and a single cloned peptide. The control protein used in this experiment has the amino acid sequence shown in SEQ ID NO: 333 (). Flow cytometry plots of cells expressing the peptide of SEQ ID NO: 1 (x-axis, GFP) and the control protein (y-axis, stained with fluorescent streptavidin) are shown. [Figure 2C]Figure 1 shows cells presenting TfR and a single cloned peptide. The control protein used in this experiment has the amino acid sequence shown in SEQ ID NO: 333(). Flow cytometry plots of cells expressing the peptide of SEQ ID NO: 1 (x-axis, GFP) and TfR (y-axis, stained with fluorescent anti-His antibody) are shown. [Figure 2D]Figure 1 shows cells presenting TfR and a single cloned peptide. The control protein used in this experiment has the amino acid sequence shown in SEQ ID NO: 333(). Flow cytometry plots of cells expressing the peptide of SEQ ID NO: 1 (x-axis, GFP) and TfR (y-axis, stained with fluorescent streptavidin) are shown. [Figure 3A]This figure shows TfR binding to peptide variants generated by permuting variants enriched by site-saturation mutagenesis (SSM). Each graph represents the completion of one round of SSM, and the colored bars in the graph indicate the number of mutations in the variant peptide compared to the respective peptide sequence at the start of the SSM round. The data represent the final step of SSM, after which the next generation molecule is determined. This figure shows the amount of hTfR bound to variants containing the sequences of SEQ ID NOs: 3 to 23, respectively, obtained from site-saturation mutagenesis (SSM) for affinity maturation of a peptide with the sequence of SEQ ID NO: 1. [Figure 3B] This figure shows TfR binding to peptide variants generated by permuting variants enriched by site-saturation mutagenesis (SSM). Each graph represents the completion of one round of SSM, and the colored bars in the graph indicate the number of mutations in the variant peptide compared to the respective peptide sequence at the start of the SSM round. The data represent the final step of SSM, after which the next generation molecule is determined. This figure shows the amount of hTfR bound to peptide variants with sequences SEQ ID NOs: 24 to 31, respectively, obtained from site-saturation mutagenesis (SSM) for affinity maturation of the starting peptide with the sequence SEQ ID NO: 2. [Figure 4A] Figure 1 shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC plots of five soluble TfR-binding peptide variants, each having the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 30, and SEQ ID NO: 32, under reducing (DTT) or non-reducing (PBS) conditions. The data indicate that the TfR-binding peptides are highly soluble. Comparison of the gels under reducing (10 mM DTT) and non-reducing (PBS) conditions indicates that the peptides form disulfide bonds. Figure 2 shows SDS-PAGE and RP-HPLC chromatograms of SEQ ID NO: 1. [Figure 4B]Figure 1 shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC plots of five soluble TfR-binding peptide variants, each having the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 30, and SEQ ID NO: 32, under reducing (DTT) or non-reducing (PBS) conditions. The data indicate that the TfR-binding peptides are highly soluble. Comparison of the gels under reducing (10 mM DTT) and non-reducing (PBS) conditions indicates that the peptides form disulfide bonds. Figure 2 shows SDS-PAGE and RP-HPLC chromatograms of SEQ ID NO: 2. [Figure 4C] Figure 1 shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC plots of five soluble TfR-binding peptide variants, each having the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 30, and SEQ ID NO: 32, under reducing (DTT) or non-reducing (PBS) conditions. The data indicate that the TfR-binding peptides are highly soluble. Comparison of the gels under reducing (10 mM DTT) and non-reducing (PBS) conditions indicates that the peptides form disulfide bonds. Figure 2 shows SDS-PAGE and RP-HPLC chromatograms of SEQ ID NO: 4. [Figure 4D] Figure 1 shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC plots of five soluble TfR-binding peptide variants, each having the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 30, and SEQ ID NO: 32, under reducing (DTT) or non-reducing (PBS) conditions. The data indicate that the TfR-binding peptides are highly soluble. Comparison of the gels under reducing (10 mM DTT) and non-reducing (PBS) conditions indicates that the peptides form disulfide bonds. Figure 2 shows SDS-PAGE and RP-HPLC chromatograms of SEQ ID NO: 30. [Figure 4E]Figure 1 shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC plots of five soluble TfR-binding peptide variants, each having the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 30, and SEQ ID NO: 32, under reducing (DTT) or non-reducing (PBS) conditions. The data indicate that the TfR-binding peptides are highly soluble. Comparison of the gels under reducing (10 mM DTT) and non-reducing (PBS) conditions indicates that the peptides form disulfide bonds. Figure 2 shows SDS-PAGE and RP-HPLC chromatograms of SEQ ID NO: 32. [Figure 5A] Figure 1 shows that later generation TfR-binding peptides have improved resistance to glutathione (GSH) reduction. Overlays of reverse-phase HPLC traces (one for each peptide tested) of peptides subjected to either no treatment (red), 10 μM GSH (blue), or 10 μM DTT (green) are shown. The resistance of the TfR-binding peptide having the sequence of SEQ ID NO: 1 to glutathione (GSH) reduction is shown. [Figure 5B] Figure 1 shows that later generation TfR-binding peptides have improved resistance to glutathione (GSH) reduction. Shown is an overlay of reverse-phase HPLC traces (one for each peptide tested) of peptides subjected to either no treatment (red), 10 μM GSH (blue), or 10 μM DTT (green). Improved TfR-binding peptide resistance to glutathione (GSH) reduction is shown for the peptide having the sequence of SEQ ID NO: 2. [Figure 5C] Figure 1 shows that later generation TfR-binding peptides have improved resistance to glutathione (GSH) reduction. Shown is an overlay of reverse-phase HPLC traces (one for each peptide tested) of peptides subjected to either no treatment (red), 10 μM GSH (blue), or 10 μM DTT (green). Improved TfR-binding peptide resistance to glutathione (GSH) and dithiothreitol (DTT) reduction for the peptide having the sequence of SEQ ID NO: 32 is shown. [Figure 5D]Figure 1 shows that later generation TfR-binding peptides have improved resistance to glutathione (GSH) reduction. Shown is an overlay of reverse-phase HPLC traces (one for each peptide tested) of peptides subjected to either no treatment (red), 10 μM GSH (blue), or 10 μM DTT (green). Improved TfR-binding peptide resistance to glutathione (GSH) reduction is shown for the peptide having the sequence of SEQ ID NO: 30. [Figure 6A] Figure 1 shows that the resistance of affinity-matured TfR-binding peptides to pepsin improves with peptide maturation. Shown is an overlay of reverse-phase HPLC traces (one for each peptide tested) of TfR-binding peptides subjected to either no treatment (red), trypsin (blue), or pepsin (green). Shown is treatment of the peptide having the sequence of SEQ ID NO: 1 with trypsin (blue) and pepsin (green). [Figure 6B] Figure 1 shows that the resistance of affinity-matured TfR-binding peptides to pepsin improves with peptide maturation. Shown is an overlay of reverse-phase HPLC traces (one for each peptide tested) of TfR-binding peptides subjected to either no treatment (red), trypsin (blue), or pepsin (green). Shown is treatment of the peptide having the sequence of SEQ ID NO: 2 with trypsin (blue) and pepsin (green). [Figure 6C] Figure 1 shows that the resistance of affinity-matured TfR-binding peptides to pepsin improves with peptide maturation. Shown is an overlay of reverse-phase HPLC traces (one for each peptide tested) of TfR-binding peptides subjected to either no treatment (red), trypsin (blue), or pepsin (green). Shown is treatment of the peptide having the sequence of SEQ ID NO: 32 with trypsin (blue) and pepsin (green). [Figure 6D] Figure 1 shows that the resistance of affinity-matured TfR-binding peptides to pepsin improves with peptide maturation. Shown is an overlay of reverse-phase HPLC traces (one for each peptide tested) of TfR-binding peptides subjected to either no treatment (red), trypsin (blue), or pepsin (green). Shown is treatment of the peptide having the sequence of SEQ ID NO: 30 with trypsin (blue) and pepsin (green). [Figure 7] 1 shows surface plasmon resonance (SPR) curves showing binding of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 32 to hTfR. [Figure 8] 1 shows surface plasmon resonance (SPR) traces showing hTfR binding at varying concentrations of a peptide having the sequence of SEQ ID NO:2. [Figure 9] 1 shows surface plasmon resonance (SPR) traces showing hTfR binding at varying concentrations of a peptide having the sequence of SEQ ID NO:4. [Figure 10] Binding and single-cycle kinetic data for SEQ ID NO: 32 binding to captured biotinylated hTfR by surface plasmon resonance (SPR) are shown. Five concentrations of the peptide having the sequence of SEQ ID NO: 32 (0.037 nM, 0.11 nM, 0.33 nM, 1 nM, and 3 nM) were injected over two densities of captured biotinylated (Bt)-hTfR and analyzed globally. [Figure 11] Binding and single-cycle kinetic data of SEQ ID NO: 30 binding to captured biotinylated hTfR by SPR are shown. Five concentrations of the peptide having the sequence of SEQ ID NO: 30 (0.037 nM, 0.11 nM, 0.33 nM, 1 nM, 3 nM) were injected over two densities of captured Bt-hTfR and analyzed globally. [Figure 12] 1 shows a bar graph quantitating flow cytometry data from a competitive binding assay in which peptides having the sequences of SEQ ID NO:2 and SEQ ID NO:32 were incubated with hTfR in the presence of 10 μM iron-loaded human holotransferrin or empty human apotransferrin. [Figure 13A]Figure 1 shows the co-crystallization of hTfR with an hTfR-binding peptide having the sequence of SEQ ID NO: 32. Figure 2 shows the first view of hTfR co-crystallized with a peptide having the sequence of SEQ ID NO: 32. The structure shows that the binding site of the hTfR-binding peptide overlaps with the binding site of the endogenous binding substance, transferrin. The connecting sequence between the two helices, depicted by the solid black coiled line, was not resolved in the crystal structure and is therefore based on structural modeling. The crystal structure of the transferrin receptor (TfR) is displayed as a space-filling electron density map. It appears to be a large globular protein with two prominent lobes. Between the two lobes, the crystal structure of a short peptide corresponding to SEQ ID NO: 32 is shown interacting with the globular structure of the transferrin receptor. The peptide of SEQ ID NO: 32 is shown as a cartoon model with two antiparallel α-helices oriented diagonally from the upper left to the lower right. Stick models corresponding to the amino acid side chains of SEQ ID NO: 32 are shown protruding from the α-helices, some of which interact with the globular protein of TfR. [Figure 13B] 13A shows co-crystallization of hTfR with an hTfR-binding peptide having the sequence of SEQ ID NO: 32. A close-up of FIG. 13A is shown, depicting hTfR co-crystallized with a peptide having the sequence of SEQ ID NO: 32. Any amino acid side chains that were mutated from the endogenous CDP or that are part of the interface are shown as sticks. Residues adjacent to the interface are shown in black, while residues distal to the interface are shown in cyan. A portion of the space-filling electron density map of TfR shown in FIG. 13A interacting with the peptide of SEQ ID NO: 32 is shown as a cartoon model with two antiparallel α-helices, as shown in FIG. 13A, positioned directly above the structure of TfR, with the α-helices oriented horizontally. The amino acid side chains of SEQ ID NO: 32 are shown as sticks. The side chains of SEQ ID NO: 32 that project downward from the α-helix toward the globular structure of TfR interact with TfR. [Figure 13C]Figure 1 shows co-crystallization of hTfR with an hTfR-binding peptide having the sequence of SEQ ID NO:32. Shown are a structural model of SEQ ID NO:32 (top row, N-terminus blue, C-terminus red, visible side chains colored by atom type), a cartoon representation of the crystal structure of free SEQ ID NO:32 (interrupted, blue; 2.55 Å resolution, 4-fold overlay), and a cartoon representation of the crystal structure of SEQ ID NO:32 when complexed with TfR (bottom row, red; 1.85 Å resolution, 2-fold overlay). Side chains with atoms within 4 Å of TfR in the complex structure are shown as sticks. The N- and C-termini are labeled. Red arrows indicate the most notable structural difference near the N-terminus between the model, unbound, and bound structures of SEQ ID NO:32. A gray cross indicates the location of the disordered interhelical linker. The top structure, labeled "Model," shows an in silico model of the peptide corresponding to SEQ ID NO:32, displayed as a cartoon model. The horizontally oriented antiparallel α-helices are connected by a short loop on the right side. The N-terminus of the peptide is located to the left of the lower helix, and the C-terminus is located to the left of the upper helix. Amino acid side chains are shown as sticks protruding from the α-helices. The middle structure, labeled "4x free," shows the crystal structure of the peptide corresponding to SEQ ID NO: 32 crystallized in the absence of TfR, shown as a cartoon model. Loops observed in the in silico model are not shown in this structure and are indicated by Xs to the right of the two helices. The N-terminus of the peptide is located to the left of the lower helix, and the C-terminus is located to the left of the upper helix. Amino acid side chains are shown as sticks protruding from the α-helices. The bottom structure, labeled "2x TfR bound," shows the crystal structure of the peptide corresponding to SEQ ID NO: 32 crystallized in the presence of TfR, shown as a cartoon model. Loops observed in the in silico model are not shown in this structure and are indicated by Xs to the right of the two helices. The N-terminus of the peptide is located to the left of the lower helix and the C-terminus is located to the left of the upper helix. Amino acid side chains are shown as sticks protruding from the α-helix.Three triangular arrows pointing to the N-terminal region at the left end of the lower helix of each structure indicate the region of most significant structural difference between the three structures. [Figure 13D] Figure 1 shows co-crystallization of hTfR with an hTfR-binding peptide having the sequence of SEQ ID NO: 32. A close-up of SEQ ID NO: 32 bound to TfR is shown, demonstrating the numerous polar and non-polar interactions that drive binding. Non-polar interactions are primarily Leu and Met, while polar interactions are a mix of several charged and uncharged residues. A portion of the crystal structure of TfR is shown as a space-filling electron density map. The peptide corresponding to SEQ ID NO: 2 is shown as a ribbon diagram above the TfR structure. The α-helices are oriented vertically, and a loop connects the tops of the two helices. Amino acid side chains are shown as sticks, with the side chains facing the TfR structure interacting with TfR. The amino acid residues of SEQ ID NO: 32 are written as follows (top to bottom, then left to right): E16, M9, S6, N12, L43, L15, L36, and D44. [Figure 13E]Figure 14 shows the co-crystallization of hTfR with an hTfR-binding peptide having the sequence of SEQ ID NO: 32. Alignment of the TfR:SEQ ID NO: 32 co-crystal with the published co-crystal of the TfR:transferrin complex (PDB 3S9L; TfR not shown) is shown, expanding on the binding interactions shown in Figure 14A. The structural alignment (left) shows the overlap of the binding sites of SEQ ID NO: 32 (blue) and transferrin (SEQ ID NO: 353, green).The binding site of SEQ ID NO: 32 is colored to match the homology of the human (SEQ ID NO: 353) and mouse (SEQ ID NO: 354) transferrin receptors, showing that most of the binding surface on the TfR is identical or similar between the two species (right). The left panel shows a portion of the structure of TfR as a space-filling electron density map. A ribbon diagram of transferrin derived from the 3S9L crystal structure (labeled "3S9L-derived Tf") is positioned to the right of the TfR and superimposed on the TfR structure. The Tf structure contains numerous α-helices that match the TfR structure.This structure also contains a β-sheet structure in the lower right region, as well as numerous loops protruding from the ends of the α-helices. The structure of SEQ ID NO: 32 crystallized with TfR is shown in the upper right as a cartoon model interacting with TfR. It consists of two vertically oriented α-helices, overlapping with the superimposed Tf structure. The right panel shows a rotated view of the TfR and SEQ ID NO: 32 structures shown in the left panel. TfR is shown as a space-filling electron density map, and SEQ ID NO: 32 is shown as a cartoon model containing two α-helices oriented diagonally from the upper left to the lower right. Shaded regions on the TfR structure indicate regions where the binding sites of HsTfR and MmTfR are similar or dissimilar. Exemplary similar and dissimilar regions are indicated by arrows. The remaining regions of the structure not marked as similar or dissimilar, which occupy most of the surface of the TfR structure, have identical binding sites. A small region just above the right helix and a small region just below the top end of the right helix of SEQ ID NO: 32 are shown to have similar binding sites. A small region just above the right helix, a small region just below the bottom end of the right helix, and a small region just below the bottom end of the left helix of SEQ ID NO: 32 are shown to have dissimilar binding sites. [Figure 14A]1 shows the TfR:transferrin complex and the TfR:peptide complex, showing the respective superpositions of transferrin (data taken from the public literature (RCSB PDB 1SUV)) and the peptide having the sequence of SEQ ID NO: 32 bound to hTfR, indicating that transferrin and the peptide having the sequence of SEQ ID NO: 32 overlap in their binding sites on hTfR.

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[0081] [0 The arrow labeled "SEQ ID NO: 32" points to the site just above transferrin on the right side of the TfR, indicating the binding site for the peptide corresponding to SEQ ID NO: 32. [Figure 14B]14A and 14B show the TfR:transferrin complex and the TfR:peptide complex, respectively, showing the superposition of transferrin (data obtained from the public literature (RCSB PDB 1SUV)) and the peptide having the sequence of SEQ ID NO: 32 bound to hTfR, indicating that transferrin and the peptide having the sequence of SEQ ID NO: 32 have overlapping binding sites on hTfR. A close-up of the peptide having the sequence of SEQ ID NO: 32 bound to TfR is shown, and compared to the binding site of endogenous transferrin in FIG. 14A, it shows that both the peptide having the sequence of SEQ ID NO: 32 and transferrin have overlapping binding sites on hTfR. Any amino acid side chains that have been mutated from endogenous CDP or are part of the interface are indicated by sticks. Residues adjacent to the interface are indicated in black, while residues distal to the interface are indicated in cyan. A portion of the space-filling electron density map of TfR shown in FIG. 13A interacting with the peptide of SEQ ID NO: 32 is shown. The peptide of SEQ ID NO:32 is shown as a cartoon model with two antiparallel α-helices positioned directly above the TfR structure, with the α-helices oriented horizontally, as shown in Figure 13A. The amino acid side chains of SEQ ID NO:32 are shown as sticks. The side chains of SEQ ID NO:32 that project downward from the α-helix toward the globular structure of TfR interact with TfR. [Figure 15A]We demonstrate that the TfR-binding peptide is cross-reactive with mouse TfR (mTfR) in cell surface binding assays. 293F cells expressing either human or mouse TfR on their surface were stained with a soluble TfR-binding peptide directly labeled with AlexaFluor 647 dye. This demonstrates the species specificity of the TfR used in these experiments, in this case, human TfR. Data are presented as two topographic density maps, showing flow cytometry data for transferrin stained with an anti-hTfR (CD71) antibody. The upper density map, arranged diagonally from bottom left to top right, shows 293ST + SDGF-hTfR. The lower density map, arranged horizontally, shows 293ST + SDGF-mTfR. The y-axis shows hTfR + streptavidin on a logarithmic scale of 0–107 in 10 increments. The x-axis shows GFP on a logarithmic scale of 0–106 in 10 increments. [Figure 15B] We demonstrate that the TfR-binding peptide is cross-reactive with mouse TfR (mTfR) in cell surface binding assays. 293F cells expressing either human or mouse TfR on their surface were stained with a soluble TfR-binding peptide directly labeled with AlexaFluor 647 dye. This demonstrates the species specificity of the TfR used in these experiments, in this case, mouse TfR. Data are presented as two topographic density maps, showing flow cytometry data for transferrin stained with an anti-mTfR (CD71) antibody. The upper density map, arranged diagonally from bottom left to top right, shows 293ST + SDGF-mTfR. The lower density map, with three lobes, shows 293ST + SDGF-hTfR. The y-axis shows hTfR + streptavidin on a logarithmic scale from 10-4 to 10-7 in 10-step increments. The x-axis shows GFP on a logarithmic scale from 0 to 10-6 in 10-step increments. [Figure 15C]This shows that TfR-binding peptides are cross-reactive with mouse TfR (mTfR) in cell surface binding assays. 293F cells expressing either human or mouse TfR on their surface were stained with soluble TfR-binding peptides directly labeled with AlexaFluor 647 dye. The peptides having the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 32 bind to human TfR. Data are presented as four topographic density maps, representing flow cytometry data using 293ST cells + SDGF-hTFR. Three density maps are nearly overlapping and are positioned on top of the fourth density map. The bottom density map is horizontally aligned and represents SEQ ID NO: 1 (first generation). The top three density maps are diagonally aligned from bottom left to top right. The density map slightly above the other two corresponds to SEQ ID NO: 32 (third generation). The density map slightly below the other two corresponds to SEQ ID NO: 2 (second generation). The third density map corresponds to SEQ ID NO: 30 (third generation). The y-axis shows hTfR+streptavidin on a logarithmic scale from 0 to 10 in increments of 10. The x-axis shows GFP on a logarithmic scale from 0 to 10 in increments of 10. [Figure 15D]This shows that TfR-binding peptides are cross-reactive with mouse TfR (mTfR) in cell surface binding assays. 293F cells expressing either human or mouse TfR on their surface were stained with soluble TfR-binding peptides directly labeled with AlexaFluor 647 dye. The peptides having the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 32 bind to mouse TfR. Data are presented as four topographic density maps, representing flow cytometry data using 293ST cells + SDGF-mTFR. Three density maps are nearly overlapping and positioned on top of the fourth. The bottom density map is horizontally aligned and represents SEQ ID NO: 1 (first generation). The top three density maps are diagonally aligned from bottom left to top right. The density map slightly above the other two corresponds to SEQ ID NO: 32 (third generation). The density map slightly below the other two corresponds to SEQ ID NO: 2 (second generation). The third density map corresponds to SEQ ID NO: 30 (third generation). The y-axis shows hTfR+streptavidin on a logarithmic scale from 0 to 10 in increments of 10. The x-axis shows GFP on a logarithmic scale from 0 to 10 in increments of 10. [Figure 16A] Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled TfR-binding peptides (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 32) to assess biodistribution. Images were taken 3 hours after administration of a 100 nmol dose of peptide (20 mg / kg, assuming approximately 25 g body weight) to mice bearing xenograft tumors (subcutaneous human astrocytoma U87 cells). These images show significant accumulation in the spleen, liver, and kidney, with high accumulation also in muscle, bone marrow, and skin. While CNS accumulation is less than in other tissues, it is still significant (>25% of serum) compared to historical values ​​for compounds and biologics that do not penetrate the blood-brain barrier (BBB). Background blood levels observed in the brain are 3% of the cardiac blood signal. Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled SEQ ID NO: 1. [Figure 16B]Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled TfR-binding peptides (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 32) to assess biodistribution. Images were taken 3 hours after administration of a 100 nmol dose of peptide (20 mg / kg, assuming approximately 25 g body weight) to mice bearing xenograft tumors (subcutaneous human astrocytoma U87 cells). These images show significant accumulation in the spleen, liver, and kidney, with high accumulation also in muscle, bone marrow, and skin. While CNS accumulation is less than in other tissues, it is still significant (>25% of serum) compared to historical values ​​for compounds and biologics that do not penetrate the blood-brain barrier (BBB). Background blood levels observed in the brain are 3% of the cardiac blood signal. Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled SEQ ID NO: 2. [Figure 16C] Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled TfR-binding peptides (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 32) to assess biodistribution. Images were taken 3 hours after administration of a 100 nmol dose of peptide (20 mg / kg, assuming approximately 25 g body weight) to mice bearing xenograft tumors (subcutaneous human astrocytoma U87 cells). These images show significant accumulation in the spleen, liver, and kidney, with high accumulation also in muscle, bone marrow, and skin. While CNS accumulation is less than in other tissues, it is still significant (>25% of serum) compared to historical values ​​for compounds and biologics that do not penetrate the blood-brain barrier (BBB). Background blood levels observed in the brain are 3% of the cardiac blood signal. Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled SEQ ID NO: 30. [Figure 16D]Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled TfR-binding peptides (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 32) to assess biodistribution. Images were taken 3 hours after administration of a 100 nmol dose of peptide (20 mg / kg, assuming approximately 25 g body weight) to mice bearing xenograft tumors (subcutaneous human astrocytoma U87 cells). These images show significant accumulation in the spleen, liver, and kidney, with higher accumulation in muscle, bone marrow, and skin. While CNS accumulation is less than in other tissues, it is still significant (>25% of serum) compared to historical values ​​for compounds and biologics that do not penetrate the blood-brain barrier (BBB). Background blood levels observed in the brain are 3% of the cardiac blood signal. Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled SEQ ID NO: 32. [Figure 17A] Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled peptides (SEQ ID NO: 1 and SEQ ID NO: 32) to assess biodistribution. Significant accumulation was also observed in the flank tumor. Accumulation within the tumor was dispersed throughout rather than concentrated in blood-rich areas, suggesting extravasation and distribution throughout the tumor parenchyma. Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled SEQ ID NO: 1. [Figure 17B] Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled peptides (SEQ ID NO: 1 and SEQ ID NO: 32) to assess biodistribution. Significant accumulation was also observed in the flank tumor. Accumulation within the tumor was dispersed throughout rather than concentrated in blood-rich areas, suggesting extravasation and distribution throughout the tumor parenchyma. Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled SEQ ID NO: 1. [Figure 17C]Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled peptides (SEQ ID NO: 1 and SEQ ID NO: 32) to assess biodistribution. Significant accumulation was also observed in the flank tumor. Accumulation within the tumor was dispersed throughout rather than concentrated in blood-rich areas, suggesting extravasation and distribution throughout the tumor parenchyma. Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled SEQ ID NO: 32. [Figure 17D] Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled peptides (SEQ ID NO: 1 and SEQ ID NO: 32) to assess biodistribution. Significant accumulation was also observed in the flank tumor. Accumulation within the tumor was dispersed throughout rather than concentrated in blood-rich areas, suggesting extravasation and distribution throughout the tumor parenchyma. Figure 1 shows whole-body autoradiography of mice intravenously injected with 14C-labeled SEQ ID NO: 32. [Figure 18] Quantification of peptide biodistribution and organ accumulation after a single IV dose of 20 mg / kg of either a TfR-binding peptide having the sequence of SEQ ID NO: 1 or a peptide having the sequence of SEQ ID NO: 32 is shown. This dose reached levels of over 150 nM in the CNS and approximately 1 μM in the tumor after 3 hours. Bar graphs quantify the levels of each C-labeled peptide relative to known radioactivity in the same image, as measured by whole-body autoradiography, and the peptide specific activity in various tissues obtained 3 hours after administration from mice intravenously injected with the peptide. [Figure 19] Quantification of peptide biodistribution and organ accumulation is shown for peptides having the sequences of SEQ ID NO: 1 and SEQ ID NO: 32. Values ​​represent tissue levels versus blood levels, with no brain penetration resulting in a 3% level, assuming the brain is 3% blood. Bar graphs quantify the levels of each C-labeled peptide normalized to a control of known radioactivity in the same image, as measured by whole-body autoradiography, and to the peptide's specific activity in various tissues obtained 3 hours post-dose from mice intravenously injected with the peptide. [Figure 20A]Figure 1 shows scintillation counts over time in various tissues after administration of 20 mg / kg of a TfR-binding peptide having the sequence of SEQ ID NO: 32 with a specific activity of 147 Ci / mol, verifying whole-body autoradiography (WBA) tissue accumulation. Figure 1 shows scintillation counts over time in the kidney. The y-intercept at time 0 (Y0) is 1900 pCi, the half-life (t1 / 2) is 0.89 hours, and the goodness of fit R2 is 0.89. [Figure 20B] Figure 1 shows scintillation counts over time in various tissues after administration of 20 mg / kg of a TfR-binding peptide having the sequence of SEQ ID NO: 32 with a specific activity of 147 Ci / mol, verifying whole-body autoradiography (WBA) tissue accumulation. Figure 1 shows scintillation counts over time in the liver. The y-intercept at time 0 (Y0) is 400 pCi, the half-life (t1 / 2) is 7.22 hours, and the goodness of fit R2 is 0.97. [Figure 20C] Figure 1 shows scintillation counts over time in various tissues after administration of 20 mg / kg of a TfR-binding peptide having the sequence of SEQ ID NO: 32 with a specific activity of 147 Ci / mol, verifying whole-body autoradiography (WBA) tissue accumulation. Figure 1 shows scintillation counts over time in the brain. The y-intercept at time 0 (Y0) is 7.3 pCi, the half-life (t1 / 2) is 0.72 hours, and the goodness of fit R2 is 0.45. [Figure 20D] Figure 1 shows scintillation counts over time in various tissues after administration of 20 mg / kg of a TfR-binding peptide having the sequence of SEQ ID NO: 32 with a specific activity of 147 Ci / mol, verifying whole-body autoradiography (WBA) tissue accumulation. Figure 1 shows scintillation counts over time in the spleen. The y-intercept at time 0 (Y0) is 168 pCi, the half-life (t1 / 2) is 12.1 hours, and the goodness of fit R2 is 0.93. [Figure 20E]Figure 1 shows scintillation counts over time in various tissues after administration of 20 mg / kg of a TfR-binding peptide having the sequence of SEQ ID NO: 32 with a specific activity of 147 Ci / mol, verifying whole-body autoradiography (WBA) tissue accumulation. Figure 1 shows scintillation counts over time in muscle. The y-intercept at time 0 (Y0) is 31.4 pCi, the half-life (t1 / 2) is 2.54 hours, and the goodness of fit R2 is 0.85. [Figure 20F] Figure 1 shows scintillation counts over time in various tissues after administration of 20 mg / kg of a TfR-binding peptide having the sequence of SEQ ID NO: 32 with a specific activity of 147 Ci / mol, verifying whole-body autoradiography (WBA) tissue accumulation. Figure 1 shows scintillation counts over time in skin. The y-intercept at time 0 (Y0) is 68.3 pCi, the half-life (t1 / 2) is 0.33 hours, and the goodness of fit R2 is 0.31. [Figure 21] 1 shows a serum elimination plot in mice intravenously administered 20 mg / kg of a peptide having the sequence of SEQ ID NO: 32, including a biphasic elimination regression analysis showing the kinetics of the fast phase (95%, t1 / 2 15.6 min) and slow phase (5%, t1 / 2 10.3 hr). [Figure 22A] 1 shows multiple time regression and capillary depletion analyses of TfR-binding peptides having the sequences of SEQ ID NO: 1 and SEQ ID NO: 32. The multiple regression analyses of SEQ ID NO: 1 and SEQ ID NO: 32 are shown in a single plot, with the y-axis representing the brain to serum ratio. [Figure 22B] 1 shows multiple time regression analysis and capillary depletion analysis of TfR-binding peptides having the sequences of SEQ ID NO: 1 and SEQ ID NO: 32. The parenchymal and capillary distribution of TfR-binding peptides having the sequences of SEQ ID NO: 1 and SEQ ID NO: 32 is shown, with the y-axis representing the tissue-to-serum ratio (μL / g). [Figure 23A]Purification and testing of soluble transferrin receptor (TfR) ectodomain. Surface plasmon resonance (SPR) of holo- or apo-transferrin (Tf) binding to purified TfR is shown. Holo-Tf binds to TfR, but apo-Tf does not, as evidenced by an increase in response (RU) over time. [Figure 23B] Figure 1 shows the purification and testing of soluble transferrin receptor (TfR) ectodomain. A schematic diagram of vector display scaffolds and target binding used to screen and optimize peptide binding properties is shown. A surface display vector (SDGF) encoding a GFP-tagged construct of a binding agent (e.g., SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 32) is expressed on the cell surface. A target protein (e.g., TfR) labeled with a fluorescent dye ("co-stain") binds to the surface-expressed binding agent. [Figure 23C] Figure 23C shows the purification and testing of soluble transferrin receptor (TfR) ectodomain. Figure 23C shows flow cytometry screening for specific TfR binding, as measured by the amount of Alexa Fluor 647-TfR (costained in Figure 23B). Cells transfected with the Machupo virus glycoprotein (SDGF-MaCV) are tested with biotinylated TfR in combination with Alexa Fluor 647-labeled streptavidin (Strep-647), SDGF-MaCV cells in combination with Alexa Fluor 647-labeled elastase, or SDGF-elafin cells in combination with TfR and Strep-647. Cell conjugates of elastase and elafin do not bind to cells. [Figure 24] Whole-body autoradiography (WBA) of 14C peptide variant distribution is shown. Histological sections of mice treated with 14C peptide variants at 30 and 180 minutes post-administration are shown. Representative WBA images of the brain and heart (inset) of three peptide generations are shown, along with images of a control, non-TfR-binding CDP. Whole-body images of the same sections are shown in Figure 25, and some of the 180-minute sections are also shown in Figure 16. [Figure 25]Whole-body radiographs taken 30 minutes (left column) and 180 minutes (right column) after administration are shown in Figure 24. A portion of the 180-minute section is shown in Figure 16. [Figure 26] Figure 1 shows the thermal stability of SEQ ID NO: 32 under reducing and non-reducing conditions as measured using circular dichroism. Relative ellipticity at 210 nm (mdeg) is plotted against temperature in °C. NR indicates non-reduced. DTT indicates reduction of the peptide with DTT. Vertical lines represent calculated melting points (approximately 74 °C for NR and approximately 38 °C for 10 mM DTT treatment) based on non-linear curve fitting (sigmoidal, variable slope, constrained base = 0; R2 > 0.98 in both cases). Note that the NR sample does not reach equilibrium until 95 °C, so the protein may not be completely "melted" at high temperatures and may simply be in a disordered state, especially in the extended loop region. [Figure 27] Figure 1 shows the purification and characterization of mouse transferrin, mouse transferrin-NT fusion, and various CDP-neurotensin (NT) conjugates of the present disclosure. SDS-PAGE and Coomassie dye-stained gels under reducing (DTT) and non-reducing (PBS) conditions demonstrate the purity of the peptides after purification. As shown are mouse endogenous transferrin (designated MmTf, SEQ ID NO: 354), mouse endogenous transferrin conjugated to neurotensin (designated MmTf-NT, SEQ ID NO: 355), and CDP-NT peptide constructs (SEQ ID NOs: 33-35). Gels are run under reducing (DTT) or non-reducing (PBS) conditions. Reduction with 10 mM DTT indicated that the peptides formed disulfide bonds. [Figure 28A]Figure 1 shows a CDP-NT peptide construct that induces an IP1 response downstream of the neurotensin receptor (NTSR) in both CRE-luciferase (CRE-Luc) mice and mammalian cells. Related pathways affecting CRE-driven luciferase in CRE-Luc mice are shown. PLC indicates phospholipase C. AC indicates adenylyl cyclase. CaMK indicates calmodulin-dependent protein kinase. CREB indicates cAMP response element-binding protein. PKA indicates protein kinase A. PDE indicates cAMP phosphodiesterase. FS indicates forskolin. Rol indicates rolipram. GPCR indicates G protein-coupled receptor. [Figure 28B] This figure shows the CDP-NT peptide construct inducing an IP1 response downstream of the neurotensin receptor (NTSR) in both CRE-luciferase (CRE-Luc) mice and mammalian cells. This figure demonstrates in vitro neurotensin (NT) receptor binding, and demonstrates IP1 accumulation only in response to NT or the NT peptide construct in HEK-293 cells expressing NTSR1. IP1 was measured using a FRET ratio readout using an assay kit (CisBio 62IPAPEB). All wells were 3, except for vehicle, which was 36. Horizontal bars indicate sample means. mTF = mouse transferrin. Baseline HEK293 = mean assay value of HEK293 cells not expressing NTSR1 (N = 36 wells) was included as a reference. [Figure 29] Immunohistochemical staining of tissue samples from mice administered with NT peptide constructs is shown. Tissue staining demonstrates increased luciferase expression in the cortex, striatum, and thalamus of mice 4 hours after administration of CDP-NT of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. Mice administered with transferrin-NT ("transferrin-NT," SEQ ID NO: 355) or no peptide ("no stimulation") are shown as controls. The scale bar (upper left panel) is 100 μm, and all panels are at the same magnification. [Figure 30A]Quantification of the effect of CDP-NT peptide constructs in CRE-luciferase (CRE-Luc) mice is shown. Luminescence (via intraperitoneal luciferin administration) is shown either before (unstimulated) or 4 hours after (labeled "NT fusion" or "parent") intravenous administration of a parent TfR-binding peptide ("Parent") (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 32, or SEQ ID NO: 354, as indicated) or a cysteine-dense peptide (CDP)-NT construct or a mouse transferrin-NT construct (e.g., comprising SEQ ID NO: 33-SEQ ID NO: 35 or SEQ ID NO: 355, as indicated) ("NT fusion") using a matched cohort. Horizontal bars represent sample means. Significance was determined using an unpaired, two-tailed t-test. (*: P<0.05; **: P<0.01; #: P<0.0001). Mouse transferrin was used for both the parental (SEQ ID NO: 354) and NT transferrin peptide constructs (SEQ ID NO: 355). [Figure 30B] 30A shows the quantification of the effect of CDP-NT peptide constructs in CRE-luciferase (CRE-Luc) mice. Images of the SEQ ID NO: 32 cohort quantified in Figure 30A are shown in pseudocolor luminescence intensity. All images have been equally background-subtracted and contrast-enhanced for clarity only. [Figure 31] Figure 3 shows in vivo CRE-luciferase luminescence 4 hours after induction with forskolin and rolipram. The images show luciferase fluorescence images of mice and serve as a positive control for the experiment shown in Figure 30. Forskolin and rolipram activate CRE-dependent luciferase expression independent of the neurotensin receptor pathway. Forskolin and rolipram induce CRE expression via activation of adenylyl cyclase and inhibition of cAMP phosphodiesterase, respectively. N=8 mice. Panels show replicate animals treated identically with rolipram and forskolin. One mouse was discontinued due to missed intraperitoneal luciferin injection. [Figure 32]Luciferase expression in CRE-Luc mice after intravenous administration of free neurotensin peptide is shown. Intravenous free neurotensin fails to induce luciferase expression in CRE-Luc mice. Four hours after intravenous administration of a high dose of free neurotensin peptide (300 nmol) or vehicle (10% DMSO in PBS), in vivo luminescence in CRE-Luciferase mice was assessed by intraperitoneal luciferin injection and imaged with an IVIS imager. "ns" indicates that the results were not significant (P = 0.48) as calculated by unpaired, two-tailed t-test. [Figure 33]Figure 18 shows quantification of peptide biodistribution and organ accumulation after a single 20 mg / kg IV dose of either a TfR-binding peptide having the sequence of SEQ ID NO: 1 or a peptide having the sequence of SEQ ID NO: 32. Peptide levels were measured at 30 minutes and 3 hours (the 3-hour data is also shown in Figure 18). At this dose, levels of over 150 nM were achieved in the CNS and approximately 1 μM in the tumor after 3 hours. The bar graph quantifies the levels of each C-labeled peptide relative to known radioactivity within the same image, as measured by whole-body autoradiography, and the peptide specific activity in various tissues obtained 3 hours after administration from mice intravenously injected with the peptide. Tissue data are plotted in seven clusters of four bars each. The clusters of four bars correspond to different tissue types, from left to right, as indicated on the x-axis: skin, fat, muscle, spleen, kidney, liver, brain, and blood. The four bars in each cluster correspond, from left to right, to SEQ ID NO: 1 30 min (N = 10 slices), SEQ ID NO: 1 180 min (N = 14 slices), SEQ ID NO: 32 30 min (N = 12 slices), and SEQ ID NO: 32 180 min (N = 21 slices). The y-axis shows nmol g-1 tissue peptide on a logarithmic scale from 0.01 to 100 in increments of 10. The values ​​for each bar, from left to right, are: skin: 1.33, 1.12, 1.70, 1.02; fat: 0.79, 0.52, 0.52, 0.27; muscle: 0.63, 0.35, 0.44, 0.35; spleen: 4.37, 20.03, 1.17, 3.60; kidney: 29.05, 9.71, 16.91, 3.10; liver: 4.79, 22.18, 1.80, 8.54; brain: 0.23, 0.16, 0.21, 0.09; and blood: 1.58, 0.62, 0.86, 0.34. The bars corresponding to spleen, kidney, and liver are significantly higher than the other sets. The inset shows the same data from the brain normalized to blood. The four bars, from left to right, correspond to SEQ ID NO: 1 30 min (N=10 sections), SEQ ID NO: 1 180 min (N=14 sections), SEQ ID NO: 32 30 min (N=12 sections), and SEQ ID NO: 32 180 min (N=21 sections). The y-axis shows peptide normalized to blood on a logarithmic scale from 1 to 100 in increments of 10. The values ​​for each bar, from left to right, are 14%, 26%, 25%, and 27% of the blood peptide levels. [Figure 34A]

[0033] Figure 1 shows the purification of the SA21 fusion peptide. This figure shows the purification of a TfR-binding peptide fused to a serum albumin peptide (SA21) corresponding to SEQ ID NO: 373. Purity was verified by SDS-PAGE (left) and RP-HPLC (right) under DTT-reducing ("R") or non-reducing ("NR") conditions. SDS-PAGE was also performed on the uncleaved ("U") siderocalin-CDP fusion peptide. [Figure 34B] Purification of SA21 fusion peptide. Purification of a peptide fused to SA21 corresponding to SEQ ID NO: 456 (GSRLIEDICLPRWGCLWEDDGGGGSGGGGSVRIPVSCKHSGQCLKPCKDAGMRFGKCMNGKCDCTPK) is shown. Purity was verified by SDS-PAGE (left) and RP-HPLC (right) under DTT-reducing ("R") or non-reducing ("NR") conditions. SDS-PAGE was also performed on the uncleaved ("U") siderocalin-CDP fusion peptide. DETAILED DESCRIPTION OF THE INVENTION

[0122] Drug delivery to targets located in the central nervous system (CNS) can be hindered by the blood-brain barrier (BBB), a term referring to the vascular endothelial cells of CNS capillaries. This system exists to prevent harmful metabolites and pathogens from entering the CNS (e.g., the brain), but it also makes treating CNS disorders particularly challenging with conventional drugs. This may explain why primary CNS tumors (e.g., gliomas) and neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer's disease respond particularly poorly to treatments that can be highly effective for similar diseases affecting peripheral tissues, where drug delivery to target cells is less inhibited. CNS disorders are myriad, ranging from brain cancer to neurodegeneration to age-related inflammatory processes, necessitating a variety of approaches for drug delivery to the CNS. The BBB allows osmolytes and nutrients to enter the brain from serum, and CNS astrocytes provide many of the growth and survival signals required by neurons. However, several large hormones and proteins, such as transferrin (an iron chaperone), insulin, and leptin, can cross the BBB. CNS transport of large molecules, such as transferrin, can be achieved by receptor-mediated transcytosis or vesicular transcytosis (e.g., transport of cargo from the apical to the basolateral side or vice versa in intracellular vesicles). For example, variants of the endogenous receptors for insulin, leptin, and transferrin (InsR, ObR, and TfR, respectively) that contain normal ectodomains can be utilized by CNS vascular endothelial cells to facilitate transport of these molecules into the CNS. In this highly selective manner, certain large molecules, such as transferrin (molecular weight approximately 75 kDa), can access the brain parenchyma.

[0123] In various embodiments, the present disclosure provides compositions and methods for transporting cargo molecules or active agents (e.g., small molecules, peptides, or proteins) across cell layers or barriers, including endothelial cell layers (e.g., the BBB) or epithelial cell layers, and methods for using these compositions. In some embodiments, the present disclosure provides compositions and methods for delivering various molecules to the CNS that cannot cross the BBB. In some cases, the present disclosure provides compositions and methods for delivering therapeutic and / or diagnostic molecules to the CNS, e.g., the brain. Thus, in various embodiments, the present disclosure provides peptides capable of crossing the BBB. These peptides may have affinity and selectivity for the transferrin receptor (TfR). The TfR-binding peptide may be a cysteine-dense peptide (CDP). In some cases, the peptides of the present disclosure can cross the BBB via TfR-mediated transcytosis.

[0124] In some embodiments, the peptides described herein may be peptide conjugates, peptide constructs, fusion peptides, or fusion molecules (e.g., peptide constructs) linked by chemical conjugation of any type of molecule, such as a small molecule, peptide, or protein, or by recombinant fusion of a peptide or protein, respectively. The terms "fusion peptide" and "peptide fusion" are used interchangeably herein. In some embodiments, peptide constructs can be produced biologically or synthetically. Thus, in some cases, a TfR-binding peptide may comprise a TfR-binding peptide domain linked to another molecule or group of molecules, such as a small molecule, peptide, or protein, or to another macromolecule, such as a nanoparticle.

[0125] In some embodiments, the present disclosure provides methods and compositions that enable transport across cellular or molecular barriers. In various embodiments, the present disclosure provides methods and compositions that enable TfR-mediated transport across a cell layer (e.g., endothelial or epithelial cells) or cell membrane. In some cases, the TfR-binding peptides of the present disclosure enable transport across the blood-brain barrier (BBB). In various aspects, the peptides of the present disclosure can be used to target any cell that expresses TfR. In addition to the BBB, various other cells, tissues, and organs express TfR. Cells that express TfR can include hepatocytes, erythrocytes and erythroid progenitor cells in the bone marrow, immune cells, stem cells, and rapidly dividing cells. Tissues and organs that express TfR can include the brain (e.g., cerebral cortex, hippocampus, caudate, cerebellum), endocrine tissues (e.g., thyroid, parathyroid, and adrenal glands), bone marrow and immune system (e.g., appendix, lymph nodes, tonsils, spleen), muscle tissues (e.g., heart, skeletal, and smooth muscle), liver, gallbladder, pancreas, gastrointestinal tract (e.g., oral mucosa, esophagus, stomach, duodenum, small intestine, colon, rectum), kidney, bladder, gynecological tissues (e.g., fallopian tubes, breast, vagina, cervix, endometrium, ovaries, and placenta), adipose and soft tissue, and skin. Thus, the TfR-binding peptides of the present disclosure can be used to target and deliver active agents to these cells, tissues, and organs, for example, via TfR-mediated transcytosis (e.g., across a cellular barrier such as the BBB) or via TfR-mediated endocytosis (e.g., across a cell membrane and into a cell).

[0126] In various embodiments, the present disclosure provides methods and compositions that enable TfR-mediated trafficking and delivery to cancer cells that express TfR or CMYC-overexpressing cancers, as in some cases CMYC overexpression can result in TfR overexpression. Cancers that express TfR can include ovarian cancer, colon cancer, lung cancer, cancer located in the bone or bone marrow, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma (DIPG), breast cancer, liver cancer, colon cancer, brain cancer, splenic cancer, cancer of the salivary glands, renal cancer, muscle cancer, myeloid cell carcinoma, skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, neuroblastoma, prostate cancer, bladder cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or a CMYC-overexpressing cancer.

[0127] In some embodiments, the peptides described herein may be peptide constructs comprising one or more TfR-binding peptides described herein conjugated, linked, or fused to one or more active agents. The peptide constructs described herein may include chemical conjugates and recombinant fusion molecules. In some cases, chemical conjugates may comprise a TfR-binding peptide described herein chemically conjugated or linked to another molecule. The molecule may include small molecules, peptides, polypeptides, proteins, or other macromolecules (e.g., nanoparticles) and polymers (e.g., nucleic acids, polylysine, or polyethylene glycol). In some cases, the TfR-binding peptides of the present disclosure are conjugated to another molecule via a linker. The linker moiety may comprise a cleavable (e.g., pH-sensitive linker or enzyme-labile linker) or stable linker. In some embodiments, the peptide construct is a fusion molecule (e.g., a fusion peptide or fusion protein) that can be recombinantly expressed, and the fusion molecule can include one or more TfR-binding peptides fused to one or more other peptides, polypeptides, proteins, or other macromolecules that can be recombinantly expressed.

[0128] In some cases, the TfR-binding peptides of the present disclosure are conjugated, linked, or fused to a therapeutic (also called an "active agent") or diagnostic (also called a "detectable agent") cargo molecule capable of exerting a specific biological effect, resulting in a peptide construct. In some cases, the therapeutic or diagnostic cargo molecule used herein can be transported across the BBB via TfR-mediated transcytosis. Once inside the CNS, the cargo molecule can target specific cells, cell populations, or tissues. In some cases, the therapeutic or diagnostic cargo molecule is a known compound that, when used in combination with the compositions and methods disclosed herein, can be effectively transported across the BBB to exert extremely high efficacy within the CNS.

[0129] The peptides of the present disclosure, or derivatives, fragments, or variants thereof, may have affinity and selectivity for TfR, or derivatives or analogs thereof. In some cases, peptides of the present disclosure can be engineered using site-saturation mutagenesis (SSM) to exhibit improved TfR binding properties or more effectively promote transcytosis. In some cases, peptides of the present disclosure are knot peptides or cysteine-dense peptides (CDPs) related to hittin- or knottin-derived peptides. The TfR-binding peptide can be a cysteine-dense peptide (CDP). The terms "peptide," "CDP," "TfR-binding peptide," "TfR-binding CDP," and "TfR-binding peptide" are used interchangeably herein. Hitchins may be a subclass of CDPs, in which six cysteine ​​residues form disulfide bonds according to the connectivity [1-4], 2-5, 3-6, which indicates that the first cysteine ​​residue forms a disulfide bond with the fourth residue, the second cysteine ​​residue with the fifth residue, and the third cysteine ​​residue with the sixth residue. The brackets in this nomenclature indicate that the cysteine ​​residues form a knotting disulfide bond. (See, e.g., Correnti (See, e.g., [Crossref], [Delta ... The amino acid sequence of the peptide disclosed herein may contain multiple cysteine ​​residues. In some cases, at least one of the multiple cysteine ​​residues present in the amino acid sequence of the peptide participates in disulfide bond formation. In some cases, all of the multiple cysteine ​​residues present in the amino acid sequence of the peptide participate in disulfide bond formation. As used herein, the term "knot peptide" may be used interchangeably with the terms "cysteine-dense peptide," "CDP," or "peptide."

[0130] Provided herein are methods for identifying, maturing, characterizing, and utilizing CDPs that bind to the transferrin receptor and enable the accumulation of bioactive molecules at therapeutically relevant concentrations in subjects (e.g., humans or non-human animals). This disclosure demonstrates the utility of CDPs as a diverse family of scaffolds that can be screened for potential applications in modern drug discovery strategies. CDPs are an alternative to existing biologics, primarily antibodies, and can avoid some of the disadvantages of immunoglobulin scaffolds, including poor tissue penetration, immunogenicity, and long serum half-lives, which can be problematic in the event of toxicity. The disclosed peptides, ranging from 20 to 80 amino acids, are medium-sized and possess many of the favorable binding specificity and affinity characteristics of antibodies, yet offer improved stability, reduced immunogenicity, and simpler manufacturing methods for medically relevant therapeutics. The intramolecular disulfide structure of CDPs confers a particularly high stability index, thereby reducing fragmentation and immunogenicity, while their small size allows for improved tissue or cellular penetration and easier tuning of serum half-lives. Disclosed herein are peptides that represent peptide candidates that can function as CNS drug delivery vehicles.The peptides of the present disclosure can be cell-penetrating peptides (CPPs).CPPs can be cell-penetrating, tissue-penetrating, or both.

[0131] In some embodiments, the CPP may be a CDP. A CPP may contain peptides that facilitate cellular uptake or incorporation of various moieties and drugs, and may itself translocate across the cell membrane. A CPP may contain a protein transduction domain, a type of short peptide sequence that can translocate across the cell membrane. Cell-penetrating peptides can directly or indirectly enter the cytosol, nucleus, or other cellular components of a cell. Cell-penetrating peptides can be used as suitable carriers for various cargoes, including nucleic acids, peptides, proteins, siRNA, dsRNA, nucleic acids containing alternative backbone chemistries (e.g., linked nucleic acids (LNA) or peptide nucleic acids (PNA)), radionuclides, contrast agents, fluorescent agents, therapeutic agents, nanoparticles, etc. A CPP may be an artificial or engineered sequence (e.g., a synthetic sequence). A CPP may contain multiple protein sequences, whether natural, synthetic, or variant (e.g., chimeric). A CPP may be derived from a single protein sequence, whether natural, synthetic, or variant (e.g., a protein-derived sequence). CPPs can exhibit a variety of physiochemical properties, such as cationic, amphipathic, or hydrophobic. Some mechanisms of CPP internalization include direct cell penetration, use of endocytic pathways, and translocation via the formation of transient structures. It is understood that the description and use of cell penetrating peptides (CPPs) herein are not limiting. Non-limiting examples of CPPs are described in Derakhshankhah and Jafari, "Cell penetrating peptides: A concise review with emphasis on "Biomedicine & Pharmacotherapy: A Review of Novel Drugs and Their Application to Biomedical Applications" (Biomedicine & Pharmacotherapy; Volume 108, December 2018, Pages 1090-1096), which is incorporated by reference in its entirety.

[0132] Some therapeutic molecules that can be used in combination with the methods and compositions disclosed herein can target one or more specific cells or tissues that are part of the CNS. For example, the neurotensin receptor (NTSR), a G protein-coupled receptor, is targeted using a fusion protein containing a CDP linked to neurotensin (abbreviated herein as "NT"; ELYENKPRRPYIL (SEQ ID NO: 350)), or its derivative, a neuropeptide abbreviated as NTSR. In some cases, the CDP-NT peptide construct is used to prevent or treat chronic pain or neuropathic pain in a subject (e.g., a human).

[0133] Also described herein are peptides that selectively home to, target, direct, migrate, deliver, retain, accumulate, and / or bind to specific regions, tissues, structures, or cells of the central nervous system (CNS) involved in the perception, modulation, management, reduction, elimination, or reduction of pain, including nociceptive pain or other therapeutic indications described herein. Peptides that home to, target, migrate, direct, retain, accumulate, and / or bind to one or more specific regions, tissues, structures, or cells of an affected area may have fewer off-target and potential adverse effects, such as side effects, that often limit the use and effectiveness of analgesic drugs. In addition, such peptides can deliver active agents to areas such as the CNS, even when the active agents cannot reach the CNS at therapeutic levels due to the blood-brain barrier. Such peptides can also reduce the need for other analgesics, including opioid drugs. In addition, such peptides can reduce the dosage and increase the effectiveness of existing drugs by directly targeting them to specific regions, tissues, structures, or cells in the affected area and facilitating contact with the affected area or increasing the local concentration of the drug. The peptides themselves can regulate pain, or they can be conjugated to pain-regulating drugs. Such pain regulation can work through various mechanisms, such as regulating inflammation, autoimmune responses, direct or indirect effects on pain receptors, cell killing, or programmed cell death (whether through apoptotic and / or non-apoptotic pathways in affected cells or tissues, etc. (Tait et al. J Cell Sci 127(Pt 10):2135-44(2014)).

[0134] CDPs may be advantageous for delivery to the CNS due to their small size, high tissue or cellular penetration, and rapid serum clearance compared to other molecules, such as antibodies, and their resistance to proteases compared to small peptides (both stability and reduced immunogenicity). In some embodiments, the TfR-binding peptides (e.g., CDPs, knot peptides, or hitchins), TfR-binding peptide conjugates (e.g., comprising one or more TfR-binding peptides and one or more active agents), or engineered TfR-binding fusion peptides (e.g., comprising one or more TfR-binding peptides and one or more peptides) of the present disclosure may have superior properties to TfR-binding antibodies. For example, the peptides and constructs described herein may provide better, deeper, and / or faster tissue or cellular penetration into cells and target tissues (e.g., penetration into brain parenchyma, penetration into solid tumors), as well as faster clearance from non-target tissues and serum. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure may have a lower molecular weight than TfR-binding antibodies. The lower molecular weight may confer advantageous properties to the TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure compared to TfR-binding antibodies. For example, the TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure may penetrate cells or tissues more easily than anti-TfR antibodies or have lower molar dose toxicity than anti-TfR antibodies. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure may be advantageous because they lack the Fc function of antibodies. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure may be advantageous because they allow for higher concentrations of formulations on a molar basis.

[0135] The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure may have a wider therapeutic window (e.g., a dosage above the amount at which a therapeutic pharmacodynamic response is observed but below the amount at which toxicity is observed) compared to TfR-binding antibody-based therapeutics. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure may be used at higher molar dosages with less risk of toxicity compared to TfR-binding antibody-based therapeutics. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure may be less immunogenic than TfR-binding antibody-based therapeutics because they contain fewer epitopes that elicit an adaptive immune response. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure may exhibit easier and less disruptive incorporation of active agents into protein fusion constructs compared to TfR-binding antibody-based therapeutics. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure may have a smaller surface area and therefore a lower risk of off-target binding compared to TfR-binding antibody-based therapeutics. An exemplary comparison is described in Example 54.

[0136] In some embodiments, the TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure exhibit lower on-target toxicity than anti-TfR antibodies when administered to a subject at the same molar dose or a similarly effective dose. In some embodiments, the TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides exhibit lower off-target toxicity than antibodies when administered to a subject at the same molar dose or a similarly effective dose. For example, the TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure can be administered to a subject at a molar dose that is about 1-, 2-, 3-, 4-, 5-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or 100-fold higher than an antibody, yet exhibit comparable or lower toxicity. In some embodiments, the TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of the present disclosure exhibit greater efficacy than anti-TfR antibodies when administered to a subject at the same weight dose as the anti-TfR antibody. When fused to a half-life extending moiety (e.g., Fc, SA21, PEG), the TfR-binding peptides of the present disclosure can be delivered at even lower doses while maintaining activity and efficacy, thus far superior to administration of anti-TfR antibodies.

[0137] When fused to an active agent as a peptide construct, the TfR-binding peptides of the present disclosure may exhibit higher efficacy than engineered anti-TfR bispecific antibodies against the same target. For example, the engineered TfR-binding peptides disclosed herein may be further fused to a BACE inhibitor. These engineered TfR-binding peptide-BACE inhibitor constructs can be administered at higher molar doses than bispecific anti-TfR / BACE1 antibodies while maintaining the same or higher efficacy. For example, engineered TfR-binding peptide-BACE inhibitor constructs are less likely to cause reticulocytopenia, can effectively inhibit amyloid beta formation, and exhibit low or no toxicity to subjects administered the engineered TfR-binding peptide-BACE inhibitor constructs. In contrast, bispecific anti-TfR / BACE1 antibodies may cause reticulocytopenia and / or be less effective at inhibiting amyloid beta formation and may be more toxic to subjects, resulting in symptoms of lethargy, distress, and hemolysis.

[0138] In some embodiments, the present disclosure provides peptides (e.g., CDPs, knot peptides, or hitchins), chemical conjugates (e.g., comprising one or more TfR-binding peptides and one or more active agents), or recombinantly expressed fusion molecules (e.g., comprising one or more TfR-binding peptides and one or more active agents) that bind to TfR. The TfR-binding peptide may be a cysteine-dense peptide (CDP). The terms "peptide," "CDP," "TfR-binding peptide," "TfR-binding CDP," "TfR-binding peptide," and "engineered TfR-binding peptide" are used interchangeably herein. Binding of the peptides described in this disclosure to TfR can promote transcytosis of the peptide or peptide construct (e.g., a fusion protein, or a peptide conjugated, linked, or fused to an agent) across a cellular barrier (e.g., the BBB). Also disclosed herein is the use of a mammalian surface-display screening platform to screen a diverse library of CDPs to identify CDPs that specifically bind to human TfR. Further affinity maturation can then be performed to obtain an allelic series of TfR-binding CDPs with varying affinities. In some embodiments, TfR-binding CDPs are identified and binding can be determined by crystallography. The peptides of the present disclosure may have cross-reactivity across species. For example, the peptides disclosed herein bind to human and mouse TfR in some cases. The peptides disclosed herein, including those using neurotensin peptide constructs to induce the cAMP response element signaling cascade, accumulate in the CNS after intravenous administration and can penetrate the BBB via TfR binding. Disclosed herein are TfR-binding CDPs for use as therapeutic delivery agents in oncology, autoimmune diseases, acute and chronic neurodegeneration, and pain management. Delivery of active or medicinal agents via TfR-binding CDPs may be advantageous over conventional anti-TfR antibodies due to easier manufacturing (peptides can be produced by biological or synthetic means), improved stability, and smaller size (less potential steric hindrance of cargo activity).Thus, the disclosed methods and compositions can provide a solution to the problem of effectively transporting cargo molecules (e.g., therapeutic and / or diagnostic small molecules, peptides, or proteins) into the CNS (e.g., the brain). For example, the disclosed peptides aid in drug delivery to tumors located in the brain.

[0139] In some embodiments of the present disclosure, CDPs, knot peptides, hitchins, or diverse libraries of knot peptide or hitchin-derived peptides can be used in conjunction with mammalian surface display screening platforms to identify peptides that specifically bind to human TfR. (See, e.g., Crook et al. (2017) Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets. Nat Commun 8:2244). In some embodiments, CDPs, knot peptides, hitchins, or diverse libraries of knot peptide or hitchin-derived peptides are mutated from endogenous peptide sequences to generate novel peptide sequences. Once TfR-binding peptides are identified, affinity maturation (e.g., site-saturation mutagenesis) can be performed to generate an allelic series of binders with varying (e.g., improved) affinities for TfR. These techniques can be used in conjunction with various other analytical methods (e.g., crystallography or spectroscopy) to identify the nature of the peptide-receptor interaction (e.g., amino acid residues important for receptor binding, etc.). In some cases, peptides of the present disclosure are developed to bind to human TfR, but the peptides may exhibit cross-reactivity with TfR from other species, such as mouse TfR.

[0140] The peptides of the present disclosure may have various biodistribution patterns in vivo. In some cases, for example, radiolabeled peptides (e.g., 14 Mouse biodistribution and pharmacokinetic studies are performed using C-labeled peptides.

[0141] In various embodiments, the present disclosure provides peptides that accumulate in the CNS (e.g., the brain). In some cases, CNS accumulation of the peptides is due to penetration across the BBB. In some cases, the engineered TfR-binding peptide variants described herein cross the BBB by TfR-mediated transcytosis. In some cases, the TfR-binding peptides of the present disclosure accumulate in tumor tissue located in the CNS. In some cases, the tumor in which the peptide accumulates is a brain tumor (e.g., a glioma). As used herein, the terms "accumulate" and "accumulate in" are used interchangeably and may be used to refer to accumulation in or on cells and / or tissues, and it may be understood that the concentration of the respective molecule (e.g., TfR-binding peptide or peptide construct) gradually increases over time.

[0142] In some embodiments, engineered peptides (e.g., histidine-containing or histidine-rich TfR-binding peptides) of the present disclosure have high TfR-binding affinity at physiological pH, but the binding affinity may be significantly reduced at low pH levels, such as endosomal pH 5.4. In some cases, TfR-binding peptides of the present disclosure may be optimized to improve intravesicular (e.g., intraendosomal) and / or intracellular delivery function while retaining high TfR-binding ability. In some cases, histidine scanning and comparative binding experiments can be performed to develop and screen such peptides. In addition, some peptides may contain (e.g., conjugated, linked, or fused) motifs that promote low-pH endosomal escape of the peptide or peptide construct to enhance delivery function (e.g., intracellular delivery of therapeutic agents). In some embodiments, amino acid residues in the peptides of the present disclosure are substituted with different amino acid residues to alter the pH-dependent binding affinity to TfR. The amino acid substitutions can include those that increase binding affinity at low pH, those that increase binding affinity at high pH, ​​those that decrease binding affinity at low pH, those that decrease binding affinity at high pH, ​​or a combination thereof.

[0143] Exemplary peptides of the present disclosure are set forth in Table 1, which include the amino acid sequences set forth in SEQ ID NOs: 1 to 32, 36 to 67, 136 to 167, and 171 to 202.

[0144] In various embodiments, peptides of the present disclosure are part of a peptide construct, such as a fusion peptide, designed to transport therapeutic and / or diagnostic molecules (e.g., small molecules, peptides, or proteins) across a cell layer or a cellular barrier, such as the BBB (e.g., having the amino acid sequence of any one of SEQ ID NOs: 33-35, 68-70, 168-170, 203-205, 225-332, 359-361, 373, 375, 381, 384, 385, 388, 389, 393, 396, 397, 400, and 401). Exemplary peptide constructs are shown in Tables 2, 5, and 6. Peptide constructs of the present disclosure can include a TfR-targeting peptide linked (e.g., chemically conjugated or fused) to a therapeutic and / or diagnostic molecule or compound. TfR-binding peptide constructs can cross the BBB via TfR-mediated transcytosis, thus delivering therapeutic and / or diagnostic molecules or compounds to the CNS. Therefore, the disclosed methods and compositions may have a significant impact on the diagnosis, prevention, and treatment of various diseases or conditions. Disease areas in which the disclosed methods and compositions can be used include, but are not limited to, oncology, autoimmune diseases, acute and chronic neurodegeneration, muscle disorders, neuropsychological disorders, epilepsy, schizophrenia, depression, anxiety, bipolar disorder, brain development disorders (e.g., autism spectrum disorder), pain, epilepsy, mood disorders, and pain management.

[0145] Thus, subjects with CNS disorders who are unable to benefit from drugs or drug classes limited by poor BBB penetration can still benefit from the compositions and methods described herein, since therapeutically effective drug concentrations in the CNS can be achieved by conjugating the drugs to the TfR-binding peptides provided herein. In addition to pharmacological advantages, the compositions and methods disclosed herein, including TfR-binding peptides, may be superior to conventional CNS and TfR-targeted agents (e.g., anti-TfR antibodies) in terms of production, quality control, and safety. For example, the peptides and peptide constructs of the present disclosure can be produced in a simpler, less expensive, and more resource-efficient manner (e.g., peptides can be synthesized via biological or synthetic approaches), the peptides and peptide constructs of the present disclosure may exhibit improved ex vivo and in vivo stability, be small in size (e.g., potentially resulting in less steric hindrance to cargo activity, the ability to penetrate dense tissues such as solid tumors, and potentially faster clearance from the systemic circulation), exhibit high tissue or cellular penetration, and have low immunogenicity. The peptides of the present disclosure can be engineered to have low immunogenicity by combining crystallographic data with major histocompatibility complex (MHC) or human leukocyte antigen (HLA) peptide fragment binding experiments or computational predictions thereof, or a combination of the foregoing.

[0146] Identifying therapeutic or diagnostic agents capable of crossing cell layers or cellular barriers such as the BBB has been challenging because such methods require high specificity or targeting, high-affinity binders for receptors (e.g., TfR) that can promote transcytosis, and the ability to target cells and act on target proteins after transcytosis (with few adverse off-target effects).With few exceptions, high-throughput screening projects using small molecule libraries have failed to provide specific compounds capable of crossing endothelial or epithelial layers and / or transporting cargo across those layers.

[0147] Described herein, in some embodiments, are peptides and peptide constructs that target a protein of interest, such as TfR, as well as methods for screening peptides and peptide constructs. Compared to wild-type or endogenous molecules, such as transferrin, the methods and compositions described herein can provide peptides with improved TfR binding ability, peptides that exhibit improved transport ability across the BBB, or any combination thereof. In some cases, the peptides described herein efficiently transport cargo molecules (e.g., therapeutic or diagnostic small molecules or proteins) across an endothelial cell layer (e.g., the BBB) or an epithelial layer. In some embodiments, the TfR-binding peptides of the present disclosure bind to TfR and promote vesicular transcytosis. In some cases, the TfR-binding peptides of the present disclosure bind to cells that overexpress TfR (e.g., cancer cells) and promote cellular uptake of the peptide. In some embodiments, the TfR-binding peptides or peptide constructs described herein promote vesicular transcytosis and uptake by TfR-overexpressing cells, such as cancer cells, or a combination thereof.

[0148] The TfR-binding peptides of the present disclosure can bind to TfRs from different species, including human, monkey, mouse, and rat TfRs. In some cases, changes or mutations in any of the amino acid residues of the TfR-binding peptides can affect cross-reactivity. In some cases, changes or mutations in any of the amino acid residues of the TfR-binding peptides that interact with the binding site of the TfR can affect cross-reactivity.

[0149] Described herein are peptides, including but not limited to, designed or engineered peptides, recombinant peptides, and cysteine-dense peptides (CDPs) / small disulfide-knot peptides (e.g., knot peptides, hitchins, and peptides derived therefrom), that can be large enough to deliver cargo molecules while retaining the ability to bind to target proteins (e.g., TfR) with high affinity, yet small enough to access cellular compartments such as the cytosol or nucleus, or tissues such as the center of cell aggregates (e.g., solid tumors). In some cases, the peptides described herein deliver cargo molecules across the BBB to the CNS (e.g., parenchyma) via vascular transcytosis. In some cases, the transcytosis is TfR-mediated.

[0150] Additionally, described herein are methods and compositions for determining the nature of peptide-receptor interactions (e.g., using X-ray crystallography) and determining in vivo pharmacodynamic and pharmacokinetic properties, including accumulation in the CNS (e.g., brain). Some of the peptides described herein have the ability to target and accumulate in tumor cells. In some cases, the tumor cells overexpress TfR. In some embodiments, the peptides of the present disclosure have high in vivo stability, e.g., high protease stability, high resistance to reducing agents such as glutathione (GSH), and can withstand high temperatures (e.g., up to 95°C).

[0151] In some embodiments, the present disclosure provides a peptide or protein design approach based on a 3D protein or receptor structure to identify peptides or proteins capable of binding to such receptors. In some cases, the receptor is a transferrin receptor.

[0152]

[0013] Further aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0153] As used herein, the abbreviations for natural L-enantiomer amino acids are conventional and are as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine ​​(C, Cys); glutamic acid (E, Glu); glutamine (Q, Gln); glycine (G, Gly); histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Typically, Xaa can represent any amino acid. In some embodiments, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).

[0154] Some embodiments of the present disclosure contemplate D-amino acid residues of any standard or non-standard amino acid or analog thereof. When an amino acid sequence is represented by a series of three-letter or one-letter amino acid abbreviations, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0155] The terms "peptide," "polypeptide," "protein," "hittin," "cysteine-dense peptide," "knot peptide," or "CDP" may be used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, a "peptide," "polypeptide," and "protein" may be a chain of amino acids whose alpha carbons are linked via peptide bonds. Thus, the terminal amino acid at one end of the chain (e.g., the amino terminus) may have a free amino group, and the terminal amino acid at the other end of the chain (e.g., the carboxy terminus) may have a free carboxyl group. As used herein, the term "amino terminus" (e.g., abbreviated N-terminus) may refer to the free α-amino group on the amino terminal amino acid of a peptide, or the α-amino group (e.g., an imino group when involved in a peptide bond) of an amino acid located anywhere else within the peptide. Similarly, the term "carboxy terminus" may refer to the free carboxyl group on the carboxy terminal amino acid of a peptide, or the carboxyl group of an amino acid located anywhere else within the peptide. Peptides also include essentially any polyamino acid, including, but not limited to, peptidomimetics such as amino acids linked by ether or thioether to an amide bond.

[0156] As used herein, the term "peptide construct" may refer to a molecule comprising one or more peptides of the present disclosure that can be conjugated, linked, or fused to one or more cargo molecules. In some cases, the cargo molecule is an active agent. The term "active agent" may refer to any molecule, for example, any molecule capable of eliciting a biological and / or physical effect (e.g., emission of radiation) that can enable the localization, detection, or visualization of the respective peptide construct. In various embodiments, the term "active agent" refers to a therapeutic and / or diagnostic agent. The peptide constructs of the present disclosure may comprise a TfR-binding peptide linked to one or more active agents via one or more linker moieties (e.g., cleavable or stable linkers) described herein.

[0157] As used herein, the terms "comprise" and "have" may be used interchangeably. For example, the terms "a peptide comprising the amino acid sequence of SEQ ID NO: 32" and "a peptide having the amino acid sequence of SEQ ID NO: 32" may be used interchangeably.

[0158] As used herein, unless otherwise specified, the term "TfR" or "transferrin receptor" is a type of protein used herein and can refer to transferrin receptor from any species (e.g., human or mouse TfR or any human or non-human animal TfR). In some cases, the term "TfR" or "transferrin receptor" as used herein refers to human TfR (hTfR) and can include TfR or any of the known TfR homologs or orthologs, including TfR1, TfR2, soluble TfR, or any combination or fragment (e.g., ectodomain) thereof.

[0159] The term "engineered," when applied to a polynucleotide, indicates that the polynucleotide has been removed from its natural genetic environment, and therefore is free of other unrelated or undesired coding sequences, and is in a form suitable for use in a genetically engineered protein production system. Such engineered molecules are those that have been separated from their natural environment and include cDNA and genomic clones (i.e., prokaryotic or eukaryotic cells containing vectors containing fragments of DNA from different organisms). Engineered DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally or non-natively occurring 5' and 3' untranslated regions, such as enhancers, promoters, and terminators.

[0160] An "engineered" polypeptide or protein is one that exists in a state other than its native environment, such as away from blood and animal tissue. In a preferred form, the engineered polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. The polypeptide is preferably provided in a highly purified form, e.g., greater than 95% pure, more preferably greater than 98% pure, or greater than 99% pure. As used in this context, the term "engineered" does not exclude the existence of the same polypeptide in alternative physical forms, such as dimers, heterodimers and multimers, or glycosylated, carboxylated, modified, or derivatized forms.

[0161] An "engineered" peptide or protein is a polypeptide that differs distinctly in structure, sequence, or composition from a naturally occurring polypeptide. Engineered peptides include non-naturally occurring peptides, artificial peptides, isolated peptides, synthetic peptides, designed peptides, modified peptides, or recombinantly expressed peptides. Engineered TfR-binding peptides, variants, or fragments thereof are provided herein. These engineered TfR-binding peptides can be further linked to an active agent or a detectable agent. The active agent can be a half-life extending moiety.

[0162] Polypeptides of the present disclosure include polypeptides that have been modified in some way to, for example, (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) impart or alter other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) are made in the native sequence (e.g., in a portion of the polypeptide other than the domain(s) that form intermolecular contacts). A "conservative amino acid substitution" may refer to the substitution of an amino acid in a polypeptide with a functionally similar amino acid. The following six groups contain amino acids that can be conservatively substituted for one another: i) alanine (A), serine (S), and threonine (T); ii) aspartic acid (D) and glutamic acid (E); iii) asparagine (N) and glutamine (Q); iv) arginine (R) and lysine (K); v) isoleucine (I), leucine (L), methionine (M), and valine (V); vi) phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0163] As used herein, the terms "polypeptide fragment" and "truncated polypeptide" can refer to a polypeptide having an amino-terminal and / or carboxy-terminal deletion compared to the corresponding full-length peptide or protein. In various embodiments, a fragment is at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids in length. In various embodiments, a fragment can also be, for example, up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, up to 25, up to 10, or up to 5 amino acids in length. A fragment may further comprise one or more additional amino acids at either or both of its termini, such as a sequence of amino acids derived from a different naturally occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0164] In various embodiments, the interface residues of the TfR-binding peptides of the present disclosure (e.g., amino acid residues that interact with TfR for receptor binding) can be separated into the two primary helical domains of the peptide. In some cases, the interface residues can include residues corresponding to residues 5-25 relative to SEQ ID NO: 32 (e.g., including residues corresponding to G5, A7, S8, M11, N14, L17, E18, and E21), or residues corresponding to residues 35-51 relative to SEQ ID NO: 32 (e.g., including residues corresponding to L38, L41, L42, L45, D46, H47, H49, S50, and Q51), or both. For example, the interface residues may include residues corresponding to residues 5-25 based on SEQ ID NO: 32 (e.g., including the corresponding residues of G5, A7, S8, M11, N14, L17, E18, and E21), or residues corresponding to residues 35-51 based on SEQ ID NO: 32 (e.g., including the corresponding residues of L38, L41, L42, L45, D46, H47, H49, S50, and Q51). In some embodiments, the TfR-binding peptide can include a fragment of a peptide provided herein, wherein the fragment includes the minimum interface residues for binding, e.g., residues corresponding to residues 5-25 relative to SEQ ID NO: 32 (e.g., including residues corresponding to G5, A7, S8, M11, N14, L17, E18, and E21), or residues corresponding to residues 35-51 relative to SEQ ID NO: 32 (e.g., including residues corresponding to L38, L41, L42, L45, D46, H47, H49, S50, and Q51). In some cases, the TfR-binding peptide is a peptide having the sequence set forth in SEQ ID NO: 32, which includes TfR-binding residues corresponding to residues G5, A7, S8, M11, N14, L17, E18, and E21 of the first domain, and TfR-binding residues corresponding to residues L38, L41, L42, L45, D46, H47, H49, S50, and Q51 of the second domain, based on SEQ ID NO: 32.

[0165] As used herein, the terms "peptide" or "polypeptide," in connection with "variant," "mutant," or "enriched variant," or "permutation-enriched variant," can refer to a peptide or polypeptide that can include an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted relative to another polypeptide sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues is at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. Variants of the present disclosure include peptide conjugates or fusion molecules (e.g., peptide constructs).

[0166] A "derivative" of a peptide or polypeptide can be a peptide or polypeptide that may have been chemically modified, e.g., conjugated to another chemical moiety, such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

[0167] The term "% sequence identity" can be used interchangeably herein with "% identity" and can refer to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity determined by a defined algorithm, meaning that a given sequence is at least 80% identical to another sequence of a different length. In various embodiments, the % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In various embodiments, the percent identity is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0168] The terms "% sequence homology" or "percent sequence homology" or "percent sequence identity" may be used interchangeably herein with the terms "% homology," "% sequence identity," or "% identity," and may refer to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same as 80% sequence homology determined by a defined algorithm; thus, a homolog of a given sequence has greater than 80% sequence homology over the length of the given sequence. In various embodiments, the % homology is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence homology to a given sequence. In various embodiments, the percent homology is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0169] A protein or polypeptide may be "substantially pure," "substantially homogeneous," or "substantially purified" if at least about 60%-75% of the sample represents a single polypeptide species. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein may typically comprise about 50%, 60%, 70%, 80%, or 90% w / w of a protein sample, more commonly about 95%, e.g., greater than 99% pure. Protein purity or homogeneity can be demonstrated by a number of means well known in the art, such as subjecting a protein sample to polyacrylamide gel electrophoresis followed by staining the gel with stains well known in the art to visualize a single polypeptide band. For certain purposes, high resolution can be achieved by using high-performance liquid chromatography (e.g., HPLC) or other high-resolution analytical techniques (e.g., LC-mass spectrometry).

[0170] As used herein, the term "pharmaceutical composition" may generally refer to a composition suitable for pharmaceutical use in a subject, such as an animal (e.g., a human or a mouse). A pharmaceutical composition may include a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. The term "pharmacologically effective amount" may refer to an amount of an agent effective to produce an intended biological or pharmacological result.

[0171] As used herein, the term "pharmaceutically acceptable carrier" can refer to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as phosphate-buffered saline, 5% aqueous dextrose, and emulsions such as oil / water or water / oil emulsions, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co., Easton. A "pharmaceutically acceptable salt" can be a salt that can be formulated into a compound for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0172] As used herein, the terms "treat," "treating," and "treatment" can refer to a method of alleviating or alleviating a biological disorder and / or at least one of its associated symptoms. As used herein, "alleviating" a disease, disorder, or condition means, for example, reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" can include references to curative, palliative, and prophylactic or diagnostic treatment.

[0173] Generally, cells of the present disclosure can be eukaryotic or prokaryotic. Cells can be epithelial cells. Cells can be animal or plant cells. Animal cells can include cells derived from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. Mammalian cells can be obtained from primates, apes, equines, bovines, porcines, canines, felines, or rodents. Mammals can be primates, apes, dogs, cats, rabbits, ferrets, etc. Rodents can be mice, rats, hamsters, gerbils, chinchillas, or guinea pigs. Avian cells can be derived from canaries, parakeets, or parrots. Reptile cells can be derived from turtles, lizards, or snakes. Fish cells can be derived from tropical fish. For example, fish cells can be derived from zebrafish (e.g., Danino rerio). Helminth cells can be derived from nematodes (e.g., C. elegans), amphibian cells can be derived from frogs, and arthropod cells can be derived from tarantulas or hermit crabs.

[0174] Mammalian cells can also include cells obtained from primates (e.g., humans or non-human primates). Mammalian cells can include blood cells, stem cells, epithelial cells, connective tissue cells, hormone-secreting cells, nerve cells, skeletal muscle cells, or immune system cells. In preferred embodiments, the methods and compositions of the present disclosure are used in combination with one or more mammalian blood cells.

[0175] As used herein, the term "vector" generally refers to a DNA molecule capable of replication in a host cell and / or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector.

[0176] As used herein, the term "subject" generally refers to a human or another animal. A subject can be of any age, for example, a subject can be an infant, toddler, child, pre-adolescent, adolescent, adult, or geriatric individual.

[0177] Ranges may be expressed herein from "approximately" one particular value and / or to "approximately" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another embodiment. It is further understood that each endpoint of a range corresponds to, and is independent of, the other endpoint. As used herein, the term "about" refers to a range of plus or minus 15% from the specified numerical value, within the scope of particular use. For example, about 10 may include a range of 8.5 to 11.5.

[0178] peptide Disclosed herein are peptide sequences, such as those listed in Table 1, that are capable of binding to TfR or any of the known TfR homologs, including TfR1, TfR2, soluble TfR, or any combination or fragment (e.g., ectodomain) thereof. Peptides capable of binding to transferrin receptor or TfR homologs may be referred to herein as transferrin receptor-binding peptides or TfR-binding peptides. In some embodiments, the peptides disclosed herein are capable of penetrating, passing through, or entering target cells in a TfR-mediated manner. These cell layers or cells may include TfR-expressing endothelial cells, TfR-expressing epithelial cells, and TfR-expressing cells of various tissues or organs, such as tumor cells, brain cells, cancerous or tumor cells, liver cells, pancreatic cells, colon cells, ovarian cells, breast cells, and / or lung cells, or any combination thereof.

[0179] In various embodiments, the present disclosure provides methods and compositions that enable TfR-mediated transport across a cell layer (e.g., endothelial or epithelial cells) or cell membrane. In addition to the BBB, various other cells, tissues, and organs express TfR. Single cells that express TfR can include hepatocytes, red blood cells and erythroid progenitor cells in the bone marrow, immune cells, stem cells, and rapidly dividing cells. Tissues and organs that express TfR can include the brain (e.g., cerebral cortex, hippocampus, caudate, cerebellum), endocrine tissues (e.g., thyroid, parathyroid, and adrenal glands), bone marrow and immune system (e.g., appendix, lymph nodes, tonsils, spleen), muscle tissues (e.g., heart, skeletal, and smooth muscle), liver, gallbladder, pancreas, gastrointestinal tract (e.g., oral mucosa, esophagus, stomach, duodenum, small intestine, colon, rectum), kidney, bladder, gynecological tissues (e.g., fallopian tubes, breast, vagina, cervix, endometrium, ovaries, and placenta), adipose and soft tissue, and skin. Thus, the TfR-binding peptides of the present disclosure can be used to target and deliver active agents to these cells, tissues, and organs, for example, via TfR-mediated transcytosis (e.g., across a cell barrier such as the BBB), or via TfR-mediated endocytosis (e.g., across a cell membrane into the cell), or via TfR-mediated accumulation in a tissue, to treat and / or prevent a disease or condition in one or more of these cells, tissues, or organs.

[0180] In various embodiments, the present disclosure provides methods and compositions that enable TfR-mediated trafficking and delivery to cancer cells that express TfR. TfR-expressing cancers can include ovarian cancer, colon cancer, lung cancer, cancers located in the bone or bone marrow, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma (DIPG), breast cancer, liver cancer, colon cancer, brain cancer, spleen cancer, salivary gland cancer, renal cancer, muscle cancer, myeloid cell carcinoma, skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, neuroblastoma, prostate cancer, bladder cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or CMYC-overexpressing cancers.

[0181] TfR-binding peptide constructs that can be used to prevent and / or treat cancer include those comprising a TfR-binding peptide fused to an active agent with anti-tumor activity, such as an IL15 / IL15Ra complex, IFN-gamma, and an anti-CD3 agent. Examples of such peptide constructs (e.g., fusion peptides) include those having the amino acid sequence set forth in any one of SEQ ID NOs: 33-35, 68-70, 168-170, 203-205, 225-332, or 359-361. In some embodiments, the TfR-binding peptide construct is fused to IL-15 to recruit and stimulate immune T cells or NK cells. In some embodiments, the TfR-binding peptide construct is fused to IFN-gamma to activate macrophages and upregulate MHC antigens on tumor cells. In some embodiments, the TfR-binding peptide construct is fused to CD3 so that, in addition to binding to TfR on cancer cells, it also binds to T cells to form an immune synapse and activate the T cells to target the cancer.

[0182] In general, the TfR-binding peptides of the present disclosure can be used in combination with a variety of active agents, and the TfR-binding peptides of the present disclosure can be conjugated, linked, or fused to one or more of these active agents. In various embodiments, the active agent is an immunotherapeutic agent, a CTLA-4 targeted agent, a PD-1 targeted agent, a PDL-1 targeted agent, an IL15 agent, a fused IL-15 / IL-15Ra combination agent, an IFN gamma agent, an anti-CD3 agent, an ion channel modulator, a Kv1.3 inhibitor, an auristatin, MMAE, a maytansinoid, DM1, DM4, doxorubicin, a calicheamicin, a platinum compound, cisplatin, a taxane, paclitaxel, SN-38, a BACE inhibitor, a Bcl-xL inhibitor, WEHI-539, venetoclax, ABT-199, navitoclax, AT-101, obatoclax, a pyrrolobenzodiazepine or pyrrolobenzodiazepine dimer, a dolastatin, or a neurotransmitter such as neurotensin.

[0183] In some embodiments, the peptides disclosed herein can cross a cell layer or barrier (e.g., the BBB) or cell membrane, for example, via TfR-mediated vesicular transcytosis and TfR-mediated endocytosis, respectively. In addition to binding to TfR and promoting transcytosis and / or endocytosis, the peptides of the present disclosure can also bind to additional target proteins on cells, such as cancer cells. In some cases, the peptide is a peptide or peptide construct comprising a TfR-binding peptide conjugated, linked, or fused to a targeting moiety or active agent (e.g., a therapeutic or diagnostic agent), such as a small molecule or peptide having affinity for the additional target protein (e.g., a receptor or enzyme). In some cases, the TfR-binding peptide is linked to a cargo molecule, enabling or promoting TfR-mediated transcytosis of the cargo molecule across the BBB or TfR-mediated endocytosis into the cell. In some examples, after transcytosis, peptide constructs comprising a TfR-binding peptide and a cargo moiety can target specific cells or tissues within the CNS and exert a biological effect (e.g., binding to a target protein) upon reaching the cells or tissues. In some cases, peptide constructs of the present disclosure exert a biological effect mediated by the TfR-binding peptide, cargo molecule, or active agent, or a combination thereof. In some cases, TfR-binding peptide constructs of the present disclosure comprising one or more active agents (e.g., therapeutic agents) can transport and / or deliver the one or more active agents into cells expressing TfR. In some cases, TfR-binding peptides accumulate in tissues of the CNS. In some cases, CNS-specific accumulation reduces off-target effects. In some cases, TfR-binding peptides accumulate in tissues other than the CNS (e.g., liver, kidney, spleen, or skin). In some cases, the cells expressing TfR are tumor cells, and the TfR-binding peptide constructs deliver anti-tumor agents to these tumor cells.In some cases, anti-tumor agents alone have no or very limited therapeutic effect on tumor cells, but when the anti-tumor agents are combined with the TfR-binding peptides of the present disclosure, e.g., as a peptide construct, the therapeutic effect of these anti-tumor agents is greatly improved.

[0184] In some embodiments, the TfR-binding peptides of the present disclosure can induce a biologically relevant response. In some embodiments, the biologically relevant response can be induced after intravenous administration, and in some embodiments, after a single intravenous administration. In some embodiments, the TfR-binding peptides can be used in combination with various other classes of therapeutic compounds used to treat and / or prevent pain, neuropathic pain, or other neurological disorders, such as neurodegenerative disorders, infectious diseases, and immune disorders (e.g., autoimmune diseases). The binding of the peptides and peptide constructs (e.g., peptide conjugates, fusion peptides, or recombinantly produced peptide constructs) described herein to TfR and subsequent transport across a cell barrier, such as a cell layer or the BBB (e.g., via TfR-mediated vesicular transcytosis) or across a cell membrane (e.g., via TfR-mediated endocytosis) can affect numerous diseases, conditions, or disorders in subjects (e.g., humans) associated with chronic pain (e.g., headache or migraine), neuropathic pain, obesity, insulin resistance, opioid addiction, or other neurological or psychiatric disorders.

[0185] Binding of the peptides and peptide constructs described herein (e.g., peptide conjugates, fusion peptides, or recombinantly produced peptide constructs) to TfR and subsequent transport across a cellular barrier, such as a cell layer or the BBB (e.g., via vesicular transcytosis) or across a cell membrane (e.g., via endocytosis) can impact a number of diseases, conditions, or disorders associated with neurodegeneration. Neurodegenerative diseases that can be treated, prevented, or diagnosed with the TfR-binding peptides described herein can include Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, disease with Lewy bodies, Parkinson's disease, spinal muscular atrophy, motor neuron disease, Lyme disease, ataxia-telangiectasia, autosomal dominant cerebellar ataxia, Batten disease, corticobasal syndrome, Creutzfeldt-Jakob disease, parafragile X tremor / ataxia syndrome, Kufour-Rakeb syndrome, Machado-Joseph disease, multiple sclerosis, chronic traumatic encephalopathy, or frontotemporal dementia.

[0186] The binding of the peptides and peptide constructs (e.g., peptide conjugates, fusion peptides, or recombinantly produced peptide constructs) described herein to TfR and subsequent transport across a cell barrier, such as a cell layer or the BBB (e.g., via vesicular transcytosis) or across a cell membrane (e.g., via endocytosis) can impact numerous autoimmune diseases and therapeutically address autoimmune or inflammatory disorders of the brain. In some cases, autoimmune diseases can be treated and / or prevented by conjugating, linking, or fusing a TfR-binding peptide to an active agent, which may be an ion channel modulator, such as a Kv1.3 potassium channel inhibitor. Additional diseases that can be treated and / or prevented using ion channel modulators, such as Kv1.3 potassium channel inhibitors, include psoriasis and other autoimmune diseases outside the brain due to effects on effector T cells, as well as neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, traumatic brain injury, or radiation therapy toxicity. In some cases, the Kv1.3 potassium channel inhibitor can be a small molecule (e.g., domatinostat tosylate), or a peptide (e.g., Vm24 or an ShK peptide such as ShK-170, ShK-186, or ShK-192, or any fragment or derivative thereof), or any combination thereof (see, e.g., Bartok et al., "An engineered scorpion toxin analogue with improved Kv1.3 selectivity displays reduced conformational flexibility," Sci Rep. 2015;5:18397).

[0187] In some embodiments, the TfR-binding peptides of the present disclosure can be used to treat and prevent various neurological disorders, including, but not limited to, epilepsy, schizophrenia, depression, anxiety, bipolar disorder, disorders of brain development (e.g., autism spectrum), or mood disorders.

[0188] In some embodiments, the TfR-binding peptides of the present disclosure can be used to treat and prevent Crohn's disease or, more generally, inflammatory bowel disease. In some cases, the TfR-binding peptides of the present disclosure exhibit high uptake and retention in intestinal glandular cells, which may express high amounts of TfR.

[0189] In various embodiments, the therapeutic efficacy of these drugs, when used in combination with the TfR-binding peptides of the present disclosure, may be significantly improved compared to administration without conjugation to the TfR-binding peptides described herein. In various embodiments, efficacy may be improved by delivering and achieving higher levels of the drug in the tissue or cell of interest. In various embodiments, efficacy may be improved by increasing the relative level of the drug in the tissue or cell of interest and decreasing the level of the drug in other tissues or compartments, thereby improving the therapeutic window or reducing toxic side effects.

[0190] In other embodiments, the peptides described herein compete with endogenous molecules for binding to the TfR. As described herein, "competition" or peptide competition for binding to a target protein, such as the TfR, includes, but is not limited to, steric hindrance, occupancy of the target protein's binding site, non-covalent interactions such as salt bridges or hydrophobic interactions, cross-linking, covalent interactions, sequestration, allosteric modulation, or any combination thereof.

[0191] In some embodiments, the peptides of the present disclosure can bind to any of the known TfR homologs, including TfR1, TfR2, soluble TfR, or any combination or fragment thereof (e.g., ectodomain). Thus, as used herein, "TfR" can refer to any known homolog, derivative, fragment, or member of the TfR family, including TfR1, TfR2, and soluble TfR. In other embodiments, the peptides are capable of binding to one, more than one, or all TfR homologs. In some embodiments, the peptides of the present disclosure can bind to TfR and promote a specific biological effect, such as vesicular transcytosis. In some embodiments, the peptides of the present disclosure, including peptides and peptide constructs comprising the amino acid sequences set forth in SEQ ID NOs:1-70, 136-205, 225-332, and 356-361, and any derivatives or variants thereof, block or reduce binding of endogenous TfR binding substances (e.g., transferrin or any derivatives such as apotransferrin or holotransferrin) to TfR. In some embodiments, peptides or peptide constructs of the present disclosure include derivatives and variants having at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to the amino acid sequences set forth in SEQ ID NOs:1 to 70, 136 to 205, 225 to 332, and 356 to 361.

[0192] In some embodiments, the peptides have equal, similar, or higher affinity (e.g., lower dissociation constant K) than endogenous molecules (e.g., transferrin, holotransferrin (iron-bound transferrin), apotransferrin (iron-unbound transferrin), or any other endogenous TfR ligand) or other exogenous molecules. D In some embodiments, the peptide binds to TfR with a K of less than 50 μM, less than 5 μM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 1 nM, or less than 0.1 nM. D In some embodiments, peptide transport by the TfR may have a lower affinity (e.g., a higher dissociation constant K) compared to the endogenous molecule. D In some embodiments, peptide transport by TfR is improved by having a faster off-rate or higher k than the endogenous molecule. off In some embodiments, the off-rate or k off is similar to transferrin. In some embodiments, peptide transport is enhanced by a faster on-rate or a higher k on , optionally with a k higher than transferrin on In other embodiments, one or more conserved residues at the transferrin (Tf)-TfR binding interface are also present in the amino acid sequences of the peptides described herein.

[0193] In some embodiments, peptides that exhibit improved TfR receptor binding exhibit improved transcytosis function. In some embodiments, peptides that exhibit improved TfR receptor binding exhibit no or only a small change in transcytosis function. In some embodiments, peptides that exhibit improved TfR receptor binding exhibit decreased transcytosis function. In some embodiments, the peptides bind to a site that is highly homologous between human TfR and mouse TfR, including one or more or all of the amino acid domains corresponding to residues 506-510, 523-531, and 611-662 of human TfR (SEQ ID NO: 363). In some embodiments, the region of TfR to which the peptides disclosed herein or variants thereof bind is all or part of such TfR domain. In some embodiments, the peptides disclosed herein bind to any one, any two, or all three highly homologous regions of the TfR, including the amino acid domains corresponding to residues 506-510, 523-531, and 611-662 of the human TfR (SEQ ID NO: 363). In some embodiments, the peptides disclosed herein bind to at least the domain corresponding to residues 611-662 of the human TfR.

[0194] In some embodiments, the K A value and K D Values ​​can be adjusted and optimized (eg, via amino acid substitution) to provide the optimal ratio of TfR binding affinity and efficient transcytosis function.

[0195] In some embodiments, the peptides disclosed herein or variants thereof bind to TfR at residues found in the binding interface (e.g., binding domain or binding pocket) between TfR and other exogenous or endogenous ligands (e.g., transferrin (Tf), Tf derivatives, or Tf-like peptides or proteins). In some embodiments, the TfR-binding peptides disclosed herein or variants thereof have at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology, or at least 100% homology to sequences that bind to residues of TfR that comprise the binding pocket. In some embodiments, the TfR-binding peptides or variants thereof disclosed herein have at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to an endogenous or exogenous polypeptide known to bind to TfR, e.g., endogenous transferrin, or any one of the peptides listed in Table 1. In other embodiments, the peptides described herein bind to a protein of interest that has at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to TfR, a fragment, homolog, or variant thereof.

[0196] In some embodiments, a peptide disclosed herein or a variant thereof binds to a region of TfR comprising amino acid residues corresponding to residues 506-510, 523-531, and 611-662 (the numbering of these amino acid residues is based on the following Uniprot reference protein sequence for endogenous human TfR: UniProtKB-P02786 (SEQ ID NO: 363, TFR1_HUMAN)). In some embodiments, the region of TfR to which a peptide disclosed herein or a variant thereof binds overlaps with that of Tf, a fragment, homolog, or variant thereof.

[0197] In other embodiments, the nucleic acid, vector, plasmid, or donor DNA comprises a sequence encoding a peptide, peptide construct, or variant or functional fragment thereof described in this disclosure. In further embodiments, a specific portion or fragment of a TfR binding motif (e.g., a conserved binding motif) may be grafted onto a peptide or peptide construct comprising any one of the sequences set forth in SEQ ID NOs: 1-70, 136-205, 225-332, or 356-361.

[0198] In some embodiments, the peptide inhibits binding between TfR and Tf, or between TfR and any other protein. In some embodiments, the peptide blocks protein-protein interactions of TfR and / or blocks TfR localization to the nucleus of a cell. In some cases, the peptide inactivates TfR. In some embodiments, the peptide can cause degradation of TfR, block localization of TfR to the nucleus of a cell, or block the interaction of TfR with Tf or a Tf-like protein.

[0199] In some embodiments, peptides competitively bind to TfR by binding to a specific amino acid residue or amino acid residue motif in the TfR, compared to endogenous Tf or any other endogenous or exogenous TfR-binding substance. Furthermore, peptides can be selected for further testing or use based on their ability to bind to a specific amino acid residue or amino acid residue motif. A specific amino acid residue or amino acid residue motif in the TfR can be identified by the amino acid residue or amino acid residue sequence involved in TfR binding to Tf. A specific amino acid residue or amino acid residue motif can be identified from the crystal structure of the TfR:Tf complex. In some embodiments, peptides (e.g., CDPs) exhibit resistance to heat, proteases (pepsin), and reduction.

[0200] Peptides and peptide constructs (e.g., peptide conjugates or fusion peptides) comprising one or more of the amino acid sequences set forth in SEQ ID NOs: 1 to 70, 136 to 205, 225 to 332, and 356 to 361 can bind to proteins of interest. In some embodiments, the protein of interest is TfR. In some embodiments, peptides and peptide constructs (e.g., peptide conjugates or fusion peptides) that bind to TfR comprise at least one of the amino acid sequences set forth in SEQ ID NOs: 1 to 70, 136 to 205, 225 to 332, and 356 to 361. In some embodiments, TfR-binding peptides, peptide constructs (e.g., peptide conjugates and fusion molecules) of the present disclosure include peptide derivatives or variants having at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology, or at least 100% homology, to the amino acid sequences set forth in SEQ ID NOs:1-70, 136-205, 225-332, and 356-361.

[0201] In some embodiments, a peptide or library of peptides is designed in silico rather than derived from a natural scaffold of knotted peptides. In other embodiments, a peptide or library of peptides is designed in silico by grafting relevant protein-binding residues or conserved residues of the protein-binding interface onto a natural peptide or protein known to bind to a protein or receptor of interest. In some embodiments, the peptides (e.g., SEQ ID NOs: 1-32, 36-67, 136-167, or 171-202) are simple helix-turn-helices. In some embodiments, helix-turn-helices can be used to transfer pharmacophores onto other scaffolds; for example, fusion tags can be used to graft desired TfR-engaging surfaces onto a helix-turn-helix scaffold.

[0202] In some embodiments, a peptide comprising SEQ ID NO: 1 is used as a scaffold or base sequence for further modification, including addition, deletion, or amino acid substitution. In some embodiments, a short sequence of amino acid residues, such as GS, is added to the N-terminus of the peptide. In some embodiments, the peptide lacks GS at the N-terminus. In some examples, the peptide undergoes one or more post-translational modifications.

[0203] Table 1 lists exemplary peptide sequences according to the methods and compositions of the present disclosure. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0204] X can be independently selected from D, E, N, Q, S, or T; X can be independently selected from D, E, H, K, R, N, Q, S, or T; and X can be independently selected from H, K, R, N, Q, S, or T.

[0205] In some embodiments, the TfR-binding peptide disclosed herein comprises GSREX1CX2X3RCX4KYX5DEX6X7KCX8ARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 207) or REX1CX2X3RCX4KYX5DEX6X7KCX8ARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 335), wherein X1, X2, X3, X4, X5, X6, X7, and X8 are TfR-binding interface residues and may independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises GSREGCASRCMKYNDELEKCEARMMSMSNTEEDCEQEX1EDX2X3YCX4X5X6CX7X8X9 (SEQ ID NO: 208) or REGCASRCMKYNDELEKCEARMMSMSNTEEDCEQEX1EDX2X3YCX4X5X6CX7X8X9 (SEQ ID NO: 336), where X1, X2, X3, X4, X5, X6, X7, X8, and X9 are TfR-binding interface residues and may independently be any amino acid. 10 X 11 YCX 12 X 13 X 13 CX 15 X 16 X 17 (SEQ ID NO: 209) or REX1CX2X3RCX4KYX5DEX6X7KCX8ARMMSMSNTEEDCEQEX9EDX 10 X 11 YCX 12 X 13 X 13 CX 15 X 16 X 17 (SEQ ID NO: 337), wherein: X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16and X 17 are TfR binding interface residues and can independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises GSREGCASRCMKYNDELEKCEARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 32).

[0206] In some embodiments, the TfR-binding peptide disclosed herein is 10 X 11 LEX 12 X 13 EX 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 (SEQ ID NO: 210), wherein: X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X25 , X 26 , X 27 , X 28 , X 29 , X 30 , X 31 , X 32 , X 33 , X 34 , X 35 , X 36 , X 37 , X 38 , X 39 , X 40 , X 41 , X 42 , and X 43 can independently be any amino acid.

[0207] In some embodiments, the TfR-binding peptide disclosed herein is 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 LX 38 X 39 LLX 40 X 41 LDHX 42 HSQ (SEQ ID NO: 211), wherein: X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , X 27 , X 28 , X 29 , X 30 , X 31 , X 32 , X 33 , X 34 , X 35 , X 36 , X 37 , X 38 , X 39 , X 40 , X 41 , and X 42 can independently be any amino acid.

[0208] In some embodiments, the TfR-binding peptide disclosed herein is 10 X 11 LEX 12 X 13 EX 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 LX 30 X 31 LLX 32 X 33 LDHX 34 HSQ (SEQ ID NO: 212), wherein: X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , X 27 , X 28 , X 29 , X 30 , X 31 , X 32 , X 33 , and X 34 can independently be any amino acid.

[0209] In some embodiments, the TfR-binding peptide or peptide construct disclosed herein has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 1-70, 136-205, 225-332, or 356-361, or any variant, homolog, or functional fragment thereof. In some embodiments, the TfR-binding peptide or peptide construct disclosed herein comprises any one of SEQ ID NOs: 1-70, 136-205, 225-332, or 356-361, or any variant, homolog, or functional fragment thereof. In some embodiments, the peptide that binds to TfR comprises the amino acid sequence set forth in SEQ ID NO:32.

[0210] In some embodiments, the TfR-binding peptide comprises a canonical amino acid residue as a surface interface residue at any one of positions corresponding to 5, 7, 8, 14, 17, 18, 21, 38, 42, 45, 46, 47, 50, 51, or a combination thereof, relative to SEQ ID NO: 32. In some embodiments, the TfR-binding peptide comprises a canonical amino acid residue as a surface interface residue at any one of positions corresponding to G5, A7, S8, N14, L17, E18, E21, L38, L42, L45, D46, H47, S50, Q51, or a combination thereof, relative to SEQ ID NO: 32. In some embodiments, a peptide or peptide construct of the present disclosure comprises at least one or more of these corresponding residues in SEQ ID NOs: 1-70, 136-205, 225-332, or 356-361. Thus, such peptides can be engineered to enhance binding to TfR. In some embodiments, the TfR-binding peptides disclosed herein have the structure: X1X2X3X4GX5ASX6X7X8X9X 10 NX 11 X 12 LEX 13 X 14 EX 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 LX 31 X 32 X 33 LX 34 X 35 LDHX 36 X 37 SQ (SEQ ID NO: 213), wherein: X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13, X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , X 27 , X 28 , X 29 , X 30 , X 31 , X 32 , X 33 , X 34 , X 35 , X 36 , and X 37 can independently be any amino acid.

[0211] In some embodiments, surface-distal hydrophilic amino acid residues (e.g., D, E, H, K, R, N, Q, S, or T) present in the amino acid sequence of the peptide contribute to the solubility of the peptide. In some embodiments, the peptides disclosed herein comprise a hydrophilic amino acid residue at any one of positions corresponding to 3, 4, 9, 11, 15, 16, 19, 23, 26, 28, 29, 30, 31, 32, 33, 35, 36, 37, 39, 40, or any combination thereof, relative to SEQ ID NO: 32. In some examples, the peptides of the present disclosure comprise hydrophilic amino acid residues at the following corresponding positions relative to SEQ ID NO: 32: R3, E4, R9, K12, D15, E16, K19, R23, S26, S28, N29, T30, E31, E32, D33, E35, Q36, E37, E39, D40, or any combination thereof. In some embodiments, relative to SEQ ID NO: 32, any one or any combination of the corresponding positions R3, E4, R9, K12, D15, E16, K19, R23, S26, S28, N29, T30, E31, E32, D33, E35, Q36, E37, E39, D40 can be changed to another hydrophilic residue without significantly affecting solubility or TfR binding. 10 DEX 11 X 12 KX 13 X 14 X 15 RX 16 X 17 SX 18 SNTEEDX 19 EQEX 20 EDX 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 (SEQ ID NO: 214), wherein: X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , X 27 , X 28 , X 29 , X 30 , and X 31 can be independently any amino acid. In some embodiments, the TfR-binding peptide disclosed herein is 10 X 11 X 12 X 13 X 14 X 15 CX 16 X 17 X 18 LX 19 X 20 LLYCLDHCHSQ (SEQ ID NO: 215) or X1X2GCASX3CMX4YNX5X6LEX7CEAX8MMX9MX 10 X 11 X 12 X 13 X 14 X 15 CX 16 X 17 X 18 LX 19 X 20 LLYCLDHCHSQ (SEQ ID NO: 338), wherein: X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , and X 20can be independently selected from D, E, H, K, R, N, Q, S, or T.

[0212] In some embodiments, the peptides of the present disclosure comprise a hydrophilic residue (e.g., D, E, H, K, R, N, Q, S, or T) at corresponding positions 15, 35, 39, 49, or any combination thereof, relative to SEQ ID NO: 32. In some examples, the peptides of the present disclosure comprise a hydrophilic amino acid residue at the following corresponding positions relative to SEQ ID NO: 32: D15, E35, E39, H49, or any combination thereof. In some embodiments, any one or any combination of corresponding positions D15, E35, E39, H49 relative to SEQ ID NO: 32 can be changed to another hydrophilic residue without significantly affecting solubility or TfR binding. In some embodiments, the TfR-binding peptides disclosed herein comprise: XI X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 DX 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 EX 34 X 35 X 36 EX 37 X 38 X 39 X 40 X 41 X 42 X 43 X 44 X 45 HX 46 X47 (SEQ ID NO: 216), wherein: X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , X 27 , X 28 , X 29 , X 30 , X 31 , X 32 , X 33 , X 34 , X 35 , X 36 , X 37 , X 38 , X 39 , X 40 , X 41 , X 42 , X 43 , X 44 , X 45 , X 46 , and X 47 can independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises GSREGCASRCMKYNX1ELEKCEARMMSMSNTEEDCX2QELX3DLLYCLDHCX4SQ (SEQ ID NO: 217) or REGCASRCMKYNX1ELEKCEARMMSMSNTEEDCX2QELX3DLLYCLDHCX4SQ (SEQ ID NO: 339), wherein X1, X2, X3, and X4 can be independently selected from D, E, H, K, R, N, Q, S, or T.

[0213] In some embodiments, a peptide of the present disclosure comprises a hydrophobic residue (e.g., A, M, I, L, V, F, W, or Y) at corresponding position 15, 35, 39, 49, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, a TfR-binding peptide disclosed herein comprises GSREGCASRCMKYNX1ELEKCEARMMSMSNTEEDCX2QELX3DLLYCLDHCX4SQ (SEQ ID NO: 218) or REGCASRCMKYNX1ELEKCEARMMSMSNTEEDCX2QELX3DLLYCLDHCX4SQ (SEQ ID NO: 340), wherein X1, X2, X3, and X4 can be independently selected from A, M, I, L, V, F, W, or Y. In some embodiments, a hydrophilic amino acid residue at any one of corresponding positions 15, 35, 39, and 49, relative to SEQ ID NO: 32, is associated with higher binding affinity (e.g., target binding) to TfR and higher solubility. In some embodiments, mutation of any one of the amino acid residues at corresponding positions 15, 35, 39, and 49 of SEQ ID NO: 32 from a hydrophobic residue to a hydrophilic residue can result in higher binding affinity (e.g., target binding) to TfR and higher solubility.

[0214] In some embodiments, a peptide of the present disclosure comprises a hydrophobic residue (e.g., A, M, I, L, V, F, W, or Y) at corresponding position 11, 25, 27, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, a peptide of the present disclosure comprises a hydrophilic residue (e.g., D, E, H, K, R, N, Q, S, or T) at corresponding position 11, 25, 27, or any combination thereof, relative to SEQ ID NO: 32. In some embodiments, a hydrophobic amino acid residue at any one of corresponding positions 11, 25, and 27, relative to SEQ ID NO: 32, is associated with higher binding affinity (e.g., target binding) to TfR and higher solubility. In some embodiments, mutation of an amino acid residue at any one of corresponding positions 11, 25, and 27, relative to SEQ ID NO: 32, from a hydrophilic residue to a hydrophobic residue can result in higher binding affinity (e.g., target binding) to TfR and higher solubility. In some embodiments, the peptides of the present disclosure comprise hydrophobic amino acid residues at corresponding positions M11, M25, M27, or any combination thereof, relative to SEQ ID NO: 32. In some examples, the peptides comprise hydrophobic amino acid residues at corresponding positions M11, M25, and M27, relative to SEQ ID NO: 32. In some embodiments, any combination of corresponding positions M11, M25, and M27, relative to SEQ ID NO: 32, can be changed to another hydrophobic residue without significantly affecting solubility or TfR binding. In some embodiments, the TfR-binding peptides disclosed herein comprise a TfR-binding peptide of the formula X1X2X3X4X5X6X7X8X9X 10 MX 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 MX 24 MX 25 X 26 X 27 X 28X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 X 47 X 48 (Arrangement number 219)をincluding, in arrangement, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 X 47 , and びX 48can independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises GSREGCASRCX1KYNDELEKCEARMX2SX3SNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 220) or REGCASRCX1KYNDELEKCEARMX2SX3SNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 341), wherein X1, X2, and X3 can be independently selected from A, M, I, L, V, F, W, or Y. In some embodiments, a TfR-binding peptide disclosed herein comprises GSREGCASRCX1KYNDELEKCEARMX2SX3SNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 221) or REGCASRCX1KYNDELEKCEARMX2SX3SNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 342), wherein X1, X2, and X3 can be independently selected from D, E, H, K, R, N, Q, S, or T. In some embodiments, a peptide of the present disclosure comprises an aliphatic amino acid residue (e.g., A, M, I, L, or V) at corresponding position 45 relative to SEQ ID NO: 32. In some embodiments, a peptide of the present disclosure comprises an aromatic amino acid residue (e.g., F, W, or Y) at corresponding position 45. In some embodiments, an aliphatic amino acid residue at corresponding position 45 is associated with higher binding affinity to TfR. In some examples, a peptide comprises an aliphatic amino acid residue corresponding to L45 relative to SEQ ID NO: 32. In some embodiments, mutation of the amino acid residue at corresponding position 45 from an aromatic residue to an aliphatic residue can result in higher binding affinity (e.g., target binding) to TfR and higher solubility. In some embodiments, changing the corresponding position L45 to another aliphatic residue does not significantly affect solubility or TfR binding. In some embodiments, the TfR-binding peptides disclosed herein are 10 X 11 X 12 X 13 X 14 X 15 X16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 X 44 LX 45 X 46 X 47 X 48 X 49 X 50 (Screen 222) There are X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 、X 11 、X 12 、X 13 、X 14 、X 15 、X 16 、X 17 、X 18 、X 19 、X 20 、X 21 、X 22 、X 23 、X 24 、X 25 、X 26 、X 27 、X 28 、X 29 、X 30 、X 31 、X 32 、X 33 、X 34 、X 35 、X 36 、X 37 、X 38 、X 39 、X 40 、X41 , X 42 , X 43 , X 44 , X 45 , X 46 , X 47 , X 48 , X 49 , and X 50 can be independently any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises GSREGCASRCMKYNDELEKCEARMMSMSNTEEDCEQELEDLLYCX1DHCHSQ (SEQ ID NO: 223) or REGCASRCMKYNDELEKCEARMMSMSNTEEDCEQELEDLLYCX1DHCHSQ (SEQ ID NO: 343), wherein X1 can be independently selected from A, M, I, L, or V.

[0215] In some embodiments, a peptide of the present disclosure comprises GSREGCASRCMX1YNDELEX2CEARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 224) or REGCASRCMX1YNDELEX2CEARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 344), wherein X1 and X2 can be independently selected from K or R. In some embodiments, these residues at corresponding positions 12 and 19, relative to SEQ ID NO: 32, can be used for chemical conjugation to another molecule (e.g., an active agent or a detectable agent). In some embodiments, both X1 and X2 are R, and chemical conjugation is at the N-terminus of the peptide.

[0216] In some embodiments, mutation of any one or more amino acid residues of a peptide of the present disclosure can improve the binding affinity of the peptide to TfR. In some embodiments, mutation of 5 to 80% of the amino acid residues of a peptide of the present disclosure can improve the binding affinity of the peptide to TfR. In some embodiments, mutation of 1 to 100%, 5 to 100%, or 5 to 50% of the amino acid residues of a peptide of the present disclosure can improve the binding affinity of the peptide to TfR. In some embodiments, mutation of 15 to 50% of the amino acid residues of a peptide of the present disclosure can improve the binding affinity of the peptide to TfR. In some embodiments, mutation of 15 to 30% of the amino acid residues of a peptide of the present disclosure can improve the binding affinity of the peptide to TfR. In some embodiments, mutation of 25 to 30% of the amino acid residues of a peptide of the present disclosure can improve the binding affinity of the peptide to TfR. For example, mutation of 14 of the 51 amino acid residues (27.5%) of a peptide having the sequence of SEQ ID NO: 32 can improve the binding affinity of the peptide to TfR.

[0217] In some embodiments, mutations in any one or more amino acid residues of the peptides of the present disclosure may be at the binding interface of TfR. In some embodiments, mutations to the peptide can improve binding affinity, which may be beneficial for the binding and transcytosis of the peptides or peptide constructs disclosed herein. In some embodiments, the peptides provided herein may have many or few mutations to achieve optimal activity, where optimal activity is sufficient binding to TfR but not necessarily strong enough to prevent release of the peptide and / or peptide construct after transcytosis. Thus, the peptides of the present disclosure may include multiple mutations (also referred to as % mutated amino acid residues) that adjust binding affinity and off-rate to achieve optimal binding, function (e.g., transcytosis, BBB penetration, cell membrane penetration, transport across biological barriers), and release of the peptide or peptide construct. Therefore, mutations that result in maximum affinity may not necessarily correlate with a superior peptide with optimal binding and transcytosis.

[0218] In some embodiments, 1-100% or 5-100% of the amino acid residues of a peptide of the present disclosure are located in the binding interface of the TfR. In some embodiments, 10-90% of the amino acid residues of a peptide of the present disclosure are located in the binding interface of the TfR. In some embodiments, 20-80% of the amino acid residues of a peptide of the present disclosure are located in the binding interface of the TfR. In some embodiments, 30-70% of the amino acid residues of a peptide of the present disclosure are located in the binding interface of the TfR. In some embodiments, 40-60% of the amino acid residues of a peptide of the present disclosure are located in the binding interface of the TfR. In some embodiments, 30-35% of the amino acid residues of a peptide of the present disclosure are located in the binding interface of the TfR. For example, 17 of the 51 amino acid residues (33%) of a peptide having the sequence of SEQ ID NO: 32 may be located in the binding interface of the TfR.

[0219] In some embodiments, mutation of any one or more of the amino acid residues of a peptide of the present disclosure located in the binding interface of TfR can improve the binding affinity of the peptide to TfR. In some embodiments, mutation of 1 to 100% or 5 to 100% of the amino acid residues of a peptide of the present disclosure located in the binding interface of TfR improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 5 to 80% of the amino acid residues of a peptide of the present disclosure located in the binding interface of TfR improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 10 to 70% of the amino acid residues of a peptide of the present disclosure located in the binding interface of TfR improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 15 to 60% of the amino acid residues of a peptide of the present disclosure located in the binding interface of TfR improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 20 to 50% of the amino acid residues of a peptide of the present disclosure located in the binding interface of TfR improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 25-30% of the amino acid residues of a peptide of the present disclosure located at the binding interface of TfR improves the binding affinity of the peptide to TfR. For example, mutation of 5 of 17 amino acid residues (29%) of a peptide having the sequence of SEQ ID NO: 32 located at the binding interface of TfR can improve the binding affinity of the peptide to TfR.

[0220] In some embodiments, any one or more mutations in amino acid residues of a peptide of the present disclosure are distal to the binding interface of the TfR. In some embodiments, 1-100% or 5-100% of the amino acid residues of a peptide of the present disclosure are distal to the binding interface of the TfR. In some embodiments, 10-90% of the amino acid residues of a peptide of the present disclosure are distal to the binding interface of the TfR. In some embodiments, 20-80% of the amino acid residues of a peptide of the present disclosure are distal to the binding interface of the TfR. In some embodiments, 30-70% of the amino acid residues of a peptide of the present disclosure are distal to the binding interface of the TfR. In some embodiments, 40-60% of the amino acid residues of a peptide of the present disclosure are distal to the binding interface of the TfR. In some embodiments, 65-70% of the amino acid residues of a peptide of the present disclosure are distal to the binding interface of the TfR. For example, 34 of the 51 amino acid residues (66%) of the peptide having the sequence of SEQ ID NO: 32 may be in the binding interface of the TfR.

[0221] In some embodiments, mutation of any one or more of the amino acid residues of the peptides of the present disclosure that are distal to the TfR binding interface improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 1 to 100% or 5 to 100% of the amino acid residues of the peptides of the present disclosure that are distal to the TfR binding interface improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 5 to 80% of the amino acid residues of the peptides of the present disclosure that are distal to the TfR binding interface improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 10 to 70% of the amino acid residues of the peptides of the present disclosure that are distal to the TfR binding interface improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 15 to 60% of the amino acid residues of the peptides of the present disclosure that are distal to the TfR binding interface improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 20-50% of the amino acid residues of a peptide of the present disclosure distal to the TfR binding interface improves the binding affinity of the peptide to TfR. In some embodiments, mutation of 25-30% of the amino acid residues of a peptide of the present disclosure distal to the TfR binding interface improves the binding affinity of the peptide to TfR. For example, mutation of 5 of 17 amino acid residues distal to the TfR binding interface can improve the binding affinity of the peptide to TfR. For example, mutation of 9 of 34 amino acid residues (26.5%) of a peptide having the sequence of SEQ ID NO: 32 distal to the TfR binding interface can improve the binding affinity of the peptide to TfR. In some embodiments, without being bound by any theory, mutation of one or more amino acid residues of a peptide distal to the TfR binding interface can improve protein folding, increase protein solubility, and / or alter the backbone geometry, which can improve binding through optimized interface shape complementation.

[0222] In some embodiments, peptides of the present disclosure may comprise a sequence having a cysteine ​​residue at one or more of positions 11, 12, 13, 14, 19, 20, 21, 22, 36, 38, 39, and 41. In some embodiments, peptides comprise a Cys at position 11, 12, 19, 20, 36, 39, or any combination thereof. For example, in certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 11. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 12. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 13. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 14. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 19. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 20. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 21. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 22. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 36. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 38. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 39. In certain embodiments, a peptide may comprise a sequence having a cysteine ​​residue at position 41. In some embodiments, the first cysteine ​​residue in the sequence may be disulfide bonded to the fourth cysteine ​​residue in the sequence, the second cysteine ​​residue in the sequence may be disulfide bonded to the fifth cysteine ​​residue in the sequence, and the third cysteine ​​residue in the sequence may be disulfide bonded to the sixth cysteine ​​residue in the sequence. Optionally, the peptide may comprise one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a "two-and-through" structural system. In some embodiments, the peptides disclosed herein may have one or more cysteines mutated to serine.

[0223] In some embodiments, the peptides of the present disclosure comprise at least one cysteine ​​residue. In some embodiments, the peptides of the present disclosure comprise at least two cysteine ​​residues. In some embodiments, the peptides of the present disclosure comprise at least three cysteine ​​residues. In some embodiments, the peptides of the present disclosure comprise at least four cysteine ​​residues. In some embodiments, the peptides of the present disclosure comprise at least five cysteine ​​residues. In some embodiments, the peptides of the present disclosure comprise at least six cysteine ​​residues. In some embodiments, the peptides of the present disclosure comprise at least ten cysteine ​​residues. In some embodiments, the peptides of the present disclosure comprise six cysteine ​​residues.

[0224] In some embodiments, the peptides of the present disclosure comprise an amino acid sequence having a cysteine ​​residue at one or more positions. In some embodiments, the one or more cysteine ​​residues are located at any one of amino acid positions 6, 10, 20, 34, 44, 48, or any combination thereof. In some aspects of the present disclosure, the one or more cysteine ​​(C) residues participate in disulfide bonds with various pairing patterns (e.g., C10-C20). In some embodiments, the pairing patterns are C6-C48, C10-C44, and C20-C34. In some embodiments, the peptides described herein comprise at least one, at least two, or at least three disulfide bonds. In some embodiments, the at least one, at least two, or at least three disulfide bonds are arranged according to the C6-C48, C10-C44, and C20-C34 pairing patterns, or a combination thereof. In some embodiments, the peptides described herein contain three disulfide bonds with pairing patterns C6-C48, C10-C44, and C20-C34.

[0225] In certain embodiments, the peptide comprises a sequence having a cysteine ​​residue at position 6. In certain embodiments, the peptide comprises a sequence having a cysteine ​​residue at position 10. In certain embodiments, the peptide comprises a sequence having a cysteine ​​residue at position 20. In certain embodiments, the peptide comprises a sequence having a cysteine ​​residue at position 34. In certain embodiments, the peptide comprises a sequence having a cysteine ​​residue at position 44. In certain embodiments, the peptide comprises a sequence having a cysteine ​​residue at position 50. In some embodiments, the first cysteine ​​residue in the sequence forms a disulfide bond with the last cysteine ​​residue in the sequence. In some embodiments, the second cysteine ​​residue in the sequence forms a disulfide bond with the penultimate cysteine ​​residue in the sequence. In some embodiments, the third cysteine ​​residue in the sequence forms a disulfide bond with the third penultimate cysteine ​​residue in the sequence, etc.

[0226] In some embodiments, the first cysteine ​​residue in the sequence is disulfide bonded to the sixth cysteine ​​residue in the sequence, the second cysteine ​​residue in the sequence is disulfide bonded to the fifth cysteine ​​residue in the sequence, and the third cysteine ​​residue in the sequence is disulfide bonded to the fourth cysteine ​​residue in the sequence. Optionally, the peptide may contain one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a "two-and-through" structural system. In some embodiments, the peptides disclosed herein have one or more cysteines mutated to serine.

[0227] In some embodiments, the peptide contains no cysteines or disulfides. In some embodiments, the peptide contains 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more cysteines or disulfides. In other embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more cysteine ​​residues are substituted with serine residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more cysteine ​​residues are substituted with threonine residues.

[0228] In some embodiments, the peptide contains no Cys or disulfides. In some embodiments, the peptide contains 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more Cys or disulfides. In other embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues are substituted with Ser residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues are substituted with Thr residues.

[0229] In some examples, one or more or all methionine residues in the peptide are substituted with leucine or isoleucine. In some examples, one or more or all tryptophan residues in the peptide are substituted with phenylalanine or tyrosine. In some examples, one or more or all asparagine residues in the peptide are substituted with glutamine. In some embodiments, the N-terminus of the peptide is blocked with an acetyl group or the like. Alternatively, or in combination, in some examples, the C-terminus of the peptide is blocked with an amide group or the like. In some embodiments, the peptide is modified by methylation on the free amine.

[0230] For example, complete methylation can be achieved through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.

[0231] In some embodiments, a peptide or peptide construct described herein targets and / or penetrates a TfR-expressing cell layer or barrier and / or the membrane of a TfR-expressing cell. In some embodiments, the peptide targets and / or penetrates the plasma membrane of a cell located in the CNS, such as the brain. For example, a peptide construct comprising a TfR-binding peptide and one or more active agents (e.g., therapeutic or diagnostic compounds) crosses a cell barrier (e.g., the BBB) via vesicular transcytosis and then targets and / or penetrates the plasma membrane of a cell located in the CNS, delivering the one or more active agents to the cell.

[0232] In various embodiments, peptide constructs comprising a TfR-binding peptide described herein and one or more active agents (e.g., therapeutic or diagnostic compounds) target and / or penetrate the cell membrane of TfR-expressing cells located in the gastrointestinal tract, spleen, liver, kidney, muscle, bone marrow, brain, or skin. In some cases, the TfR-expressing cells are tumor cells, immune cells, erythrocytes, erythroid progenitor cells, stem cells, bone marrow cells, or stem cells. In some cases, for example, when the cells overexpress TfR, the TfR-binding peptide is involved in cell targeting. In various embodiments, peptide constructs described herein comprising a TfR-binding peptide conjugated, linked, or fused to one or more cargo molecules (e.g., active agents and / or detectable agents) target and / or penetrate the cell membrane of cells located in various organs, such as the spleen, brain, liver, kidney, muscle, bone marrow, gastrointestinal tract, or skin.

[0233] In some cases, the cargo molecule facilitates targeting of a specific cell, cell population, or tissue. In some cases, it is a combination of TfR-mediated cell targeting and a cargo molecule that facilitates cell targeting. In some embodiments, the peptides or peptide constructs (e.g., peptide conjugates or fusion peptides) of the present disclosure are used to target the cells, cell populations, or tissues to exert a specific biological (e.g., therapeutic) effect. In some embodiments, the peptides or peptide constructs (e.g., peptide conjugates or fusion peptides) of the present disclosure are used to deliver cargo molecules into the cells to exert a specific biological effect.

[0234] In some embodiments, the peptide may include at least one or more tag peptide sequences to improve cell penetration. For example, the peptide may include at least one or more Arg residues or residues derived from the Tat protein to improve cell penetration properties. Additional tag peptide sequences include CysTat (CYRKKRRQRRR; SEQ ID NO: 71), S19-TAT (PFVIGAGVLGALGTGIGGIGRKKRRQRRR; SEQ ID NO: 72), R8 (RRRRRRRR; SEQ ID NO: 73), pAntp (RQIKIWFQNRRMKWKK; SEQ ID NO: 74), Pas-TAT (FFLIPKGGRKKRRQRRR; SEQ ID NO: 75), Pas-R8 (FFLIPKGRRRRRRRR; SEQ ID NO: 76), PasFHV (FFL IPKGRRRRNRTRRNRRRVR; SEQ ID NO: 77), Pas-pAntP (FFLIPKGRQIKIWFQNRRMKWKK; SEQ ID NO: 78), F2R4 (FFRRRR; SEQ ID NO: 79), B55 (KAVLGATKIDLPVDINDPYDLGLLLRHLRHHSNLLANIGDPAVREQVLSAMQEEE; SEQ ID NO: 80), ausulin (LSTAADMQGVVTDGMASGLDKDYLKPDD; SEQ ID NO: 81), IMT-P8 (RRWRRWNR FNRRRCR; SEQ ID NO: 82), BR2 (RAGLQFPVGRLLRRLLR; SEQ ID NO: 83), OMOTAG1 (KRAHHNALERKRR; SEQ ID NO: 84), OMOTAG2 (RRMKANARERNRM; SEQ ID NO: 85), pVEC (LLIILRRRIRKQAHAHSK; SEQ ID NO: 86), SynB3 (RRLSYSRRRF; SEQ ID NO: 87), DPV1047 (VKRGLKLRHVRPRVTRMDV; SEQ ID NO: 88), CY105Y (CSIPPEVKFN KPFVYLI; SEQ ID NO: 89), transportan (GWTLNSAGYLLGKINLKALAALAKKIL; SEQ ID NO: 90), MTS (KGEGAAVLLPVLLAAPG; SEQ ID NO: 91), hLF (KCFQWQRNMRKVRGPPVSCIKR; SEQ ID NO: 92), PFVYLI (PFVYLI; SEQ ID NO: 93), yBBR (VLDSLEFIASKL; SEQ ID NO: 94), DRI-TAT31 (rrrqrrkkrgy; lowercase letters in the sequences indicate D-amino acids;SEQ ID NO: 95), cyclic heptapeptide cyclo(cFΦR4) (FΦRRRRQ, where Φ in the sequence represents 1-2-naphthylalanine, the entire moiety is cyclized, and Gln acts as a conjugation handle and can be replaced with other functional groups such as Lys (for amine coupling) or Cys (for sulfhydryl coupling); SEQ ID NO: 96), DPV3 (RKKRRRESRKKRRRES; SEQ ID NO: 403), CH (RKGFYKRKQCKPSRGRKR; SEQ ID NO: 404), VP22 (NAATATRGRSAASRPTQRPRAPARSASRPRRPVQ; SEQ ID NO: 405), TP10 (AGYLLGKINLKALAALAKKIL; SEQ ID NO: 406), MAP (KLALKLALKALKAALKLA; SEQ ID NO: 407), BPrPp (MVKSKIGSWILVLFVAMWSDVGLCKKRP; SEQ ID NO: 408), sequence number 408), ARF (MVRRFLVTLRIRRACGPPRVRV; SEQ ID NO: 409), GALA (WEAALAEALAEALAEHLAEALAEALEALAA; SEQ ID NO: 410), SAP (VRLPPPVRLPPPVRLPPP; SEQ ID NO: 411), MPG (GLAFLGFLGAAGSTMGAWSQPKKKRKV; SEQ ID NO: 412), Pep-1 (KETWWETWWTEWSQPKKKRKV; SEQ ID NO: 413), [WR]4 (WRWRWRWR; SEQ ID NO: 414), Ig(v) (MGLGLHLLVLAAALQGAKKKRKV; SEQ ID NO: 415), K-FGF (AAVALLPAVLLAHLLAP; SEQ ID NO: 416), melittin (GIGAVLKVLTTGLPALISWIKRKRQQ; SEQ ID NO: 417), gH625 (HGLASTLTRWAHYNALIRAF; SEQ ID NO: 418), HIV-1 TAT protein (48-60) (GRKKRRQRRRPPQ; SEQ ID NO: 419), MPG HIV-gp41 / SV40 T-antigen (GALFLGFLGAAGSTMGAWSQPKKKRKV; SEQ ID NO: 420), R6W3 (RRWWRRWRR; SEQ ID NO: 421), NLS (CGYGPKKKRKVGG; SEQ ID NO: 422), 8-lysine (KKKKKKKK; SEQ ID NO: 423), HRSV (RRIPNRRPRR; SEQ ID NO: 424), AIP6 (RLRWR; SEQ ID NO: 425), Pep-1 (KETWWETWWTEWSQPKKRKV;SEQ ID NO: 426), MAP17 (QLALQLALQALQAALQLA; SEQ ID NO: 427), VT5 (DPKGDPKGVTVTVTVTVTGKGDPKPD; SEQ ID NO: 428), Bac7 (RRIRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO: 429), (PPR)n ((PPRPPRPPR; SEQ ID NO: 430), (PPRPPRPPRPPRPR; SEQ ID NO: 431), (PPRPPRPPRPPRPPR; SEQ ID NO: 432), (PPRPPRPPRPPRPPRPPR; SEQ ID NO: 433)), INF7 (GLF EAIEGFIENGWEGMIDGWYGC; SEQ ID NO: 434), CADY (GLWRALWRLLRSLWRLLWRA; SEQ ID NO: 435), Pep-7 (SDLWEMMMVSLACQY; SEQ ID NO: 436), TGN (TGNYKALHPHNG; SEQ ID NO: 437), Ku-70 (VPMLK; SEQ ID NO: 438), CPP (RRRRRGGRRRRRG; SEQ ID NO: 452) (RRRRRRGGRRRRRG; SEQ ID NO: 439), SVS-1 (KVKVKVKVDPPTKVKVKVK; SEQ ID NO: 440), L-CPP (L AGRRRRRRRRRK; SEQ ID NO: 441), RLW (RLWMRWYSPRTRAYG; SEQ ID NO: 442), K16ApoE (KKKKKKKKKKKKKKKKLRVRLASHLRKLRKRLLRDA; SEQ ID NO: 443), Angiopep-2 (TFFYGGSRGKRNNFKTEEY; SEQ ID NO: 444), ACPP (EEEEEEEEPLGLAGRRRRRRRRN; SEQ ID NO: 445), KAFAK (KAFAKLAARLYRKALARQLGVAA; SEQ ID NO: 446), hCT(9-32)(LGTY TQDFNKFHTFPQTAIGVGAP; SEQ ID NO: 447), VP22 (version 2) (DAATATRGRSAASRPTQRPRAPARSASRPRRPVE; SEQ ID NO: 448), MPG (GALFLGFLGAAGSTMGAWSQPKSKRKV; SEQ ID NO: 449), hPP3 (KPKRKRRKKKGHGWSR; SEQ ID NO: 450), PepNeg (SGTQEEY; SEQ ID NO: 451), CM18-TAT (KWKLFKKIGAVLKVLTTG; SEQ ID NO: 453), PTD4 (YARAAARQARA;Examples of suitable peptides include WaTx (MKYFTLALTLLFLLLINPCKDMNFAWAESSEKVERASPQQAKYCYEQCNVNKVPFDQCYQMCSPLERS; SEQ ID NO: 454), or WaTx (MKYFTLALTLLFLLLINPCKDMNFAWAESSEKVERASPQQAKYCYEQCNVNKVPFDQCYQMCSPLERS; SEQ ID NO: 455). For example, in some embodiments, the peptide may include an arginine patch (Arg patch), e.g., RRRRRRRR (SEQ ID NO: 73), or a variant or fragment thereof, wherein the sequence may be added to either the N-terminus or C-terminus of the peptide. In some embodiments, the Arg patch may include two or more Arg residues, or Arg; n(where n is an integer and can be 2, 3, 4, 5, 6, 7, 8, 9, or 10) (SEQ ID NO: 98). In other embodiments, the peptide can include a Tat peptide (Tat protein is reviewed in Gump et al. TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med. 2007 Oct; 13(10):443-8 and Harada et al. Antitumor protein therapy; application of the protein transduction domain to the development of a protein drug for cancer treatment. Breast Cancer. 2006; 13(1):16-26). The Tat peptide can have the sequence, for example, YGRKKRRQRRR (SEQ ID NO: 99), GRKKRRQRRR (SEQ ID NO: 100), or any modification, variant, or fragment thereof, and can be added to the N- or C-terminus of any TfR-binding peptide of the present disclosure. In some embodiments, the Tat peptide sequence can be GRKKRRQRRRPQ (SEQ ID NO: 101), GRKKRRQRRR (SEQ ID NO: 100), or a fragment or variant thereof. In some embodiments, the Tat peptide can be added to the N-terminus of any TfR-binding peptide of the present disclosure after the N-terminal GS dipeptide and before the spacer, e.g., GGGS (SEQ ID NO: 103) spacer. In some embodiments, a cell-penetrating tag peptide, such as any one of SEQ ID NOs: 71-96, 98-101, 116-124, 126, or 403-455, is added to the N-terminus of any TfR-binding peptide of the present disclosure after the N-terminal GS dipeptide and before the spacer, e.g., GGGS (SEQ ID NO: 103). x S y(SEQ ID NO: 104) peptide linker (where x and y can be any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) can be used to add to either the N-terminus or C-terminus of any peptide disclosed herein. In other embodiments, a cell-penetrating peptide, such as a Tat peptide or an Arg patch, or any other moiety, can be added to either the N-terminus or C-terminus of any peptide disclosed herein. x S y(SEQ ID NO: 104) peptide linker (wherein x and y can be any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) can be used to append to either the N-terminus or C-terminus of any peptide disclosed herein. In some embodiments, the peptide linker comprises (GS)x (SEQ ID NO: 105), where x can be any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 106), GGGGG (SEQ ID NO: 107), GSGSGSGS (SEQ ID NO: 108), GSGG (SEQ ID NO: 109), GGGGS (SEQ ID NO: 110), GGGS (SEQ ID NO: 103), GGS (SEQ ID NO: 112), GGGSGGGSGGGS (SEQ ID NO: 113), or a variant or fragment thereof. Additionally, KKYKPYVPVTTN (SEQ ID NO: 114) derived from DkTx and EPKSSDKTHT (SEQ ID NO: 115) derived from human IgG3 can be used as peptide linkers. In other embodiments, the tag peptide can be added to the peptide at any amino acid residue. In further embodiments, the tag peptide can be added to the peptide at any amino acid residue without interfering with TfR binding activity. In some embodiments, the tag peptide is added via conjugation, linkage, or fusion techniques. In other embodiments, the Tat peptide can be added to the peptide at any amino acid residue. In further embodiments, the Tat peptide can be added to the peptide at any amino acid residue without interfering with TfR binding activity. In some embodiments, the Tat peptide is added to the TfR-binding peptide via conjugation, linkage, or fusion techniques to obtain a TfR-binding cell-penetrating peptide fusion (CPP fusion).

[0235] Cell-penetrating peptides include, but are not limited to, short, amphipathic or cationic peptides with a net positive charge that can penetrate cell membranes and transport molecules or cargoes attached to the peptide, either covalently or non-covalently, into cells. Such cell-penetrating peptides can be synthesized or derived from known proteins, such as penetratin, Tat peptide, pVEC, or chimeric peptides, such as transportan, MPG, and Pep-1, or synthetic peptides, such as polyarginine, MAP, and R6W3.

[0236] In some embodiments, the peptide may include at least one cell-penetrating peptide sequence to improve cell penetration. For example, cell-penetrating peptides include maurocalin (GDCLPHLKLCKENKDCCSKKCKRRGTNIEKRCR; SEQ ID NO: 116), imperatoxin (GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR; SEQ ID NO: 117), hadrucalcin (SEKDCIKHLQRCRENKDCCSKKCSRRGTNPEKRCR; SEQ ID NO: 118), hemicalcin (GDCLPHLKLCKADKDCCSKKCKRRGTNPEKRCR; SEQ ID NO: 119), opicalcin-1 (GDCLPHLKRCKENNDCCSKKCKRRGTNPEKRCR; SEQ ID NO: 120), opicalcin-2 (GDCLPHLKRCKENNDCCSKKCKRRGANPEKRCR; SEQ ID NO: 121), and micalcin-3 (GDCLPHLKRCKENNDCCSKKCKRRGANPEKRCR; SEQ ID NO: 122). Examples of suitable cell-penetrating peptides include cytochrome P450 (CKYKFENWGACDGGTGTKVRQGTLKKARYNAQCQETIRVTKPC; SEQ ID NO: 122), MCoTI-II (SGSDGGVCPKILKKCRRDSDCPGACICRGNGYCG; SEQ ID NO: 123), or chlorotoxin (MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR; SEQ ID NO: 124). In some embodiments, the cell-penetrating peptide may have at least 80%, 90%, 95%, or 99% sequence identity to any of SEQ ID NOs: 71 to 96, 98 to 101, 116 to 124, 126, or 403 to 455.

[0237] In some embodiments, peptides are conjugated, linked, or fused to one or more cell-penetrating peptides, such as arginine-rich, amphipathic and lysine-rich, and hydrophobic residues or peptides capable of penetrating the plasma membrane or nucleus, for in vivo delivery of proteins or macromolecular cargoes. The conjugation or fusion can be direct or separated by a spacer (chemical or peptide-based). The spacer can be any peptide linker. For example, the spacer can be GGGSGGSGGGS (SEQ ID NO: 125), KKYKPYVPVTTN (SEQ ID NO: 114) derived from DkTx, EPKSSDKTHT (SEQ ID NO: 115) derived from human IgG3, or any variant or fragment thereof. In some embodiments, the cell-penetrating peptide sequence can be added to either the N-terminus or C-terminus of the peptide. In some embodiments, the cell-penetrating peptide can be added to the N-terminus of any TfR-binding peptide of the present disclosure after the N-terminal GS dipeptide and before the spacer, e.g., the GGGS (SEQ ID NO: 103) spacer. In some embodiments, the cell-penetrating tag peptide, such as any one of SEQ ID NOs: 71 to 96, 98 to 101, 116 to 124, 126, or 403 to 455, is G x S y(SEQ ID NO: 104) peptide linker (wherein x and y can be any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) can be used to attach a peptide to either the N-terminus or C-terminus of any peptide disclosed herein. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 106), GGGGG (SEQ ID NO: 107), GSGSGSGS (SEQ ID NO: 108), GSGG (SEQ ID NO: 109), GGGGS (SEQ ID NO: 110), GGGS (SEQ ID NO: 103), GGS (SEQ ID NO: 112), GGGSGGGSGGGS (SEQ ID NO: 113), GGGSGGSGGGS (SEQ ID NO: 225), or a variant or fragment thereof. Additionally, KKYKPYVPVTTN (SEQ ID NO: 114) derived from DkTx and EPKSSDKTHT (SEQ ID NO: 115) derived from human IgG3 can be used as peptide linkers. In other embodiments, a cell-penetrating peptide can be attached to a peptide at any amino acid residue. In further embodiments, the cell-penetrating peptide can be added to the peptide at any amino acid residue without interfering with TfR binding activity. In some embodiments, the cell-penetrating peptide is added via conjugation, linkage, or fusion techniques. In other embodiments, the cell-penetrating peptide can be added to the peptide at any amino acid residue.

[0238] In other embodiments, cell penetration can be increased by using a high dose of the peptides described herein, such as up to 10 μM or more of the peptide. In some cases, an Arg patch can be fused, conjugated, linked, or co-delivered with the peptide. Up to 10 μM or more of the Arg patch can be co-delivered with the peptide to promote cell penetration. Protein transfection agents can also be used to increase cell penetration of the peptide. In some embodiments, direct cytosolic expression of the peptide can be used. In other embodiments, physical disruption methods such as electroporation can be used to improve intracellular delivery of the peptide.

[0239] In some embodiments, the cell permeability of the peptides described herein can be improved. For example, the binding interface of the peptides described herein can be grafted onto a scaffold, such as calcine, which is known to be cell-permeable. Similarly, sequences known to be cell-permeable can be grafted onto the peptides of the present disclosure. Some non-limiting examples of calcines can be imperatoxin-A, maurocalcine, hemicalcine, opiclasin 1, opicalcine 2, and hadrcalcine. The scaffold can comprise at least 60%, 70%, 80%, 90%, 95%, or 98% of any one of SEQ ID NOs: 116 to 124. In some embodiments, the cell-permeable peptide can be calcine, a modified calcine, a derivative of calcine, or a fragment thereof, and can be used to increase cell permeability. Modified calcine, a derivative of calcine, or a fragment thereof can be used to increase cell permeability by activating the sarcoplasmic reticulum ryanodine receptor, ryanodine-sensitive Ca2+ receptors, and the like. 2+ The calcines can be screened for cell-penetrating activity, such as activity against RyR1, RyR2, or both of the channels, or as selective agonists. Furthermore, modified calcines can include substitution, addition, or reduction of lysine residues or other charged residues within calcin to alter activity and optimize the calcin's cell-penetrating activity or activity against the RyR1 or RyR2 receptor. As an example, grafting a 6-amino acid portion of helix 3 (MLICLF; SEQ ID NO: 126) onto a calcin or modified calcin scaffold can generate a bifunctional peptide that retains the cell-penetrating function of calcin with the novel TfR-binding function of the TfR-binding peptide. As another example, the peptides described herein can be improved in cell-penetrating capacity through the use of cis-acting elements, including the inclusion of K / R-rich sequences such as TAT ​​or octa-arginine, intrahelical arginine patches, or fusion to larger fragments of proteins identified in cell-penetrating screens, such as penetratin or melittin.

[0240] In some embodiments, a nuclear localization signal may be linked, conjugated, linked, or fused to the peptides described herein to promote nuclear localization. In some embodiments, the TfR-binding peptide is conjugated, linked, or fused to a nuclear localization signal such as the four-residue sequence KK / RXK / R (SEQ ID NO: 129) (wherein X can be any amino acid or variant thereof). In some embodiments, the TfR-binding peptide is conjugated, linked, or fused to a nuclear localization signal described in Lange et al., J Biol Chem. 2007 Feb 23;282(8):5101-5, such as PKKKRRV (SEQ ID NO: 130) or KRPAATKKAGQAKKKK (SEQ ID NO: 131). In some embodiments, the peptides described herein are conjugated, linked, or fused to a nuclear localization signal comprising KxRy (SEQ ID NO: 132), where x and y can independently be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, such as KKRR (SEQ ID NO: 133), KKKRR (SEQ ID NO: 134), or KKKK (SEQ ID NO: 135). Other cell-penetrating moieties can be linked, conjugated, linked, or fused to the peptides described herein, including, but not limited to, polycations, polyorganic acids, endosomal release polymers, poly(2-propylacrylic acid), poly(2-ethylacrylic acid), or any combination thereof.

[0241] In some embodiments, peptides of the present disclosure (e.g., histidine-containing or histidine-rich TfR-binding peptides) have high TfR-binding affinity at physiological pH, but the binding affinity may be significantly reduced at low pH levels, such as endosomal pH 5.4. In some cases, TfR-binding peptides of the present disclosure may be optimized to improve intravesicular (e.g., intraendosomal) and / or intracellular delivery function while retaining high TfR-binding ability. In some cases, histidine scanning and comparative binding experiments can be performed to develop and screen such peptides. In addition, some peptides may contain (e.g., conjugated, linked, or fused) motifs that promote low-pH endosomal escape of the peptide or peptide construct to enhance delivery function (e.g., intracellular delivery of a therapeutic agent). In some embodiments, amino acid residues in the peptides of the present disclosure are substituted with different amino acid residues to alter the pH-dependent binding affinity to TfR. The amino acid substitutions can include those that increase binding affinity at low pH, those that increase binding affinity at high pH, ​​those that decrease binding affinity at low pH, those that decrease binding affinity at high pH, ​​or a combination thereof.

[0242] In some embodiments, peptides of the present disclosure are capable of vesicular transcytosis across a cell barrier, such as a cell layer or the BBB. In some embodiments, peptides target and / or penetrate a cell or the nucleus of a cell (e.g., a brain cell). Examples of cells or tissues that can be targeted include cells or tissues associated with a disease or condition, such as cells or tissues of the CNS, brain cells, cancerous cells, and other cell types in which a particular biological pathway in the cell is dysregulated. Further examples of cells or tissues that can be targeted include cells or tissues of the spleen, liver, kidney, muscle, bone marrow, or skin. Cells that can be targeted using peptides of the present disclosure can be human cells, mammalian cells, human or mammalian cell lines, cancer cell lines, or cells extracted from a subject in vivo or in vitro.

[0243] In some examples, the peptides disclosed herein contain only one lysine residue or no lysine residues. In some examples, one or more or all of the lysine residues in the peptide are substituted with arginine residues. In some examples, one or more or all of the methionine residues in the peptide are substituted with leucine or isoleucine. One or more or all of the tryptophan residues in the peptide can be substituted with phenylalanine or tyrosine. In some examples, one or more or all of the asparagine residues in the peptide can be substituted with glutamine. In some embodiments, one or more or all of the aspartic acid residues can be substituted with glutamic acid residues. In some examples, one or more or all of the lysine residues in the peptide can be substituted with alanine or arginine. In some embodiments, the N-terminus of the peptide can be blocked or protected, such as with an acetyl group or a tert-butyloxycarbonyl group. Alternatively, or in combination, the C-terminus of the peptide can be blocked or protected, such as with an amide group or by forming an ester (e.g., a butyl or benzyl ester). In some embodiments, peptides are modified by methylation on free amines, for example, complete methylation is achieved through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.

[0244] In some embodiments, the dipeptide GS can be added as the first two N-terminal amino acids, as shown in SEQ ID NO:1 to SEQ ID NO:35, or SEQ ID NO:136 to SEQ ID NO:170, or such N-terminal dipeptide GS can be absent or replaced by any other one or two amino acids, as shown in SEQ ID NO:36 to SEQ ID NO:70, or SEQ ID NO:171 to SEQ ID NO:205. In some embodiments, the dipeptide GS is used as a linker or is attached to a linker and used to form a peptide conjugate or fusion molecule, such as a peptide construct. In some embodiments, the linker is a G x S y (SEQ ID NO: 104) peptide, wherein x and y are independently any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises (GS)x (SEQ ID NO: 105), wherein x can be any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 106), GGGGG (SEQ ID NO: 107), GSGSGSGS (SEQ ID NO: 108), GGGGS (SEQ ID NO: 110), GGGS (SEQ ID NO: 103), or a variant or fragment thereof.

[0245] In some embodiments of the present disclosure, a peptide or peptide construct described herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-70, 136-205, 225-332, or 356-361. The peptides disclosed herein may comprise at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least The peptide fragment may be a contiguous fragment of any one of SEQ ID NOs: 1-32, 36-67, 136-167, or 171-202 that is 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, or at least 65 residues in length, and the peptide fragment may be selected from any portion of the peptide. In some embodiments, the peptide sequence is flanked by additional amino acids. The one or more additional amino acids may, for example, confer a particular in vivo charge, isoelectric point, chemical conjugation site, stability, or physiological property to the peptide.

[0246] In some examples, peptides described herein capable of targeting and binding to TfR include peptides that are 80 amino acids or less in length, or 70 amino acids or less in length, 60 amino acids or less in length, 40 amino acids or less in length, 35 amino acids or less in length, 30 amino acids or less in length, 25 amino acids or less in length, 20 amino acids or less in length, 15 amino acids or less in length, or 10 amino acids or less in length.

[0247] In other embodiments, the peptide may be conjugated, linked, or fused to a carrier or molecule that has targeting or homing function to a cell of interest or target cell, hi other embodiments, the peptide may be conjugated, linked, or fused to a molecule that extends the half-life or alters the pharmacodynamic and / or pharmacokinetic properties of the peptide, or any combination thereof.

[0248] In some examples, the peptide contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged residues, such as Arg or Lys, or any combination thereof. In some examples, one or more lysine residues in the peptide are substituted with arginine residues. In some embodiments, the peptide contains one or more Arg patches. In some embodiments, the Arg patch is located at the N-terminus of the peptide. In other aspects, the Arg patch is located at the C-terminus of the peptide. In some embodiments, the Arg patch comprises eight consecutive Arg residues (SEQ ID NO: 73). In some embodiments, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more Arg or Lys residues are solvent-exposed in the peptide. In some embodiments, the Arg patch can be two or more consecutive Arg residues. In some embodiments, the Arg patch comprises one or more Arg substituted with Lys.

[0249] The peptides of the present disclosure may further comprise neutral amino acid residues. In some embodiments, the peptides have 35 or fewer neutral amino acid residues. In other embodiments, the peptides have 81 or fewer neutral amino acid residues, 70 or fewer neutral amino acid residues, 60 or fewer neutral amino acid residues, 50 or fewer neutral amino acid residues, 40 or fewer neutral amino acid residues, 36 or fewer neutral amino acid residues, 33 or fewer neutral amino acid residues, 30 or fewer neutral amino acid residues, 25 or fewer neutral amino acid residues, or 10 or fewer neutral amino acid residues.

[0250] The peptides of the present disclosure may further comprise negative amino acid residues. In some embodiments, the peptide has six or fewer negative amino acid residues, five or fewer negative amino acid residues, four or fewer negative amino acid residues, three or fewer negative amino acid residues, two or fewer negative amino acid residues, or one or fewer negative amino acid residues. The negative amino acid residues can be selected from any negatively charged amino acid residue, but in some embodiments, the negative amino acid residues are either E or D, or a combination of E and D.

[0251] In some embodiments of the present disclosure, the three-dimensional or tertiary structure of the peptide is primarily composed of beta sheets and / or alpha helices. In some embodiments, the designed or engineered TfR-binding peptides of the present disclosure are small, compact peptides or polypeptides stabilized by intrachain disulfide bonds (e.g., mediated by cysteines) forming cysteines and a hydrophobic core. In some embodiments, the engineered TfR-binding peptide has a structure comprising a helical bundle with at least one disulfide bridge between each of the alpha helices, thereby stabilizing the peptide. In other embodiments, the engineered TfR-binding peptide comprises a structure with three alpha helices and three intrachain disulfide bonds, one between each of the three alpha helices in the alpha helical bundle.

[0252] In other embodiments, the peptide may be conjugated, linked, or fused to a molecule (e.g., a small molecule, peptide, or protein) that has a targeting or homing function to a cell of interest or a target protein located on the surface or within the cell. In other embodiments, the peptide may be conjugated, linked, or fused to a molecule that extends the plasma half-life and / or biological half-life of the peptide, or alters the pharmacodynamics (e.g., improved binding to the target protein) and / or pharmacokinetic properties (e.g., rate and mode of clearance), or any combination thereof.

[0253] Generally, nuclear magnetic resonance (NMR)-resolved or X-ray crystallographic structures of related structural homologs can be used to inform mutational strategies that can improve the folding, stability, and manufacturability of the peptides described herein while maintaining specific biological functions (e.g., TfR binding). These techniques can be used to predict 3D pharmacophores of structurally homologous scaffolds and to predict potential graftable regions of related proteins to generate chimeras with improved properties (e.g., binding characteristics). For example, this strategy can be used to identify key amino acid positions and loops that can be used to design peptides with improved TfR receptor binding and transcytosis properties, high expression, high in vivo stability, or any combination of these properties.

[0254] In some embodiments, a peptide capable of TfR binding and transcytosis across a cell membrane comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the exemplary peptide sequences listed in Table 1 (SEQ ID NO:1-32, SEQ ID NO:36-67, SEQ ID NO:136-167, and SEQ ID NO:171-202), or a functional fragment thereof. Two or more peptides may have a degree of sequence identity or homology and may have similar properties in vivo. For example, the peptide may have a degree of sequence identity or homology to any one of the peptides set forth in SEQ ID NO:1 to SEQ ID NO:32, SEQ ID NO:36 to SEQ ID NO:67, SEQ ID NO:136 to SEQ ID NO:167, or SEQ ID NO:171 to SEQ ID NO:202.In some embodiments, one or more peptides of the present disclosure have up to about 20% pairwise sequence identity or homology, up to about 25% pairwise sequence identity or homology, up to about 30% pairwise sequence identity or homology, up to about 35% pairwise sequence identity or homology, up to about 40% pairwise sequence identity or homology, up to about 45% pairwise sequence identity or homology, up to about 50% pairwise sequence identity or homology, up to about 55% pairwise sequence identity or homology, up to about 60% pairwise sequence identity or homology, up to about 65% pairwise sequence identity or homology, up to about 70% pairwise sequence identity. up to about 75% pairwise sequence identity or homology, up to about 80% pairwise sequence identity or homology, up to about 85% pairwise sequence identity or homology, up to about 90% pairwise sequence identity or homology, up to about 95% pairwise sequence identity or homology, up to about 96% pairwise sequence identity or homology, up to about 97% pairwise sequence identity or homology, up to about 98% pairwise sequence identity or homology, up to about 99% pairwise sequence identity or homology, up to about 99.5% pairwise sequence identity or homology, or up to about 99.9% pairwise sequence identity or homology.In some embodiments, one or more peptides of the present disclosure share with a second peptide at least about 20% pairwise sequence identity or homology, at least about 25% pairwise sequence identity or homology, at least about 30% pairwise sequence identity or homology, at least about 35% pairwise sequence identity or homology, at least about 40% pairwise sequence identity or homology, at least about 45% pairwise sequence identity or homology, at least about 50% pairwise sequence identity or homology, at least about 55% pairwise sequence identity or homology, at least about 60% pairwise sequence identity or homology, at least about 65% pairwise sequence identity or homology, at least about 70% pairwise sequence identity or homology, at least about 75% pairwise sequence identity or homology, at least about 80% pairwise sequence identity or homology, at least about 85 ... The pairwise sequence identity or homology may be at least about 75% pairwise sequence identity or homology, at least about 80% pairwise sequence identity or homology, at least about 85% pairwise sequence identity or homology, at least about 90% pairwise sequence identity or homology, at least about 95% pairwise sequence identity or homology, at least about 96% pairwise sequence identity or homology, at least about 97% pairwise sequence identity or homology, at least about 98% pairwise sequence identity or homology, at least about 99% pairwise sequence identity or homology, at least about 99.5% pairwise sequence identity or homology, or at least about 99.9% pairwise sequence identity or homology.

[0255] In some embodiments, peptides that exhibit improved TfR receptor binding exhibit improved transcytosis function. In some cases, peptides that exhibit improved TfR receptor binding exhibit no or only a small change in transcytosis function. In some cases, peptides that exhibit improved TfR receptor binding exhibit decreased transcytosis function. In some embodiments, the K of the TfR-binding peptide A value and K D Values ​​can be adjusted and optimized (eg, via amino acid substitution) to provide the optimal ratio of TfR binding affinity and efficient transcytosis function.

[0256] Various methods and software programs, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm, can be used to determine homology between two or more peptides. Pairwise sequence alignment can be used to identify regions of similarity that may indicate functional, structural, and / or evolutionary relationships between two biological sequences (e.g., amino acid or nucleic acid sequences). Additionally, multiple sequence alignment (MSA) is the alignment of three or more biological sequences. From the results of MSA applications, homology can be estimated and evolutionary relationships between sequences can be assessed. As used herein, "sequence homology," "sequence identity," "percent sequence identity," and "percent sequence homology" are used interchangeably and refer to the relatedness or amount of variation of a sequence relative to a reference polynucleotide or amino acid sequence, as appropriate.

[0257] In some examples, the peptide or peptide construct is any one of SEQ ID NOs:1-70, 136-205, 225-332, or 356-361, or a functional fragment thereof. In other embodiments, the peptide or peptide construct of the present disclosure further comprises a peptide having 99%, 95%, 90%, 85%, or 80% sequence identity or homology to any one of SEQ ID NOs:1-70, 136-205, 225-332, or 356-361, or a functional fragment thereof.

[0258] In other examples, the peptide or peptide construct may be a peptide that is homologous to any one of SEQ ID NOs: 1-70, 136-205, 225-332, or 356-361, or a functional fragment thereof. As further described herein, the term "homologous" may be used herein to refer to a peptide or peptide construct that has at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% sequence identity or homology to any one of SEQ ID NOs: 1-70, 136-205, 225-332, or 356-361, or a functional fragment thereof.

[0259] In yet another example, a nucleic acid molecule encoding a peptide or peptide construct of any one of SEQ ID NOs:1 to 70, 136 to 205, 225 to 332, or 356 to 361 can be identified by determining either the sequence identity or homology between the encoded peptide amino acid sequence and the amino acid sequence of any one of SEQ ID NOs:1 to 70, 136 to 205, 225 to 332, or 356 to 361, or by a nucleic acid hybridization assay. Such peptide variants or peptide construct variants of any one of SEQ ID NOs: 1 to 70, 136 to 205, 225 to 332, or 356 to 361 are (1) purified under high stringency washing conditions of 0.1×-0.2×SSC, 0.1% SDS, and a washing stringency equivalent to 50 to 65°C, and are capable of cleaving the peptide or peptide construct variants of any one of SEQ ID NOs: 1 to 70, 136 to 205, 225 to 332, or 356 to 361. 361 (or its complement); and (2) encoding a peptide having at least 70%, at least 80%, at least 90%, at least 95%, or greater than 95% sequence identity or homology to the amino acid sequence of any one of SEQ ID NOs:1-70, SEQ ID NOs:136-205, SEQ ID NOs:225-332, or SEQ ID NOs:356-361.

[0260] Knot Peptide In some embodiments, the TfR-binding peptides of the present disclosure contain one or more Cys or one or more disulfide bonds. In some embodiments, the TfR-binding peptide is derived from a cysteine-dense peptide (CDP), a knot peptide, or a hitchin. As used herein, the term "peptide" is considered synonymous with the terms "knot peptide," "cysteine-dense peptide," "CDP," and "hitchin." (See, e.g., Correnti et al. Screening, large-scale production, and structure-based classification for cystine-dense peptides. Nat Struct Mol Biol. 2018 Mar;25(3):270-278).

[0261] In some embodiments, the TfR-binding peptides of the present disclosure can bind to TfR, thereby blocking TfR interactions, such as interactions between TfR and other exogenous or endogenous ligands (e.g., Tf or its homologs or fragments). In some embodiments, the TfR-binding peptides of the present disclosure can bind to TfR without affecting the binding of TfR to other exogenous or endogenous ligands (e.g., Tf or its homologs or fragments). In some embodiments, the TfR-binding peptides can be engineered peptides. Engineered peptides can be non-natural, artificial, isolated, synthetic, designed, or recombinantly expressed peptides. In some embodiments, the TfR-binding peptides of the present disclosure comprise one or more properties of CDPs, knot peptides, or hitchins, such as stability, resistance to proteolysis, resistance to reducing conditions, and / or the ability to cross the blood-brain barrier.

[0262] In some embodiments, CDPs or knot peptides, including engineered, non-naturally occurring CDPs and those found in nature, can be conjugated, linked, or fused to TfR-binding peptides of the present disclosure, such as those described in Table 1, to provide additional homing or targeting functionality to target cells, such as cancer cells, pancreatic cells, liver cells, colon cells, ovarian cells, breast cells, lung cells, or any combination thereof. In some embodiments, CDPs or knot peptides, including engineered, isolated, and naturally occurring CDPs, can be conjugated, linked, or fused to TfR-binding peptides of the present disclosure, such as those described in Table 1, to provide additional homing or targeting functionality to target cells, such as cancer cells, pancreatic cells, liver cells, colon cells, ovarian cells, breast cells, lung cells, or any combination thereof. Engineered peptides can be non-naturally occurring, artificial, synthetic, designed, or recombinantly expressed peptides. In some embodiments, the TfR-binding peptides of the present disclosure enable TfR-mediated transcytosis and / or cellular endocytosis, and an additional CDP or knot peptide conjugated, linked, or fused to the TfR-binding peptide can selectively target an enzyme or other protein of interest within a cell associated with a disease or condition. In some cases, the cell is a cancer cell. The cancer may include breast cancer, liver cancer, colon cancer, brain cancer, spleen cancer, salivary gland cancer, kidney cancer, muscle cancer, ovarian cancer, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma, lung cancer, myeloid cell carcinoma, or skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, neuroblastoma, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, and diffuse intrinsic pontine glioma (DIPG), or CMYC-overexpressing cancer. In some cases, other CDP or knot peptides (e.g., those found in nature) can be conjugated, linked, or fused to the TfR-binding peptide to localize the TfR-binding peptide across the blood-brain barrier and deliver the TfR-binding peptide to target cells in the central nervous system.

[0263] CDPs (e.g., knot peptides) are a class of peptides typically ranging from about 11 to about 81 amino acids in length that often fold into compact structures. Knot peptides are typically characterized by numerous intramolecular disulfide bridges and assemble into complex tertiary structures that may include beta-strands, alpha-helices, and other secondary structures. The presence of disulfide bonds confers remarkable environmental stability to knot peptides, allowing them to withstand extreme temperatures and pH and resist proteolytic enzymes in the bloodstream. The presence of disulfide knots can also provide resistance to reduction by reducing agents. The rigidity of knot peptides also allows them to bind to targets without incurring the "entropy penalty" that occurs when flexible peptides bind to target cells. For example, binding can be adversely affected by the entropy loss that occurs when a peptide binds to a target and forms a complex. Thus, the "entropy penalty" is a detrimental effect on binding, and the greater the entropy loss that occurs during binding, the greater the "entropy penalty." Furthermore, flexible unbound molecules lose more entropy when forming complexes than rigidly structured molecules because they lose flexibility when bound into complexes. Meanwhile, the rigidity of unbound molecules also generally limits the number of complexes a molecule can form, thereby increasing specificity. Peptides can bind to targets with affinity similar to that of antibodies, or with nanomolar or picomolar affinities. Close examination of the sequence structure and sequence identity or homology of knot peptides reveals that knot peptides have arisen through convergent evolution in all kinds of animals and plants. In animals, knot peptides are often found in venoms, such as those of spiders and scorpions, and are involved in regulating ion channels. Plant knot proteins can inhibit animal proteolytic enzymes or possess antibacterial activity, suggesting that knot peptides may function in molecular defense systems found in plants.

[0264] The peptides of the present disclosure contain cysteine ​​amino acid residues. In some embodiments, the peptides have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 cysteine ​​amino acid residues. In some embodiments, the peptides have at least 8 cysteine ​​amino acid residues. In other embodiments, the peptides have at least 10 cysteine ​​amino acid residues, at least 12 cysteine ​​amino acid residues, at least 14 cysteine ​​amino acid residues, or at least 16 cysteine ​​amino acid residues.

[0265] The knot peptide may contain a disulfide bridge. The knot peptide may be a peptide in which 5% or more of the residues are cysteines that form intramolecular disulfide bonds. The disulfide-linked peptide may be a drug scaffold. In some embodiments, the disulfide bridge forms a knot. The disulfide bridge may be formed between cysteine ​​residues, for example, between cysteines 1 and 4, 2 and 5, or 3 and 6. In some embodiments, one disulfide bridge passes through a loop formed by two other disulfide bridges, for example, to form a knot. In other embodiments, the disulfide bridge may be formed between any two cysteine ​​residues.

[0266] The present disclosure further includes peptide scaffolds, which can be used, for example, as a starting point for generating additional peptides. In some embodiments, these scaffolds can be derived from various knot peptides (e.g., CDPs or knot peptides). In certain embodiments, CDPs (e.g., knot peptides) feature multiple intramolecular disulfide bridges and are assembled into complex tertiary structures, optionally including beta strands and other secondary structures such as alpha helices. For example, CDPs (e.g., knot peptides) include, in some embodiments, small disulfide-rich proteins characterized by disulfides mediated by a disulfide knot. This knot can be obtained, for example, when one disulfide bridge passes through a macrocyclic polymer formed by two other disulfides and their interconnected backbones. In some embodiments, the knot peptide can include a growth factor cysteine ​​knot or an inhibitory factor cysteine ​​knot. Other possible peptide structures include peptides with two parallel helices connected by two disulfide bridges without a beta sheet (e.g., hefutoxin).

[0267] Some peptides of the present disclosure may contain at least one amino acid residue that is in the L-configuration. Peptides may contain at least one amino acid residue that is in the D-configuration. In some embodiments, peptides are 15-75 amino acid residues in length. In other embodiments, peptides are 11-55 amino acid residues in length. In yet other embodiments, peptides are 11-65 amino acid residues in length. In further embodiments, peptides are at least 20 amino acid residues in length.

[0268] Some CDPs (e.g., knot peptides) can be derived or isolated from a type of protein known to be present in or associated with toxins or venoms. In some cases, peptides can be obtained from toxins or venoms associated with scorpions or spiders. Peptides can be obtained from the venoms and toxins of various genera and species of spiders and scorpions. For example, peptides can be obtained from the venoms and toxins of Leiurus quinquestriatus hebraeus, Buthus occitanus tunetanus, Hottentotta judaicus, Mesobuthus eupeus, Buthus occitanus israelis, Hadrurus gertschi, Androctonus australis, Centruroides noxius, Heterometrus laoticus, Opistophthalmus carinatus, Haplopelma schmidti, Isometrus maculatus, Haplopelma huwenum, Haplopelma The peptide may be derived from the venom or toxin of another suitable genus or species of scorpion or spider, such as Buthus martensii Karsh (scorpion) venom, or from the venom of another suitable genus or species of scorpion or spider, such as Buthus martensii Karsh (scorpion) venom.

[0269] Sequence identity and homology Percent (%) sequence identity or homology is determined by conventional methods. (See, e.g., Altschul et al. (1986), Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff (1992), Proc. Natl. Acad. Sci. USA 89:10915.) Briefly, two amino acid sequences can be aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.). Sequence identity or homology is then calculated as follows: ([total number of identical matches] / [length of the longer sequence and the number of gaps introduced into the longer sequence to align the two sequences])(100).

[0270] Furthermore, several established algorithms are available for aligning two amino acid sequences. For example, the Pearson and Lipman "FASTA" similarity search algorithm may be a suitable protein alignment method for determining the level of sequence identity or homology shared between the amino acid sequence of a peptide disclosed herein and the amino acid sequence of a peptide variant. The FASTA algorithm is described, for example, by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988) and Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA characterizes sequence similarity by first identifying regions shared by a query sequence (e.g., SEQ ID NO: 1) and a test sequence with either the highest density of matches (when the ktup variable is 1) or match pairs (when ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. Next, the 10 regions with the highest density of matches are rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only the residues that contribute the highest score. If there are multiple regions with scores greater than a "cutoff" value (calculated using a predetermined formula based on the sequence length and ktup value), the trimmed initial regions are examined to determine whether these regions can be combined to form a gapped approximate alignment. Finally, the highest-scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, Siam J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. For example, exemplary parameters for FASTA analysis are ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62.These parameters can be introduced into the FASTA program by modifying the scoring matrix file ("SMATRIX") as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

[0271] FASTA can also be used to determine sequence identity or homology of nucleic acid sequences or molecules using the ratios disclosed above. When comparing nucleic acid sequences, the ktup value can range from 1 to 6, preferably 2 to 6, and most preferably 3, with other parameters set as described herein.

[0272] Some examples of common amino acids that are "conservative amino acid substitutions" are exemplified by substitutions between amino acids within the following groups: (1) glycine, alanine, valine, leucine, and isoleucine; (2) phenylalanine, tyrosine, and tryptophan; (3) serine and threonine; (4) aspartic acid and glutamic acid; (5) glutamine and asparagine; and (6) lysine, arginine, and histidine. The BLOSUM62 table is an amino acid substitution matrix representing highly conserved regions of over 500 related protein groups derived from approximately 2,000 local multiple alignments of protein sequence segments (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915 (1992)). Therefore, BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely on chemical properties (as described above), the term "conservative amino acid substitution" preferably refers to a substitution represented by a BLOSUM62 value greater than -1. For example, an amino acid substitution is conservative if it is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2, or 3), and more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

[0273] Amino acid residues within regions or domains that are important for maintaining structural integrity can be determined. Within these regions, specific residues that are tolerant to change and maintain the overall tertiary structure of the molecule can be determined. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity or homology and computer analysis using available software (e.g., Insight II.RTM. Viewer and Homology Modeling Tools; MSI, San Diego, Calif.), secondary structure propensity, binary patterns, complementary packing, and buried polar interactions (Barton, GJ, Current Opin. Struct. Biol. 5:372-6 (1995) and Cordes, MH et al., Current Opin. Struct. Biol. 6:3-10 (1996)). In general, when designing modifications to a molecule or identifying specific fragments, structural determination can typically be achieved by evaluating the activity of the modified molecule.

[0274] Physicochemical properties of peptides In some embodiments, the TfR-binding peptides of the present disclosure can include a wide range of physicochemical properties, such as molecular size and structure, pH, isoelectric point, and overall molecular net charge, which can affect the ability of the peptide to bind to TfR, promote transcytosis, and transport cargo molecules across cellular barriers such as the BBB.

[0275] The peptides of the present disclosure may comprise at least one amino acid residue in the D configuration. In some embodiments, the peptide is about 5 to 100 amino acid residues in length. In some embodiments, the peptide is about 10 to 90 amino acid residues in length. In some embodiments, the peptide is about 15 to 80 amino acid residues in length. In some embodiments, the peptide is about 15 to 75 amino acid residues in length. In some embodiments, the peptide is about 15 to 70 amino acid residues in length. In some embodiments, the peptide is about 20 to 65 amino acid residues in length. In some embodiments, the peptide is about 20 to 60 amino acid residues in length. In some embodiments, the peptide is about 25 to 55 amino acid residues in length. In some embodiments, the peptide is about 25 to 50 amino acid residues in length. In some embodiments, the peptide is about 25 to 40 amino acid residues in length. In some embodiments, the peptide is about 11 to 35 amino acid residues in length. In some embodiments, the peptide is about 10 to 25 amino acid residues in length.

[0276] In some embodiments, the peptide is at least 5 amino acid residues long. In some embodiments, the peptide is at least 10 amino acid residues long. In some embodiments, the peptide is at least 15 amino acid residues long. In some embodiments, the peptide is at least 20 amino acid residues long. In some embodiments, the peptide is at least 25 amino acid residues long. In some embodiments, the peptide is at least 30 amino acid residues long. In some embodiments, the peptide is at least 35 amino acid residues long. In some embodiments, the peptide is at least 40 amino acid residues long. In some embodiments, the peptide is at least 45 amino acid residues long. In some embodiments, the peptide is at least 50 amino acid residues long. In some embodiments, the peptide is at least 55 amino acid residues long. In some embodiments, the peptide is at least 60 amino acid residues long. In some embodiments, the peptide is at least 65 amino acid residues long. In some embodiments, the peptide is at least 70 amino acid residues long. In some embodiments, the peptide is at least 75 amino acid residues long.

[0277] In some embodiments, the amino acid sequence of the peptides described herein is at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 9 at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58 residues, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, or at least 81 amino acid residues.

[0278] In some embodiments of the present disclosure, the three-dimensional or tertiary structure of the peptide is primarily composed of beta sheets and / or alpha helices. In some embodiments, the designed or engineered TfR-binding peptides of the present disclosure are small, compact peptides or polypeptides stabilized by intrachain disulfide bonds (e.g., mediated by cysteines) and a hydrophobic core. In some embodiments, the engineered TfR-binding peptide has a structure comprising a helical bundle with at least one disulfide bridge between each of the alpha helices, thereby stabilizing the peptide. In other embodiments, the engineered TfR-binding peptide comprises a structure with three alpha helices and three intrachain disulfide bonds, one between each of the three alpha helices in the alpha helical bundle.

[0279] At physiological pH, the peptides described herein can have an overall molecular net charge of, for example, −5, −4, −3, −2, −1, 0, +1, +2, +3, +4, or +5. When the net charge is zero, the peptide can be uncharged or zwitterionic. In some embodiments, the peptide contains one or more disulfide bonds and has a net positive charge at physiological pH, which may be +0.5 or less, +1 or less, +1.5 or less, +2 or less, +2.5 or less, +3 or less, +3.5 or less, +4 or less, +4.5 or less, +5 or less, +5.5 or less, +6 or less, +6.5 or less, +7 or less, +7.5 or less, +8 or less, +8.5 or less, +9 or less, or +10 or less. In some embodiments, the peptide has a net negative charge at physiological pH, which may be -0.5 or less, -1 or less, -1.5 or less, -2 or less, -2.5 or less, -3 or less, -3.5 or less, -4 or less, -4.5 or less, -5 or less, -5.5 or less, -6 or less, -6.5 or less, -7 or less, -7.5 or less, -8 or less, -8.5 or less, -9 or less, -9.5, or -10 or less.

[0280] In some embodiments, peptides of the present disclosure may have an isoelectric point (pI) value of 3 to 10. In other embodiments, peptides of the present disclosure may have a pI value of 4.3 to 8.9. In some embodiments, peptides of the present disclosure may have a pI value of 3 to 4. In some embodiments, peptides of the present disclosure may have a pI value of 3 to 5. In some embodiments, peptides of the present disclosure may have a pI value of 3 to 6. In some embodiments, peptides of the present disclosure may have a pI value of 3 to 7. In some embodiments, peptides of the present disclosure may have a pI value of 3 to 8. In some embodiments, peptides of the present disclosure may have a pI value of 3 to 9. In some embodiments, peptides of the present disclosure may have a pI value of 4 to 5. In some embodiments, peptides of the present disclosure may have a pI value of 4 to 6. In some embodiments, peptides of the present disclosure may have a pI value of 4 to 7. In some embodiments, peptides of the present disclosure may have a pI value of 4 to 8. In some embodiments, peptides of the present disclosure may have a pI value of 4 to 9. In some embodiments, peptides of the present disclosure may have a pI value of 4 to 10. In some embodiments, peptides of the present disclosure may have a pI value of 5 to 6. In some embodiments, peptides of the present disclosure may have a pI value of 5 to 7. In some embodiments, peptides of the present disclosure may have a pI value of 5 to 8. In some embodiments, peptides of the present disclosure may have a pI value of 5 to 9. In some embodiments, peptides of the present d...

Claims

[Claim 1] The invention described in this specification.

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