Blood-brain barrier-transporting peptides and related molecules and methods of their use

Abstract

Peptides capable of binding to receptors that mediate receptor-mediated transcytosis (RMT) across the blood-brain barrier (BBB), as well as binding molecules incorporating such peptides, are provided. Conjugates are also provided that include fusion proteins composed of the peptide or binding molecule and a therapeutic or diagnostic agent. In some embodiments, the conjugate can, after parenteral administration, cross the blood-brain barrier and enable the therapeutic or diagnostic agent to function in the central nervous system. Methods for making and using the provided peptides and molecules are also provided.

Description

Technical Field

[0001] This application claims priority based on U.S. Provisional Application No. 63 / 342,622, filed on May 16, 2022, with the title "BLOOD-BRAIN BARRIER TRANSLOCATING PEPTIDES AND RELATED MOLECULES AND METHODS OF USE THEREOF", the entire content of which is incorporated herein by reference.

[0002] Incorporation by reference of the Sequence Listing This application is filed with a Sequence Listing in electronic form. The Sequence Listing is provided as a file titled 166302000240SeqList.xml, created on May 15, 2023, and having a size of 253,440 kilobytes. The information in the electronic form of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] Field The present disclosure provides peptides capable of binding to receptors that mediate receptor-mediated transcytosis (RMT) across the blood-brain barrier (BBB), as well as binding molecules incorporating such peptides. The present disclosure also provides conjugates comprising fusion proteins composed of such peptides or binding molecules and therapeutic or diagnostic agents. In some embodiments, the conjugate can pass through the blood-brain barrier after parenteral administration and enable the therapeutic or diagnostic agent to function in the central nervous system. The present disclosure also provides methods of making and using the peptides and molecules provided.

Background Art

[0004] Background The blood-brain barrier (BBB) serves a neuroprotective function by tightly controlling access to the brain, and as a result, also impedes access of pharmacological agents to brain tissue. Therefore, the use of vectors is required to allow such agents to cross. BBB permeability frequently limits the penetration of drugs or peptides into the central nervous system (CNS) (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999); Bickel, U., Yoshikawa, T. & Pardridge, W. M. Adv. Drug Deliv. Rev. 46: 247-279 (2001)). The brain is shielded from potentially toxic substances by the BBB, which is formed by brain capillary endothelial cells sealed by tight junctions. Furthermore, brain capillaries have few fenestrae and few intracellular vesicles compared to capillaries in other organs (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999)). Except for some specific proteins taken up by receptor-mediated endocytosis, such as transferrin, lactoferrin, and low-density lipoprotein, large hydrophilic molecules hardly cross the BBB (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999); Tsuji, A. & Tamai, I. Adv. Drug Deliv. Rev. 36: 277-290 (1999); Kusuhara, H. & Sugiyama, Y. Drug Discov. Today 6: 150-156 (2001); Dehouck, B. et al. J. Cell. Biol. 138: 877-889 (1997); and Fillebeen, C. et al. J. Biol. Chem. 274: 7011-7017 (1999)). Thus, improved molecules are needed to enable certain agents, such as therapeutic and diagnostic agents, to cross the BBB. Molecules that meet such needs are provided.

PRIOR ART DOCUMENTS

Non-Patent Literature

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Summary of the Invention

Means for Solving the Problems

[0006] Abstract Provided herein are: i) a peptide that binds to a blood-brain barrier transcytosis receptor (BBB-R) selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), leptin receptor (ObR), low density lipoprotein receptor-related protein 1 (LRP-1), and receptor for advanced glycation end products (RAGE), the peptide having an amino acid sequence of 2 to 50 amino acid residues; and ii) a modified cyclotide comprising a cyclotide scaffold containing the peptide of i), having structure (I):

Chemical Formula

[0007] A modified cyclotide comprising a peptide that binds to a blood-brain barrier transcytosis (BBB-R) receptor, wherein the peptide is inserted into at least one loop of the cyclotide scaffold shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or one or more amino acids in at least one loop are replaced, the peptide is about 2 to 50 amino acid residues, and the BBB-R is selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), leptin receptor (ObR), low density lipoprotein receptor-related protein 1 (LRP-1), and the modified cyclotide is provided herein.

[0008] In some parts of any of the provided embodiments, the cyclotide scaffold is shown in SEQ ID NO: 2. In some parts of any of the provided embodiments, at least one loop is loop 1, loop 5, or loop 6, or a combination thereof. In some parts of any of the provided embodiments, at least one loop is loop 1. In some parts of any of the provided embodiments, the peptide is inserted into only one loop of the cyclotide scaffold and replaces the amino acids within that loop. In some parts of any of the provided embodiments, the only one loop is loop 1. In some parts of any of the provided embodiments, the cyclotide scaffold is as shown in SEQ ID NO: 2, and the peptide replaces the loop 1 amino acids between cysteine 4 and cysteine 11 of SEQ ID NO: 2. In some parts of any of the provided embodiments, all amino acids in at least one loop are replaced by the peptide.

[0009] A modified cyclotide comprising a peptide inserted into Loop 1 that replaces all amino acids between cysteine 4 and cysteine 11 of array number 2, wherein the peptide is 2 to 50 amino acid residues and binds to a blood-brain barrier transcytosis receptor (BBB-R) selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), leptin receptor (ObR), and low density lipoprotein receptor-related protein 1 (LRP-1). Modified cyclotides are provided herein.

[0010] In some embodiments provided, the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 amino acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids. In some embodiments provided, the peptide is 2 to 30 amino acids, for example, 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids. In some embodiments provided, the peptide is 14 to 20 amino acids. In some embodiments provided, the peptide is 10 amino acids. In some embodiments provided, the peptide is 12 amino acids.

[0011] In some of the provided embodiments, BBB-R is expressed on brain endothelial cells. In some of the provided embodiments, the peptide has blood-brain barrier translocation activity. In some of the provided embodiments, BBB-R is a transferrin receptor.

[0012] In some of the provided embodiments, the peptide comprises a sequence shown in any one of SEQ ID NOs: 26-34 and 49-54. In some embodiments, the peptide is shown in any one of SEQ ID NOs: 26-34 and 49-54. In some of the provided embodiments, the peptide has a consensus motif (SEQ ID NO: 177) shown as xxxxxHxxSWGx. In some of the provided embodiments, the modified cyclotide comprises a sequence shown in any one of SEQ ID NOs: 72-80 and 95-100. In some embodiments, the modified cyclotide is shown in any one of SEQ ID NOs: 72-80 and 95-100. In some of the provided embodiments, the peptide comprises the sequence shown in SEQ ID NO: 26. In some embodiments, the peptide is shown in SEQ ID NO: 26. In some of the provided embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 72. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 72. In some embodiments, the peptide comprises the sequence shown in SEQ ID NO: 49. In some embodiments, the peptide is shown in SEQ ID NO: 49. In some embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 95. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 95.

[0013] In some embodiments of any of the provided embodiments, the peptide comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 26-34 and 49-54. In some embodiments, the amino acid substitution(s) is a substitution of one amino acid with another amino acid selected from histidine or alanine. In some embodiments, the peptide comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in SEQ ID NO: 26. In some embodiments of any of the provided embodiments, the peptide comprises the sequence shown in any one of SEQ ID NOs: 55 and 117-128. In some embodiments of any of the provided embodiments, the peptide is shown in any one of SEQ ID NOs: 55 and 117-128. In some embodiments, the modified cyclotide comprises the sequence shown in any one of SEQ ID NOs: 101 and 105-116. In some embodiments, the modified cyclotide is shown in any one of SEQ ID NOs: 101 and 105-116. In some embodiments of any of the provided embodiments, the peptide comprises the sequence shown in SEQ ID NO: 55. In some embodiments, the peptide is shown in SEQ ID NO: 55. In some embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 101. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 101.

[0014] In some embodiments of any of the provided embodiments, the BBB-R is the leptin receptor. In some embodiments of any of the provided embodiments, the peptide comprises the sequence shown in any one of SEQ ID NOs: 10-23. In some embodiments of any of the provided embodiments, the peptide is shown in any one of SEQ ID NOs: 10-23. In some embodiments of any of the provided embodiments, the modified cyclotide comprises the sequence shown in any one of SEQ ID NOs: 56-69. In some embodiments, the modified cyclotide is shown in any one of SEQ ID NOs: 56-69. In some embodiments of any of the provided embodiments, the peptide comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 10-23. In some embodiments, the amino acid substitution(s) is a substitution of one amino acid with another amino acid selected from histidine or alanine.

[0015] In some of the provided embodiments, the BBB-R is ErbB3. In some of the provided embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 24 or 25. In some of the provided embodiments, the peptide is set forth in SEQ ID NO: 24 or 25. In some of the provided embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO: 70 or 71. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 70 or 71. In some of the provided embodiments, the peptide comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence set forth in SEQ ID NO: 24 or 25. In some embodiments, the amino acid substitution(s) is a substitution of one amino acid for another amino acid selected from histidine or alanine.

[0016] In some of the provided embodiments, the BBB-R is the insulin-like growth factor type 1 receptor (IGFR). In some of the provided embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 35 or 36. In some of the provided embodiments, the peptide is set forth in SEQ ID NO: 35 or 36. In some of the provided embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 36. In some embodiments, the peptide is set forth in SEQ ID NO: 36. In some of the provided embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO: 81 or 82. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 81 or 82. In some of the provided embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO: 82. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 82. In some of the provided embodiments, the peptide comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence set forth in SEQ ID NO: 35 or 36. In some embodiments, the amino acid substitution(s) is a substitution of one amino acid for another amino acid selected from histidine or alanine.

[0017] In some of the provided embodiments, the BBB-R is RAGE. In some of the provided embodiments, the peptide comprises the sequence shown in SEQ ID NO: 37 or 38. In some of the provided embodiments, the peptide is shown in SEQ ID NO: 37 or 38. In some of the provided embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 83 or 84. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 83 or 84. In some of the provided embodiments, the peptide comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in SEQ ID NO: 37 or 38. In some embodiments, the amino acid substitution(s) is / are substitution(s) to histidine or alanine.

[0018] In some of the provided embodiments, the BBB-R is lipoprotein receptor-related protein 1 (LRP-1). In some of the provided embodiments, the peptide comprises a sequence shown in any one of SEQ ID NOs: 39-48. In some of the provided embodiments, the peptide is shown in any one of SEQ ID NOs: 39-48. In some embodiments, the peptide comprises the sequence shown in SEQ ID NO: 39. In some embodiments, the peptide is shown in SEQ ID NO: 39. In some embodiments, the peptide comprises the sequence shown in SEQ ID NO: 43. In some embodiments, the peptide is shown in SEQ ID NO: 43. In some embodiments, the peptide comprises the sequence shown in SEQ ID NO: 47. In some embodiments, the peptide is shown in SEQ ID NO: 47. In some of the provided embodiments, the modified cyclotide comprises a sequence shown in any one of SEQ ID NOs: 85-94. In some embodiments, the modified cyclotide is shown in any one of SEQ ID NOs: 85-94. In some embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 85. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 85. In some embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 89. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 89. In some embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 93. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 93. In some of the provided embodiments, the peptide comprises a sequence having one, two, three or four amino acid substitutions compared to a sequence shown in any one of SEQ ID NOs: 39-48. In some embodiments, the amino acid substitution(s) is / are the substitution of an amino acid with another amino acid selected from histidine or alanine.

[0019] A peptide comprising an amino acid sequence shown in any one of SEQ ID NOs: 10-55 or 117-128, wherein the peptide is 6 to 50 amino acids in length and binds to a receptor (BBB-R) involved in blood-brain barrier transcytosis, is provided herein.

[0020] In some of the provided embodiments, the peptide has blood-brain barrier translocation activity. In some of the provided embodiments, the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 amino acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids. In some of the provided embodiments, the peptide is 2 to 30 amino acids, for example, 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids. In some of the provided embodiments, the peptide is 14 to 20 amino acids. In some of the provided embodiments, the peptide is 10 amino acids. In some of the provided embodiments, the peptide is 12 amino acids.

[0021] Peptides consisting of the sequences shown in any one of SEQ ID NOs: 10 to 55 or 117 to 128 are provided herein. In some of the provided embodiments, the peptide binds to a receptor involved in blood-brain barrier transcytosis.

[0022] Peptides shown in any of the sequences of SEQ ID NOs: 26-34, 49-55, and 117-128, which bind to the transferrin receptor, are provided herein. In some of any of the provided embodiments, the peptide is shown in SEQ ID NO: 26. In some of any of the provided embodiments, the peptide is shown in SEQ ID NO: 49. In some of any of the provided embodiments, the peptide is shown in SEQ ID NO: 55.

[0023] Peptides shown in any of the sequences of SEQ ID NOs: 10-23, which bind to the leptin receptor, are provided herein. Peptides shown in any of the sequences of SEQ ID NO: 24 or 25, which bind to ErbR3, are provided herein.

[0024] Peptides shown in any of the sequences of SEQ ID NO: 35 or 36, which bind to IGFR, are provided herein. In some of any of the provided embodiments, the peptide is shown in SEQ ID NO: 35. In some of any of the provided embodiments, the peptide is shown in SEQ ID NO: 36.

[0025] Peptides shown in the sequence of SEQ ID NO: 37 or 38, which bind to RAGE, are provided herein.

[0026] Peptides shown in any of the sequences of SEQ ID NOs: 39-48, which bind to lipoprotein receptor-related protein 1 (LRP-1), are provided herein. In some of any of the provided embodiments, the peptide is shown in SEQ ID NO: 39. In some of any of the provided embodiments, the peptide is shown in SEQ ID NO: 43. In some of any of the provided embodiments, the peptide is shown in SEQ ID NO: 47.

[0027] In some of any of the provided embodiments, the peptide is synthetic. In some of any of the provided embodiments, the peptide is isolated.

[0028] Binding molecules are provided herein that include either a binding scaffold and a provided peptide. In some of the provided embodiments, the binding scaffold is a cyclotide. In some of the provided embodiments, the peptide is inserted into a loop of the cyclotide backbone or replaces one or more amino acids of a loop of the cyclotide backbone. In some embodiments, at least one loop is Loop 1. In some of the provided embodiments, the binding scaffold is shown in any one of SEQ ID NOs: 1-3. In some of the provided embodiments, the binding scaffold is shown in SEQ ID NO: 2.

[0029] Nucleic acid molecules encoding any of the provided modified cyclotides or any of the provided binding molecules are provided herein.

[0030] Vectors are provided herein that include any of the provided nucleic acids. In some of the provided embodiments, the vector is an expression vector.

[0031] Host cells are provided herein that include any of the provided nucleic acid molecules or any of the provided vectors.

[0032] Methods for producing a modified cyclotide or a binding molecule, comprising introducing the nucleic acid according to claim 87 or the vector according to claim 88 or claim 89 into a host cell, and culturing the host cell under conditions in which the protein is expressed in the cell, are provided herein. In some embodiments, the method further comprises purifying the protein from the cell.

[0033] Purified binding molecules or modified cyclotides produced by any of the provided methods are provided herein.

[0034] Provided herein are conjugates comprising any of the provided modified cyclotides, or any of the provided binding molecules, and an agent having biological activity. In some of the provided embodiments, the agent having biological activity is a small molecule, peptide or protein. In some of the provided embodiments, the agent having biological activity is a diagnostic agent or a therapeutic agent. In some of the provided embodiments, any of the provided conjugates is a fusion protein comprising a modified cyclotide operably linked to an agent having biological activity that is a protein or peptide.

[0035] Provided herein are fusion proteins comprising any of the provided modified cyclotides or any of the provided binding molecules, and an agent having biological activity that is a protein or peptide.

[0036] In some of the provided embodiments, the agent having biological activity is an antibody. In some embodiments, the antibody is against an antigen selected from the group consisting of human epidermal growth factor receptor 2 (HER2), beta-secretase 1 (BACE1), amyloid beta (A beta), epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), P75 neurotrophin receptor (P75NTR), caspase 6 and TNF-alpha.

[0037] In some of the provided embodiments, the antibody is trastuzumab, adalimumab or aducanumab. In some of the provided embodiments, the agent having biological activity is a growth factor or a hormone. In some of the provided embodiments, the agent having biological activity is a growth factor, and the growth factor is nerve growth factor (NGF) or granulocyte colony-stimulating factor (GCSF).

[0038] In some of the provided embodiments, the agent having biological activity is an enzyme. In some of the provided embodiments, the enzyme is a ceramide-degrading enzyme, lipase, hydrolase-type enzyme or sulfatase. In some of the provided embodiments, the enzyme is a ceramide-degrading enzyme, and the ceramide-degrading enzyme is glucocerebrosidase, galactocerebrosidase or alpha-galactosidase. In some embodiments, the enzyme is glucocerebrosidase having an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 144 or SEQ ID NO: 145. In some embodiments, the glucocerebrosidase is a variant containing 1 to 5 amino acid substitutions compared to the sequence shown in SEQ ID NO: 144 or SEQ ID NO: 145. Non-limiting mutations include any of those described herein. In some embodiments, the enzyme is glucocerebrosidase having the amino acid sequence shown in SEQ ID NO: 144. In some embodiments, the enzyme is glucocerebrosidase having the amino acid sequence shown in SEQ ID NO: 145. In some of the provided embodiments, the enzyme is a lipase or a hydrolase-type enzyme, and the enzyme is sphingomyelinase, cell liponase or alpha-glucosidase.

[0039] In some of the provided embodiments, the conjugate or fusion protein comprises a single modified cyclotide. In some of the provided embodiments, the conjugate or fusion protein comprises two, three, or four modified cyclotides. In some of the provided embodiments, each modified cyclotide is the same. In some of the provided embodiments, each modified cyclotide is different.

[0040] In some of the provided embodiments, the conjugate or fusion protein is monovalent with respect to binding to the BBB-R.

[0041] In some of the provided embodiments, the conjugate is divalent with respect to binding to the BBB-R. In some of the provided embodiments, the conjugate or fusion protein comprises at least two different modified cyclotides that bind to different BBB-Rs.

[0042] In some of the provided embodiments, the conjugate or fusion protein is bispecific and binds to two different BBB-Rs. In some embodiments, the conjugate is bispecific and binds to two different BBB-Rs and comprises at least two different modified cyclotides that each bind to a different BBB-R.

[0043] In some embodiments of the provided embodiments, the modified cyclotide is linked to a biologically active agent via a linker. In some embodiments, the linker is at least 10 amino acids in length. In some embodiments, the linker is at least 15 amino acids in length. In some embodiments, the linker is 10 to 20 amino acids in length. In some embodiments of the provided embodiments, the linker is a flexible peptide linker. In some embodiments, the peptide linker comprises the sequence GGGGS (SEQ ID NO: 148), (GGGGS)2 (SEQ ID NO: 154), or (GGGGS)3 (SEQ ID NO: 154). In some embodiments of the provided embodiments, the linker is shown in SEQ ID NO: 104. In some embodiments of the provided embodiments, the linker is a cleavable linker that includes an endosome-specific protease cleavage site. In some embodiments of the provided embodiments, the endosome-specific protease cleavage site is a cathepsin cleavage site. In some embodiments of the provided embodiments, the cathepsin cleavage site is a cathepsin B cleavage site. In some embodiments of the provided embodiments, the linker comprises the sequence shown in SEQ ID NO: 133.

[0044] Also provided herein is a pharmaceutical composition comprising any of the provided conjugates or fusion proteins and a pharmaceutical carrier.

[0045] Also provided herein is a method for transporting a biologically active agent across the blood-brain barrier of an individual, the method comprising administering to the individual in need thereof any of the provided conjugates or fusion proteins or any of the provided pharmaceutical compositions. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0046] In some of the provided embodiments, an individual such as a mammal (e.g., a human) has a neurological disorder. In some of the provided embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, stroke, brain tumor, and brain metastasis. In some of the provided embodiments, the neurological disorder is a congenital disorder selected from the group consisting of Austin disease, Canavan disease, Gaucher disease, Hunter syndrome, Hurler-Scheie syndrome, Jansky-Bielschowsky disease, Krabbe disease, LCAT deficiency, Lowe syndrome, Maroteaux-Lamy syndrome, Morquio syndrome type A, Morquio syndrome type B, Sanfilippo syndrome type A, Sanfilippo syndrome type B, Sanfilippo syndrome type C, Sanfilippo syndrome type D, spinal muscular atrophy, Tay-Sachs disease, and Walker-Warburg syndrome.

[0047] A method for treating a patient having a neurological disorder, the method comprising administering to the patient any of the provided conjugates or any of the provided pharmaceutical compositions is provided herein. A method for diagnosing a neurological disorder in a patient in need thereof, the method comprising administering to the patient any of the provided conjugates or a fusion protein or any of the provided pharmaceutical compositions, wherein the conjugate comprises a radiolabel is provided herein. In some of the provided embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, stroke, brain tumor, and brain metastasis. In some of the provided embodiments, the neurological disorder is a congenital disorder selected from the group consisting of Austin disease, Canavan disease, Gaucher disease, Hunter syndrome, Hurler-Scheie syndrome, Jansky-Bielschowsky disease, Krabbe disease, LCAT deficiency, Lowe syndrome, Maroteaux-Lamy syndrome, Morquio syndrome type A, Morquio syndrome type B, Sanfilippo syndrome type A, Sanfilippo syndrome type B, Sanfilippo syndrome type C, Sanfilippo syndrome type D, spinal muscular atrophy, Tay-Sachs disease, and Walker-Warburg syndrome.

[0048] Also provided is any of the compositions provided herein for use in the treatment of neurological disorders. Also provided is the use of any of the pharmaceutical compositions provided in the manufacture of a medicament for use in the treatment of neurological disorders. Also provided is any of the compositions provided herein for use in the diagnosis of neurological disorders. Also provided herein is the use of any of the pharmaceutical compositions provided in the manufacture of a medicament for use in the diagnosis of neurological conditions. In some of the provided embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, stroke, brain tumor, and brain metastases. In some of the provided embodiments, the neurological disorder is a congenital disorder selected from the group consisting of Austin disease, Canavan disease, Gaucher disease, Hunter syndrome, Hurler-Scheie syndrome, Jansky-Bielschowsky disease, Krabbe disease, LCAT deficiency, Lowe syndrome, Maroteaux-Lamy syndrome, Morquio syndrome type A, Morquio syndrome type B, Sanfilippo syndrome type A, Sanfilippo syndrome type B, Sanfilippo syndrome type C, Sanfilippo syndrome type D, spinal muscular atrophy, Tay-Sachs disease, and Walker-Warburg syndrome.

Brief Description of the Drawings

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[0060] Detailed Description For example, provided herein are peptides that exhibit blood-brain barrier translocation activity (including the ability to facilitate crossing of the BBB via transcytosis) by binding to receptors involved in blood-brain barrier transcytosis. The provided peptides can be inserted into a protein scaffold to provide a larger binding molecular backbone or framework, thereby, in some embodiments, improving the stability or half-life of the peptide. In some embodiments, the scaffold is a cyclotide backbone for providing a modified cyclotide with improved or enhanced binding to receptors involved in blood-brain barrier translocation activity and / or blood-brain barrier transcytosis. The provided modified cyclotide molecules do not occur naturally and exhibit certain advantages with respect to crossing the blood-brain barrier. The provided modified scaffolds, e.g., the provided modified cyclotides, into which the provided peptides are inserted, can be attached to or linked with other molecules such as polypeptides, proteins and small molecules including therapeutic or diagnostic agents and used as carriers for delivering such other molecules across the blood-brain barrier. To treat neurological conditions, the provided modified cyclotides can be used to deliver therapeutic and other biological agents across the BBB.

[0061] Problems related to the delivery of certain therapeutic or other biological agents are due to the restricted passive transfer of substances from capillaries into the brain, making it difficult to deliver parenterally administered biological agents, such as therapeutic and diagnostic agents, to the central nervous system (CNS). Unlike capillaries in other tissues such as muscle, the capillaries that supply most of the brain tissue are different in that the endothelial cells forming the endothelium are interconnected by tight junctions between cells. This system that restricts the exchange of substances between the blood and the brain interstitial fluid through the endothelium of the brain capillaries is called the blood-brain barrier or BBB. Thus, since therapeutic or other biological agents do not always pass through the BBB, problems arise due to the BBB with respect to the access of therapeutic or other biological agents to the CNS. For example, when the BBB is intact, only approximately 0.2% of the monoclonal antibody dose administered intravenously reaches the brain.

[0062] To enable the passage of biological agents through the BBB, various methods have been attempted. Such attempts include, for example, encapsulating the agent in liposomes, directly delivering the biological agent to the brain, or conjugating the agent with an antibody molecule that binds to a membrane protein on the endothelial cells of the brain capillaries. There is no doubt that these methods can increase the transcytosis efficiency, but the existing methods are invasive and / or complex. For example, antibodies can confer affinity for receptors involved in blood-brain barrier transcytosis, but their large size may reduce the manufacturability of therapeutic fusion proteins due to problems associated with, for example, expression, purification, or stability.

[0063] The provided embodiments address these problems. The provided embodiments relate to novel peptides that exhibit binding to receptors involved in blood-brain barrier transcytosis. These peptides can be incorporated into a binding molecular scaffold to confer stability to the molecule and the ability to bind to receptors that mediate RMT across the blood-brain barrier (BBB). In some embodiments, the binding molecular scaffold is a cyclotide, which is a cysteine knot protein. The ability to incorporate peptides into a stable scaffold such as a cyclotide results in a highly developable platform that utilizes small, highly stable peptides that are well-expressed as fusion proteins. Furthermore, due to the smaller size of the scaffold platform, it is also possible to create multi-domain molecules using the provided embodiments that have the ability to target more than one transcytosis pathway.

[0064] In particular, the provided binding molecular scaffolds include cyclotides, which are members of a family of naturally occurring cysteine knot microproteins found in various plant species. Cysteine knot microproteins (cyclotides) are small peptides, generally consisting of about 30 to 40 amino acids, and can be found naturally in cyclic or linear forms, with the cyclic form having no free N-terminal or C-terminal amino or carboxyl termini. These cyclotides have a defined structure based on three intramolecular disulfide bonds and a small triple-stranded β-sheet (Craik et al., 2001, Toxicon 39, 43-60). Cyclic proteins exhibit conserved cysteine residues that define a structure herein referred to as the "cysteine knot". This family includes naturally occurring cyclic molecules and their linear derivatives, as well as linear molecules that have undergone cyclization. These molecules are useful as molecular framework structures with enhanced stability compared to less structured peptides (Colgrave and Craik, 2004, Biochemistry 43, 5965-5975). However, these molecules cannot cross the blood-brain barrier to any extent on their own, cannot be used as neurotherapeutic agents on their own, and cannot function as carriers for therapeutic peptide or protein agents.

[0065] The main features of cyclotides are the remarkable stability due to their small size, which enables easy access to chemical synthesis, and the excellent tolerance to sequence variations. Thus, cyclotides seem to be an attractive lead or scaffold for peptide drug design. Cyclotide scaffolds have been found in almost 30 different protein families, among which the conotoxin, spider venom, squash inhibitor, agouti-related protein, and plant cyclotide families are the most abundant. Cyclotides derived from plants of the Rubiaceae and Violaceae families are most often found as head-to-tail cyclic peptides. However, within the cyclotide squash inhibitor family, both cyclic and linear cyclotides have been identified from Momordica cochinchinensis: cyclic trypsin inhibitors (MCoTI)-I and (MCoTI)-II and their linear counterparts MCoTI-III. It is now clear that both cyclic and linear variants can exist in different cyclotide families, but the impact of cyclization is not fully understood. Cyclic peptides are expected to show improved stability, better resistance to proteases, and reduced flexibility compared to their linear counterparts, leading to enhanced biological activity. However, linear cyclotides have the advantage of being more easily linked to other peptides or proteins.

[0066] In some embodiments, the cyclotide sequence is characterized as having a cystine knot backbone moiety, the cystine knot backbone having structure (I):

Chemical formula

[0067] In some embodiments, cyclotides also exhibit the characteristic of having a low immunogenic risk for the molecule in addition to the stability of the molecule. Cyclotides are proteins that originate from non-human sequences and are heavily disulfide-bonded. Evidence has shown that non-human disulfide-bonded peptides do not exhibit detectable immunogenicity. For example, ziconitide is a disulfide-bonded non-human peptide similar to cyclotides that has been approved by the FDA, and in preclinical mouse and rat immunogenicity studies, no anti-drug antibodies (ADA) were detectable even after repeated dosing (Skov M. et al. Int. J. Toxicology, 2007 26: 411-421).

[0068] Since cyclotides have not been proven to easily cross the BBB in their native forms, it is considered desirable to combine the structural stability of the cyclotide scaffold and the ability to cross the blood-brain barrier, and link other biological activities to the cyclotide molecule. Therefore, it is considered desirable to identify cyclotides suitable for maintaining the structure and blood-brain transfer ability as either linear or cyclic molecules. It is further considered desirable that such cyclotides can be used to increase the ability to improve the in vivo activity and bioavailability of biological therapeutics.

[0069] The provided molecules, including the modified cyclotides, meet these needs. In some embodiments, the provided modified cyclotide molecules are useful as carrier microprotein peptides capable of crossing the BBB and can be fused with a desired biological agent, such as a therapeutic molecule, for delivery to the brain.

[0070] The embodiments provided herein relate to a cyclotide molecular framework for generating a modified cyclotide into which a peptide sequence that confers enhanced BBB translocation activity compared to the parent cyclotide is inserted. In some embodiments, the modified cyclotide is composed of a cystine knot backbone having sufficient disulfide bonds or their chemical equivalents to confer a topology having a knot in the three-dimensional structure of the cystine knot backbone, and compared to the naturally occurring cyclotide amino acid sequence, for example, at least one exposed amino acid residue on one or more beta-turns and / or within one or more loops has been inserted or substituted (replaced) by a receptor-binding peptide. In some embodiments, the modified cyclotide has enhanced translocation behavior compared to the unmodified parent cyclotide. In some embodiments, the modified cyclotide has the desired properties of high enzymatic stability and translocation, and thus, blood-brain barrier entry of the modified cyclotide is feasible.

[0071] In some embodiments, the modified cyclotide sequences provided herein can be defined as having a cystine knot backbone portion and a peptide that is a blood-brain barrier translocation portion, and the modified cyclotide comprises: i) a peptide having an amino acid sequence of about 2 to 50 amino acid residues, which is a peptide having blood-brain barrier translocation activity; and ii) a cystine knot backbone grafted onto the peptide of item i), having structure (I):

Chemical formula

[0072] In some embodiments, the provided modified cyclotide sequence can be either linear or cyclic.

[0073] In some embodiments, the cysteine knot backbone is a cyclotide of the Momordicae species including the cucurbitacin protease inhibitor family. Exemplary cyclotides of the Momordica (Momoridicase) species include those shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the peptide is inserted into at least one loop of the cyclotide scaffold shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or replaces one or more amino acids of at least one loop.

[0074] Accordingly, the cyclotides provided herein are modified cyclotides as compared to natural or wild-type unmodified cyclotides, and the modified cyclotides have one or more amino acid sequences, such as peptides of 2 to 50 amino acids, inserted into one or more loops, or one or more amino acid sequences of one or more loops are replaced by the peptides. In the provided embodiments, one or more amino acids, such as a peptide sequence of 2 to 50 amino acids, inserted into or incorporated by substitution into the loops of the unmodified or wild-type cyclotide can bind to receptors involved in transcytosis across the blood-brain barrier and / or are blood-brain barrier translocation moieties that mediate the blood-brain barrier translocation or permeation of the cyclotide. In certain embodiments, the peptide binds to the transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), leptin receptor (ObR), low density lipoprotein receptor-related protein 1 (LRP-1), or receptor for advanced glycation end products (RAGE). In aspects of the provided embodiments, a sufficient amino acid structure is incorporated into the modified cyclotides of the invention to provide high enzymatic stability and good translocation or permeation behavior. In some embodiments, the provided modified cyclotides can be used as novel carrier cyclotides that facilitate or mediate the translocation of an attached polypeptide, such as a therapeutic or diagnostic agent.

[0075] In some embodiments, the peptide-modified cyclotide can be conjugated to another agent or polypeptide, or, in certain embodiments, one or more modified cyclotides can be incorporated into another polypeptide to form a synthetic chimeric (or fusion) polypeptide / protein. Suitably, the peptide of the modified cyclotide can be included within the loop region of the scaffold polypeptide. The resulting modified scaffold itself can then be conjugated to or incorporated into a larger molecule. Suitably, the cyclotide into which the receptor-binding peptide provided herein is inserted can be enriched in cysteine residues. In certain embodiments, the provided modified cyclotide exhibits the ability to cross the blood-brain barrier, suitably allowing the brain to access the modified cyclotide or any molecule contained therein or conjugated thereto.

[0076] Using the provided binding molecules such as modified cyclotides, an agent having biological activity, such as a peptide, polypeptide, protein or small molecule, can be delivered across the blood-brain barrier. In some embodiments, the agent having biological activity is a therapeutic agent, such as a therapeutic agent having agonist or antagonist activity. In some embodiments, the agent having biological activity is a diagnostic agent.

[0077] In some embodiments, the provided modified cyclotide, such as a linear or cyclic cyclotide, is operably linked to the N-terminus or C-terminus of an agent having biological activity, such as a peptide or protein moiety. In some embodiments, the linkage can be direct or indirect via a peptide linker. In some embodiments, the linkage results in a conjugate of the modified cyclotide and the agent having biological activity, such as a fusion protein. Also provided herein are conjugates, such as fusion proteins, comprising the provided modified cyclotide operably linked to the N-terminus or C-terminus of an agent having biological activity, such as a peptide or protein moiety.

[0078] In some embodiments, the biologically active agent is a therapeutic agent, for example, an agent that is an agonist or antagonist. The ligation or fusion of the biologically active agent with the provided modified cyclotide enables the translocation and entry of the biologically active agent, such as a peptide or protein moiety, across the BBB. In the provided embodiments, such ligation enables the translocation and entry across the BBB of a pharmacologically meaningful dose of the biologically active agent, such as a peptide or protein moiety.

[0079] In some embodiments, more than one receptor-binding molecule is incorporated, thereby providing herein bispecific molecules that target more than one transcytosis pathway. For example, in some embodiments, at least two different modified cyclotides can be linked to a biologically active agent to yield a multi-domain protein that can further improve the transcytosis ability of the linked biological agent across the BBB. In some embodiments, one, two, three, or four different modified cyclotides, each modified with a different receptor-binding peptide, can be linked or conjugated to a biological agent to enable the involvement of one or more BBB transporter pathways.

[0080] All publications, including patent documents, scientific papers, and databases, referred to in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were incorporated by reference separately. If the definitions set forth herein conflict with or are otherwise inconsistent with the definitions set forth in patents, applications incorporated herein by reference, published applications, and other publications, the definitions set forth herein shall control over the definitions incorporated herein by reference.

[0081] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0082] I. Receptor-Binding Peptides and Modified Cyclotides Peptides 2 to 50 amino acids in length that bind to receptors involved in blood-brain barrier transcytosis are provided herein. In some embodiments, peptides 6 to 50 amino acids in length that bind to receptors involved in blood-brain barrier transcytosis are provided herein. In some aspects, the provided peptides exhibit blood-brain barrier translocation activity. Also provided herein are binding molecules in which any of the provided peptides are incorporated into a binding molecule scaffold. In some embodiments, the binding molecule scaffold is a small cysteine knot protein. In some embodiments, the cysteine knot protein is a cyclotide. Accordingly, modified cyclotides are also provided herein as compared to the parent or native cyclotide backbone (also referred to as the cysteine knot backbone), where the provided modified cyclotides exhibit an improvement or enhancement in the ability to bind to receptors involved in transcytosis across the blood-brain barrier (BBB; hereinafter “BBB transcytosis receptor”), and / or exhibit an improvement or enhancement in blood-brain barrier translocation characteristics.

[0083] A. Receptor-Binding Peptides and Selection Methods Isolated or synthetic peptides that bind to the BBB transcytosis receptor (also referred to herein as BBB-R) are provided herein. In some embodiments, the peptide is inserted into or can be inserted into the backbone of a cysteine knot scaffold, such as a cyclotide, a binding molecule scaffold. Exemplary modified cyclotides containing the receptor-binding peptide are described in Subsection I.B.

[0084] In some embodiments, BBB-R is expressed on brain endothelial cells. In some embodiments, BBB-R is selected from the transferrin receptor (TrfR); lactoferrin receptor (LtfR); leptin receptor (LEP-R, also known as OB-R); receptor tyrosine-protein kinase (ErbB3), insulin receptor such as insulin receptor A (IRA), IGF-1 receptor (IGF-1R), IGF-II receptor (IGF-IIR), RXFP1, RXFP2, RXFP3 and RXFP4; low density lipoprotein receptor-related protein 1 (LRP-1), receptor for advanced glycation end products (RAGE); heparin-binding EGF-like growth factor receptor (HB EGFR); intercellular adhesion molecule 1 (ICAM1), intercellular adhesion molecule 2 (ICAM2) or neural cell adhesion molecule (NCAM); or any other receptor having receptor-mediated transcytosis activity in brain endothelial cells.

[0085] In some embodiments, BBB-R is a mammalian receptor. In some embodiments, BBB-R is a mouse BBB-R. In some embodiments, BBB-R is a human BBB-R.

[0086] In some embodiments, BBB-R is selected from the human transferrin receptor (human TrfR); human lactoferrin receptor (human LtFR); human leptin receptor (human Ob-R); human ErbB3 receptor, human insulin receptor such as insulin receptor A (IRA), IGF-1 receptor (IGF-1R), IGF-II receptor (IGF-IIR), RXFP1, RXFP2, RXFP3 and RXFP4; human low density lipoprotein receptor-related protein 1 (human LRP-1); human receptor for advanced glycation end products (human RAGE); human heparin-binding EGF-like growth factor receptor (human HB EGFR); human intercellular adhesion molecule 1 (human ICAM1), intercellular adhesion molecule 2 (human ICAM2) or neural cell adhesion molecule (human NCAM).

[0087] In some embodiments, BBB-R is Erb-B2 receptor tyrosine kinase 3 (ErbB3), such as human ErbB3. In some embodiments, BBB-R is TrfR, such as human TrfR. In some embodiments, BBB-R is the leptin receptor (ObR), such as human ObR. In some embodiments, BBB-R is the insulin-like growth factor type 1 receptor (IGFR), such as human IgFR. In some embodiments, BBB-R is low density lipoprotein related protein 1 (LRP-1), such as human LRP-1. In some embodiments, BBB-R is the receptor for advanced glycation end products (RAGE), such as human RAGE.

[0088] In some embodiments, the peptide is a synthetic peptide. In some embodiments, the peptide is an isolated peptide.

[0089] In some embodiments, the peptide has an amino acid sequence that is about 2 to 50 amino acid residues in length. In some embodiments, the peptide is 2 to 50 amino acid residues. In some embodiments, the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 amino acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids. In some embodiments, the peptide is 2 to 30 amino acids, for example, 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids.

[0090] In some embodiments, the peptide is 10 to 25 amino acids. In some embodiments, the peptide is 10 amino acids. In some embodiments, the peptide is 11 amino acids. In some embodiments, the peptide is 12 amino acids. In some embodiments, the peptide is 13 amino acids. In some embodiments, the peptide is 14 amino acids. In some embodiments, the peptide is 15 amino acids. In some embodiments, the peptide is 16 amino acids. In some embodiments, the peptide is 17 amino acids. In some embodiments, the peptide is 18 amino acids. In some embodiments, the peptide is 19 amino acids. In some embodiments, the peptide is 20 amino acids. In some embodiments, the peptide is 21 amino acids. In some embodiments, the peptide is 22 amino acids. In some embodiments, the peptide is 23 amino acids. In some embodiments, the peptide is 24 amino acids. In some embodiments, the peptide is 25 amino acids.

[0091] The BBB-R antigen used for peptide production or screening may be in a soluble form or a portion thereof (e.g., extracellular domain) containing the desired epitope. The BBB-R target molecule may be isolated from a natural source or prepared by recombinant methods by procedures known in the art. The purified target molecule can be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyalkyl methacrylate gels, polyacrylic copolymers and polymethacrylic copolymers, nylon, neutral and ionic carriers. Attachment of the target protein to the matrix can be achieved by the methods described in Methods in Enzymology, 44 1976 or by other means known in the art. Alternatively or in addition, cells expressing BBB-R on the cell surface can be used to generate or screen for binding molecules. In some embodiments, the binding molecules can be screened for membrane translocation activity.

[0092] In some embodiments, a method for selecting or identifying a peptide that binds to BBB-R is provided. In some embodiments, random peptides can be inserted into a scaffold sequence, such as any of those described in Section I.B, including the backbone of a cyclotide, and the scaffold library can be used to screen for binding to BBB-R. Any known method for generating a library containing variant polynucleotides and / or polypeptides can be used with the methods and vectors provided for generating a display library, such as a phage display library, and for selecting binding proteins from the library. The library can be used in a screening assay for selecting binding proteins from the library for binding to BBB-R. To facilitate screening, a library of variant binding molecules (e.g., a cyclotide library into which random peptides have been inserted) is generally screened using display techniques, and thus there is a physical linkage between the individual molecules (phenotypes) of the library and the genetic information (genotype) encoding them. These methods include, but are not limited to, cell display, including bacterial display, yeast display, mammalian display, phage display (Smith, G. P. (1985) Science 228: 1315-1317), mRNA display, ribosome display, and DNA display.

[0093] In some embodiments, a cyclotide library into which random peptides have been inserted is screened to identify individual members of the library that exhibit a desired biological activity, such as binding to BBB-R. The method for screening for binding to BBB-R, according to one embodiment, a) constructing a cyclotide sequence display library in which random peptides are inserted into a cyclotide scaffold (e.g., into its loop, such as loop 1); b) expressing an array library to obtain an expressed cyclotide or a polypeptide containing a cyclotide; c) selecting the expressed array library against a suitable purified BBB-R; d) recovering all cyclotides that bind to the receptor; e) determining the sequence of the recovered cyclotide or the peptide contained therein. It includes.

[0094] For example, the display library is a phage display library of a plurality of modified cyclotide scaffolds (or other binding molecule scaffolds) into which random peptides are inserted respectively. In some embodiments, each modified cyclotide scaffold of the library is fused to a phage coat protein and displayed on the surface of phagemid particles containing DNA encoding the polypeptide, usually as a single copy of each associated polypeptide on average. These phagemid particles are then contacted with the BBB-R target, and the particles with the highest affinity for the target are separated from the particles with low affinity. Then, the high-affinity binding particles (binders) are amplified by infection of a bacterial host, and the competitive binding step is repeated. This process is repeated until a polypeptide with the desired affinity is obtained.

[0095] In some embodiments, the provided method includes contacting any of the display libraries provided herein with a target molecule under conditions that allow binding of the display particles, such as phagemid particles, to the target molecule. In some embodiments, the method further includes separating the binding display particles, such as phagemid particles, from the non-binding particles, thereby selecting display particles, such as phagemid particles, that contain an antibody-binding protein that binds to the target molecule. In some embodiments, the method includes determining the sequence of the fusion gene in the selected particles to identify the antibody-binding protein.

[0096] In some embodiments, the BBB-R target molecule is contacted with a library of display particles, such as phagemid particles, under conditions suitable for binding of at least a portion of the display particles to the target molecule. Typically, the conditions, including pH, ionic strength, temperature, etc., are such that physiological conditions are mimicked. Exemplary "contact" conditions can include incubating at 4°C to 37°C, such as at room temperature, for 15 minutes to 4 hours, such as for 1 hour. However, these can be varied as needed depending on, for example, the nature of the interacting binding partners. The mixture can be subjected to gentle rocking, mixing, or rotation. Additionally, other suitable reagents, such as blocking agents to reduce non-specific binding, can be added. For example, 1-4% BSA or other suitable blocking agents (e.g., milk) can be used. However, it will be understood that the contact conditions can be varied and adapted by those skilled in the art according to the purpose of the screening method. For example, when the incubation temperature is, for example, room temperature or 37°C, the likelihood of identifying binding particles that are stable under these conditions, such as when incubated at 37°C, and stable under conditions found in the human body, can be increased. Such properties can be highly advantageous when one or both of the binding partners are candidates for use in some therapeutic application, such as an antibody. Again, adaptation to such conditions is within the purview of those skilled in the art.

[0097] Bound display particles ("binding particles") having a high affinity for the immobilized target molecule can be separated from display particles with low affinity (and thus not binding to the target) by washing. The binding particles can be dissociated from the immobilized target molecule by various methods. These methods include competitive dissociation using a wild-type ligand, alteration of pH and / or ionic strength, as well as methods known in the art.

[0098] In another embodiment, peptides can be screened for those activities that confer blood-brain barrier translocation activity. In such an embodiment, a cyclotide library into which random peptides have been inserted, respectively, is screened to identify individual members of the library that exhibit a desired biological activity, such as membrane translocation activity, particularly translocation across the BBB when delivered to an animal. "Translocation across the blood-brain barrier" or "crossing the blood-brain barrier" or other variations thereof means that a polypeptide can be delivered to an animal and enter the animal's brain by crossing the blood vessel wall within the brain. According to one embodiment, the method for screening for BBB translocation is a) constructing a cyclotide sequence display library in which a random peptide is inserted into a cyclotide scaffold (e.g., into its loop, such as loop 1); b) expressing the sequence library to obtain the expressed cyclotide or a polypeptide containing the cyclotide; c) administering the expressed cyclotide or a polypeptide containing the cyclotide to an animal; d) recovering all cyclotides from the brain of the mammal; e) determining the sequence of the recovered cyclotide or the peptide contained therein and comprising.

[0099] The animals used for such screening are generally birds or mammals, and can be selected from humans, primates, cows, sheep, rodents, cats, dogs, and rabbits. In the case of non-human animals, the cyclotide library can be appropriately administered by oral gavage or by including it in normal animal feed. The recovery of cyclotides from the animal body can be by biopsy or, in the case of non-human animals, by slaughter of the animal and histological and pathological analysis of the tissues in the body. Thus, it is also possible to identify members of the cyclotide library that exhibit tissue specificity and / or availability across various additional barriers in the animal body. As an example, modified cyclotides found in a screened non-human animal brain biopsy are considered to show the ability to cross the BBB. In certain embodiments of the invention, the cyclotides of the invention are included in a phage display library and a determination of BBB entry is made by analyzing whether any cyclotide is capable of facilitating the transport of the associated phage particles as a whole into the brain of an animal that has been fed or intravenously injected with the phage display library as part of the feed.

[0100] Once one or more sets of cyclotides or peptides have been selected or isolated according to the provided method, they can be subjected to further analysis. In some embodiments, the further analysis involves isolating the cyclotide by infection of bacteria as an amplification step, isolating phage or phagemid DNA, and cloning the DNA sequence encoding the candidate cyclotide contained in the phage or phagemid DNA into a suitable expression vector. Such an infection step can also enable the amplification of the cyclotide. Alternatively, the cyclotide can be amplified at this stage by other suitable methods, for example, by PCR of the nucleic acid encoding the cyclotide or by transformation of the nucleic acid into a suitable host cell (under the circumstances of a suitable expression vector).

[0101] Once the DNA encoding the cyclotide is cloned into an appropriate expression vector, the sequence of the DNA encoding the cyclotide can be determined, or the protein can be expressed in a soluble form, for example, by following the methods provided herein, and subjected to appropriate binding assays to further characterize the candidate at the protein level. In some embodiments, the peptides contained in the selected cyclotide can be identified and the binding to BBB-R can be evaluated. Appropriate binding assays depend on the nature of the binding particles and include, but are not limited to, ELISA, filter screening assays, FACS, or immunofluorescence assays, BiaCore affinity measurements or other methods for quantifying binding constants, tissue slide or cell staining, and other immunohistochemical methods. One or more of these binding assays can be used to analyze the binding particles.

[0102] In some embodiments, the identified peptide can be modified by histidine or alanine scanning to identify peptides with altered binding or affinity. For example, a library of mutant peptides can be generated and the binding of each variant to the BBB-R target molecule can be evaluated. Any binding assay known to those skilled in the art, including any of the above, can be used. In some embodiments, histidine scanning is performed to mutate or change the amino acid residues within the identified peptide to histidine. In some embodiments, for example, histidine scanning can be used to identify pH-sensitive antigen binding by identifying peptides that have reduced binding at acidic pH compared to neutral pH. Without wishing to be bound by theory, receptor-binding peptides bind to the BBB-R at neutral pH (e.g., pH 7.0 - 7.4), but do not bind or have reduced binding at pH 5.0, such as that present in endosomes, and thus it is desirable that binding molecules containing the peptide (e.g., modified cyclotide) are released into the blood-brain barrier and not recycled. In some embodiments, alanine scanning is performed to mutate or change the amino acid residues within the identified peptide to alanine. In some embodiments, alanine scanning is useful for identifying residues that can be mutated while retaining the binding function.

[0103] Also provided herein is a method for identifying a peptide that binds to the BBB-R by an amino acid sequence, including those from a sequence library.

[0104] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence shown in any one of SEQ ID NOs: 26 to 34 or 49 to 54. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids in length and comprises a sequence shown in any one of SEQ ID NOs: 26 to 34 or 49 to 54. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence shown in SEQ ID NO: 26. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence shown in SEQ ID NO: 49. In some embodiments, the peptide is shown in any one of SEQ ID NOs: 26 to 34 or 49 to 54. In some embodiments, the peptide is shown in SEQ ID NO: 26. In some embodiments, the peptide is shown in SEQ ID NO: 49. In some embodiments, the peptide moiety binds to the transferrin receptor (TrfR). In some embodiments, the TrfR is the mouse TrfR. In some embodiments, the TrfR is the human TrfR. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0105] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having at least 85%, at least 90%, or at least 95% amino acid sequence identity to the sequence set forth in any one of SEQ ID NOs: 26-34 or 49-54. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 26-34 or 49-54. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs: 26-34 or 49-54. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with alanine. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with histidine. In some embodiments, the peptide moiety binds to the transferrin receptor (TrfR). In some embodiments, the TrfR is a mouse TrfR. In some embodiments, the TrfR is a human TrfR. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0106] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having at least 85%, at least 90%, or at least 95% amino acid sequence identity to any one of the sequences set forth in SEQ ID NO: 26. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having one, two, three, or four amino acid substitutions compared to any one of the sequences set forth in SEQ ID NO: 26. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with alanine. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with histidine. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence set forth in any one of SEQ ID NO: 55 or SEQ ID NOs: 117 - 128. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids in length and comprises a sequence set forth in any one of SEQ ID NO: 55 or SEQ ID NOs: 117 - 128. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence set forth in SEQ ID NO: 55. In some embodiments, the peptide is set forth in any one of SEQ ID NO: 55 or SEQ ID NOs: 117 - 128. In some embodiments, the peptide is set forth in SEQ ID NO: 55. In some embodiments, the peptide moiety binds to the transferrin receptor (TrfR). In some embodiments, the TrfR is the mouse TrfR. In some embodiments, the TrfR is the human TrfR. In some embodiments, the peptide has blood - brain barrier translocation activity.

[0107] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence shown in any one of SEQ ID NOs: 10 to 23. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids in length and comprises a sequence shown in any one of SEQ ID NOs: 10 to 23. In some embodiments, the peptide is shown in any one of SEQ ID NOs: 10 to 23. In some embodiments, the peptide moiety binds to the leptin receptor (ObR). In some embodiments, ObR is the human ObR. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0108] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having at least 85%, at least 90%, at least 95% amino acid sequence identity to a sequence shown in any one of SEQ ID NOs: 10 to 23. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having one, two, three or four amino acid substitutions compared to a sequence shown in any one of SEQ ID NOs: 10 to 23. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises a sequence having one, two, three or four amino acid substitutions compared to a sequence shown in any one of SEQ ID NOs: 210 to 23. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with alanine. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with histidine. In some embodiments, the peptide moiety binds to the leptin receptor (ObR). In some embodiments, ObR is the human ObR. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0109] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises the sequence shown in any one of SEQ ID NO: 24 or 25. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids in length and comprises the sequence shown in any one of SEQ ID NO: 24 or 25. In some embodiments, the peptide is shown in SEQ ID NO: 24. In some embodiments, the peptide is shown in SEQ ID NO: 25. In some embodiments, the peptide moiety binds to ErbB3. In some embodiments, ErBB3 is human ErBB3. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0110] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having at least 85%, at least 90%, or at least 95% amino acid sequence identity to the sequence shown in any one of SEQ ID NO: 24 or 25. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 24 or 25. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 24 or 25. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with alanine. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with histidine. In some embodiments, the peptide moiety binds to ErbB3. In some embodiments, ErbB3 is human ErbB3. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0111] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises the sequence shown in any one of SEQ ID NO: 35 or 36. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids in length and comprises the sequence shown in any one of SEQ ID NO: 35 or 36. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence shown in SEQ ID NO: 35. In some embodiments, the peptide is shown in SEQ ID NO: 35. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence shown in SEQ ID NO: 36. In some embodiments, the peptide is shown in SEQ ID NO: 36. In some embodiments, the peptide moiety binds to IGFR. In some embodiments, the IGFR is human IGFR. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0112] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having at least 85%, at least 90%, at least 95% amino acid sequence identity to the sequence shown in any one of SEQ ID NO: 35 or 36. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having one, two, three or four amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 35 or 36. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises a sequence having one, two, three or four amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 35 or 36. In some embodiments, the amino acid substitution(s) is / are substitution of an amino acid residue with alanine. In some embodiments, the amino acid substitution(s) is / are substitution of an amino acid residue with histidine. In some embodiments, the peptide moiety binds to IGFR. In some embodiments, the IGFR is human IGFR. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0113] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises the sequence shown in either SEQ ID NO: 37 or SEQ ID NO: 38. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids in length and comprises the sequence shown in either SEQ ID NO: 37 or SEQ ID NO: 38. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence shown in SEQ ID NO: 37. In some embodiments, the peptide is shown in SEQ ID NO: 37. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence shown in SEQ ID NO: 38. In some embodiments, the peptide is shown in SEQ ID NO: 38. In some embodiments, the peptide moiety binds to RAGE. In some embodiments, RAGE is human RAGE. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0114] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having at least 85%, at least 90%, or at least 95% amino acid sequence identity to the sequence shown in either SEQ ID NO: 37 or SEQ ID NO: 38. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in either SEQ ID NO: 37 or SEQ ID NO: 38. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in either SEQ ID NO: 37 or SEQ ID NO: 38. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with alanine. In some embodiments, the amino acid substitution(s) is / are a substitution of an amino acid residue with histidine. In some embodiments, the peptide moiety binds to RAGE. In some embodiments, RAGE is human RAGE. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0115] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises the sequence shown in any one of SEQ ID NOs: 39 to 48. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids in length and comprises the sequence shown in any one of SEQ ID NOs: 39 to 48. In some embodiments, the peptide is shown in any one of SEQ ID NOs: 39 to 48. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence shown in SEQ ID NO: 39. In some embodiments, the peptide is shown in SEQ ID NO: 39. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence shown in SEQ ID NO: 41. In some embodiments, the peptide is shown in SEQ ID NO: 41. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence shown in SEQ ID NO: 43. In some embodiments, the peptide is shown in SEQ ID NO: 43. In some embodiments, the peptide moiety binds to LRP-1. In some embodiments, LRP-1 is human LRP-1. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0116] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having at least 85%, at least 90%, or at least 95% amino acid sequence identity to the sequence shown in any one of SEQ ID NOs: 39-48. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 39-48. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 39, 41, or 43. In some embodiments, the amino acid substitution(s) is a substitution of an amino acid residue with alanine. In some embodiments, the amino acid substitution(s) is a substitution of an amino acid residue with histidine. In some embodiments, the peptide moiety binds to LRP-1. In some embodiments, LRP-1 is human LRP-1. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0117] In some portions of any of the foregoing embodiments, the peptide has blood-brain barrier translocation activity. In some portions of any of the foregoing embodiments, the peptide binds to a receptor involved in blood-brain barrier transcytosis. B. Binding Molecules (e.g., Cyclotides) Scaffolds and Modified Cyclotides

[0118] Also provided herein are binding molecules comprising a peptide that binds to a BBB-R, including any of those described in Section I.A. above. In some embodiments, the binding molecule provides a stable scaffold (the term "backbone" is also used interchangeably) into which the peptide can be inserted. In some embodiments, the binding molecule is a cysteine knot protein. In some embodiments, the binding molecule is a cyclotide.

[0119] Techniques using protein "scaffolds" and engineering manipulations of loops or regions within the scaffold to alter activity are of most interest in the field of antibodies and antibody fragments that have a natural repertoire of variable regions or loops. Antibody variable loops have been extensively engineered to create peptides with improved binding properties (e.g., affinity and / or specificity) for known ligands and also to expand the binding substrate of a particular antibody framework (see, e.g., Knappik et al., (2000) J. Mol. Biol., 296, 57-86; and EP1025218). Engineering manipulations of non-antibody frameworks are reviewed, for example, in Hosse et al., (2006), Protein Sci., 15, 14-27. Such non-antibody or alternative scaffold proteins have advantages to consider compared to conventional antibodies due to their small size, high stability, and ability to be expressed in prokaryotic hosts. Suitable scaffolds include, for example, modified whey acidic protein (WAP) domain-containing polypeptides such as those described in the international patent application published as WO-A-2012073045. Suitable scaffolds are not limited to just the WAP domain of human elafin (trappin-2), and it will be understood by those skilled in the art that other human trappins (e.g., SLPI) or non-human trappins (e.g., from porcine, bovine subfamily animal or monkey sources) may also be included. Furthermore, the provided embodiments extend to cyclotides included in other non-trappin members of the WAP domain family. Additionally, the provided embodiments also relate to utilizing plant cyclotides as scaffolds.

[0120] In some embodiments, the molecules provided herein that utilize cysteine knot proteins such as cyclotides as scaffolds for inserting peptides can be used as carriers for delivering biological agents, such as protein therapeutics, across the blood-brain barrier. In some embodiments, the cyclotide is a linear or cyclic amino acid sequence that contains a structure referred to herein as a "cysteine knot." A cysteine knot results when a disulfide bond passes through a closed cyclic loop formed by two other disulfide bonds and amino acids within the backbone. However, references herein to "cysteine knot" include references to structural equivalents of cysteine knot that impose similar constraints on the three-dimensional structure of the cyclic backbone. For example, appropriate turns and loops in the "cysteine knot" backbone can also be achieved by engineering manipulations of appropriate covalent or other forms of molecular association. All such modifications to the cyclic backbone that retain the topology with a three-dimensional knot imparted by the cysteine knot are encompassed by the provided embodiments. Further, while cysteine knot is characterized by a knot formed by three disulfide bonds, the provided embodiments also extend to molecules that contain only two disulfide bonds. In such cases, the molecular framework may need to be further stabilized using other means, or the molecular framework may retain appropriate activity despite a change in three-dimensional structure due to the absence of a third disulfide bond. In yet another modification, the cysteine knot backbone can contain more than three disulfide bonds, such as those that occur in, for example, a double or multiple cysteine knot arrangement, or a single cysteine knot arrangement supplemented with one or two additional disulfide bonds.

[0121] Cystine knot microproteins (such as cyclotides) include naturally occurring cystine knot microprotein families or cyclotides found in various plant species. Cystine knot microproteins (cyclotides) are small peptides, generally consisting of about 30 to 40 amino acids, and can be found naturally in cyclic or linear forms, with the cyclic form having no free N-terminal or C-terminal amino or carboxyl termini. These cyclotides have a defined structure based on three intramolecular disulfide bonds and a small triple-stranded β-sheet (Craik et al., 2001; Toxicon 39, 43-60). Cyclic proteins exhibit conserved cysteine residues that define a structure herein referred to as the "cystine knot". This family includes both naturally occurring cyclic molecules and their linear derivatives as well as linear molecules that have undergone cyclization. These molecules are useful as molecular framework structures with enhanced stability compared to less structured peptides (Colgrave and Craik, 2004; Biochemistry 43, 5965-5975). However, these molecules cannot, on their own, cross the blood-brain barrier to any extent, cannot be used as neurotherapeutic agents on their own, and cannot function as carriers for therapeutic peptide or protein agents.

[0122] The main features of cyclotides are the remarkable stability due to their small size, which makes chemical synthesis readily available, and the excellent tolerance to sequence variations. Thus, the provided embodiments are based on the understanding herein that cyclotides seem to be an attractive entry point or scaffold for peptide drug design. Cyclotide scaffolds have been found in almost 30 different protein families, among which the families with the highest abundance of conotoxins, spider toxins, squash inhibitors, agouti-related proteins, and plant cyclotides. Cyclotides derived from plants of the Rubiaceae and Violaceae families are most often found as head-to-tail cyclic peptides (Craik et al. 2010. Cell. Mol. Life Sci. 67: 9-16). However, within the squash inhibitor family of cyclotides, both the following cyclic and linear cyclotides have been identified from Momordica cochinchinensis: cyclic trypsin inhibitors (MCoTI)-I and (MCoTI)-II and their linear counterparts MCoTI-III (Hernandez et al. 2000. Biochemistry, 39, 5722-5730). It is now clear that both cyclic and linear variants can exist in different cyclotide families, but the effects of cyclization are not fully understood. Cyclic peptides are expected to show improved stability, better resistance to proteases, and reduced flexibility compared to their linear counterparts, leading to enhanced biological activity being expected. However, linear cyclotides have the advantage that they can be more easily ligated to other peptides or proteins.

[0123] For example, cyclotides are commonly found in plants. In aspects of the provided embodiments, the cyclotides of the present invention are derived from linear or cyclic forms of cyclotides of Momordica, Rubiaceae, and Violaceae plant species. In a preferred aspect, the cyclotides of the present invention are derived from linear or cyclic forms of cyclotides of Momordicae species, including the Momordica charantia protease inhibitor family (Otlewski & Korowarsch Acta Biochim Pol. 1996;43(3):431-44), and in a more preferred aspect, are derived from Momordica cochinchinensis trypsin inhibitor. In some embodiments, the Momordica cochinschinenis trypsin inhibitor includes the following inhibitors MCoTI-I (SEQ ID NO: 1) and -II (SEQ ID NO: 2) (naturally cyclic) and MCoTI-III (naturally linear) (SEQ ID NO: 3). Mcoti-I GGVCPKILQRCRRDSDSPGACICRGNGYCGSGSD (SEQ ID NO: 1) Mcoti-II GGVCPKILKKCRRDSDSPGACICRGNGYCGSGSD (SEQ ID NO: 2) Mcoti-III ERACPRILKKCRRDSDSPGACICRGNGYCG (SEQ ID NO: 3)

[0124] Modified cyclotides containing peptides of 2 to 50 amino acids that bind to BBB-R are provided herein. In some embodiments, the peptide is any of those described in Section I.A. In some embodiments, the peptide is inserted within the loop of an unmodified or wild-type cyclotide. In some embodiments, the modified cyclotide sequences provided herein can be defined as having a cystine knot backbone moiety and a peptide that is a blood-brain barrier translocation moiety, and the modified cyclotide comprises: i) a peptide that binds to BBB-R and is about 2 to 50 amino acid residues in length, and ii) a cystine knot backbone grafted onto the peptide of item i) above, having structure (I):

Chemical formula

[0125] In some embodiments, the unmodified or wild-type cyclotide has one or more amino acid sequences, such as a peptide of 2 to 50 amino acids, such as any of those described in Section I.A, inserted into one or more of its loops, or one or more loops are replaced by the peptide, and may be a cyclotide as described in any one of SEQ ID NOs: 1 to 3. In some embodiments, the unmodified or wild-type cyclotide has one or more amino acid sequences, such as a peptide of 2 to 50 amino acids, such as any of those described in Section I.A, inserted into one or more of its loops, or one or more loops are replaced by the peptide, and may be a cyclotide as described in any one of SEQ ID NOs: 1. In some embodiments, the unmodified or wild-type cyclotide has one or more amino acid sequences, such as a peptide of 2 to 50 amino acids, such as any of those described in Section I.A, inserted into one or more of its loops, or one or more loops are replaced by the peptide, and may be a cyclotide as described in SEQ ID NO: 2. In some embodiments, the unmodified or wild-type cyclotide has one or more amino acid sequences, such as a peptide of 2 to 50 amino acids, such as any of those described in Section I.A, inserted into one or more of its loops, or one or more loops are replaced by the peptide, and may be a cyclotide as described in any one of SEQ ID NOs: 3. In some embodiments, the modified cyclotides provided by the present invention exhibit improved or enhanced binding to receptors involved in transcytosis across the blood-brain barrier, such as BBB-R, such as the human transferrin receptor. In some embodiments, the modified cyclotides provided have enhanced blood-brain barrier translocation characteristics. The improvement or enhancement is relative to the unmodified cyclotide from which the modified cyclotide is derived, such as a wild-type or native cyclotide including the cyclotides described in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0126] The provided cyclotide contains an amino acid sequence or an analog thereof that forms a cystine knot backbone, where the cystine knot backbone contains sufficient disulfide bonds or their chemical equivalents to confer a topology having a knot in the three-dimensional structure of the cystine knot backbone, and compared to the naturally occurring cyclotide amino acid sequence, there are insertions or substitutions in at least one exposed amino acid residue, for example, on one or more beta turns and / or within one or more loops. In some embodiments, the loop with the insertion or substitution is loop 1 of the native or naturally occurring cyclotide sequence. In some embodiments, the loop with the insertion or substitution is loop 2 of the native or naturally occurring cyclotide sequence. In some embodiments, the loop with the insertion or substitution is loop 3 of the native or naturally occurring cyclotide sequence. In some embodiments, the loop with the insertion or substitution is loop 5 of the native or naturally occurring cyclotide sequence. In some embodiments, the loop with the insertion or substitution is loop 6 of the native or naturally occurring cyclotide sequence. In some embodiments, the provided cyclotide exhibits enhanced migration behavior compared to the unmodified or native parent cyclotide.

[0127] In certain embodiments, the modified cyclotide provided is derived from a loop replacement library based on Mcoti-I (SEQ ID NO: 1). In certain embodiments, provided herein are modified cyclotides comprising a blood-brain barrier translocation peptide moiety inserted into or substituting for or replacing one or more amino acids of the loop of cyclotide Mcoti-I (SEQ ID NO: 1). In some embodiments, the peptide is from about 2 to 50 amino acid residues, for example having any of the amino acid sequences described in Section I.A. In some embodiments, the loop into which the peptide is inserted or incorporated by substitution is Loop 1. In some embodiments, the modified cyclotide has the sequence of SEQ ID NO: 1 in which the residues between the first two cysteines within Loop 1 are replaced by a binding peptide. In some embodiments, the peptide is inserted between cysteine 4 (Cys4) and cysteine 11 (Cys11) of SEQ ID NO: 1. In some embodiments, the loop into which the peptide is inserted or incorporated by substitution is Loop 5. In some embodiments, the modified cyclotide has the sequence of SEQ ID NO: 1 in which the residues between the last two cysteines within Loop 5 are replaced by a binding peptide. In some embodiments, the peptide is inserted between cysteine 23 (Cys23) and cysteine 29 (Cys29) of SEQ ID NO: 1. In some embodiments, the loop into which the peptide is inserted or incorporated by substitution is, for example, Loop 6 formed by subjecting to cyclization. In some embodiments, the modified cyclotide has the sequence of SEQ ID NO: 1 in which the residues between the first cysteine and the last cysteine (after cyclization) within Loop 6 are replaced by a binding peptide. In some embodiments, the peptide is inserted between cysteine 29 (Cys29) and cysteine 4 (Cys4) of SEQ ID NO: 1.

[0128] In certain embodiments, the modified cyclotide provided is derived from a loop replacement library based on Mcoti-II (SEQ ID NO: 2). In certain embodiments, a modified cyclotide is provided herein that comprises a blood-brain barrier translocation peptide moiety inserted into or substituting or replacing one or more amino acids of the loop of cyclotide Mcoti-II (SEQ ID NO: 2). In some embodiments, the peptide is about 2 to 50 amino acid residues, for example having any of the amino acid sequences described in Section I.A. In some embodiments, the loop into which the peptide is inserted or incorporated by substitution is Loop 1. In some embodiments, the modified cyclotide has the sequence of SEQ ID NO: 2 in which the residues between the first two cysteines within Loop 1 are replaced by a linker peptide. In some embodiments, the peptide is inserted between cysteine 4 (Cys4) and cysteine 11 (Cys11) of SEQ ID NO: 2. In some embodiments, the loop into which the peptide is inserted or incorporated by substitution is Loop 5. In some embodiments, the modified cyclotide has the sequence of SEQ ID NO: 2 in which the residues between the last two cysteines within Loop 5 are replaced by a linker peptide. In some embodiments, the peptide is inserted between cysteine 23 (Cys23) and cysteine 29 (Cys29) of SEQ ID NO: 2. In some embodiments, the loop into which the peptide is inserted or incorporated by substitution is, for example, Loop 6 formed by subjecting to cyclization. In some embodiments, the modified cyclotide has the sequence of SEQ ID NO: 2 in which the residues between the first and last cysteines (after cyclization) within Loop 6 are replaced by a linker peptide. In some embodiments, the peptide is inserted between cysteine 29 (Cys29) and cysteine 4 (Cys4) of SEQ ID NO: 2. In certain embodiments, the modified cyclotide provided is derived from a loop replacement library based on Mcoti-III (SEQ ID NO: 3).In certain embodiments, provided herein are modified cyclotides comprising a blood-brain barrier translocation peptide moiety inserted into or substituting for or replacing one or more amino acids of one or more loops of cyclotide Mcoti-III (SEQ ID NO: 3). In some embodiments, the peptide is from about 2 to 50 amino acid residues, for example having any of the amino acid sequences described in Section I.A. In some embodiments, the loop into which the peptide is inserted or incorporated by substitution is Loop 1. In some embodiments, the modified cyclotide has the sequence of SEQ ID NO: 3 in which the residues between the first two cysteines within Loop 1 are replaced by a linker peptide. In some embodiments, the peptide is inserted between cysteine 4 (Cys4) and cysteine 11 (Cys11) of SEQ ID NO: 3. In some embodiments, the loop into which the peptide is inserted or incorporated by substitution is Loop 5. In some embodiments, the modified cyclotide has the sequence of SEQ ID NO: 3 in which the residues between the last two cysteines within Loop 5 are replaced by a linker peptide. In some embodiments, the peptide is inserted between cysteine 23 (Cys23) and cysteine 29 (Cys29) of SEQ ID NO: 3. In some embodiments, the loop into which the peptide is inserted or incorporated by substitution is, for example, Loop 6 formed by subjecting to cyclization. In some embodiments, the modified cyclotide has the sequence of SEQ ID NO: 3 in which the residues between the first cysteine and the last cysteine (after cyclization) within Loop 6 are replaced by a linker peptide. In some embodiments, the peptide is inserted between cysteine 29 (Cys29) and cysteine 4 (Cys4) of SEQ ID NO: 3.

[0129] In some embodiments, the peptide inserted into or incorporated by replacement into a non-modified cyclotide, such as cyclotide Mcoti-I (SEQ ID NO: 1), is 2 to 50 amino acid residues. In some embodiments, the peptide inserted into or incorporated by replacement into a non-modified cyclotide, such as cyclotide Mcoti-II (SEQ ID NO: 2), is 2 to 50 amino acid residues. In some embodiments, the peptide inserted into or incorporated by replacement into a non-modified cyclotide, such as cyclotide Mcoti-III (SEQ ID NO: 3), is 2 to 50 amino acid residues. In some embodiments, the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 amino acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids. In some embodiments, the peptide is 2 to 30 amino acids, such as 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids. In some embodiments, the peptide is 14 to 20 amino acids. In some embodiments, the peptide is 10 amino acids. In some embodiments, the peptide is 11 amino acids. In some embodiments, the peptide is 12 amino acids. In some embodiments, the peptide is 13 amino acids. In some embodiments, the peptide is 14 amino acids. In some embodiments, the peptide is 15 amino acids. In some embodiments, the peptide is 16 amino acids.In some embodiments, the peptide is 17 amino acids. In some embodiments, the peptide is 18 amino acids. In some embodiments, the peptide is 19 amino acids. In some embodiments, the peptide is 20 amino acids.

[0130] In some embodiments, the provided modified cyclotide has an amino acid sequence that exhibits at least 85% sequence identity to any of SEQ ID NOs: 56-101 or 105-116, wherein the modified cyclotide has blood-brain barrier translocation activity and / or binds to a receptor involved in blood-brain barrier transcytosis. In some embodiments, the provided modified cyclotide has an amino acid sequence that exhibits at least 90% sequence identity to any of SEQ ID NOs: 56-101 or 105-116, wherein the modified cyclotide has blood-brain barrier translocation activity and / or binds to a receptor involved in blood-brain barrier transcytosis. In some embodiments, the provided modified cyclotide has an amino acid sequence that exhibits at least 95% sequence identity to any of SEQ ID NOs: 56-101 or 105-116, wherein the modified cyclotide has blood-brain barrier translocation activity and / or binds to a receptor involved in blood-brain barrier transcytosis. In some embodiments, the sequence identity is 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% to any of SEQ ID NOs: 56-101 or 105-116. In some portions of any of the above embodiments, the modified cyclotide binds to a receptor involved in blood-brain barrier transcytosis, such as BBB-R. In some embodiments, the modified cyclotide has blood-brain barrier translocation activity, for example, as measured after subcutaneous administration to a subject. In some embodiments, the modified cyclotide binds to a receptor involved in blood-brain barrier transcytosis, namely BBB-R, and has blood-brain barrier translocation activity when administered subcutaneously to a subject.

[0131] In some parts of any of the foregoing embodiments, the modified cyclotide binds to the BBB-R expressed on the brain endothelial cells. In some embodiments, the BBB-R is a transferrin receptor (TrfR); a lactoferrin receptor (LtfR); a leptin receptor (LEP-R, also known as OB-R); a receptor tyrosine-protein kinase (ErbB3), an insulin receptor, such as insulin receptor A (IRA), an IGF-1 receptor (IGF-1R), an IGF-II receptor (IGF-IIR), RXFP1, RXFP2, RXFP3, and RXFP4; a low density lipoprotein receptor-related protein 1 (LRP-1), a receptor for advanced glycation end products (RAGE); a heparin-binding EGF-like growth factor receptor (HB EGFR); an intercellular adhesion molecule 1 (ICAM1), an intercellular adhesion molecule 2 (ICAM2), or a neural cell adhesion molecule (NCAM); or any other receptor having receptor-mediated transcytosis activity in brain endothelial cells.

[0132] In some embodiments, the modified cyclotide is derived from a loop replacement library based on Mcoti-II (SEQ ID NO: 2) and binds to the transferrin receptor (e.g., the human transferrin receptor). In some embodiments, the loop replacement is replacement of at least one loop, e.g., loop 1, with a peptide comprising a sequence shown in any one of SEQ ID NOs: 26-34 and 49-54. In some embodiments, the peptide is a peptide shown in any one of SEQ ID NOs: 26-34 and 49-54. In some embodiments, the peptide is the peptide shown in SEQ ID NO: 26. In some embodiments, the peptide is the peptide shown in SEQ ID NO: 49. In some embodiments, the modified cyclotide comprises a sequence shown in any one of SEQ ID NOs: 72-80 and 95-100. In some embodiments, the modified cyclotide is shown in any one of SEQ ID NOs: 72-80 and 95-100. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 72. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 95. In some embodiments, the peptide contained in the modified cyclotide has one, two, three, or four amino acid substitutions compared to a peptide shown in any one of SEQ ID NOs: 26-34 and 49-54. In some embodiments, the amino acid substitution(s) is a substitution to histidine. In some embodiments, the amino acid substitution(s) is a substitution to alanine. In some embodiments, the modified cyclotide comprises a peptide sequence shown in any one of SEQ ID NOs: 55 and 117-128. In some embodiments, the peptide sequence is shown in any one of SEQ ID NOs: 55 and 117-128. In some embodiments, the modified cyclotide comprises a sequence shown in any one of SEQ ID NOs: 101 and 105-116. In some embodiments, the modified cyclotide is shown in any one of SEQ ID NOs: 101 and 105-116. In some embodiments, the peptide sequence is the one shown in SEQ ID NO: 55 and the modified cyclotide is shown in SEQ ID NO: 101.

[0133] In some embodiments, the modified cyclotide binds to transferrin but has an altered binding to the transferrin receptor compared to any one of the cyclotides shown in SEQ ID NOs: 72-80 and 95-100. In some embodiments, the modified cyclotide has an altered binding to the transferrin receptor compared to the cyclotide shown in SEQ ID NO: 72. In some embodiments, the modified cyclotide has a decreased binding affinity to the transferrin receptor compared to the modified cyclotide shown in SEQ ID NO: 72, for example, decreased by 1 / 1.5, 1 / 2.0, 1 / 3.0, 1 / 4.0, 1 / 5.0, 1 / 10.0 or more. In some embodiments, the modified cyclotide has a difference of 1, 2, 3, 4 or 5 amino acids compared to the cyclotide shown in SEQ ID NO: 72. In some embodiments, the modified cyclotide has one or more amino acid substitutions compared to the cyclotide shown in SEQ ID NO: 72, and the one or more amino acid substitutions are selected from the group consisting of W9H, L11H, S13H, W14H and G15H. In some embodiments, the peptide is any one of the peptides shown in SEQ ID NOs: 55, 122, 124, 126 or 127. In certain embodiments, the peptide is the one shown in SEQ ID NO: 55. In some embodiments, the modified cyclotide is modified by replacement of a loop (e.g., loop 1) of Mcoti-II (SEQ ID NO: 2) with the peptide shown in SEQ ID NO: 122, for example, the modified cyclotide comprises the sequence shown in SEQ ID NO: 110. In some embodiments, the modified cyclotide is modified by replacement of a loop (e.g., loop 1) of Mcoti-II (SEQ ID NO: 2) with the peptide shown in SEQ ID NO: 124, for example, the modified cyclotide comprises the sequence shown in SEQ ID NO: 112. In some embodiments, the modified cyclotide is modified by replacement of a loop (e.g., loop 1) of Mcoti-II (SEQ ID NO: 2) with the peptide shown in SEQ ID NO: 126, for example, the modified cyclotide comprises the sequence shown in SEQ ID NO: 114.In some embodiments, the modified cyclotide is modified by replacement of a loop (e.g., loop 1) of Mcoti-II (SEQ ID NO: 2) with the peptide shown in SEQ ID NO: 127, and for example, the modified cyclotide comprises the sequence shown in SEQ ID NO: 115. In some embodiments, the modified cyclotide is modified by replacement of a loop (e.g., loop 1) of Mcoti-II (SEQ ID NO: 2) with the peptide shown in SEQ ID NO: 55, and for example, the modified cyclotide comprises the sequence shown in SEQ ID NO: 101. In some embodiments, the binding affinity for the transferrin receptor is pH-dependent, and thus the modified cyclotide exhibits a higher binding affinity for the transferrin receptor at neutral pH (e.g., 7.0 - 7.4) than at the acidic pH of the endosome (e.g., pH 4.5 - 6.5, e.g., pH 5.0 or about pH 5.0). In some embodiments, such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics. Without wishing to be bound by theory, in some aspects, modified cyclotides with reduced binding to the transferrin receptor are released into the blood-brain barrier and not recycled.

[0134] In some embodiments, the modified cyclotide is derived from a loop replacement library based on Mcoti-II (SEQ ID NO: 2) and binds to the leptin receptor (e.g., the human leptin receptor, also called Hu ObR). In some embodiments, the loop replacement is replacement with a peptide comprising the sequence shown in any one of SEQ ID NOs: 10 - 23 of at least one loop, e.g., loop 1. In some embodiments, the peptide is the peptide shown in any one of SEQ ID NOs: 10 - 23. In some embodiments, the modified cyclotide comprises the sequence shown in any one of SEQ ID NOs: 56 - 69. In some embodiments, the modified cyclotide is shown in any one of SEQ ID NOs: 56 - 69.

[0135] In some embodiments, the modified cyclotide binds to the leptin receptor but has an altered binding to the leptin receptor as compared to any one of the cyclotides set forth in SEQ ID NOs: 56-69. In some embodiments, the binding affinity to the leptin receptor is decreased, for example, 1 / 1.5, 1 / 2.0, 1 / 3.0, 1 / 4.0, 1 / 5.0, 1 / 10.0 or more significantly decreased. In some embodiments, the binding affinity to the leptin receptor is pH-dependent, and thus the modified cyclotide exhibits a higher binding affinity to the leptin receptor at neutral pH (e.g., 7.0-7.4) than at the acidic pH of the endosome (e.g., pH 4.5-6.5, e.g., pH 5.0 or about pH 5.0). In some embodiments, the modified cyclotide has a loop having a peptide with one, two, three or four amino acid substitutions as compared to the peptide set forth in any one of SEQ ID NOs: 10-23. In some embodiments, the modified cyclotide is modified by replacement of a loop of Mcoti-II (SEQ ID NO: 2) (e.g., loop 1) with a peptide having one, two, three or four amino acid substitutions as compared to the peptide set forth in any one of SEQ ID NOs: 10-23. In some embodiments, the amino acid substitution(s) is a substitution to histidine. In some embodiments, the amino acid substitution(s) is a substitution to alanine. In some embodiments, such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.

[0136] In some embodiments, the modified cyclotide is derived from a loop replacement library based on Mcoti-II (SEQ ID NO: 2) and binds to ErbB3 (e.g., human ErbB3). In some embodiments, the loop replacement is replacement of at least one loop, e.g., loop 1, with a peptide comprising the sequence set forth in SEQ ID NO: 24 or 25. In some embodiments, the peptide is the peptide set forth in SEQ ID NO: 24 or 25. In some embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO: 70 or 71. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 70 or 71.

[0137] In some embodiments, the modified cyclotide binds to ErbB3 but has an altered binding to ErbB3 compared to the cyclotide shown in SEQ ID NO: 70 or 71. In some embodiments, the binding affinity for ErbB3 is decreased, e.g., 1 / 1.5, 1 / 2.0, 1 / 3.0, 1 / 4.0, 1 / 5.0, 1 / 10.0 or more significantly decreased. In some embodiments, the binding affinity for ErbB3 is pH-dependent, and thus the modified cyclotide exhibits a higher binding affinity for ErbB3 at neutral pH (e.g., 7.0 - 7.4) than at the acidic pH of the endosome (e.g., pH 4.5 - 6.5, e.g., pH 5.0 or about pH 5.0). In some embodiments, the modified cyclotide has a loop with a peptide having one, two, three or four amino acid substitutions compared to the peptide shown in SEQ ID NO: 24 or 25. In some embodiments, the modified cyclotide is modified by replacement of a loop of Mcoti-II (SEQ ID NO: 2) (e.g., loop 1) with a peptide having one, two, three or four amino acid substitutions compared to either one of the peptides shown in SEQ ID NO: 24 or 25. In some embodiments, the amino acid substitution(s) is / are substitution to histidine. In some embodiments, the amino acid substitution(s) is / are substitution to alanine. In some embodiments, such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.

[0138] In some embodiments, the modified cyclotide is derived from a loop replacement library based on Mcoti-II (SEQ ID NO: 2) and binds to IGFR (e.g., human IgFR). In some embodiments, the loop replacement is replacement of at least one loop, e.g., loop 1, with a peptide comprising the sequence shown in SEQ ID NO: 35 or 36. In some embodiments, the peptide is the peptide shown in SEQ ID NO: 35 or 36. In some embodiments, the peptide is the peptide shown in SEQ ID NO: 36. In some embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 81 or 82. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 81 or 82. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 81. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 82.

[0139] In some embodiments, the modified cyclotide binds to IGFR but has an altered binding to IGFR compared to the cyclotide shown in SEQ ID NO: 81 or 82. In some embodiments, the modified cyclotide has an altered binding to IGFR compared to the cyclotide shown in SEQ ID NO: 82. In some embodiments, the binding affinity to IGFR is reduced, for example, 1 / 1.5, 1 / 2.0, 1 / 3.0, 1 / 4.0, 1 / 5.0, 1 / 10.0 or more significantly reduced. In some embodiments, the binding affinity to IGFR is pH-dependent, and thus the modified cyclotide exhibits a higher binding affinity to IGFR at neutral pH (e.g., 7.0 - 7.4) than at the acidic pH of the endosome (e.g., pH 4.5 - 6.5, e.g., pH 5.0 or about pH 5.0). In some embodiments, the modified cyclotide has a loop with a peptide having one, two, three or four amino acid substitutions compared to the peptide shown in SEQ ID NO: 35 or 36. In some embodiments, the modified cyclotide is modified by replacement of a loop of Mcoti-II (SEQ ID NO: 2) (e.g., loop 1) with a peptide having one, two, three or four amino acid substitutions compared to any one of the peptides shown in SEQ ID NO: 35 or 36. In some embodiments, the amino acid substitution(s) is / are substitution to histidine. In some embodiments, the amino acid substitution(s) is / are substitution to alanine. In some embodiments, such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.

[0140] In some embodiments, the modified cyclotide is derived from a loop replacement library based on Mcoti-II (SEQ ID NO: 2) and binds to RAGE (e.g., human RAGE). In some embodiments, the loop replacement is replacement of at least one loop, e.g., loop 1, with a peptide comprising the sequence shown in SEQ ID NO: 37 or 38. In some embodiments, the peptide is the peptide shown in SEQ ID NO: 37 or 38. In some embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 83 or 84. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 83 or 84. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 83. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 84.

[0141] In some embodiments, the modified cyclotide binds to RAGE and is a modified cyclotide with an altered binding to RAGE as compared to the cyclotide shown in SEQ ID NO: 83 or 84. In some embodiments, the binding affinity to RAGE is decreased, for example, 1 / 1.5, 1 / 2.0, 1 / 3.0, 1 / 4.0, 1 / 5.0, 1 / 10.0 or more significantly decreased. In some embodiments, the binding affinity to RAGE is pH-dependent, and thus the modified cyclotide exhibits a higher binding affinity to ErB3 at neutral pH (e.g., 7.0 - 7.4) than at the acidic pH of the endosome (e.g., pH 4.5 - 6.5, e.g., pH 5.0 or about pH 5.0). In some embodiments, the modified cyclotide has a loop with a peptide having one, two, three or four amino acid substitutions as compared to the peptide shown in SEQ ID NO: 37 or 38. In some embodiments, the modified cyclotide is modified by replacement of a loop of Mcoti-II (SEQ ID NO: 2) (e.g., loop 1) with a peptide having one, two, three or four amino acid substitutions as compared to any one of the peptides shown in SEQ ID NO: 37 or 38. In some embodiments, the amino acid substitution(s) is a substitution to histidine. In some embodiments, the amino acid substitution(s) is a substitution to alanine. In some embodiments, such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.

[0142] In some embodiments, the modified cyclotide is derived from a loop replacement library based on Mcoti-II (SEQ ID NO: 2) and binds to LRP-1 (e.g., human LRP-1). In some embodiments, the loop replacement is replacement by a peptide comprising a sequence shown in any one of SEQ ID NOs: 39-48, for example loop 1. In some embodiments, the peptide is a peptide shown in any one of SEQ ID NOs: 39-48. In some embodiments, the modified cyclotide comprises a sequence shown in any one of SEQ ID NOs: 85-94. In some embodiments, the modified cyclotide is shown in any one of SEQ ID NOs: 85-94. In some embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 87. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 87. In some embodiments, the modified cyclotide comprises the sequence shown in SEQ ID NO: 89. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 89.

[0143] In some embodiments, the modified cyclotide binds to LRP-1 and is a modified cyclotide with an altered binding to LRP-1 compared to any one of the cyclotides set forth in SEQ ID NOs: 85-94. In some embodiments, the binding affinity to LRP-1 is decreased, for example, 1 / 1.5, 1 / 2.0, 1 / 3.0, 1 / 4.0, 1 / 5.0, 1 / 10.0 or more significantly decreased. In some embodiments, the binding affinity to the leptin receptor is pH-dependent, and thus the modified cyclotide exhibits a higher binding affinity to LRP-1 at neutral pH (e.g., 7.0-7.4) than at the acidic pH of the endosome (e.g., pH 4.5-6.5, e.g., pH 5.0 or about pH 5.0). In some embodiments, the modified cyclotide has a loop having a peptide with one, two, three or four amino acid substitutions compared to the peptide shown in any one of SEQ ID NOs: 39-48. In some embodiments, the modified cyclotide is modified by replacement of a loop of Mcoti-II (SEQ ID NO: 2) (e.g., loop 1) with a peptide having one, two, three or four amino acid substitutions compared to the peptide shown in any one of SEQ ID NOs: 39-48. In some embodiments, the amino acid substitution(s) is a substitution to histidine. In some embodiments, the amino acid substitution(s) is a substitution to alanine. In some embodiments, such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.

[0144] In some embodiments, any of the modified cyclotides provided binds to the BBB-R targeted by the peptide. Methods for determining binding affinity, or relative binding affinity, are known in the art and include solid-phase ELISA immunoassays, ForteBio Octet, Biacore measurements, or flow cytometry. See, for example, Larsen et al., American Journal of Transplantation, vol. 5: 443-453 (2005); Linsley et al., Immunity, Vol 1 (9): 793-801 (1994). In some embodiments, binding affinity can be measured by flow cytometry, for example, based on Mean Fluorescence Intensity (MFI) in a flow binding assay. In some embodiments, the conjugate provided, such as a fusion protein, has a binding affinity for the BBB-R determined, for example, by solid-phase ELISA immunoassay, flow cytometry, or surface plasmon resonance (Biacore) assay.

[0145] In some embodiments, the conjugate (e.g., fusion protein) has a binding affinity for the BBB-R that is higher than 10 nM and lower than 1000 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R that is higher than 20 nM and lower than 800 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of about 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM or any value between any of the foregoing. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 50 nM to about 500 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 50 nM to about 250 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 50 nM to about 100 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 100 nM to about 500 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 100 nM to about 250 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 250 nM to 500 nM.

[0146] The cyclotide molecules provided herein can include certain functionality, generally including at least some type of membrane translocation activity. In certain embodiments of the invention, the membrane translocation activity is the ability to translocate across the intestinal wall (e.g., intestinal mucosa); the ability to translocate across the blood-brain barrier; the ability to translocate across the cell membrane; the ability to translocate into intracellular compartments; the ability to translocate into the nucleus of a cell; and the ability to translocate into organelles including mitochondria, and includes activities selected from one or more of these. In some embodiments, the modified cyclotides provided exhibit improved or enhanced transcytosis across the blood-brain barrier as compared to the parent or scaffold cyclotide, e.g., cyclotide Mcoti-II (SEQ ID NO: 2). In some embodiments, the improvement or enhancement of transcytosis is due to binding to receptors involved in blood-brain barrier transcytosis. The membrane translocation activity can be appropriately included in peptides or polypeptides present within functional modules of larger proteins. Thus, the cyclotides provided represent synthetic polypeptide scaffolds. Desirable physical properties of potential scaffold molecules include high thermal stability and reversibility of heat-induced folding and unfolding.

[0147] II. Cyclotide Conjugates and Fusion Proteins and Biological Agents In some embodiments, conjugates are also provided herein that include a modified cyclotide or binding molecule of any of the foregoing embodiments and an agent having biological activity. In some embodiments, the modified cyclotide or binding molecule is any of those described in Section I.B. In some embodiments, the agent having biological activity is a small molecule, peptide, or protein. In some embodiments, the agent having biological activity is a diagnostic agent or a therapeutic agent.

[0148] In some embodiments, the biologically active agent is any agent having therapeutic potential for treating a disorder of the central nervous system (CNS). In some embodiments, the biologically active agent can be selected from neuroleptics, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibodies directed against brain targets, monoclonal antibodies directed against brain targets, or peptides directed against brain targets.

[0149] The cyclotide of the present invention can also be conjugated or otherwise linked to a biologically active agent or therapeutic agent selected from the group consisting of small molecules; antibodies or antibody fragments; hormones, cytokines; nucleic acids; bioactive peptides; glycosylated peptides; contrast agents; and radiolabeled compounds.

[0150] In some embodiments, the cyclotide moiety may be an amino acid extension at the C-terminus or N-terminus of a biologically active agent, such as a polypeptide. In some aspects, a short amino acid linker sequence may be present between the biologically active agent, such as a polypeptide, and the cyclotide moiety. Suitable linker groups can include amides, esters, disulfides, sulfides, ketals, succinates, oximes, carbamates, carbonates, sialyl ethers, or triazoles. In some embodiments, the linker may be a peptide linker. In some embodiments, the molecules provided include molecules in which a biologically active agent, such as a polypeptide, is linked to the cyclotide moiety via a linker sequence, if necessary, for example by chemical conjugation. Generally, a modified polypeptide is linked to another moiety via a site that does not interfere with the activity of either moiety.

[0151] In some of any of the foregoing embodiments, the conjugate is a fusion protein comprising a modified cyclotide operably linked to a biologically active agent that is a protein, polypeptide or peptide.

[0152] In some of the foregoing embodiments, the biologically active agent is selected from the group consisting of an antibody or an antibody fragment thereof. In some of the foregoing embodiments, the biologically active agent is selected from the group consisting of a growth factor or a hormone. In some of the foregoing embodiments, the biologically active agent is an enzyme.

[0153] In other embodiments of the invention, a cyclotide is conjugated to or included within a desired biologically therapeutic agent and then administered to a test animal to determine the BBB entry of the modified biologically therapeutic agent.

[0154] Accordingly, the therapeutic use and application of the cyclotides of the invention encompasses any disease or condition that requires a repetitive treatment regimen or frequent administration of a biologically active agent. This includes applications to therapies where it may be beneficial to conjugate a cyclotide to the N-terminus or C-terminus of a therapeutic agent and thus make the therapeutic agent more readily available to the recipient's brain or cerebrospinal fluid. Diseases suitable for treatment include, but are not limited to, various neoplastic diseases and disorders as well as non-neoplastic diseases and disorders (e.g., cancer / neoplastic diseases and related conditions); chronic degenerative and neurodegenerative diseases or disorders (e.g., multiple sclerosis, Parkinson's disease, and Alzheimer's disease), stroke, or other conditions where the brain has been damaged. In a further embodiment, the cyclotides of the invention are fused with a therapeutic agent and used as an intravenously administered therapeutic treatment for Alzheimer's disease or an intracranial neoplasm. In a further embodiment, the cyclotides of the invention are conjugated to a therapeutic agent capable of enhancing cognitive abilities such as memory.

[0155] In a further aspect, the invention relates to a conjugate that can include a carrier selected from the group consisting of any one of the modified cyclotides of the invention conjugated to a bioactive agent or therapeutic agent selected from the group consisting of, for example, a drug (e.g., a small molecule drug, e.g., an antibiotic), a medicament, a detectable label, a protein (e.g., an enzyme), a protein-based compound (e.g., a protein complex comprising one or more polypeptide chains), and a polypeptide (peptide). The agent may more particularly be a molecule that is active at the level of the central nervous system. The agent may be any agent for treating or detecting a neurological disorder.

[0156] According to the invention, the detectable label may be a radiopaque agent. Examples of labels included herein that can be conjugated to the carrier of the invention include, for example, without limitation thereto, isotopes, fluorescent labels (e.g., rhodamine), reporter molecules (e.g., biotin), and the like. Other examples of detectable labels include, for example, green fluorescent protein, biotin, his-tag protein, and beta-galactosidase.

[0157] In some embodiments, the biologically active agent is a therapeutic protein agent. Examples of therapeutic proteins or protein-based compounds included herein that can be conjugated to the carrier of the present invention include, but are not limited to, antibodies, antibody fragments (e.g., antibody-binding fragments such as Fv fragments, F(ab)2, F(ab)2’ and Fab), single-domain antibodies (e.g., camelid VHH domains, shark new antigen receptors (NAR), or human VH or VL domains), peptide-based drugs or protein-based drugs (e.g., positive pharmacological modulators (agonists) or pharmacological inhibitors (antagonists)), and the like. Other examples of agents included herein are growth factors (e.g., fibroblast growth factors and related proteins, nerve growth factor, glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4), cytotoxins (e.g., monomethyl auristatin E (MMAE), toxins derived from bacterial endotoxins and exotoxins; diphtheria toxin, botunilum toxin, tetanus toxin, pertussis toxin, Staphylococcus enterotoxin, toxic shock syndrome toxin TSST-1, adenylate cyclase toxin, Shiga toxin, cholera enterotoxin, and others), soluble receptors (e.g., TNF receptor 1 or 2) and anti-angiogenic compounds (endostatin, catechins, nutriceuticals, chemokine IP-10, matrix metalloproteinase inhibitors (MMPI), anastellin, vironectin, antithrombin, tyrosine kinase inhibitors, VEGF inhibitors, antibodies against receptors, Herceptin®, avastin and panitumumab and others).

[0158] In some embodiments, the biologically active agent is an agent for treating neuropathy. In some embodiments, agents for treating neuropathy include, but are not limited to, small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified versions of natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small interfering RNA (siRNA) and small hairpin RNA (shRNA)), ribozymes, and small molecules, or active fragments of any of the foregoing. Exemplary neuropathic drugs of the present invention are described herein and include, but are not limited to, antibodies, aptamers, proteins, peptides, inhibitory nucleic acids, and small molecules that are themselves CNS antigens or target molecules, or that specifically recognize and / or act on (i.e., inhibit, activate, or detect) CNS antigens or target molecules, as well as active fragments of any of the foregoing. In some embodiments, CNS antigen target molecules include, but are not limited to, amyloid precursor protein or a portion thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophins. Non-limiting examples of neuropathic drugs and the corresponding disorders that can be treated using them include brain-derived neurotrophic factor (BDNF) for treating chronic brain injury (neurogenesis); anti-EGFR antibody for treating brain cancer; glial cell line-derived neurotrophic factor (GDNF) for treating Parkinson's disease; brain-derived neurotrophic factor (BDNF) for treating amyotrophic lateral sclerosis or depression; lysosomal enzymes for treating lysosomal storage diseases of the brain; ciliary neurotrophic factor (CNTF) for treating amyotrophic lateral sclerosis; neuregulin-1 for treating schizophrenia; anti-HER2 antibody (e.g., trastuzumab) for treating brain metastases of HER2-positive cancer.

[0159] In some embodiments, the biologically active agent is an antibody. In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is a full-length antibody targeting a brain target. In some embodiments, the antibody is a full-length IgG. Previous studies have demonstrated that a very small percentage (approximately 0.1%) of the IgG injected into the bloodstream can penetrate into the CNS compartment (Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)). Thus, the provided embodiments provide an improved therapeutic agent with enhanced blood-brain barrier translocation activity.

[0160] In some embodiments, the antibody targets and, for example, specifically binds to an antigen and / or molecule expressed in the CNS, including the brain, that can be targeted using the antibody or a small molecule. Examples of such antigens and / or molecules include, without limitation, beta-secretase 1 (BACE1), amyloid beta (A-beta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), P75 neurotrophin receptor (P75NTR), and caspase 6.

[0161] In some embodiments, the antibody targets or binds to HER2. In some embodiments, the antibody is trastuzumab (Herceptin®). In some embodiments, the antibody has a heavy chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the heavy chain sequence shown in SEQ ID NO: 134 and a light chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the light chain sequence shown in SEQ ID NO: 137. In some embodiments, the antibody contains the heavy chain sequence shown in SEQ ID NO: 134 and the light chain sequence shown in SEQ ID NO: 137. In some embodiments, the HER2-targeting antibody and the provided conjugate fusion containing the same can be used to treat metastatic breast cancer. In some embodiments, the HER2-targeting antibody and the provided conjugate fusion containing the same can be used to treat neurological diseases such as Alzheimer's disease.

[0162] In some embodiments, the antibody targets or binds to amyloid beta. In some embodiments, the antibody is aducanumab (Aduhelm). In some embodiments, the antibody has a heavy chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the heavy chain sequence shown in SEQ ID NO: 129 and a light chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the light chain sequence shown in SEQ ID NO: 132. In some embodiments, the antibody contains the heavy chain sequence shown in SEQ ID NO: 129 and the light chain sequence shown in SEQ ID NO: 132. In some embodiments, the amyloid beta-targeting antibody and the provided conjugate fusion containing the same can be used to treat Alzheimer's disease.

[0163] In some embodiments, the biologically active agent is a TNF inhibitor. In some embodiments, the inhibitor is a small molecule, peptide or protein. In some embodiments, the inhibitor is an antibody. In some embodiments, the antibody targets or binds to TNF-alpha. In some embodiments, the antibody is adalimumab (Humira). In some embodiments, the antibody contains a heavy chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the heavy chain sequence shown in SEQ ID NO: 102 and a light chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the light chain sequence shown in SEQ ID NO: 103. In some embodiments, the antibody contains the heavy chain sequence shown in SEQ ID NO: 102 and the light chain sequence shown in SEQ ID NO: 103. In some embodiments, antibodies that target TNF-alpha, and conjugate fusions provided therewith, can be used to treat neurological diseases such as stroke, traumatic brain injury or Alzheimer's disease.

[0164] In some embodiments, the biologically active agent is a growth factor or a hormone. In some embodiments, the biologically active agent is a growth factor. In particular, for treating various neurological disorders such as stroke, Alzheimer's, Parkinson's disease, and multiple sclerosis, the cyclotide of the present invention can be operably linked to growth factors including ADNP, BDNF, CNTF, GCSF, GDNF, IFN beta, IL-10, insulin, MANF, NGF, NT-3, progranulin. In some embodiments, the biologically active agent is a nerve growth factor (NGF) for treating, for example, Parkinson's disease, stroke, or Alzheimer's disease. In some embodiments, the biologically active agent is a granulocyte colony-stimulating factor (GCSF) for use in treating, for example, stroke or traumatic brain injury (TBI). In some embodiments, the biologically active agent is IL-10 for use in treating, for example, Alzheimer's disease, heart disease, or brain cancer. In some embodiments, the biologically active agent is BDNF for use in treating, for example, ALS or depression. In some embodiments, the biologically active agent is ADNP for use in treating, for example, autism spectrum disorder.

[0165] In some embodiments, the biologically active agent is NGF and has the sequence shown in SEQ ID NO: 147 or a sequence having at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 147. In some embodiments, the biologically active agent is shown in SEQ ID NO: 147.

[0166] In some embodiments, the biologically active agent is an enzyme. In some embodiments, the enzyme is a ceramide-degrading enzyme, a lipase, a hydrolase-type enzyme, or a sulfatase. In some embodiments, the enzyme is a ceramide-degrading enzyme, and the ceramide-degrading enzyme is glucocerebrosidase, galactocerebrosidase, or alpha-galactosidase. In some embodiments, the enzyme is a lipase or a hydrolase-type enzyme, and the enzyme is sphingomyelinase, cell liponase, or alpha-glucosidase. In some embodiments, the enzyme is, for example, heparin N-sulfatase for treating Sanfilippo A syndrome. In some embodiments, the enzyme is, for example, glucocerebrosidase for treating Gaucher disease. In some embodiments, the enzyme is, for example, glucocerebrosidase for treating Parkinson's disease. In some embodiments, the enzyme is, for example, galactocerebrosidase for use in treating Krabbe disease. In some embodiments, the enzyme is, for example, alpha-galactosidase for use in treating Fabry disease. In some embodiments, the enzyme is, for example, sphingomyelinase for treating Niemann Pick disease. In some embodiments, the enzyme is, for example, cell liponase alpha for use in treating Jansky Bielschowsky disease. In some embodiments, the enzyme is, for example, alpha-glucosidase for treating Pompe’s disease. In some embodiments, the enzyme is, for example, tripeptidyl peptidase I for use in treating Jansky Bielschowsky disease or Batten disease. In some embodiments, the enzyme is, for example, galactosamine 6-sulfatase for use in treating Marquio syndrome.

[0167] In some embodiments, the biologically active agent is glucocerebrosidase (GCase). In some embodiments, the enzyme has an amino acid sequence shown in SEQ ID NO: 144 or an amino acid sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 144. In some embodiments, the sequence is shown in SEQ ID NO: 144. In some embodiments, the enzyme has an amino acid sequence shown in SEQ ID NO: 145 or an amino acid sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 145. In some embodiments, the sequence is shown in SEQ ID NO: 145. In some embodiments, the glucocerebrosidase is a glucocerebrosidase mutant GCase molecule that exhibits increased stability and enhanced function. Exemplary GCase mutants have one or more amino acid substitutions selected from I5N, F31Y, L34Q, M53T, P55T, H145F, H145L, H223N, H223Y, E233Q, H274N, W312C, F316A, L317F, K321V, K321A, K321N, A341C, H365K, I368C, D443C, D445C, H451K, S455C, S464C, R495C, R495N, and combinations thereof, compared to the sequence shown in SEQ ID NO: 144 or SEQ ID NO: 145. In some embodiments, the GCase mutant contains one, two, three, four, five, six, or seven amino acid substitutions from I5N, F31Y, L34Q, M53T, P55T, H145F, H145L, H223N, H223Y, E233Q, H274N, W312C, F316A, L317F, K321V, K321A, K321N, A341C, H365K, I368C, D443C, D445C, H451K, S455C, S464C, R495C, R495N, compared to the sequence shown in SEQ ID NO: 144 or SEQ ID NO: 145. In some embodiments, the GCase mutant has the amino acid substitutions M53T and P55T compared to the sequence shown in SEQ ID NO: 144 or SEQ ID NO: 145.In some embodiments, the GCase variant has the amino acid substitutions H145L / K321N, D443C / S464C, S455C / R495C, W312C / A341C, I368C / D445C, and E233Q / W312C / A341C compared to the sequences shown in SEQ ID NO: 144 or SEQ ID NO: 145. In some embodiments, the GCase variant has the amino acid substitutions F316A / L317F, F316A / L317F / K321N, or H145L / K321N compared to the sequences shown in SEQ ID NO: 144 or SEQ ID NO: 145. Non-limiting examples of GCase variants are described in PCT Published Application Nos. WO2012 / 064709; WO2021 / 048034; and WO2022 / 023761.

[0168] Among the conjugates provided are modified cyclotides linked to glucocerebrosidase. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 101, which targets TrfR, for example. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 81, which targets IgFR, for example. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 82, which targets IgFR, for example. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 85, which targets LRP-1, for example. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 87, which targets LRP-1, for example. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 89, which targets LRP-1, for example. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 83, which targets RAGE, for example. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 84, which targets RAGE, for example. In some embodiments, the modified cyclotide targets TrfR, and the conjugate has the amino acid sequence shown in SEQ ID NO: 146 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 146. In some embodiments, the conjugate has the sequence shown in SEQ ID NO: 146. In some embodiments, the modified cyclotide binds to a BBB-R (e.g., TrfR) and transports glucocerebrosidase across the blood-brain barrier.

[0169] In some embodiments, a conjugate of a modified cyclotide and a biological protein agent, such as a fusion protein, contains 1, 2, 3, 4, 5 or more copies of the modified cyclotide. In some embodiments, the conjugate provided, such as a fusion protein, contains one modified cyclotide, and thus the conjugate (e.g., fusion protein) is monovalent with respect to binding to a BBB-R. In certain embodiments, the conjugate (e.g., fusion protein) is monovalent with respect to binding to any one type of BBB-R.

[0170] In some embodiments, a conjugate (e.g., a fusion protein) may contain multiple copies of a modified cyclotide, e.g., 2, 3, 4, or 5 copies of the modified cyclotide. In such embodiments, the conjugate (e.g., a fusion protein) may be referred to as a multivalent binding protein. Also provided are multivalent binding proteins that contain multiple copies of any of the provided modified cyclotides. In some embodiments, each of the modified cyclotides may be linked tandemly, directly or indirectly, on the same polypeptide chain. In some embodiments, the fusion protein is a multi-chain polypeptide, and each chain of the polypeptide contains at least 1 copy of the modified cyclotide. For example, in some embodiments, provided is a fusion protein of an antibody (e.g., a therapeutic antibody) and a modified cyclotide, wherein the first heavy or light chain of the antibody is linked to the first copy of the modified cyclotide and the second heavy or light chain is linked to the second copy of the modified cyclotide. In some embodiments, the binding protein is bivalent and contains 2 copies of the modified cyclotide.

[0171] In some embodiments, a conjugate (e.g., a fusion protein) may contain multiple different modified cyclotides, e.g., 2, 3, 4, or 5 different modified cyclotides. In such embodiments, the conjugate (e.g., a fusion protein) may be referred to as a multispecific binding protein. Also provided are multispecific binding proteins that include multiple different cyclotides from any of the provided modified cyclotides. In some embodiments, the binding protein is multispecific and contains at least two different modified cyclotides that each bind to a different BBB-R. In some embodiments, the multispecific binding protein contains 2, 3, or 4 different modified cyclotides, where each modified cyclotide binds to a different BBB-R. In some embodiments, each of the modified cyclotides may be linked tandemly, directly or indirectly, on the same polypeptide chain. In some embodiments, the fusion protein is a multichain polypeptide, and each chain of the polypeptide contains a modified cyclotide, and those modified cyclotides may be different. For example, in some embodiments, provided is a fusion protein of an antibody (e.g., a therapeutic antibody) and a modified cyclotide, wherein the first heavy or light chain of the antibody is linked to a first modified cyclotide, and the second heavy or light chain is linked to a second modified cyclotide that is different from the first modified cyclotide. In some embodiments, the binding protein is bispecific and contains two different modified cyclotides that each bind to a different BBB-R. By way of example, in some embodiments, the first modified cyclotide binds to the transferrin receptor and the second modified cyclotide binds to IGF1R.

[0172] Multiple modified cyclotides (same or different) need not be directly covalently linked to each other. In some embodiments, one or more amino acid residues span between and the modified cyclotides (e.g., a first modified cyclotide and a second modified cyclotide) are indirectly covalently linked to each other. The linkage may be by way of an N-terminal residue and a C-terminal residue. In some embodiments, the linkage may be made through the side chain of an amino acid residue that is not located at the N-terminus or the C-terminus of the modified cyclotide. Thus, the linkage may be made through terminal or internal amino acid residues or combinations thereof.

[0173] In some embodiments, a plurality (same or different) of any of the modified cyclotides may be linked with a biological agent (or a chain thereof) separated by a peptide linker such as a flexible linker. In some embodiments, the peptide linker may be GGGS or other similar flexible linkers including longer linkers of (GGGS)n where n is from 1 to 3 in the sequence. In some embodiments, a conjugate (e.g., a fusion protein) containing multiple modified cyclotides may include two, three, four or more modified cyclotides. In some embodiments, the modified cyclotides may be the same or different. In some embodiments, the modified cyclotides may be the same to create a divalent, trivalent or other multivalent molecule. In some embodiments, the modified cyclotides may be different to provide a bispecific, trispecific or other multispecific molecule.

[0174] In some embodiments, any plurality (same or different) of the modified cyclotides may be tandemly linked to a biological agent, separated by a peptide linker such as a flexible linker in a single polypeptide chain. In some embodiments, the peptide linker may be GGGS or other similar flexible linkers, including longer linkers of (GGGS)n (where n is from 1 to 3 in the sequence). In some embodiments, the tandem single polypeptide may include two, three, four or more modified cyclotides to create a divalent, trivalent, tetravalent or other multivalent molecule. In some embodiments, one or more of the modified cyclotides may be the same or different. In some embodiments, the tandem single polypeptide may include two, three, four or more different cyclotides to provide a bispecific, trispecific or other multispecific molecule.

[0175] In some embodiments, the fusion protein is composed of an antibody (e.g., a therapeutic antibody) and at least one modified cyclotide. In some embodiments, one, two, three or four modified cyclotides may be linked to the antibody, and the modified cyclotides may all be the same or different. In some embodiments, the modified cyclotide may be linked to the heavy chain or the light chain of the antibody. In some embodiments, the modified cyclotide may be linked to the N-terminus of the heavy chain or the light chain of the antibody. In some embodiments, the modified cyclotide may be linked to the C-terminus of the heavy chain or the light chain of the antibody. In some embodiments, each of the modified cyclotides may be linked to the heavy chain of the antibody. In some embodiments, each chain of the antibody may be linked to a modified cyclotide.

[0176] In some embodiments, a bivalent fusion protein composed of an antibody (e.g., a therapeutic antibody) and two copies of a modified cyclotide is provided. In some embodiments, the modified cyclotide is linked to both heavy chains of the antibody. In some embodiments, the modified cyclotide is linked to both light chains of the antibody. In some embodiments, the modified cyclotide may be linked to the N-terminus of the heavy or light chain of the antibody. In some embodiments, the modified cyclotide may be linked to the C-terminus of the heavy or light chain of the antibody. Methods for creating antibody fusions are known. In some embodiments, the heavy and light chains are co-expressed in a cell. In some embodiments, when produced in a cell, a double-stranded polypeptide is formed by dimer formation resulting from disulfide formation between two heavy chain molecules. In some embodiments, the antibody fusion protein is a homodimer containing two identical copies of the modified cyclotide.

[0177] In some embodiments, a monovalent fusion protein composed of an antibody linked to a single modified cyclotide is provided. In such embodiments, the antibody fusion protein is monovalent with respect to binding to BBB-R. In some embodiments, the modified cyclotide is linked to one heavy chain of the antibody. In some embodiments, the modified cyclotide is linked to one light chain of the antibody. In some embodiments, the modified cyclotide may be linked to the N-terminus of the heavy or light chain of the antibody. In some embodiments, the modified cyclotide may be linked to the C-terminus of the heavy or light chain of the antibody. In some embodiments, the fusion protein is a heterodimer composed of two different heavy chains and light chains paired with each heavy chain, with only one of the heavy chains linked to the modified cyclotide.

[0178] In some embodiments, provided is a multispecific fusion protein composed of an antibody linked to at least two different modified cyclotides. In some embodiments, the antibody fusion protein is bispecific and contains two different modified cyclotides for binding to two different BBB-Rs. In some embodiments, each modified cyclotide is linked to the heavy chain of the antibody. In some embodiments, the modified cyclotide may be linked tandemly with the heavy chain. In some embodiments, the modified cyclotides may each be linked to a different heavy chain of the antibody. In some embodiments, each modified cyclotide is linked to the light chain of the antibody. In some embodiments, the modified cyclotide may be linked tandemly with the light chain. In some embodiments, the modified cyclotides may each be linked to a different light chain of the antibody. In some embodiments, the modified cyclotide may be linked to the N-terminus of the heavy chain or light chain of the antibody. In some embodiments, the modified cyclotide may be linked to the C-terminus of the heavy chain or light chain of the antibody. In some embodiments, the fusion protein is a heterodimer composed of two different heavy chains and light chains paired with each heavy chain, wherein one heavy chain is linked tandemly with a different modified cyclotide and the other heavy chain is not linked to a modified cyclotide. In some embodiments, the fusion protein is a heterodimer composed of two different heavy chains and light chains paired with each heavy chain, wherein each of the heavy chains is linked to a different modified cyclotide.

[0179] Methods for producing heterodimeric antibody fusion proteins are known. In some embodiments, knob-into-hole engineering strategies or other strategies can be used to co-express two different heavy chains in a cell to produce a heterodimer, where the two different heavy chains, e.g., heavy chains each having different modified cyclotides, can interact to form a heterodimer. In some embodiments, residues of the constant chain are modified by amino acid substitution so as to facilitate heterodimer formation. In some parts of any embodiment, one or more amino acid modifications are selected from knob-into-hole modifications and charge mutations to reduce or prevent self-association due to charge repulsion. The heterodimer can be formed by transforming a cell with both a first nucleic acid molecule encoding a first heavy chain polypeptide subunit (e.g., a knob sequence) and a second nucleic acid molecule encoding a second different heavy chain polypeptide subunit (e.g., a hole sequence). In some aspects, the heterodimer, when expressed, is produced as a result of covalent or non-covalent interactions between residues of the two polypeptide subunits that mediate dimer formation and is secreted from the cell. In such a process, generally, a mixture of dimer molecules, including homodimers and heterodimers, is formed. Additional purification steps may be required to generate the heterodimer. For example, the first polypeptide and the second polypeptide can be engineered to include tags using metal chelates or other epitopes, where the tags are different. The tagged domains can be used for rapid purification by metal chelate chromatography and / or by antibodies to enable detection by activity depletion / blocking in Western blot, immunoprecipitation, or bioassay. In other embodiments, methods for promoting heterodimer formation can be implemented.

[0180] The methods include those described in U.S. Patent No. 10,995,127. For example, if there is an amino acid modification at Thr366 within the CH3 domain, and it is a replacement with a larger amino acid, such as Try (T366W), it can preferentially pair with a second CH3 domain having amino acid modifications to smaller amino acids, such as Ser, Ala, and Val at positions Thr366, Leu368, and Tyr407 respectively (T366S / L368A / Y407V). In some embodiments, the "knob" Fc domain contains the T366W mutation. In some embodiments, the "hole" Fc domain contains the T366S mutation, the L368A mutation, and the Y407V mutation. Heterodimer formation by CH3 modification can be further stabilized by introducing disulfide bonds, for example, by changing Ser354 to Cys (S354C) on the opposite CH3 domain and changing Y349 to Cys (Y349C) (as outlined in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, the Fc domains used for heterodimer formation contain additional mutations, such as the S354C mutation on the first member of the heterodimer Fc pair that forms an asymmetric disulfide with the corresponding Y349C mutation on the second member of the heterodimer Fc pair. In some embodiments, one member of the heterodimer Fc pair contains the H435R or H435K modification to prevent binding to Protein A while maintaining binding to FcRn. In some embodiments, one member of the heterodimer Fc pair contains the H435R or H435K modification, while the second member of the heterodimer Fc pair is unmodified at H435. In various embodiments, the hole Fc domain contains the H435R or H435K modification (in some cases, when the modification is H435R, it is referred to as "hole-R"), while the knob Fc domain does not contain the modification. In some cases, the hole-R mutation improves the purification of the heterodimer relative to the possible homodimeric hole Fc domain.

[0181] Performing appropriate knob and hole modifications to the heavy chain of any antibody is within the scope of those skilled in the art. By way of example, the knob and hole sequences of the exemplary antibody trastuzumab are shown in SEQ ID NO: 135 and SEQ ID NO: 136, respectively. In another example, the knob and hole sequences of the exemplary antibody aducanumab are shown in SEQ ID NO: 130 and SEQ ID NO: 131, respectively. To promote heterodimer formation, any antibody can similarly be modified in the CH3 domain to create knob and hole chains. In some embodiments, a modified cyclotide is linked to the knob heavy chain and / or the hole heavy chain of the antibody. In some embodiments, the knob heavy chain, the hole heavy chain and the light chain are co-expressed in a cell, in which case a heterodimeric antibody containing two different heavy chains (knob and hole), each forming a complex with the light chain, is produced.

[0182] In some embodiments, one or more “peptide linkers” are used to link a modified cyclotide to one or more other modified cyclotides or biological agents. In some embodiments, the peptide linker may be a single amino acid residue or may be longer. In some embodiments, the peptide linker has at least one amino acid residue, but is 20 amino acid residues or less, 19 amino acid residues or less, 18 amino acid residues or less, 17 amino acid residues or less, 16 amino acid residues or less, 15 amino acid residues or less, 14 amino acid residues or less, 13 amino acid residues or less, 12 amino acid residues or less, 11 amino acid residues or less, 10 amino acid residues or less, 9 amino acid residues or less, 8 amino acid residues or less, 7 amino acid residues or less, 6 amino acid residues or less, 5 amino acid residues or less, 4 amino acid residues or less, 3 amino acid residues or less, 2 amino acid residues or less, or 1 amino acid residue or less in length. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (in one-letter amino acid code) GGGGS (“4GS”), or a multimer of the 4GS linker, for example, a repeat of two, three, four, or five 4GS linkers. In some embodiments, the linker is or includes GGGGS (SEQ ID NO: 148). In some embodiments, the peptide linker is or includes (GGGGS)2 or (GGGGS)3 as shown in SEQ ID NOs: 154 and 155, respectively. In some embodiments, the linker may also include a series of alanine residues alone or in addition to another peptide linker (e.g., a 4GS linker or a multimer thereof). In some embodiments, the linker is GSGGGSGGGGSGGGGS (SEQ ID NO: 104).

[0183] In some embodiments, the linker is a GS linker that is at least 10 amino acids in length, such as at least 15 amino acids in length. In some embodiments, the GS linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length, or any value in between any of the foregoing amino acid lengths. In some embodiments, the GS linker is 10 to 25 amino acids. In some embodiments, the GS linker is 10 to 20 amino acids. In some embodiments, the GS linker is GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 186); GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 187); GGGGGGSGGGGGSGGGGGS, i.e., (G5S)3 (SEQ ID NO: 188), GGSGGGGSGGGGSGGGGS (SEQ ID NO: 189) and GGGGSGGGGSGGGGS (SEQ ID NO: 155), or GSGGGSGGGGSGGGGS (SEQ ID NO: 104). In some embodiments, the GS linker is as set forth in SEQ ID NO: 104.

[0184] In some embodiments, the linker is a cleavable linker. In some embodiments, the use of a cleavable linker can be used to reliably release a free protein, i.e., a biological agent (e.g., a therapeutic agent) into the brain. Examples of cleavable linkers include acid-labile linkers. Acid-labile linkers include cis-aconitic acid, cis-carboxyalkadiene, cis-carboxyalkatriene, and poly-maleic anhydride. Other cleavable linkers are linkers that can attach to a primary alcohol group.

[0185] In some embodiments, the linker is a cleavable linker containing an endosome-specific protease cleavage site. In some embodiments, the endosome-specific protease cleavage site is a cathepsin cleavage site, and the linker is a linker cleavable by cathepsin. In some embodiments, the cathepsin is cathepsin B, cathepsin D, cathepsin K, cathepsin S, or cathepsin L. In some embodiments, the cathepsin cleavage site may be any of those described in PCT Publication No. WO2016 / 050934. In some embodiments, the linker containing the cathepsin cleavage site is shown by any one of SEQ ID NOs: 133 and 156 to 176. In some embodiments, the linker has a cathepsin B cleavage site. In some embodiments, the cathepsin B linker is shown by SEQ ID NO: 133.

[0186] Among the conjugates provided are modified cyclotides, such as bivalent cyclotide antibody fusions composed of an antibody linked to any of the modified cyclotides described herein. In some embodiments, the modified cyclotide is linked to the C-terminus of both heavy chains of the antibody. In some embodiments, the antibody is adalimumab. In some embodiments, the antibody fusion contains a heavy chain, a linker, and a modified cyclotide, such as any of those described in Section I.B, composed of the adalimumab heavy chain shown in SEQ ID NO: 102, and a light chain shown in SEQ ID NO: 103. In some embodiments, the antibody fusion contains a heavy chain, a linker, and a modified cyclotide, such as any of those described in Section I.B, composed of the trastuzumab heavy chain shown in SEQ ID NO: 103, and a light chain shown in SEQ ID NO: 137. In some embodiments, the antibody is a homodimer composed of two identical heavy chains and two identical light chains. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 72, for example, targeting TrfR. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 101, for example, targeting TrfR. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 81, for example, targeting IgFR. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 82, for example, targeting IgFR. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 85, for example, targeting LRP-1. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 87, for example, targeting LRP-1. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 89, for example, targeting LRP-1. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 83, for example, targeting RAGE. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 84, for example, targeting RAGE. In some embodiments, the linker is a GS linker that is at least 10 amino acids in length, such as at least 15 amino acids in length. In some embodiments, the GS linker is 10 to 25 amino acids. In some embodiments, the GS linker is shown in SEQ ID NO: 104.Antibody fusion conjugate having a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to any of the foregoing amino acid sequences is also provided herein. In some embodiments, the modified cyclotide binds to a BBB-R recognized by the modified cyclotide to transport the antibody across the blood-brain barrier.

[0187] Among the conjugates provided are monovalent cyclotide antibody fusions composed of a modified cyclotide, such as an antibody linked to any of the modified cyclotides described herein. In some embodiments, the modified cyclotide is linked to the C-terminus of one of the heavy chains of a heterodimeric antibody. In some embodiments, the antibody fusion contains a knob heavy chain of the antibody, a second heavy chain composed of a hole heavy chain of the antibody, and a light chain of the antibody, wherein a modified cyclotide, such as any of those described in Section I.B, is linked by a linker to the C-terminus of either the knob heavy chain or the hole heavy chain. The antibody can be a heterodimeric antibody (e.g., a knob and hole variant) of any desired therapeutic or diagnostic antibody that is desired to be transported across the blood-brain barrier. Non-limiting examples of antibodies include any of those described herein. In one embodiment, the antibody is aducanumab. In some embodiments, the antibody fusion contains a first heavy chain that is the aducanumab hole heavy chain shown in SEQ ID NO: 131, a second heavy chain composed of the aducanumab knob heavy chain shown in SEQ ID NO: 130, a linker and a modified cyclotide, such as any of those described in Section I.B, and a light chain shown in SEQ ID NO: 132. In another embodiment, the antibody fusion contains a first heavy chain that is the aducanumab hole heavy chain shown in SEQ ID NO: 131, a linker and a modified cyclotide, such as any of those described in Section I.B, a second heavy chain that is the aducanumab knob heavy chain shown in SEQ ID NO: 130, and a light chain shown in SEQ ID NO: 132. In a further embodiment, the antibody fusion contains a first heavy chain that is the trastuzumab hole heavy chain shown in SEQ ID NO: 136 or 178, a second heavy chain composed of the trastuzumab knob heavy chain shown in SEQ ID NO: 135, a linker and a modified cyclotide, such as any of those described in Section I.B, and a light chain shown in SEQ ID NO: 137. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 72, for example, targeting TrfR. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 101, for example, targeting TrfR. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 81, for example, targeting IgFR.In some embodiments, the modified cyclotide is shown in SEQ ID NO: 82, which targets, for example, IgFR. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 85, which targets, for example, LRP-1. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 87, which targets, for example, LRP-1. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 89, which targets, for example, LRP-1. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 83, which targets, for example, RAGE. In some embodiments, the modified cyclotide is shown in SEQ ID NO: 84, which targets, for example, RAGE. In some embodiments, the linker is a GS linker that is at least 10 amino acids in length, for example at least 15 amino acids in length. In some embodiments, the GS linker is 10 to 25 amino acids. In some embodiments, the GS linker is shown in SEQ ID NO: 104. Antibody fusion conjugate having a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to any of the foregoing amino acid sequences are also provided herein. In some embodiments, the modified cyclotide binds to the BBB-R recognized by the modified cyclotide and transports the antibody across the blood-brain barrier.

[0188] Among the conjugates provided are bispecific cyclotide antibody fusions composed of two different modified cyclotides, e.g., an antibody linked to any of the modified cyclotides described herein. In some embodiments, one modified cyclotide is linked to the C-terminus of one heavy chain of a heterodimeric antibody, and the other modified cyclotide is linked to the C-terminus of the other heavy chain of the heterodimeric antibody. In some embodiments, the antibody fusion comprises a knob heavy chain of an antibody linked to a first modified cyclotide by a linker at the C-terminus, a second heavy chain composed of a hole heavy chain of the antibody linked to a second modified cyclotide by a linker at the C-terminus, and a light chain of the antibody. The antibody can be a heterodimeric antibody (e.g., a knob and hole variant) of any desired therapeutic or diagnostic antibody that is desired to be transported across the blood-brain barrier. Non-limiting examples of antibodies include any of those described herein. In one embodiment, the antibody is aducanumab. In some embodiments, the antibody fusion comprises a first heavy chain that is the aducanumab hole heavy chain shown in SEQ ID NO: 131, a linker and a first modified cyclotide, a second heavy chain composed of the aducanumab knob heavy chain shown in SEQ ID NO: 130, a linker and a second modified cyclotide, and a light chain shown in SEQ ID NO: 132. In some embodiments, the antibody fusion comprises a first heavy chain that is the trastuzumab hole heavy chain shown in SEQ ID NO: 136, a linker and a first modified cyclotide, a second heavy chain composed of the trastuzumab knob heavy chain shown in SEQ ID NO: 135, a linker and a second modified cyclotide, and a light chain shown in SEQ ID NO: 137. In some embodiments, the first modified cyclotide and the second modified cyclotide are different and are each independently any of those described in Section I.B. In some embodiments, the modified cyclotides are independently selected from modified cyclotides that target TrfR (e.g., shown in SEQ ID NO: 72 or SEQ ID NO: 101), IgFR (e.g., shown in SEQ ID NO: 81 or SEQ ID NO: 82), LRP-1 (e.g., SEQ ID NO: 85, SEQ ID NO: 87 or SEQ ID NO: 89), or RAGE (e.g., shown in SEQ ID NO: 83 or SEQ ID NO: 84).In some embodiments, one of the first modified cyclotide and the second modified cyclotide is the modified cyclotide shown in SEQ ID NO: 72, and the other of the first modified cyclotide and the second modified cyclotide is the cyclotide shown in SEQ ID NO: 82. In some embodiments, one of the first modified cyclotide and the second modified cyclotide is the modified cyclotide shown in SEQ ID NO: 101, and the other of the first modified cyclotide and the second modified cyclotide is the cyclotide shown in SEQ ID NO: 82. Any of the various combinations of modified cyclotides, such as those described in Section I.B, can be used. In some embodiments, the linker is a GS linker that is at least 10 amino acids in length, such as at least 15 amino acids in length. In some embodiments, the GS linker is 10 to 25 amino acids. In some embodiments, the GS linker is shown in SEQ ID NO: 104. Also provided herein are antibody fusion conjugate having a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to any of the foregoing amino acid sequences. In some embodiments, the modified cyclotide binds to the BBB-R recognized by the modified cyclotide and transports the antibody across the blood-brain barrier.

[0189] In some embodiments, any of the conjugates provided, such as a fusion protein, binds to the BBB-R, and the binding is conferred by the peptide of the modified cyclotide of the conjugate. Methods for determining binding affinity, or relative binding affinity, are known in the art and include solid-phase ELISA immunoassay, ForteBio Octet, Biacore measurement, or flow cytometry. See, for example, Larsen et al., American Journal of Transplantation, vol. 5: 443-453 (2005); Linsley et al., Immunity, Vol 1 (9): 793-801 (1994). In some embodiments, the binding affinity can be measured by flow cytometry, for example, based on the mean fluorescence intensity (MFI) in a flow binding assay. In some embodiments, the conjugate provided, such as a fusion protein, has a binding affinity for the BBB-R determined by, for example, a solid-phase ELISA immunoassay, flow cytometry, or surface plasmon resonance (Biacore) assay.

[0190] In some embodiments, the conjugate (e.g., a fusion protein) has a binding affinity for the BBB-R that is greater than 10 nM and less than 1000 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R that is greater than 20 nM and less than 800 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of about 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, or any value between any of the foregoing. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 50 nM to about 500 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 50 nM to about 250 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 50 nM to about 100 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 100 nM to about 500 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 100 nM to about 250 nM. In some embodiments, the conjugate has a binding affinity for the BBB-R of from about 250 nM to 500 nM. In some embodiments, the binding affinity is from about 50 nM to 500 nM.

[0191] In embodiments where the conjugate (e.g., a fusion protein) contains a cleavable linker, such as an endosome-specific cleavable linker (e.g., a linker cleavable by cathepsin), the conjugate may have a higher binding affinity for the BBB-R. In some embodiments, the conjugate (e.g., a fusion protein) has a binding affinity for the BBB-R that is 50 nM or lower, e.g., from 1 pM to 10 nM.

[0192] In some embodiments, a conjugate containing a modified cyclotide exhibits improved or enhanced delivery of a bioactive agent or therapeutic agent to the brain as compared to the unconjugated form of the bioactive agent or therapeutic agent that is not conjugated to the modified cyclotide. In some embodiments, the delivery is improved or enhanced by about 1.2-fold, about 1.3-fold, 1.4-fold, 1.5-fold, 1.8-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold or greater. In some embodiments, greater than 0.1% of the bioactive agent or therapeutic agent injected into the bloodstream (e.g., by subcutaneous or intravenous injection) is delivered to the brain by the conjugate provided. In some embodiments, greater than 0.2%, greater than 0.25%, greater than 0.3%, greater than 0.35%, greater than 0.40% or more of the dose injected into the bloodstream (e.g., by subcutaneous or intravenous injection) is delivered into the brain by the conjugate provided.

[0193] III. Nucleic Acids and Methods of Making Further embodiments provided herein provide a nucleic acid sequence encoding the amino acid sequence of any of the provided binding molecules, such as any of the provided modified cyclotides or a fusion protein containing the same. In certain embodiments, the nucleic acid sequence may be contained in a vector, suitably an expression vector, optionally a display vector (including phage or cis-display vectors), in which case the coding DNA is operably linked to a peptide.

[0194] Methods of making provided binding molecules, such as modified cyclotides, or fusion proteins containing the same are also provided herein. In some embodiments, the method includes culturing a host cell under conditions capable of expressing the fusion protein. In some embodiments, the provided method further includes, after the culturing step, isolating the expressed modified cyclotide or fusion protein from the supernatant or from the lysate of the host cell.

[0195] In any of the provided embodiments herein, a nucleic acid encoding a binding molecule or fusion protein, such as any of the provided modified cyclotides provided herein, can be introduced into cells using recombinant DNA and cloning techniques. To do so, a recombinant DNA molecule encoding the polypeptide is prepared. Methods for preparing such DNA molecules are well known in the art. In some embodiments, the DNA molecule can be synthesized using chemical synthesis techniques such as the phosphoramidite method. In some cases, recombinant or synthetic nucleic acids can be generated by polymerase chain reaction (PCR). In some embodiments, the DNA can be cloned into a suitable transfection / transformation vector known to those of skill in the art. An expression vector containing the nucleic acid molecule is also provided.

[0196] In some embodiments, the expression vector can express a binding molecule such as a modified cyclotide or a fusion protein containing the same in a suitable cell under conditions suitable for protein expression. In some aspects, the nucleic acid molecule or expression vector comprises a DNA molecule encoding a modified cyclotide or fusion protein operatively linked to a suitable expression control sequence. Methods for making this operable linkage either before or after insertion of the DNA molecule into the vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, initiation signals, termination signals, cap signals, polyadenylation signals, and other signals involved in the control of transcription or translation.

[0197] Vectors are also provided that contain any of the nucleic acid sequences encoding binding molecules such as modified cyclotides or fusion proteins. Vectors suitable for use include, for example, those commonly used in genetic engineering techniques such as bacteriophages, plasmids, cosmids, viruses, or retroviruses. Vectors suitable for use may also contain other expression control elements, such as transcription initiation sites, transcription termination sites, ribosome binding sites, RNA splicing sites, polyadenylation sites, translation termination sites, etc. Vectors suitable for use may further contain additional regulatory elements such as transcription / translation enhancer sequences, and at least a marker gene or reporter gene that enables screening of the vector under appropriate conditions. Examples of marker genes suitable for use include the dihydrofolate reductase gene useful in eukaryotic cell culture and the G418 or neomycin resistance genes, as well as the ampicillin, streptomycin, tetracycline, or kanamycin resistance genes useful in E. coli and other bacterial cultures. Vectors suitable for use in the present invention may further contain a nucleic acid sequence encoding a secretion signal. These sequences are well known to those skilled in the art.

[0198] In some embodiments, the expression vector further contains a promoter sequence for controlling the expression of the modified cyclotide or fusion protein. As used herein, the term "promoter sequence" generally refers to a DNA sequence located upstream of a gene present within a DNA polymer, which provides the start site for transcription of the gene into mRNA. Promoter sequences suitable for use can be derived from viruses, bacteriophages, prokaryotic cells, or eukaryotic cells, and can be constitutive promoters or inducible promoters.

[0199] In some embodiments, the promoter sequence is operatively linked to a sequence encoding a modified cyclotide or a fusion protein. As used herein, the term "operatively linked" means that the first sequence is placed sufficiently close to the second sequence such that the first sequence can affect the second sequence or a region under the control of the second sequence. For example, a promoter sequence may be operatively linked to a gene sequence, and is typically located at the 5' end of the gene sequence such that the expression of the gene sequence is placed under the control of the promoter sequence. Further, regulatory sequences can be operatively linked to the promoter sequence to enhance the ability of the promoter sequence to promote transcription. In such cases, the regulatory sequences are generally located at the 5' end of the promoter sequence.

[0200] Examples of promoter sequences suitable for use include any one of the following: viral, bacterial cell, yeast cell, fungal cell, algal cell, plant cell, insect cell, animal cell, and human cell. For example, promoters useful in bacterial cells include, but are not limited to, the tac promoter, T7 promoter, T7 A1 promoter, lac promoter, trp promoter, trc promoter, araBAD promoter, and λPRPL promoter. Examples of promoters useful in plant cells include, for example, the 35S CaMV promoter, actin promoter, ubiquitin promoter, and the like. Examples of regulatory elements suitable for use in mammalian cells include the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter, CMV enhancer, or SV40 enhancer.

[0201] Depending on the vector and host cell line used, the recombinant gene product (protein) produced remains within the recombinant cell, is secreted into the culture medium, is secreted into the periplasm, or is retained on the outer surface of the cell membrane. The recombinant gene product (protein) produced by this method can be purified using various standard protein purification techniques, including but not limited to affinity chromatography, ion exchange chromatography, gel filtration, electrophoresis, reverse phase chromatography, chromatofocusing, etc. The recombinant gene product (protein) produced by this method is preferably recovered in a "substantially pure" form. As used herein, the term "substantially pure" refers to the purity of the purified protein that enables effective use of the purified protein as a commercial product.

[0202] In some embodiments, the provided method for producing a binding molecule such as a modified cyclotide or a fusion protein containing the same can be carried out using any host organism or cell capable of expressing a heterologous polypeptide and capable of genetic modification. For clarity, the term "host cell" is used throughout this specification, but it should be understood that, unless technically infeasible, the host organism can be used in place of the host cell.

[0203] In some embodiments, the host cell may be a yeast cell or various eukaryotic cells such as mammalian cells, e.g., Chinese hamster ovary (CHO) or HEK293 cells. In some embodiments, the host cell is a floating cell, and the polypeptide is engineered or produced in a culture suspension, e.g., a cultured floating CHO cell, e.g., CHO-S cells. In some examples, the cell line is a DHFR-deficient (DHFR-) CHO cell line, e.g., DG44 and DUXB11. In some embodiments, the cells are deficient in glutamine synthetase (GS), e.g., CHO-S cells, CHOK1 SV cells, and CHOZN® GS- / - cells. In some embodiments, CHO cells such as floating CHO cells may be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpiCHO-S cells.

[0204] In some embodiments, the host cell may be a prokaryotic cell such as E. coli. The transformed recombinant host is cultured under polypeptide expression conditions and then purified to obtain a soluble protein. The recombinant host cell can be cultured under conventional fermentation conditions such that the desired polypeptide is expressed. Such fermentation conditions are well known in the art. Finally, the polypeptides provided herein can be recovered and purified from the recombinant cell culture by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography. A protein refolding step can be used as desired upon completion of the mature protein construct. Finally, high performance liquid chromatography (HPLC) can be utilized in the final purification step.

[0205] In some embodiments, the polypeptides provided herein can also be made by synthetic methods. Solid-phase synthesis is a preferred technique for making individual peptides because it is the most cost-effective method for making small peptides. For example, well-known solid-phase synthesis techniques include the use of protecting groups, linkers, and solid-phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, the use of scavengers, and other aspects of solid-phase peptide synthesis. The peptides can then be assembled into the polypeptides provided herein.

[0206] IV. Pharmaceutical Compositions In some embodiments, pharmaceutical compositions are also provided herein that comprise any of the provided molecules of any of the foregoing embodiments, including any modified cyclotide or conjugate or fusion protein containing the same, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is used for treating a neurological disorder. In some embodiments, the pharmaceutical composition is used for diagnosing a neurological disorder.

[0207] Such compositions generally contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with the administration of pharmaceuticals. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the art, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional media or agent is intended, provided that it is not incompatible with the active compounds in the composition. Supplementary active compounds can also be incorporated into the composition.

[0208] The agents and pharmaceutical compositions of the present invention may take the form of solutions, suspensions, lotions, gels, tablets, pills, pellets, powders, controlled release formulations (e.g., sustained release or extended release), suppositories, emulsions, aerosols, sprays, capsules (e.g., capsules containing solutions or powders), liposomes, microparticles, or any other suitable formulation known in the art. Other examples of suitable pharmaceutical vehicles are described in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, see, for example, pages 1447-1676.

[0209] To assist in the dissolution of therapeutic agents (including cyclotides) in an aqueous environment, a surfactant can be added as a wetting agent. Examples of surfactants include anionic surfactants such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic surfactants can also be used, examples of which include benzalkonium chloride or benzethonium chloride. Potential non-ionic surfactants that can be included as surfactants in the formulation include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, polysorbate 20, 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. These surfactants, when used, may be present in the formulation of the peptide or nucleic acid or derivative either alone or as a mixture in different ratios.

[0210] To further enhance the intracellular uptake of the cyclotides of the present invention, additives such as the fatty acids oleic acid, linoleic acid, and linolenic acid can be included.

[0211] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration such as intravenous administration, intradermal administration, subcutaneous administration, intratumoral administration, oral administration (e.g., inhalation), transdermal administration (i.e., topical), transmucosal administration, and rectal administration. In certain embodiments, the composition is formulated for parenteral administration. In some embodiments, the composition is formulated for subcutaneous or intravenous administration.

[0212] Solutions or suspensions for parenteral, intradermal, or subcutaneous use may contain the following components: a sterile diluent such as water for injection, aqueous saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules made of glass or plastic, disposable syringes or multi-dose vials.

[0213] A pharmaceutical composition suitable for use in injection comprises a sterile injectable solution or dispersion, a sterile aqueous solution (in the case of water solubility) or dispersion for immediate preparation of the injectable solution or dispersion, and sterile powders. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injectability exists. The composition must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable that the composition contains isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride. By including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition, sustained absorption of the injectable composition can be achieved.

[0214] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent, with one or a combination of the ingredients listed above, and then, if necessary, filtering the solution through a sterile filter for sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the necessary other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and lyophilization to obtain the powder of the active ingredient and any additional desired ingredients, if any, from its previously sterile filtered solution.

[0215] Oral compositions generally include an inert diluent or an edible carrier. Oral compositions can also be enclosed in gelatin capsules or compressed into tablets. For therapeutic oral administration, the active compounds can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which case the compounds in the fluid carrier are applied orally, swirled in the mouth, and spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients or compounds of similar nature: binding particles such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0216] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or a nebulizer.

[0217] Systemic administration may be by transmucosal or transdermal means. For transmucosal or transdermal administration, permeation enhancers suitable for permeation across the body orifice are used in the formulation. Such permeation enhancers are generally known in the art and include, for example, surfactants, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using a spray-type nasal drug or a suppository. For transdermal administration, as is generally known in the art, the active compounds are formulated as ointments, plasters, gels, or creams.

[0218] The compounds can also be prepared in the form of suppositories (using conventional suppository bases such as cocoa butter and other glycerides, for example) or retention enemas for rectal delivery.

[0219] In one embodiment, the active compounds are prepared with a carrier that protects the compounds from rapid elimination from the body, such as, for example, controlled release formulations including implant and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, as described, for example, in U.S. Patent No. 4,522,811.

[0220] It is particularly advantageous to formulate the oral or parenteral compositions in unit dosage form (for ease of administration and uniformity of dosage). As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present disclosure are determined by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the inherent limitations in the art of compounding such active compounds for treating individuals.

[0221] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser, together with instructions for administration. These pharmaceutical compositions can be included in a diagnostic kit, together with instructions for use.

[0222] The pharmaceutical composition is administered in an effective amount for the treatment or prevention of a specific indication. The therapeutically effective amount generally depends on the body weight of the subject being treated, the physical or health condition of that subject, the extent of the condition being treated, or the age of the subject being treated. In some embodiments, the pharmaceutical composition can be administered in an amount in the range of about 50 μg per kg of body weight to about 50 mg per kg of body weight per dose. In some embodiments, the pharmaceutical composition can be administered in an amount in the range of about 100 μg per kg of body weight to about 50 mg per kg of body weight per dose. In some embodiments, the pharmaceutical composition can be administered in an amount in the range of about 100 μg per kg of body weight to about 20 mg per kg of body weight per dose. In some embodiments, the pharmaceutical composition can be administered in an amount in the range of about 0.5 mg per kg of body weight to about 20 mg per kg of body weight per dose.

[0223] In some embodiments, the pharmaceutical composition can be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition can be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition can be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition can be administered in an amount in the range of about 20 mg to about 200 mg per dose.

[0224] The pharmaceutical composition can be administered to a subject as needed. In some embodiments, an effective dose of the pharmaceutical composition is administered to the subject once or multiple times. In various embodiments, an effective dose of the pharmaceutical composition is administered to the subject once a month, less than once a month, for example, once every two months, once every three months, or once every six months. In other embodiments, an effective dose of the pharmaceutical composition is administered more than once a month, for example, once every two weeks, once a week, twice a week, three times a week, daily, or multiple times per day. An effective dose of the pharmaceutical composition is administered to the subject at least once. In some embodiments, an effective dose of the pharmaceutical composition can be administered multiple times, including over a period of at least one month, at least six months, or at least one year. In some embodiments, the pharmaceutical composition is administered to the subject if necessary to alleviate one or more symptoms of the condition.

[0225] V. Method of Use and Treatment In some embodiments, provided herein is a method for transporting an agent having biological activity across the blood-brain barrier of an individual, the method comprising administering to a mammal in need thereof a conjugate (e.g., a fusion protein) or a pharmaceutical composition of any of the foregoing embodiments.

[0226] In some embodiments, provided herein is also a method for treating a subject having a neurological disorder, the method comprising administering to the subject a conjugate (e.g., a fusion protein) or a pharmaceutical composition of any of the foregoing embodiments. In some embodiments, the conjugate (e.g., a fusion protein) or a pharmaceutical composition comprising the same is administered to the subject in an effective amount to effect treatment of the neurological disorder. Also provided herein is the use of a conjugate (e.g., a fusion protein) or a pharmaceutical composition comprising the same in such methods and treatments and in the preparation of a medicament for practicing such therapies. In some embodiments, the method is carried out by administering a conjugate (e.g., a fusion protein) or a composition comprising the same to a subject having, having had, or suspected of having a neurological disorder. In some embodiments, the method results in treatment of the neurological disorder in the subject. Also provided herein is the use of any of the compositions provided herein, e.g., pharmaceutical compositions, for treating a neurological disorder.

[0227] In some embodiments, provided herein is also a method for diagnosing a neurological disorder in a patient in need thereof, the method comprising administering to the patient a conjugate or a pharmaceutical composition of any of the foregoing embodiments, wherein the conjugate comprises a radiolabel.

[0228] In some embodiments, the mammal has a neurological disorder. In some embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Rett syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury. In some embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, stroke, brain tumor, brain metastasis, or traumatic brain injury (TBI). In some embodiments, the neurological disorder is a congenital disorder selected from the group consisting of Austin disease, Canavan disease, Gaucher disease, Hunter syndrome, Hurler - Scheie syndrome, Jansky - Bielschowsky disease, Krabbe disease, LCAT deficiency, Lowe syndrome, Maroteaux - Lamy syndrome, Morquio syndrome type A, Morquio syndrome type B, Sanfilippo syndrome type A, Sanfilippo syndrome type B, Sanfilippo syndrome type C, Sanfilippo syndrome type D, spinal muscular atrophy, Tay - Sachs disease, and Walker - Warburg syndrome.

[0229] In some embodiments, the neurological disorder includes non - neoplastic diseases and disorders, such as cancer / neoplastic diseases and related conditions. In some embodiments, the neurological disorder is an intracranial neoplasm.

[0230] In some embodiments, the neurological disorder or condition is for treating a stroke or other condition in which the brain has been damaged. In some embodiments, the cyclotide of the present invention is conjugated to a therapeutic agent capable of enhancing cognitive abilities such as memory.

[0231] In some embodiments, to treat a neurodegenerative disease, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to glucocerebrosidase is administered to a subject. In some embodiments, the neurological disease is Gaucher disease. In some embodiments, the neurological disease is Parkinson's disease. Glucocerebrosidase and conjugates containing it may be any of those described in Section II. Glucocerebrosidase is a lysosomal lumen membrane-bound hydrolase that interacts with glycolipids in the lysosomal lumen. Hereditary autosomal recessive mutations in the GBA gene that encodes glucocerebrosidase (GCase) result in a lack of activity and the clinical picture of Gaucher disease (GD) (Beutler and Grabowski, 1994). Mutations in GBA are also frequently found in patients with Parkinson's disease. In the case of dysfunction, the accumulation of substrates in tissue macrophages (Gaucher cells) and other related cells induces an altered inflammatory response (Komhaber et al, 2008; Schetz & Shankar, 2004; Smith et al, 2017). Gaucher cells are the most prominent pathological feature in the brain along with mononuclear phagocytes and neuronal cells and are involved in the pathology of GD (Beutler & Grabowski, 2001).

[0232] In some embodiments, the neurological disorder is Gaucher disease. Symptoms of GD include anemia, hepatosplenomegaly, bone lesions, and severe cases of neurological manifestation broadly classified into three clinical subtypes (Beutler et al, 1994; Komhaber et al, 2008). The most common form is classified as type I Gaucher disease, which has little neuronal disorder effect and is currently treatable by using enzyme replacement therapy (ERT). Types II and III are more severe forms of GD. Type II presents symptoms near birth and has an acute neuronal disorder phenotype that progresses until death during early infancy. Type III causes chronic severe neurological disorder symptoms including learning disabilities, cardiac abnormalities, and myoclonic epilepsy (Beutler et al, 1994; Sidransky et al, 2007; Sidransky & Lopez, 2012). In some embodiments, the provided method is for treating type I Gaucher disease. In some embodiments, the provided method is for treating type II Gaucher disease. In some embodiments, the provided method is for treating type III Gaucher disease.

[0233] In some embodiments, the subject to be treated has a mutation in the native glucocerebrosidase gene (also referred to as the GCase or GBA gene), and the mutation is associated with a neurological disorder such as GD in the subject. Approximately 300 mutations have been identified and are directly associated with the progression of GD, which results in a wide range of pathological disorders (Alfonso et al, 2007; Grabowski & Horowitz, 1997). Hereditary missense and nonsense mutations in the GBA gene can lead to misfolding, mistransport, and destabilization of the glucocerebrosidase enzyme. Two of the most frequently found mutations in the literature are the N370S mutation and the L444P mutation, which together account for 50% of all known GBA mutations. In some embodiments of the provided method, the subject has the N370S mutation and / or the L444P mutation of GCase. Patients with the mutated GCase variant N370S are typically diagnosed with type I GD and have a wide range of symptoms. N370S is involved in preserving and stabilizing the proper conformation of the active binding pocket (Lieberman et al, 2007). L444P presents a more severe form that is categorized as type II or III, and when this GBA gene mutation is present, in some cases, the hydrophobic structure is disrupted, thereby altering the domain II function (Lieberman et al, 2007).

[0234] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to an anti-HER2 antibody is administered to a subject. The anti-HER2 antibody and the conjugate containing it may be any of those described in Section II. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the conjugate is used to treat metastatic brain cancer. In some embodiments, the conjugate is used to treat Alzheimer's disease.

[0235] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to an anti-TNF-alpha antibody is administered to a subject. The anti-TNF-alpha antibody and the conjugate containing it may be any of those described in Section II. In some embodiments, the anti-TNF-alpha antibody is adalimumab. In some embodiments, the neurological disease or disorder is stroke, traumatic brain injury or Alzheimer's disease.

[0236] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to an anti-amyloid beta antibody is administered to a subject. The anti-amyloid beta antibody and the conjugate containing it may be any of those described in Section II. In some embodiments, the anti-amyloid beta antibody is aducanumab. In some embodiments, the neurological disease or disorder is Alzheimer's disease.

[0237] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to nerve growth factor (NGF) is administered to a subject. The NGF and the conjugate containing it may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Parkinson's disease, stroke or Alzheimer's disease.

[0238] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to granulocyte colony-stimulating factor (GCSF) is administered to a subject. The GCSF and the conjugate containing it may be any of those described in Section II. In some embodiments, the neurological disease or disorder is stroke or traumatic brain injury.

[0239] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to interleukin 10 (IL-10) is administered to a subject. IL-10 and conjugates containing it may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Alzheimer's disease.

[0240] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to brain-derived neurotrophic factor (BDNF) is administered to a subject. BDNF and conjugates containing it may be any of those described in Section II. In some embodiments, the neurological disease or disorder is ALS or depression.

[0241] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to activity-dependent neuroprotective protein (ADNP) is administered to a subject. ADNP and conjugates containing it may be any of those described in Section II. In some embodiments, the neurological disease or disorder is an autism spectrum disorder.

[0242] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to heparin N-sulfatase is administered to a subject. Heparin N-sulfatase and conjugates containing it may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Sanfilippo syndrome type A.

[0243] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to galactosylceramidase is administered to a subject for treating a neurological disease or disorder. The galactosylceramidase and the conjugate containing the same may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Krabbe disease.

[0244] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to alpha-galactosidase is administered to a subject for treating a neurological disease or disorder. The alpha-galactosidase and the conjugate containing the same may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Fabry disease.

[0245] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to sphingomyelinase is administered to a subject for treating a neurological disease or disorder. The sphingomyelinase and the conjugate containing the same may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Niemann-Pick disease.

[0246] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to cell lipase alpha is administered to a subject for treating a neurological disease or disorder. The cell lipase alpha and the conjugate containing the same may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Jansky-Bielschowsky disease.

[0247] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to alpha-glucosidase is administered to a subject. The alpha-glucosidase and the conjugate containing it may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Pompe disease.

[0248] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to tripeptidyl peptidase I is administered to a subject. The tripeptidyl peptidase I and the conjugate containing it may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Jansky-Bielschowsky disease or Batten disease.

[0249] In some embodiments, to treat a neurological disease or disorder, a conjugate (e.g., a fusion protein) containing a modified cyclotide linked to galactosamine-6-sulfatase is administered to a subject. The galactosamine-6-sulfatase and the conjugate containing it may be any of those described in Section II. In some embodiments, the neurological disease or disorder is Morquio syndrome.

[0250] In some embodiments, the methods and uses include administering to a subject (e.g., a human) a conjugate (e.g., a fusion protein) provided or a pharmaceutical composition comprising it. In some embodiments, it is administered to the subject by parenteral administration. In some embodiments, the administration is by intramuscular administration, subcutaneous administration, intravenous administration, topical administration, oral administration, or administration by inhalation. In some embodiments, the administration is by intramuscular administration. In some embodiments, the administration is by subcutaneous administration.

[0251] In some embodiments, an effective amount or a therapeutically effective amount of the provided conjugate (e.g., fusion protein) is administered to a subject. In some embodiments, the effective dose or therapeutically effective dose is a dose for treating neuropathy. In some embodiments, the effective dose or therapeutically effective dose is an amount sufficient to alleviate one or more signs and / or symptoms of neuropathy in the subject being treated, whether the alleviation is by inducing regression or elimination of such signs and / or symptoms or by inhibiting the progression of such signs and / or symptoms. The dose can vary depending on the age and size of the subject being administered, the target disease, condition, route of administration, etc. In certain embodiments, for example, the effective dose or therapeutically effective dose of the provided conjugate (e.g., fusion protein) for treating neuropathy in an adult human subject is from about 0.001 mg / kg to about 200 mg / kg, such as 0.01 mg / kg to 200 mg / kg or 0.1 mg / kg to 200 mg / kg. Depending on the severity of the infection, the frequency and duration of treatment can be adjusted.

[0252] In certain embodiments of the invention, the conjugate of the cyclotide of the invention and the therapeutic molecule is utilized as a separately administered composition or in combination with other therapeutic agents. These additional agents can include various immunotherapeutic drugs such as cyclosporine, methotrexate, adriamycin or cisplatin, and immunotoxins, or chemotherapeutic drugs such as tamoxifen, paclitaxel, oxaliplatin, vincristine and fluorouracil. The pharmaceutical composition can include a combination of various cytotoxic agents or other agents in combination with the polypeptide of the invention.

[0253] VI. Definitions Unless otherwise defined, all technical terms, notations, and other scientific and technical terms or terminology used in this specification are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined in this specification for clarity and / or for ready reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0254] Some definitions are provided to assist in understanding the present invention. All references cited in this specification are incorporated by reference in their entirety. Unless otherwise defined, all scientific and technical terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

[0255] As used in this specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". It is understood that the aspects and variations described in this specification include "consisting of" and / or "consisting essentially of" aspects and variations.

[0256] Throughout this disclosure, various aspects of the claimed subject matter are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the recitation of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values falling within that range. For example, when a range of values is presented, each intervening value, between the upper and lower limits of that range, and any other expressly stated or intervening range, is to be considered as included within the claimed subject matter. The upper and lower limits of these smaller ranges are also independently included within the smaller ranges and are to be considered as included within the claimed subject matter, subject to any specifically excluded limits within the expressly stated range. When either or both of the limits of an expressly stated range are included, ranges excluding either or both of those included limits are also to be considered as included within the claimed subject matter. This applies regardless of the breadth of the range.

[0257] As used herein, the term "about" refers to the normal error range for each respective value that would be readily understood in the art. References herein to "about" values or parameters include (and describe) embodiments that are directed to the value or parameter itself. For example, a recitation of "about X" includes a recitation of "X".

[0258] The term "conjugate" is used in its broadest sense to encompass all attachment or joining methods known in the art. Such conjugates include fusion proteins made by chemical conjugation and fusion proteins made by any other method. The term "conjugated" is used interchangeably with terms such as "linked", "bound", "associated", "fused" or "attached". A wide range of covalent and non-covalent forms of conjugation are known to those skilled in the art and fall within the scope of the present invention. For example, disulfide bonds, chemical linkages and peptide chains are all forms of covalent linkage. When non-covalent means of conjugation are preferred, the attachment means may be, for example, biotin-(strept)avidin linkage. Antibody (or antibody fragment)-antigen interactions can also be appropriately utilized to conjugate the cyclotide of the present invention to another moiety, such as a polypeptide, peptide, non-polypeptide moiety, small molecule, or biological drug.

[0259] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens rapidly and tend to remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of these methods can be used for the purposes of the present disclosure.

[0260] "Percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific peptide or polypeptide sequence, after aligning the candidate sequence with the specific peptide or polypeptide sequence and introducing gaps if necessary to achieve the maximum percent sequence identity, where any conservative substitutions are not considered part of the sequence identity. The alignment for determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MegAlign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0261] "Amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds having a structure different from the general chemical structure of an amino acid, but which function in a manner similar to a naturally occurring amino acid.

[0262] "Conservative variant" applies to both amino acid and nucleic acid sequences. An "amino acid variant" refers to an amino acid sequence. For a particular nucleic acid sequence, a conservative variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or, in the case of a nucleic acid that does not encode an amino acid sequence, refers to an essentially identical or attendant (e.g., naturally contiguous) sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at all positions where alanine is specified by a codon, the codon can be changed to another corresponding codon that is listed, and the encoded polypeptide is not changed. Such nucleic acid variations are "silent variations" and are one type of conservative modification variation. All nucleic acid sequences herein that encode polypeptides also describe silent variations of the nucleic acids. In certain circumstances, it will be understood by those skilled in the art that each codon in a nucleic acid (except for the AUG, which is usually the only codon for methionine, and the TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, with respect to the expression product, silent variations of the nucleic acids encoding the polypeptide are implicitly meant in the described sequences, but not with respect to the actual probe sequences. With respect to amino acid sequences, individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that change, add, or delete a single amino acid or a small percentage of amino acids of the encoded sequence are "conservative variant", including amino acid substitutions by chemically similar amino acids as a result of the change. Conservative substitution tables presenting functionally similar amino acids are well known in the art. Such conservative variants are in addition to, and not excluding, the polymorphic variants, interspecies homologs, and alleles disclosed herein.Generally, as conservative substitutions, 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) can be mentioned (for example, see Creighton, Proteins (1984)).

[0263] A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether naturally occurring or made in vitro by synthetic means. In some embodiments, polymers of amino acids of 2 to 50 amino acids are generally referred to as "peptides". The term "polypeptide" as used herein refers to naturally occurring polypeptides, precursor forms or products of proteins. This term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Polypeptides may be subject to maturation or post-translational modification processes including, but not limited to, glycosylation, proteolytic cleavage, lipid addition, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. The term "protein" as used herein refers to large polypeptide molecules as well as macromolecules containing one or more polypeptide chains.

[0264] The term "peptide" as used herein refers to, for example, a plurality of amino acids joined in a linear or cyclic chain. The term oligopeptide is generally used to refer to peptides having from 2 amino acids to about 50 amino acids or more. Peptides larger than about 50 are often referred to as polypeptides or proteins.

[0265] "Non-polypeptide moiety", as used herein, refers to an entity that contains neither a polypeptide sequence nor a three-dimensional folding. One of ordinary skill in the art can understand and determine whether a polypeptide molecule is a polypeptide or a peptide sequence, for example, by sequence homology or prediction or determination of structure. Such non-polypeptide moieties include, among others, nucleic acids and other polymers, peptides, proteins, peptide nucleic acids (PNAs), antibodies, antibody fragments, and small molecules. Appropriately, the non-polypeptide moiety is a biomolecule (e.g., including polynucleotides or peptides) and advantageously is a therapeutic or targeting molecule.

[0266] The term "antibody" is used herein in the broadest sense and includes intact antibodies and functional (antigen-binding) antibody fragments, including antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V H ) regions capable of specifically binding, and fragments such as single-chain variable fragments (scFv), including polyclonal and monoclonal antibodies.

[0267] "Antibody fragment" includes a portion of an intact antibody, the antigen-binding and / or variable regions of an intact antibody. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments; single-chain antibody molecules including single-chain Fvs (scFv) or single-chain Fabs (scFab); antigen-binding fragments of any of the above and multispecific antibodies derived from antibody fragments.

[0268] "Fab fragment" is an antibody fragment produced by papain digestion of a full-length immunoglobulin or a fragment having the same structure produced synthetically, e.g., by recombinant methods. The Fab fragment contains the light chain (V L and C L containing) and the variable domain of the heavy chain (V H ) and the heavy chain C HIt contains another chain containing one of the steady-state domain domains of (1).

[0269] An "scFv fragment" is a variable light chain (V) covalently connected by a polypeptide linker in any order L ) and a variable heavy chain (V H ). The linker is of a length such that the two variable domains are crosslinked without substantial interference. An exemplary linker is (Gly-Ser) n residues, and some Glu residues or Lys residues are dispersed throughout to increase solubility.

[0270] The term "operably linked", when applied to DNA sequences in an expression vector or construct, for example, means that those sequences are arranged so as to function cooperatively to achieve the intended purpose, i.e., the promoter sequence enables the initiation of transcription that proceeds through the linked coding sequence to the termination sequence.

[0271] The term "nucleic acid sequence", as used herein, is a sequence of nucleotides linked by covalent bonds, either single-stranded or double-stranded, with the 3' and 5' ends of each nucleotide joined by a phosphodiester bond. The polynucleotide may be composed of deoxyribonucleotide bases or ribonucleotide bases. The nucleic acid sequence may include DNA and RNA and may be manufactured synthetically in vitro or isolated from a natural source. The size of the nucleic acid sequence, also referred to herein as a "polynucleotide", is generally expressed as the number of base pairs (bp) for double-stranded polynucleotides or as the number of nucleotides (nt) for single-stranded polynucleotides. 1,000 bp or nt is equal to a kilobase (kb). Polynucleotides less than approximately 40 nucleotides in length are generally referred to as "oligonucleotides" and may include primers for use in the manipulation of DNA, such as by polymerase chain reaction (PCR).

[0272] The term "vector" is used to refer to either a linear or circular DNA molecule that can incorporate another nucleic acid (generally DNA) sequence fragment of appropriate size. Such DNA fragment(s) may contain additional segments that result in transcription of the gene(s) encoded by the DNA sequence fragment. Examples of additional segments include, but are not limited to, promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selectable markers, origins of replication, etc. A variety of suitable promoters for prokaryotic hosts (e.g., β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, lac, tac, T3, T7 promoters of E. coli) and a variety of suitable promoters for eukaryotic hosts (e.g., simian virus 40 early or late promoters, Rous sarcoma virus long terminal repeat promoter, cytomegalovirus promoter, adenovirus late promoter, EG-1a promoter) are available. Expression vectors often derive from plasmids, cosmids, viral vectors and yeast artificial chromosomes, and vectors are often recombinant molecules containing DNA sequences from several sources.

[0273] Certain embodiments provide an expression vector encoding a modified polypeptide or a fragment / domain thereof that includes the molecule of the invention. Thus, DNA encoding a relevant peptide of the invention can be inserted, according to conventional techniques, into a suitable expression vector (e.g., pGEM®, Promega Corp., USA) such that it is operably linked to a suitable expression sequence and transformed into a suitable host cell for protein expression (Sambrook J. et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY). Suitable host cells include bacteria, fungal cells, and cells of higher eukaryotic origin, preferably mammalian cells, generally human cells and cell lines, that can be grown in culture and are suitable for transformation with exogenous DNA. To facilitate purification of the peptides of the invention, the polypeptides (and corresponding nucleic acids) of the invention can include a purification sequence such as a His tag. In addition or alternatively, the modified polypeptide can be grown, for example, as a fusion with another protein and purified from bacterial cells as an insoluble inclusion body. This is particularly convenient when the modified polypeptide being synthesized can be toxic to the host cell in which it is expressed. Alternatively, the modified polypeptide can be synthesized in vitro using a suitable in vitro (transcription and) translation system (e.g., E. coli S30 extract system: Promega corp., USA).

[0274] In one embodiment of the invention, the vector is suitable as a polypeptide library display vector that allows the polypeptide gene product of the cyclotide-encoding gene to remain associated with the vector post-transcription.

[0275] As used herein, the term "polypeptide library display" refers to a system in which a collection of polypeptides or peptides that can form part or all of a library is available for selection based on a particular characteristic. The particular characteristic can be a physical, chemical, or functional characteristic. In a suitable display system, a cellular expression system is utilized, for example, the expression of a library of nucleic acids in a cell appropriately transformed, infected, transfected, or transduced, and the display of the encoded polypeptide on the surface of the cell. Alternative cellular expression systems can include compartmentalization and display in an emulsion. In a display system as needed, the coding function of a nucleic acid is associated with the physical, chemical, and / or functional characteristics of the polypeptide or peptide encoded by that nucleic acid. When utilizing such a display system, a polypeptide or peptide having the desired physical, chemical, and / or functional characteristics can be selected, and the nucleic acid encoding the selected polypeptide is readily isolated. Several display systems that associate the coding functionality of a nucleic acid with the attendant polypeptide product are known in the art, for example, bacteriophage display (phage display), ribosome display, compartmentalization and display in an emulsion, yeast display, puromycin display, bacterial display, display on a plasmid, covalent display, CIS display, etc. (see, for example, EP0436597 (Dyax), U.S. Patent No. 6,172,197 (McCafferty et al.), U.S. Patent No. 6,489,103 (Griffiths et al.).

[0276] The term "library" refers to a mixture of heterogeneous polypeptides or nucleic acids. A library is composed of a plurality of members, each having a substantially unique polypeptide or nucleic acid sequence. The differences in sequence among the members of the library give rise to the diversity present in the library. In the present invention, the library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form of an organism or cell transformed with a library of nucleic acids, such as a bacterium, virus, animal or plant cell. Usually, each individual organism (e.g., phage) or cell contains only one or a very limited number of members of the library. It is advantageous to incorporate the nucleic acid into an expression vector in order to enable the expression of the polypeptide encoded by the nucleic acid. Thus, in a preferred embodiment, the library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in the form of a nucleic acid capable of expressing and producing its corresponding polypeptide member, i.e., the polypeptide gene product. Thus, the population of host organisms has the potential to encode a wide variety of polypeptides. Certain embodiments of the present invention provide a library of polypeptides based on a modified version of a cyclobody polypeptide, wherein the diversity or differences among the members of the library are located in one or more polypeptide sequences of one or more loop or variable regions of one or more functional modules within the protein.

[0277] "Derived from" means that the corresponding cyclotide contains one or more amino acid modifications such as insertions, deletions or mutations as compared to the primary amino acid sequence of the cyclotide from which it is based. Thus, derivatives of cyclotides are also referred to as "modified cyclotides". For example, derivatives of cyclotide polypeptides may contain one or more (e.g., one, two, three, four, five or more) amino acid mutations, substitutions or deletions relative to the primary sequence of a selected modified polypeptide. Thus, the present invention encompasses the results of maturation experiments performed on cyclotide polypeptides to improve or alter one or more characteristics of the original or unmodified cyclotide. By way of example, one or more amino acid residues of a selected cyclotide polypeptide sequence can be randomly or specifically mutated (or substituted) using procedures known in the art (e.g., by modifying the coding DNA or RNA sequence). A library or population of the resulting derivatized polypeptides can be selected according to predetermined requirements such as improvement in specificity for a particular target receptor (e.g., a receptor involved in transcytosis); improvement in transcytosis across the blood-brain barrier; or improvement in drug properties (e.g., solubility, bioavailability, immunogenicity, etc.) by any method known in the art.

[0278] The term "isolated", with respect to a binding molecule such as a peptide or an antibody or other binding molecule, or a polynucleotide and vector encoding the same, means that it is at least partially free of other biomolecules from the cells or cell culture from which it is derived. Such biomolecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other substances such as cell debris and growth media. An isolated binding molecule may further be at least partially free of the components of the expression system, such as biomolecules from the host cell or from the growth media itself. In general, the term "isolated" does not mean that such biomolecules are completely absent, nor does it mean that water, buffer, or salts, or the components of a pharmaceutical formulation containing the antibody or fragment are absent.

[0279] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of a pharmaceutical composition sufficient to significantly and positively modify the symptoms and / or condition being treated (e.g., result in a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary depending on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of combination therapy, the particular active ingredient(s) utilized, the particular pharmaceutically acceptable excipient(s) and / or carrier(s) utilized, and like factors, and is within the knowledge and expertise of the attending physician.

[0280] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not inhibit the biological activity or properties of a compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting harmfully with any of the components of the composition in which it is contained.

[0281] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. The composition may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or a combination thereof.

[0282] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound of the present invention and other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition facilitates the administration of the compound to an organism. Multiple techniques for administering the compound exist in the art, including but not limited to intravenous administration, oral administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration, and topical administration.

[0283] As used herein, "disease or disorder" refers to a pathological condition in an organism that is caused by or results from a cause or condition, including but not limited to infection, acquired conditions, genetic conditions, and is characterized by identifiable symptoms.

[0284] As used herein, the terms "treat", "treating", or "treatment" refer to ameliorating a disease or disorder, e.g., slowing or arresting or reducing the onset of at least one of the root cause of the disease or disorder, e.g., the disorder, or its clinical symptoms.

[0285] As used herein, the term "subject" refers to an animal, including mammals (e.g., rats, mice, cats, dogs, cows, pigs, sheep, horses, goats, rabbits), e.g., humans. Generally, the subject is a human subject. The terms subject and patient may be used interchangeably.

[0286] As used herein, "as needed" or "as required" means that the event or situation described thereafter may or may not occur, and that the description encompasses both the case where the event or situation occurs and the case where it does not occur. For example, a group that is optionally substituted means that the group is either unsubstituted or substituted.

[0287] VII. Exemplary Embodiments The provided embodiments include the following: Embodiment 1. i) A peptide that binds to a blood-brain barrier transcytosis receptor (BBB-R) selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), leptin receptor (ObR), low density lipoprotein receptor-related protein 1 (LRP-1), and receptor for advanced glycation end products (RAGE), the peptide having an amino acid sequence of 2 to 50 amino acid residues, and ii) a cyclotide scaffold comprising the peptide of i) and a modified cyclotide comprising Structure (I):

Chemical formula

Examples

[0288] VIII. Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

[0289] (Example 1) Cyclotide (CYC) library construction A cyclotide library was created by inserting random peptides into the cyclotide MCOTII scaffold shown in SEQ ID NO: 2 as the backbone. The library was constructed based on the MCOTII native DNA sequence (SEQ ID NO: 4) as the cyclotide scaffold to contain a variant sequence between the first two cysteine residues of SEQ ID NO: 2 (referred to as "Loop 1"), and the library had different numbers of randomized peptides. To create the library, oligonucleotides were designed such that the variant sequence was inserted into Loop 1 and, when expressed, generated libraries with loops having 10 amino acids ("10-amino acid Loop 1"; SEQ ID NO: 5) or 12 amino acids ("12-amino acid Loop 1", SEQ ID NO: 6). Additional libraries were similarly generated to encode 14-20 randomized amino acids within Loop 1. All randomized amino acid positions in the library were encoded such that they would result in an equal mixture of codons for the 19 natural amino acids excluding cysteine.

[0290] The PCR products were cloned as NcoI-NotI digested fragments into the pSP1 phagemid pIII fusion vector derived from the similarly digested pHEN1 pIII vector (Hoogenboom et al., 1991, Nucleic Acids Res., 19: 4133-4137).

[0291] (i) PCR amplification of the cyclotide library For the primary PCR amplification, 10 × 50 μl amplifications were prepared using appropriate oligonucleotide primers (the "10 amino acid loop 1" or "12 amino acid loop 1" SEQ ID NO: 5 or 6 and pIIIseqrev SEQ ID NO: 7) for the loop library. Each 50 μl reaction mixture contained 10 ng of pCyclo1, 25 pmol of the appropriate forward and reverse primers, 0.1 mM dNTPs, 2.5 units of Taq DNA polymerase, and 1× NEB PCR reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton® X-100; NEB Ltd, Cambridge, UK). The reaction was carried out for 30 cycles of a PCR cycle of 94°C for 20 s; 60°C for 40 s; 72°C for 30 s, followed by 5 min at 72°C. The reaction products were purified using two Wizard PCR clean-up columns (Promega Ltd, Southampton, UK) per repertoire and eluted in 50 μl of water per column.

[0292] (ii) Pull-through reamplification of the selected DNA To prepare the final cyclotide library DNA product, for each cyclotide loop library, 40 × 50 μl amplifications were prepared using the oligonucleotide primers "Loop1PTrev" and "Loop5PTrev" (SEQ ID NOs: 8 and 9). Each 50 μl reaction mixture contained approximately 25 ng of the primary cyclotide loop library, 25 pmol of the appropriate forward and reverse primers, 0.1 mM dNTPs, 2.5 units of Taq DNA polymerase, and 1× NEB PCR reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton® X-100; NEB Ltd, Cambridge, UK). The reaction was carried out for 25 cycles of 94 °C, 20 s; 60 °C, 40 s; 72 °C, 30 s, followed by 5 min at 72 °C. The reaction products were purified using four Wizard PCR clean-up columns (Promega Ltd, Southampton, UK) per library and eluted in 100 μl of water per column. (iii) Cloning into vector pSP1

[0293] Each of the libraries, and 250 μg of pSP1 vector DNA, were digested with the enzymes NcoI and NotI (100 units each) at 37 °C for 5 h (NEB, Cambridge, UK), and purified using one Wizard PCR clean-up column per library, and four Wizard PCR clean-up columns for the digested vector DNA (Promega Ltd, Southampton, UK). Each DNA sample was then eluted in 100 μl of water. Half of each digested library DNA was ligated overnight at 16 °C in 400 μl with 50 μg of NcoI-NotI cut pSP1 vector and 4000 U of T4 DNA ligase (NEB Ltd, Southampton, UK). After incubation, the ligation was adjusted to 200 μl with nuclease-free water, and the DNA was precipitated using 1 μl of 20 mg / ml glycogen, 100 μl of 7.5 M ammonium acetate and 900 μl of ice-cold (−20 °C) absolute ethanol, mixed by vortexing, and spun at high speed for 20 min at 13,000 RPM in a microcentrifuge tube to pellet the DNA. The pellet was washed by centrifuging at 13,000 RPM for 2 min using 500 μl of ice-cold 70% ethanol, then vacuum dried and resuspended in 100 μl of DEPC-treated water. An aliquot of 1 μl of each library was electroporated into 80 μl of E. coli (TG1). The bacterial cells were grown for 1 h at 37 °C in 1 ml of SOC medium per cuvette and plated onto 2×TY agar plates supplemented with 2% glucose and 100 μg / ml ampicillin. 10 -4 dilutions, 10 -5 dilutions and 10 -6 dilutions were also plated to assess the library size. The colonies were grown overnight at 30 °C. The combined library size was approximately 2×10 10 clones, and the in-frame inserts exceeded 95%.

[0294] (iv) Phage amplification Separate phage stocks were prepared for each nucleotide library. Next, bacteria were scraped from the plate and placed into 50 ml of 2×TY broth supplemented with 20% glycerol, 2% glucose, and 100 μg / ml ampicillin. 1 ml of the bacterial culture was added to 50 ml of 2×TY culture broth supplemented with 1% glucose and 100 μg / ml ampicillin, and 10 11 kanamycin resistance units (kru) of M13K07 helper phage were added without shaking at 37°C for 30 minutes, and then infected while shaking at 200 RPM for 30 minutes. The infected bacteria were transferred to 200 ml of 2×TY broth supplemented with 25 μg / ml kanamycin, 100 μg / ml ampicillin, and 20 μM IPTG, and then incubated overnight while shaking at 30°C and 200 RPM. The bacteria were pelleted in a 50 ml Falcon tube at 4000 RPM for 20 minutes, 40 ml of 2.5 M NaCl / 20% PEG6000 was added to 400 ml of the particle supernatant, mixed vigorously, and incubated on ice for 1 hour to precipitate the phage particles. The particles were pelleted in a 250 ml Oakridge tube at 4°C in a Sorvall RC5B centrifuge at 11000 RPM for 30 minutes, then resuspended in 40 ml of water, 8 ml of 2.5 M NaCl / 20% PEG6000 was added to precipitate the particles again, and then incubated on ice for 20 minutes. The particles were pelleted again in a 50 ml Oakridge tube at 4°C in a Sorvall RC5B centrifuge at 11000 RPM for 30 minutes, then all traces of PEG / NaCl were removed using a pipette and resuspended in 5 ml of PBS buffer. Bacterial debris was removed by centrifugation at 13500 RPM for 5 minutes in a microcentrifuge. The supernatant was filtered through a 0.45 μm polysulfone syringe filter, adjusted to 20% glycerol, and stored at -70°C.

[0295] (Example 2) Library Selection for Blood-Brain Barrier Transporter Receptors The cyclotide phage library described in Example 1 was screened for receptor-binding peptides capable of binding to receptors that can induce receptor-mediated transcytosis (RMT) across the blood-brain barrier (BBB), including human HER3 / ErbB3 (Hu ErbB3), human and mouse transferrin receptors (Hu TrfR or Mu TrfR), human leptin receptor (Hu ObR), human insulin-like growth factor type 1 receptor (Hu IGFR), human low density lipoprotein receptor-related protein 1 (Hu LRP-1), and human receptor for advanced glycation end products (Hu RAGE).

[0296] Selection was performed using standard phage display selection methods on a solid phase coated with a recombinant protein composed of the extracellular domain of each human (Hu) or mouse (Mu) receptor (recombinant Fc fusion protein obtained from R&D Systems, or His-tagged receptor from Sino Biologicals).

[0297] For TfR cyclotide selection, one round of selection was performed even in the presence of human holotransferrin, and thus selection for human TfR was carried out when human holotransferrin was bound to the receptor. In this selection, CYC40 (SEQ ID NO: 95) was the most dominant peptide hit in this selection.

[0298] After phage ELISA to identify receptor-binding cyclotides, plasmid DNA was then prepared and the sequences of individual cyclotide clones were determined by sequencing. Binding specificity was confirmed by ELISA.

[0299] Table E1 shows the sequences of the selected cyclotides and the receptors to which they bind.

Table E1-1

Table E1-2

Table E1-3

[0300] (Example 3) Generation and Evaluation of Adalimumab Antibody-Cyclotide Fusion Proteins The selected cyclotide peptides described in Table E1 were fused with the antibody adalimumab. The cyclotide was linked to the C-terminus of the polynucleotide encoding the heavy chain (SEQ ID NO: 102) or light chain (SEQ ID NO: 103) of adalimumab via a Gly-Ser linker (e.g., SEQ ID NO: 104). An exemplary depiction of the adalimumab-cyclotide fusion protein is shown in Figure 1A.

[0301] A gene construct for expressing an antibody-cyclotide fusion was prepared by cloning a cyclotide gene fused to the 3'-end of a nucleotide sequence encoding an antibody heavy chain gene (having a 5' IL2 secretion signal peptide sequence) into a mammalian expression vector. The adalimumab light chain gene (having a 5' secretion signal peptide sequence) was also cloned into a mammalian expression vector. These two vector constructs were used to co-transfect HEK293F cells, and the cells were grown for 4 - 6 days while shaking at 37 °C, 8% CO2, and 130 rpm. After removing the cells and filtering the supernatant, the resulting adalimumab-cyclotide fusion was purified and concentrated by protein A affinity chromatography. For two exemplary different adalimumab-cyclotide fusion proteins, no signs of enhanced aggregation were shown as determined by Western blot compared to the antibody in the version not fused to the cyclotide (data not shown).

[0302] (Example 4) Internalization of a Cyclotide-Antibody Fusion Targeting the Transferrin Receptor (TfR) An exemplary cyclotide CYC17 (also called HT2; shown in SEQ ID NO: 72) that binds to the human transferrin receptor was fused with adalimumab. The adalimumab-CYC17 cyclotide fusion was compared to adalimumab for internalization via the transferrin receptor.

[0303] Cloning, Expression, and Purification of Adalimumab and Adalimumab-HT2 Cyclotide Fusion To express the adalimumab heavy chain (SEQ ID NO: 102) and light chain (SEQ ID NO: 103), gene constructs were prepared by cloning the adalimumab heavy chain gene (having a 5’ IL2 secretion signal peptide sequence) and the adalimumab light chain gene (having a 5’ secretion signal peptide sequence) into mammalian expression vectors, respectively.

[0304] The CYC17 cyclotide shown in SEQ ID NO: 72 was linked to the C-terminus of the adalimumab heavy chain (SEQ ID NO: 102) via a Gly-Ser linker (e.g., SEQ ID NO: 104). A gene construct for expressing the adalimumab-CYC17 cyclotide fusion protein was prepared by cloning the cyclotide gene fused to the 3’ end of the nucleotide sequence encoding the antibody heavy chain gene (having a 5’ IL2 secretion signal peptide sequence) into a mammalian expression vector.

[0305] HEK293F cells were co-transfected with the expression constructs of the adalimumab heavy chain-CYC17 cyclotide fusion protein (or adalimumab heavy chain alone) and the adalimumab light chain (amino acid sequence shown in SEQ ID NO: 103), and the cells were grown for 4 - 6 days while shaking at 37 °C, 8% CO2, and 130 rpm. After removing the cells and filtering the supernatant, the obtained adalimumab-CYC17 cyclotide fusion protein was purified by protein A affinity chromatography and concentrated using a centrifugal filtration unit (Amicon) with a fractional molecular weight of 50 KDa.

[0306] Fluorophore Labeling of Adalimumab and Adalimumab-HT2 Cyclotide Fusion The purified adalimumab and adalimumab-CYC17 cyclotide fusion protein were fluorescently labeled by incubating with a 5- to 10-fold molar excess of Alexafluor 488 N-hydroxysuccinimide (NHS) ester at room temperature, pH approximately 8.3, in the dark for 1 hour. The free dye was then removed by passing through one or two desalting columns. Preparation of human cells expressing human or mouse transferrin receptor

[0307] HEK293F cells were first passaged continuously two to three times and then transfected with an expression construct encoding the human or mouse transferrin receptor (hTfR and mTfR, respectively), and grown for 2 - 3 days at 37 °C, 8% CO2, with shaking at 130 rpm in preparation for the assay.

[0308] Assay for internalization of the labeled adalimumab and adalimumab-CYC17 cyclotide fusion by human cells expressing transferrin receptor. After growing for approximately 64 hours, 1×10 5 ~5×10 5The transfected cells (and untransfected and mock-transfected controls) were transferred to a 96-well V-bottom plate, pelleted, the supernatant discarded, resuspended in 40 μL of fresh medium containing 50 nM of labeled adalimumab or adalimumab-CYC17 cyclotide fusion, and grown for 4 hours with shaking at 37 °C, 8% CO2, 500 rpm. The cells were then pelleted, the supernatant discarded, resuspended in 40 μL of fresh medium containing 250 nM of anti-488 quenching antibody (or medium lacking the 488 quenching antibody as a control), incubated on ice for 5 minutes, pelleted, the supernatant discarded, washed with 200 μL of cold FACS buffer, pelleted, the supernatant discarded, and resuspended in 200 μL of cold FACS buffer. Each sample was then subjected to FACS analysis using the FITC fluorescence detection channel, normal-shaped live cells were selected using gate 1 (forward scatter, linear and side scatter, linear), single cells were selected using gate 2 (side scatter, area and side scatter, linear), and the logarithmic FITC signal was measured for 20,000 - 50,000 cells per sample. Subsequent data analysis was performed using the FlowJo software package. The mean FITC fluorescence intensity of each sample was monitored as an indication of the extent to which the labeled adalimumab or adalimumab-CYC17 cyclotide fusion had been internalized. From the results shown in Figure 2, it was demonstrated that the adalimumab-CYC17 cyclotide fusion was internalized by both mouse TfR (mTfR) and human TfR (hTfR), whereas adalimumab was not internalized.

[0309] (Example 5) Affinity modulation of transferrin receptor-binding cyclotides The selected TfR-binding cyclotides identified in Table E1 were further evaluated for their affinity for TfR, and mutant cyclotides with reduced affinity were generated.

[0310] A gene construct was prepared for expressing histidine-scanning mutants and alanine-scanning mutants of adalimumab-CYC17 cyclotide fusions. Variants of CYC17 (SEQ ID NO: 72) were prepared by PCR-based assembly of overlapping oligonucleotide primers encoding each CYC17 variant. Each mutant CYC17 cyclotide gene was fused to the 3' end of the nucleotide sequence encoding the adalimumab heavy chain gene (having a 5' secretory signal peptide sequence) via a sequence encoding a flexible linker and cloned into a mammalian expression vector. A vector construct encoding each adalimumab heavy chain-mutant CYC17 cyclotide fusion and a vector construct encoding the adalimumab light chain were co-transfected into HEK293F cells, and the cells were grown for 4 - 6 days while shaking at 37 °C, 8% CO2, 130 rpm. After removing the cells and filtering the supernatant, the resulting adalimumab-cyclotide fusion was purified and concentrated by protein A affinity chromatography.

[0311] Exemplary CYC17 mutants are described in Table E2. Changes in binding to the human transferrin receptor for the exemplary CYC17 mutants were evaluated by ELISA. Briefly, His-tagged human transferrin receptor (TrFR) was coated onto a microtiter plate at 1 μg / mL at 4 °C overnight, blocked with 2% non-fat milk / PBS for 1 hour at room temperature, and various concentrations of purified antibody-cyclotide fusions from 0.001 nM to 100 nM were contacted with the coated transferrin receptor. Binding of the antibody cyclotide fusion to the coated human transferrin receptor was monitored using a 1:5000 diluted anti-human IgG-HRP conjugate (Jackson Laboratories) and TMB substrate reagent. As shown in Figure 3, CYC17 mutants having the amino acid substitutions W9H, L11H, S13H, W14H, and G15H exhibited a decrease in affinity for the transferrin receptor.

Table E2-1

Table E2-2

[0312] (Example 6) Evaluation of the binding affinity of aducanumab and adalimumab antibody-cyclotide fusions

[0313] Monovalent, bivalent, and bispecific antibody-cyclotide fusions were created by engineering the knob (K) and hole (H) of the Fc domain. The heavy chain of the antibody aducanumab (Aduhelm™; SEQ ID NO: 129) was engineered by CH3 mutation to contain either a knob mutation (K; heavy chain-K shown in SEQ ID NO: 130) or a hole mutation (H; heavy chain-H shown in SEQ ID NO: 131). For IGF1R targeting, the heavy chain-K of aducanumab was fused to the cyclotide CYC27 (SEQ ID NO: 82). For hTfR targeting, the heavy chain-K or heavy chain-H of aducanumab was fused to the cyclotide CYC17 (SEQ ID NO: 72). For the expression and production of monovalent and bispecific heterodimeric aducanumab-cyclotide fusions, nucleic acids encoding the heavy chain-K fusion and the heavy chain-H fusion were introduced into HEK293F together with the nucleic acid encoding the light chain of aducanumab (SEQ ID NO: 132). Schematic diagrams of monovalent and bispecific heterodimeric aducanumab-cyclotide fusions are shown in FIGS. 1B-1D.

[0314] For hTfR targeting, the heavy chain of the antibody adalimumab (Humira™; SEQ ID NO: 102) was fused to the cyclotide CYC17 as described above. For the expression and production of the bivalent adalimumab-cyclotide fusion, HEK293F cells were co-transfected using the expression construct of the adalimumab heavy chain-CYC17 cyclotide fusion and the expression construct of the adalimumab light chain (SEQ ID NO: 103). A schematic diagram of the bivalent adalimumab-CYC17 cyclotide fusion is shown in FIG. 1A.

[0315] By a standard receptor-substrate binding assay, the affinity of the monovalent variant of the aducanumab-CYC17 cyclotide fusion to hTfR was reduced, while the divalent adalimumab-CYC17 cyclotide fusion and the bispecific aducanumab-H-CYC17-K-CYC27 were shown to bind to hTfR with higher affinity. The results for the binding affinity are shown in Figure 4. The EC50 binding affinity is shown in Table E3. Without wishing to be bound by theory, antibody-cyclotide fusions with higher affinity may not be released from endosomes after RMT, while antibody-cyclotide fusions with lower affinity may be released from endosomes and transported to the brain after interaction with receptors at the blood-brain barrier.

Table E3

[0316] (Example 7) Generation and evaluation of monovalent and bispecific trastuzumab antibody-cyclotide fusions Monovalent and bispecific antibody-cyclotide fusions were generated by fusion with the exemplary antibody trastuzumab, where cyclotide was also fused to the antibody using a flexible linker (SEQ ID NO: 104) with or without a cathepsin B cleavage sequence (CB; SEQ ID NO: 133; Taha TA et al. FEBS Lett. 2006 Nov 13; 580 (26): 6047-54). Adding a cleavable cathepsin B cleavage site between the linker and the cyclotide was to facilitate cleavage of the cyclotide peptide portion from the fusion protein after the fusion protein had translocated into the endosome, enhancing the release of the antibody from the endosome and the transport of the antibody across the blood-brain barrier.

[0317] The heavy chain of the antibody trastuzumab (Herceptin®, SEQ ID NO: 134) was engineered by CH3 mutations to contain either a knob mutation (K; heavy chain-K shown in SEQ ID NO: 135) or a hole mutation (H; heavy chain-H shown in SEQ ID NO: 136). For human transferrin receptor (hTfR) targeting, the heavy chain-K or heavy chain-H of trastuzumab was fused with cyclotide CYC17 (SEQ ID NO: 72) or the lower affinity variant CYC17-15H (SEQ ID NO: 101). For each, the cyclotide was linked to the C-terminus of the heavy chain-K (SEQ ID NO: 135) of trastuzumab via a Gly-Ser linker (e.g., SEQ ID NO: 104), and in some cases further included a cathepsin B cleavage sequence (SEQ ID NO: 133). For the bispecific construct, for IGF1R targeting, the heavy chain-K of trastuzumab was fused with cyclotide CYC27 (SEQ ID NO: 82).

[0318] Table E4 shows the sequences of the generated constructs.

Table E4-1

Table E4-2

[0319] For the expression and production of monovalent and bispecific heterodimeric trastuzumab-cyclotide fusions, nucleic acids encoding the respective full heavy chain-K fusions (e.g., SEQ ID NO: 139, 140, 141 or 142) and full heavy chain-H fusions (e.g., SEQ ID NO: 136 or 143) were introduced into HEK293F together with a nucleic acid encoding the light chain of trastuzumab (SEQ ID NO: 137). For the trastuzumab control, nucleic acids encoding the wild-type (WT) trastuzumab heavy chain (SEQ ID NO: 134) and a nucleic acid encoding the light chain of trastuzumab (SEQ ID NO: 137) were introduced into HEK293F cells. The cells were grown for 4 - 6 days while shaking at 37 °C, 8% CO2, 130 rpm. After removing the cells and filtering the supernatant, the resulting trastuzumab-cyclotide fusion protein was purified and concentrated by protein A affinity chromatography.

[0320] Results for the expression of exemplary constructs are shown in Figure 5. As determined by ELISA, the trastuzumab fusion proteins (C, D, E and F in Table E4) were expressed at levels similar to the parental antibody (A in Table E4), and no signs of aggregation were observed from SDS-PAGE gel electrophoresis.

[0321] (Example 8) Expression and Activity of Glucosylceramidase Fusion Cyclotides An exemplary human transferrin receptor (hTfR)-binding cyclotide CYC17 (SEQ ID NO: 72) was fused to a glucosylceramidase enzyme (GlcCSase; SEQ ID NO: 144), an enzyme that cleaves the beta-glucosidic linkage of the chemical glucosylceramide by hydrolysis. The cyclotide was linked to the C-terminus of glucosylceramidase via a Gly-Ser linker (e.g., SEQ ID NO: 104). A nucleic acid sequence encoding the CYC17-glucosylceramidase fusion cyclotide shown in SEQ ID NO: 146 was expressed in HEK293 cells.

[0322] The expression and integrity of the glucocerebrosidase-CYC17 fusion were evaluated by Western blot using an anti-glucocerebrosidase antibody (data not shown). Western blot analysis showed the expression of a 61.1 kDa protein corresponding to the size of the GlcCSase-CYC17 fusion cyclotide (data not shown).

[0323] Glucocerebroside Activity Assay A standard glucocerebroside activity assay based on the ability of the GlcCSase-CYC17 fusion cyclotide to react with its substrate was performed to evaluate the functional integrity / activity of the fusion cyclotide. Specifically, enzyme activity was assayed using the SensoLyte Red Glucocerebrosidase Fluorometric Assay Kit (AnaSpec) according to the manufacturer's protocol. In this assay, a resorufin-β-glucoside substrate that releases resorufin, which is excited at 570 nm and emits fluorescence at 610 nm when the glycosidic linkage is cleaved by hGCSase, is used. This kinetic assay was performed in triplicate at room temperature in an opaque 96-well plate using a SpectraMax fluorescence plate reader over a 1-hour period with 10 μL of filtered and concentrated supernatant from HEK293F hGCSase-CYC17-expressing cultures, 40 μL of assay buffer, and 50 μL of substrate solution. Positive control (GCSase enzyme), inhibitor control, vehicle-only control, and substrate-only control were assayed in triplicate in parallel.

[0324] As shown in Figure 6A, after incubation with the substrate, the GlcCSase-CYC17 fusion cyclotide showed a time-dependent increase in the fluorescence emitted, indicating higher enzyme activity than levels corresponding to the negative control.

[0325] Binding to the Transferrin Receptor The binding of the GlcCSase-CYC17 fusion cyclotide was evaluated by enzyme-linked immunosorbent assay (ELISA) of hGCSase-HT2 using the human transferrin receptor (hTfR) antigen. Wells of a 96-well plate with high binding half area in duplicate (Corning) were directly coated with 125 ng of hTfR in 50 μL of Tris-buffered saline + 2% skim milk containing 0.1% (v / v) Tween® 20 (TBST) (or TBST containing only 2% skim milk as a negative control) overnight at 4 °C. After removing the supernatant, the wells were blocked with 50 μL of TBST containing 2% skim milk for 2 hours at room temperature. The blocking agent was removed, and 25 μL of the filtered and concentrated supernatant from the HEK293F hGCSase-CYC17 fusion expression culture (or 25 μL of the filtered and concentrated supernatant from the HEK293F expression culture without hGCSase expression as a negative control) + 25 μL of TBST containing 4% skim milk were added to the a...

Claims

1. A modified cyclotide containing a peptide that binds to the blood-brain barrier transcytosis receptor (BBB-R), The peptide is inserted into at least one loop of the cyclotide scaffold shown in SEQ ID NO: 2, SEQ ID NO: 1, or SEQ ID NO: 3, or replaces one or more amino acids in at least one loop, and the peptide consists of about 2 to 50 amino acid residues. A modified cyclotide in which the BBB-R is selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), leptin receptor (ObR), low-density lipoprotein receptor-related protein 1 (LRP-1), and receptor for advanced glycation end products (RAGE).

2. (A) The at least one loop is loop 1, loop 5, or loop 6, or a combination thereof; (B) The at least one loop is loop 1, If necessary, the peptide replaces the loop 1 amino acid between cysteine ​​4 and cysteine ​​11 of the cyclotide scaffold; (C) The peptide is inserted into only one loop of the cyclotide scaffold and replaces an amino acid within that loop. as needed, (a) The one loop is Loop 1; and / or (b) The peptide replaces the loop 1 amino acid between cysteine ​​4 and cysteine ​​11 of the cyclotide scaffold. Furthermore, if necessary, the cyclotide scaffold is SEQ ID NO: 2, and the peptide is 2 to 50 amino acid residues; and / or (D) The modified cyclotide according to claim 1, wherein the peptide comprises 10 to 25 amino acids.

3. (A) The BBB-R is a transferrin receptor (TrfR); (B) The peptide comprises the sequence shown in any one of SEQ ID NOs: 26-34 and 49-54; and / or (C) (a) (i) The peptide has a consensus motif represented as xxxxxHxxSWGx (SEQ ID NO: 177); (ii) The peptide comprises a sequence having one, two, three, or four amino acid substitutions compared to any one of the sequences shown in SEQ ID NOs: 26-34 and 49-54. If necessary, the peptide may include a sequence having one, two, three, or four amino acid substitutions compared to the sequence shown in SEQ ID NO:

26. (iii) The amino acid substitution(s) is a substitution of one amino acid to another amino acid selected from histidine or alanine; (iv) The peptide comprises the sequence shown in any one of SEQ ID NOs. 55 and 117-128; and / or (v) The modified cyclotide comprises the sequence shown in any one of SEQ ID NOs: 101 and 105-116; or (b) The modified cyclotide according to claim 1, wherein the modified cyclotide comprises the sequence shown in any one of SEQ ID NOs. 72-80 and 95-100.

4. (A) (a) The BBB-R is a leptin receptor; (b) The peptide comprises the sequence shown in any one of SEQ ID NOs: 10 to 23; and / or (c) The modified cyclotide comprises the sequence shown in any one of SEQ ID NOs: 56 to 69; (B) (a) The BBB-R is ErbB3; (b) The peptide comprises the sequence shown in SEQ ID NO: 24 or 25; and / or (c) The modified cyclotide comprises the sequence shown in Sequence ID No. 70 or 71; (C) (a) The BBB-R is an insulin-like growth factor type 1 receptor (IGFR); (b) The peptide comprises the sequence shown in SEQ ID NO: 35 or 36; and / or (c) The modified cyclotide comprises the sequence shown in SEQ ID NO: 81 or 82; or (D) (a) The BBB-R is RAGE; (b) The peptide comprises the sequence shown in SEQ ID NO: 37 or 38; and / or (c) The modified cyclotide according to claim 1, wherein the modified cyclotide comprises the sequence shown in SEQ ID NO: 83 or 84.

5. (A) The BBB-R is the low-density lipoprotein receptor-related protein 1 (LRP-1); (B) The peptide comprises the sequence shown in any one of SEQ ID NOs: 39 to 48; (C) The peptide is (a) Sequence ID 39; (b) Sequence ID 43; or (c) Sequence ID 47 Includes the sequence shown; and / or (D) The modified cyclotide may contain one of the sequence numbers 85 to 94, if necessary. (i) Sequence ID 85, (ii) Sequence ID 89 or (iii) Sequence ID 93 A modified cyclotide according to claim 1, comprising the sequence shown in [image / figure].

6. A peptide having a length of 6 to 50 amino acids and binding to the receptor (BBB-R) involved in blood-brain barrier transcytosis, comprising the amino acid sequence shown in any of SEQ ID NOs: 10-55 or 117-128, If necessary, the peptide is a synthetic peptide.

7. A binding molecule comprising a binding scaffold and the peptide described in claim 6, If necessary, the binding scaffold is a binding molecule that is a cysteine ​​knot protein.

8. A nucleic acid molecule encoding a modified cyclotide according to any one of claims 1 to 5 or a binding molecule according to claim 7.

9. A vector comprising the nucleic acid molecule described in claim 8.

10. A host cell comprising the nucleic acid molecule described in claim 8 or a vector comprising the nucleic acid molecule described in claim 8.

11. A method for producing a modified cyclotide or a binding molecule, comprising the steps of: introducing a nucleic acid according to claim 8 or a vector containing a nucleic acid molecule according to claim 8 into a host cell; culturing the host cell under conditions in which a protein is expressed in the cell; and, if necessary, purifying the protein from the cell.

12. A purified modified cyclotide or binding molecule produced by the method of claim 11.

13. A conjugate comprising a modified cyclotide according to any one of claims 1 to 5 or a binding molecule according to claim 7, and a biologically active substance, optionally, (A) The biologically active substance is a small molecule, peptide, or protein; (B) The biologically active substance is a diagnostic substance or a therapeutic substance; (C) A fusion protein comprising the modified cyclotide operably linked to a biologically active substance which is a protein or peptide; and / or (D) The biologically active substance is (a) It is an antibody, If necessary, the antibody targets antigens selected from the group consisting of human epidermal growth factor receptor 2 (HER2), beta-secretase 1 (BACE1), amyloid beta (A-beta), epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, cell death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, and TNF-alpha; (b) A growth factor or hormone, If necessary, the biologically active substance is a growth factor, and the growth factor is nerve growth factor (NGF) or granulocyte colony-stimulating factor (GCSF); or (c) It is an enzyme, as needed, (i) The enzyme is a ceramide-degrading enzyme, lipase, hydrolase-type enzyme or sulfatase; and / or (ii) A conjugate in which the enzyme is a ceramide-degrading enzyme, and the ceramide-degrading enzyme is glucocerebrosidase, galactocerebrosidase, or alpha-galactosidase.

14. A pharmaceutical composition comprising the conjugate and pharmaceutical carrier described in claim 13.

15. (A) Treatment of neurological disorders; or (B) Diagnosis of neurological disorders The pharmaceutical composition according to claim 14 for use in [the specified area].

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