Blood brain barrier translocation peptides and related molecules and methods of use thereof

Abstract

Peptides capable of binding to receptors that mediate receptor-mediated endocytosis transport (RMT) across the blood-brain barrier (BBB), as well as binding molecules incorporating the peptides, are provided. Conjugates, including fusion proteins, comprised of the peptide or the binding molecule and a therapeutic or diagnostic agent are also provided. In some embodiments, the conjugate is capable of crossing the blood-brain barrier after parenteral administration to allow the therapeutic agent or the diagnostic agent to function in the central nervous system. Methods of making and using the provided peptides and molecules are also provided.

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 342,622, filed on May 16, 2022, entitled “Blood-Brain Barrier Translocation Peptides and Related Molecules and Methods of Use Thereof,” the contents of which are incorporated by reference in their entirety. Incorporation by Reference to the Sequence Listing

[0002] This application is filed with a sequence listing in electronic form. The sequence listing is provided as a file named 166302000240SeqList.xml, created on May 15, 2023, and having a size of 253,440 kilobytes. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. Technical Field

[0003] The disclosure provides peptides capable of binding to the following receptors and binding molecules incorporating the peptides, the receptors mediate receptor-mediated transcytosis (RMT) across the blood-brain barrier (BBB). The disclosure also provides conjugates consisting of the peptides or binding molecules and therapeutic agents or diagnostic agents, including fusion proteins. In some embodiments, the conjugates can pass through the blood-brain barrier after parenteral administration to allow the therapeutic agents or diagnostic agents to function in the central nervous system. The disclosure also provides methods for preparing and using the peptides and molecules provided. Background Art

[0004] The blood-brain barrier (BBB) ​​exerts neuroprotective functions by strictly controlling access to the brain; therefore, it also hinders pharmacological agents from entering brain tissue, making it necessary to use carriers to transport them. BBB permeability is usually the rate-limiting factor for drugs or peptides to penetrate into the central nervous system (CNS) (see Pardridge, WMJ Neurovirol.5:556-569 (1999); Bickel, U., Yoshikawa, T. and Pardridge, WM Adv. Drug Deliv. Rev. 46:247-279 (2001)). The brain is shielded by the BBB to resist potential toxic substances, and the BBB is formed by brain capillary endothelial cells tightly sealed by tight junctions. In addition, compared with the capillaries of other organs, brain capillaries have few fenestrations and few endocytic vesicles (see Pardridge, WMJ Neurovirol.5:556-569 (1999)). Except for some specific proteins such as transferrin, lactoferrin and low-density lipoprotein, which are taken up by receptor-mediated endocytosis, few large hydrophilic molecules are transported across the BBB (see Pardridge, WM J Neurovirol. 5:556-569 (1999); Tsuji, A. and Tamai, I. Adv. Drug Deliv. Rev. 36:277-290 (1999); Kusuhara, H. and 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, there is a need for improved molecules, for example, to allow transport of certain agents (such as therapeutic agents and diagnostic agents) across the BBB. Molecules that meet such needs are provided. Summary of the invention

[0005] Provided herein is a modified cyclic peptide, the modified cyclic peptide comprising 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), wherein the peptide has an amino acid sequence of 2 to 50 amino acid residues; and ii) a cyclic peptide scaffold containing the peptide of i), wherein the modified cyclic peptide comprises structure (I): (XV1…XVf)C1(X1…Xa)C2(XI1…XIb)C3(XII1…XIIc)C4(XIII1…XIIId)C5(XIV1…XIVe)C6(XV 1…XVf) ring 6 ring 1 ring 2 ring 3 ring 4 ring 5 ring 6, wherein C1 to C6 are cysteine ​​residues; wherein each of C1 and C4, C2 and C5, and C3 and C6 are linked by a disulfide bond to form a cysteine ​​knot; wherein each X represents an amino acid residue in a ring, wherein the amino acid residues are the same or different; wherein d is about 1-2; wherein at least one ring from ring 1, 2, 3, 5 or 6 has an amino acid sequence comprising the sequence of the peptide of clause i), wherein any ring comprising the sequence of the peptide of clause i) comprises 2 to about 30 amino acids, and wherein for any of rings 1, 2, 3, 5 or 6 without the sequence of the peptide of clause i), a, b, c, e and f are the same or different and are each any number from 3 to 10, and b, c, e and f are each any number from 1 to 20.

[0006] In some any embodiments of the provided embodiments, the BBB-R is human. In some any embodiments of the provided embodiments, the cyclic peptide scaffold is selected from plant cyclic peptides. In some any embodiments of the provided embodiments, the cyclic peptide scaffold is a cyclic peptide scaffold of a species of the genus Momordicae. In some any embodiments of the provided embodiments, the cyclic peptide scaffold is a Momordica cochinchinensis trypsin inhibitor. In some any embodiments of the provided embodiments, the Momordica cochinchinensis trypsin inhibitor is MCoTI-I shown in SEQ ID NO:1, MCoTI-II shown in SEQ ID NO:2, or MCoTI-III shown in SEQ ID NO:3. In some any embodiments of the provided embodiments, the sequence of the peptide replaces or substitutes one or more amino acids in one of the one or more rings of the cyclic peptide scaffold.

[0007] Provided herein is a modified cyclic peptide comprising a peptide that binds to a blood-brain barrier transcytosis receptor (BBB-R), wherein: the peptide is inserted into at least one loop of the cyclic peptide scaffold shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or replaces one or more amino acids of the at least one loop, and wherein the peptide is about 2 to 50 amino acid residues; and the BBB-R is selected from 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).

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

[0009] Provided herein is a modified cyclic peptide comprising a peptide inserted into loop 1 to replace all amino acids between cysteine ​​4 and cysteine ​​11 of SEQ ID NO: 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).

[0010] In some any of the 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 any embodiments of the provided 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 any embodiments of the provided embodiments, the peptide is 14 to 20 amino acids. In some any embodiments of the provided embodiments, the peptide is 10 amino acids. In some any embodiments of the provided embodiments, the peptide is 12 amino acids.

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

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

[0013] In some any embodiments of the provided embodiments, the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 26-34 and 49-54. In some embodiments, the one or more amino acid substitutions are substitutions of an amino acid for another amino acid selected from histidine or alanine. In some embodiments, the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in SEQ ID NO: 26. In some any embodiments of the provided embodiments, the peptide comprises a sequence shown in any one of SEQ ID NOs: 55 and 117-128. In some any embodiments of the provided embodiments, the peptide is as shown in any one of SEQ ID NOs: 55 and 117-128. In some embodiments, the modified cyclic peptide comprises a sequence shown in any one of SEQ ID NOs: 101 and 105-116. In some embodiments, the modified cyclic peptide is as shown in any one of SEQ ID NOs: 101 and 105-116. In some any embodiments of the provided embodiments, the peptide comprises a sequence shown in SEQ ID NO: 55. In some embodiments, the peptide is as shown in SEQ ID NO: 55. In some embodiments, the modified cyclic peptide comprises the sequence shown in SEQ ID NO: 101. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 101.

[0014] In some any embodiments of the provided embodiments, the BBB-R is the leptin receptor. In some any embodiments of the provided embodiments, the peptide comprises a sequence as shown in any one of SEQ ID NOs: 10-23. In some any embodiments of the provided embodiments, the peptide is as shown in any one of SEQ ID NOs: 10-23. In some any embodiments of the provided embodiments, the modified cyclic peptide comprises a sequence as shown in any one of SEQ ID NOs: 56-69. In some embodiments, the modified cyclic peptide is as shown in any one of SEQ ID NOs: 56-69. In some any embodiments of the provided embodiments, the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 10-23. In some embodiments, the one or more amino acid substitutions are substitutions of an amino acid for another amino acid selected from histidine or alanine.

[0015] In some any embodiments of the provided embodiments, the BBB-R is ErbB3. In some any embodiments of the provided embodiments, the peptide comprises the sequence shown in SEQ ID NO:24 or 25. In some any embodiments of the provided embodiments, the peptide is as shown in SEQ ID NO:24 or 25. In some any embodiments of the provided embodiments, the modified cyclic peptide comprises the sequence shown in SEQ ID NO:70 or 71. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO:70 or 71. In some any embodiments of the provided embodiments, the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in SEQ ID NO:24 or 25. In some embodiments, the one or more amino acid substitutions are substitutions of an amino acid for another amino acid selected from histidine or alanine.

[0016] In some any embodiments of the provided embodiments, the BBB-R is an insulin-like growth factor type 1 receptor (IGFR). In some any embodiments of the provided embodiments, the peptide comprises the sequence shown in SEQ ID NO:35 or 36. In some any embodiments of the provided embodiments, the peptide is as shown in SEQ ID NO:35 or 36. In some any embodiments of the provided embodiments, the peptide comprises the sequence shown in SEQ ID NO:36. In some embodiments, the peptide is as shown in SEQ ID NO:36. In some any embodiments of the provided embodiments, the modified cyclic peptide comprises the sequence shown in SEQ ID NO:81 or 82. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO:81 or 82. In some any embodiments of the provided embodiments, the modified cyclic peptide comprises the sequence shown in SEQ ID NO:82. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO:82. In some any embodiments of the provided embodiments, the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in SEQ ID NO:35 or 36. In some embodiments, the one or more amino acid substitutions are substitutions of an amino acid for another amino acid selected from histidine or alanine.

[0017] In some any embodiments of the provided embodiments, the BBB-R is RAGE. In some any embodiments of the provided embodiments, the peptide comprises the sequence shown in SEQ ID NO:37 or 38. In some any embodiments of the provided embodiments, the peptide is as shown in SEQ ID NO:37 or 38. In some any embodiments of the provided embodiments, the modified cyclic peptide comprises the sequence shown in SEQ ID NO:83 or 84. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO:83 or 84. In some any embodiments of the provided embodiments, the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in SEQID NO:37 or 38. In some embodiments, the one or more amino acid substitutions are substituted with histidine or alanine.

[0018] In some embodiments of any of the provided embodiments, the BBB-R is the 1 lipoprotein receptor-related protein 1 (LRP-1). In some embodiments of any of the provided embodiments, the peptide comprises the sequence shown in any one of SEQ ID NOs: 39-48. In some embodiments of any of the provided embodiments, the peptide is as 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 as 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 as 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 as shown in SEQ ID NO: 47. In some embodiments of any of the provided embodiments, the modified cyclic peptide comprises the sequence shown in any one of SEQ ID NOs: 85-94. In some embodiments, the modified cyclic peptide is as shown in any one of SEQ ID NOs: 85-94. In some embodiments, the modified cyclic peptide comprises the sequence shown in SEQ ID NO:85. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO:85. In some embodiments, the modified cyclic peptide comprises the sequence shown in SEQ ID NO:89. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO:89. In some embodiments, the modified cyclic peptide comprises the sequence shown in SEQ ID NO:93. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO:93. In some any embodiments of the provided embodiments, the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NO:39-48. In some embodiments, the one or more amino acid substitutions are substitutions of an amino acid for another amino acid selected from histidine or alanine.

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

[0020] In some any of the embodiments provided, the peptide has blood-brain barrier translocation activity. In some any of the 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 any embodiments of the provided 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 any embodiments of the provided embodiments, the peptide is 14 to 20 amino acids. In some any embodiments of the provided embodiments, the peptide is 10 amino acids. In some any embodiments of the provided embodiments, the peptide is 12 amino acids.

[0021] Provided herein is a peptide consisting of a sequence set forth in any one of SEQ ID NOs: 10-55 or 117-128. In some any of the embodiments provided, the peptide binds to a receptor involved in transcytosis at the blood-brain barrier.

[0022] Provided herein is a peptide, the peptide being set forth in any of SEQ ID NOs:26-34 and 49-55 and 117-128, wherein the peptide binds to a transferrin receptor. In some embodiments of the provided embodiments, the peptide is set forth in SEQ ID NO:26. In some embodiments of the provided embodiments, the peptide is set forth in SEQ ID NO:49. In some embodiments of the provided embodiments, the peptide is set forth in SEQ ID NO:55.

[0023] Provided herein is a peptide represented by any one of SEQ ID NOs: 10-23, wherein the peptide binds to the leptin receptor. Provided herein is a peptide represented by any one of SEQ ID NOs: 24 or 25, wherein the peptide binds to ErbR3.

[0024] Provided herein is a peptide represented by the sequence of any one of SEQ ID NO:35 or 36, wherein the peptide binds to IGFR. In some any of the embodiments provided, the peptide is represented by SEQ ID NO:35. In some any of the embodiments provided, the peptide is represented by SEQ ID NO:36.

[0025] Provided herein is a peptide represented by the sequence of SEQ ID NO: 37 or 38, wherein the peptide binds to RAGE.

[0026] Provided herein is a peptide represented by the sequence of any one of SEQ ID NOs:39-48, wherein the peptide binds to the lipoprotein receptor-related protein 1 (LRP-1). In some any of the embodiments provided, the peptide is represented by SEQ ID NO:39. In some any of the embodiments provided, the peptide is represented by SEQ ID NO:43. In some any of the embodiments provided, the peptide is represented by SEQ ID NO:47.

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

[0028] Provided herein is a binding molecule comprising a binding scaffold and any provided peptide. In some embodiments of the provided embodiments, the binding scaffold is a cyclic peptide. In some embodiments of the provided embodiments, the peptide is inserted into a ring of the cyclic peptide backbone or replaces one or more amino acids of the ring. In some embodiments, the at least one ring is ring 1. In some embodiments of the provided embodiments, the binding scaffold is as shown in any one of SEQ ID NO: 1-3. In some embodiments of the provided embodiments, the binding scaffold is as shown in SEQ ID NO: 2.

[0029] Provided herein is a nucleic acid molecule encoding any of the provided modified cyclic peptides or any of the provided binding molecules.

[0030] Provided herein is a vector comprising any of the provided nucleic acids. In some any of the provided embodiments, the vector is an expression vector.

[0031] Provided herein is a host cell comprising any of the provided nucleic acid molecules or any of the provided vectors.

[0032] Provided herein is a method for producing a modified cyclic peptide or binding molecule, the method 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 certain conditions to express the protein in the cell. In some embodiments, the method further comprises purifying the protein from the cell.

[0033] Provided herein is a purified binding molecule or modified cyclic peptide produced by any of the provided methods.

[0034] Provided herein is a conjugate comprising any of the provided modified cyclic peptides or any of the provided binding molecules and a bioactive agent. In some any of the provided embodiments, the bioactive agent is a small molecule, a peptide or a protein. In some any of the provided embodiments, the bioactive agent is a diagnostic agent or a therapeutic agent. In some any of the provided embodiments, any of the provided conjugates is a fusion protein comprising the modified cyclic peptide operably linked to a bioactive agent, the bioactive agent being a protein or a peptide.

[0035] Provided herein is a fusion protein comprising any of the provided modified cyclic peptides or any of the provided binding molecules and a bioactive agent, which is a protein or a peptide.

[0036] In some any embodiments of the provided embodiments, the bioactive agent is an antibody. In some embodiments, the antibody is directed against an antigen selected from the group consisting of human epidermal growth factor receptor 2 (HER2), β-secretase 1 (BACE1), amyloid β (Aβ), epidermal growth factor receptor (EGFR), tau protein, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin protein, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, and TNF-α.

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

[0038] In some embodiments of the provided embodiments, the bioactive agent is an enzyme. In some embodiments of the provided embodiments, the enzyme is a ceramide degrading enzyme, a lipase, a hydrolase-type enzyme, or a sulfatase. In some embodiments of the provided embodiments, the enzyme is a ceramide degrading enzyme, and the ceramide degrading enzyme is a glucocerebrosidase, a galactocerebrosidase, or an alpha galactosidase. In some embodiments, the enzyme is a glucocerebrosidase having an amino acid sequence 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 1-5 amino acid substitutions compared to the sequence shown in SEQ ID NO: 144 or SEQ ID NO: 145. Non-limiting mutations include any mutations as described herein. In some embodiments, the enzyme is a glucocerebrosidase having an amino acid sequence shown in SEQ ID NO: 144. In some embodiments, the enzyme is a glucocerebrosidase having an amino acid sequence shown in SEQ ID NO: 145. In some any of the embodiments provided, the enzyme is a lipase or hydrolase type enzyme, and the enzyme is a sphingomyelinase, a cerliponase, or an alpha glucosidase.

[0039] In some any embodiments of the provided embodiments, the conjugate or fusion protein comprises a single modified cyclic peptide. In some any embodiments of the provided embodiments, the conjugate or fusion protein comprises 2, 3 or 4 modified cyclic peptides. In some any embodiments of the provided embodiments, each modified cyclic peptide is the same. In some any embodiments of the provided embodiments, each modified cyclic peptide is different.

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

[0041] In some any of the provided embodiments, the conjugate is bivalent for binding to BBB-R.In some any of the provided embodiments, the conjugate or fusion protein comprises at least two different modified cyclic peptides that bind to different BBB-Rs.

[0042] In some any of the provided embodiments, the conjugate or fusion protein is bispecific for binding to two different BBB-Rs. In some embodiments, the conjugate is bispecific for binding to two different BBB-Rs and comprises at least two different modified cyclic peptides, each of which binds to a different BBB-R.

[0043] In some any embodiments of the provided embodiments, the modified cyclic peptide is connected to the bioactive agent via a linker. In some embodiments, the length of the linker is at least 10 amino acids. In some embodiments, the length of the linker is at least 15 amino acids. In some embodiments, the length of the linker is 10 to 20 amino acids. In some any 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 any embodiments of the provided embodiments, the linker is as shown in SEQ ID NO: 104. In some any embodiments of the provided embodiments, the linker is a cleavable linker comprising an endosomal specific protease cleavage site. In some any embodiments of the provided embodiments, the endosomal specific protease cleavage site is a cathepsin cleavage site. In some any embodiments of the provided embodiments, the cathepsin cleavage site is a cathepsin B cleavage site. In some any embodiments of the provided embodiments, the linker comprises the sequence shown in SEQ ID NO: 133.

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

[0045] Provided herein is a method for transporting a bioactive agent across the blood-brain barrier of an individual, the method comprising administering any provided conjugate or fusion protein or any provided pharmaceutical composition to an individual in need thereof. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0046] In some any embodiments of the provided embodiments, the individual (such as mammals (e.g., people)) suffers from a nervous system disease. In some any embodiments of the provided embodiments, the nervous system disease is selected from Alzheimer's disease, Parkinson's disease, apoplexy, brain tumors and brain metastasis. In some any embodiments of the provided embodiments, the nervous system disease is selected from the following congenital diseases: Austin disease (Austen Disease), Canavan disease, Gaucher disease, Hunter syndrome, Hurler-Scheie syndrome, Bi-Young disease, Krabbe disease, LCAT deficiency, Lowe syndrome, Maroteaux-Lamy syndrome, Morquio syndrome A, Morquio syndrome B, Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome C, Sanfilippo syndrome D, spinal muscular atrophy, Tay-Sachs disease and Walker-Warburg syndrome.

[0047] Provided herein is a method for treating a patient with a neurological disease, the method comprising administering any provided conjugate or any provided pharmaceutical composition to the patient. Provided herein is a method for diagnosing a neurological disease in a patient in need, the method comprising administering any provided conjugate or fusion protein or any provided pharmaceutical composition to the patient, and wherein the conjugate comprises a radioactive label. In some any embodiments of the provided embodiments, the neurological disease is selected from Alzheimer's disease, Parkinson's disease, stroke, brain tumors and brain metastases. In some any embodiments of the provided embodiments, the neurological disease is a congenital disease selected from the following: Austin disease, Canavan disease, Gaucher disease, Hunter syndrome, Hurler-Scheie syndrome, Bi-Young disease, Krabbe disease, LCAT deficiency, Lowe syndrome, Maroteaux-Lamy syndrome, Moquio syndrome A, Moquio syndrome B, Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome C, Sanfilippo syndrome D, spinal muscular atrophy, Tay-Sachs disease and Walker-Warburg syndrome.

[0048] Also provided are any compositions provided herein for treating a nervous system disease. Also provided are uses of any provided pharmaceutical compositions in the manufacture of medicaments for treating a nervous system disease. Also provided are any compositions provided herein for diagnosing a nervous system disease. Also provided herein are uses of any provided pharmaceutical compositions in the manufacture of medicaments for diagnosing a nervous system disease. In some any embodiments of the provided embodiments, the nervous system disease is selected from Alzheimer's disease, Parkinson's disease, stroke, brain tumors and brain metastases. In some any embodiments of the provided embodiments, the nervous system disease is a congenital disease selected from the following: Austin disease, Canavan disease, Gaucher disease, Hunter syndrome, Hurler-Scheie syndrome, Bee-Young disease, Krabbe disease, LCAT deficiency, Lowe syndrome, Maroteaux-Lamy syndrome, Morquio syndrome A, Morquio syndrome B, Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome C, Sanfilippo syndrome D, spinal muscular atrophy, Tay-Sachs disease and Walker-Warburg syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1A-Figure 1D Depicts an exemplary configuration of a provided antibody fusion protein, the fusion protein consisting of an antibody and at least one modified cyclic peptide, the fusion protein comprising: a bivalent molecule ( Figure 1A ); Bispecific molecules ( Figure 1B ); a monovalent molecule in which the heavy chain is modified with a modified cyclic peptide ( Figure 1C ); and monovalent molecules in which the knob heavy chain is modified with a modified cyclic peptide ( Figure 1D ).

[0050] Figure 2 Depicted is a flow cytometry-based assay for the internalization of fluorescently labeled adalimumab-CYC17 cyclic peptide fusion and adalimumab by human cells expressing the transferrin receptor.

[0051] Figure 3 Depicted are ELISA results of binding of various adalimumab-cyclic peptide fusions to transferrin receptor (TfR) coated on microwells, wherein the cyclic peptide portion is CYC17 or various mutants thereof containing amino acid substitutions at the indicated positions (e.g., 5H refers to a histidine (H) substitution at position 5).

[0052] Figure 4 Describes different cyclic peptide antibody fusion molecules (including Figure 1A-Figure 1DThe molecules shown in Figure 1 show binding of CYC17 to human transferrin receptor. The molecules include: monovalent molecules in which the exemplary CYC17 is fused to the knob or hole chain of the heterodimeric Aduhelm antibody; bispecific fusions in which CYC17 (directed to TfR) is fused to the hole of the heterodimeric Aduhelm antibody and CYC27 (directed to IGF1R) is fused to the knob; and bivalent adalimumab (Humira) containing two copies of the CYC17 cyclic peptide.

[0053] Figure 5 Depicted is the expression of cyclic peptide-trastuzumab antibody fusion molecules in supernatants from transfected host cells.

[0054] Fig. 6A The enzymatic activity of the GlcCSase-CYC17 fusion cyclic peptide in the glucocerebroside activity assay is depicted.

[0055] Figure 6B Depicted are ELISA results for binding of the GlcCSase-CYC17 fusion cyclic peptide to human transferrin receptor (hTfR) coated on microwells.

[0056] Figure 7 Depicted are ELISA results for binding of NGF-CYC17 fusion cyclic peptide to human transferrin receptor (hTfR) coated on microwells.

[0057] Fig. 8A Depicts ELISA results of binding of exemplary trastuzumab fusion cyclic peptides to human LPR1 coated on microwells. The fusion molecules include monovalent molecules in which the exemplary cyclic peptides CYC30, CYC32 or CYC34 bind to the heterodimeric trastuzumab Antibody knob chain fusion.

[0058] Figure 8B Depicted are the quantitative results of in vivo cyclic peptide-mediated trastuzumab delivery to mouse brain at 24 hours compared to trastuzumab alone. Seven antibody-monovalent cyclic peptide fusion molecules targeting IGF1R, LRP1 or RAGE receptors were administered to mice and quantified from fresh brain homogenates. The fusion molecules include monovalent molecules in which the exemplary cyclic peptides CYC26, CYC27, CYC28, CYC29, CYC30, CYC32 or CYC34 are combined with heterodimeric trastuzumab Antibody knob chain fusion.

[0059] Fig. 9Depicted are the quantitative results of in vivo cyclic peptide-mediated trastuzumab delivery to mouse brain at 24 hours compared to trastuzumab alone. Two antibody-monovalent cyclic peptide fusion molecules targeting human transferrin receptor (hTfR) were administered to mice and quantified from fresh brain homogenates. The fusion molecules include monovalent molecules in which the exemplary cyclic peptides CYC26 or CYC27 are combined with heterodimeric trastuzumab Antibody knob chain fusion. DETAILED DESCRIPTION

[0060] Provided herein are peptides that exhibit blood-brain barrier translocation activity (including the ability to mediate endocytosis to promote the passage through the BBB), such as via binding to receptors involved in blood-brain barrier endocytosis. The peptide provided can be inserted into a protein scaffold to provide a larger binding molecule skeleton or framework, which in some aspects can improve the stability or half-life of the peptide. In some embodiments, the scaffold is a cyclic peptide skeleton to provide a modified cyclic peptide with improved or enhanced blood-brain barrier translocation activity and / or binding to a receptor involved in blood-brain barrier endocytosis. The modified cyclic peptide molecules provided are not naturally occurring and show special advantages in passing through the blood-brain barrier. The modified scaffold provided (such as the modified cyclic peptide provided) inserted into the provided peptide can be used as a carrier attached or connected to other molecules (such as polypeptides, proteins and small molecules, including therapeutic agents or diagnostic agents) to deliver such other molecules through the blood-brain barrier. The modified cyclic peptide provided can be used to deliver therapeutic agents and other biological agents through the BBB for the treatment of neurological disorders.

[0061] The problem of delivering some therapeutic agents or other biological agents is that it is difficult to deliver parenterally administered biological agents (such as therapeutic agents and diagnostic agents) to the central nervous system (CNS) because the passive transfer of substances from capillaries to the brain is limited. Unlike the capillaries in other tissues such as muscles, the difference between the capillaries supplying blood to most brain tissues is that the endothelial cells forming their endothelium are interconnected by tight intercellular junctions. This system that limits the exchange of substances between blood and brain tissue fluid through the brain capillary endothelium is called the blood-brain barrier or BBB. Therefore, the BBB causes problems in terms of the entry of therapeutic agents or other biological agents into the CNS because they are not always able to pass through the BBB. For example, when the BBB is intact, only about 0.2% of the monoclonal antibody dose administered intravenously will reach the brain.

[0062] Attempts have been made to develop various methods to allow for the transport of biologics across the BBB. Such attempts include, for example, encapsulating the agent in liposomes, delivering the biologic directly into the brain, or by conjugating the agent to an antibody molecule that binds to a membrane protein on brain capillary endothelial cells. Although some of these methods can improve transcytosis efficiency, existing methods are invasive and / or complex. For example, although antibodies can confer affinity for receptors involved in blood-brain barrier transcytosis, the larger size of the antibody may reduce the manufacturability of the therapeutic fusion protein, such as due to expression, purification or stability issues.

[0063] The embodiments provided solve these problems. The embodiments provided relate to novel peptides, which exhibit binding to receptors involved in blood-brain barrier endocytic transport. These peptides can be incorporated into binding molecule scaffolds to provide stability to the molecule and to confer the ability of the molecule to bind to the receptors that mediate RMT across the blood-brain barrier (BBB). In some embodiments, the binding molecule scaffold is a cyclic peptide, which is a cysteine ​​knot protein. The ability to incorporate peptides into stable scaffolds (such as cyclic peptides) provides a highly developable platform, which relates to highly stable small peptides that are well expressed as fusion proteins. In addition, due to the smaller size of the scaffold platform, the embodiments provided can also be used to produce multi-domain molecules with the ability to target more than one endocytic transport pathway.

[0064] In particular, the binding molecule scaffold provided includes cyclic peptides, which are members of the naturally occurring cysteine ​​knot microprotein (microprotein) family found in various plant species. Cysteine ​​knot microprotein (cyclic peptide) is a small peptide, usually composed of about 30-40 amino acids, which can be naturally present in a cyclic or linear form, wherein the cyclic form does not have a free N-terminal or C-terminal amino or carboxyl end. They have a limited structure based on three intramolecular disulfide bonds and a small three-chain β-fold (Craik et al., 2001, Toxicon 39, 43-60). The cyclic protein exhibits a conservative cysteine ​​residue, which is defined as a structure referred to herein as "cysteine ​​knot". This family includes both naturally occurring cyclic molecules and their linear derivatives and linear molecules that have undergone cyclization. These molecules can be used as molecular framework structures, and they have stability (Colgrave and Craik, 2004, Biochemistry 43, 5965-5975) that is enhanced compared to lower structured peptides. However, these molecules themselves are unable to cross the blood-brain barrier to any significant extent and cannot be used as neurotherapeutics themselves or as carriers for therapeutic peptide or protein agents.

[0065] The main cyclic peptide characteristics are the remarkable stability due to the cysteine ​​knot, the small size that makes them easy to chemically synthesize, and the extremely high tolerance to sequence changes. Therefore, cyclic peptides appear to be attractive leads or scaffolds for peptide drug design. Cyclic peptide scaffolds have been found in nearly 30 different protein families, among which conotoxins, spider toxins, squash inhibitors, spiny mouse-related proteins and plant cyclic peptides are the families with the most members. It was found that cyclic peptides from Rubiaceae and Violaceae plants are mostly head-to-tail cyclic peptides. However, within the squash inhibitor family of cyclic peptides, both cyclic and linear cyclic peptides have been identified from Momordica cochinchinensis: cyclic trypsin inhibitor (MCoTI)-I and -II and its linear counterpart MCoTI-III. It is now clear that both cyclic and linear variants can exist in different cyclic peptide families, but little is known about the effects of cyclization. When compared to their linear counterparts, cyclic peptides are expected to show improved stability, better resistance to proteases and reduced flexibility, hopefully leading to enhanced biological activity. However, linear cyclic peptides have the advantage of being able to be more easily linked to other peptides or proteins.

[0066] In some embodiments, the cyclic peptide sequence is characterized by having a cysteine ​​knot backbone portion, wherein the cysteine ​​knot backbone comprises structure (I): (X V 1…X V f )C1(X1…X a )C2(X I 1…X I b )C3(X II 1…X II c )C4(X III 1…X III d )C5(X IV 1…X IV e )C6(X V 1…X V f ) wherein C1 to C6 are cysteine ​​residues; wherein each of C1 and C4, C2 and C5, and C3 and C6 are linked by a disulfide bond to form a cysteine ​​knot; wherein each X represents an amino acid residue in the ring, wherein the amino acid residues are the same or different; wherein d is about 1-2; and wherein a, b, c, e and f are the same or different and are each any number from 3 to 10, and b, c, e and f are each any number from 1 to 20.

[0067] In some embodiments, in addition to the stability of the molecule, the cyclic peptide also exhibits characteristics indicating that the molecule has a low risk of immunogenicity. Although the cyclic peptide originates from a non-human sequence, they are highly disulfide-bonded proteins. Evidence shows that non-human disulfide-bonded peptides do not exhibit detectable immunogenicity. For example, Ziconitide is a disulfide-bonded non-human peptide similar to a cyclic peptide approved by the FDA, and in preclinical mouse and rat immunogenicity tests, even after repeated doses, no anti-drug antibodies (ADA) were detected (Skov M. et al. Int. J. Toxicology, 2007 26: 411-421).

[0068] Since cyclic peptides have not been shown to readily cross the BBB in their native form, it is desirable to combine the structural stability of cyclic peptide scaffolds with the ability to cross the blood-brain barrier and to associate other biological activities with cyclic peptide molecules. Therefore, it is desirable to identify cyclic peptides that are suitable for retaining both structure and blood-brain translocation capabilities as linear or cyclic molecules. It is also desirable to be able to use such cyclic peptides to improve the ability to improve the activity and bioavailability of biotherapeutics in vivo.

[0069] The provided molecules, including modified cyclic peptides, meet these needs. In some aspects, the provided modified cyclic peptide molecules can be used as carrier microprotein peptides that can cross the BBB and can be fused with desired biological agents (such as therapeutic molecules) for delivery to the brain.

[0070] The embodiments provided herein relate to cyclic peptide molecular frameworks to produce modified cyclic peptides, into which peptide sequences are inserted to confer enhanced BBB translocation activity relative to the parent cyclic peptide. In some embodiments, the modified cyclic peptide is composed of a cysteine ​​knot skeleton, and the cysteine ​​knot skeleton has enough disulfide bonds or their chemical equivalents to confer a knotted topological structure on the three-dimensional structure of the cysteine ​​knot skeleton, and wherein relative to the naturally occurring cyclic peptide amino acid sequence, at least one exposed amino acid residue (such as on one or more β turns and / or in one or more loops) is inserted or replaced (substituted) by a receptor binding peptide. In some embodiments, compared with the unmodified parent cyclic peptide, the modified cyclic peptide has enhanced translocation behavior. In some embodiments, the modified cyclic peptide has the desired characteristics of high enzyme stability and translocation, making the blood-brain barrier transfer of the modified cyclic peptide feasible.

[0071] In some embodiments, the modified cyclic peptide sequence provided herein can be defined as a peptide having a cysteine ​​knot backbone portion and as a blood-brain barrier translocation portion, the modified cyclic peptide comprising: i) a peptide having the blood-brain barrier translocation activity, wherein the peptide has an amino acid sequence of about 2 to 50 amino acid residues; and ii) a cysteine ​​knot backbone grafted to the peptide of clause i), wherein the cysteine ​​knot backbone comprises structure (I): (X V 1…X V f )C1(X1…X a )C2(X I 1…X I b )C3(X II 1…X II c )C4(X III 1…X III d )C5(X IV 1…X IV e )C6(X V 1…X V f ) wherein C1 to C6 are cysteine ​​residues; wherein each of C1 and C4, C2 and C5, and C3 and C6 are linked by a disulfide bond to form a cysteine ​​knot; wherein each X represents an amino acid residue in a ring, wherein the amino acid residues are the same or different; wherein d is about 1-2; wherein one or more of loops 1, 2, 3, 5 or 6 has an amino acid sequence comprising the sequence of the peptide of clause i), wherein any loop of the sequence of the peptide comprising clause i) comprises 2 to about 50 amino acids, and wherein for any of loops 1, 2, 3, 5 or 6 without the sequence of the peptide of clause i), a, b, c, e and f are the same or different and are each any number from 3 to 10, and b, c, e and f are each any number from 1 to 20.

[0072] In some embodiments, the provided modified cyclic peptide sequences can be linear or cyclic.

[0073] In some embodiments, the cysteine ​​knot scaffold is a cyclic peptide of a species of Momordica genus that includes the pumpkin serine protease inhibitor family. Exemplary cyclic peptides of Momordica genus 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 cyclic peptide scaffold shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or replaces one or more amino acids of the at least one loop.

[0074] Therefore, compared with natural or wild-type unmodified cyclic peptides, the cyclic peptides provided herein are modified cyclic peptides, wherein one or more rings of the modified cyclic peptides are inserted or substituted by one or more amino acid sequences (e.g., peptides of 2 to 50 amino acids). In the provided embodiments, one or more amino acids (e.g., peptide sequences of 2 to 50 amino acids) inserted or substituted into the rings of unmodified or wild-type cyclic peptides are blood-brain barrier translocation moieties that are capable of binding to receptors involved in endocytic transport across the blood-brain barrier and / or mediating blood-brain barrier translocation or penetration of the cyclic peptides. In specific embodiments, the peptide is a peptide that binds to the following: 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 the aspects of the provided embodiments, the modified cyclic peptides of the present invention incorporate sufficient amino acid structures to provide high enzyme stability and good translocation or penetration behavior. In some embodiments, provided modified cyclic peptides can be used as novel carrier cyclic peptides to facilitate or mediate translocation of attached polypeptides, such as therapeutic or diagnostic agents.

[0075] In some embodiments, the peptide-modified cyclic peptide can be conjugated to another agent or polypeptide, or in certain embodiments, one or more modified cyclic peptides are incorporated into another polypeptide to form a synthetic chimeric (or fusion) polypeptide / protein. Suitably, the peptide of the modified cyclic peptide can be contained in the ring region of the scaffold polypeptide. Then, the resulting modified scaffold itself can be conjugated or contained in a larger molecule. Suitably, the cyclic peptide inserted into the receptor binding peptide as provided herein can be enriched with cysteine ​​residues. In specific embodiments, the modified cyclic peptide provided exhibits the ability to pass through the blood-brain barrier, and the modified cyclic peptide or any molecule contained or conjugated thereto can be used for the brain.

[0076] The provided binding molecules (such as modified cyclic peptides) can be used to deliver bioactive agents (such as peptides, polypeptides, proteins or small molecules) across the blood-brain barrier. In some embodiments, the bioactive agent is a therapeutic agent, such as a therapeutic agent with therapeutic agonist or antagonist activity. In some embodiments, the bioactive agent is a diagnostic agent.

[0077] In some embodiments, the modified cyclic peptide (such as a linear or cyclic cyclic peptide) provided is operably connected to the N-terminus or C-terminus of a bioactive agent (such as a peptide or protein portion). In some embodiments, the connection can be a direct connection or an indirect connection via a peptide linker. In some embodiments, the connection provides a conjugate of a modified cyclic peptide and a bioactive agent, such as a fusion protein. Also provided herein is a conjugate, such as a fusion protein, comprising a provided modified cyclic peptide operably connected to the N-terminus or C-terminus of a bioactive agent (such as a peptide or protein portion).

[0078] In some embodiments, the bioactive agent is a therapeutic agent, such as an agent as an agonist or antagonist. The connection or fusion of the bioactive agent to the modified cyclic peptide provided allows the bioactive agent (such as a peptide or protein moiety) to translocate and enter across the BBB. In the aspects provided, this connection allows a pharmacologically relevant dose of the bioactive agent (such as a peptide or protein moiety) to translocate and enter across the BBB.

[0079] In some embodiments, bispecific molecules are also provided herein that incorporate more than one receptor binding molecule to target more than one transcytosis pathway. For example, in some embodiments, at least two different modified cyclic peptides can be connected to a bioactive agent to provide a multi-domain protein that can further improve the ability of the connected biological agent to cross the BBB. In some embodiments, 1, 2, 3 or 4 different modified cyclic peptides, each modified with a different receptor binding peptide, can be connected or conjugated to a biological agent to allow one or more BBB transport protein pathways to be connected.

[0080] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If definitions set forth herein are contrary to or otherwise inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over 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. I. Receptor Binding Peptides and Modified Cyclic Peptides

[0082] Provided herein is a peptide of 2-50 amino acids in length, which binds to a receptor involved in blood-brain barrier endocytosis. In some embodiments, provided herein is a peptide of 6-50 amino acids in length, which binds to a receptor involved in blood-brain barrier endocytosis. In some aspects, the peptide provided exhibits blood-brain barrier translocation activity. Also provided herein is a binding molecule incorporating any provided peptide 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 cyclic peptide. Therefore, also provided herein is a modified cyclic peptide compared to a parent or natural cyclic peptide skeleton (also referred to as a cysteine ​​knot skeleton), wherein the modified cyclic peptide provided exhibits improved or enhanced binding to a receptor (BBB; hereinafter "BBB endocytosis receptor") involved in endocytosis through the blood-brain barrier and / or exhibits improved or enhanced blood-brain barrier translocation characteristics. A. Receptor Binding Peptides and Selection Methods

[0083] Provided herein are isolated or synthetic peptides that bind to BBB transcytosis receptors (also referred to herein as BBB-R). In some embodiments, the peptide is inserted or can be inserted into the backbone of a binding molecule scaffold (such as a cysteine ​​knot scaffold, such as a cyclic peptide). Exemplary modified cyclic peptides containing receptor binding peptides are described in subsection IB.

[0084] In some embodiments, BBB-R is expressed on brain endothelial cells. In some embodiments, BBB-R is selected from transferrin receptor (TrfR); lactoferrin receptor (LtfR); leptin receptor (LEP-R, or also known as OB-R); receptor tyrosine-protein kinase (ErbB3), insulin receptors 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, the BBB-R is a mammalian receptor. In some embodiments, the BBB-R is murine. In some embodiments, the BBB-R is human.

[0086] In some embodiments, the BBB-R is selected from 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), human intercellular adhesion molecule 2 (human ICAM2) or neural cell adhesion molecule (human NCAM).

[0087] In some embodiments, the BBB-R is Erb-B2 receptor tyrosine kinase 3 (ErbB3), such as human ErbB3. In some embodiments, the BBB-R is TrfR, such as human TrfR. In some embodiments, the BBB-R is a leptin receptor (ObR), such as human ObR. In some embodiments, the BBB-R is an insulin-like growth factor type 1 receptor (IGFR), such as human IgFR. In some embodiments, the BBB-R is a low-density lipoprotein-related protein 1 (LRP-1), such as human LRP-1. In some embodiments, the BBB-R is a 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 In some embodiments, the present invention relates to a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of 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.

[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 for producing or screening peptides can be a soluble form of the antigen or a part (for example, extracellular domain) containing a desired epitope. BBB-R target molecules can be separated from natural sources or prepared by recombinant methods by procedures known in the art. The target molecules of purification can be attached to suitable substrates, such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic acid copolymers, hydroxyalkyl methacrylate gels, polyacrylic acid and polymethacrylic acid copolymers, nylon, neutral and ion carriers, etc. The attachment of target protein to substrate can be achieved by the method described in Methods in Enzymology, 441976 or by other means known in the art. Alternatively or additionally, cells expressing BBB-R on the cell surface can be used to produce or screen binding molecules. In some embodiments, binding molecules can be screened for membrane translocation activity.

[0092] In some aspects, a method for selecting or identifying a peptide binding to BBB-R is provided. In some embodiments, random peptides can be inserted into the skeleton of a scaffold sequence (such as a cyclic peptide, including any one as described in Section IB), and a 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 together with the provided method and carrier to generate 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 to select binding proteins from the library for binding to BBB-R. For ease of screening, a library of variant binding molecules (for example, a cyclic peptide library with random peptides inserted therein) is generally screened using display technology, so that there is a physical connection between the individual molecules (phenotype) 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, GP (1985) Science 228: 1315-1317), mRNA display, ribosome display, and DNA display.

[0093] In some embodiments, a library of cyclic peptides each having a random peptide inserted therein is screened to identify individual members of the library that exhibit a desired biological activity (e.g., binding to BBB-R). According to one embodiment, a method for screening for BBB-R binding comprises the following steps: a) constructing a cyclic peptide sequence display library, wherein random peptides are inserted into a cyclic peptide scaffold (e.g., into a loop thereof, such as loop 1); b) expressing the sequence library to obtain the expressed cyclic peptide or a polypeptide comprising the cyclic peptide; c) selecting the expressed sequence library against a suitable purified BBB-R; d) recovering any cyclic peptide bound to the receptor; e) determining the sequence of the recovered cyclic peptide or the peptide contained therein.

[0094] For example, the display library is a phage display library of multiple modified cyclic peptide scaffolds (or other binding molecule scaffolds), wherein each scaffold is inserted with a random peptide. In some embodiments, each modified cyclic peptide scaffold of the library is fused to a phage coat protein, and is usually displayed on the surface of a phagemid particle containing a DNA encoding the polypeptide as a single copy of each related polypeptide on average. These phagemid particles are then contacted with the BBB-R target, and those particles with the highest affinity for the target are separated from those with lower affinity. The higher affinity binders are then amplified by infecting 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 method provided includes contacting any display library provided herein with a target molecule under conditions that allow display particles (e.g., phasmid particles) to bind to the target molecule. In some embodiments, the method further includes separating the bound display particles (e.g., phasmid particles) from those that are not bound, thereby selecting display particles (e.g., phasmid particles) that include antibody-binding proteins that bind to the target molecule. In some embodiments, the method includes sequencing the fusion gene in the selected particle to identify the antibody-binding protein.

[0096] In some embodiments, the BBB-R target molecule is contacted with a library of display particles (e.g., phasmid particles) under conditions suitable for binding at least a portion of the display particles to the target molecule. Typically, the conditions (including pH, ionic strength, temperature, etc.) will simulate physiological conditions. Exemplary "contact" conditions may include incubation at 4°C-37°C (e.g., at room temperature) for 15 minutes to 4 hours (e.g., 1 hour). However, these conditions may vary as needed, depending on the nature of the interacting binding partner, etc. The mixture may be subjected to gentle shaking, mixing, or rotation. In addition, other appropriate reagents, such as blocking agents, may be added to reduce nonspecific binding. For example, 1%-4% BSA or other suitable blocking agents (e.g., milk) may be used. However, it should be understood that the contact conditions may be changed and adjusted by the technician according to the purpose of the screening method. For example, if the incubation temperature is, for example, room temperature or 37°C, this may increase the possibility of identifying a binder that is stable under these conditions (e.g., stable under conditions found in the human body when incubated at 37°C). This property may be extremely advantageous if one or both of the binding partners is a candidate for some therapeutic application (eg, an antibody). Again, such adjustments to the conditions are within the capabilities of the skilled artisan.

[0097] The bound display particles ("conjugates") with high affinity for the fixed target molecule can be separated from those with low affinity (and therefore not binding the target) by washing. The conjugates can be dissociated from the fixed target molecule by a variety of methods. These methods include competitive dissociation using wild-type ligands, changing pH and / or ionic strength, and methods known in the art.

[0098] In another embodiment, the peptide can be screened for the activity of the blood-brain barrier translocation activity conferred by the peptide. In this embodiment, the library of the cyclic peptides each inserted with a random peptide is screened to identify the individual members that exhibit the desired biological activity (such as membrane translocation activity, and particularly when delivered to an animal, transfer across the BBB). "Transfer across the blood-brain barrier" or "passing through the blood-brain barrier" or other variants thereof means that the polypeptide is delivered to an animal and can enter the animal brain by passing through the blood vessel wall in the brain. According to one embodiment, the method for screening BBB transfer comprises the following steps: a) constructing a cyclic peptide sequence display library, wherein random peptides are inserted into a cyclic peptide scaffold (e.g., into a loop thereof, such as loop 1); b) expressing the sequence library to obtain the expressed cyclic peptide or a polypeptide comprising the cyclic peptide; c) administering the expressed cyclic peptide or a polypeptide comprising the cyclic peptide to an animal; d) recovering any cyclic peptide from the brain of a mammal; e) determining the sequence of the recovered cyclic peptide or the peptide contained therein.

[0099] The animals used in this screening are usually birds or mammals, and can be selected from humans, primates, cattle, sheep, rodents, cats, dogs and rabbits. In the case of non-human animals, the cyclic peptide library can be appropriately administered by oral gavage or contained in normal animal feed. Recovering the cyclic peptide from the animal body can be performed via biopsy, or in the case of non-human animals, the histology and pathological analysis of the killed animals and the in vivo tissues can be performed. In this way, members of the availability of the cyclic peptide library that exhibit tissue specificity and / or pass through various other barriers in the animal body can also be identified. For example, the modified cyclic peptides found in the brain biopsy of the non-human animal screened are considered to show the ability to pass through the BBB. In a specific embodiment of the present invention, the cyclic peptides of the present invention are included in the phage display library, and the determination of BBB transfer is performed in the following manner: which cyclic peptides are analyzed to promote the relevant phage particles to be transported to the animal brain as a whole, and the animal has been fed or intravenously injected with at least a portion of the phage display library.

[0100] Once one or more groups of cyclic peptides or peptides are selected or separated according to the provided method, these cyclic peptides or peptides can be further analyzed. In some embodiments, the further analysis includes separating the cyclic peptides, separating the phage or phagemid DNA by bacterial infection as an amplification step, and cloning the DNA sequence encoding the candidate cyclic peptide contained in the phage or phagemid DNA into a suitable expression vector. This infection step can also allow the amplification of the cyclic peptide. Alternatively, the cyclic peptide can be amplified by other appropriate methods at this stage, for example, by PCR of the nucleic acid encoding the cyclic peptide or by converting the nucleic acid into a suitable host cell (in the case of a suitable expression vector).

[0101] Once the DNA encoding the cyclic peptide is cloned in a suitable expression vector, the DNA encoding the cyclic peptide can be sequenced, or the protein can be expressed in a soluble form (e.g., including according to the method provided herein) and undergo appropriate binding studies to further characterize the candidate at the protein level. In some embodiments, the peptide contained in the selected cyclic peptide can be identified, and its combination with BBB-R can be assessed. Appropriate binding studies depend on the properties of the conjugate, and include but are not limited to ELISA, filtration screening assays, FACS or immunofluorescence assays, BiaCore affinity measurements or other methods of quantitatively binding constants, staining of tissue slides or cells and other immunohistochemical methods. One or more analytical conjugates in these binding studies can be used.

[0102] In some embodiments, the identified peptides can be modified by histidine or alanine scanning to identify peptides with changed binding or affinity. For example, a library of mutant peptides can be produced, and each variant can be evaluated for binding to BBB-R target molecules. Any binding assay known to technicians can be used, including any of the above. In some embodiments, histidine scanning is performed to mutate or change the amino acid residues in the identified peptides to histidine. In some embodiments, histidine scanning can be used to identify pH-sensitive antigen binding, such as identifying peptides with reduced binding at acidic pH compared to neutral pH. It is not desirable to be bound by theory, and it is expected that receptor binding peptides bind to 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 present in endosomes), so that the binding molecules containing the peptides (e.g., modified cyclic peptides) are released into the blood-brain barrier and are not recycled. In some embodiments, alanine scanning is performed to mutate or change the amino acid residues in the identified peptides to alanine. In some embodiments, alanine scanning can be used to identify residues that can be mutated and retain binding function.

[0103] Also provided herein are methods for identifying peptides that bind to the BBB-R by amino acid sequence, including from sequence libraries.

[0104] 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: 26-34 or 49-54. 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 and comprises the sequence shown 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 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 as shown in any one of SEQ ID NO:26-34 or 49-54. In some embodiments, the peptide is as shown in SEQ ID NO:26. In some embodiments, the peptide is as shown in SEQ ID NO:49. In some embodiments, the peptide portion binds to transferrin receptor (TrfR). In some embodiments, TrfR is mouse. In some embodiments, TrfR is human. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0105] In some embodiments, the length of the peptide is 6 to 50 amino acids, and comprises a sequence with at least 85%, at least 90%, at least 95% amino acid sequence identity to the sequence shown in any one of SEQ ID NO:26-34 or 49-54. In some embodiments, the length of the peptide is 6 to 50 amino acids, and comprises a sequence with 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NO:26-34 or 49-54. In some embodiments, the length of the peptide is 12 to 18 amino acids, and comprises a sequence with 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NO:26-34 or 49-54. In some embodiments, one or more amino acid substitutions are amino acid residues substituted with alanine. In some embodiments, one or more amino acid substitutions are amino acid residues substituted with histidine. In some embodiments, the peptide portion binds to transferrin receptor (TrfR). In some embodiments, TrfR is mouse. In some embodiments, TrfR is human. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0106] In some embodiments, the length of the peptide is 6 to 50 amino acids and comprises a sequence having at least 85%, at least 90%, at least 95% amino acid sequence identity with the sequence shown in any one of SEQ ID NO: 26. In some embodiments, the length of the peptide is 6 to 50 amino acids and comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 26. In some embodiments, one or more amino acid substitutions are amino acid residues substituted with alanine. In some embodiments, one or more amino acid substitutions are amino acid residues substituted with histidine. In some embodiments, the length of the peptide is 6 to 50 amino acids and comprises a sequence shown in any one of SEQ ID NO: 55 or 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, and comprises the sequence shown in any one of SEQ ID NO:55 or 117-128. In some embodiments, the length of the peptide is 12 to 18 amino acids and comprises the sequence shown in SEQ ID NO:55. In some embodiments, the peptide is as shown in any one of SEQ ID NO:55 or 117-128. In some embodiments, the peptide is as shown in SEQ ID NO:55. In some embodiments, the peptide portion binds to transferrin receptor (TrfR). In some embodiments, TrfR is mouse. In some embodiments, TrfR is human. In some embodiments, the peptide has blood-brain barrier translocation activity.

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

[0108] In some embodiments, the length of the peptide is 6 to 50 amino acids and comprises a sequence having at least 85%, at least 90%, at least 95% amino acid sequence identity with the sequence shown in any one of SEQ ID NOs: 10-23. In some embodiments, the length of the peptide is 6 to 50 amino acids and comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 10-23. In some embodiments, the length of the peptide is 12 to 18 amino acids and comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 210-23. In some embodiments, one or more amino acid substitutions are amino acid residues substituted with alanine. In some embodiments, one or more amino acid substitutions are amino acid residues substituted with histidine. In some embodiments, the peptide portion binds to the leptin receptor (ObR). In some embodiments, ObR is human. 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 set forth 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 and comprises the sequence set forth in any one of SEQ ID NO: 24 or 25. In some embodiments, the peptide is as set forth in SEQ ID NO: 24. In some embodiments, the peptide is as set forth in SEQ ID NO: 25. In some embodiments, the peptide portion binds to ErbB3. In some embodiments, ErBB3 is human. 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%, 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 1, 2, 3 or 4 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 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 24 or 25. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues to alanine. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues to histidine. In some embodiments, the peptide portion binds to ErbB3. In some embodiments, ErbB3 is human. 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 set forth in any one of SEQ ID NOs: 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 and comprises the sequence set forth in any one of SEQ ID NOs: 35 or 36. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence set forth in SEQ ID NO: 35. In some embodiments, the peptide is as set forth in SEQ ID NO: 35. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence set forth in SEQ ID NO: 36. In some embodiments, the peptide is as set forth in any one of SEQ ID NOs: 36. In some embodiments, the peptide portion binds to IGFR. In some embodiments, IGFR is human. 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 with 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 1, 2, 3 or 4 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 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 35 or 36. In some embodiments, one or more amino acid substitutions are amino acid residues substituted with alanine. In some embodiments, one or more amino acid substitutions are amino acid residues substituted with histidine. In some embodiments, the peptide portion binds to IGFR. In some embodiments, IGFR is human. 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 set forth in any one of SEQ ID NOs: 37 or 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 and comprises the sequence set forth in any one of SEQ ID NOs: 37 or 38. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence set forth in SEQ ID NO: 37. In some embodiments, the peptide is as set forth in SEQ ID NO: 37. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence set forth in SEQ ID NO: 38. In some embodiments, the peptide is as set forth in SEQ ID NO: 38. In some embodiments, the peptide portion binds to RAGE. In some embodiments, RAGE is human. 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%, at least 95% amino acid sequence identity with the sequence shown in any one of SEQ ID NO: 37 or 38. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 37 or 38. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 37 or 38. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues to alanine. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues to histidine. In some embodiments, the peptide portion binds to RAGE. In some embodiments, RAGE is human. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0115] In some embodiments, the length of the peptide is 6 to 50 amino acids and comprises the sequence shown in any one of SEQ ID NOs: 39-48. 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 and comprises the sequence shown in any one of SEQ ID NOs: 39-48. In some embodiments, the peptide is as shown in any one of SEQ ID NOs: 39-48. In some embodiments, the length of the peptide is 12 to 18 amino acids and comprises the sequence shown in SEQ ID NO: 39. In some embodiments, the peptide is as shown in SEQ ID NO: 39. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence set forth in SEQ ID NO: 41. In some embodiments, the peptide is as set forth in SEQ ID NO: 41. In some embodiments, the peptide is 12 to 18 amino acids in length and comprises the sequence set forth in SEQ ID NO: 43. In some embodiments, the peptide is as set forth in SEQ ID NO: 43. In some embodiments, the peptide portion binds to LRP-1. In some embodiments, LRP-1 is human. 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%, at least 95% amino acid sequence identity with 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 1, 2, 3 or 4 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 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 39, 41 or 43. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues to alanine. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues to histidine. In some embodiments, the peptide portion binds to LRP-1. In some embodiments, LRP-1 is human. In some embodiments, the peptide has blood-brain barrier translocation activity.

[0117] In some any of the foregoing embodiments, the peptide has blood-brain barrier translocation activity. In some any of the foregoing embodiments, the peptide binds to a receptor involved in blood-brain barrier transcytosis. B. Binding Molecular (eg, Cyclic Peptide) Scaffolds and Modified Cyclic Peptides

[0118] Also provided herein are binding molecules comprising peptides that bind to BBB-R, including any of those shown in Section IA above. In some embodiments, the binding molecule provides a stable scaffold (also interchangeable with the term "skeleton") 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 cyclic peptide.

[0119] The use of protein "scaffolds" and the engineering of loops or regions within the scaffold to change the technology of activity is most noteworthy in terms of antibodies and antibody fragments, which have natural repertoires of variable regions or loops. The variable loops of antibodies have been extensively engineered to produce peptides with improved binding (e.g., affinity and / or specificity) to known ligands, and also to expand the binding substrates of specific antibody frameworks (see, e.g., Knappik et al., (2000) J. Mol. Biol., 296, 57-86; and EP 1025218). The engineering of non-antibody frameworks has been reviewed by, e.g., Hosse et al., (2006), Protein Sci., 15, 14-27. Such non-antibodies or alternative scaffold proteins have considerable advantages over traditional antibodies due to their small size, high stability, and ability to be expressed in prokaryotic hosts. Suitable scaffolds may include polypeptides containing modified whey acid protein (WAP) domains, such as those described in the international patent application published as WO-A-2012073045. Those skilled in the art will appreciate that suitable scaffolds are not limited to the WAP domain of human elastin (trappin-2), but may also include other human trappins (e.g., SLPI) or non-human trappins (e.g., from porcine, bovine or simian sources). In addition, the embodiments provided extend to cyclic peptides contained within other non-trappin members of the WAP domain family. In addition, the embodiments provided also relate to the use of plant cyclic peptides as scaffolds.

[0120] In some embodiments, the molecules provided herein that use cysteine ​​knot proteins (such as cyclic peptides) 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 cyclic peptide is a linear or cyclic amino acid sequence containing a structure referred to herein as a "cysteine ​​knot". When a disulfide bond passes through a closed cyclic ring formed by two other disulfide bonds and the amino acids in the skeleton, a cysteine ​​knot appears. However, the reference to "cysteine ​​knot" herein includes reference to its structural equivalents, which provide similar constraints on the three-dimensional structure of the cyclic skeleton. For example, the appropriate turns and loops in the "cysteine ​​knot" skeleton can also be achieved by engineering suitable covalent bonds or other forms of molecular association. The embodiments provided cover all such modifications made to the cyclic skeleton, which result in retaining the three-dimensional knotted topology imparted by the cysteine ​​knot. In addition, although the cysteine ​​knot is characterized by a knot formed by three disulfide bonds, the embodiments provided extend to molecules that only contain two disulfide bonds. In this case, it may be necessary to further stabilize the molecular framework using other means, or the molecular framework may retain suitable activity despite the change in three-dimensional structure caused by the absence of the third disulfide bond. In yet other modifications, the cysteine ​​knot backbone may contain more than three disulfide bonds, such as appearing in a double cysteine ​​knot or multi-cysteine ​​knot arrangement, or in a single cysteine ​​knot arrangement with one or two additional disulfide bonds.

[0121] Cysteine ​​knot microproteins (such as cyclic peptides) include naturally occurring cysteine ​​knot microproteins or cyclic peptide families found in various plant species. Cysteine ​​knot microproteins (cyclic peptides) are small peptides, usually composed of about 30-40 amino acids, which can exist naturally in cyclic or linear form, wherein the cyclic form does not have a free N-terminal or C-terminal amino or carboxyl end. They have a defined structure based on three intramolecular disulfide bonds and a small three-chain β-fold (Craik et al., 2001; Toxicon 39, 43-60). The cyclic protein exhibits a conserved cysteine ​​residue, which defines a structure referred to herein as "cysteine ​​knot". This family includes both naturally occurring cyclic molecules and their linear derivatives and linear molecules that have undergone cyclization. These molecules can be used as molecular framework structures, and they have enhanced stability compared to lower structured peptides (Colgrave and Craik, 2004; Biochemistry 43, 5965-5975). However, these molecules themselves are unable to cross the blood-brain barrier to any significant extent and cannot be used as neurotherapeutics themselves or as carriers for therapeutic peptide or protein agents.

[0122] The main cyclic peptide features are the remarkable stability due to the cysteine ​​knot, the small size that makes them easy to chemically synthesize, and the extremely high tolerance to sequence changes. The embodiments provided are based on the recognition herein that cyclic peptides therefore appear to be attractive leads or scaffolds for peptide drug design. Cyclic peptide scaffolds have been found in nearly 30 different protein families, among which conotoxins, spider toxins, pumpkin inhibitors, spiny mouse-related proteins and plant cyclic peptides are the families with the most members. It was found that cyclic peptides from Rubiaceae and Violaceae plants are mostly head-to-tail cyclic peptides (Craik et al. 2010. Cell. Mol. Life Sci. 67: 9-16). However, within the pumpkin inhibitor family of cyclic peptides, both cyclic and linear cyclic peptides have been identified from Momordica cochinchinensis: cyclic trypsin inhibitor (MCoTI)-I and -II and its linear counterpart MCoTI-III (Hernandez et al. 2000. Biochemistry, 39, 5722-5730). It is now clear that both cyclic and linear variants can exist in different cyclic peptide families, but little is known about the effects of cyclization. When compared to their linear counterparts, cyclic peptides are expected to show improved stability, better resistance to proteases, and reduced flexibility, hopefully leading to enhanced biological activity. However, linear cyclic peptides have the advantage of being able to be more easily linked to other peptides or proteins.

[0123] For example, cyclic peptides are commonly found in plants. In the aspect of the provided embodiment, the cyclic peptides of the present invention are derived from linear or cyclic forms of cyclic peptides of species of Momordica, Rubiaceae and Violaceae. In a preferred aspect, the cyclic peptides of the present invention are derived from linear or cyclic forms of cyclic peptides of species of Momordica, including the pumpkin serine protease inhibitor family (Otlewski and Korowarsch Acta Biochim Pol. 1996; 43 (3): 431-44), and in a more preferred aspect from Momordica trypsin inhibitor. In some embodiments, Momordica trypsin inhibitors include the following inhibitors MCoTI-I (SEQ ID NO: 1) and -II (SEQ ID NO: 2) (natural cyclic) and MCoTI-III (natural 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] Provided herein are modified cyclic peptides containing peptides of 2 to 50 amino acids that bind to BBB-R. In some embodiments, the peptide is any peptide as described in Section IA. In some embodiments, the peptide is inserted into the ring of an unmodified or wild-type cyclic peptide. In some embodiments, the modified cyclic peptide sequence provided herein can be defined as a peptide having a cysteine ​​knot backbone portion and as a blood-brain barrier translocation portion, the modified cyclic peptide comprising: i) a peptide that binds to BBB-R, wherein the length of the peptide is about 2 to 50 amino acid residues; and ii) a cysteine ​​knot backbone grafted to the peptide of clause i), wherein the cysteine ​​knot backbone comprises structure (I): (X V 1…X V f )C1(X1…X a )C2(X I 1…X I b )C3(X II 1…X II c )C4(X III 1…X III d )C5(X IV 1…X IV e )C6(X V 1…X V f ) wherein C1 to C6 are cysteine ​​residues; wherein each of C1 and C4, C2 and C5, and C3 and C6 are linked by a disulfide bond to form a cysteine ​​knot; wherein each X represents an amino acid residue in a loop, wherein the amino acid residues are the same or different; wherein d is about 1-2; wherein one or more of loops 1, 2, 3, 5 or 6 have an amino acid sequence comprising the sequence of the peptide of clause i), wherein any loop of the sequence of the peptide of clause i) comprises 2 to about 50 amino acids, and wherein for any of loops 1, 2, 3, 5 or 6 of the sequence of the peptide without clause i), a, b, c, e and f are the same or different and are each any number from 3 to 10, and b, c, e and f are each any number from 1 to 20. In some embodiments, the peptide is any peptide described in Section IA. In some embodiments, the peptide is any peptide shown in Table E1 or Table E2.

[0125] In some embodiments, the unmodified or wild-type cyclic peptide may be a cyclic peptide as shown in any one of SEQ ID NOs: 1-3, one or more loops of which are inserted or substituted by one or more amino acid sequences (e.g., a peptide of 2 to 50 amino acids, such as any of those described in Section IA). In some embodiments, the unmodified or wild-type cyclic peptide may be a cyclic peptide as shown in any one of SEQ ID NOs: 1, one or more loops of which are inserted or substituted by one or more amino acid sequences (e.g., a peptide of 2 to 50 amino acids, such as any of those described in Section IA). In some embodiments, the unmodified or wild-type cyclic peptide may be a cyclic peptide as shown in SEQ ID NO: 2, one or more loops of which are inserted or substituted by one or more amino acid sequences (e.g., a peptide of 2 to 50 amino acids, such as any of those described in Section IA). In some embodiments, the unmodified or wild-type cyclic peptide may be a cyclic peptide as shown in any one of SEQ ID NOs: 3, one or more loops of which are inserted or substituted by one or more amino acid sequences (e.g., a peptide of 2 to 50 amino acids, such as any of those described in Section IA). In some embodiments, the modified cyclic peptides provided by the present invention exhibit improved or enhanced binding to receptors involved in endocytic transport across the blood-brain barrier (such as BBB-R, such as human transferrin receptor). In some embodiments, the modified cyclic peptides provided have enhanced blood-brain barrier translocation characteristics. The improvement or enhancement is compared to the unmodified cyclic peptide (such as wild-type or natural cyclic peptide, including cyclic peptides shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3) from which the modified cyclic peptide is derived.

[0126] The provided cyclic peptide contains a sequence of amino acids or analogs thereof that form a cysteine ​​knot backbone, wherein the cysteine ​​knot backbone contains sufficient disulfide bonds or chemical equivalents thereof to confer a knotted topology on the three-dimensional structure of the cysteine ​​knot backbone, and wherein at least one exposed amino acid residue (such as at one or more β turns and / or within one or more loops) is inserted or substituted relative to a naturally occurring cyclic peptide amino acid sequence. In some embodiments, the inserted or substituted loop is loop 1 of a natural or naturally occurring cyclic peptide sequence. In some embodiments, the inserted or substituted loop is loop 2 of a natural or naturally occurring cyclic peptide sequence. In some embodiments, the inserted or substituted loop is loop 3 of a natural or naturally occurring cyclic peptide sequence. In some embodiments, the inserted or substituted loop is loop 5 of a natural or naturally occurring cyclic peptide sequence. In some embodiments, the inserted or substituted loop is loop 6 of a natural or naturally occurring cyclic peptide sequence. In some embodiments, the provided cyclic peptide exhibits enhanced translocation behavior compared to an unmodified or natural parent cyclic peptide.

[0127] In a specific embodiment, the modified cyclic peptide provided is derived from a ring replacement library based on Mcoti-I (SEQ ID NO: 1). In a specific embodiment, a modified cyclic peptide is provided herein, the modified cyclic peptide comprising a blood-brain barrier translocation peptide portion, the blood-brain barrier translocation peptide portion being inserted into the ring of the cyclic peptide Mcoti-I (SEQ ID NO: 1) or replacing or replacing one or more amino acids of the ring. In some embodiments, the peptide has an amino acid sequence of about 2 to 50 amino acid residues, such as any amino acid sequence described in Section IA. In some embodiments, the ring inserted or replaced by the peptide is ring 1. In some embodiments, the modified cyclic peptide has the sequence of SEQ ID NO: 1, wherein the residue between the first two cysteines in ring 1 is 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 ring inserted or replaced by the peptide is ring 5. In some embodiments, the modified cyclic peptide has the sequence of SEQ ID NO: 1, wherein the residue between the last two cysteines in loop 5 is 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 inserted or replaced by the peptide is loop 6, such as formed by cyclization. In some embodiments, the modified cyclic peptide has the sequence of SEQ ID NO: 1, wherein the residue between the first and last cysteines (after cyclization) in loop 6 is 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 a specific embodiment, the modified cyclic peptide provided is derived from a ring replacement library based on Mcoti-II (SEQ ID NO: 2). In a specific embodiment, a modified cyclic peptide is provided herein, the modified cyclic peptide comprising a blood-brain barrier translocation peptide portion, the blood-brain barrier translocation peptide portion being inserted into the ring of the cyclic peptide Mcoti-II (SEQ ID NO: 2) or replacing or replacing one or more amino acids of the ring. In some embodiments, the peptide has an amino acid sequence of about 2 to 50 amino acid residues, such as any amino acid sequence described in Section IA. In some embodiments, the ring inserted or replaced by the peptide is ring 1. In some embodiments, the modified cyclic peptide has the sequence of SEQ ID NO: 2, wherein the residue between the first two cysteines in ring 1 is replaced by a binding 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 ring inserted or replaced by the peptide is ring 5. In some embodiments, the modified cyclic peptide has the sequence of SEQ ID NO:2, wherein the residue between the last two cysteines in loop 5 is replaced by a binding 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 ring inserted or replaced by the peptide is loop 6, such as formed by cyclization. In some embodiments, the modified cyclic peptide has the sequence of SEQ ID NO:2, wherein the residue between the first and last cysteines (after cyclization) in loop 6 is replaced by a binding peptide. In some embodiments, the peptide is inserted between cysteine ​​29 (Cys29) and cysteine ​​4 (Cys4) of SEQ IDNO:2. In a specific embodiment, the modified cyclic peptide provided is derived from a ring replacement library based on Mcoti-III (SEQ ID NO:3). In a specific embodiment, a modified cyclic peptide is provided herein, comprising a blood-brain barrier translocation peptide portion, which is inserted into the ring of the cyclic peptide Mcoti-III (SEQ ID NO: 3) or replaces or replaces one or more amino acids of the ring. In some embodiments, the peptide has an amino acid sequence of about 2 to 50 amino acid residues, such as any amino acid sequence described in Section IA. In some embodiments, the ring inserted or replaced by the peptide is ring 1. In some embodiments, the modified cyclic peptide has the sequence of SEQ ID NO: 3, wherein the residue between the first two cysteines in ring 1 is replaced by a binding 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 ring inserted or replaced by the peptide is ring 5.In some embodiments, the modified cyclic peptide has the sequence of SEQ ID NO: 3, wherein the residue between the last two cysteines in loop 5 is replaced by a binding 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 inserted or replaced by the peptide is loop 6, such as formed by cyclization. In some embodiments, the modified cyclic peptide has the sequence of SEQ ID NO: 3, wherein the residue between the first and last cysteines (after cyclization) in loop 6 is replaced by a binding 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 or substituted into the unmodified cyclic peptide (e.g., cyclic peptide Mcoti-I (SEQ ID NO: 1)) is 2 to 50 amino acid residues. In some embodiments, the peptide inserted or substituted into the unmodified cyclic peptide (e.g., cyclic peptide Mcoti-II (SEQ ID NO: 2)) is 2 to 50 amino acid residues. In some embodiments, the peptide inserted or substituted into the unmodified cyclic peptide (e.g., cyclic peptide 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 In some embodiments, the present invention relates to a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of amino acids, preferably a polypeptide having a plurality of 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 modified cyclic peptide provided has an amino acid sequence that exhibits at least 85% sequence identity to any one of SEQ ID NOs: 56-101 or 105-116, wherein the modified cyclic peptide has blood-brain barrier translocation activity and / or binds to a receptor involved in blood-brain barrier endocytosis. In some embodiments, the modified cyclic peptide provided has an amino acid sequence that exhibits at least 90% sequence identity to any one of SEQ ID NOs: 56-101 or 105-116, wherein the modified cyclic peptide has blood-brain barrier translocation activity and / or binds to a receptor involved in blood-brain barrier endocytosis. In some embodiments, the modified cyclic peptide provided has an amino acid sequence that exhibits at least 95% sequence identity to any one of SEQ ID NOs: 56-101 or 105-116, wherein the modified cyclic peptide has blood-brain barrier translocation activity and / or binds to a receptor involved in blood-brain barrier endocytosis. In some embodiments, the sequence identity to any one of SEQ ID NO: 56-101 or 105-116 is or about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments of any of the above embodiments, the modified cyclic peptide binds to a receptor involved in blood-brain barrier transcytosis, such as BBB-R. In some embodiments, the modified cyclic peptide has blood-brain barrier translocation activity, such as measured after subcutaneous administration to a subject. In some embodiments, the modified cyclic peptide binds to a receptor involved in blood-brain barrier transcytosis, i.e., BBB-R, and has blood-brain barrier translocation activity when subcutaneously administered to a subject.

[0131] In some any embodiments of the foregoing embodiments, the modified cyclic peptide binds to BBB-R expressed on brain endothelial cells. In some embodiments, BBB-R is selected from transferrin receptor (TrfR); lactoferrin receptor (LtfR); leptin receptor (LEP-R, or also known as OB-R); receptor tyrosine-protein kinase (ErbB3), insulin receptors 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.

[0132] In some embodiments, the modified cyclic peptide is derived from a ring replacement library based on Mcoti-II (SEQ ID NO: 2) and binds to a transferrin receptor (e.g., human transferrin receptor). In some embodiments, the ring replacement is to replace at least one ring (such as ring 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 as shown in any one of SEQ ID NOs: 26-34 and 49-54. In some embodiments, the peptide is a peptide as shown in SEQ ID NO: 26. In some embodiments, the peptide is a peptide as shown in SEQ ID NO: 49. In some embodiments, the modified cyclic peptide comprises a sequence as shown in any one of SEQ ID NOs: 72-80 and 95-100. In some embodiments, the modified cyclic peptide is as shown in any one of SEQ ID NOs: 72-80 and 95-100. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 72. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 95. In some embodiments, the peptide contained in the modified cyclic peptide has 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NOs: 26-34 and 49-54. In some embodiments, one or more amino acid substitutions are substituted with histidine. In some embodiments, one or more amino acid substitutions are substituted with alanine. In some embodiments, the modified cyclic peptide comprises a peptide sequence shown in any one of SEQ ID NOs: 55 and 117-128. In some embodiments, the peptide sequence is as shown in any one of SEQ ID NOs: 55 and 117-128. In some embodiments, the modified cyclic peptide comprises a sequence shown in any one of SEQ ID NOs: 101 and 105-116. In some embodiments, the modified cyclic peptide is as shown in any one of SEQ ID NOs: 101 and 105-116. In some embodiments, the peptide sequence is as shown in SEQ ID NOs: 55, and the modified cyclic peptide is as shown in SEQ ID NOs: 101.

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

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

[0135] In some embodiments, the modified cyclic peptide is a modified cyclic peptide that binds to a leptin receptor but has an altered binding to a leptin receptor compared to the cyclic peptide shown in any one of SEQ ID NOs: 56-69. In some embodiments, the binding affinity to the leptin receptor is reduced, such as by 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 10.0 times or more. In some embodiments, the binding affinity to the leptin receptor is pH-dependent, such that the modified cyclic peptide exhibits a greater binding affinity to the leptin receptor at a neutral pH (e.g., 7.0-7.4) than at an acidic pH of the endosome (e.g., a pH of 4.5 to 6.5, such as at or about pH 5.0). In some embodiments, the modified cyclic peptide has a loop with a peptide having 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NOs: 10-23. In some embodiments, the modified cyclic peptide is modified by ring replacement (e.g., ring 1) of Mcoti-II (SEQ ID NO: 2) with the following peptide, which has 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NO: 10-23. In some embodiments, one or more amino acid substitutions are substituted with histidine. In some embodiments, one or more amino acid substitutions are substituted with alanine. In some embodiments, such modified cyclic peptides exhibit enhanced blood-brain barrier translocation characteristics.

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

[0137] In some embodiments, the modified cyclic peptide is a modified cyclic peptide that binds ErbB3 but has an altered binding to ErB3 compared to the cyclic peptide shown in SEQ ID NO: 70 or 71. In some embodiments, the binding affinity to ErbB3 is reduced, such as 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 10.0 times or more. In some embodiments, the binding affinity to ErbB3 is pH-dependent, such that the modified cyclic peptide exhibits a greater binding affinity to ErB3 at a neutral pH (e.g., 7.0-7.4) than at an acidic pH of the endosomal pH (e.g., a pH of 4.5 to 6.5, such as at or about pH 5.0). In some embodiments, the modified cyclic peptide has a loop with a peptide having 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NO: 24 or 25. In some embodiments, the modified cyclic peptide is modified by ring replacement (e.g., ring 1) of Mcoti-II (SEQ ID NO: 2) with a peptide having 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NO: 24 or 25. In some embodiments, one or more amino acid substitutions are substituted with histidine. In some embodiments, one or more amino acid substitutions are substituted with alanine. In some embodiments, such modified cyclic peptides exhibit enhanced blood-brain barrier translocation characteristics.

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

[0139] In some embodiments, the modified cyclic peptide is a modified cyclic peptide that binds to IGFR but has an altered binding to IGFR compared to the cyclic peptide shown in SEQ ID NO: 81 or 82. In some embodiments, the modified cyclic peptide has an altered binding to IGFR compared to the cyclic peptide shown in SEQ ID NO: 82. In some embodiments, the binding affinity to IGFR is reduced, such as by 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 10.0 times or more. In some embodiments, the binding affinity to IGFR is pH-dependent, such that the modified cyclic peptide exhibits a greater binding affinity to IGFR at a neutral pH (e.g., 7.0-7.4) than at an acidic pH of the endosomal pH (e.g., a pH of 4.5 to 6.5, such as or about pH 5.0). In some embodiments, the modified cyclic peptide has a loop with a peptide having 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NO: 35 or 36. In some embodiments, the modified cyclic peptide is modified by ring replacement (e.g., loop 1) of Mcoti-II (SEQ ID NO: 2) with a peptide having 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NO: 35 or 36. In some embodiments, one or more amino acid substitutions are substituted with histidine. In some embodiments, one or more amino acid substitutions are substituted with alanine. In some embodiments, such modified cyclic peptides exhibit enhanced blood-brain barrier translocation characteristics.

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

[0141] In some embodiments, the modified cyclic peptide is a modified cyclic peptide that binds to RAGE but has an altered binding to RAGE compared to the cyclic peptide shown in SEQ ID NO: 83 or 84. In some embodiments, the binding affinity to RAGE is reduced, such as by 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 10.0 times or more. In some embodiments, the binding affinity to RAGE is pH-dependent, such that the modified cyclic peptide exhibits a greater binding affinity to ErB3 at a neutral pH (e.g., 7.0-7.4) than at an acidic pH of the endosomal pH (e.g., a pH of 4.5 to 6.5, such as at or about pH 5.0). In some embodiments, the modified cyclic peptide has a loop with a peptide having 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NO: 37 or 38. In some embodiments, the modified cyclic peptide is modified by ring replacement (e.g., ring 1) of Mcoti-II (SEQ ID NO: 2) with a peptide having 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NO: 37 or 38. In some embodiments, one or more amino acid substitutions are substituted with histidine. In some embodiments, one or more amino acid substitutions are substituted with alanine. In some embodiments, such modified cyclic peptides exhibit enhanced blood-brain barrier translocation characteristics.

[0142] In some embodiments, the modified cyclic peptide is derived from a ring replacement library based on Mcot-II (SEQ ID NO: 2) and binds to LRP-1 (e.g., human LRP-1). In some embodiments, the ring replacement is to replace at least one ring (such as ring 1) with a peptide comprising a sequence shown in any one of SEQ ID NOs: 39-48. In some embodiments, the peptide is a peptide as shown in any one of SEQ ID NOs: 39-48. In some embodiments, the modified cyclic peptide comprises a sequence as shown in any one of SEQ ID NOs: 85-94. In some embodiments, the modified cyclic peptide is as shown in any one of SEQ ID NOs: 85-94. In some embodiments, the modified cyclic peptide comprises a sequence as shown in SEQ ID NO: 87. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 87. In some embodiments, the modified cyclic peptide comprises a sequence as shown in SEQ ID NO: 89. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 89.

[0143] In some embodiments, the modified cyclic peptide is a modified cyclic peptide that binds to LRP-1 but has an altered binding to LRP-1 compared to the cyclic peptide shown in any one of SEQ ID NOs: 85-94. In some embodiments, the binding affinity to LRP-1 is reduced, such as by 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 10.0 times or more. In some embodiments, the binding affinity to the leptin receptor is pH-dependent, so that the modified cyclic peptide exhibits a greater binding affinity to LRP-1 at a neutral pH (e.g., 7.0-7.4) than at an acidic pH of the endosome (e.g., a pH of 4.5 to 6.5, such as being or about pH 5.0). In some embodiments, the modified cyclic peptide has a loop with a peptide that has 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NOs: 39-48. In some embodiments, the modified cyclic peptide is modified by ring replacement (e.g., ring 1) of Mcoti-II (SEQ ID NO: 2) with the following peptide, which has 1, 2, 3 or 4 amino acid substitutions compared to the peptide shown in any one of SEQ ID NO: 39-48. In some embodiments, one or more amino acid substitutions are substituted with histidine. In some embodiments, one or more amino acid substitutions are substituted with alanine. In some embodiments, such modified cyclic peptides exhibit enhanced blood-brain barrier translocation characteristics.

[0144] In some embodiments, any provided modified cyclic peptide binding peptide targeting BBB-R. Methods for determining binding affinity or relative binding affinity are known in the art, solid phase ELISA immunoassay, ForteBioOctet, 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, binding affinity can be measured by flow cytometry, such as measured 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 to BBB-R as determined by, for example, solid phase ELISA immunoassay, flow cytometry or surface plasmon resonance (Biacore) assays.

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

[0146] The cyclic peptide molecules provided herein may include specific functionality, which generally include at least a certain type of membrane translocation activity. In a specific embodiment of the present invention, the membrane translocation activity includes one or more activities selected from the following: the ability to translocate through the intestinal wall (e.g., intestinal mucosa); the ability to translocate through the blood-brain barrier; the ability to translocate through the cell membrane; the ability to translocate into subcellular compartments; the ability to translocate into the nucleus; and the ability to translocate into organelles (including mitochondria). In some embodiments, compared with the parent or scaffold cyclic peptide (such as cyclic peptide Mcoti-II (SEQ ID NO: 2)), the modified cyclic peptide provided exhibits improved or enhanced endocytic transport through the blood-brain barrier. In some embodiments, the improved or enhanced endocytic transport is due to the binding of receptors involved in the endocytic transport of the blood-brain barrier. The membrane translocation activity may be appropriately contained in a peptide or polypeptide, which is located in a functional module of a larger protein. Therefore, the cyclic peptide provided represents a synthetic polypeptide scaffold. The desired physical properties of potential scaffold molecules include high thermal stability and the reversibility of thermal folding and unfolding. II. Conjugates and Fusion Proteins of Cyclic Peptides and Biological Agents

[0147] In some embodiments, a conjugate is also provided herein, comprising: a modified cyclic peptide or binding molecule in any of the foregoing embodiments, and a bioactive agent. In some embodiments, the modified cyclic peptide or binding molecule is any one of those described in Section IB. In some embodiments, the bioactive agent is a small molecule, a peptide, or a protein. In some embodiments, the bioactive agent is a diagnostic agent or a therapeutic agent.

[0148] In some embodiments, the bioactive agent is any agent with therapeutic potential for the treatment of pathologies in the central nervous system (CNS). In some embodiments, the bioactive agent can be selected from neurological disorder drugs, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibodies to brain targets, monoclonal antibodies to brain targets, or peptides to brain targets.

[0149] The cyclic peptides of the invention may also be conjugated or otherwise linked to a biologically active or therapeutic agent selected from the group consisting of: small molecules; antibodies or antibody fragments; hormones; cytokines; nucleic acids; biologically active peptides; glycosylated peptides; imaging agents; and radioactive compounds.

[0150] In some embodiments, the cyclic peptide portion can be an amino acid extension of the C-terminal or N-terminal of a biological agent (e.g., polypeptide). In some respects, a short amino acid linker sequence can be located between a biological agent (e.g., polypeptide) and the cyclic peptide portion. Suitable linker groups can include amides, esters, disulfides, sulfides, ketals, succinates, oximes, carbamates, carbonates, sialyl ethers, or triazoles. In some embodiments, the joint can be a peptide jointer. In some embodiments, the molecule provided includes following molecules, wherein the biological agent (e.g., polypeptide) is, for example, connected to the cyclic peptide portion by chemical conjugation, optionally connected via a linker sequence. Typically, modified polypeptides are connected to another portion via a site that does not interfere with the activity of any portion.

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

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

[0153] In other embodiments of the invention, the cyclic peptides are conjugated to or contained within a desired biotherapeutic, which is then administered to test animals to determine BBB translocation of the modified biotherapeutic.

[0154] Therefore, the therapeutic uses and applications of the cyclic peptides of the present invention include any disease or condition that requires repeated treatment regimens or frequent administration of biologically active agents. This includes the following therapeutic applications, which can benefit from the conjugation of the cyclic peptide to the N-terminus or C-terminus of the therapeutic agent, thereby making the therapeutic agent more easily available to the recipient's brain or cerebrospinal fluid. Diseases suitable for treatment include, but are not limited to: treatment of various neoplastic and non-neoplastic diseases and conditions (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 brain injury conditions. In another embodiment, the cyclic peptide of the present invention is fused with a therapeutic agent and used as an intravenously administered therapeutic treatment for Alzheimer's disease or intracranial tumors. In another embodiment, the cyclic peptide of the present invention is conjugated to a therapeutic agent that can enhance cognitive abilities such as memory.

[0155] In another aspect, the present invention relates to a conjugate, which may include a carrier selected from any one of the modified cyclic peptides of the present invention, and the carrier is connected to a biologically active agent or therapeutic agent selected from, for example, a drug (e.g., a small molecule drug, such as an antibiotic), a medicament, a detectable marker, a protein (e.g., an enzyme), a protein-based compound (e.g., a protein complex comprising a chain or polypeptide chain) and a polypeptide (peptide). More particularly, the medicament may be a molecule active at the level of the central nervous system. The medicament may be any medicament for treating or detecting a neurological disease.

[0156] According to the present invention, the detectable label can be a radioactive imaging agent. Examples of labels that can be conjugated to the carrier of the present invention and are encompassed herein include, for example, but not limited to, isotopes, fluorescent labels (e.g., rhodamine), reporter molecules (e.g., biotin), etc. Other examples of detectable labels include, for example, green fluorescent protein, biotin, his-tagged proteins, and β-galactosidase.

[0157] In some embodiments, the bioactive agent is a therapeutic protein agent. Examples of therapeutic proteins or protein-based compounds that can be conjugated to the carrier of the present invention and are encompassed herein 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, etc.), single domain antibodies (e.g., camelid VHH domains, shark novel antigen receptors (NARs) or human VH or VL domains), peptide or protein-based drugs (e.g., positive pharmacological modulators (agonists) or pharmacological inhibitors (antagonists)), etc. Other examples of agents contemplated herein include growth factors (e.g., fibroblast growth factor 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 from bacterial endotoxins and exotoxins; diphtheria toxin, botulinum toxin, tetanus toxin, pertussis toxin, staphylococcus enterotoxin, toxic shock syndrome toxin TSST-1, adenylate cyclase toxin, Shiga toxin, cholera enterotoxin, etc.), soluble receptors (such as TNF receptor 1 or 2), and anti-angiogenic compounds (endostatin, catechins, nutritional drugs, chemokine IP-10, matrix metalloproteinase (MMPI) inhibitors, anastellin, vironectin, antithrombin, tyrosine kinase inhibitors, VEGF inhibitors, antibodies to receptors, avastin and panitumumab, etc.).

[0158] In some embodiments, the bioactive agent is a therapeutic agent for nervous system disorders. In some embodiments, therapeutic agents for nervous system disorders include but are not limited to small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified forms of natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small inhibitory RNA (siRNA) and short hairpin RNA (shRNA)), ribozymes and small molecules, or active fragments of any of the foregoing. Exemplary nervous system disorder 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 and active fragments of any of the foregoing, which themselves are or specifically recognize and / or act on (i.e., inhibit, activate or detect) CNS antigens or target molecules. 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 protein, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers and neurotrophins. Non-limiting examples of neurological disorder drugs and the corresponding disorders they can be used to treat include brain-derived neurotrophic factor (BDNF) for the treatment of chronic brain injury (neurogenesis); anti-EGFR antibodies for the treatment of brain cancer; glial cell line-derived neurotrophic factor (GDNF) for the treatment of Parkinson's disease; brain-derived neurotrophic factor (BDNF) for the treatment of amyotrophic lateral sclerosis or depression; lysosomal enzymes for the treatment of lysosomal storage disorders of the brain; ciliary neurotrophic factor (CNTF) for the treatment of amyotrophic lateral sclerosis; neuregulin-1 for the treatment of schizophrenia; anti-HER2 antibodies (e.g., trastuzumab) for the treatment of brain metastases of HER2-positive cancers.

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

[0160] In some embodiments, the antibody is directed against (e.g., specifically binds to) antigens and / or molecules expressed in the CNS (including the brain) that can be targeted by antibodies or small molecules. Examples of such antigens and / or molecules include, but are not limited to, β-secretase 1 (BACE1), amyloid β (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau protein, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin protein, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6.

[0161] In some embodiments, the antibody is directed against or binds to HER2. In some embodiments, the antibody is trastuzumab 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 with the heavy chain sequence set forth 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 with the light chain sequence set forth in SEQ ID NO: 137. In some embodiments, the antibody contains a heavy chain sequence set forth in SEQ ID NO: 134 and a light chain sequence set forth in SEQ ID NO: 137. In some embodiments, antibodies targeting HER2 and provided conjugate fusions containing the antibodies can be used to treat metastatic breast cancer. In some embodiments, antibodies targeting HER2 and provided conjugate fusions containing the antibodies can be used to treat neurological diseases, such as Alzheimer's disease.

[0162] In some embodiments, the antibody is directed against or binds to amyloid beta. In some embodiments, the antibody is Aduhelm. 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 with 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 with the light chain sequence shown in SEQ ID NO: 132. In some embodiments, the antibody contains a heavy chain sequence shown in SEQ ID NO: 129 and a light chain sequence shown in SEQ ID NO: 132. In some embodiments, antibodies targeting amyloid beta and provided conjugate fusions containing the antibodies can be used to treat Alzheimer's disease.

[0163] In some embodiments, the bioactive agent is a TNF inhibitor. In some embodiments, the inhibitor is a small molecule, a peptide or a protein. In some embodiments, the inhibitor is an antibody. In some embodiments, the antibody is directed to or in conjunction with TNF-α. In some embodiments, the antibody is adalimumab (Humira). In some embodiments, the antibody contains a heavy chain sequence showing at least about 85%, at least about 90% or at least about 95% sequence identity with the heavy chain sequence shown in SEQ ID NO: 102 and a light chain sequence showing at least about 85%, at least about 90% or at least about 95% sequence identity with the light chain sequence shown in SEQ ID NO: 103. In some embodiments, the antibody contains a heavy chain sequence shown in SEQ ID NO: 102 and a light chain sequence shown in SEQ ID NO: 103. In some embodiments, the antibody targeting TNF-α and the conjugate fusion provided containing the antibody can be used to treat neurological diseases, such as stroke, craniocerebral injury or Alzheimer's disease.

[0164] In some embodiments, the bioactive agent is a growth factor or hormone. In some embodiments, the bioactive agent is a growth factor. The cyclic peptide of the present invention can be operably connected to a growth factor, and the growth factor especially includes ADNP, BDNF, CNTF, GCSF, GDNF, IFNβ, IL-10, insulin, MANF, NGF, NT-3, progranulin, to treat a variety of nervous system disorders, such as stroke, Alzheimer's disease, Parkinson's disease and multiple sclerosis. In some embodiments, the bioactive agent is nerve growth factor (NGF), such as for the treatment of Parkinson's disease, stroke or Alzheimer's disease. In some embodiments, the bioactive agent is granulocyte colony stimulating factor (GCSF), such as for the treatment of stroke or craniocerebral injury (TBI). In some embodiments, the bioactive agent is IL-10, such as for the treatment of Alzheimer's disease, heart disease or brain cancer. In some embodiments, the bioactive agent is BDNF, such as for the treatment of ALS or depression. In some embodiments, the bioactive agent is ADNP, such as for the treatment of autistic disorders.

[0165] In some embodiments, the bioactive agent is NGF and has the sequence set forth in SEQ ID NO: 147 or a sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 147. In some embodiments, the bioactive agent is set forth in SEQ ID NO: 147.

[0166] In some embodiments, the bioactive 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 a glucocerebrosidase, a galactocerebrosidase, or an alpha galactosidase. In some embodiments, the enzyme is a lipase or a hydrolase-type enzyme, and the enzyme is a sphingomyelinase, a serripeptase, or an alpha glucosidase. In some embodiments, the enzyme is heparin N sulfatase, such as for the treatment of Sanfilippo syndrome A. In some embodiments, the enzyme is a glucocerebrosidase, such as for the treatment of Gaucher disease. In some embodiments, the enzyme is a glucocerebrosidase, such as for the treatment of Parkinson's disease. In some embodiments, the enzyme is a galactocerebrosidase, such as for the treatment of Krabbe disease. In some embodiments, the enzyme is an alpha galactosidase, such as for the treatment of Fabry disease. In some embodiments, the enzyme is a sphingomyelinase, such as for the treatment of Niemann-Pick disease. In some embodiments, the enzyme is serripase alpha, such as for the treatment of Bi-Young disease. In some embodiments, the enzyme is alpha glucosidase, such as for the treatment of Pompe disease. In some embodiments, the enzyme is tripeptidyl peptidase I, such as for the treatment of Bi-Young disease or Batten disease. In some embodiments, the enzyme is galactosamine 6-sulfatase, such as for the treatment of Morquio syndrome.

[0167] In some embodiments, the bioactive agent is glucocerebrosidase (GCase). In some embodiments, the enzyme has the amino acid sequence set forth 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 with SEQ ID NO: 144. In some embodiments, the sequence is as set forth in SEQ ID NO: 144. In some embodiments, the enzyme has the amino acid sequence set forth 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 with SEQ ID NO: 145. In some embodiments, the sequence is as set forth in SEQ ID NO: 145. In some embodiments, the glucocerebrosidase is a glucocerebrosidase mutant GCase molecule that exhibits increased stability and enhanced function. Relative to the sequence set forth in SEQ ID NO: 144 or SEQ ID NO: 145, exemplary GCase mutants have one or more amino acid substitutions selected from the group consisting of 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. In some embodiments, the GCase mutant contains 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions from the following: 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, relative to the sequence shown in SEQ ID NO: 144 or SEQ ID NO: 145. In some embodiments, the GCase mutant has amino acid substitutions M53T and P55T relative to the sequence shown in SEQ ID NO: 144 or SEQ ID NO: 145. In some embodiments, the GCase mutant has amino acid substitutions H145L / K321N, D443C / S464C, S455C / R495C, W312C / A341C, I368C / D445C, and E233Q / W312C / A341C relative to the sequence shown in SEQ ID NO:144 or SEQ ID NO:145.In some embodiments, the GCase mutant has an amino acid substitution F316A / L317F, F316A / L317F / K321N or H145L / K321N relative to the sequence shown in SEQ ID NO: 144 or SEQ ID NO: 145. Non-limiting examples of GCase mutants are described in published PCT application numbers WO 2012 / 064709; WO 2021 / 048034; and WO 2022 / 023761.

[0168] The conjugates provided include a modified cyclic peptide linked to a glucocerebrosidase. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 101, such as for targeting TrfR. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 81, such as for targeting IgFR. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 82, such as for targeting IgFR. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 85, such as for targeting LRP-1. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 87, such as for targeting LRP-1. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 89, such as for targeting LRP-1. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 83, such as for targeting RAGE. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 84, such as for targeting RAGE. In some embodiments, the modified cyclic peptide targets TrfR, and the conjugate has the amino acid sequence set forth in SEQ ID NO: 146, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 146. In some embodiments, the conjugate has the sequence set forth in SEQ ID NO: 146. In some embodiments, the modified cyclic peptide binds to the BBB-R (e.g., TrfR) to transport glucocerebrosidase across the blood-brain barrier.

[0169] In some embodiments, the conjugate (such as a fusion protein of a modified cyclic peptide and a biological protein agent) contains 1, 2, 3, 4, 5 or more copies of the modified cyclic peptide. In some embodiments, the conjugate (such as a provided fusion protein) contains a modified cyclic peptide so that the conjugate (e.g., fusion protein) exhibits monovalence for binding to BBB-R. In a specific embodiment, the conjugate (e.g., fusion protein) is monovalent for binding to any BBB-R.

[0170] In some embodiments, the conjugate (e.g., fusion protein) may contain multiple copies of modified cyclic peptides, such as 2, 3, 4 or 5 copies of modified cyclic peptides. In this embodiment, the conjugate (e.g., fusion protein) can be referred to as a multivalent binding protein. A multivalent binding protein comprising a plurality of any provided modified cyclic peptides is also provided. In some embodiments, each modified cyclic peptide can be directly or indirectly connected in series on the same polypeptide chain. In some embodiments, the fusion protein is a multichain polypeptide, and each chain of the polypeptide contains at least one copy of the modified cyclic peptide. For example, in some embodiments, a fusion protein of an antibody (e.g., a therapeutic antibody) and a modified cyclic peptide is provided, wherein the first heavy chain or light chain of the antibody is connected to the first copy of the modified cyclic peptide, and the second heavy chain or light chain is connected to the second copy of the modified cyclic peptide. In some embodiments, the binding protein is divalent and contains two copies of the modified cyclic peptide.

[0171] In some embodiments, the conjugate (e.g., fusion protein) may contain a plurality of different modified cyclic peptides, such as 2, 3, 4 or 5 different modified cyclic peptides. In this embodiment, the conjugate (e.g., fusion protein) may be referred to as a multispecific binding protein. A multispecific binding protein comprising a plurality of different cyclic peptides is also provided, and the cyclic peptides are from any provided modified cyclic peptides. In some embodiments, the binding protein is multispecific and contains at least two different modified cyclic peptides each binding to a different BBB-R. In some embodiments, the multispecific binding protein contains 2, 3 or 4 different modified cyclic peptides, wherein each modified cyclic peptide binds to a different BBB-R. In some embodiments, each modified cyclic peptide can be directly or indirectly connected in series on the same polypeptide chain. In some embodiments, the fusion protein is a multi-chain polypeptide, and each chain of the polypeptide contains a possibly different modified cyclic peptide. For example, in some embodiments, a fusion protein of an antibody (e.g., a therapeutic antibody) and a modified cyclic peptide is provided, wherein the first heavy chain or light chain of the antibody is connected to the first modified cyclic peptide, and the second heavy chain or light chain is connected to the second modified cyclic peptide different from the first modified cyclic peptide. In some embodiments, the binding protein is bispecific and contains two different modified cyclic peptides each binding to a different BBB-R. As an example, in some embodiments, the first modified cyclic peptide binds to the transferrin receptor, and the second modified cyclic peptide binds to IGF1R.

[0172] A plurality of modified cyclic peptides (same or different) do not need to be directly covalently linked to each other. In some embodiments, the intermediate span of one or more amino acid residues causes the modified cyclic peptides (e.g., the first modified cyclic peptide and the second modified cyclic peptide) to be indirectly covalently linked to each other. The connection can be via the N-terminal to the C-terminal residue. In some embodiments, the connection can be carried out via the side chain of the amino acid residue that is not located at the N-terminal or C-terminal of the modified cyclic peptide. Therefore, the connection can be carried out via the terminal or internal amino acid residue or a combination thereof.

[0173] In some embodiments, any of a plurality of modified cyclic peptides (same or different) can be a biological agent (or its chain) separated by a peptide linker (such as a flexible linker). In some embodiments, the peptide linker can be GGGS or other similar flexible linkers, including longer linkers of (GGGS)n, where n is 1-3. In some embodiments, a conjugate (e.g., a fusion protein) containing a plurality of modified cyclic peptides can include 2, 3, 4 or more modified cyclic peptides. In some embodiments, the modified cyclic peptides can be the same or different. In some embodiments, the modified cyclic peptides can be the same to produce divalent, trivalent or other multivalent molecules. In some embodiments, the modified cyclic peptides can be different to provide bispecific, trispecific or other multispecific molecules.

[0174] In some embodiments, any of a plurality of modified cyclic peptides (the same or different) can be connected in series with a biological agent separated by a peptide linker (such as a flexible linker) in a single polypeptide chain. In some embodiments, the peptide linker can be GGGS or other similar flexible linkers, including longer linkers of (GGGS)n, where n is 1-3. In some embodiments, a single polypeptide in series can contain 2, 3, 4 or more modified cyclic peptides to produce a divalent, trivalent, tetravalent or other multivalent molecule. In some embodiments, one or more modified cyclic peptides can be the same or different. In some embodiments, a single polypeptide in series can contain 2, 3, 4 or more different cyclic peptides 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 cyclic peptide. In some embodiments, the antibody can be connected to 1, 2, 3 or 4 modified cyclic peptides, any of which can be the same or different. In some embodiments, the modified cyclic peptide can be connected to the heavy chain or light chain of the antibody. In some embodiments, the modified cyclic peptide can be connected to the N-terminus of the heavy chain or light chain of the antibody. In some embodiments, the modified cyclic peptide can be connected to the C-terminus of the heavy chain or light chain of the antibody. In some embodiments, each modified cyclic peptide is connected to the heavy chain of the antibody. In some embodiments, each chain of the antibody is connected to the modified cyclic peptide.

[0176] In some embodiments, a bivalent fusion protein consisting of an antibody (e.g., a therapeutic antibody) and two copies of a modified cyclic peptide is provided. In some embodiments, the modified cyclic peptide is connected to the two heavy chains of the antibody. In some embodiments, the modified cyclic peptide is connected to the two light chains of the antibody. In some embodiments, the modified cyclic peptide can be connected to the N-terminus of the heavy chain or light chain of the antibody. In some embodiments, the modified cyclic peptide can be connected to the C-terminus of the heavy chain or light chain of the antibody. Methods for preparing antibody fusions are known. In some embodiments, the heavy chain and the light chain are co-expressed in a cell. In some embodiments, when produced in a cell, a double-chain polypeptide is formed by dimerization caused by the formation of a disulfide bond between two heavy chain molecules. In some embodiments, the antibody fusion protein is a homodimer containing two identical copies of a modified cyclic peptide.

[0177] In some embodiments, a monovalent fusion protein consisting of an antibody is provided, wherein a single modified cyclic peptide is connected. In this embodiment, the antibody fusion protein is monovalent for binding to BBB-R. In some embodiments, the modified cyclic peptide is connected to a heavy chain of the antibody. In some embodiments, the modified cyclic peptide is connected to a light chain of the antibody. In some embodiments, the modified cyclic peptide can be connected to the N-terminus of the heavy chain or light chain of the antibody. In some embodiments, the modified cyclic peptide can be connected to the C-terminus of the heavy chain or light chain of the antibody. In some embodiments, the fusion protein is a heterodimer consisting of two different heavy chains and a light chain paired with each heavy chain, wherein only one heavy chain is connected to the modified cyclic peptide.

[0178] In some embodiments, a multi-specific fusion protein consisting of an antibody is provided, wherein at least two different modified cyclic peptides are connected. In some embodiments, the antibody fusion protein is bispecific and contains two different modified cyclic peptides for binding to two different BBB-Rs. In some embodiments, each modified cyclic peptide is connected to the heavy chain of the antibody. In some embodiments, the modified cyclic peptide can be connected in series with the heavy chain. In some embodiments, the modified cyclic peptide can be connected to different heavy chains of the antibody. In some embodiments, each modified cyclic peptide is connected to the light chain of the antibody. In some embodiments, the modified cyclic peptide can be connected in series with the light chain. In some embodiments, the modified cyclic peptide can be connected to different light chains of the antibody. In some embodiments, the modified cyclic peptide can be connected to the N-terminus of the heavy chain or light chain of the antibody. In some embodiments, the modified cyclic peptide can be connected to the C-terminus of the heavy chain or light chain of the antibody. In some embodiments, the fusion protein is a heterodimer consisting of two different heavy chains and a light chain paired with each heavy chain, wherein one heavy chain is connected in series with a different modified cyclic peptide, and the other heavy chain is not connected to a modified cyclic peptide. In some embodiments, the fusion protein is a heterodimer consisting of two different heavy chains and a light chain paired with each heavy chain, wherein each heavy chain is connected to a different modified cyclic peptide.

[0179] Methods for producing heterodimeric antibody fusion proteins are known. In some embodiments, two different heavy chains can be co-expressed in cells using knob-into-hole engineering strategies or other strategies to produce heterodimers, wherein two different heavy chains (e.g., each carrying a different modified cyclic peptide) can interact to form heterodimers. In some embodiments, the residues of the constant chain are modified by amino acid substitution to promote heterodimer formation. In some any embodiments, the 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. Heterodimers can be formed by transforming 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 different second heavy chain polypeptide subunit (e.g., a hole sequence) into a cell. In some aspects, heterodimers are formed by covalent or non-covalent interactions between the residues of the two polypeptide subunits mediating dimer formation, which is produced from cells after expression and secretion. In such a process, a mixture of dimeric molecules is generally formed, the mixture comprising homodimers and heterodimers. For the production of heterodimers, additional steps for purification may be required. For example, the first and second polypeptides can be engineered to include tags with metal chelates or other epitopes, wherein the tags are different. The tagged domains can be used for rapid purification by metal chelate chromatography and / or by antibodies to allow detection by Western blot, immunoprecipitation, or activity depletion / blocking in bioassays. In other embodiments, methods to promote heterodimerization can be performed.

[0180] Methods include those described in U.S. Pat. No. 10,995,127. For example, having an amino acid modification at Thr366 within a CH3 domain when replaced with a bulkier amino acid (e.g., Try (T366W)) can preferentially pair with a second CH3 domain having amino acid modifications at positions Thr366, Leu368, and Tyr407, respectively, to smaller amino acids, e.g., Ser, Ala, and Val (T366S / L368A / Y407V). In some embodiments, the "knob" Fc domain comprises the mutation T366W. In some embodiments, the "hole" Fc domain comprises the mutations T366S, L368A, and Y407V. The heterodimerization carried out via CH3 modification can be further stabilized by introducing a disulfide bond, for example, by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on the relative CH3 domain (reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, the Fc domain for heterodimerization comprises other mutations, such as the mutation S354C on the first member of the heterodimer Fc pair, which forms an asymmetric disulfide bond with the corresponding mutation Y349C on the second member of the heterodimer Fc pair. In some embodiments, one member of the heterodimer Fc pair comprises modification H435R or H435K to prevent protein A from binding while maintaining FcRn binding. In some embodiments, one member of the heterodimer Fc pair comprises modification H435R or H435K, and the second member of the heterodimer Fc pair is unmodified at H435. In various embodiments, the hole Fc domain comprises the modification H435R or H435K (when the modification is H435R, referred to as "hole-R" in some cases), while the knob Fc domain does not contain the modification. In some cases, the hole-R mutation improves the purification of heterodimers compared to the homodimeric hole Fc domain that may be present.

[0181] It is within the level of the skilled artisan to make appropriate knob and hole modifications in the heavy chain of any antibody. As an example, the knob and hole sequences of the exemplary antibody trastuzumab are shown as 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 as SEQ ID NO: 130 and SEQ ID NO: 131, respectively. Any antibody can be similarly modified in the CH3 domain to generate knob and hole chains to promote heterodimerization. In some embodiments, the modified cyclic peptide is linked to the knob heavy chain and / or hole heavy chain of the antibody. In some embodiments, the knob heavy chain, hole heavy chain and light chain are co-expressed in a cell, wherein a heterodimeric antibody containing two different heavy chains (knob and hole) is produced, each of which is complexed with a light chain.

[0182] In some embodiments, one or more "peptide linkers" connect the modified cyclic peptide and one or more other modified cyclic peptides or biological agents. In some embodiments, the length of the peptide linker can be a single amino acid residue or longer. In some embodiments, the peptide linker has at least one amino acid residue, but the length does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (by single-letter amino acid code): GGGGS ("4GS") or a polymer of a 4GS linker, such as 2, 3, 4 or 5 repeats of 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 NO: 154 and 155, respectively. In some embodiments, the linker may also include a single series of alanine residues or may also include another peptide linker (such as a 4GS linker or a polymer thereof). In some embodiments, the linker is GSGGGSGGGGSGGGGS (SEQ ID NO: 104).

[0183] In some embodiments, the linker is a GS linker of 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 between any of the foregoing values. 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); GGGGGSGGGGGSGGGGGS, i.e. (G5S)3 (SEQ ID NO: 188); GGSGGGGSGGGGSGGGGS (SEQ ID NO: 189) and GGGGSGGGGSGGGGS (SEQ ID NO: 155) or GSGGGSGGGGSG GGGS (SEQ ID NO: 104). In some embodiments, the GS linker is as shown 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 ensure that free protein (i.e., a biological agent (e.g., a therapeutic agent)) is released to the brain. Examples of cleavable linkers include acid-labile linkers. Acid-labile linkers include cis-aconitic acid, cis-carboxylic acid dienes, cis-carboxylic acid trienes, and polymaleic anhydride. Other cleavable linkers are linkers that can be attached to a primary alcohol group.

[0185] In some embodiments, the joint is a cleavable joint containing an endosome-specific protease cleavage site. In some embodiments, the endosome-specific protease cleavage site is a cathepsin cleavage site, and the joint is a cathepsin cleavable joint. In some embodiments, the cathepsin is cathepsin B, cathepsin D, cathepsin K, cathepsin S or cathepsin L. In some embodiments, the cathepsin cleavage site can be any site as described in PCT Publication No. WO 2016 / 050934. In some embodiments, the joint containing the cathepsin cleavage site is shown in any one of SEQ ID NO: 133 and 156-176. In some embodiments, the joint has a cathepsin B cleavage site. In some embodiments, the cathepsin B joint is shown in SEQ ID NO: 133.

[0186] The conjugates provided include bivalent cyclic peptide antibody fusions, which are composed of antibodies connected to modified cyclic peptides (such as any modified cyclic peptides described herein). In some embodiments, the modified cyclic peptide is connected to the C-terminus of the two heavy chains of the antibody. In some embodiments, the antibody is adalimumab. In some embodiments, the antibody fusion contains a heavy chain consisting of an adalimumab heavy chain, a linker, and a modified cyclic peptide (such as any of those described in Section IB) as shown in SEQ ID NO: 102; and a light chain as shown in SEQ ID NO: 103. In some embodiments, the antibody fusion contains a heavy chain consisting of a trastuzumab heavy chain, a linker, and a modified cyclic peptide (such as any of those described in Section IB) as shown in SEQ ID NO: 103; and a light chain as shown in SEQ ID NO: 137. In some embodiments, the antibody is a homodimer consisting of two identical heavy chains and two identical light chains. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 72, such as for targeting TrfR. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 101, such as for targeting TrfR. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 81, such as for targeting IgFR. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 82, such as for targeting IgFR. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 85, such as for targeting LRP-1. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 87, such as for targeting LRP-1. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 89, such as for targeting LRP-1. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 83, such as for targeting RAGE. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 84, such as for targeting RAGE. In some embodiments, the linker is a GS linker of 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 as shown in SEQ ID NO: 104. Also provided herein are antibody fusion conjugates having a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the aforementioned amino acid sequences. In some embodiments, the modified cyclic peptide binds to the BBB-R recognized by the modified cyclic peptide to transport the antibody across the blood-brain barrier.

[0187] The conjugates provided include monovalent cyclic peptide antibody fusions consisting of an antibody connected to a modified cyclic peptide (such as any modified cyclic peptide described herein). In some embodiments, the modified cyclic peptide is connected to the C-terminus of one heavy chain of a heterodimeric antibody. In some embodiments, the antibody fusion contains a knob heavy chain of the antibody; a second heavy chain consisting of a mortar heavy chain of the antibody; and a light chain of the antibody, wherein the knob heavy chain or the mortar heavy chain is connected to a modified cyclic peptide (such as any of those described in Section IB) at its C-terminus by a linker. The antibody can be a heterodimeric antibody (e.g., knob and mortar variants) of any desired therapeutic or diagnostic antibody desired for transport across the blood-brain barrier. Non-limiting examples of antibodies include any of the antibodies described herein. In one embodiment, the antibody is aducanumab. In some embodiments, the antibody fusion contains a first heavy chain, which is the aducanumab knob heavy chain set forth in SEQ ID NO: 131; a second heavy chain, which is composed of the aducanumab knob heavy chain set forth in SEQ ID NO: 130, a linker, and a modified cyclic peptide (such as any of those described in Section IB); and a light chain set forth in SEQ ID NO: 132. In another embodiment, the antibody fusion contains a first heavy chain composed of the aducanumab knob heavy chain set forth in SEQ ID NO: 131, a linker, and a modified cyclic peptide (such as any of those described in Section IB); a second heavy chain, which is the aducanumab knob heavy chain set forth in SEQ ID NO: 130; and a light chain set forth in SEQ ID NO: 132. In additional embodiments, the antibody fusion contains a first heavy chain that is a trastuzumab hole heavy chain as set forth in SEQ ID NO: 136 or 178; a second heavy chain that is composed of a trastuzumab knob heavy chain as set forth in SEQ ID NO: 135, a linker, and a modified cyclic peptide (such as any of those described in Section IB); and a light chain as set forth in SEQ ID NO: 137. In some embodiments, the modified cyclic peptide is as set forth in SEQ ID NO: 72, such as for targeting TrfR. In some embodiments, the modified cyclic peptide is as set forth in SEQ ID NO: 101, such as for targeting TrfR. In some embodiments, the modified cyclic peptide is as set forth in SEQ ID NO: 81, such as for targeting IgFR. In some embodiments, the modified cyclic peptide is as set forth in SEQ ID NO: 82, such as for targeting IgFR. In some embodiments, the modified cyclic peptide is as set forth in SEQ ID NO: 85, such as for targeting LRP-1. In some embodiments, the modified cyclic peptide is as set forth in SEQ ID NO: 87, such as for targeting LRP-1. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 89, such as for targeting LRP-1. In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO: 83, such as for targeting RAGE.In some embodiments, the modified cyclic peptide is as shown in SEQ ID NO:84, such as for targeting RAGE. In some embodiments, the linker is a GS linker of 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 as shown in SEQ ID NO:104. Also provided herein is an antibody fusion conjugate having a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of the aforementioned amino acid sequences. In some embodiments, the modified cyclic peptide binds to the BBB-R recognized by the modified cyclic peptide to transport the antibody across the blood-brain barrier.

[0188] The conjugates provided include bispecific cyclic peptide antibody fusions, which are composed of antibodies connected to two different modified cyclic peptides (such as any modified cyclic peptides described herein). In some embodiments, one modified cyclic peptide is connected to the C-terminus of one heavy chain of a heterodimeric antibody, and the other modified cyclic peptide is connected to the C-terminus of the other heavy chain of the heterodimeric antibody. In some embodiments, the antibody fusion contains a knob heavy chain of an antibody, which is connected to the first modified cyclic peptide at its C-terminus by a linker; a second heavy chain, which is composed of a mortise heavy chain of an antibody, which is connected to the second modified cyclic peptide at its C-terminus by a linker; and a light chain of an antibody. The antibody can be a heterodimeric antibody (e.g., a knob and mortise variant) of any desired therapeutic or diagnostic antibody that is desired for transport across the blood-brain barrier. Non-limiting examples of antibodies include any antibody as described herein. In one embodiment, the antibody is aducanumab. In some embodiments, the antibody fusion contains a first heavy chain, which is a mortal aducanumab heavy chain as set forth in SEQ ID NO: 131, a linker, and a first modified cyclic peptide; a second heavy chain, which is composed of a knob aducanumab heavy chain as set forth in SEQ ID NO: 130, a linker, and a second modified cyclic peptide; and a light chain as set forth in SEQ ID NO: 132. In some embodiments, the antibody fusion contains a first heavy chain, which is a mortal trastuzumab heavy chain, a linker, and a first modified cyclic peptide as set forth in SEQ ID NO: 136; a second heavy chain, which is composed of a knob trastuzumab heavy chain as set forth in SEQ ID NO: 135, a linker, and a second modified cyclic peptide; and a light chain as set forth in SEQ ID NO: 137. In some embodiments, the first modified cyclic peptide and the second modified cyclic peptide are different and are each independently any one of those described in Section IB. In some embodiments, the modified cyclic peptide is independently selected from the following modified cyclic peptides: it targets TrfR (e.g., as shown in SEQ ID NO: 72 or SEQ ID NO: 101), targets IgFR (e.g., as shown in SEQ ID NO: 81 or SEQ ID NO: 82), targets LRP-1 (e.g., SEQ ID NO: 85, SEQ ID NO: 87 or SEQ ID NO: 89), or is used to target RAGE (e.g., SEQ ID NO: 83 or SEQ ID NO: 84). In some embodiments, one of the first modified cyclic peptide and the second modified cyclic peptide is a modified cyclic peptide as shown in SEQ ID NO: 72, and the other of the first modified cyclic peptide and the second modified cyclic peptide is a cyclic peptide as shown in SEQ ID NO: 82.In some embodiments, one of the first modified cyclic peptide and the second modified cyclic peptide is a modified cyclic peptide as shown in SEQ ID NO: 101, and the other of the first modified cyclic peptide and the second modified cyclic peptide is a cyclic peptide as shown in SEQ ID NO: 82. Any of the various combinations of modified cyclic peptides (such as any of those described in Section IB) can be used. In some embodiments, the linker is a GS linker of 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 as shown in SEQ ID NO: 104. Also provided herein is an antibody fusion conjugate having a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of the aforementioned amino acid sequences. In some embodiments, the modified cyclic peptide binds to the BBB-R recognized by the modified cyclic peptide to transport the antibody across the blood-brain barrier.

[0189] In some embodiments, any provided conjugate (such as fusion protein) binds to BBB-R, and the binding is conferred by the peptide of the modified cyclic peptide of the conjugate. The method for determining binding affinity or relative binding affinity is known in the art, 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, binding affinity can be measured by flow cytometry, such as measured based on mean fluorescence intensity (MFI) in a flow binding assay. In some embodiments, the conjugate provided, such as fusion protein, has a binding affinity to BBB-R as determined by, for example, solid phase ELISA immunoassay, flow cytometry or surface plasmon resonance (Biacore) assay.

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

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

[0192] In some embodiments, compared to the non-conjugated form of the bioactive agent or therapeutic agent not conjugated to the modified cyclic peptide, the conjugate containing the modified cyclic peptide exhibits improved or enhanced delivery of the bioactive agent or therapeutic agent to the brain. In some embodiments, the delivery is improved or enhanced by about 1.2 times, about 1.3 times, 1.4 times, 1.5 times, 1.8 times, 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times or more. In some embodiments, the conjugate provided delivers greater than 0.1% of the bioactive agent or therapeutic agent injected into the bloodstream (e.g., by subcutaneous or intravenous injection) to the brain. In some embodiments, the conjugate provided delivers 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) to the brain. III. Nucleic Acids and Preparation Methods

[0193] Additional embodiments provided herein provide nucleic acid sequences encoding the amino acid sequence of any provided binding molecule (e.g., any provided modified cyclic peptide or fusion protein containing the modified cyclic peptide). In specific embodiments, the nucleic acid sequence can be contained in a vector, a suitable expression vector, optionally a display vector (including a phage or cis display vector), wherein the encoding DNA is operably linked to the peptide.

[0194] Also provided herein are methods for preparing the provided binding molecules (e.g., modified cyclic peptides or fusion proteins containing the modified cyclic peptides). In some embodiments, the methods include culturing host cells under conditions that allow expression of the fusion protein. In some embodiments, the provided methods further include separating the expressed modified cyclic peptide or fusion protein from the supernatant or from the lysate of the host cells after culturing.

[0195] In any of the embodiments provided above, nucleic acids encoding the binding molecules provided herein (such as any modified cyclic peptides or fusion proteins provided) can be introduced into cells using recombinant DNA and cloning techniques. For this reason, recombinant DNA molecules encoding polypeptides are prepared. Methods for preparing such DNA molecules are well known in the art. In some embodiments, chemical synthesis techniques such as the phosphoramidite method can be used to synthesize DNA molecules. In some cases, recombinant or synthetic nucleic acids can be produced by polymerase chain reaction (PCR). In some embodiments, DNA can be cloned into suitable transduction / transfection vectors as known to those skilled in the art. Expression vectors containing nucleic acid molecules are also provided.

[0196] In some embodiments, the expression vector is capable of expressing a binding molecule (such as a modified cyclic peptide or a fusion protein containing the modified cyclic peptide) in an appropriate cell under conditions suitable for expressing the protein. In some aspects, the nucleic acid molecule or expression vector comprises a DNA molecule encoding a modified cyclic peptide or fusion protein operably linked to an appropriate expression control sequence. Methods for achieving such operative connection before or after the DNA molecule is inserted into the vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, termination signals, capping signals, polyadenylation signals, and other signals related to transcription or translation control.

[0197] Also provided is a vector, the vector comprising any nucleic acid sequence encoding a binding molecule (such as a modified cyclic peptide or fusion protein). Suitable vectors include those commonly used in genetic engineering technology, such as bacteriophages, plasmids, cosmids, viruses or retroviruses. Suitable vectors may include other expression control elements, such as transcription start sites, transcription termination sites, ribosome binding sites, RNA splicing sites, polyadenylation sites, translation termination sites, etc. Suitable vectors may further include other regulatory elements, such as transcription / translation enhancer sequences, and at least marker genes or reporter genes that allow screening of vectors under suitable conditions. Suitable marker genes include, for example, dihydrofolate reductase genes and G418 or neomycin resistance genes that can be used for eukaryotic cell culture, and ampicillin, streptomycin, tetracycline or kanamycin resistance genes that can be used for Escherichia coli (E.coli) and other bacterial cultures. Suitable vectors for the present invention may further include nucleic acid sequences encoding secretion signals. These sequences are well known to those skilled in the art.

[0198] In some embodiments, the expression vector further comprises a promoter sequence to control the expression of the modified cyclic peptide or fusion protein. As used herein, the term "promoter sequence" refers to a DNA sequence that is generally located upstream of a gene present in a DNA polymer and provides a site for the initiation of transcription of the gene into mRNA. Promoter sequences suitable for use can be derived from viruses, bacteriophages, prokaryotes or eukaryotic cells, and can be constitutive promoters or inducible promoters.

[0199] In some embodiments, the promoter sequence is operably connected to the sequence encoding the modified cyclic peptide or fusion protein. As used herein, the term "operably connected" means that the first sequence is arranged close enough to the second sequence so that the first sequence can affect the second sequence or the region under the control of the second sequence. For example, the promoter sequence can be operably connected to a gene sequence, and is generally located at the 5' end of the gene sequence so that the expression of the gene sequence is under the control of the promoter sequence. In addition, a regulatory sequence can be operably connected to a promoter sequence to enhance the ability of the promoter sequence in promoting transcription. In this case, the regulatory sequence is generally located at the 5' end of the promoter sequence.

[0200] Promoter sequences suitable for use include any of the following: viruses, bacterial cells, yeast cells, fungal cells, algae cells, plant cells, insect cells, animal cells and human cells. For example, promoters that can be used for bacterial cells include but are not limited to tac promoter, T7 promoter, T7 A1 promoter, lac promoter, trp promoter, trc promoter, araBAD promoter and λPRPL promoter. Promoters that can be used in plant cells include, for example, 35SCaMV promoter, actin promoter, ubiquitin promoter, etc. Regulatory elements suitable for mammalian cells include CMV-HSV thymidine kinase promoter, SV40, RSV promoter, CMV enhancer or SV40 enhancer.

[0201] Depending on the vector and host cell system used, the recombinant gene product (protein) produced can be retained in the recombinant cell, secreted into the culture medium, secreted into the periplasm, or retained on the outer surface of the cell membrane. The recombinant gene product (protein) produced by the method can be purified by using a variety of 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 the method is preferably recovered in a "substantially pure" form. As used herein, the term "substantially pure" refers to a purity of the purified protein that allows the effective use of the purified protein as a commercial product.

[0202] In some embodiments, the methods provided for producing binding molecules (such as modified cyclic peptides or fusion proteins containing the modified cyclic peptides) can be performed using any host organism or cell that can express heterologous polypeptides and can be genetically modified. For the purpose of clarity, the term "host cell" is used throughout this article, but it should be understood that unless it is not feasible due to technical reasons, the host organism can replace the host cell.

[0203] In some embodiments, the host cell can be a variety of eukaryotic cells, such as yeast cells, or mammalian cells, such as Chinese hamster ovary (CHO) or HEK293 cells. In some embodiments, the host cell is a suspension cell, and the polypeptide is engineered or produced in a culture suspension, such as in a cultured suspension CHO cell (e.g., CHO-S cell). In some examples, the cell line is a CHO cell line lacking DHFR (DHFR-), such as DG44 and DUXB11. In some embodiments, the cell lacks glutamine synthase (GS), for example, CHO-S cells, CHOK1 SV cells, and CHOZN ((R)) GS- / - cells. In some embodiments, the CHO cell (e.g., suspension CHO cell) can be a CHO-S-2H2 cell, a CHO-S-clone 14 cell, or an ExpiCHO-S cell.

[0204] In some embodiments, the host cell can also be a prokaryotic cell, such as Escherichia coli. The transformed recombinant host is cultured under polypeptide expression conditions and then purified to obtain soluble protein. The recombinant host cell can be cultured under conventional fermentation conditions so that the desired polypeptide is expressed. Such fermentation conditions are well known in the art. Finally, the polypeptide provided herein can be recovered and purified from recombinant cell culture by any of many 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. When completing the configuration of mature protein, a protein refolding step can be used as needed. Finally, high performance liquid chromatography (HPLC) can be used in the final purification step.

[0205] In some embodiments, polypeptide provided herein can also be prepared by synthetic method.Solid phase synthesis is the preferred technology for preparing independent peptides, because it is the most cost-effective method for preparing small peptides.For example, known solid phase synthesis technology comprises the use of blocking group, joint and solid support, and specific protection and deprotection reaction conditions, joint cutting conditions, use of scavenger and other aspects of solid phase peptide synthesis.Then peptide can be assembled into polypeptide as provided in this paper. IV. Pharmaceutical Compositions

[0206] In some embodiments, a pharmaceutical composition is also provided herein, comprising any of the molecules provided in any of the foregoing embodiments, the molecules including any modified cyclic peptide or a conjugate or fusion protein containing the modified cyclic peptide, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is used to treat a nervous system disease. In some embodiments, the pharmaceutical composition is used to diagnose a nervous system disease.

[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 agents and absorption delaying agents compatible with drug administration. Suitable carriers are described in the latest edition of the standard reference text Remington's Pharmaceutical Sciences 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. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and medicaments for pharmaceutically active substances is well known in the art. Unless any conventional media or medicaments are incompatible with the active compound, its use in the composition is envisioned. Supplementary active compounds may also be incorporated into the composition.

[0208] The medicaments and pharmaceutical compositions of the present invention can take the following forms: liquid, solution, suspension, lotion, gel, tablet, pill, pellet, powder, modified release formulation (such as slow release or sustained release), suppository, emulsion, aerosol, spray, capsule (for example, capsule containing liquid or powder), liposome, microparticle 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 edition, 1995, see, for example, pages 1447-1676.

[0209] In order to help therapeutic agent (comprising cyclic peptide) to be dissolved in aqueous environment, surfactant can be added as wetting agent.Surfactant can include anionic detergent, such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.Cationic detergent can be used, and it can include benzalkonium chloride or benzethonium chloride.Potential nonionic detergent that can be included in preparation as surfactant includes: lauromacrogol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10,50 and 60, glyceryl monostearate, polysorbate 20,40,60,65 and 80, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose.When using, these surfactants can be present in the preparation of peptide or nucleic acid or derivative alone or as the mixture of different ratios.

[0210] Additives may be included to further enhance cellular uptake of the cyclic peptides of the invention, such as the fatty acids oleic acid, linoleic acid, and linolenic acid.

[0211] The pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration. The example of route of administration includes parenteral, such as intravenous, intradermal, subcutaneous, intratumoral, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal and rectal administration. In a specific embodiment, 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 application may contain the following components: a sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. pH may be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0213] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMO PHOR EL TM (BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under manufacturing and storage conditions and must be preserved against the contaminating effects of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Preventing the effects of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. Extended absorption of the injectable composition can be achieved by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0214] Sterile injectable solutions can be prepared by incorporating the active compound in the desired amount into an appropriate solvent optionally with one or a combination of the ingredients enumerated above, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient and any additional desired ingredients from previously sterile filtered solutions.

[0215] Oral compositions generally include an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and gargled, and is spit out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: a binder such as microcrystalline cellulose, tragacanth or gelatin; an excipient such as starch or lactose; a disintegrant such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as mint, methyl salicylate or orange flavor.

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

[0217] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art, and for example, include detergents, bile salts and fusidic acid derivatives with respect to transmucosal administration. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream as generally known in the art.

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

[0219] In one embodiment, the active compound is prepared with a carrier that protects the compound from being rapidly eliminated from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. The preparation method of such formulations will be clear to those skilled in the art. These materials can also be commercially available from Alza and Nova Pharmaceuticals. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art (e.g., as described in U.S. Patent No. 4,522,811).

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

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

[0222] The pharmaceutical composition is administered in an amount effective for treating or preventing a specific indication. The therapeutically effective amount generally depends on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the illness to be treated, or the age of the subject being treated. In some embodiments, the pharmaceutical composition may be administered in an amount ranging from about 50 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount ranging from about 100 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose.

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

[0224] The pharmaceutical composition can be applied to the subject as needed. In some embodiments, the pharmaceutical composition of an effective dose is applied to the subject once or repeatedly. In various embodiments, once a month, less than once a month (such as, for example, once every two months, once every three months, or once every six months) the pharmaceutical composition of an effective dose is applied to the subject. In other embodiments, more than once a month (such as, for example, once every two weeks, once a week, twice a week, three times a week, once a day, or multiple times a day) the pharmaceutical composition of an effective dose is applied. The pharmaceutical composition of an effective dose is applied to the subject at least once. In some embodiments, the pharmaceutical composition of an effective dose can be applied multiple times, including a time period that lasts at least one month, at least six months, or at least one year. In some embodiments, the pharmaceutical composition is applied to the subject as needed, to alleviate one or more symptoms of illness. V. Method of use and treatment

[0225] In some embodiments, also provided herein are methods for transporting a bioactive agent across the blood-brain barrier of a subject, the method comprising administering to a mammal in need thereof a conjugate (eg, fusion protein) or pharmaceutical composition of any of the foregoing embodiments.

[0226] In some embodiments, the present invention also provides a method for treating a subject suffering from a nervous system disease, the method comprising administering a conjugate (e.g., fusion protein) or a pharmaceutical composition of any of the foregoing embodiments to the subject. In some embodiments, a conjugate (e.g., fusion protein) or a pharmaceutical composition comprising it is administered to a subject in an effective amount to achieve the treatment of a nervous system disease. Also provided herein is a conjugate (e.g., fusion protein) or a pharmaceutical composition containing it in such methods and treatments and in the preparation of a medicament for such treatment methods. In some embodiments, the method is performed by administering a conjugate (e.g., fusion protein) or a composition comprising it to a subject suffering from, having suffered from, or suspected of suffering from a nervous system disease. In some embodiments, the method thus treats a nervous system disease in a subject. Also provided herein is the use of any composition (such as a pharmaceutical composition provided herein) for the treatment of a nervous system disease.

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

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

[0229] In some embodiments, the nervous system disease includes non-neoplastic diseases and disorders, such as cancer / neoplastic diseases and related conditions. In some embodiments, the nervous system disease is an intracranial tumor.

[0230] In some embodiments, the neurological disease or disorder is directed to treating stroke or other brain damaging conditions.In some embodiments, the cyclic peptides of the invention are conjugated to therapeutic agents capable of enhancing cognitive abilities such as memory.

[0231] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclic peptide connected to a glucocerebrosidase is administered to a subject to treat a neurodegenerative disease. In some embodiments, the neurological disease is Gaucher disease. In some embodiments, the neurological disease is Parkinson's disease. Glucocerebrosidase and a conjugate containing it can be any of those described in Section II. Glucocerebrosidase is a hydrolase bound to the luminal membrane of the lysosome, where it interacts with glycolipids. Inherited autosomal recessive mutations in the GBA gene encoding glucocerebrosidase (GCase) lead to clinical manifestations of activity deficiency and Gaucher disease (GD) (Beutler and Grabowski, 1994). Mutations in GBA are also often found in Parkinson's patients. When dysfunction occurs, the accumulation of substrates induces altered inflammatory responses in tissue macrophages (Gaucher cells) and other related cells (Komhaber et al., 2008; Schetz and Shankar, 2004; Smith et al., 2017). Gaucher cells are the most important pathological hallmarks along with mononuclear phagocytes and neuronal cells involved in the pathology of GD in the brain (Beutler and Grabowski, 2001).

[0232] In some embodiments, the neurological disease is Gaucher disease. Symptoms of GD include anemia, hepatomegaly and splenomegaly, bone lesions, and in severe cases neurological manifestations, which are roughly divided 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 almost no neuropathic effects and can currently be treated with enzyme replacement therapy (ERT). Type II and type III are more severe types of GD, where type II has an acute neuropathic phenotype, indicating symptoms close to birth, and progresses in early infancy until death. Type III causes chronic severe neuropathic symptoms, including learning disabilities, cardiac abnormalities, and myoclonic epilepsy (Beutler et al., 1994; Sidransky et al., 2007; Sidransky and Lopez, 2012). In some embodiments, the methods provided are used to treat type I Gaucher disease. In some embodiments, the methods provided are used to treat type II Gaucher disease. In some embodiments, the methods provided are used to treat type III Gaucher disease.

[0233] In some embodiments, the subject to be treated has a mutation in a native glucocerebrosidase gene (also referred to as GCase or GBA gene) that is associated with a neurological disease (e.g., GD) in the subject. Approximately 300 mutations have been identified and are directly associated with the progression of GD, which results in a wide spectrum of pathology for this disorder (Alfonso et al., 2007; Grabowski and Horowitz, 1997). Inherited missense and nonsense mutations in the GBA gene can lead to misfolding, mistransport, and destabilization of glucocerebrosidase. The two most common mutations found in the literature are mutations N370S and L444P, accounting for 50% of all known GBA mutations. In some embodiments of the provided methods, the subject carries the GCase mutations N370S and / or L444P. Patients carrying the mutant GCase variant N370S are often diagnosed with type I GD, with a wide variety of symptoms. N370S is responsible for preserving and stabilizing the proper conformation of the active binding pocket (Lieberman et al., 2007). L444P revealed a more severe type classified as type II or type III when carrying this GBA gene mutation, which may disrupt the hydrophobic structure and alter domain II function (Lieberman et al., 2007).

[0234] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclic peptide connected to an anti-HER2 antibody is administered to a subject to treat a neurological disease or disorder. Anti-HER2 antibodies and conjugates containing them can be any of those described in Chapter 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, a conjugate (e.g., a fusion protein) containing a modified cyclic peptide connected to an anti-TNF-α antibody is administered to a subject to treat a neurological disease or disorder. The anti-TNF-α antibody and the conjugate containing it can be any of those described in Section II. In some embodiments, the anti-TNF-α antibody is adalimumab. In some embodiments, the neurological disease or disorder is a stroke, craniocerebral injury, or Alzheimer's disease.

[0236] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclic peptide connected to an anti-amyloid beta antibody is administered to a subject to treat a neurological disease or disorder. The anti-amyloid beta antibody and the conjugate containing it can 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, a conjugate (e.g., a fusion protein) containing a modified cyclic peptide connected to a nerve growth factor (NGF) is administered to a subject to treat a neurological disease or disorder. NGF and conjugates containing it can 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, a conjugate (e.g., fusion protein) containing a modified cyclic peptide linked to granulocyte colony stimulating factor (GCSF) is administered to a subject to treat a neurological disease or disorder. GCSF and conjugates containing it can be any of those described in Section II. In some embodiments, the neurological disease or disorder is a stroke or craniocerebral injury.

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

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

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

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

[0243] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclic peptide linked to a galactocerebrosidase is administered to a subject to treat a neurological disease or disorder. The galactocerebrosidase and the conjugate containing it can 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 cyclic peptide linked to an alpha galactosidase is administered to a subject to treat a neurological disease or disorder. The alpha galactosidase and conjugate containing it can 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 cyclic peptide connected to a sphingomyelinase is administered to a subject to treat a nervous system disease or disorder. Sphingomyelinase and a conjugate containing it can be any of those described in Section II. In some embodiments, the nervous system disease or disorder is Niemann-Pick disease.

[0246] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclic peptide linked to serripagal is administered to a subject to treat a neurological disease or disorder. Serripagal and conjugates containing it can be any of those described in Section II. In some embodiments, the neurological disease or disorder is Bi-Young's disease.

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

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

[0249] In some embodiments, a conjugate (e.g., a fusion protein) containing a modified cyclic peptide linked to a galactosamine 6 sulfatase is administered to a subject to treat a neurological disease or disorder. The galactosamine 6 sulfatase and the conjugate containing it can 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 a provided conjugate (e.g., fusion protein) or a pharmaceutical composition comprising the same to a subject (e.g., human). In some embodiments, administration is by parenteral administration to a subject. In some embodiments, administration is intramuscular, subcutaneous, intravenous, topical, oral, or by inhalation. In some embodiments, administration is intramuscular administration. In some embodiments, administration is subcutaneous administration.

[0251] In some embodiments, the conjugate (for example, fusion protein) provided is administered to the subject in an effective amount or a therapeutically effective amount. In some embodiments, the effective dose or therapeutically effective dose is a dose for treating a nervous system disorder. In some embodiments, the effective dose or therapeutically effective dose is an amount sufficient to alleviate one or more signs and / or symptoms of the nervous system disorder of the treated subject, whether by inducing the disappearance or elimination of such signs and / or symptoms or by suppressing the progress of such signs and / or symptoms. The amount of the dosage can vary according to the age and body shape of the subject to be administered, the target disease, the condition, the route of administration, etc. In an embodiment, the effective dose or therapeutically effective dose of the conjugate (for example, fusion protein) provided for treating a nervous system disorder (for example, in an adult subject) is 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. According to the severity of the infection, the frequency and duration of treatment can be adjusted.

[0252] In a specific embodiment of the invention, the cyclic peptide-conjugated therapeutic molecule of the invention is used as a composition administered alone or in combination with other therapeutic agents. These additional agents may include various immunotherapeutic drugs (such as cyclosporine, methotrexate, doxorubicin or cisplatin), and immunotoxins or chemotherapeutic drugs (such as tamoxifen, paclitaxel, oxaliplatin, vincristine and fluorouracil). Pharmaceutical compositions may include combinations of various cytotoxic agents or other agents combined with the polypeptides of the invention. VI. Definitions

[0253] Unless otherwise defined, all art terms, symbols and other technical and scientific terms or nomenclature used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and such definitions contained herein should not be construed as representing a substantial difference from what is generally understood in the art.

[0254] A number of definitions are provided to aid in understanding the present invention. All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0255] Unless the context clearly dictates otherwise, as used herein, the singular forms "a", "an", and "the" include plural referents. For example, "a" or "an" means "at least one" or "one or more". It should be understood that the aspects and variations described herein include "consisting of" and / or "consisting essentially of" the aspects and variations.

[0256] Throughout this disclosure, various aspects of the claimed subject matter are presented in the form of ranges. It should be understood that the description of the range form is only for convenience and brevity, and should not be interpreted as a rigid limitation on the scope of the claimed subject matter. Therefore, it should be considered that the description of the range specifically discloses all possible sub-ranges and each numerical value in the range. For example, in the case of providing a series of values, it should be understood that each intermediate value between the upper and lower limits of the range and any other described or intermediate value in the range are included in the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in the claimed subject matter, subject to any exact exclusion of the limitations in the stated range. In the case where the stated range includes one or both of the limitations, the scope excluding any one or both of the limitations included is also included in the claimed subject matter. Regardless of the width of the range, this applies.

[0257] As used herein, the term "about" refers to the usual error range of the corresponding value that is readily known in the art. References herein to "about" a certain value or parameter include (and describe) embodiments involving the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0258] The term "conjugate" is used in its broadest sense to cover all attachment or connection methods known in the art. Such conjugates include fusion proteins, i.e., those produced by chemical conjugates and those produced by any other method. The term "conjugated" can be used interchangeably with terms such as "connected", "bound", "associated", "fused" or "attached". A variety of covalent and non-covalent forms of conjugation are known to those skilled in the art and are within the scope of the present invention. For example, disulfide bonds, chemical connections and peptide chains are all forms of covalent connection. In the case where non-covalent conjugation is preferred, the attachment method can be, for example, biotin-(streptavidin) avidin connection, etc. It is also possible to suitably use antibody (or antibody fragment)-antigen interactions to conjugate the cyclic peptide of the present invention to another part (such as a polypeptide, peptide, non-polypeptide part, small molecule or biopharmaceutical).

[0259] "Binding affinity" generally refers to the strength of the sum 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 indicated, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which may be used for the purposes of the present disclosure.

[0260] "Percentage (%) of 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 particular peptide or polypeptide sequence, after aligning the sequences and introducing spaces (if necessary) to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be achieved in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0261] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are the amino acids encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (e.g., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid but function similarly to a naturally occurring amino acid.

[0262] "Conservatively modified variants" apply to both amino acids and nucleic acid sequences. "Amino acid variants" refer to amino acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids encoding identical or substantially identical amino acid sequences, or, in the case where the nucleic acid does not encode an amino acid sequence, refer to substantially identical or related (e.g., naturally contiguous) sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is specified by a codon, the codon can be changed to another of the described corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes silent variations of nucleic acids. The skilled person will recognize that, in certain contexts, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, the silent variations of nucleic acids encoding polypeptides are implicit in the described sequences about the expression product rather than about the actual probe sequence. With respect to amino acid sequences, the skilled artisan will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence that alter, add or delete a single amino acid or a small percentage of amino acids in the coding sequence are "conservatively modified variants," including where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude the polymorphic variants, interspecies homologs and alleles disclosed herein. Typical conservative substitutions include: 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) (see, e.g., Creighton, Proteins (1984)).

[0263] "Polypeptide" is a polymer of amino acid residues connected by peptide bonds, whether naturally occurring or produced in vitro by synthetic means. In some embodiments, amino acid polymers of 2 to 50 amino acids are generally referred to as "peptides". As used herein, the term "polypeptide" refers to a naturally occurring polypeptide, a precursor form, or a product of a proprotein. The term is also applicable to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics 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 also undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. The term "protein" is used herein to refer to large polypeptide molecules and macromolecules comprising one or more polypeptide chains.

[0264] As used herein, the term "peptide" refers to a plurality of amino acids linked together, such as in a linear or circular chain. The term oligopeptide is generally used to describe peptides having between 2 and about 50 or more amino acids. Peptides greater than about 50 are generally referred to as polypeptides or proteins.

[0265] As used herein, "non-polypeptide moiety" refers to an entity that does not contain a polypeptide sequence or three-dimensional folding. One skilled in the art understands and can determine whether a polypeptide molecule is a polyprotein or a peptide sequence, for example, by sequence homology or structural prediction or determination. Such non-polypeptide moieties include, among others, nucleic acids and other polymers, peptides, proteins, peptide nucleic acids (PNA), antibodies, antibody fragments, and small molecules. Suitably, the non-polypeptide moiety is a biomolecule (e.g., comprising a polynucleotide or a peptide), and advantageously is a therapeutic or targeting molecule.

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

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

[0268] A "Fab fragment" is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment of the same structure produced synthetically (e.g., by recombinant methods). A Fab fragment contains the light chain (containing the V L and C L ) and containing the heavy chain variable domain (V H ) and a constant region domain of the heavy chain (C H 1) Another chain.

[0269] A "scFv fragment" refers to a fragment comprising variable light chains (V L ) and variable heavy chain (V H ). The length of the linker allows the two variable domains to be bridged without being seriously disturbed. An exemplary linker is (Gly-Ser) n residues, some of which are Glu or Lys residues scattered throughout to increase solubility.

[0270] When applied to DNA sequences, for example in an expression vector or construct, the term "operably linked" indicates that the sequences are arranged so that they function together to achieve their intended purpose, i.e., a promoter sequence allows initiation of transcription, which proceeds through the linked coding sequence to the termination sequence.

[0271] As used herein, the term "nucleic acid sequence" is a nucleotide sequence covalently linked to a single or double strand, wherein the 3' and 5' ends on each nucleotide are linked by a phosphodiester bond. Polynucleotides can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acid sequences can include DNA and RNA, and can be synthesized in vitro or separated from natural sources. The size of a nucleic acid sequence (also referred to herein as "polynucleotide") is usually expressed as a base pair (bp) number for a double-stranded polynucleotide, or as a nucleotide (nt) number in the case of a single-stranded polynucleotide. One thousand bp or nt is equal to a kilobase (kb). Polynucleotides less than about 40 nucleotides in length are commonly referred to as "oligonucleotides", and can include primers for DNA manipulations such as via polymerase chain reaction (PCR).

[0272] The term "vector" is used to refer to a linear or circular DNA molecule into which another nucleic acid (usually DNA) sequence fragment of appropriate size can be integrated. Such one or more DNA fragments may include additional segments that provide for transcription of the gene encoded by the DNA sequence fragment. The additional segments may include and are not limited to: promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selectable markers, replication origins, etc. Various promoters suitable for prokaryotic hosts (e.g., β-lactamase and lactose promoter systems for Escherichia coli, alkaline phosphatase, tryptophan (trp) promoter systems, lac, tac, T3, T7 promoters) and various promoters suitable 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 are usually derived from plasmids, cosmids, viral vectors and yeast artificial chromosomes; vectors are usually recombinant molecules containing DNA sequences from several sources.

[0273] Specific embodiments provide expression vectors encoding modified polypeptides or fragments / domains thereof comprising the molecules of the present invention. Thus, DNA encoding the relevant peptides of the present invention can be inserted into a suitable expression vector (e.g. Promega Corp., USA), wherein the DNA is operably linked to an appropriate expression sequence, and the expression vector is transformed into a suitable host cell for protein expression according to conventional techniques (Sambrook J. et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY). Suitable host cells are those that can be grown in culture and are suitable for transformation with exogenous DNA, including bacteria, fungal cells and cells of higher eukaryotic origin, preferably mammalian cells, usually human cells and cell lines. In order to help purify the peptides of the present invention, the polypeptides of the present invention (and corresponding nucleic acids) may include purification sequences, such as His tags. In addition, or alternatively, the modified polypeptides can be grown, for example, fused with another protein and purified from bacterial cells as insoluble inclusion bodies. This is particularly convenient when the modified polypeptide to be synthesized may be toxic to the host cell in which it is to be expressed. Alternatively, the modified polypeptides can be synthesized in vitro using a suitable in vitro (transcription and) translation system (e.g., E. coli S30 extraction system: Promega Corp., USA).

[0274] In one embodiment of the present invention, the vector is suitable as a polypeptide library display vector, so that the polypeptide gene product of the cyclic peptide encoding gene can remain associated with the vector after transcription.

[0275] As used herein, the term "polypeptide library display" refers to a system in which a collection of part or all of the polypeptides or peptides that may form a library can be used to select based on a specified feature. The specified feature can be a physical, chemical or functional feature. Suitable display systems utilize cell expression systems, such as expression of nucleic acid libraries in cells that are appropriately transformed, infected, transfected or transduced, and display of encoded polypeptides on cell surfaces. Alternative cell expression systems can include emulsion compartmentalization and display. Optional display systems link the encoding function of nucleic acids to the physical, chemical and / or functional features of the polypeptides or peptides encoded by the nucleic acids. When using this display system, polypeptides or peptides with desired physical, chemical and / or functional features can be selected, and the nucleic acid encoding the selected polypeptide is easy to separate. Several display systems that link the encoding functionality of nucleic acids to related polypeptide products are known in the art, for example, bacteriophage display (phage display), ribosome display, emulsion compartmentalization and display, yeast display, puromycin display, bacterial display, plasmid display, covalent display, CIS display, etc. (See, e.g., EP 0436597 (Dyax), U.S. Pat. No. 6,172,197 (McCafferty et al.), U.S. Pat. No. 6,489,103 (Griffiths et al.)).

[0276] The term "library" refers to a mixture of heterogeneous polypeptides or nucleic acids. The library is composed of multiple members, each of which has a substantially unique polypeptide or nucleic acid sequence. The sequence differences between the library members lead to the diversity present in the library. In the present invention, the library can take the form of a simple mixture of polypeptides or nucleic acids, or can be in the form of an organism or cell transformed with a nucleic acid library, such as bacteria, viruses, animals or plant cells, etc. Usually, each individual organism (such as phage) or cell contains only one or a very limited number of library members. Advantageously, nucleic acids are incorporated into expression vectors to allow the expression of polypeptides encoded by the nucleic acids. Therefore, in preferred aspects, the library can take the form of a host organism group, each organism containing one or more copies of expression vectors, the expression vector containing a single member of the library in the form of nucleic acids, the member can be expressed to produce its corresponding polypeptide member, i.e., polypeptide gene product. Therefore, the host organism group has the potential to encode a wide range of different numbers of polypeptides. Embodiments of the present invention provide polypeptide libraries based on modified forms of cyclobody polypeptides, wherein the diversity or differences between library members are located in the polypeptide sequence of one or more loops or variable regions of one or more functional modules within the protein.

[0277] "Derived from" means that the cyclic peptide involved includes one or more amino acid modifications, such as insertions, deletions or mutations, compared to the primary amino acid sequence of the cyclic peptide on which it is based. Therefore, derivatives of cyclic peptides are also referred to as "modified cyclic peptides". For example, a derivative of a cyclic peptide polypeptide may contain one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid mutations, substitutions or deletions of the primary sequence of the selected modified polypeptide. Therefore, the present invention encompasses the results of maturation experiments on cyclic peptide polypeptides to improve or change one or more characteristics of the initial or unmodified cyclic peptide. For example, one or more amino acid residues of the selected cyclic peptide polypeptide sequence can be randomly or specifically mutated (or substituted) using procedures known in the art (e.g., by modifying the encoding DNA or RNA sequence). The resulting library or population of derivatized polypeptides can be selected by any known method in the art according to predetermined requirements: such as improved specificity for a specific target receptor (e.g., a receptor involved in endocytic transport); improved endocytic transport across the blood-brain barrier; or improved drug properties (e.g., solubility, bioavailability, immunogenicity, etc.).

[0278] With respect to binding molecules (such as peptides or antibodies or other binding molecules, or polynucleotides and vectors encoding them), the term "isolated" refers to being at least partially free of other biomolecules from the cells or cell cultures that produce them. Such biomolecules include nucleic acids, proteins, other antibodies or antigen binding fragments, lipids, carbohydrates or other materials, such as cell debris and growth medium. The isolated binding molecules may further be at least partially free of expression system components, such as biomolecules, from the host cell or its growth medium. Generally, the term "isolated" is not intended to refer to being completely free of such biomolecules, or is not intended to refer to components of a pharmaceutical formulation that does not contain water, buffers or salts, or includes antibodies or fragments.

[0279] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of a pharmaceutical composition sufficient to significantly and positively alter the symptoms and / or condition to be treated (e.g., to provide a positive clinical response). The effective amount of the active ingredient used in the pharmaceutical composition will vary with the specific condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the specific active ingredient(s) employed, the specific pharmaceutically acceptable excipient(s) and / or carrier(s) used, and similar factors to 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, that does not abrogate the biological activity or properties of the compound and is relatively nontoxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any component 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). It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

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

[0283] As used herein, "disease or disorder" refers to a pathological condition in an organism resulting from an etiology or condition (including but not limited to infection, acquired condition, genetic condition) and 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 progression of a disease or disorder, e.g., the underlying cause of the disorder or at least one of its clinical symptoms.

[0285] As used herein, the term "subject" refers to an animal, including a mammal (e.g., rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit), such as a human. Typically, the subject is a human subject. The terms subject and patient can be used interchangeably.

[0286] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, an optionally substituted group means that the group is unsubstituted or substituted. VII. Exemplary Embodiments

[0287] The provided implementations include: 1. A modified cyclic peptide, comprising: 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), wherein the peptide has an amino acid sequence of 2 to 50 amino acid residues; and ii) a cyclic peptide scaffold comprising the peptide of i), The modified cyclic peptide comprises structure (I): (X V 1…X V f )C1(X1…X a )C2(X I 1…X I b )C3(X II 1…X II c )C4(X III 1…X III d )C5(X IV 1…X IV e )C6(X V 1…X V f ) Ring 6 Ring 1 Ring 2 Ring 3 Ring 4 Ring 5 Ring 6 wherein C1 to C6 are cysteine ​​residues; wherein each of C1 and C4, C2 and C5, and C3 and C6 are linked by a disulfide bond to form a cysteine ​​knot; wherein each X represents an amino acid residue in a loop, wherein the amino acid residues are the same or different; wherein d is about 1-2; wherein at least one loop from loop 1, 2, 3, 5 or 6 has an amino acid sequence comprising the sequence of the peptide of clause i), wherein any loop of the sequence of the peptide comprising clause i) comprises 2 to about 30 amino acids, and wherein for any of loops 1, 2, 3, 5 or 6 of the sequence of the peptide not containing clause i), a, b, c, e and f are the same or different and are each any number from 3 to 10, and b, c, e and f are each any number from 1 to 20. 2. The modified cyclic peptide according to embodiment 1, wherein the BBB-R is human. 3. The modified cyclic peptide according to embodiment 1 or embodiment 2, wherein the cyclic peptide scaffold is selected from plant cyclic peptides. 4. The modified cyclic peptide according to any one of embodiments 1-3, wherein the cyclic peptide scaffold is a cyclic peptide scaffold of Momordica species. 5. The modified cyclic peptide according to embodiment 4, wherein the cyclic peptide scaffold is Momordica cochinchinensis trypsin inhibitor. 6. The modified cyclic peptide according to embodiment 5, wherein the Momordica cochinchinensis trypsin inhibitor is MCoTI-I shown in SEQ ID NO: 1, MCoTI-II shown in SEQ ID NO: 2, or MCoTI-III shown in SEQ ID NO: 3. 7. A modified cyclic peptide according to any one of embodiments 1-6, wherein the sequence of the peptide replaces or substitutes one or more amino acids in one of the one or more rings of the cyclic peptide scaffold. 8. A modified cyclic peptide comprising a peptide that binds to a blood-brain barrier transcytosis receptor (BBB-R), wherein: The peptide is inserted into at least one loop of the cyclic peptide scaffold shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or replaces one or more amino acids of the at least one loop, and wherein the peptide is about 2 to 50 amino acid residues; and The BBB-R is selected from 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 9. The modified cyclic peptide according to any one of embodiments 1-8, wherein the cyclic peptide scaffold is as shown in SEQ ID NO: 2. 10. The modified cyclic peptide according to any one of embodiments 1-9, wherein the at least one ring is ring 1, ring 5 or ring 6, or a combination thereof. 11. The modified cyclic peptide according to any one of embodiments 1-10, wherein the at least one ring is ring 1. 12. The modified cyclic peptide according to any one of embodiments 1-11, wherein the peptide is inserted into only one ring of the cyclic peptide scaffold and replaces the amino acids of the only one ring. 13. The modified cyclic peptide according to embodiment 12, wherein the only one ring is ring 1. 14. The modified cyclic peptide according to any one of embodiments 11-13, wherein the cyclic peptide scaffold is as shown in SEQ ID NO: 2, and the peptide replaces the loop 1 amino acid between cysteine ​​4 and cysteine ​​11 of SEQ ID NO: 2. 15. The modified cyclic peptide according to any one of embodiments 1-14, wherein all amino acids of the at least one ring are replaced by the peptide. 16. A modified cyclic peptide comprising a peptide inserted into loop 1 to replace all amino acids between cysteine ​​4 and cysteine ​​11 of SEQ ID NO: 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). 17. The modified cyclic peptide of any one of embodiments 1-16, wherein 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, 1 0 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. 18. The modified cyclic peptide of any one of embodiments 1-16, wherein 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. 19. The modified cyclic peptide according to any one of embodiments 1-16, wherein the peptide is 14 to 20 amino acids. 20. The modified cyclic peptide according to any one of embodiments 1-18, wherein the peptide is 10 amino acids. 21. The modified cyclic peptide according to any one of embodiments 1-18, wherein the peptide is 12 amino acids. 22. The modified cyclic peptide of any one of embodiments 1-21, wherein the BBB-R is expressed on brain endothelial cells. 23. The modified cyclic peptide according to any one of embodiments 1-22, wherein the peptide has blood-brain barrier translocation activity. 24. The modified cyclic peptide of any one of embodiments 1-23, wherein the BBB-R is the transferrin receptor. 25. A modified cyclic peptide according to any one of embodiments 1-24, wherein the peptide comprises a sequence as shown in any one of SEQ ID NOs: 26-34 and 49-54, optionally wherein the peptide is as shown in any one of SEQ ID NOs: 26-34 and 49-54. 26. The modified cyclic peptide according to any one of embodiments 1-24, wherein the peptide has a consensus motif as shown in CxxxxxHxxSWGxC (SEQ ID NO: 177). 27. A modified cyclic peptide according to any one of embodiments 1-26, wherein the modified cyclic peptide comprises a sequence as shown in any one of SEQ ID NOs: 72-80 and 95-100, optionally wherein the modified cyclic peptide is as shown in any one of SEQ ID NOs: 72-80 and 95-100. 28. The modified cyclic peptide according to any one of embodiments 1-24 and 26, wherein the peptide comprises the sequence shown in SEQ ID NO: 26, optionally wherein the peptide is as shown in SEQ ID NO: 26. 29. The modified cyclic peptide according to any one of embodiments 1-28, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO:72, optionally wherein the modified cyclic peptide is as shown in SEQ ID NO:72. 30. A modified cyclic peptide according to any one of embodiments 1-29, wherein the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 26-34 and 49-54, optionally wherein the one or more amino acid substitutions are substitutions to histidine or alanine. 31. The modified cyclic peptide according to any one of embodiments 1-24 and 30, wherein the peptide comprises the sequence shown in any one of SEQ ID NOs: 55 and 117-128. 32. The modified cyclic peptide according to any one of embodiments 1-24 and 30, wherein the peptide is as shown in any one of SEQ ID NO: 55 and 117-128. 33. The modified cyclic peptide according to any one of embodiments 1-24 and 30-32, wherein the peptide comprises the sequence shown in SEQ ID NO:55, optionally wherein the peptide is as shown in SEQ ID NO:55. 34. The modified cyclic peptide of any one of embodiments 1-23, wherein the BBB-R is the leptin receptor. 35. The modified cyclic peptide according to any one of embodiments 1-23 and 34, wherein the peptide comprises the sequence shown in any one of SEQ ID NOs: 10-23. 36. The modified cyclic peptide according to any one of embodiments 1-23, 34 and 35, wherein the peptide is as shown in any one of SEQ ID NOs: 10-23. 37. A modified cyclic peptide according to any one of embodiments 1-23 and 34-36, wherein the modified cyclic peptide comprises a sequence as shown in any one of SEQ ID NOs: 56-69, optionally wherein the modified cyclic peptide is as shown in any one of SEQ ID NOs: 56-69. 38. A modified cyclic peptide according to any one of embodiments 1-23 and 34, wherein the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 10-23, optionally wherein the one or more amino acid substitutions are substitutions to histidine or alanine. 39. The modified cyclic peptide of any one of embodiments 1-23, wherein the BBB-R is ErbB3. 40. The modified cyclic peptide according to any one of embodiments 1-23 and 39, wherein the peptide comprises the sequence shown in SEQ ID NO: 24 or 25. 41. The modified cyclic peptide according to any one of embodiments 1-23, 39 and 40, wherein the peptide is as shown in SEQ ID NO: 24 or 25. 42. A modified cyclic peptide according to any one of embodiments 1-23 and 39-41, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO: 70 or 71, optionally wherein the modified cyclic peptide is as shown in SEQ ID NO: 70 or 71. 43. A modified cyclic peptide according to any one of embodiments 1-23 and 39, wherein the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in SEQ ID NO: 24 or 25, optionally wherein the one or more amino acid substitutions are substitutions to histidine or alanine. 44. The modified cyclic peptide of any one of embodiments 1-23, wherein the BBB-R is insulin-like growth factor type 1 receptor (IGFR). 45. The modified cyclic peptide according to any one of embodiments 1-23 and 44, wherein the peptide comprises the sequence shown in SEQ ID NO: 35 or 36. 46. ​​The modified cyclic peptide according to any one of embodiments 1-23, 44 and 45, wherein the peptide is as shown in SEQ ID NO: 35 or 36. 47. The modified cyclic peptide according to any one of embodiments 1-23 and 44-46, wherein the peptide comprises the sequence shown in SEQ ID NO:36, optionally wherein the peptide is as shown in SEQ ID NO:36. 48. A modified cyclic peptide according to any one of embodiments 1-23 and 44-47, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO:81 or 82, optionally wherein the modified cyclic peptide is as shown in SEQ ID NO:81 or 82. 49. The modified cyclic peptide according to any one of embodiments 1-23 and 44-48, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO:82, optionally wherein the modified cyclic peptide is as shown in SEQ ID NO:82. 50. A modified cyclic peptide according to any one of embodiments 1-23 and 44, wherein the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in SEQ ID NO: 35 or 36, optionally wherein the one or more amino acid substitutions are substitutions to histidine or alanine. 51. The modified cyclic peptide of any one of embodiments 1-23, wherein the BBB-R is RAGE. 52. The modified cyclic peptide according to any one of embodiments 1-23 and 51, wherein the peptide comprises the sequence shown in SEQ ID NO: 37 or 38. 53. The modified cyclic peptide according to any one of embodiments 1-23, 51 and 52, wherein the peptide is as shown in SEQ ID NO: 37 or 38. 54. A modified cyclic peptide according to any one of embodiments 1-23 and 51-53, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO: 83 or 84, optionally wherein the modified cyclic peptide is as shown in SEQ ID NO: 83 or 84. 55. A modified cyclic peptide according to any one of embodiments 1-23 and 51, wherein the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in SEQ ID NO: 37 or 38, optionally wherein the one or more amino acid substitutions are substitutions to histidine or alanine. 56. The modified cyclic peptide of any one of embodiments 1-23, wherein the BBB-R is the 1 lipoprotein receptor-related protein 1 (LRP-1). 57. The modified cyclic peptide according to any one of embodiments 1-23 and 56, wherein the peptide comprises the sequence shown in any one of SEQ ID NOs: 39-48. 58. The modified cyclic peptide according to any one of embodiments 1-23, 56 and 57, wherein the peptide is as shown in any one of SEQ ID NOs: 39-48. 59. A modified cyclic peptide according to any one of embodiments 1-23 and 56-58, wherein the modified cyclic peptide comprises a sequence as shown in any one of SEQ ID NOs: 85-94, optionally wherein the modified cyclic peptide is as shown in any one of SEQ ID NOs: 85-94. 60. A modified cyclic peptide according to any one of embodiments 1-23 and 56, wherein the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 39-48, optionally wherein the one or more amino acid substitutions are substitutions to histidine or alanine. 61. A peptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 10-55 or 117-128, wherein the peptide is 6-50 amino acids in length and binds to a receptor involved in blood-brain barrier transcytosis (BBB-R). 62. A peptide according to embodiment 61, wherein the peptide has blood-brain barrier translocation activity. 63. The peptide of embodiment 61 or embodiment 62, wherein 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 or 40 to 50 amino acids. 64. A peptide according to any of embodiments 61-63, wherein 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. 65. A peptide according to any one of embodiments 61-64, wherein the peptide is 14 to 20 amino acids. 66. A peptide according to any one of embodiments 61-64, wherein the peptide is 10 amino acids. 67. A peptide according to any one of embodiments 61-64, wherein the peptide is 12 amino acids. 68. A peptide consisting of the sequence shown in any one of SEQ ID NOs: 10-55 or 117-128. 69. A peptide according to embodiment 68, wherein the peptide has blood-brain barrier translocation activity. 70. A peptide according to embodiment 68 or embodiment 69, wherein the peptide binds to a receptor involved in blood-brain barrier endocytosis. 71. A peptide represented by the sequence of any one of SEQ ID NOs: 26-34 and 49-55 and 117-128, wherein the peptide binds to transferrin receptor. 72. A peptide according to any one of embodiments 61-71, wherein the peptide is as shown in SEQ ID NO:26. 73. A peptide according to any one of embodiments 61-71, wherein the peptide is as shown in SEQ ID NO:55. 74. A peptide represented by the sequence of any one of SEQ ID NOs: 10-23, wherein the peptide binds to the leptin receptor. 75. A peptide represented by the sequence of any one of SEQ ID NO: 24 or 25, wherein the peptide binds to ErbR3. 76. A peptide represented by the sequence of any one of SEQ ID NO: 35 or 36, wherein the peptide binds to IGFR. 77. A peptide according to embodiment 76, wherein the peptide is as shown in SEQ ID NO:36. 78. A peptide represented by the sequence of SEQ ID NO: 37 or 38, wherein the peptide binds to RAGE. 79. A peptide represented by the sequence of any one of SEQ ID NOs: 39-48, wherein the peptide binds to the lipoprotein receptor-related protein 1 (LRP-1). 80. A peptide according to any one of embodiments 61-79, wherein the peptide is synthetic. 81. A peptide according to any one of embodiments 61-79, wherein the peptide is isolated. 82. A binding molecule comprising a binding scaffold and a peptide according to any one of embodiments 61-81. 83. A binding molecule according to embodiment 82, wherein the binding scaffold is a cyclic peptide. 84. A binding molecule according to embodiment 83, wherein the peptide is inserted into a ring of a cyclic peptide backbone or replaces one or more amino acids of the ring, optionally wherein at least one of the rings is ring 1. 85. A binding molecule according to embodiment 83 or embodiment 84, wherein the binding scaffold is as shown in any one of SEQ ID NOs: 1-3. 86. A binding molecule according to any one of embodiments 83-85, wherein the binding scaffold is as shown in SEQ ID NO:2. 87. A nucleic acid molecule encoding a modified cyclic peptide according to any one of embodiments 1-60 or a binding molecule according to any one of embodiments 82-86. 88. A vector comprising the nucleic acid molecule according to embodiment 87. 89. The vector of embodiment 88, which is an expression vector. 90. A host cell comprising the nucleic acid molecule of embodiment 87 or the vector of embodiment 87 or embodiment 89. 91. A method for producing a modified cyclic peptide or binding molecule, the method comprising introducing the nucleic acid according to embodiment 87 or the vector according to embodiment 88 or embodiment 89 into a host cell and culturing the host cell under certain conditions to express the protein in the cell, optionally purifying the protein from the cell. 92. A purified binding molecule or modified cyclic peptide produced by the method according to embodiment 91. 93. A conjugate comprising: a modified cyclic peptide according to any one of embodiments 1-60 or a binding molecule according to any one of embodiments 82-86 and a bioactive agent. 94. The conjugate of embodiment 93, wherein the bioactive agent is a small molecule, a peptide or a protein. 95. The conjugate of embodiment 93 or embodiment 94, wherein the bioactive agent is a diagnostic agent or a therapeutic agent. 96. The conjugate of any one of embodiments 93-95, wherein the conjugate is a fusion protein comprising the modified cyclic peptide operably linked to a bioactive agent, wherein the bioactive agent is a protein or a peptide. 97. A fusion protein comprising the modified cyclic peptide according to any one of embodiments 1-60 or the binding molecule according to any one of embodiments 82-86 and a biologically active protein agent. 98. The conjugate of any one of embodiments 93-96 or the fusion protein of embodiment 97, wherein the bioactive agent is an antibody. 99. The conjugate or fusion protein of embodiment 98, wherein the antibody is trastuzumab, adalimumab or aducanumab. 100. The conjugate of any one of embodiments 93-96 or the fusion protein of embodiment 97, wherein the bioactive agent is a growth factor or a hormone. 101. A conjugate or fusion protein according to embodiment 100, wherein the bioactive agent is a growth factor, and the growth factor is nerve growth factor (NGF) or granulocyte colony stimulating factor (GCSF). 102. The conjugate of any one of embodiments 93-96 or the fusion protein of embodiment 97, wherein the bioactive agent is an enzyme. 103. The conjugate or fusion protein of embodiment 102, wherein the enzyme is a ceramide degrading enzyme, a lipase, a hydrolase-type enzyme, or a sulfatase. 104. The conjugate or fusion protein of embodiment 102 or 103, wherein the enzyme is a ceramide degrading enzyme, and the ceramide degrading enzyme is glucocerebrosidase, galactocerebrosidase or alpha-galactosidase. 105. The conjugate or fusion protein of embodiment 102 or embodiment 103, wherein the enzyme is a lipase or hydrolase type enzyme and the enzyme is a sphingomyelinase, a serripeptase or an alpha glucosidase. 106. The conjugate or fusion protein of any one of embodiments 93-105, comprising a single modified cyclic peptide. 107. The conjugate or fusion protein of any one of embodiments 93-105, comprising 2, 3 or 4 modified cyclic peptides. 108. The conjugate or fusion protein of embodiment 107, wherein each modified cyclic peptide is identical. 109. The conjugate or fusion protein of embodiment 107, wherein each modified cyclic peptide is different. 110. The conjugate or fusion protein of any one of embodiments 93-107 and 108, which is monovalent for binding to the BBB-R. 111. The conjugate or fusion protein of any one of embodiments 93-105, 107 and 109, comprising at least two different modified cyclic peptides that bind to different BBB-Rs. 112. The conjugate or fusion protein of embodiment 111, which is bispecific for binding to two different BBB-Rs. 113. A conjugate or fusion protein according to any one of embodiments 93-112, wherein the modified cyclic peptide is linked to the biologically active agent via a linker. 114. A conjugate or fusion protein according to embodiment 113, wherein the linker is a flexible peptide linker, and the flexible peptide linker optionally comprises the sequence GGGGS (SEQ ID NO: 148), (GGGGS)2 (SEQ ID NO: 154) or (GGGGS)3 (SEQ ID NO: 154). 115. A conjugate or fusion protein according to embodiment 113 or embodiment 114, wherein the linker is as shown in SEQ ID NO:104. 116. A conjugate or fusion protein according to embodiment 113, wherein the linker is a cleavable linker comprising an endosomal-specific protease cleavage site. 117. A conjugate or fusion protein according to embodiment 116, wherein the endosomal-specific protease cleavage site is a tissue protease cleavage site. 118. A conjugate or fusion protein according to embodiment 117, wherein the cathepsin cleavage site is a cathepsin B cleavage site. 119. A conjugate or fusion protein according to any one of embodiments 116-118, wherein the linker comprises the sequence shown in SEQ ID NO:133. 120. A pharmaceutical composition comprising the conjugate or fusion protein according to any one of embodiments 93-119 and a drug carrier. 121. A method for transporting a biologically active agent across the blood-brain barrier of an individual, the method comprising administering to a mammal in need thereof a conjugate or fusion protein according to any one of embodiments 93-119 or a pharmaceutical composition according to embodiment 120. 122. The method of embodiment 121, wherein the mammal suffers from a neurological disease. 123. The method of embodiment 121, wherein the neurological disease is selected from Alzheimer's disease, Parkinson's disease, stroke, brain tumor and brain metastasis. 124. The method of embodiment 121 or embodiment 123, wherein the neurological disease is a congenital disease selected from the group consisting of Austin disease, Canavan disease, Gaucher disease, Hunter syndrome, Hurler-Scheie syndrome, Beecham-Young disease, Krabbe disease, LCAT deficiency, Lowe syndrome, Maroteaux-Lamy syndrome, Morquio syndrome A, Morquio syndrome B, Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome C, Sanfilippo syndrome D, spinal muscular atrophy, Tay-Sachs disease, and Walker-Warburg syndrome. 125. A method for treating a patient suffering from a neurological disease, the method comprising administering to the patient a conjugate according to any one of embodiments 46-52 or a pharmaceutical composition according to embodiment 53. 126. A method for diagnosing a neurological disease in a patient in need thereof, the method comprising administering to the patient a conjugate or fusion protein according to any one of embodiments 93-119 or a pharmaceutical composition according to embodiment 53, and wherein the conjugate comprises a radioactive label. 127. A pharmaceutical composition according to embodiment 120, which is used to treat a nervous system disease. 128. A pharmaceutical composition according to embodiment 120, which is used to diagnose neurological diseases. VIII. Examples

[0288] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention. Example 1: Construction of cyclic peptide (CYC) library

[0289] A cyclic peptide library is created by inserting random peptides into the skeleton cyclic peptide MCOTII scaffold shown in SEQ ID NO:2. The library is based on the MCOTII natural DNA sequence (SEQ ID NO:4) as a cyclic peptide scaffold, and is constructed to contain a mutant sequence between the first two cysteine ​​residues of SEQ ID NO:2 (referred to as "loop 1"), wherein the library differs in the number of randomized peptides. In order to create the library, oligonucleotides are designed to insert the mutant sequence into loop 1 to produce a library with a loop of 10 amino acids ("Loop1for10"; SEQ ID NO:5) or a loop of 12 amino acids ("Loop1for12", SEQ ID NO:6) when expressed. Another library is similarly produced to encode 14-20 randomized amino acids in loop 1. All randomized amino acid positions are encoded in the library to represent an equal mixture of codons for 19 naturally occurring amino acids except cysteine.

[0290] The PCR product was cloned as a NcoI-NotI digested fragment into a similarly digested pSP1 phagemid pill fusion vector derived from the pHEN1 pill vector (Hoogenboom et al., 1991, Nucleic Acids Res., 19:4133-4137). (i) PCR amplification of cyclic peptide library

[0291] For primary PCR amplification, 10x 50 μl amplification was set up for the loop library using appropriate oligonucleotide primers ("Loop1for10" or "Loop1for12" SEQ ID NO: 5 or 6 and pIIIseqrev SEQ ID NO: 7). Each 50 μl reaction mixture contained 10 ng pCyclo1, 25 pmol appropriate forward and reverse primers, 0.1 mM dNTP, 2.5 units of Taq DNA polymerase and 1x NEB PCR reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM (NH4) 2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Trit on X-100; NEB Ltd, Cambridge, UK). The reaction was performed for 30 PCR cycles as follows: 94°C, 20 s; 60°C, 40 s; 72°C, 30 s, then at 72°C for 5 minutes. The reaction products were purified using two Wizard PCR clean-up columns (Promega Corp., Southampton, UK) per library and eluted into 50 μl of water per column. (ii) Pull-through reamplification of selected DNA

[0292] To prepare the final cyclic peptide library DNA product, 40x 50 μl amplification was set for each cyclic peptide ring library using oligonucleotide primers "ring 1 PTrev" and "ring 5 PTrev" (SEQ ID NO: 8 and 9). Each 50 μl reaction mixture contained approximately 25 ng of the primary cyclic peptide ring library, 25 pmol of appropriate forward and reverse primers, 0.1 mM dNTP, 2.5 units of Taq DNA polymerase and 1x 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 performed for 25 cycles as follows: 94°C, 20 s; 60°C, 40 s; 72°C, 30 s, then at 72°C for 5 minutes. The reaction products were purified using four Wizard PCR clean-up columns (Promega Corp., Southampton, UK) per library and eluted into 100 μl of water per column. (iii) Cloning into vector pSP1

[0293] Each library and 250 μg of pSP1 vector DNA were digested with enzymes NcoI and NotI (100 units of each enzyme) at 37°C for 5 hours (NEB, Cambridge, UK), and each library was purified using one Wizard PCR cleanup column and the digested vector DNA was purified using four Wizard PCR cleanup columns (Promega Corp., Southampton, UK). Each DNA sample was then eluted into 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 4000U 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 with 1 μl 20 mg / ml glycogen, 100 μl 7.5 M ammonium acetate and 900 μl ice-cold (-20°C) anhydrous ethanol, vortexed and spun at 13,000 RPM for 20 minutes in a microcentrifuge to precipitate the DNA. The precipitate was washed with 500 μl ice-cold 70% ethanol by centrifugation at 13,000 RPM for 2 minutes, then vacuum dried and resuspended in 100 μl DEPC-treated water. 1 μl aliquots of each library were electroporated into 80 μl Escherichia coli (TG1). Bacterial cells were grown in 1 ml SOC medium in each cuvette for 1 hour at 37°C and plated onto 2x TY agar plates supplemented with 2% glucose and 100 μg / ml ampicillin. 10 of the electroporated bacteria were also electroporated. -4 , 10 -5 and 10-6 Dilutions were plated to estimate library size. Colonies were allowed to grow overnight at 30°C. The combined library size was approximately 2 x 10 10 clones, >95% of which had in-frame inserts. (iv) Phage amplification

[0294] Prepare a separate phage stock for each cyclic peptide library. Then scrape the bacteria from the plate into 50 ml 2x TY broth supplemented with 20% glycerol, 2% glucose, and 100 μg / ml ampicillin. Add 1 ml of bacterial culture to 50 ml 2x TY culture broth supplemented with 1% glucose and 100 μg / ml ampicillin and add 10 11 Kanamycin resistance unit (kru) M13K07 helper phage was infected at 37°C without shaking for 30 minutes, then infected at 200RPM shaking for 30 minutes. The infected bacteria were transferred to 200ml 2xTY broth supplemented with 25μg / ml kanamycin, 100μg / ml ampicillin and 20μM IPTG, then incubated overnight at 30°C at 200RPM shaking. The bacteria were precipitated at 4000RPM in a 50ml Falcon tube for 20 minutes, and 40ml 2.5M NaCl / 20% PEG 6000 was added to 400ml of the pellet supernatant, vigorously mixed and incubated on ice for 1 hour to precipitate the phage particles. The particles were precipitated at 11000RPM for 30 minutes at 4°C in a 250ml Oakridge tube in a Sorvall RC5B centrifuge, then resuspended in 40ml water, and 8ml 2.5M NaCl / 20% PEG 6000 was added to reprecipitate the particles, then incubated on ice for 20 minutes. The particles were precipitated again at 11000RPM for 30 minutes at 4°C in a 50ml Oakridge tube in a Sorvall RC5B centrifuge, then resuspended in 5ml PBS buffer after removing all traces of PEG / NaCl with a pipette. Bacterial debris was removed via centrifugation at 13500RPM 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. Example 2: Library selection targeting blood-brain barrier transport receptors

[0295] The cyclic peptide phage library described in Example 1 was screened for receptor binding peptides capable of binding to the following receptors, which are capable of inducing receptor-mediated transcytosis (RMT) across the blood-brain barrier (BBB), including human HER3 / ErbB3 (Hu ErbB3), human and mouse transferrin receptor (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] Selections were performed using standard phage display selection methods on a solid phase coated with recombinant proteins consisting of the extracellular domain of each of the human (Hu) or mouse (Mu) receptors (recombinant Fc fusion proteins obtained from R&D Systems, or His-tagged receptors from Sino Biologicals).

[0297] For the TfR cyclic peptide selection, a round of selection was also performed in the presence of human iron-saturated transferrin, so that selection was performed against human TfR when human iron-saturated transferrin was bound to the receptor. In this selection, CYC40 (SEQ ID NO: 95) was the top peptide hit in this selection.

[0298] After performing phage ELISA to identify receptor-binding cyclic peptides, plasmid DNA was then prepared and the sequence of individual cyclic peptide clones was confirmed by sequencing. Binding specificity was confirmed by ELISA.

[0299] Table E1 lists the sequences of selected cyclic peptides and the receptors to which they bind. Table E1. Cyclic peptide and receptor binding peptide targets and sequences Example 3: Production and evaluation of adalimumab antibody-cyclic peptide fusions

[0300] The selected cyclic peptides described in Table E1 were fused to the antibody adalimumab. The cyclic peptide was connected 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-cyclic peptide fusion protein is shown in Figure 1A middle.

[0301] Gene constructs for expressing antibody-cyclic peptide fusions were prepared in the following manner: the cyclic peptide gene fused to the 3' end of the nucleotide sequence encoding the antibody heavy chain gene (with 5'IL2 secretion signal peptide sequence) was cloned into a mammalian expression vector. The adalimumab light chain gene (with 5' secretion signal peptide sequence) was also cloned into a mammalian expression vector. HEK293F cells were co-transfected using these two vector constructs and grown for 4 to 6 days at 37°C, 8% CO2, 130rpm shaking. After removing the cells and filtering the supernatant, the resulting adalimumab-cyclic peptide fusions were purified and concentrated by protein A affinity chromatography. When compared with the non-cyclic peptide fusion form of the antibody, two different exemplary adalimumab-cyclic peptide fusion proteins did not show signs of enhanced aggregation, as determined by Western blotting (data not shown). Example 4: Internalization of Cyclic Peptide-Antibody Fusions Targeting Transferrin Receptor (TfR)

[0302] An exemplary cyclic peptide CYC17 (also known as HT2; set forth in SEQ ID NO: 72) that binds to the human transferrin receptor was fused to adalimumab. The adalimumab-CYC17 cyclic peptide fusion was compared to adalimumab for internalization via the transferrin receptor. Cloning, expression, and purification of adalimumab and adalimumab-HT2 cyclic peptide fusions

[0303] Gene constructs for expressing adalimumab heavy chain (SEQ ID NO: 102) and light chain (SEQ ID NO: 103) were prepared by cloning adalimumab heavy chain gene (with 5' IL2 secretion signal peptide sequence) and adalimumab light chain gene (with 5' secretion signal peptide sequence) into mammalian expression vectors, respectively.

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

[0305] HEK293F cells were co-transfected with expression constructs of adalimumab heavy chain-CYC17 cyclic peptide fusion protein (or only adalimumab heavy chain) and adalimumab light chain (amino acid sequence shown in SEQ ID NO: 103) and grown for 4 to 6 days at 37° C., 8% CO 2 , and 130 rpm shaking. After removing the cells and filtering the supernatant, the resulting adalimumab-CYC17 cyclic peptide fusion protein was purified by protein A affinity chromatography and concentrated using a 50 KDa molecular weight cutoff centrifugal filter unit (Amicon). Fluorophore labeling of adalimumab and adalimumab-HT2 cyclic peptide fusion

[0306] Purified adalimumab and adalimumab-CYC17 cyclic peptide fusion proteins were fluorescently labeled by incubation with 5- to 10-fold molar excess of Alexafluor 488 N-hydroxysuccinamide (NHS) ester at room temperature, pH about 8.3 in the dark for 1 hour. Free dye was then removed by passing through one to two desalting columns. Preparation of human cells expressing human or mouse transferrin receptor

[0307] In preparation for the assay, HEK293F cells were first serially passaged two to three times and then transfected with expression constructs encoding human or mouse transferrin receptor (hTfR, mTfR, respectively) and grown for 2-3 days at 37°C, 8% CO2, shaking at 130 rpm. Internalization of labeled adalimumab and adalimumab-CYC17 cyclic peptide fusions by human cells expressing the transferrin receptor was determined.

[0308] After about 64 h of growth, 1x10 5 -5x10 5The transfected cells (as well as 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 labeled adalimumab or adalimumab-CYC17 cyclic peptide fusion, and grown for 4 h at 37°C, 8% CO2, and 500 rpm shaking. The cells were then pelleted, the supernatant discarded, and resuspended in 40 μL of fresh medium containing 250 nM anti-488 quenching antibody (or medium lacking 488 quenching antibody as a control), incubated on ice for 5 min, 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, live cells with normal morphology were selected using gate 1 (forward scatter linear vs. side scatter linear), single cells were selected using gate 2 (side scatter area vs. side scatter linear), and the logarithmic FITC signal of 20,000-50,000 cells in each sample was measured. Subsequent data analysis was performed using the FlowJo software package. The average FITC fluorescence intensity of each sample was monitored as an indication of the degree of internalization of the labeled adalimumab or adalimumab-CYC17 cyclic peptide fusion. Figure 2 The results shown in demonstrate that the adalimumab-CYC17 cyclic peptide fusion, but not adalimumab, is internalized by both mouse TfR (mTfR) and human TfR (hTfR). Example 5: Affinity modulation of transferrin receptor binding cyclic peptides

[0309] The selected TfR binding cyclic peptides identified in Table El were further evaluated for their affinity to TfR, and mutant cyclic peptides with reduced affinity were generated.

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

[0311] Exemplary CYC17 mutants are shown in Table E2. The binding changes of exemplary CYC17 mutants to human transferrin receptor were evaluated by ELISA. Briefly, His-tagged human transferrin receptor (TrFR) was coated on a microwell plate at 1 μg / mL at 4°C overnight, blocked with 2% milk powder / PBS for 1 hour at room temperature, and purified antibody-cyclic peptide fusions at different concentrations of 0.001nM to 100nM were contacted with the coated transferrin receptor, and the binding of the antibody-cyclic peptide fusion to the coated human transferrin receptor was monitored using a 1:5000 dilution of anti-human IgG-HRP conjugate (Jackson Laboratories) and TMB substrate reagent. As Figure 3 As shown, CYC17 mutants with amino acid substitutions W9H, L11H, S13H, W14H and G15H exhibited reduced affinity for transferrin receptor. Example 6: Evaluation of Binding Affinity of Aducanumab and Adalimumab Antibody-Cyclic Peptide Fusions

[0312] Monovalent, bivalent, and bispecific antibody-cyclic peptide fusions were prepared by engineering the knob (K) and hole (H) of the Fc domain.

[0313] The heavy chain of the antibody Aduhelm TM ; SEQ ID NO: 129) engineering: CH3 mutations were made to contain a knob mutation (K; heavy chain-K shown in SEQ ID NO: 130) mutation or a hole mutation (H; heavy chain-H shown in SEQ ID NO: 131). The heavy chain-K of aducanumab was fused to the cyclic peptide CYC27 (SEQ ID NO: 82) for targeting IGF1R. The heavy chain-K or heavy chain-H of aducanumab was fused to the cyclic peptide CYC17 (SEQ ID NO: 72) for targeting hTfR. Nucleic acids encoding the heavy chain-K and heavy chain-H fusions were introduced into HEK293F together with nucleic acids encoding the light chain of aducanumab (SEQ ID NO 132) for expression and production of monovalent and bispecific heterodimeric aducanumab-cyclic peptide fusions. Schematic diagrams of monovalent and bispecific heterodimeric aducanumab-cyclic peptide fusions are depicted in Figure 1B-Figure 1D middle.

[0314] As described above, the heavy chain of the antibody adalimumab (Humira TM; SEQ ID NO: 102) was fused with the cyclic peptide CYC17 for targeting hTfR. HEK293F cells were co-transfected with expression constructs of the adalimumab heavy chain-CYC17 cyclic peptide fusion and the adalimumab light chain (SEQ ID NO: 103) for expression and production of the bivalent adalimumab-cyclic peptide fusion. A schematic diagram of the bivalent adalimumab-CYC17 cyclic peptide fusion is depicted in Figure 1A middle.

[0315] Standard receptor-substrate binding assays showed that monovalent variants of the aducanumab-CYC17 cyclic peptide fusion had reduced affinity for hTfR, while the bivalent adalimumab-CYC17 cyclic peptide fusion and the bispecific aducanumab-H-CYC17-K-CYC27 bound hTfR with higher affinity. The binding affinity results are shown in Figure 4 EC50 binding affinities are depicted in Table E3. Without wishing to be bound by theory, it is believed that after RMT, higher affinity antibody-cyclic peptide fusions may not be released from endosomes, while lower affinity antibody-cyclic peptide fusions are released from endosomes and can be transported to the brain after interacting with receptors at the blood-brain barrier. Example 7: Generation and evaluation of monovalent and bispecific trastuzumab antibody-cyclic peptide fusions

[0316] Monovalent and bispecific antibody-cyclic peptide fusions were generated by fusion to the exemplary antibody trastuzumab, wherein the cyclic peptide was also fused to the antibody with 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). A cleavable cathepsin B cleavage site was added between the linker and the cyclic peptide to facilitate cleavage of the cyclic peptide portion from the fusion protein after the fusion protein is internalized into endosomes to enhance endosomal antibody release and antibody transport across the blood-brain barrier.

[0317] The heavy chain of the antibody trastuzumab ( SEQ ID NO: 134) engineering: CH3 mutation to contain a knob mutation (K; heavy chain-K as shown in SEQ ID NO: 135) or a hole mutation (H; heavy chain-H as shown in SEQ ID NO: 136). Heavy chain-K or heavy chain-H of trastuzumab was fused to the cyclic peptide CYC17 (SEQ ID NO: 72) or the lower affinity mutant CYC17-15H (SEQ ID NO: 101) for targeting human transferrin receptor (hTfR). For each, the cyclic peptide was linked to the C-terminus of heavy chain-K (SEQ ID NO: 135) of trastuzumab via a Gly-Ser linker (e.g., SEQ ID NO: 104), and, in some cases, the linker further comprised a cathepsin B cleavage sequence (SEQ ID NO: 133). For bispecific constructs, heavy chain-K of trastuzumab was fused to the cyclic peptide CYC27 (SEQ ID NO: 82) for targeting IGF1R.

[0318] Table E4 lists the sequences of the resulting constructs.

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

[0320] The expression results of exemplary constructs are shown in Figure 5 The trastuzumab fusion proteins (C, D, E, and F in Table E4) were expressed at similar levels to the parental antibody (A in Table E4) as determined by ELISA, with no evidence of aggregation as determined by SDS-PAGE gel electrophoresis. Example 8: Expression and activity of glucocerebrosidase fusion cyclic peptide

[0321] The exemplary human transferrin receptor (hTfR) binding cyclic peptide CYC17 (SEQ ID NO: 72) was fused to the enzyme glucocerebrosidase (GlcCSase; SEQ ID NO: 144), which cleaves by hydrolyzing the β-glycosidic bond of chemical glucocerebroside. The cyclic peptide was linked to the C-terminus of glucocerebrosidase via a Gly-Ser linker (e.g., SEQ ID NO: 104). The nucleic acid sequence encoding the CYC17-glucocerebrosidase fusion cyclic peptide shown in SEQ ID NO: 146 was expressed in HEK293 cells.

[0322] The expression and integrity of the glucocerebrosidase-CYC17 fusion was evaluated by Western blotting 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 cyclic peptide (data not shown). Glucocerebroside activity assay

[0323] Standard glucocerebroside activity assay based on the ability of GlcCSase-CYC17 fusion cyclic peptide to react with substrate is carried out to evaluate the functional integrity / activity of fusion cyclic peptide. Specifically, according to the manufacturer's protocol, enzyme activity is determined using SensoLyte Red glucocerebrosidase fluorescence assay kit (AnaSpec). The assay uses resorufin-β-glucoside substrate, which releases resorufin after hGCSase cuts the glycosidic bond, and the resorufin emits fluorescence at 610nm after excitation at 570nm. In a SpectraMax fluorescence plate reader, in an opaque 96-well plate at room temperature during 1 hour, 10 μL of filtered concentrated supernatant, 40 μL of assay buffer and 50 μL of substrate solution from HEK293FhGCSase-CYC17 expression culture are used to perform kinetic assays in triplicate. Positive (GCSase enzyme), inhibitor, vehicle only and substrate only controls are measured in triplicate.

[0324] like Fig. 6A As shown, after incubation with substrate, the GlcCSase-CYC17 fusion cyclic peptide showed a time-dependent increase in the emitted fluorescence indicative of enzyme activity, which was above the level corresponding to the negative control. Binding to transferrin receptor

[0325] The binding of GlcCSase-CYC17 fusion cyclic peptide was evaluated by enzyme-linked immunosorbent assay (ELISA) of hGCSase-HT2 using human transferrin receptor (hTfR) antigen. Replicate wells of high binding half-area 96-well plates (Corning) were directly coated with 125ng of hTfR (or TBST containing only 2% milk powder as a negative control) in 50μL Tris buffered saline plus 0.1% (v / v) Tween 20 (TBST) containing 2% milk powder at 4°C overnight. After removing the supernatant, the wells were blocked with 50μL of TBST containing 2% milk powder at room temperature for 2 hours. Remove the blocking agent and add 25 μL of filtered concentrated supernatant from HEK293F hGCSase-CYC17 fusion expression culture (or 25 μL of filtered concentrated supernatant from non-hGCSase expressing HEK293F expression culture as negative control) plus 25 μL of TBST containing 4% milk powder to the appropriate wells and incubate for 1 hour at room temperature. Remove the supernatant and wash the wells 4 times with TBST. Dilute approximately 1 mg / mL rabbit anti-GCSase primary antibody (Sigma) 1:300 in TBST containing 2% milk powder, add 50 μL to each well, and incubate the plate for 1 hour at room temperature. Remove the supernatant and wash the wells 4 times with TBST. Dilute approximately 1 mg / mL goat anti-rabbit IgG-HRP secondary antibody 1:5,000 in TBST containing 2% milk powder, add 50 μL to each well, and incubate the plate for 30 minutes at room temperature. The supernatant was removed and the wells were washed 4 times with TBST and then once with Tris-buffered saline (TBS). 50 μL of tetramethylbenzidine (TMB) substrate was added to each well and incubated for 5 minutes at room temperature, then quenched with 50 μL of 1N sulfuric acid and the absorbance was read at 450 nm using a SpectraMax absorbance plate reader. GlcCSase-CYC17 fusion cyclic peptide bound to hTfR was Figure 6B shown. Example 9: Preparation of NGF cyclic peptide fusion

[0326] The exemplary human transferrin receptor (hTfR) binding cyclic peptide CYC17 (SEQ ID NO:72) is fused to the C-terminus of nerve growth factor (NGF). A gene construct for expressing therapeutic human NGF-cyclic peptide fusions is prepared by cloning a cyclic peptide gene fused to the 3' end of the human Pro-NGF gene (encoding the amino acid sequence shown in SEQ ID NO:147) via a sequence encoding a flexible linker. The construct contains a 5'IL2 secretion signal peptide sequence for expression. The sequence of the Pro-NGF-CYC17 fusion construct encodes the amino acid sequence shown in SEQ ID NO:149. The gene construct also contains nucleotide sequences encoding the V5 epitope tag (SEQ ID NO:150) and the 6-His tag (SEQ ID NO...

Claims

1. A modified cyclic peptide comprising a peptide that binds to a blood-brain barrier transcytosis receptor (BBB-R), wherein: The peptide is inserted into at least one loop of the cyclic peptide scaffold shown in SEQ ID NO: 2, SEQ ID NO: 1 or SEQ ID NO: 3 or replaces one or more amino acids of the at least one loop, and wherein the peptide is about 2 to 50 amino acid residues; and The BBB-R is selected from 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 modified cyclic peptide, comprising: 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), wherein the peptide has an amino acid sequence of 2 to 50 amino acid residues; and ii) a cyclic peptide scaffold comprising the peptide of i), The modified cyclic peptide comprises structure (I): Ring 6 Ring 1 Ring 2 Ring 3 Ring 4 Ring 5 Ring 6 wherein C1 to C6 are cysteine ​​residues; wherein each of C1 and C4, C2 and C5, and C3 and C6 are linked by a disulfide bond to form a cysteine ​​knot; wherein each X represents an amino acid residue in a loop, wherein the amino acid residues are the same or different; wherein d is about 1-2; wherein at least one loop from loop 1, 2, 3, 5 or 6 has an amino acid sequence comprising the sequence of the peptide of clause i), wherein any loop of the sequence of the peptide comprising clause i) comprises 2 to about 30 amino acids, and wherein for any of loops 1, 2, 3, 5 or 6 of the sequence of the peptide not containing clause i), a, b, c, e and f are the same or different and are each any number from 3 to 10, and b, c, e and f are each any number from 1 to 20.

3. The modified cyclic peptide according to claim 1, wherein the cyclic peptide scaffold is selected from plant cyclic peptides.

4. The modified cyclic peptide according to claim 2 or claim 3, wherein the cyclic peptide scaffold is a cyclic peptide scaffold of a species of the genus Momordicae.

5. The modified cyclic peptide according to claim 4, wherein the cyclic peptide scaffold is Momordica cochinchinensis trypsin inhibitor. 6 . The modified cyclic peptide according to claim 5 , wherein the Momordica cochinchinensis trypsin inhibitor is MCoTI-II shown in SEQ ID NO: 2, MCoTI-I shown in SEQ ID NO: 1, or MCoTI-III shown in SEQ ID NO:

3.

7. The modified cyclic peptide according to any one of claims 1 to 6, wherein the BBB-R is human.

8. The modified cyclic peptide according to any one of claims 1 to 7, wherein the sequence of the peptide replaces or substitutes one or more amino acids in one of the one or more loops of the cyclic peptide scaffold.

9. The modified cyclic peptide according to any one of claims 1 to 8, wherein the cyclic peptide scaffold is as shown in SEQ ID NO:

2.

10. The modified cyclic peptide according to any one of claims 1 to 9, wherein the at least one ring is ring 1, ring 5 or ring 6, or a combination thereof.

11. The modified cyclic peptide according to any one of claims 1 to 10, wherein the at least one ring is ring 1.

12. The modified cyclic peptide according to any one of claims 1 to 11, wherein the peptide is inserted into only one loop of the cyclic peptide scaffold and replaces amino acids of the only one loop.

13. The modified cyclic peptide according to claim 12, wherein the one ring is ring 1.

14. The modified cyclic peptide according to any one of claims 11 to 13, wherein the peptide replaces the loop 1 amino acid between cysteine ​​4 and cysteine ​​11 of the cyclic peptide scaffold.

15. A modified cyclic peptide comprising a peptide inserted into loop 1 to replace all amino acids between cysteine ​​4 and cysteine ​​11 of SEQ ID NO: 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), low-density lipoprotein receptor-related protein 1 (LRP-1), and receptor for advanced glycation end products (RAGE).

16. The modified cyclic peptide according to any one of claims 1-15, wherein the peptide is 10 to 25 amino acids.

17. The modified cyclic peptide according to any one of claims 1 to 16, wherein the BBB-R is expressed on brain endothelial cells.

18. The modified cyclic peptide according to any one of claims 1 to 17, wherein the peptide has blood-brain barrier translocation activity.

19. The modified cyclic peptide according to any one of claims 1 to 18, wherein the BBB-R is transferrin receptor (TrfR).

20. The modified cyclic peptide according to any one of claims 1-19, wherein the peptide comprises the sequence shown in any one of SEQ ID NOs: 26-34 and 49-54.

21. The modified cyclic peptide according to any one of claims 1 to 20, wherein the peptide has a consensus motif as shown in xxxxxHxxSWGx (SEQ ID NO: 177).

22. The modified cyclic peptide according to any one of claims 1-20, wherein the modified cyclic peptide comprises the sequence shown in any one of SEQ ID NOs: 72-80 and 95-100.

23. The modified cyclic peptide according to any one of claims 1 to 21, wherein the peptide comprises the sequence shown in SEQ ID NO:

26.

24. The modified cyclic peptide according to any one of claims 1 to 23, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO:

72.

25. The modified cyclic peptide according to any one of claims 1-21, wherein the peptide comprises the sequence shown in SEQ ID NO:

49.

26. The modified cyclic peptide according to any one of claims 1-22 and 25, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO:

95.

27. The modified cyclic peptide according to any one of claims 1-21, wherein the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in SEQ ID NOs: 26-34 and 49-54.

28. The modified cyclic peptide according to claim 27, wherein the peptide comprises a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the sequence shown in SEQ ID NO:

26.

29. The modified cyclic peptide according to any one of claims 1 to 28, wherein the one or more amino acid substitutions are substitutions of an amino acid for another amino acid selected from histidine or alanine.

30. The modified cyclic peptide according to any one of claims 1-21 and 27-29, wherein the peptide comprises the sequence shown in any one of SEQ ID NOs: 55 and 117-128.

31. The modified cyclic peptide according to any one of claims 1-21 and 27-30, wherein the modified cyclic peptide comprises the sequence shown in any one of SEQ ID NOs: 101 and 105-116.

32. The modified cyclic peptide according to any one of claims 1-21 and 27-29, wherein the peptide comprises the sequence shown in SEQ ID NO:

55.

33. The modified cyclic peptide according to any one of claims 1-21 and 27-32, wherein the modified cyclic peptide comprises the sequence shown in any one of SEQ ID NO:

101.

34. The modified cyclic peptide of any one of claims 1-18, wherein the BBB-R is the leptin receptor.

35. The modified cyclic peptide according to any one of claims 1-18 and 34, wherein the peptide comprises the sequence shown in any one of SEQ ID NOs: 10-23.

36. The modified cyclic peptide according to any one of claims 1-18 and 34-35, wherein the modified cyclic peptide comprises the sequence shown in any one of SEQ ID NOs: 56-69.

37. The modified cyclic peptide of any one of claims 1-18, wherein the BBB-R is ErbB3.

38. The modified cyclic peptide according to any one of claims 1-18 and 37, wherein the peptide comprises the sequence shown in SEQ ID NO: 24 or 25.

39. The modified cyclic peptide according to any one of claims 1-18, 37 and 38, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO: 70 or 71.

40. The modified cyclic peptide of any one of claims 1-18, wherein the BBB-R is insulin-like growth factor type 1 receptor (IGFR).

41. The modified cyclic peptide according to any one of claims 1-18 and 40, wherein the peptide comprises the sequence shown in SEQ ID NO: 35 or 36.

42. The modified cyclic peptide according to any one of claims 1-18, 40 and 41, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO: 81 or 82.

43. The modified cyclic peptide of any one of claims 1-18, wherein the BBB-R is RAGE.

44. The modified cyclic peptide according to any one of claims 1-18 and 43, wherein the peptide comprises the sequence shown in SEQ ID NO: 37 or 38.

45. The modified cyclic peptide according to any one of claims 1-18, 43 and 44, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO: 83 or 84.

46. ​​The modified cyclic peptide of any one of claims 1-18, wherein the BBB-R is the 1 lipoprotein receptor-related protein 1 (LRP-1).

47. The modified cyclic peptide according to any one of claims 1-18 and 46, wherein the peptide comprises the sequence shown in any one of SEQ ID NOs: 39-48.

48. The modified cyclic peptide according to any one of claims 1-18, 46 and 47, wherein the peptide comprises the sequence shown in SEQ ID NO:

39.

49. The modified cyclic peptide according to any one of claims 1-18, 46 and 47, wherein the peptide comprises the sequence shown in SEQ ID NO:

43.

50. The modified cyclic peptide according to any one of claims 1-18, 46 and 47, wherein the peptide comprises the sequence shown in SEQ ID NO:

47.

51. The modified cyclic peptide according to any one of claims 1-18 and 46-50, wherein the modified cyclic peptide comprises the sequence shown in any one of SEQ ID NOs: 85-94.

52. The modified cyclic peptide according to any one of claims 1-18, 46-48 and 51, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO:

85.

53. The modified cyclic peptide according to any one of claims 1-18, 46, 47, 49 and 51, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO:

89.

54. The modified cyclic peptide according to any one of claims 1-18, 46, 47, 50 and 51, wherein the modified cyclic peptide comprises the sequence shown in SEQ ID NO:

93.

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

56. The peptide according to claim 55, wherein the peptide has blood-brain barrier translocation activity.

57. The peptide according to claims 55 and 56, wherein the peptide is 10 to 25 amino acids.

58. A peptide consisting of the sequence shown in any one of SEQ ID NOs: 10-55 or 117-128.

59. The peptide according to claim 58, wherein the peptide has blood-brain barrier translocation activity.

60. A peptide according to claim 58 or claim 59, wherein the peptide binds to a receptor involved in blood-brain barrier transcytosis.

61. A peptide represented by the sequence of any one of SEQ ID NOs: 26-34 and 49-55 and 117-128, wherein the peptide binds to the transferrin receptor.

62. The peptide according to any one of claims 55-61, wherein the peptide is shown as SEQ ID NO:

26.

63. The peptide according to any one of claims 55-61, wherein the peptide is shown as SEQ ID NO:

49.

64. The peptide according to any one of claims 55-61, wherein the peptide is shown as SEQ ID NO:

55.

65. A peptide represented by the sequence of any one of SEQ ID NOs: 10-23, wherein the peptide binds to the leptin receptor.

66. A peptide represented by the sequence of any one of SEQ ID NO: 24 or 25, wherein the peptide binds to ErbR3.

67. A peptide represented by the sequence of any one of SEQ ID NO: 35 or 36, wherein the peptide binds to IGFR.

68. The peptide according to any one of claims 55-60 and 67, wherein the peptide is shown as SEQ ID NO:

35.

69. The peptide according to any one of claims 55-60 and 67, wherein the peptide is shown as SEQ ID NO:

36.

70. A peptide represented by the sequence of SEQ ID NO: 37 or 38, wherein the peptide binds to RAGE.

71. A peptide represented by the sequence of any one of SEQ ID NOs: 39-48, wherein the peptide binds to the lipoprotein receptor-related protein 1 (LRP-1).

72. The peptide according to any one of claims 55-60 and 71, wherein the peptide is shown as SEQ ID NO:

39.

73. The peptide according to any one of claims 55-60 and 71, wherein the peptide is shown as SEQ ID NO:

47.

74. The peptide according to any one of claims 55-60 and 71, wherein the peptide is shown as SEQ ID NO:

43.

75. The peptide of any one of claims 55-74, wherein the peptide is synthetic.

76. A binding molecule comprising a binding scaffold and a peptide according to any one of claims 55-75.

77. The binding molecule of claim 76, wherein the binding scaffold is a cysteine ​​knot protein.

78. A binding molecule according to claim 76 or claim 77, wherein the binding scaffold is a cyclic peptide and the peptide is inserted into a loop of the cyclic peptide backbone or replaces one or more amino acids of the loop.

79. A nucleic acid molecule encoding a modified cyclic peptide according to any one of claims 1-54 or a binding molecule according to any one of claims 76-78.

80. A vector comprising the nucleic acid molecule according to claim 79.

81. The vector of claim 80, which is an expression vector.

82. A host cell comprising a nucleic acid molecule according to claim 79 or a vector according to claim 80 or claim 81.

83. A method for producing a modified cyclic peptide or binding molecule, the method comprising introducing a nucleic acid according to claim 79 or a vector according to claim 80 or claim 81 into a host cell and culturing the host cell under conditions to express the protein in the cell, and optionally purifying the protein from the cell.

84. A purified modified cyclic peptide or binding molecule produced by the method of claim 83.

85. A conjugate comprising the modified cyclic peptide according to any one of claims 1-54 or the binding molecule according to any one of claims 76-78 and a bioactive agent.

86. The conjugate of claim 85, wherein the bioactive agent is a small molecule, a peptide or a protein.

87. The conjugate of claim 85 or claim 86, wherein the bioactive agent is a diagnostic agent or a therapeutic agent.

88. The conjugate of any one of claims 85-87, which is a fusion protein comprising the modified cyclic peptide operably linked to a bioactive agent, which is a protein or a peptide.

89. The conjugate of any one of claims 85-88, wherein the bioactive agent is an antibody.

90. The conjugate of claim 89, wherein the antibody is directed against an antigen selected from the group consisting of human epidermal growth factor receptor 2 (HER2), β-secretase 1 (BACE1), amyloid β (Aβ), epidermal growth factor receptor (EGFR), tau protein, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin protein, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, and TNF-α.

91. The conjugate of claim 90, wherein the antibody is trastuzumab, adalimumab or aducanumab.

92. The conjugate of any one of claims 85-88, wherein the bioactive agent is a growth factor or a hormone.

93. The conjugate of claim 92, wherein the bioactive agent is a growth factor, and the growth factor is nerve growth factor (NGF) or granulocyte colony stimulating factor (GCSF).

94. The conjugate of any one of claims 85-88, wherein the bioactive agent is an enzyme.

95. The conjugate of claim 94, wherein the enzyme is a ceramide degrading enzyme, a lipase, a hydrolase-type enzyme, or a sulfatase.

96. The conjugate of claim 94 or claim 95, wherein the enzyme is a ceramide degrading enzyme, and the ceramide degrading enzyme is glucocerebrosidase, galactocerebrosidase or alpha-galactosidase.

97. The conjugate of any one of claims 94-96, wherein the enzyme is a glucocerebrosidase having an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO: 144 or SEQ ID NO:

145.

98. The conjugate of any one of claims 94-97, wherein the enzyme is a glucocerebrosidase having an amino acid sequence as shown in SEQ ID NO: 144 or SEQ ID NO:

145.

99. The conjugate of claim 94 or claim 95, wherein the enzyme is a lipase or hydrolase type enzyme and the enzyme is a sphingomyelinase, serripase or alpha glucosidase.

100. The conjugate of any one of claims 85-99, wherein the conjugate is monovalent for binding to BBB-R.

101. The conjugate of any one of claims 85-99, wherein the conjugate is bivalent for binding to the BBB-R.

102. The conjugate of any one of claims 85-99, wherein the conjugate is bispecific for binding to two different BBB-Rs and comprises at least two different modified cyclic peptides, each of which binds to a different BBB-R.

103. The conjugate of any one of claims 85-102, wherein the modified cyclic peptide is linked to the biologically active agent via a linker.

104. The conjugate of claim 103, wherein the linker is at least 10 amino acids in length.

105. The conjugate of claim 103, wherein the linker is at least 15 amino acids in length.

106. The conjugate of any one of claims 103-105, wherein the linker is 10 to 20 amino acids.

107. The conjugate of any one of claims 103-106, wherein the linker is a flexible GS peptide linker comprising the sequence GGGGS (SEQ ID NO: 148), (GGGGS)2 (SEQ ID NO: 154), or (GGGGS)3 (SEQ ID NO: 155).

108. The conjugate of any one of claims 103-107, wherein the linker is as shown in SEQ ID NO:

104.

109. The conjugate of any one of claims 103-106, wherein the linker is a cleavable linker comprising an endosomal-specific protease cleavage site.

110. The conjugate of claim 109, wherein the endosomal-specific protease cleavage site is a cathepsin cleavage site.

111. The conjugate of claim 110, wherein the cathepsin cleavage site is a cathepsin B cleavage site.

112. The conjugate of any one of claims 103-106 and 109-111, wherein the linker comprises the sequence shown in SEQ ID NO:

133.

113. A pharmaceutical composition comprising the conjugate according to any one of claims 85-112 and a pharmaceutical carrier.

114. A method for transporting a bioactive agent across the blood-brain barrier of a subject, the method comprising administering to a subject in need thereof a conjugate according to any one of claims 85-112 or a pharmaceutical composition according to claim 113.

115. The method of claim 114, wherein the individual suffers from a neurological disease.

116. A method for treating a patient suffering from a neurological disease, the method comprising administering to the patient a conjugate according to any one of claims 85-112 or a pharmaceutical composition according to claim 113.

117. A method for diagnosing a neurological disease in a patient in need thereof, the method comprising administering to the patient a conjugate according to any one of claims 85-112 or a pharmaceutical composition according to claim 113, and wherein the conjugate comprises a radiolabel or a detectable agent / diagnostic agent.

118. The pharmaceutical composition of claim 113, for use in treating a neurological disease.

119. The pharmaceutical composition of claim 113, which is used for diagnosing a neurological disease.

120. Use of the pharmaceutical composition according to claim 113 in the manufacture of a medicament for treating a nervous system disease.

121. Use of the pharmaceutical composition according to claim 113 in the manufacture of a medicament for diagnosing a nervous system disease.

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