Brain penetrating peptides and methods of use thereof

Abstract

Polypeptides crossing the blood brain barrier (BBB) are disclosed. The peptides can act as transport carriers across the BBB for delivering active agents from circulation to across the BBB to contact the CNS and / or cells and tissues within the brain. Accordingly, provided are conjugate compositions comprising a polypeptide conjugated to one or more active agents that traverses the BBB. The BBB-crossing peptide may be a fusion protein comprising one or more additional domains, such as a targeting moiety, a linker, an active agent, a purification tag, or any combination thereof. An exemplary BBB crossing peptide comprises a polypeptide having the amino acid sequence of RRVISRAKLAAAL (SEQ ID NO: 1), or a functional variant thereof, or the amino acid sequence of CVGTNCY (SEQ ID NO: 2).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 373,160, filed on August 22, 2022, the contents of which are incorporated herein in their entirety. Technical Field

[0003] The field of the invention relates generally to compositions for crossing the healthy blood-brain-barrier (BBB) ​​and methods of their use for delivering therapeutic agents from the circulation to the brain. Background Art

[0004] Brain cancer is a devastating disease. Despite surgical and medical advances, the prognosis of most brain cancers remains poor. The median survival times of glioblastoma, the most common malignant glioma in adults (Scott CB, et al., International Journal of Radiation Oncology, Biology, Physics, 40 (1): 51-55 (1998)), diffuse intrinsic pontine glioma, the most common type of brainstem glioma in children (Khatua S, et al., Childrens Nerv Syst, 27 (9): 1391-1397 (2011)) and brain metastasis (Jaboin JJ, et al., Radiat Oncol, 8 (2013)) are 14 months, 9 months and 12 months, respectively. New treatments with improved efficacy for these tumors are urgently needed.

[0005] Gene therapy is an effective method for treating a variety of tumors. However, its application in gene therapy for brain tumors is limited by the lack of an effective delivery platform that can simultaneously overcome the blood-brain barrier (BBB) ​​and cell barriers. Although local BBB destruction is observed in large brain tumors, these "leaky" blood vessels are mainly located in the center of the tumor, and the capillaries supplying the edge of the proliferative tumor are still impermeable (Blakeley J., Curr Neurol Neurosci Rep, 8 (3): 235-241 (2008)).

[0006] The BBB can potentially be bypassed using invasive methods such as surgical implantation of degradable Wafers, or the more recently developed locally administered poly(lactic-co-glycolic acid) (PLGA) brain-penetrating nanoparticles (NPs) (Strohbehn G, et al., Journal of Neuro-oncology, 121(3):441-449 (2015); Zhou J, et al., Proc Natl Acad Sci USA, 110(29):11751-11756 (2013)). Unfortunately, the clinical utility of these approaches is hampered by their highly invasive nature. In addition, limited drug penetration to distant tumor cells separated from the tumor bulk limits their therapeutic efficacy (Fung LK, et al., Pharmaceutical Research, 13(5):671-682 (1996); Fung LK, et al., Cancer Research, 58(4):672-684 (1998)). Therefore, the next generation of brain therapeutics requires the development of new technologies that are suitable for systemic delivery and can target tissues throughout the brain.

[0007] Nanotechnology represents one of the most promising approaches for intravenous delivery of therapeutic agents to the brain (Deeken JF, et al., Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 13(6):1663-1674 (2007); Patel T, et al., Advanced Drug Delivery Reviews, 64(7):701-705 (2012); Zhou J, et al., Cancer J, 18(1):89-99 (2012)). A major benefit of nanotechnology is that particles and / or macromolecules can be engineered to exploit a variety of mechanisms for brain-targeted delivery, including: 1) receptor-mediated transcytosis (Qiao R, et al., ACS Nano, 6(4):3304-3310 (2012)); 2) carrier-mediated transcytosis (Li J, et al., Biomaterials, 34(36):9142-9148 (2013)); 3) adsorption-mediated transcytosis (Liu L, et al., Biopolymers, 90(5):617-623 (2008)); 4) physical disruption of the BBB (Nance E, et al., Journal of Controlled Release: Official Journal of the Controlled Release Society, 189:123-132 (2014)); and 5) disease microenvironment-targeted delivery (Kievit FM, et al., ACS Nano, 6(4):3304-3310 (2012)); Nano, 4(8):4587-4594(2010)).

[0008] Although this is promising, nanotechnology for systemic delivery of active agents to the brain is still in its infancy. Existing engineering methods are generally unable to enhance systemic delivery of therapeutic agents to the brain to a degree sufficient to achieve therapeutic purposes (Deeken JF, et al., Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 13(6): 1663-1674 (2007); Patel T, et al., Advanced Drug Delivery Reviews, 64(7): 701-705 (2012); Zhou J, et al., Cancer J, 18(1): 89-99 (2012)).

[0009] Current methods of delivering therapeutic agents across the blood-brain barrier to treat a wide range of pathological conditions associated with diseases and disorders of the brain and CNS are inadequate.

[0010] Therefore, one of the objects of the present invention is to provide compositions that cross the healthy blood-brain barrier.

[0011] Another object of the present invention is to use compositions that cross the healthy blood-brain barrier to deliver active agents from the circulation to the brain and CNS.

[0012] It is yet another object of the present invention to treat or prevent one or more symptoms of a disease or disorder in the brain or CNS in a subject.

[0013] It is yet another object of the present invention to identify or monitor in a subject one or more physiological markers of a disease or condition in the brain or CNS.

[0014] It is yet another object of the present invention to treat or prevent one or more symptoms of neurological diseases and disorders in a subject. Summary of the invention

[0015] Polypeptides have been developed that specifically and selectively cross the healthy blood-brain barrier (BBB) ​​with high efficiency and without toxicity. The BBB-penetrating peptides comprise a BBB-crossing domain containing 5 to 50 amino acids (inclusive).

[0016] Provided are brain-penetrating peptides and conjugates thereof and compositions that cross the blood-brain barrier (BBB). In one exemplary form, the composition comprises a BBB-crossing peptide domain having an amino acid sequence comprising RRVISRAKLAAAL (SEQ ID NO: 1) or RRVISRAKLAAAL (SEQ ID NO: 1), or a functional variant thereof. In another exemplary form, the composition comprises a BBB-crossing peptide domain having an amino acid sequence comprising CVGTNCY (SEQ ID NO: 2) (referred to as a "CVG" peptide) or CVGTNCY (SEQ ID NO: 2), or a functional variant thereof.

[0017] In some forms, the length of the BBB-crossing domain is at least 13 amino acids. For example, in some forms, the brain-penetrating peptide comprises 5 to 20 consecutive amino acids of any one or more of SEQ ID NOs: 3 to 10. In some forms, the brain-penetrating peptide domain is any one of SEQ ID NOs: 3 to 10 or a functional variant thereof, which has an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to any one of SEQ ID NOs: 1 or 3 to 10. In other forms, the length of the BBB-crossing domain is at least 7 amino acids. For example, in some forms, the brain-penetrating peptide comprises 5 to 10 consecutive amino acids of one or more of SEQ ID NOs: 11 to 20 or 22 to 23, or a functional variant of one or more of SEQ ID NOs: 11 to 20 or 22 to 23. In some forms, the brain-penetrating peptide domain is any one of SEQ ID NOs: 11 to 20 or 22 to 23. Typically, the amino acid sequence of the BBB-crossing domain is not CAGALCY (SEQ ID NO: 21).

[0018] In some forms, the brain penetrating peptide or its conjugate or composition selectively home to the brain parenchyma, endothelial cells or the whole brain when administered in vivo. In some forms, when the BBB-crossing domain is or comprises the following, the brain penetrating peptide selectively home to cells of the brain parenchyma: SRRVISRAKLAAALE (SEQ ID NO: 3) or MLGDPILASRRVISRAKLAAALE (SEQ ID NO: 4).

[0019] BBB-penetrating peptide conjugates are also described, comprising (a) a brain-penetrating peptide that is or comprises an amino acid sequence of any one of SEQ ID NO: 1 to 20, 22 to 23 or a functional variant thereof and (b) a carrier molecule that is directly or indirectly conjugated or compounded with the brain-penetrating peptide. Typically, the carrier molecule does not cross the BBB in the absence of the brain-penetrating peptide. Exemplary carrier molecules include an active agent selected from the group including: a therapeutic agent, a diagnostic agent, a preventive agent, and a nutrient. In some forms, the carrier molecule is encapsulated in a carrier or conjugated with a carrier. In some forms, the carrier encapsulates one or more active agents or is compounded with one or more active agents, and is directly or indirectly conjugated with the brain-penetrating peptide. Exemplary carriers include polymer particles, lipid particles, liposomes, gels, inorganic particles, viral particles, nucleic acid nanostructures, and virus-like particles. In some forms, one or more active agents and / or carriers are conjugated with the brain-penetrating peptide through one or more joints. In some specific forms, one or more joints are cleavable joints.

[0020] In some forms, the active agent is a therapeutic agent selected from the group including: nucleic acids, peptides, lipids, glycolipids, glycoproteins and small molecules. Exemplary nucleic acid therapeutic agents include antisense molecules, aptamers, ribozymes, triplex-forming oligonucleotides, external guide sequences, RNAi, CRISPR / Cas, zinc finger nucleases and transcription activator-like effector nucleases (TALEN). In other forms, the therapeutic agent is a small molecule. Exemplary therapeutic agents include anticancer agents, anti-inflammatory agents, immunomodulators and antimicrobial agents.

[0021] Also provided are pharmaceutical compositions comprising the peptide conjugates together with a pharmaceutically acceptable excipient for in vivo administration to a subject.Exemplary pharmaceutical compositions are formulated for mucosal, pulmonary, intravenous (iv) or intramuscular (im) delivery.

[0022] Also provided is a method of treating or preventing one or more symptoms of a disease or condition in the brain or CNS of a subject by administering to the subject a pharmaceutical composition comprising a BBB-crossing peptide conjugate. Generally, the method comprises administering to the subject an effective amount of a pharmaceutical composition comprising a BBB-crossing peptide conjugate to prevent or alleviate one or more symptoms of a disease or condition in the brain or CNS of the subject.

[0023] In some forms, the active agent is a diagnostic agent selected from the group consisting of a dye, a radionuclide, a fluorescent label, a magnetic label, and a nanoparticle. Also provided is a pharmaceutical composition comprising a BBB-crossing peptide conjugated to a diagnostic agent, together with a pharmaceutically acceptable excipient for in vivo administration to a subject. Exemplary pharmaceutical compositions are formulated for mucosal, pulmonary, intravenous (iv) or intramuscular (im) delivery.

[0024] Also provided is a method for detecting or monitoring a disease or condition in the brain or CNS of a subject by administering to the subject an effective amount of a pharmaceutical composition comprising a BBB-crossing peptide conjugated to a diagnostic agent. The method detects or monitors a disease or condition in the brain or CNS of a subject in a subject.

[0025] Typically, the object is a human. In some forms, the object is an infant or child. In some forms, the object suffers from or is suspected of having a disease or illness. In some forms, the object suffers from a potential disease or illness, which aggravates or presents a disease to be treated by the method. Exemplary diseases and illnesses include cancer, inflammatory diseases, neuronal disorders, HIV / AIDS, diabetes, cardiovascular diseases, infectious diseases (including diseases of viruses, protozoa, bacteria, and allergies), autoimmune diseases and autoimmune diseases, Alzheimer's disease, Parkinson's disease, ischemia, neurodegenerative disorders, and hereditary disorders. In some specific forms, the disease is cancer, such as glioma. In other forms, the disease is a neurodegenerative disease, such as Alzheimer's disease. In some specific forms, the disease is Parkinson's disease. In some forms, the object suffers from infection.

[0026] The present invention will be further understood by referring to the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1A , 1B , 1D and 1E are graphs, and Figure 1C The sequences were compared. Figure 1A Shown is the % of injected phage accumulated in the brain throughout five rounds of bio-panning (R1 to R5). Peptide-phage were injected iv and mice were perfused with PBS after a 30-minute cycle. Phage rescued from the brain were re-amplified and injected for subsequent rounds of bio-panning. In R1, N=8, and in R2 to R5, n=2. ***p≤0.0001, ****p<0.0001. Figure 1B Shown are the total amount (PFU) of phage rescued from mouse brain for each of the CD31+ and CD31- cell fractions (N=2) after R5 with an additional step to separate brain endothelial (CD31+) cells from all other brain cells (parenchymal cells; CD31-). Figure 1CThe amino acid sequence alignment of peptides identified in the screening experiment is shown, including MLGDPILASRRVISRAKLAAALE (SEQ ID NO: 4) (the functional fragment SRRVISRAKLAAALE (SEQ ID NO: 3) is called "Pep1"); MLGDPNSGGSRRVISRAKLAAAL (SEQ ID NO: 5) (the functional fragment SRRVISRAKLAAAL (SEQ ID NO: 9) is called "Cerepep"); MLGDPNSGGSPIKASRRVISRAKIAAA (SEQ ID NO: 65) (called "Pep17"); and MLGDPNSGGSRVISRAKLAAALE (SEQ ID NO: 7) (called "Pep13"). The remainder in bold text are functional components associated with penetration into the brain. Figure 1D The prevalence of Pep1 sequences in high-throughput sequencing data is shown, displayed as a percentage of all reads. Figure 1E Peptide-phage brain homing (brain / blood titer) for each of Insertless, Pep1, Cerepep, Pep17, and Pep13 peptides is shown. N=3, *p≤0.05, **p≤0.01. All data are shown as mean with SD.

[0028] Figures 2A to 2D is a graph showing brain accumulation of peptide-phage in male and female mouse and rat brains. Figure 2A Shown is the accumulation (PFU / mg) of phage expressing no peptide (no insert), Angiopep-2, and Cerepep (SRRVISRAKLAAAL; (SEQ ID NO: 9)) peptide in the brain. Figure 2B Brain distribution of Cerepep phage in each of the olfactory bulb, cerebellum, brainstem, hemispheres, spinal cord, liver, and blood is shown (0-800 fold overcontrol). Figure 2C Shown is the brain distribution of Cerepep phage in male mice in brain, lung, liver, kidney, blood, heart, muscle, testis, pancreas, and spleen (0 to 800-fold relative to control). Figure 2D Shown is the brain distribution of Cerepep phage in female mice in brain, lung, liver, kidney, blood, and heart (0 to 600-fold relative to control).

[0029] Figure 3are histograms showing the quantification of the accumulation of silver nanoparticles (AgNPs) Cerepep(CP) / biotin in each of the brain and liver compared to Biotin-NP and Cerepep(CP) / Angiopep-2. All data are shown as mean values ​​with SD, n=3.

[0030] Figure 4 is a histogram of BP1 peptide-phage binding to different cell lines, shown as fold relative to phage without insert, showing the fold binding (0 to 150) relative to the control for each of P3 stem, WT GBM, Neuro 2A, VEGF KO, 4T1, RCC-MF, MKN45P, CT26, HEK293, M21, RC124, PPC1, HBVP, PC3, MDA-MB231, U87MG, HT1080, MCF10CA1A, RAW264.7, NCH421K and B16F10.

[0031] Figure 5 Figure 1 is a graph of data from flow cytometry analysis showing the effect of cellular uptake inhibitors on the internalization of Cerepep (BP)-AgNPs into Neuro2A cells incubated with samples including a no nanoparticle control (no NP), biotinylated nanoparticles (Biot-NPs), Cerepep nanoparticles (BP_NPs), and each of chlorpromazine, nystatin, cytochalasin D, and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). Data are quantified as the mean fluorescence (FL2-A) of each sample.

[0032] Fig. 6A and 6B are data from flow cytometry for determination of peptide-directed horseradish peroxidase (HRP)-mediated biotinylation of cellular proteins (proximity ligation assay). WT GBM ( Fig. 6A ) and Neuro2A( Figure 6B ) cell count.

[0033] Figures 7A to 7D are data from surface plasmon resonance analysis of human LRP-1 immobilized on a BIAcore sensor chip. Fig. 7A Shown are the responses (0 to 500 RU) over time (0 to 500 seconds) for Cerepep (CP)-neutravidin complex and Angiopep-2-neutravidin complex (used as positive control). Figure 7B The binding of Cerepep (CP)-neutravidin complex in the presence or absence of RAP and / or EDTA is shown. RAP and Cerepep (CP)-neutravidin were used as controls. As shown, Figure 7C Shown are brain / blood readouts (0 to 25) of the Cerepep (SRRVISRAKLAAAL; (SEQ ID NO: 9) peptide, in which each residue at each position of the peptide was independently mutated to alanine (alanine to leucine). Brain homing of each mutant peptide-phage was assessed using an in vivo competitive play-off approach. Fig.7D Different peptide sequences are shown along with the fold brain homing of each listed peptide relative to control phage, including

[0034] MLGDPILASRRVISRAKLAAALE(SEQ ID NO:4);

[0035] MLGDPNSGGSPILASRRVISRAKLAAALE(SEQ ID NO:71);

[0036] MLGDPNSGGSPILASRRVISRAKLAAALEA(SEQ ID NO:72);

[0037] MLGDPNSGGSRRVISRAKLAAALE (SEQ ID NO:73); and

[0038] MLGDPNSGGSRRVISRAKLAAAL (SEQ ID NO: 5).

[0039] Residues MLGDPNS (SEQ ID NO:69) are part of the phage expression system; residues GGS are linker residues; residues PILAS (SEQ ID NO:98) were identified in the screen and are not essential for brain homing display; residues RRVISRAKLAAAL (SEQ ID NO:1) are essential for brain homing display.

[0040] Figures 8A to 8D Shown are the identification of brain-homing peptides including the CAGALCY (SEQ ID NO: 21) peptide and mutants thereof. Fig. 8AFlow chart of the experimental design of an internally controlled in vivo competitive screening experiment (play off experiment), listing the sequence of the following steps, including iv injection of phage pool, perfusion, amplification of phage from brain, and high-throughput sequencing of each of the p1, p2, p3, p4, p5 and control (c) groups (depicted as circles at the top of the workflow). The relative amount of each combined sequence is described by the relative size of the circles at the bottom of the workflow. Figure 8B is a histogram showing brain representation (0 to 80 times relative to control) for each of the listed peptides, including CAGALCY (SEQ ID NO: 21);

[0041] TPSYDTAAELR (SEQ ID NO:33; labeled "TPS"); CLSSRLDA (SEQ ID NO:25; labeled "SRL"); LSSRLDAC (SEQ ID NO:26); ACSYTSSTMCGGGS (SEQ ID NO:27; labeled "ACSYTSS"); CGHKAKGPRK (SEQ ID NO:28; labeled "B6"); RLSSVDSDLSGC (SEQ ID NO:29; labeled "RLSSVDS"); THRPPMWSPVWP (SEQ ID NO:30; labeled "THRPPMW"); TFFYGGSRGKRNNFKTEEYC (SEQ ID NO:31; labeled "Angiopep-2"); and CMPRLRGCC (SEQ ID NO:32).

[0042] Figure 8C Shown is brain homing (0 to 1.5) as a function of the CAGALCY (SEQ ID NO: 21) peptide for each mutation listed. Fig.8D Shown is the brain representation as a function of the CAGALCY (SEQ ID NO: 21) peptide (multiplier input 0 to 250) for each of the listed peptides, including

[0043] CDGALCY (SEQ ID NO:34); CEGALCY (SEQ ID NO:24); CTGSLCY (SEQ ID NO:12); and CVGTNCY (SEQ ID NO:2).

[0044] Fig. 9is a histogram of biodistribution analysis of CVGTNCY (SEQ ID NO: 2) peptide-phage in mouse CNS and control organs, showing PFU / mg wet weight normalized to G7 for each listed organ. The CVGTNCY (SEQ ID NO: 2) peptide is referred to as the "CVG" peptide.

[0045] Fig. 10A and 10B is a histogram showing the expression fold of mouse (relative to G7 peptide displaying phage, Fig. 10A ) or rat (fold expression relative to no insert, Fig. 10B ) Homing of ivCVGTNCY (SEQ ID NO: 2) peptide-displaying phage in each of the listed organs. Fig. 10C and 10D Figure 2 shows the differences in the expression of the proteins from mouse brain ( Fig. 10C ) and control organ (liver) ( Fig. 10D ) as a function of distance (μm). Mice were injected with an equimolar mixture of Ag107 nanoparticles functionalized with CVGTNCY (SEQ ID NO: 2) peptide and control nanoparticles made of Ag109, and tissue cryosections were line scanned using laser ablation ICP-MS analysis. Fig.10E and 10F CVGTNCY (SEQ ID NO: 2) peptide Ag nanoparticles or control Ag nanoparticles were respectively expressed in the whole brain volume ( Fig.10E ) and white matter ( Fig.10F ) in the boxplot.

[0046] Figures 11A to 11D are histograms showing the expression of control and CVG peptide in hippocampal CA3 ( Fig.11A )、HippocampusGD( Fig. 11B )、Leather( Fig. 11C ), cerebellum( Fig.11D ) in each of the AgNP+NeuN+ cells.

[0047] Fig.12 is a histogram showing the fold relative to the control G7 for each of a variety of different tumorigenic and non-tumorigenic cell lines.

[0048] Fig.13 are histograms showing 0 to 1,500-fold relative to control G7 for each of Reelin, NRP1, mut NRP1, and BSA, respectively.

[0049] Figures 14A to 14C is a histogram. Fig.14ACVG peptide levels in glioma and brain are shown, ranging from 0 to 20,000 PFU / mg for each of WT GBM, VEGF Ko, and normal brain, respectively. Fig. 14B G7 levels in glioma and brain are shown, ranging from 0 to 1500 PFU / mg for each of WT GBM, VEGFKo, and normal brain, respectively. Fig. 14C Shown are CVG folds (0 to 200) relative to G7 for each of WT GBM, VEGFKo, and normal brain, respectively.

[0050] Figures 15A to 15B is a histogram. Fig.15A Shown are PFU / mg brain (1,000 to 3,000) of phages conjugated to either CVGTNCY (CVG; SEQ ID NO: 2) (ex vivo), CVG (in vivo), G7 control phage only (ex vivo), and G7 (in vivo); N=3; ***p<0.0005. Fig. 15B is a graph demonstrating that ex vivo binding of CVGTNCY (CVG; SEQ ID NO: 2) peptide-phage to brain tissue homogenate was restored in the presence of whole blood samples, showing the PFU / mg brain (1,000 to 2,500) of phage coupled to each of CVG, blood+CVG, G7 alone (control), and blood+G7, respectively; N=3; *p<0.05.

[0051] Fig.16 is a histogram depicting the saturation of CVGTNCY (CVG; SEQ ID NO: 2) receptors in the brain, showing PFU / mg (0 to 5,000) for each of various concentrations (0, 10, 30, 100 μg, respectively) of CVG peptide-streptavidin complex (SA-CVG). Angiopep2-peptide streptavidin complex (SA-Angiopep) and G7 phage were used as controls, respectively; the graph shows the mean and SD. N=3, *p<0.05; **p<0.005.

[0052] Figures 17A to 17B is a histogram of homing of CVGTNCY peptide in glioblastoma. Fig.17A Shown are CVGTNCY (CVG; SEQ ID NO: 2) levels (1,000 to 5,000 PFU / mg brain) in glioma and brain for each of the different mice, including WT-GBM, VEGFko, U87MG or NCH421k GBM, respectively, and compared to normal brain (non-tumor bearing mice). Fig. 17B The ratio of CVGTNCY (CVG; SEQ ID NO: 2) to control is shown (0 to 200-fold relative to G7).

[0053] Figures 18A to 18B is a histogram showing that the binding of AgNP-Cerepep to P3 human stem cells was reduced by several inhibitors of LDL receptor protein, Receptor Associated Protein (RAP) and anti-LRP-1 antibody. Fig.18A LRP1 antibody (AB) binding is shown as a fold (0 to 1.5) relative to AgNP-Cerepep binding for each of AgNP-Cerepep+LRP1AB, AgNP-control, and AgNP-Cerepep, respectively. Fig.18B LRP1 Antibody (AB) binding as a fold relative to AgNP-Cerepep binding (0 to 1.2) for both RAP, LDLR Antibody (AB) and LRP1 Antibody (AB) + LRP1 Antibody (AB) and each of 20% glycerol, AbNP-control and AgNp-Cerepep, respectively; experiments were performed in triplicate per condition and error bars show standard deviation. *Statistical significance of condition compared to AgNP-Cerepep.

[0054] Figures 19A to 19B is a histogram showing the interaction of Cerepep with LRP-1 cluster II and cluster III. Fig.19A Shown are the mean fluorescence intensities (0 to 4,000) of beads incubated overnight with 10 nM LRP-1 Cluster II for each of the -ve control (biotin), +ve control (beads incubated with biotinylated Angiopep2 peptide, LRP-1 Cluster II protein, and AlexaFluor 647 anti-human LRP-1 antibody), Scramble peptide, and Cerepep, respectively. Fig.19B Shown are the mean fluorescence intensities (0 to 1,000) of beads incubated overnight with 10 nM LRP-1 cluster III for each of -ve control (biotin), +ve control (beads incubated with biotinylated RPAR peptide, neuropilin-1b1b2 protein (1:200), anti-neuropilin-1 antibody (1:200), and Alexa Fluor 647 anti-rabbit antibody), Angiopep2, scrambled peptide, and Cerepep, respectively; experiments were performed in triplicate for each condition, and error bars show standard deviations. *ANOVA tests for statistical significance of compared conditions versus negative control. **t-tests for statistical significance of compared scrambled sequence (SARVISRAKLARAL (SEQ ID NO:70)) versus Cerepep.

[0055] Fig. 20 Is a histogram showing colocalization of Cerepep-AgNP with lysosomes, showing the average correlation index (0-45Icorr) of P3 stem cells grown on coverslips and incubated with AgNP-control or AgNP-Cerepep and stained with anti-LAMP1 antibody and DAPI, respectively, and then images overlapped / correlated as shown. Bars represent the average correlation index (Icorr, ImageJ output). Experiments were performed in triplicate and error bars show standard deviation. * Statistically significant.

[0056] Figures 21A to 21B is a histogram showing that LRP1 antibodies inhibit the binding of Cerepep in brain. Fig.21A Shown are PFU / mg (1 to 100,000,000) for each of Cerepep control in liver, Cerepep control in brain, 25 μg LRP1-AB+Cerepep in liver, and 25 μg LRP1-AB+Cerepep in brain, respectively. Fig. 21B Folds (0 to 1.2) relative to control phage in brain are shown for each of Cerepep control and 25 μg LRP1-AB+Cerepep, respectively. Experiments were performed in triplicate for each condition and error bars show standard deviation. *Statistically significant, p value = 0.008.

[0057] Figures 22A to 22F is a histogram showing the distribution of FAM-Cerepep monomeric peptide in the brain, showing the distribution of FAM-Cerepep monomeric peptide in the hippocampus-dentate gyrus ( Fig.22A ); hippocampus-CA1( Fig. 22B ); Brain stem ( Fig. 22C ); cerebellar molecular layer ( Fig.22D ); Cortex( Fig.22E ); and cerebellar white matter ( Fig.22F ) for each of Cerepep and scrambled peptides, respectively. The fluorescence intensity signal from FAM was measured using ImageJ. The experiment was performed in triplicate. *Statistically significant.

[0058] Fig.23 are histograms demonstrating that HER2-FAM-Cerepep antibody crosses the blood-brain barrier, showing fluorescence (au) for each of Cerepep and scrambled peptide, respectively, in each of the cortex, hippocampus, and brainstem of brain sections; *statistically significant.

[0059] Figures 24A to 24B are histograms showing accumulation of the Cerepep library in mouse brain in two second rounds of biopanning. Fig.24APhage accumulation in brain after round 1 and round 2 biopanning is shown as PFU / mg brain (1 to 1800) for each of round 1 and round 2 biopanning, respectively. Fig. 24B Folds relative to control phage for three organs (liver, lung and brain) are shown for each of round 1 and round 2 biopanning, respectively, with the highest value data point for each shown. Experiments were performed in triplicate for each condition, and error bars show standard deviation.

[0060] Fig.25 is a graphic representation of compiled peptides from a restricted Cerepep library screen showing the amino acids that were ranked for characterization in the brain based on high-throughput DNA sequencing. DETAILED DESCRIPTION

[0061] I. Definitions

[0062] As used herein, "treat," "treat," "treat," or "treat" means to prevent, reduce, diminish, or ameliorate one or more symptoms, features, or complications of an age-related disease, disorder, or condition; to reverse the progression of one or more symptoms, features, or complications of an age-related condition; to halt the progression of one or more symptoms, features, or complications of an age-related condition; to prevent the onset of one or more symptoms, features, or complications of an age-related condition; to inhibit the rate of occurrence of one or more symptoms, features, or complications, or a combination thereof.

[0063] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to rodents, simians, and humans.

[0064] The terms "reduce," "inhibit," "mitigate," and "reduce" are used relative to a control. One skilled in the art will readily determine the appropriate control to use for each experiment. For example, a reduction in a response in a subject or cell treated with a compound is relative to a response in a subject or cell not treated with the compound.

[0065] The terms "enhance," "induce," "activate," and "improve" are used relative to a control. One skilled in the art will readily determine the appropriate control to use for each experiment. For example, an increased response in a subject or cell treated with a compound is relative to a response in a subject or cell not treated with the compound.

[0066] The term "polypeptide" includes proteins and fragments thereof. Polypeptides are disclosed herein as sequences of amino acid residues. These sequences are written from left to right in the direction from the amino to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are named by three-letter or one-letter codes, such as as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

[0067] "Variant" refers to a polypeptide or polynucleotide that is different from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs from another reference polypeptide in amino acid sequence. In general, the differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and identical in many regions. The amino acid sequences of the variant and the reference polypeptide may differ by one or more modifications (e.g., substitutions, additions and / or deletions). The replaced or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is unknown whether it occurs naturally.

[0068] Modifications and changes can be made in the structure of a polypeptide in the present disclosure and still obtain a molecule with similar characteristics to the polypeptide (e.g., conservative amino acid substitutions). For example, certain amino acids can replace other amino acids in the sequence without significant loss of activity. Because it is the interaction ability and properties of the polypeptide that define the biological functional activity of the polypeptide, certain amino acid sequence substitutions can be made in the polypeptide sequence and still obtain a polypeptide with similar properties.

[0069] In making such changes, the hydropathic index of the amino acid may be considered. The importance of the amino acid hydropathic index in conferring interactive biological function to a polypeptide is generally understood in the art. It is known that certain amino acids can replace other amino acids with similar hydropathic indices or scores and still produce polypeptides with similar biological activity. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0070] It is believed that the relative hydropathicity of amino acids determines the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, etc. It is known in the art that an amino acid can be replaced by another amino acid with a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, replacement of amino acids with hydropathic indices within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0071] Substitution of similar amino acids may also be made on the basis of hydrophilicity, particularly when the resulting biologically functionally equivalent polypeptide or peptide is intended for use in an immunological format. The following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It will be appreciated that an amino acid may be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, immunologically equivalent, polypeptide. In such changes, substitution of amino acids having hydrophilicity values ​​within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0072] As outlined above, amino acid substitutions can generally be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take many of the foregoing characteristics into account are well known to those skilled in the art and include (original residue: exemplary substitutions):

[0073] (Ala:Gly,Ser),(Arg:Lys),(Asn:Gln,His),(Asp:Glu,Cys,Ser),(Gln:Asn),(Glu:Asp),(Gly:Ala),(His:Asn,Gln),(Ile: Leu,Val), (Leu:Ile,Val), (Lys:Arg), (Met:Leu,Tyr), (Ser:Thr), (Thr:Ser), (Tip:Tyr), (Tyr:Trp,Phe), and (Val:Ile,Leu).

[0074] Some forms of the disclosure therefore contemplate functionally or biologically equivalent polypeptides as set forth above. In particular, some forms of the polypeptides may include variants having about 50%, 60%, 70%, 80%, 90% and 95% sequence identity with the polypeptide of interest.

[0075] "Identity" as known in the art is the relationship between two or more polypeptide sequences determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptides as determined by the match between strings of such sequences. "Identity" can also mean the degree of sequence relatedness of polypeptides compared to the full length of a reference polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, AM, Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988).

[0076] Preferred methods for determining identity are designed to give maximum matching between the sequences tested. Methods for determining identity and similarity are incorporated into publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) incorporating Needelman and Wunsch, (J. Mol. Biol., 48:443-453, 1970) algorithms (e.g., NBLAST and XBLAST). Default parameters are used to determine the identification of the polypeptides of the present disclosure.

[0077] For example, a polypeptide sequence may be identical to a reference sequence, i.e., 100% identical, or it may contain up to a certain integer number of amino acid changes compared to the reference sequence such that the % identity is less than 100%. Such changes are selected from: at least one amino acid deletion, substitution (including conservative and non-conservative substitutions), or insertion, and wherein the changes may occur at the amino or carboxyl terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed individually between the amino acids in the reference sequence, or interspersed in one or more consecutive groups within the reference sequence. The number of amino acid changes for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percentage of the corresponding % identity (divided by 100), and then subtracting the product from the total number of amino acids in the reference polypeptide.

[0078] As used herein, the term "low stringency" refers to conditions that allow a polynucleotide or polypeptide to bind to another substance with little or no sequence specificity.

[0079] As used herein, the term "purified" and similar terms refer to a molecule or compound in substantially free (at least 60% free, preferably 75% free and most preferably 90% free) form separated from other components with which the molecule or compound is normally associated in its natural environment.

[0080] As used herein, the term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carriers such as phosphate buffered saline, water, and emulsions (eg, oil / water or water / oil emulsions) and various types of wetting agents.

[0081] "Operably linked" refers to a juxtaposition in which the components are configured to perform their usual functions. For example, a control sequence or promoter operably linked to a coding sequence can affect the expression of the coding sequence, and an organelle localization sequence operably linked to a protein will help the linked protein to be localized at a specific organelle.

[0082] "Localization signal or sequence or domain" or "targeting signal or sequence or domain" are used interchangeably and refer to a signal that directs a molecule to a specific cell, tissue, organelle, intracellular region, or cellular state. The signal may be a polynucleotide, polypeptide, or carbohydrate moiety, or may be an organic or inorganic compound sufficient to direct the attached molecule to the desired location. Exemplary targeting signals include mitochondrial localization signals from precursor proteins listed in U.S. Patent No. 8,039,587, and cellular targeting signals known in the art, such as those in Wagner et al., Adv. Gen., 53:333-354 (2005). It should be understood that the complete sequence need not be included, and modifications including truncations of these sequences are within the scope of the present disclosure, provided that these sequences are operable to direct the attached molecule to a specific cell type. The targeting signal of the present disclosure may have 80% to 100% sequence identity with a mitochondrial localization signal or cellular targeting signal sequence. One class of suitable targeting signals includes those that do not interact with the target cell in a receptor-ligand mechanism. For example, targeting signals include signals having or imparting a net charge (e.g., a positive charge). Positively charged signals can be used to target negatively charged cell types, such as neurons and muscles. Negatively charged signals can be used to target positively charged cells.

[0083] "Cell surface marker" refers to any molecule, such as a moiety, peptide, protein, carbohydrate, nucleic acid, antibody, antigen and / or metabolite, present on or near the surface of a cell that is sufficient to identify the cell as unique in type or state.

[0084] As used herein, "small molecule" refers to a molecule having a molecular weight of less than about 2000 g / mol, more preferably less than about 1500 g / mol, and most preferably less than about 1200 g / mol.

[0085] As used herein, "nanoparticles" generally refer to particles having a diameter of about 1 nm to (but not including) about 1 micron, preferably 100 nm to about 1 micron. The particles may have any shape. Nanoparticles having a spherical shape may be referred to as "nanospheres."

[0086] As used herein, "average particle size" generally refers to the statistical average particle size (diameter) of particles in a population of particles. The diameter of substantially spherical particles may refer to the physical diameter or the hydrodynamic diameter. The diameter of non-spherical particles may preferentially refer to the hydrodynamic diameter. The diameter of non-spherical particles used herein may refer to the maximum straight-line distance between two points on the particle surface. The average particle size may be measured using methods known in the art, such as dynamic light scattering.

[0087] "Monodisperse" and "uniform size distribution" are used interchangeably herein and describe a population of nanoparticles or microparticles in which all particles are the same or nearly the same size. As used herein, a monodisperse distribution refers to a distribution of particles in which 90% of the distribution is within 15% of the median particle size, more preferably within 10% of the median particle size, and most preferably within 5% of the median particle size.

[0088] As used herein, the term "carrier" or "excipient" refers to an organic or inorganic ingredient, natural or synthetic, inactive ingredient in a formulation with which one or more active ingredients are combined. II. Compositions that cross the blood-brain barrier (BBB)

[0089] Compositions comprising polypeptides that cross the blood-brain barrier (BBB) ​​have been developed. The ability of proteins to cross the BBB to deliver relevant agents to the brain and CNS provides a strategy for treating diseases and conditions of the brain and CNS. Compositions and conjugates of brain-penetrating peptides and methods of using them to cross the BBB and deliver active agents across the BBB to treat symptoms of pathological conditions in the brain or CNS are disclosed. Typically, the brain-penetrating peptide conjugate comprises at least one BBB-crossing domain, optionally comprising one or more linkers and / or one or more carriers in addition to one or more active agents (e.g., therapeutic agents, diagnostic agents, or prophylactic agents).

[0090] A. BBB-crossing peptides

[0091] Polypeptides that cross the blood-brain barrier (BBB) ​​are described. A BBB-crossing polypeptide is a peptide that passes from the circulation into the brain and / or CNS in the absence of any additional molecular components.

[0092] The blood-brain barrier (BBB) ​​is formed by brain endothelial cells (BEC), which form the lumen of the brain microvasculature (Abbott et al., Neurobiol Dis., 37: 13–25 (2010)). The barrier function is achieved by tight junctions between endothelial cells, which regulate the extravasation of molecules and cells into and out of the central nervous system (CNS). It is known that regulation of receptor signaling at BECs can regulate BBB permeability and promote the entry of molecules and cells into the central nervous system (Carman, et al., The Journal of Neuroscience, 31 (37): 13272-13280 (2011)). It has been determined that receptors on the surface of BECs can bind and / or interact with the peptides that cross the BBB to initiate and complete the crossing of BBB-crossing peptides and the entry of related cargo molecules from the circulation into the brain. In some forms, the BBB-crossing peptides specifically bind to reelin on the cell surface to induce transport and / or entry across the BBB. In other forms, the BBB-crossing peptides specifically bind to low-density lipoprotein receptor-related protein 1 (Lipoprotein Receptor-Related Protein 1, LRP1) on the cell surface to induce transport and / or entry across the BBB.

[0093] Preferably, the polypeptide is non-toxic in the brain and does not damage or otherwise disrupt the integrity of the blood-brain barrier or any other tissue or structure in the brain. Thus, preferably, the peptide crosses a complete functional BBB (i.e., a "healthy BBB") by one or more active or passive transport mechanisms. In some forms, the peptide crosses the damaged BBB to the same or different extent than a healthy BBB.

[0094] 1. BBB-crossing domain

[0095] BBB-crossing polypeptides include at least one BBB-crossing domain. The term "BBB-crossing domain" as used herein means a polypeptide that is capable of crossing the BBB in a subject in the absence of another molecule targeting, accompanying or otherwise mediating the crossing of the peptide across the BBB. Typically, a BBB-crossing domain is or comprises a polypeptide having a sequence of about 5 to about 25 consecutive amino acids, the sequence comprising any one or more of SEQ ID NOs: 1 to 20, 22 to 23.

[0096] In some forms, the BBB crossing domain comprises the amino acid sequence RRVISRAKLAAAL (SEQ ID NO: 1) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain comprises the amino acid sequence CVGTNCT (SEQ ID NO: 2) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence SRRVISRAKLAAALE (SEQ ID NO: 3) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPILASRRVISRAKLAAALE (SEQ ID NO: 4) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPNSGGSRRVISRAKLAAAL (SEQ ID NO: 5) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPNSGGSRRVISRAKLAAALE (SEQ ID NO: 6) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPNSGGSRRVISRAKLAAALE (SEQ ID NO: 7) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence ILASRRVISRAKLAAALE (SEQ ID NO: 8) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence SRRVISRAKLAAAL (SEQ ID NO: 9) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence GGSPILASRRVISRAKLAAALE (SEQ ID NO: 10) or a functional fragment, variant or derivative thereof.

[0097] In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPNLASRRRVISRAKLAAALE (SEQ ID NO: 35) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPILASRRVISRAKLAAAL (SEQ ID NO: 36) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPNFAIRRVISRAKLAAALE (SEQ ID NO: 37) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPNSSSVDKLAAALE (SEQ ID NO: 38) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPILAGRRIVSRAKLAAALE (SEQ ID NO: 39) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence NFAIRRVISRAKLAAALE (SEQ ID NO: 62) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPNSGGSPILASRRVISRAKLAAALE (SEQ ID NO: 63) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence PILASRRVISRAKLAAALE (SEQ ID NO: 64) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence MLGDPNSGGSPILASRRVISRAKLAAA (SEQ ID NO: 65) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence SRVISRAKLAAALE (SEQ ID NO: 66) or a functional fragment, variant or derivative thereof. In other forms, the BBB-crossing domain is or comprises the amino acid sequence MLGDPNLASRRVISRAKLAAALE (SEQ ID NO: 67).

[0098] In other forms, the BBB crossing domain is or comprises the amino acid sequence CEGSLCY (SEQ ID NO: 11) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence CTGSLCY (SEQ ID NO: 12) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence CAGSMCY (SEQ ID NO: 13) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence CVGTLCY (SEQ ID NO: 14) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence CKGSNCY (SEQ ID NO: 15) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence CVGQLCY (SEQ ID NO: 16) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence CAGPLCY (SEQ ID NO: 17) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises the amino acid sequence CVGANCY (SEQ ID NO: 18) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain comprises the amino acid sequence CVGANCY (SEQ ID NO: 19) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain comprises the amino acid sequence CQGSNCY (SEQ ID NO: 20) or a functional fragment, variant or derivative thereof. In other forms, the BBB crossing domain is or comprises a functional fragment, variant or derivative of a polypeptide having the amino acid sequence CVGANCY (SEQ ID NO: 24). In other forms, the BBB crossing domain is or comprises the amino acid sequence CTGSMCY (SEQ ID NO: 22) or a functional fragment, variant or derivative thereof. In other forms, the BBB-crossing domain is or comprises the amino acid sequence CMGDLCY (SEQ ID NO: 23) or a functional fragment, variant or derivative thereof.

[0099] Typically, the BBB-crossing domain or a functional fragment thereof is about 5 amino acids to about 25 amino acids, including any one of SEQ ID NOs: 1 to 20 or 22 to 23 or a homolog thereof (e.g., an ortholog thereof or a paralog thereof); or any combination thereof, or any subrange thereof, or any specific integer number of amino acids therebetween, including but not limited to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. The variant may have, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: SEQ ID NOs: 1 to 20 or 22 to 23 or a functional fragment thereof; or a corresponding homolog sequence (e.g., an ortholog or paralog of any of the foregoing sequences); or any combination thereof. In a specific form, the variant BBB crossing domain has at least 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1. In a specific form, the variant BBB crossing domain has at least 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 2. A BBB crossing polypeptide variant is considered "functional" if it maintains the ability to cross the BBB in a subject (e.g., a healthy BBB in a subject) without being toxic to the subject.

[0100] In some forms, the BBB-crossing peptide specifically binds to low-density lipoprotein receptor-related protein 1 (LRP1) on the cell surface to trigger transport and / or entry across the BBB. The brain specificity of the BBB-crossing peptide may be due to the relative overexpression of LRP-1 in brain microvascular endothelial cells, or may involve another receptor that works in concert with LRP1. Therefore, in some forms, a "functional" BBB-crossing peptide is a peptide that binds to the LRP1 receptor in a manner sufficient to transport the peptide across the BBB. For example, in some forms, a BBB-crossing domain is or comprises a polypeptide having a sequence of about 5 to about 25 consecutive amino acids, the sequence including any one or more of SEQ ID NOs: 1, 3 to 10, which selectively binds to low-density lipoprotein receptor-related protein 1 (LRP1) on the cell surface to trigger transport and / or entry across the BBB.

[0101] In other forms, the BBB-crossing peptide specifically binds to the striated protein to induce transport and / or entry across the BBB. The striated protein itself binds to cell surface receptors, including apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR), which are mainly expressed on the cell membrane of cortical neurons. Therefore, in some forms, a "functional" BBB-crossing peptide is a peptide that binds to the striated protein in a manner that maintains the natural interaction of the striated protein with VLDLR and / or ApoER2, which is sufficient to transport the peptide across the BBB. For example, in some forms, the BBB-crossing domain is or comprises a polypeptide having a sequence of about 5 to about 25 consecutive amino acids, the sequence including any one or more of SEQ ID NOs 2, 11 to 20, 22 to 23, which selectively binds to the striated protein and enables striated protein receptor-mediated transport across the cell surface to induce transport and / or entry across the BBB.

[0102] In some forms, BBB-crossing peptides are identified using restricted library screening, e.g., based on accumulation in the brain. An exemplary restricted library screen is shown in Example 1, using peptides from the restricted library XRRXIXRAXLAXXX (wherein X is a random amino acid; SEQ ID NO: 76; based on the Cerepep peptide), ranked by high-throughput DNA sequencing-based characterization in the brain (ranked based on the ratio of round 2 to round 1).

[0103] In some forms, the BBB-crossing peptide is or comprises an amino acid sequence of one or more of the following:

[0104] NRRVIDRAALASL(SEQ ID NO:77);

[0105] SRRVISRAGLADNL (SEQ ID NO:78); PRRVIGRAGLASTA (SEQ ID NO:79); GRRVIGRASLAPDS (SEQ ID NO:80); GRRIIERAALALED (SEQ ID NO:81); NO:84); ARRIINRAILASDP (SEQ ID NO:85); PRRIITRATLAPPV (SEQ ID NO:86); TRRVIDRAGLANEK (SEQ ID NO:87); ARRVISRAGLAQDT (SEQ ID NO:88); ARRIIPRAPLADR (SEQ ID NO:89); ARRVISRAELARNG (SEQ ID NO:90);ARRTISRAALAQ(SEQ ID NO:91);QRRVIPRAKLALEE(SEQ ID NO:92); ARRVISRANLANPT (SEQ ID NO:93); NRRVIPRAGLAENQ (SEQ ID NO:94); GRRVIGRASLAQDE (SEQ ID NO:95); and NRRVIPRAGLASND (SEQ ID NO:96),

[0106] Or a functional fragment, variant or derivative thereof of any one of SEQ ID NOs: 77 to 96.

[0107] 2. BBB-crossing domain fusion peptide

[0108] Fusion proteins and polypeptides are described that comprise one or more additional polypeptide sequences fused to one or more BBB-crossing domain polypeptides.

[0109] The term "BBB crossing domain" is used only in the context of a fusion peptide comprising one or more BBB crossing domains, and refers to a component of a fusion peptide comprising a BBB crossing domain. In some forms, the BBB crossing domain of the fusion peptide comprises 5 to 25 amino acids of any one or more of SEQ ID NOs: 1 to 20, 22 to 23. Thus, in some forms, the BBB crossing domain of the fusion peptide comprises all amino acids of any one or more of SEQ ID NOs: 1 to 20, 22 to 23. Thus, in some forms, the BBB crossing domain fusion peptide comprises 5 to 25 amino acids of any one or more of SEQ ID NOs: 1 to 20, 22 to 23, contiguous with one or more additional heterologous polypeptide sequences.

[0110] The term "additional heterologous polypeptide sequence" refers to a heterologous sequence fused directly or indirectly to a BBB-crossing domain. Typically, the additional heterologous polypeptide sequence does not cross the BBB in the absence of a BBB-crossing domain. Thus, the additional heterologous polypeptide sequence of the BBB-crossing domain fusion peptide does not comprise 5 to 25 amino acids of any one or more of SEQ ID NOs: 1 to 20, 22 to 23. The length of the additional heterologous polypeptide sequence is typically two to two thousand consecutive amino acids, such as 5, 10, 20, 50, 100, 150, 250, 500, 600, 750, 1000 or 1,500 amino acids, or more than 1,500 amino acids.

[0111] In some forms, the BBB-crossing domain fusion protein comprises one or more additional heterologous polypeptide sequences fused to the amino (N) or carboxyl (C) terminus of the BBB-crossing domain. An exemplary schematic diagram of the domain structure of the fusion protein comprises:

[0112] N-[BBB-crossing domain]-[heterologous polypeptide]-C; or

[0113] N-[heterologous polypeptide]-[BBB-crossing domain]-C; or

[0114] N-[heterologous polypeptide]-[BBB-crossing domain]-[heterologous polypeptide]-C, wherein "N" and "C" refer to the amino (NH2) and carboxyl (COOH) termini, respectively.

[0115] The number of functional domains of the BBB-crossing domain can be varied depending on the requirements of the fusion peptide.

[0116] For example, the domain structure of the fusion protein may include:

[0117] N-[BBB-crossing domain]X-[heterologous polypeptide]YC; or

[0118] N-[heterologous polypeptide]X-[BBB-passing domain]YC; or

[0119] N-[heterologous polypeptide]X-[BBB-passing domain]Y-[heterologous polypeptide]ZC, wherein "N" and "C" refer to the amino (NH2) and carboxyl (COOH) termini, respectively, and wherein X, Y and Z are each independently an integer from 1 to 10.

[0120] In exemplary forms, X and Y and Z are independently 1, 2 or 3.

[0121] Exemplary heterologous polypeptides include any known protein or fragment thereof, such as enzymes, immunoglobulins, cell surface receptors, viral capsids, immune receptors, membrane proteins, etc. In some forms, the heterologous polypeptide is an active agent or forms an active agent, such as a therapeutic agent, a diagnostic agent, or a prophylactic agent. Therefore, in some forms, the heterologous polypeptide of the BBB crossing domain is a cargo molecule.

[0122] B. BBB-crossing polypeptide conjugates

[0123] Conjugates of BBB-crossing polypeptides complexed or conjugated to one or more additional molecules are described.

[0124] Typically, the conjugate includes one or more additional molecules as carrier molecules. The carrier molecule includes an active agent for delivery across the BBB. For example, in some forms, one or more carrier molecules are therapeutic molecules, diagnostic molecules or preventive molecules for treating or diagnosing one or more diseases or conditions in the brain or CNS of a subject. Typically, one or more agents are covalently linked to one or more groups on the BBB-crossing polypeptide. In some forms, the BBB-crossing polypeptide conjugate includes one or more therapeutic agents, preventive agents or diagnostic agents that are conjugated or compounded with the BBB-crossing polypeptide through one or more connecting parts. In other forms, the connecting part incorporates one or more spacer parts or is conjugated with one or more spacer parts. The connection and / or spacer part can be cleavable, for example, by being exposed to the intracellular compartment of the target cell in vivo. Therapeutic agents, preventive agents or diagnostic agents and / or targeting moieties can be covalently linked or dispersed or encapsulated intramolecularly.

[0125] 1. Carrier molecules

[0126] A conjugate of a BBB-crossing polypeptide and one or more carrier molecules for delivery across the BBB to the brain or CNS. The carrier molecule typically comprises one or more therapeutic, preventive or diagnostic agents. Typically, the carrier molecule does not cross the BBB in the absence of a BBB-crossing polypeptide.

[0127] Exemplary cargo molecules are tethered, complexed or otherwise conjugated to BBB-crossing polypeptides. In some forms, two, three, four or more cargo molecules are connected and / or conjugated to BBB-crossing polypeptides. Two or more different cargo molecules may be loaded into the same BBB-crossing polypeptide or different BBB-crossing polypeptides, connected to their surfaces or conjugated to them. For example, in some forms, the composition comprises two or more different types of BBB-crossing polypeptide conjugates having the same or different cargo molecules associated therewith. In some forms, additional cargo molecules are co-administered to the subject without being loaded into the disclosed BBB-crossing polypeptide conjugates, connected to their surfaces and / or encapsulated therein, and may be, for example, free or soluble, or a different carrier or dosage form. For example, such a cargo molecule may be a free or soluble active agent, or an active agent of a different carrier or dosage form, but still a part of the same pharmaceutical composition as the nanocarrier composition.

[0128] The compositions disclosed herein generally include one or more therapeutic, preventive or diagnostic active agents loaded onto, attached to, compounded and / or conjugated to a BBB-crossing polypeptide conjugate. In some forms, two, three, four or more active agents are loaded onto, attached to, compounded and / or conjugated to a BBB-crossing polypeptide conjugate. Two or more agents may be loaded onto, attached to, compounded and / or conjugated to a BBB-crossing polypeptide conjugate. In some forms, the composition comprises two or more different types of BBB-crossing polypeptide conjugates having the same or different active agents associated therewith. In some forms, additional active agents are co-administered to a subject without being loaded into, attached to, and / or encapsulated in the disclosed BBB-crossing polypeptide conjugate, and may be, for example, free or soluble, or in different carriers or in different dosage forms. For example, such an active agent may be a free or soluble active agent, or an active agent of a different carrier or dosage form, but still a part of the same pharmaceutical composition as the BBB-crossing polypeptide conjugate composition.

[0129] The activating agent can be a small molecule activating agent or a biomacromolecule, such as a protein, a polypeptide or a nucleic acid. In some forms, the nucleic acid is an expression vector encoding a protein or a functional nucleic acid. In some forms, the vector is suitable for integration into the cell genome or for extrachromosomal expression. In other forms, the nucleic acid is a functional nucleic acid. Suitable small molecule activating agents include organic compounds and organometallic compounds. Small molecule activating agents can be hydrophilic, hydrophobic or amphipathic compounds. The activating agent can be a therapeutic agent, a nutrient, a diagnostic agent or a preventive agent.

[0130] Exemplary active agents include, but are not limited to, chemotherapeutic agents, neurological agents, tumor antigens, CD4+ T cell epitopes, cytokines, imaging agents, radionuclides, small molecule signal transduction inhibitors, photothermal antennae, immune danger signaling molecules, other immunotherapies, enzymes, antibiotics, antiviral agents, antiparasitic (helminth, protozoan) agents, growth factors, growth inhibitory agents, hormones, hormone antagonists, antibodies and biologically active fragments thereof (including humanized single chain and chimeric antibodies), antigens and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatory agents, immunomodulators (including ligands that bind to Toll-like receptors for activating the innate immune system (including but not limited to CpG oligonucleotides), agents that mobilize and optimize the adaptive immune system The invention also includes molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells and helper T cells, and molecules that inactivate or downregulate inhibitory or regulatory T cells), agents that promote the uptake of nanocarriers into cells (including dendritic cells and other antigen presenting cells), nutraceuticals such as vitamins, oligonucleotide drugs (including DNA, RNA, antisense, aptamers, small interfering RNA, ribozymes, external guide sequences of ribonuclease P, and triplex forming agents) and other gene modifying agents (such as ribozymes, CRISPR / Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs)).

[0131] Exemplary diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast agents.

[0132] The optimal drug load will necessarily depend on many factors, including the choice of drug, the structure and size of the BBB-crossing polypeptide, and the tissue to be treated. In some forms, one or more therapeutic agents, prophylactic agents, or diagnostic agents are encapsulated, associated with, and / or conjugated to the following concentrations of BBB-crossing polypeptides: about 50% to about 95%, including endpoints; preferably about 50% to about 80%, including endpoints; about 40% to about 70%, including endpoints; about 50% to about 80%, including endpoints; about 1% to about 20%, including endpoints; about 1% to about 5%, including endpoints; about 3% to about 20% by weight, including endpoints; and about 3% to about 10% by weight, including endpoints. However, the optimal drug load for any given drug, BBB-crossing polypeptide, and target site can be determined by conventional methods, such as those described.

[0133] a. Therapeutic agents

[0134] In some forms, the carrier molecule comprises one or more therapeutic agents. Exemplary therapeutic agents that can be conjugated or complexed with the BBB-crossing peptide as a carrier molecule include anti-cancer molecules, such as monoclonal antibodies (mAbs) Herceptin; neuroactive agents, such as neuroprotective growth factors and interfering peptides; and immunomodulators, such as anti-inflammatory agents.

[0135] In some forms, the therapeutic agent is a monoclonal antibody or an antigen-binding fragment of mAb that is specific for one or more molecular targets associated with a disease or disorder. In other forms, the therapeutic agent is a small molecule. In other forms, the agent is a designer interference peptide (iPep). Ipep is a natural or synthetic agent that is produced to block selective interactions between protein partners that are difficult to target when used with conventional small molecule chemicals or large biological agents. Protein-protein interaction (PPI) is a promising therapeutic target, and in some cases, interference peptides (i.e., natural or synthetic peptides that can interfere with PPI) are more suitable than small molecules for interfering with large surfaces associated with PPI. In some forms, the therapeutic agent is a neurotrophic factor, such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor (GDNF), and nerve growth factor (NGF).

[0136] Supramolecular agents such as anticancer agents, neuroactive agents, and immunomodulators loaded into biological and synthetic nanoassemblies are also described. Carriers and cells comprising the conjugates are also described. Exemplary cells include CAR-T cells.

[0137] (a) Chemotherapeutic agents

[0138] In certain forms, the cargo molecule comprises one or more anticancer agents, such as cytotoxic agents (e.g., the potent cytotoxic drug monomethyl auristatin A, paclitaxel), signaling pathway modulators, antibodies and fragments thereof (e.g., HER2 and immune checkpoint inhibitor antibodies), proteins / protein fragments / peptides (e.g., interfering peptides, pro-apoptotic peptides, secondary targeting peptides for targets behind the BBB, cytokines and interleukins), polysaccharides, photosensitizers (e.g., verteporfin), haptens used as immunotherapy targets (FAM / FITC), nucleic acids (RNA and DNA), and therapeutic radioisotopes.

[0139] Representative anticancer agents include, but are not limited to, alkylating agents (e.g., cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (e.g., fluorouracil (5-FU), gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and floxuridine), antimitotic agents (including taxanes such as paclitaxel and decetaxel, and vinca alkaloids such as vincristine), , vinblastine, vinorelbine and vindesine), anthracyclines (including doxorubicin, daunorubicin, valrubicin, idarubicin and epirubicin, and actinomycins such as dactinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin and bleomycin), topoisomerase inhibitors (including camptothecins such as camptothecin, irinotecan and topotecan, and derivatives of epipodophyllotoxins such as amsacrine, etoposide, etoposide phosphate and teniposide), antibodies against vascular endothelial growth factor (VEGF) such as bevacizumab Other anti-VEGF compounds; thalidomide and its derivatives such as lenalidomide Endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors such as sunitinib Tyrosine kinase inhibitors such as sorafenib Erlotinib Pazopanib, axitinib, and lapatinib; inhibitors of transforming growth factor-alpha or transforming growth factor-beta, and antibodies against the epidermal growth factor receptor such as panitumumab and cetuximab

[0140] In some forms, particularly those used to treat cancer, one or more active agents may be chemotherapeutic agents with immune signaling properties. In some forms, the agent is a Herceptin monoclonal antibody (mAb). Herceptin is a mAb specifically designed to target the HER2 receptor, which is a transmembrane receptor on both normal cells and HER2+ tumor cells. HER2 plays an important role in the signaling network that drives cell proliferation.

[0141] (b) Neuroactive Agents

[0142] In some forms, the active agent is for neurodegeneration or for improving or enhancing conventional treatments for neuroprotection. Exemplary neuroprotectants are known in the art, including, for example, glutamate antagonists, antioxidants, and NMDA receptor stimulants. In some forms, the carrier molecules used to treat neurodegenerative diseases include neurostimulatory growth factors (CDNF, GDNF), therapeutic antibodies and interfering peptides, and neuropeptides, such as galanin and neurotensin. Other neuroprotectants and treatments include caspase inhibitors, nutritional factors, anti-protein aggregation agents, therapeutic hypothermia and erythropoietin. Amantadine and anticholinergics are used to treat motor symptoms, clozapine is used to treat psychosis, and cholinesterase inhibitors are used to treat dementia. Treatment strategies may also include the use of modafinil.

[0143] For subjects with Huntington's disease, dopamine blockers are used to help reduce abnormal behaviors and movements, and drugs such as amantadine and tetrabenazine are used to control movements, etc. Drugs that help reduce chorea include tranquilizers and benzodiazepines. Class. Compounds such as amantadine or remacemide have shown preliminary positive results. Hypokinesia and rigidity, especially in adolescent cases, can be treated with antiparkinsonian drugs, and myoclonic hyperkinesia can be treated with valproic acid. Psychiatric symptoms can be treated with drugs similar to those used in the general population. Selective serotonin reuptake inhibitors and mirtazapine are recommended for depression, while atypical antipsychotics are recommended for psychosis and behavioral problems.

[0144] Treatments for Parkinson's disease include, but are not limited to, levodopa (often in combination with a dopa decarboxylase inhibitor or COMT inhibitor), dopamine agonists, and MAO-B inhibitors.

[0145] The only compound that yielded borderline significance with respect to survival time in subjects with ALS was riluzole. (2-amino-6-(trifluoromethoxy)benzothiazole), an antiexciyotoxin. Other drugs and interventions, most used off-label, can reduce symptoms caused by ALS. Some treatments can improve quality of life and some have been shown to prolong life. Common ALS-related treatments are reviewed in Gordon, Aging and Disease, 4(5):295-310 (2013), which is specifically incorporated herein by reference in its entirety. Exemplary ALS treatments and interventions are also discussed in Gordon, Aging and Disease, 4(5):295-310 (2013) and are listed in the tables provided herein.

[0146] Many other agents have been tested in one or more clinical trials with efficacy ranging from ineffective to promising. Exemplary agents are reviewed in Carlesi, et al., Archives Italiennes de Biologie, 149:151-167 (2011) and include, for example, agents that reduce excitotoxicity, such as talampanel (8-methyl-7H-1,3-dioxol-(2,3)benzodiazepine agents that reduce oxidative stress, such as coenzyme Q10, manganoporphyrins, KNS-760704 [(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine dihydrochloride, RPPX], and edaravone (3-methyl-1-phenyl-2-pyrazol-5-one, MCI-186); agents that reduce apoptosis, such as histone deacetylase (HDAC) inhibitors including valproic acid, TCH346 (dibenzo(b,f) agents that reduce neuroinflammation, such as thalidomide and celastol; neurotropic agents such as insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF); heat shock protein inducers such as arimoclomol; or autophagy inducers such as rapamycin or lithium.

[0147] Exemplary neurological drugs include, but are not limited to (Fentanyl), (aspirin / extended-release dipyridamole), (naratriptan), (Fampridine), (Cyclobenzaprine hydrochloride extended release), (Apomorphine hydrochloride), (eslicarbazepine acetate), (donepezil hydrochloride), aspirin, (Morphine sulfate), (interferon beta 1-A), (almotriptan malate), (Octyne), (rufinamide), (Suvoreshen), (onabotulinumtoxinA) (Bromfenac), (Buprenorphine), (Diclofenac Potassium), (Carboglutamate), (carbamazepine), (synthetic conjugated estrogens, A), (tadalafil), (clonazepam), (Entacapone), (Glatiramer acetate), (glycopyrrolate), (Divalproex sodium), (Divalproex sodium), DEPAKOTE (Divalproex sodium), (Carbidopa and Levodopa), (difluprednate), (Diclofenac Sodium), (Zolpidem tartrate), (Apixaban), (morphine sulfate and naltrexone hydrochloride), (Hydromorphone hydrochloride), (rivastigmine tartrate), (rivastigmine tartrate), (Bupivacaine liposome injectable suspension), (interferon beta-1b), (Levomilnacipran), (dextrorotatory methylphenidate hydrochloride), (frovatriptan succinate), (Perampanel), (zinc acetate), (gabapentin), (Tasimeteon), (Gabapentin enacarbil), (Gabapentin enacarbil), (Sumatriptan), (Sumatriptan), (Zolpidem tartrate sublingual tablets), (Guanfacine extended-release), (Palipidone), (iontocaine), (Morphine sulfate), (clonidine hydrochloride), (Kepla), (lamotrigine), (Fentanyl citrate), (Alemtuzumab), (vardenafil), (eszopiclone), LUPRON (Leuprolide acetate), (fospropofol disodium), (pregabalin), (Rizatriptan benzoate), MERREM (meropenem), METADATE (methylphenidate hydrochloride), (dihydroergotamine), (Pramipexole), (naloxetine), (botulinum toxin B (rimabotulinumtoxinB)), (naltrexone hydrochloride), (Memantine hydrochloride), (Memantine hydrochloride extended release + donepezil hydrochloride), (Rotigotine transdermal system), (Rotigotine), (gabapentin), (Hydrocodone Bitartrate / Acetaminophen 10mg / 325mg), (droxidopa), (mitoxantrone hydrochloride), (Tapentadol), (dextromethorphan hydrobromide and quinidine sulfate), (Armodafinil), (Nimodipine), (clobazam), (Fentanyl buccal agent), (oxycodone hydrochloride), OXTELLAR (Oxcarbazepine extended release), (oxycodone), (oxycodone / aspirin), (oxycodone and acetaminophen), (peginterferon beta-1a), (mibefradil), (Izogabin), (Laixi Jue Nansam), QUDEXY (topiramate), QUILLIVANT (methylphenidate hydrochloride), (capsaicin), (interferon beta-1a), (Dexfenfluramine hydrochloride), (eletriptan hydrobromide), (Galantamine hydrobromide), (ropinirole hydrochloride), (riluzole), (Ramelteon), (Carbidopa and Levodopa), (vigabatrin), (Si Li Jilin), (Duoluping), (Zaleplon), (ketorolac tromethamine), (Valproic acid delayed release), (atomoxetine hydrochloride), (fentanyl sublingual spray), TARGINIQ (Oxycodone hydrochloride + Naloxone hydrochloride), (Tocapone), (carbamazepine), (Indomethacin), (topiramate), (oxcarbazepine), TROKENDI (topiramate), (natalizumab), (acetaminophen and tramadol hydrochloride), ULTRAJECT (midazolam hydrochloride), (vilazodone hydrochloride), (Lacosamide), (iodixanol), (naltrexone), (velaglucerase alfa), (lisdextroamphetamine dimesylate), XARTEMIS (oxycodone hydrochloride and acetaminophen), (Tetrabenazine), (Rifaximin), (Sodium Oxybate), (Tizanidine hydrochloride), (lidocaine hydrochloride monohydrate), (diclofenac potassium), ZOHYDRO (Hydrocodone Bitartrate), (zolmitriptan), (Zonisamide), (buprenorphine and naloxone).

[0148] Treatments for Lewy body dementia may include, for example, acetylcholinesterase inhibitors, such as tacrine, rivastigmine, galantamine, or donepezil; the N-methyl d-aspartate receptor antagonist memantine hydrochloride; dopaminergic treatments, such as levodopa or selegiline; antipsychotics, such as olanzapine or clozapine; REM disorder treatments, such as clonazepam, melatonin, or quetiapine; antidepressant and antianxiety treatments, such as selective serotonin reuptake inhibitors (citalopram, escitalopram, sertraline, paroxetine, etc.) or serotonin and norepinephrine reuptake inhibitors (venlafaxine, mirtazapine, and bupropion) (see, for example, Macijauskiene, et al., Medicina (Kaunas), 48(1): 1-8 (2012)).

[0149] In some forms, the therapeutic agent is a neuroprotective factor. Neuroprotective factors usually prevent neuronal cell death by intervening and inhibiting the pathogenic processes that cause neuronal dysfunction and death. Tranquilizers, antidepressants, sedatives / hypnotics and antianxiety drugs are common prescriptions for the treatment of neuropsychiatric diseases. Exemplary neuroprotective agents include, for example, glutamate antagonists, antioxidants and NMDA receptor agonists. Other neuroprotective agents and treatments include caspase inhibitors, trophic factors, anti-protein aggregation agents, therapeutic hypothermia and erythropoietin. In some forms, the medicament is a neuroprotective growth factor. Exemplary neuroprotective growth factors include brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF).

[0150] Other common therapeutic, prophylactic, or diagnostic agents used to treat neurological dysfunction include amantadine and anticholinergics for motor symptoms, clozapine for psychosis, cholinesterase inhibitors for dementia, and modafinil for daytime sleepiness.

[0151] (c) Immunomodulators

[0152] The active agent can be an immunomodulatory agent, such as an immune response stimulator or an agent that blocks immunosuppression. In some particularly preferred forms, the active agent targets a tumor checkpoint blockade or a co-stimulatory molecule.

[0153] The immune system is composed of cellular (T cell driven) and humoral (B cell driven) elements. It is generally believed that for cancer, triggering a strong cell-mediated immune response is more effective than activating humoral immunity. Cell-based immunity depends on the interaction and cooperation of a large number of different immune cell types, including antigen-presenting cells (antigen-presenting cell, APC; dendritic cells are important components thereof), cytotoxic T cells, natural killer cells and T helper cells. Therefore, an active agent can be an agent that improves a cellular (T cell driven) immune response, a humoral (B cell driven) immune response or a combination thereof. For example, in some forms, the agent enhances T cell response, improves T cell activity, increases T cell proliferation, reduces T cell inhibitory signals, enhances the production of cytokines, stimulates T cell differentiation or effector function, promotes the survival of T cells or any combination thereof.

[0154] Exemplary immunomodulatory agents include cytokines, xanthines, interleukins, interferons, oligodeoxynucleotides, dextran, growth factors (e.g., TNF, CSF, GM-CSF and G-CSF), hormones such as estrogens (diethylstilbestrol, estradiol), androgens (testosterone, (fluoxymesterone)), progesterone ( (Megestrol acetate)), (medroxyprogesterone acetate) and corticosteroids (prednisone, dexamethasone, hydrocortisone).

[0155] In some forms, the active agent is an inflammatory molecule, such as a cytokine, metalloproteinase, or other molecule, including but not limited to IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.

[0156] (1) Cytokines

[0157] In a preferred form, at least one activating agent is a proinflammatory cytokine. Cytokines generally serve as hormone regulators or signal transduction molecules of nanomolar to picomolar concentrations and contribute to cell signaling. Cytokines can be proteins, peptides or glycoproteins. Exemplary cytokines include but are not limited to interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, etc.), interferons (e.g., interferon-γ), macrophage colony stimulating factor, granulocyte colony stimulating factor, tumor necrosis factor, leukocyte inhibitory factor (Leukocyte Inhibitory Factor, LIF), chemokines, SDF-1α and cytokine CXC family.

[0158] (2) Chemokines

[0159] In another form, at least one activating agent is a proinflammatory chemokine. Chemokines are a family of small cytokines. Their names are derived from their ability to induce directional chemotaxis in nearby responsive cells. Therefore, they are chemotactic cytokines. Proteins are classified as chemokines according to shared structural features such as small size (their sizes are all about 8 to 10 kDa) and four cysteine ​​residues present in conservative positions, which are the key to forming their three-dimensional shape. Chemokines have been divided into four major subfamilies: CXC, CC, CX3C and XC. Chemokines induce cell signaling by combining with transmembrane receptors (i.e., chemokine receptors) connected to G proteins.

[0160] (d) Drugs that block immunosuppression

[0161] At least one active agent can be an agent that blocks, inhibits or reduces immunosuppression or blocks, inhibits or reduces the biological activity of factors that contribute to immunosuppression. It is becoming increasingly clear that tumor-associated immunosuppression not only greatly contributes to tumor progression, but is also one of the main factors limiting the activity of cancer immunotherapy. Antigen-specific T cell tolerance is one of the main tumor escape mechanisms, and the antigen-specific nature of tumor non-responsiveness indicates that tumor-bearing hosts cannot maintain tumor-specific immune responses while still responding to other immune stimuli (Willimsky, et al., Immunol. Rev., 220: 102–12 (2007), Wang, et al. Semin Cancer Biol., 16: 73–9 (2006), Frey, et al., Immunol. Rev., 222: 192–205 (2008), Nagaraj, et al., Clinical Cancer Research, 16 (6): 1812-23 (2010)).

[0162] b. Diagnostic agents

[0163] In some cases, the active agent conjugated to the BBB-crossing polypeptide is a diagnostic agent. When the conjugate comprises a diagnostic agent, the diagnostic agent can be a tumor imaging agent alone or in combination with one or more therapeutic agents. Exemplary reporter agents include fluorescent reporter dyes (including near-infrared dyes), radioactive reporters, and contrast agents for MRI (e.g., iron oxide nanoparticles).

[0164] Some examples of diagnostic agents that can be delivered to the brain through the BBB-crossing polypeptide conjugate include paramagnetic molecules, fluorescent compounds, magnetic molecules and radionuclides, x-ray imaging agents and contrast agents. The BBB-crossing polypeptide conjugate may include an agent that can be used to determine the position of the applied composition. Agents that can be used for this purpose include fluorescent labels, radionuclides and contrast agents.

[0165] Exemplary diagnostic agents include dyes, fluorescent dyes, near-infrared dyes, SPECT imaging agents, PET imaging agents, and radioisotopes. Representative dyes include carbocyanine, indocarbocyanine, oxacarbocyanine, thicarbocyanine and merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY),

[0166] Cy5,Cy5.5,Cy7,VivoTag-680,VivoTag-S680,VivoTag-S750,AlexaFluor660,AlexaFluor680,AlexaFluor 700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.

[0167] Exemplary SPECT or PET imaging agents include chelating agents such as di-ethylenetri-amine penta-acetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diamine dithiol, activated mercaptoacetyl-glycyl-glycyl-gylcine (MAG3), and hydrazidonicotinamide (HYNIC).

[0168] Exemplary isotopes include

[0169] Tc-94m,Tc-99m,In-111,Ga-67,Ga-68,Gd3+,Y-86,Y-90,Lu-177,Re-186,Re-188,Cu-64,Cu -67, Co-55, Co-57, F-18, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, and Dy-166.

[0170] In some forms, the BBB-crossing polypeptide conjugate comprises one or more radioisotopes suitable for positron emission tomography (PET) imaging. Exemplary positron-emitting radioisotopes include carbon-11 (11C), copper-64 (64Cu), nitrogen-13 (13N), oxygen-15 (15O), gallium-68 (68Ga) and fluorine-18 (18F), for example, 2-deoxy-2-18F-fluoro-β-D-glucose (18F-FDG).

[0171] In other forms, a single BBB-crossing polypeptide conjugate composition can simultaneously treat and / or diagnose a disease or condition at one or more locations in the body.

[0172] 2. Linkers and coupling agents

[0173] The conjugate of the complex or conjugation of the BBB-crossing polypeptide and one or more cargo molecules for delivery across the BBB to the brain or CNS may include one or more linkers. As described above, the targeting agent BBB-crossing polypeptide and / or cargo molecule may be directly or indirectly connected via one or more linkers.

[0174] An exemplary coupling agent is biotin. For example, the BBB-crossing polypeptide can be a biotinylated polypeptide. As described in the Examples, when the BBB-crossing polypeptide is biotinylated, a conjugate can be formed by specific interaction with an active agent conjugated to streptavidin.

[0175] The term "linker" as used herein refers to a carbon chain that may contain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and may be as long as

[0176] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 atoms. The joint can be substituted by the following various substituents, including but not limited to hydrogen atoms, alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocycle, aromatic heterocycle, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkyl thioether, thiol and urea group. Those skilled in the art will recognize that each of these groups can be substituted in turn. Some examples of linkers include, but are not limited to, pH-sensitive linkers, protease-cleavable peptide linkers, nuclease-sensitive nucleic acid linkers, lipase-sensitive lipid linkers, glycosidase-sensitive carbohydrate linkers, hypoxia-sensitive linkers, photocleavable linkers, thermolabile linkers, enzyme-cleavable linkers (e.g., esterase-cleavable linkers), ultrasound-sensitive linkers, and x-ray-cleavable linkers.

[0177] In some forms, the linking moiety is designed to be cleaved in vivo. The linking moiety can be designed to be cleaved hydrolytically, enzymatically, or by a combination thereof to provide sustained release of the attached agent in vivo. The composition of the linking moiety and its point of attachment to the agent are selected so that cleavage of the linking moiety releases the therapeutic, prophylactic, or diagnostic agent or a prodrug thereof. The composition of the linking moiety can also be selected in view of the desired release rate of the agent.

[0178] In some forms, the connection of one or more agents occurs through one or more of disulfide, ester, ether, thioester, carbamate, carbonate / ester, hydrazine or amide linkage. In some preferred forms, the connection occurs through a suitable spacer, which provides an ester bond or an amide bond between the agent and the polypeptide according to the desired release kinetics of the agent. In an exemplary form, the linker is a valine citrulline (VCit) dipeptide linker. For example, in some forms, the VCit linker is used as an enzymatically cleavable linker between a cargo molecule and a BBB-traversing polypeptide. In some forms, a composition comprising a VCit linker is internalized by a cell, such as a target cell, and is cut by a tissue protease in the cell, resulting in a traceless release of the cargo molecule.

[0179] 3. Targeting part

[0180] In some forms, the conjugate of the BBB-crossing polypeptide comprises one or more targeting moieties for targeting the conjugate to one or more specific tissues or cells in vivo. The targeting moiety is distinct from the BBB-crossing domain.

[0181] In an exemplary form, one or more targeting moieties target the conjugate to the brain to one or more specific tissues or cells within the brain or CNS. In other forms, one or more targeting moieties target the conjugate to brain epithelial cells and / or to the BBB. As described above, the targeting agent BBB-crossing polypeptide and / or the cargo molecule can be directly or indirectly connected via one or more linkers.

[0182] In some forms, the targeting moiety directs the conjugate to the BBB, or to tissues / cells associated with the BBB. In other forms, the targeting moiety directs the conjugate to a specific location in the brain. For example, in some forms, the targeting moiety directs the conjugate to a specific location after it crosses the BBB.

[0183] Typically, the targeting moiety utilizes surface markers that are specific to the cell group to be targeted. Exemplary targeting elements include proteins, peptides, nucleic acids, lipids, sugars or polysaccharides that bind to one or more targets, and the one or more targets associate with cells or extracellular matrix or specific types of tumors or infected cells. The targeting molecule can be selected according to the desired physical properties, such as suitable affinity and specificity for the target. Exemplary targeting molecules with high specificity and affinity include antibodies or their antigen-binding fragments. The specificity of the antibody is particularly helpful for actively targeting the composition described herein. It will be understood by those skilled in the art that any antibody that specifically binds to the desired target / antigen can be used according to the disclosed composition. Therefore, antibodies that specifically recognize one or more types of cells, tissues, organs or microenvironments are known in the art, and their use in preferentially targeting BBB-crossing polypeptide conjugates is considered herein. Therefore, in some forms, the composition of the BBB-crossing polypeptide comprises one or more antibodies or antigen-binding fragments that are specific to the epitope. The epitope can be a linear epitope. The epitope can be specific to a cell type or can be expressed by a variety of different cell types. In other forms, antibodies or their antigen-binding fragments can bind to conformational epitopes comprising 3D surface features, shapes or tertiary structures at the surface of target cells. In an exemplary form, the targeting moiety selectively binds to BBB-related cells, tissues or structures. In some forms, the targeting moiety binds to cell surface receptors such as lipoprotein receptor-related protein 1 (LRP1) or apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR). In other forms, the targeting moiety selectively binds to proteins such as skeletal protein.

[0184] In some forms, the targeting moiety is a polypeptide. For example, in some forms, the BBB described herein crosses the polypeptide and is conjugated to a polypeptide-based ligand, which helps them preferentially target one or more types of cells, tissues, organs or microenvironments. Some non-limiting examples of polypeptide-based ligands include homing peptides, protein domains and antibodies (including antibody fragments and derivatives, such as Fab, Fab', F(ab')2, Fv fragments, double antibodies, affibodies, nanobodies, linear antibodies and single-chain antibody molecules). Short homing peptides that are smaller than antibodies but larger than small molecules provide additional targeting options. In some forms, the targeting moiety is an amino acid sequence that is continuous with the amino acid sequence of the BBB-crossing domain. Therefore, in some forms, the BBB-crossing polypeptide comprises a targeting motif sequence at the carboxyl or amino terminal of the polypeptide position relative to the BBB.

[0185] In other forms, the targeting molecule is an aptamer. An aptamer is a short single-stranded DNA or RNA oligonucleotide (6 to 26 kDa) folded into a clearly defined 3D structure, which identifies a variety of biomolecules including transmembrane proteins, sugars and nucleic acids with high affinity and specificity (Yu B, et al., Mol Membr Biol., 27 (7): 286-98 (2010)). The high sequence and conformational diversity of the initial aptamer library (not yet selected for the target) makes it highly possible to find that the target binds to the aptamer. The selection of aptamers that can bind to the target of interest is referred to as "systematic evolution of ligands by exponential enrichment" (Systematic Evolution of Ligands by EXponential enrichment, SELEX). SELEX includes iterative rounds of target binding, separation of binding sequences from unbound sequences and amplification of the binding sequences of enrichment. Given their unique conformation with ligand binding characteristics, typical non-immunogenicity and non-toxicity, and ability to be modified for stability in circulation, aptamers are suitable for active targeting of the BBB-crossing polypeptides and conjugates thereof described herein.

[0186] In some forms, the aptamer has nuclease resistance. In some forms, the aptamer is an RNA aptamer modified by 2'- (for example, 2'-fluorine and 2'-O-methyl). In some forms, the aptamer (for example, RNA aptamer) shows fluorescence after binding to small molecules. For example, Spinach and Spinach2 aptamers bind and activate the fluorescence of fluorophores similar to those found in green fluorescent protein, and Broccoli is a 49nt long aptamer (Filonov GS, et al., J Am Chem Soc., 136 (46): 16299-308 (2014)) that shows bright green fluorescence after binding to DFHBI or DFHBI-1T.

[0187] In some forms, the targeting moiety is directed to cells of the nervous system (including the brain and peripheral nervous system), or to the blood-brain barrier itself. Cells in the brain include several types and states and possess unique cell surface molecules that are specific to the type. In addition, cell types and states can be further characterized and grouped by the presentation of common cell surface molecules.

[0188] The targeting moiety may be directed to a specific neurotransmitter receptor expressed on the surface of cells of the nervous system. The distribution of neurotransmitter receptors is well known in the art, and the skilled person may direct the composition by using a neurotransmitter receptor-specific antibody as a targeting signal. In addition, in view of the tropism of a neurotransmitter for its receptor, in one form, the targeting signal comprises a neurotransmitter or a ligand that can specifically bind to a neurotransmitter receptor.

[0189] The targeting moiety can be specific to cells of the nervous system, and the cells of the nervous system may include astrocytes, microglia, neurons such as mature neurons, oligodendrocytes and Schwann cells. These cells can be further divided according to their function, position, shape, neurotransmitter category and pathological state. The cells of the nervous system can also be identified by their differentiation state, such as stem cells. Exemplary markers specific to these cell types and states are well known in the art, and include but are not limited to CD133 and neurospheres. Specific preferred brain targeting moieties include, but are not limited to, the peptide mHph2 and the peptide chlorotoxin (CTX), the insulin receptor (which can be targeted by, for example, the 83-14 antibody or insulin), the EGF receptor (which can be targeted by, for example, cetuximab and its fragments (e.g., Fab')), the low-density lipoprotein receptor (which can be targeted by, for example, apolipoproteins (e.g., ApoA, ApoE, etc.)), the thiamine receptor (which can be targeted by, for example, thiamine), the transferrin receptor (which can be targeted by, for example, transferrin and OX26 antibodies and 8D3 antibodies), the folate receptor (which can be targeted by, for example, folate and its derivatives ), glycoside receptors (which can be targeted by, for example, glycosides), lactoferrin receptors (which can be targeted by, for example, lactoferrin), insulin-like growth factor receptors (IGF1R & IGF2R) (which can be targeted by, for example, insulin-like growth factor 1 & 2 (IGF-1 & IGF-2)), leptin receptors (LEPR) (which can be targeted by, for example, leptin), Fc-like growth factor receptors (FCGRT) (which can be targeted by, for example, IgG), scavenger receptor type B1 (SCARB1) (which can be targeted by, for example, (modified lipoproteins, such as acetylated low-density lipoprotein (LDL))), and other targets and targeting moieties discussed in Alam, et al., European Journal of Pharmaceutical Sciences, 40:385-403 (2010), and Wong, et al., Adv Drug Deliv Rev., 64(7):686-700 (2012)).

[0190] In other forms, the targeting moiety and marker are related to or specific for the condition being treated. For example, in some forms, the targeting moiety targets: a cancer marker; or cancer-associated stromal cells, such as M2-biased macrophages (discussed in more detail below); demyelinating diseases such as multiple sclerosis (tPA and other extracellular proteases); traumatic brain injury (e.g., the lectican family of chondroitin sulfate proteoglycans); stroke (e.g., MMP2, thrombin); epilepsy (e.g., MMP2); injury; or a neurological or neurodegenerative disease or condition.

[0191] 4. Carrier

[0192] The conjugate of the BBB-crossing polypeptide may include one or more carriers. For example, in some forms, the conjugate includes one or more BBB-crossing polypeptides and one or more active agents optionally encapsulated in a carrier, associated with a carrier or otherwise combined with a carrier. In some forms, the carrier is an inorganic carrier, such as a metal particle. Exemplary metal particles include silver or gold particles. In other forms, the carrier is an organic carrier, such as a nanogel, a nanolipid gel, a polymer particle, a lipid particle, a mixed lipid-polymer particle, an inorganic particle, a liposome (e.g., a nanoliposome), a nanosuspension, a nanoemulsion, a multilamellar vesicle, a nanofiber, a nanorobot, a solid lipid nanoparticle (solid lipidnanoparticle, SLN), a nanostructured lipid carrier (nanostructured lipid carrier, NLC) and a lipid drug conjugate (lipid drug conjugate, LDC). In some forms, the granular nanocarrier is a nanoscale composition, for example, 10nm up to but not including about 1 micron, more preferably up to about 500nm, as discussed below. However, it is understood that in some forms, and for some uses, the particles can be smaller or larger (e.g., microparticles, etc.). It is understood that in some forms, and for some uses, the conjugate / carrier composition can have nanometer-scale or micrometer-scale dimensions. Such compositions can be referred to as nanoparticles or microparticles.

[0193] In some preferred forms of treating brain diseases and disorders, it is desirable that the conjugate / carrier is a size suitable for passing through the blood-brain barrier. Therefore, in some forms, the conjugate / carrier is about 25nm to about 500nm, including endpoints, more preferably about 50nm to about 350nm, including endpoints, most preferably about 70nm to about 300nm, including endpoints. In some forms, the carrier serves as a drug carrier (e.g., a submicroscopic colloidal system with a matrix system in which a drug is dispersed, such as a nanosphere) or a nanocapsule (e.g., a reservoir surrounded therein by a single polymer film limitation).

[0194] The carrier may comprise one or more lipids or amphipathic compounds. For example, the particle may be a liposome, a lipid micelle, a solid lipid particle or a lipid-stabilized polymer particle. The lipid particle may be made of a mixture of one lipid or different lipids. The lipid particle may be formed of one or more lipids, which may be neutral, anionic or cationic at physiological pH. The lipid particle is preferably made of one or more biocompatible lipids. The lipid particle may be formed by a combination of more than one lipid, for example, at physiological pH, a charged lipid may be combined with a nonionic or uncharged lipid.

[0195] a. Moieties that enhance stability and / or half-life

[0196] In some forms, the conjugate of the BBB-crossing peptide associated with the carrier molecule includes one or more molecules for enhancing the stability and / or half-life of the conjugate in vivo. Particle size, size distribution, shape and surface characteristics are important features of BBB-crossing peptide conjugates / carrier molecules. In some forms, these features affect the distribution in vivo, biological fate (fate), toxicity, removal, uptake and targeting ability of BBB-crossing peptide conjugates / carrier molecules to serve as the delivery system of the carrier molecule. In addition, they can affect the stability of the carrier (e.g., drug) loading and release and the composition (Singh R and Lillard JW Jr. Exp Mol Pathol. 86 (3): 215-23 (2009); Bamrungsap S, et al., Nanomedicine. 7 (8): 1253-1271 (2012)). Thus, in some forms, the size, size distribution, shape, geometry, surface characteristics (e.g., surface charge, surface chemistry) of the BBB-crossing peptide conjugate / carrier molecule is modified and / or selected to enhance the stability and / or half-life of the composition in vivo.

[0197] In addition to size and shape, the surface characteristics of the composition can also determine their lifespan during circulation in the bloodstream. A major discovery is that it is found that a composition (e.g., a BBB-crossing peptide associated with a carrier molecule) coated with a hydrophilic polymer molecule such as polyethylene glycol (PEG) can resist serum protein adsorption and extend the systemic circulation of the particles. A variety of PEG variants and other hydrophilic polymers have been tested for improved circulation. The surface charge on the particles also affects other functions, such as internalization by macrophages. It has been shown that compared with neutral or negatively charged particles, positively charged particles show a higher degree of internalization by macrophages and dendritic cells, but the surface charge effect can also be cell type dependent. Therefore, in some forms, the BBB-crossing peptide associated with a carrier molecule is conjugated or compounded with one or more molecules having a negative charge to provide a molecule with a negative total surface charge in vivo. Suitable molecules include polyethylene glycol (PEG) molecules, lipids, polar groups, charged groups, amphipathic groups, and albumin binding molecules.

[0198] In order to enhance the half-life of the disclosed BBB-crossing peptide conjugate / carrier molecule, it is desirable to minimize opsonization and prolong in vivo circulation. For example, this can be achieved by coating the BBB-crossing peptide associated with the carrier molecule with a hydrophilic polymer / surfactant, or by formulating the BBB-crossing peptide with a biodegradable copolymer such as PEG, polyethylene oxide, dextran, poloxamer, poloxamine, and polysorbate 80 (Tween 80) having hydrophilic characteristics. Studies have shown that PEG on the surface of nanoparticles prevents opsonization by complement and other serum factors. PEG molecules with brush-like and intermediate configurations reduce phagocytosis and complement activation, while the surface composed of PEG with a mushroom-like structure is a potent complement activator and is conducive to phagocytosis (Singh R and Lillard JW Jr. Exp Mol Pathol. 86 (3): 215-23 (2009)).

[0199] In some forms, the BBB-crossing peptide is coated with a hydrophilic layer (e.g., PEG, polyethylene oxide, poloxamer, poloxamine, and polysorbate 80 (Tween 80)) for enhancing the stability and / or half-life of the composition in vivo. PEG-free alternatives such as poly(ethylene oxide) may also be used. Azolines, poly(amino acids), polybetaines, polyglycerols, and polysaccharide derivatives are used to enhance stability and / or half-life (Amoozgar Z, and Yeo Y. Wiley Interdiscip Rev Nanomed Nanobiotechnol., 4(2):219-33 (2012)). In some forms, the BBB-penetrating peptide is coated with a poly(amino acid). The invention relates to polyoxazolines (POZ), poly(amino acids) such as poly(hydroxyethyl l-glutamine) and poly(hydroxyethyl-l-asparagine), N-(2-hydroxypropyl)methacrylamide (HPMA) and its derivatives, polybetaines such as sulfobetaine and carboxybetaine, polyglycerol (also known as polyglycidol), and polysaccharides such as chitosan, dextran, hyaluronic acid, and derivatives of heparin.

[0200] In some forms, the BBB-crossing peptide conjugates / carrier molecules include multiple effector molecules that contribute to their physicochemical properties (e.g., enhanced stability and / or half-life). In some forms, the effector molecules are any of the above molecules that can be used to coat nucleic acid components (e.g., PEG, polyethylene oxide, poloxamer, poloxamine, and polysorbate 80 (Tween 80), polysorbate 80 (Tween 80), polysorbate 100 (Tween 100), polysorbate 2 ... In some forms, the effector molecule is a polyethylene glycol molecule, a lipid, a polar group, a charged group, an amphiphilic group, or an albumin binding molecule.

[0201] III. Pharmaceutical Compositions

[0202] Also disclosed are pharmaceutical compositions comprising conjugates of BBB-crossing polypeptides. The pharmaceutical compositions can be formulated for administration parenterally (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), by drip, or in depo form, in dosage forms suitable for each route of administration.

[0203] A. Formulations for Parenteral Administration

[0204] In some forms, the composition is administered by parenteral injection in the form of an aqueous solution. The formulation can be in the form of a suspension or an emulsion. In general, the pharmaceutical composition provided comprising an effective amount of one or more active agents optionally comprises a pharmaceutically acceptable diluent, a preservative, a solubilizing agent, an emulsifier, an adjuvant and / or a carrier. Such a composition may comprise a diluent such as sterile water, a buffered saline having a variety of buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally, additives such as detergents and solubilizing agents (e.g., 20. 80, also known as polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol). Some examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters (e.g., ethyl oleate). The formulation can be lyophilized and resuspended immediately prior to use. The formulation can be sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0205] B. Formulations for Mucosal Administration

[0206] In some forms, the compositions are formulated for mucosal administration, such as by pulmonary or intranasal delivery.

[0207] These methods of administration can be effectively performed by formulating the composition with a mucosal transport element. When delivered as an aerosol or spray-dried particle having an aerodynamic diameter of less than about 5 microns, the composition can be delivered to the lung while being inhaled and passing through the lung epithelial lining to the bloodstream.

[0208] A variety of mechanical devices designed for pulmonary delivery of therapeutic products may be used, including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which will be familiar to those skilled in the art.

[0209] Formulations for administration to the mucosa will generally be spray-dried drug particles which may be incorporated into tablets, gels, capsules, suspensions or emulsions. Standard pharmaceutical excipients are available from any formulator.

[0210] Mucosal preparations may include one or more agents for enhancing delivery through the nasal mucosa.Agents for enhancing mucosal delivery are known in the art, see, for example, US Patent Application No. 2009 / 0252672 to Eddington and US Patent Application No. 2009 / 0047234 to Touitou.

[0211] Available agents include, but are not limited to, calcium chelators (EDTA), inhibitors of nasal enzymes (boroleucine, aprotinin), inhibitors of mucociliary clearance (preservatives), solubilizers for nasal membranes (cyclodextrins, fatty acids, surfactants), and micelle formers (surfactants such as bile acids, Laureth-9, and taurodehydrofusidic acid (STDHF)). The composition may contain one or more absorption enhancers, including surfactants, fatty acids, and chitosan derivatives that can enhance delivery by regulating tight junctions (TJs) (BJ Aungst, et al., J. Pharm. Sci. 89 (4): 429-442 (2000)). In general, the best absorption enhancer should have the following qualities: its action should be reversible, it should provide rapid permeation enhancement to the cell membranes of the mucosa, and it should be non-cytotoxic and have no harmful and / or irreversible effects at effective concentration levels. Intranasal compositions can be administered using devices known in the art (e.g., nebulizers).

[0212] C. Formulations for enteral administration

[0213] The pharmaceutical composition for oral administration can be liquid or solid.Liquid dosage forms suitable for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to encapsulated or unencapsulated compounds, liquid dosage forms may include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of dehydrated sorbitan, and mixtures thereof.In addition to inert diluents, oral compositions may also include adjuvants, wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and aromatics.

[0214] Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, caplets, dragees, powders, and granules. In such solid dosage forms, the encapsulated or unencapsulated compound is typically mixed with at least one inert pharmaceutically acceptable excipient or carrier (e.g., sodium citrate or dicalcium phosphate) and / or the following: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retardants such as paraffin; f) absorption promoters such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glyceryl monostearate; h) absorbents such as kaolin and bentonite; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0215] Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols, etc. Solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings and other coatings known in the pharmaceutical preparation art that may impart enteric protection or enhance delivery through the GI tract including the intestinal epithelium and mucosa (see Samstein, et al. Biomaterials.. 29(6): 703-8 (2008)).

[0216] IV. How to use

[0217] Methods have been established for delivering conjugated active agents from the circulation to the brain using polypeptides that specifically and selectively cross the healthy blood-brain barrier (BBB).

[0218] As described in the Examples, the BBB crossing peptide domain "SRRVISRAKLAAAL" preferentially homes to the eye / brain / CNS, particularly to neurons within brain compartments expressing the low-density lipoprotein receptor-related protein 1 (LRP1) endocytic cell surface receptor.

[0219] As described in the Examples, the BBB-crossing peptide domain "CVGTNCY" (also referred to as "CVG" peptide) preferentially homes to the eye / brain / CNS, particularly to neurons within brain compartments including the hippocampus, frontal cortex, and cerebellum.

[0220] In some forms, the BBB-crossing polypeptide conjugate crosses the BBB and selectively targets neurons or is enriched in neurons, preferably in the nuclei of damaged neurons / overactive neurons. In other forms, the BBB-crossing polypeptide conjugate accumulates in one or more cell types including oligodendrocytes, microglia, and astrocytes.

[0221] Typically, an effective amount of a BBB-crossing polypeptide conjugate (comprising one or more therapeutic, prophylactic and / or diagnostic agents) is administered to an individual in need thereof. The conjugate may also comprise a targeting agent, but as demonstrated in the Examples, these are not required for delivery to neurons in the brain, spinal cord or other components of the CNS.

[0222] In some forms, the BBB-crossing polypeptide conjugate is capable of releasing a therapeutic agent, a prophylactic agent, or a diagnostic agent intracellularly under conditions found in vivo. The amount of the BBB-crossing polypeptide conjugate administered to a subject is selected to deliver an effective amount to reduce, prevent, or otherwise alleviate one or more clinical or molecular symptoms of the disease or condition to be treated compared to a control (e.g., a subject treated with a therapeutic agent, a prophylactic agent, or a diagnostic agent that does not contain a BBB-crossing polypeptide conjugate).

[0223] In some forms, the methods include the step of selecting a subject who may benefit from treatment with a BBB-crossing polypeptide conjugate composition.

[0224] A. Treatment methods

[0225] The composition is suitable for treating one or more diseases, disorders and injuries in the brain and nervous system, such as cancers and diseases associated with pathological activation of neurons. The composition can also be used to treat neurological diseases and other tissues in which nerves play a role in the disease or disorder. The composition and method are also suitable for preventive use.

[0226] Methods for treating or preventing one or more symptoms of a disease or condition in the brain or CNS of a subject are described. Generally, the methods comprise administering to the subject an effective amount of a BBB-crossing polypeptide conjugate comprising one or more therapeutic agents for treating or preventing one or more symptoms of a disease or condition in the brain or CNS, or a pharmaceutical composition thereof, in an amount effective to prevent or alleviate one or more symptoms of a disease or condition in the brain or CNS of the subject.

[0227] Also provided is the use of a BBB-crossing polypeptide conjugate comprising one or more therapeutic agents in the preparation of a medicament for treating or preventing one or more symptoms of a disease or condition in the brain or CNS of a subject. Also provided is a formulation of a BBB-crossing polypeptide conjugate or a pharmaceutical composition thereof, the formulation of a BBB-crossing polypeptide conjugate or a pharmaceutical composition thereof comprising one or more therapeutic agents for treating or preventing one or more symptoms of a disease or condition in the brain or CNS of a subject, or a pharmaceutical composition thereof, which is used to treat or prevent one or more symptoms of a disease or condition in the brain or CNS of a subject.

[0228] BBB-crossing polypeptide conjugate compositions selectively deliver active agents to the brain to treat the causes and symptoms of many disorders and conditions including neurodevelopmental diseases, neurodegenerative diseases and brain cancer. Therefore, in some forms, the BBB-crossing polypeptide conjugate compositions are administered in dosage units effective to treat or alleviate conditions associated with pathological conditions of neurons. In general, the BBB-crossing polypeptide conjugate compositions specifically deliver agents to treat diseased neurons with minimal toxicity by targeting neurons. Therefore, in some forms, the BBB-crossing polypeptide conjugate compositions are administered in amounts effective to treat pathological conditions mediated by diseased neurons in subjects in need thereof without any associated toxicity.

[0229] Typically, the subject to be treated is a human. In some forms, the subject to be treated is a child or infant. All methods may include the steps of identifying and selecting a subject in need of treatment or a subject who can benefit from the administration of the composition.

[0230] 1. Neurological and neurodegenerative diseases

[0231] BBB-crossing polypeptide conjugate compositions and preparations thereof can be used for diagnosis and / or treatment of one or more neurological diseases and neurodegenerative diseases. The composition and method are particularly suitable for treating one or more neurological diseases or neurodegenerative diseases associated with defective neurons or diseased neurons. In some forms, the disease or condition is selected from but not limited to some mental (e.g., depression, schizophrenia (schizophrenia, SZ), alcohol use disorder and morphine anti-nociceptive tolerance), neurological and neurodegenerative (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis, ALS)) conditions. In one form, the BBB-crossing polypeptide conjugate composition is used to treat Alzheimer's disease (AD) or dementia.

[0232] Neurodegenerative diseases are chronic progressive disorders of the nervous system that affect neurological and behavioral functions and involve biochemical changes, leading to different histopathological and clinical syndromes (Hardy H, et al., Science. 1998; 282: 1075–9). Abnormal proteins that are resistant to cellular degradation mechanisms accumulate within cells. The pattern of neuronal loss is selective, meaning that one group is affected while another group remains intact. Usually, there is no obvious stimulating event for the disease. Diseases typically described as neurodegenerative are Alzheimer's disease, Huntington's disease, and Parkinson's disease.

[0233] BBB-crossing polypeptide conjugate compositions and methods can also be used to deliver agents for the treatment of neurological or neurodegenerative diseases or disorders or central nervous system disorders. In some preferred forms, BBB-crossing polypeptide conjugate compositions and methods are effective in treating and / or alleviating neuroinflammation associated with neurological diseases or disorders or neurodegenerative diseases or disorders or central nervous system disorders.

[0234] In some forms, the methods comprise administering to a subject an effective amount of a BBB-crossing polypeptide conjugate composition to improve cognition or reduce cognitive decline, improve cognitive function or reduce cognitive function decline, improve memory or reduce memory decline, improve learning skill or ability or reduce learning skill or ability decline, or a combination thereof.

[0235] Neurodegeneration refers to the progressive loss of neuronal structure or function, including neuronal death. For example, in some forms, the methods administer a BBB-crossing polypeptide conjugate composition to treat a subject suffering from a disease or condition such as Parkinson's disease (PD) and PD-related disorders, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and other dementias, prion diseases such as Creutzfeldt-Jakob disease, corticobasal degeneration, frontotemporal dementia, HIV-associated cognitive impairment, mild cognitive impairment, motor neuron disease (MND), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), Friedreich's Ataxia, Lewy Body Disease, Alpers' Disease, neuronal ceroid lipofuscinosis, Batten disease, Disease), cerebro-oculofacial syndrome, corticobasal degeneration, Gerstmann-Straussler-Scheinker Disease, Kuru, Leigh's Disease, monosomic amyotrophy, multiple system atrophy, multiple system atrophy with orthostatic hypotension (Shy-Drager Syndrome), multiple sclerosis (MS), Duchenne muscular dystrophy (DMD), neurodegeneration with brain iron deposition, opsoclonus-myoclonus, posterior cortical atrophy, primary progressive aphasia, progressive supranuclear palsy, vascular dementia, progressive multifocal leukoencephalopathy, dementia with Lewy bodies (DLB), lacunar syndrome, hydrocephalus, Wernicke-Korsakoff's syndrome syndrome, postencephalitis dementia, cancer- and chemotherapy-related cognitive impairment and dementia, and depression-induced dementia and pseudodementia.

[0236] In some forms, the disease or condition is spinal muscular atrophy. In such cases, HDAC inhibitors, antisense oligonucleotide (ASO) drugs nusinersen or gene therapy drugs Can be conjugated to a BBB-crossing polypeptide for delivery to neurons or neuronal nuclei to treat spinal muscular atrophy.

[0237] In other forms, the disease or condition is injection-limited amyloidosis, cerebral amyloid angiopathy, myopathy, neuropathy, brain trauma, frontotemporal dementia, Pick's disease, multiple sclerosis, prion disease, type 2 diabetes, fatal familial insomnia, arrhythmia, isolated atrial amyloidosis, atherosclerosis, rheumatoid arthritis, familial amyloid polyneuropathy, hereditary non-neuropathic systemic amyloidosis, Finnish amyloidosis, lattice corneal dystrophy, systemic AL amyloidosis, neuronopathic Gaucher disease or Down syndrome. In some preferred forms, the disease or condition is Alzheimer's disease or dementia.

[0238] Criteria for evaluating improvements in specific neurological factors include methods for evaluating cognitive skills, motor skills, memory, etc., and methods for evaluating physical changes in selected areas of the central nervous system, such as magnetic resonance imaging (MRI) and computed tomography (CT) or other imaging methods. Such evaluation methods are well known in the fields of medicine, neurology, psychology, etc., and can be appropriately selected to diagnose the condition of specific neural damage. In order to evaluate changes in Alzheimer's disease or related neurological changes, a selected evaluation or evaluation test is performed before the start of administration of the BBB-crossing polypeptide conjugate composition. After this initial evaluation, a treatment method for administering the BBB-crossing polypeptide conjugate is started and continued at different time intervals. At a selected time interval after the initial assessment of neurological deficit damage, the same evaluation or evaluation test is used again to re-evaluate changes or improvements in selected neurological criteria.

[0239] a. Alzheimer's disease and dementia

[0240] BBB traverses polypeptide conjugate compositions suitable for reducing or preventing one or more pathological processes related to the occurrence and progress of neurological diseases such as Alzheimer's disease and dementia. Therefore, a method for treating, alleviating and preventing the pathological process related to Alzheimer's disease is provided, the method comprising effectively reducing brain and / or serum exosomes, brain and / or serum ceramide levels, serum anti-ceramide IgG, glial cell activation, total Aβ42 and plaque load, tau phosphorylation / propagation and improving the cognitive amount and dosage regimen of learning tasks (such as fear conditioning learning tasks) in individuals suffering from Alzheimer's disease or dementia. A method for reducing, preventing or reversing learning and / or memory defects in individuals suffering from Alzheimer's disease or dementia is provided.

[0241] In some forms, a BBB-crossing polypeptide conjugate composition is administered to a subject in need thereof in an amount and dosage regimen effective to induce neuroenhancement. Neuroenhancement caused by administration of a BBB-crossing polypeptide conjugate composition includes stimulation or induction of neuromitosis, causing the generation of new neurons, i.e., exhibiting a neurogenic effect, preventing or delaying neuronal loss, including a reduction in the rate of neuronal loss, i.e., exhibiting a neuroprotective effect, or one or more of these modes of action. The term "neuroprotective effect" includes preventing, delaying and / or terminating the deterioration, damage or death of neurons, neurites and neural networks of an individual. Administration of a BBB-crossing polypeptide conjugate composition results in improvement or enhancement of neurological function in an individual suffering from a neurological disease, neurological injury or age-related neuronal decline or damage.

[0242] Neurodeterioration can be the result of any condition that impairs neurological function, which may lead to neurological loss. Neurological function may be subject to, for example, the following impairments: changes in the biochemistry, physiology or anatomy of neurons including their neurites. Neuronal deterioration may include membrane, dendrite or synaptic changes that are harmful to normal neurons. The cause of neuronal deterioration, damage and / or death may be unknown. Alternatively, it may be the result of age, injury and / or disease-related neurological changes that occur in the nervous system of an individual.

[0243] In patients with Alzheimer's disease, neural loss is most significant in the hippocampus, frontal cortex, parietal cortex, anterior temporal cortex, amygdala, and olfactory system. The most significantly affected hippocampal regions include the CA1 region, subiculum, and entorhinal cortex. Memory loss is considered the earliest and most representative cognitive change because it is well known that the hippocampus plays a vital role in memory.

[0244] Nerve loss caused by disease, age-related decline or physical injury leads to neurological diseases and damage. BBB-crossing polypeptide conjugate compositions can offset the harmful effects of nerve loss by promoting the development of new neurons, new neurites and / or neural connections, causing neuroprotection of existing nerve cells, neurites and / or neural connections or one or more of these processes. Therefore, the neuro-enhancing properties of the composition provide an effective strategy to reverse the nerve loss associated with degenerative diseases, aging and physical injury or trauma in general.

[0245] Administration of a BBB-crossing polypeptide conjugate composition to a subject who is experiencing or has experienced neurological loss caused by Alzheimer's disease reduces any one or more symptoms of Alzheimer's disease or related cognitive disorders including dementia. Clinical symptoms of AD or dementia that can be treated, reduced or prevented include mild AD, moderate AD and / or severe AD or clinical symptoms of dementia.

[0246] A person with mild Alzheimer's disease may appear healthy but have increasing difficulty understanding the world around him or her. The person and family usually gradually begin to realize that something is wrong. Some exemplary symptoms of mild Alzheimer's disease / mild dementia include memory loss; poor judgment leading to poor decisions; loss of spontaneity and initiative; taking longer to complete normal daily activities; repetitive questions; difficulty handling finances and paying bills; wandering and getting lost; losing items or misplacing them in strange places; mood and personality changes, and increased anxiety and / or aggression.

[0247] Some symptoms of moderate Alzheimer's disease / moderate dementia include forgetfulness; increased memory loss and confusion; inability to learn new things; difficulty with language and with reading, writing, and handling numbers; difficulty organizing thoughts and thinking logically; reduced attention span; problems coping with new situations; difficulty performing multi-step tasks, such as getting dressed; problems recognizing family and friends; hallucinations, delusions, and paranoia; impulsive behavior, such as undressing at inappropriate times or places or using swear words; inappropriate outbursts of anger; restlessness, agitation, anxiety, crying, wandering (especially in the evening or at night); repetitive expressions or movements, and occasional muscle twitches.

[0248] Some symptoms of severe Alzheimer's disease / severe dementia include inability to communicate; weight loss; seizures; skin infections; difficulty swallowing; groaning, whimpering, or throat noises; increased sleepiness; and loss of bowel and bladder control.

[0249] Some physiological symptoms of Alzheimer's disease / dementia include a decrease in brain mass, such as a decrease in hippocampal volume. Thus, in some forms, the method of administering a BBB-crossing polypeptide conjugate composition is performed to increase brain mass, and / or reduce or prevent the rate of brain mass loss in a subject, compared to an untreated control subject; to increase hippocampal volume in a subject, and reduce or prevent the rate of hippocampal volume loss.

[0250] The BBB-crossing polypeptide conjugate composition is administered to provide an effective amount of one or more therapeutic agents after administration to an individual. As used herein, an "effective amount" of one or more therapeutic agents is an amount effective to improve or alleviate one or more symptoms associated with Alzheimer's disease or dementia, including neurological deficits or cognitive decline or impairment. Such a therapeutic effect is generally observed within about 12 to about 24 weeks of the start of administration of a composition comprising an effective amount of one or more neuroenhancing agents, although the therapeutic effect may be observed in less than 12 weeks or more than 24 weeks.

[0251] The individual is preferably an adult, and more preferably a person older than 30 years of age, who has lost a small amount of neurological function due to Alzheimer's disease or dementia. Generally speaking, neural loss means any neural loss at the cellular level, including loss of neurites, neural tissue, or neural networks.

[0252] In other forms, the method includes selecting a subject that may benefit from treatment with a BBB-crossing polypeptide conjugate composition. For example, the ceramide level in the patient's CSF is first determined and compared to the ceramide level in a healthy control. In some forms, a BBB-crossing polypeptide conjugate composition is administered to a patient who has an elevated ceramide concentration in the CSF or in the serum relative to a healthy control. In other forms, a BBB-crossing polypeptide conjugate composition is administered to a patient who has an increased amount of brain and / or serum exosomes relative to a healthy control. In other forms, a BBB-crossing polypeptide conjugate composition is administered to a patient who has an increased level of serum anti-ceramide IgG relative to a healthy control.

[0253] In some forms, the subject has a neurological disorder or is in need of neuroprotection. Exemplary disorders and / or subjects include, but are not limited to, subjects who have had, have, or are likely to develop or suffer from the following disorders: stroke, traumatic brain injury, spinal cord injury, post-traumatic stress syndrome, or a combination thereof.

[0254] In some forms, the methods administer to a subject in need thereof an effective amount to reduce or prevent one or more molecular or clinical symptoms of a neurodegenerative disease or one or more mechanisms leading to neurodegeneration.

[0255] The agents used to treat neurodegenerative diseases are well known in the art and may vary depending on the symptoms and disease to be treated. For example, conventional treatments for Parkinson's disease may include levodopa (usually in combination with a dopa decarboxylase inhibitor or a COMT inhibitor), a dopamine agonist, or a MAO-B inhibitor.

[0256] b. Huntington's disease

[0257] In some forms, the methods administer the BBB-crossing polypeptide conjugate compositions to treat Huntington's disease. Treatments for Huntington's disease may include dopamine blockers to help reduce abnormal behaviors and movements, or drugs such as amantadine and tetrabenazine to control movement. Other drugs that help reduce chorea include tranquilizers and benzodiazepines. Class. Compounds such as amantadine or remazamide have shown preliminary positive results. Hypokinesia and rigidity, especially in adolescent cases, can be treated with anti-Parkinson's drugs, and myoclonic hyperkinesia can be treated with valproic acid. Psychiatric symptoms can be treated with drugs similar to those used in the general population. Selective serotonin reuptake inhibitors and mirtazapine are recommended for depression, while atypical antipsychotics are recommended for psychosis and behavioral problems.

[0258] c. Amyotrophic lateral sclerosis (ALS)

[0259] In some forms, the methods administer a BBB-crossing polypeptide conjugate composition to treat amyotrophic lateral sclerosis (ALS). (2-amino-6-(trifluoromethoxy)benzothiazole), an excitotoxin, improved survival time in subjects with ALS. Other drugs and interventions, most used off-label, can reduce symptoms caused by ALS. Some treatments can improve quality of life and some have been shown to prolong life. Common ALS-related treatments are reviewed in Gordon, Aging and Disease, 4(5):295-310 (2013). Many other agents have been tested in one or more clinical trials with efficacy ranging from ineffective to promising. Exemplary agents are reviewed in Carlesi, et al., Archives Italiennes de Biologie, 149:151-167 (2011). For example, treatment may include an agent that reduces excitotoxicity, such as talampanel (8-methyl-7H-1,3-dioxol-(2,3)benzodiazepine agents that reduce oxidative stress, such as coenzyme Q10, manganese porphyrin, KNS-760704 [(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine dihydrochloride, RPPX] or edaravone (3-methyl-1-phenyl-2-pyrazol-5-one, MCI-186); agents that reduce apoptosis, such as histone deacetylase (HDAC) inhibitors including valproic acid, TCH346 (dibenzo(b,f) agents that reduce neuroinflammation, such as thalidomide and celastrol; neurotropic agents such as insulin-like growth factor 1 (IGF-1) or vascular endothelial growth factor (VEGF); heat shock protein inducers such as arilomol; or autophagy inducers such as rapamycin or lithium.

[0260] Treatments for Lewy body dementia may include, for example, acetylcholinesterase inhibitors, such as tacrine, rivastigmine, galantamine, or donepezil; the N-methyl d-aspartate receptor antagonist memantine hydrochloride; dopaminergic treatments, such as levodopa or selegiline; antipsychotics, such as olanzapine or clozapine; REM disorder treatments, such as clonazepam, melatonin, or quetiapine; antidepressant and antianxiety treatments, such as selective serotonin reuptake inhibitors (citalopram, escitalopram, sertraline, paroxetine, etc.) or serotonin and norepinephrine reuptake inhibitors (venlafaxine, mirtazapine, and bupropion) (see, for example, Macijauskiene, et al., Medicina (Kaunas), 48(1): 1-8 (2012)).

[0261] Other common therapeutic, prophylactic, or diagnostic agents used to treat neurological dysfunction include amantadine and anticholinergics for motor symptoms, clozapine for psychosis, cholinesterase inhibitors for dementia, and modafinil for daytime sleepiness.

[0262] d. Pain

[0263] In some forms, the method administers a BBB-crossing polypeptide conjugate composition to treat pain in a subject. The pain may be acute pain or chronic pain. The pain may be associated with one or more diseases that cause pain due to excessive tissue swelling or growth, or due to nerve damage or death. In some forms, the method administers a BBB-crossing polypeptide conjugate composition to treat or prevent pain in the brain or CNS or eye of a subject. In other forms, the method administers a BBB-crossing polypeptide conjugate composition to treat or prevent pain in one or more other areas of the body, for example, referred pain and / or neuropathic pain originating from the brain or CNS or eye of a subject.

[0264] There are three broad categories of analgesic drugs, including: (1) non-opioid analgesics, which include nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, metamizole, and others; (2) "adjunctive analgesics," which are defined as "drugs with a primary indication other than pain, but which may have analgesic effects in selected circumstances"; and (3) opioid analgesics. Thus, in some forms, the method administers to a subject one or more of a non-opioid analgesic, adjunctive analgesic, or opioid analgesic to prevent or alleviate pain in the subject. Methods for scoring and assessing pain are known in the art. Thus, in some forms, the method reduces or prevents pain by administering to a subject in need thereof one or more BBB-crossing polypeptide conjugate compositions comprising one or more analgesics to reduce or prevent pain in the subject.

[0265] (a) Neuropathic pain

[0266] In some forms, the method applies the BBB to cross the polypeptide conjugate composition to treat neuropathic pain in the subject. In some forms, the method treats or prevents neuropathic pain caused by diseases or conditions including: diabetic neuropathy, herpes zoster, postherpetic neuralgia, neuroma, phantom limb pain and trigeminal neuralgia. In some forms, the disease or condition is confirmed (established) or idiopathic chronic pain syndrome and / or disease, including fibromyalgia and complex regional pain syndrome. In some forms, the disease or condition is demyelinating myelin loss disease or demyelinating leukodystrophy. In some forms, the disease or condition is inflammatory demyelination, viral demyelination, acquired metabolic demyelination, hypoxic-ischemic demyelination or compression-induced demyelination. In some forms, the disease or disorder is diabetic neuropathy, herpes zoster, postherpetic neuralgia, neuroma, phantom limb pain, trigeminal neuralgia, multiple sclerosis, acute multiple sclerosis, neuromyelitis optica, concentric sclerosis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, progressive multifocal leukoencephalopathy, human immunodeficiency virus infection, subacute sclerosing panencephalitis, central pontine myelinolysis, extrapontine myelinolysis, fibromyalgia, or complex regional pain syndrome.

[0267] 2. Neurodevelopmental disorders

[0268] A neurodevelopmental disorder usually means that the brain was not formed normally from the beginning. Abnormal regulation of basic neurodevelopmental processes may occur, or there may be disruptions caused by injury that can take many forms. Autism and attention deficit hyperactivity disorder are typically described as neurodevelopmental disorders.

[0269] Cerebral palsy (CP) is one of the most common pediatric neurological / neurodevelopmental disorders, currently estimated to affect approximately 2 to 3 per 1,000 live births (Kirby, RS et al., Research in Developmental Disabilities, 32, 462 (2011)). CP is recognized in early childhood and the condition persists throughout life. The most common causes of CP include premature birth, hypoxia-ischemia and placental insufficiency, birth asphyxia, and maternal-fetal inflammation (Dammann, O. Acta 2007, 96, 6; Yoon, BH et al., American Journal of Obstetrics and Gynecology 2000, 182, 675; and O'Shea, TM et al., Journal of child neurology 2012, 27, 22). Although CP is heterogeneous in etiology and the mechanism of the disease is very complex, neuroinflammation is a common pathophysiological mechanism that is independent of etiology. Targeting neuroinflammation and delivering drugs directly at the site of injury may be beneficial.

[0270] Compositions and methods can also be used to deliver therapeutic agents, prophylactic agents or diagnostic agents to treat neurodevelopmental disorders, such as cerebral palsy. In some preferred forms, compositions and methods are effective in treating and / or alleviating neuroinflammation associated with neurodevelopmental disorders (e.g., cerebral palsy).

[0271] In some forms, the BBB-crossing polypeptide conjugate compositions are effective for treating, imaging and / or preventing inflammation of the brain in neurodevelopmental disorders, including, for example, Rett syndrome. In some preferred forms, the BBB-crossing polypeptide conjugate compositions will be used to deliver anti-inflammatory agents (D-NAC) and anti-excitotoxic agents and D-anti-glutamate agents. Exemplary candidates are: MK801, memantine hydrochloride, 1-MT.

[0272] In some forms, the BBB-crossing polypeptide conjugate compositions are effective in treating, imaging, and / or preventing inflammation of the brain in autism spectrum disorders. The term "spectrum" refers to the broad range of symptoms, skills, and levels of impairment or disability that a child with ASD may have. Some children are mildly impaired by their symptoms, while others are severely disabled. Although the latest edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) no longer includes Asperger's syndrome; however, the features of Asperger's syndrome are included within the broad category of ASD.

[0273] At this time, the only medications approved by the FDA to treat aspects of ASD are the antipsychotics risperidone (Risperdal) and aripiprazole (Abilify). Some medications that may be prescribed off-label for children with ASD include the following:

[0274] Antipsychotic drugs are more commonly used to treat serious neurological diseases such as schizophrenia. These drugs can help reduce aggression and other serious behavioral problems in children, including those with ASD. They can also help reduce repetitive behaviors, hyperactivity, and attention problems.

[0275] Antidepressants such as fluoxetine or sertraline are usually prescribed to treat depression and anxiety, but are sometimes prescribed to reduce repetitive behaviors. Some antidepressants may also help control aggression and anxiety in children with ASD.

[0276] Stimulant medications such as methylphenidate It is safe and effective in treating people with attention deficit hyperactivity disorder (ADHD). Methylphenidate has also been shown to be effective in treating hyperactivity in children with ASD. But not as many children with ASD respond to treatment, and those who do respond show more side effects than children with ADHD and no ASD.

[0277] 3. Brain tumors

[0278] In some forms, the method treats benign or malignant tumors by delaying or inhibiting the growth of the tumor in the subject, reducing the growth or size of the tumor, inhibiting or reducing the metastasis of the tumor and / or inhibiting or reducing the symptoms associated with tumor development or growth. In some preferred forms, the tumor to be treated is a neuronal tumor and a mixed neuronal-glial cell tumor. Neuronal tumors and mixed neuronal-glial cell tumors are the types of rare tumors that occur in the brain or spinal cord. In most cases, the tumor is not cancer (benign), but the tumor can compress nearby brain tissue and cause problems such as epilepsy.

[0279] Thus, in some forms, the BBB-crossing polypeptide conjugate composition is administered in combination with one or more additional therapeutic agents, which are known to be able to treat brain tumors or one or more symptoms associated with brain tumors. For example, the BBB-crossing polypeptide conjugate composition can be administered to the brain by intravenous administration or during surgery to remove all or part of the tumor. The BBB-crossing polypeptide conjugate composition can be used to deliver a chemotherapeutic agent, which is an agent used to enhance adjuvant therapy (e.g., adjuvant therapy for subjects undergoing radiation therapy), wherein the BBB-crossing polypeptide is linked to at least one radiosensitizer in an amount effective to block or inhibit the activity of DDX3 in a proliferative disease in the brain.

[0280] It will be appreciated by those of ordinary skill in the art that, in addition to chemotherapy, surgical intervention and radiation therapy are also used to treat cancer of the nervous system. Radiation therapy means administering ionizing radiation to a subject at a site adjacent to the cancer in the subject. In some forms, a radiosensitizer is administered in two or more doses, and then ionizing radiation is administered to the subject at a site adjacent to the cancer in the subject. In other forms, administration of a radiosensitizer followed by ionizing radiation may be repeated for 2 or more cycles. Typically, the dose of ionizing radiation varies with the size and location of the tumor, but the dose is in the range of 0.1 Gy to about 30 Gy, preferably in the range of 5 Gy to about 25 Gy.

[0281] In some forms, the ionizing radiation is in the form of stereotactic ablative radiotherapy (SABR) or stereotactic body radiation therapy (SBRT).

[0282] B. Diagnostic Methods

[0283] It has been determined that BBB-crossing polypeptide conjugate compositions comprising one or more diagnostic agents provide a means for selectively and specifically marking one or more structures in the brain or CNS. In some forms, the BBB-crossing polypeptide conjugate comprises a diagnostic agent that is a dye or marker that is specific for a particular cell type or condition. For example, in some forms, the diagnostic agent is a dye or marker that selectively binds to and marks tumor cells in the brain. Therefore, in some forms, a method for marking a cell type or condition in a subject's brain comprises administering to the subject an effective amount of a BBB-crossing polypeptide conjugate comprising a marker or dye for marking a cell type or condition in the subject's brain. In some forms, the method comprises identifying or observing one or more steps in the marker or dye, for example, by one or more imaging techniques. In some forms, a BBB-crossing polypeptide conjugate comprising a diagnostic agent is administered in combination with one or more additional therapeutically active agents, for example, known to be able to treat a brain disease or condition or one or more symptoms associated with a brain disease or condition.

[0284] Diagnostic methods can be coupled with therapeutic methods such as those disclosed herein.

[0285] C. Dosage and Effective Amount

[0286] Dosage and dosage regimen depend on the severity and location of the disease or injury and / or the method of administration, as well as the therapeutic agent or preventive agent delivered. This can be determined by those skilled in the art. The BBB-crossing polypeptide conjugate composition for treating a proliferative disease or disease in the brain in an effective amount is generally sufficient to reduce or alleviate one or more symptoms of brain cancer and / or proliferative disease in the brain. Typically, the dosage will be in the range of micrograms / kg to about 100 mg / kg body weight.

[0287] Preferably, the therapeutic agent, preventive agent or diagnostic agent does not target or otherwise regulate the activity or amount of healthy cells that are not in or associated with the diseased / damaged tissue, or targets or regulates at a reduced level compared to cells associated with a disease or disorder such as cancer and / or a proliferative disorder. In this way, by-products and other side effects associated with the composition are reduced. Therefore, in some preferred forms, the BBB-crossing polypeptide conjugate composition is administered in an amount that results in improved or enhanced function in an individual with a disease or disorder such as cancer and / or a proliferative disorder.

[0288] The actual effective amount of the BBB-crossing polypeptide conjugate composition may vary depending on factors including the specific agent being administered, the specific composition being formulated, the mode of administration, the age, weight, condition, and route of administration and the disease or disorder being treated. In general, the dose for intravenous injection or infusion will be lower than that for oral administration.

[0289] The dosage can vary and can be applied with one dose per day or more doses for one or more days. Guidance on the appropriate dosage of a given class of drug products can be found in the literature. The optimal dosage regimen can be calculated based on the measured results of drug accumulation in an object or patient. One of ordinary skill in the art can easily determine the optimal dose, method of administration, and repetition rate. The optimal dose can vary according to the relative efficacy of the individual pharmaceutical composition, and can usually be estimated based on the effective EC50 found in in vitro and in vivo animal models.

[0290] Also provided are dosage forms of pharmaceutical compositions comprising BBB-crossing polypeptide conjugate compositions. "Dosage form" refers to the physical dosage form of a therapeutic compound, such as a capsule or vial, intended for administration to a patient. The term "dosage unit" refers to the amount of a therapeutic compound administered to a patient in a single dose.

[0291] In general, the timing and frequency of administration will be adjusted to balance the efficacy of a given therapeutic or diagnostic regimen with the side effects of a given delivery system. Exemplary dosing frequencies include continuous infusion, single and multiple administrations such as hourly, daily, weekly, monthly or yearly administration.

[0292] In some forms, the dose is administered daily, twice a week, weekly, biweekly, or less frequently in an amount that provides a therapeutically effective increase in blood levels of the therapeutic agent. When administration is not by an oral route, the composition can be delivered over a period of more than one hour, such as 3 to 10 hours, to produce a therapeutically effective dose within a 24-hour period. Alternatively, the composition can be formulated for controlled release, wherein the composition is administered as a single dose, repeated in a weekly or less frequent regimen.

[0293] It will be understood by those of ordinary skill that the dosing regimen can be any length of time sufficient to treat a condition in a subject. In some forms, the regimen includes one or more cycles of one round of treatment followed by a drug holiday (e.g., no drug). The drug holiday can be 1, 2, 3, 4, 5, 6, or 7 days; or 1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5, or 6 months.

[0294] In some preferred forms, the method for treating or preventing one or more symptoms of damage, disorder or disease in the brain / CNS of a subject in need thereof comprises administering to the subject a preparation comprising a BBB-crossing polypeptide in an amount effective to treat or prevent one or more symptoms of damage, disorder or disease in the brain / CNS of the subject, wherein the BBB-crossing polypeptide is covalently conjugated, or compounded, or encapsulated with one or more therapeutic or preventive agents. It will be understood by those of ordinary skill that the amount and duration of the dosing regimen will be sufficient to treat damage, disorder or disease in the brain / CNS to alleviate one or more symptoms, such as swelling, pain or epilepsy. The physician routinely determines the length and amount of treatment to be administered.

[0295] Typically, a BBB-crossing polypeptide conjugate comprising one or more therapeutic, prophylactic or diagnostic agents is administered systemically and transported across the blood-brain barrier (BBB) ​​to enter the brain and be selectively taken up by damaged and / or diseased neurons. Typically, the BBB-crossing polypeptide conjugate composition accumulates in the nuclei of neurons and delivers the therapeutic, prophylactic or diagnostic agent to these cells.

[0296] Typically, the conjugation of an active agent to a BBB-crossing polypeptide reduces the amount of active agent that must be administered to a subject for treating or diagnosing a disease or condition in the subject's brain compared to the amount of the same active agent that must be administered in the absence of a BBB-crossing polypeptide. In some forms, when a BBB-crossing polypeptide conjugate composition is used, the effective amount of a therapeutic agent, prophylactic agent, or diagnostic agent required for treating or preventing damage, conditions, or diseases in the brain / CNS is at most one percent (100 times smaller) of the amount of a single therapeutic agent, prophylactic agent, or diagnostic agent, such as one-quarter, one-half, one-fifth, one-tenth, one-twentieth, one-thirtieth, one-fortieth, one-fiftieth, one-sixtieth, one-seventieth, one-eightieth, one-ninetieth, or one-hundredth of the amount required when a therapeutic agent, prophylactic agent, or diagnostic agent is used alone.

[0297] D. Combination treatment and manipulation

[0298] BBB-crossing polypeptide conjugate compositions can be administered alone or in combination with one or more conventional treatments. In some forms, conventional treatments include administering one or more compositions in combination with one or more additional therapeutic agents, prophylactic agents, or diagnostic agents. Combination therapy may include administering therapeutic agents, prophylactic agents, or diagnostic agents together in the form of the same mixture or in the form of different mixtures. Therefore, in some forms, the pharmaceutical composition comprises more than one therapeutic agent, prophylactic agent, or diagnostic agent. Such a preparation typically comprises an effective amount of an agent targeting a treatment site. Additional therapeutic agents, prophylactic agents, or diagnostic agents may have the same or different mechanisms of action. In some forms, the combination results in an additive effect on the treatment of a disease or condition. In some forms, the combination results in an effect that exceeds the additive effect on the treatment of a disease or condition.

[0299] In some forms, the BBB-crossing polypeptide conjugate composition is administered prior to, in combination with, after, or in alternation with treatment with one or more additional treatments or procedures. In some forms, the additional treatment is performed between drug cycles or during a drug holiday as part of the composition dosage regimen. For example, in some forms, the additional treatment or procedure is surgery, radiotherapy, or chemotherapy. Some examples of preferred additional therapeutic agents include other conventional treatments known in the art for treating the desired disease, disorder, or condition.

[0300] In the context of Alzheimer's disease, additional therapeutic agents may include one or more of the following: acetylcholinesterase inhibitors (e.g., tacrine, rivastigmine, galantamine, or donepezil), beta-secretase inhibitors such as JNJ-54861911, antibodies such as aducanumab, agonists for the 5-HT2A receptor such as pimavanserin, sargramostim, AADvac1, CAD106, CNP520, gantenerumab, solanezumab, and memantine hydrochloride.

[0301] In the context of Lewy body dementia, additional therapeutic agents may include one or more of the following: acetylcholinesterase inhibitors, such as tacrine, rivastigmine, galantamine, or donepezil; the N-methyl d-aspartate receptor antagonist memantine; dopaminergic treatments, such as levodopa or selegiline; antipsychotics, such as olanzapine or clozapine; REM disorder treatments, such as clonazepam, melatonin, or quetiapine; antidepressant and antianxiety treatments, such as selective serotonin reuptake inhibitors (citalopram, escitalopram, sertraline, paroxetine, etc.) or serotonin and norepinephrine reuptake inhibitors (venlafaxine, mirtazapine, and bupropion) (see, e.g., Macijauskiene, et al., Medicina (Kaunas), 48(1): 1-8 (2012)). Exemplary neuroprotective agents are also known in the art and include, for example, glutamate antagonists, antioxidants, and NMDA receptor agonists. Other neuroprotective agents and treatments include caspase inhibitors, trophic factors, anti-protein aggregation agents, therapeutic hypothermia, and erythropoietin.

[0302] Other common therapeutic, prophylactic, or diagnostic agents used to treat neurological dysfunction include amantadine and anticholinergics for motor symptoms, clozapine for psychosis, cholinesterase inhibitors for dementia, and modafinil for daytime sleepiness.

[0303] In the context of cancer treatment, additional treatments include one or more of the following: conventional chemotherapy, inhibition of checkpoint proteins, adoptive T-cell therapy, radiation therapy, and surgical removal of the tumor.

[0304] The composition and the additional therapeutic agent or treatment can be administered to the subject together or separately. The composition and the additional therapeutic agent or treatment can be administered to the subject on the same day, on different days, or a combination thereof.

[0305] For example, the disclosed compositions can be administered to a subject 0, 1, 2, 3, 4, 5 or more days prior to administration or exposure to an additional therapeutic agent or treatment. In some forms, one or more doses of the composition can be administered to a subject every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35 or 48 days prior to the first administration or exposure to an additional therapeutic agent or treatment.

[0306] The composition may also be administered to a subject 0, 1, 2, 3, 4, 5 or more days after administration or exposure to an additional therapeutic agent or treatment. The composition may also be administered to a subject during administration or exposure to an additional therapeutic agent or treatment. One or more doses of the composition may be administered to a subject every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35 or 48 days during or after administration of an additional therapeutic agent or treatment.

[0307] 1. Application method

[0308] The method administers the BBB-crossing polypeptide conjugate composition preventively, therapeutically, or for diagnostic / research methods, or a combination thereof. Thus, the composition may be administered during a time period before, during, or after the onset of one or more symptoms of a disease or disorder or a pathological condition, disorder, or disorder associated therewith. In some forms, the composition is administered together with one or more additional therapeutic agents as part of a co-therapy (e.g., a combination therapy comprising a BBB-crossing polypeptide conjugate composition and one or more other therapeutic agents), one or more secondary treatments (e.g., an exercise regimen, surgery, etc.), or a combination thereof.

[0309] Exemplary administration methods include systemic or topical administration to a subject, or by direct administration to a cell. As is generally known in the field of protein therapy and diagnosis, the composition can be administered to a cell or a subject. Exemplary administration forms include systemic administration, for example, by injection, such as intravenous (iv), intramuscular (im), intradermal (id), subcutaneous (sc), intravitreal, intraarticular, intraperitoneal, intraocular, and intrathecal injection. Other administration forms include oral administration, mucosal administration, and transdermal administration.

[0310] E. Control

[0311] The treatment results of the BBB-crossing polypeptide conjugate composition comprising one or more therapeutic agents, prophylactic agents or diagnostic agents can be compared with a control. Suitable controls are known in the art and include, for example, untreated subjects or subjects treated with placebos. A typical control is a comparison of the condition or symptoms of a subject before and after the administration of the BBB-crossing polypeptide conjugate. The condition or symptom can be a biochemical, molecular, physiological or pathological readout. For example, the effect of the composition on a specific symptom, a pharmacological or physiological index, can be compared with an untreated subject or the condition of the subject before treatment. In some forms, the symptom, pharmacological or physiological index is measured in the subject before treatment and is repeated one or more times after the start of treatment. In some forms, the control is a reference level, or an average value determined based on measuring symptoms, pharmacological or physiological indexes in one or more subjects (e.g., healthy subjects) who do not suffer from the disease or condition to be treated. In some forms, the effect of the treatment is compared with conventional treatments known in the art. In some forms, an untreated control subject is subjected to the same disease or condition as the treated subject.

[0312] In some forms, a control comprises an equivalent amount of a therapeutic, prophylactic, or diagnostic agent delivered alone or in combination with a carrier without a BBB crossing polypeptide conjugate.

[0313] V.Medicine Box

[0314] The composition may be packaged in a kit. The kit may include a single dose or multiple doses of a composition and instructions for administering the composition, the composition comprising one or more therapeutic agents, prophylactic agents, or diagnostic agents encapsulated in, associated with, or conjugated to a BBB-crossing polypeptide conjugate (e.g., one or more BBB-crossing polypeptides described in the Examples). Specifically, the instructions guide the administration of an effective amount of a BBB-crossing polypeptide and a therapeutic agent, prophylactic agent, or diagnostic agent to an individual suffering from the indicated specific disease / disorder. The composition may be formulated as described above with reference to a specific method of treatment and may be packaged in any convenient manner.

[0315] The present invention will be better understood by reference to the following paragraphs.

[0316] 1. A brain-penetrating peptide capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising a BBB-crossing domain comprising the amino acid sequence RRVISRAKLAAAL (SEQ ID NO: 1) or a functional variant thereof.

[0317] 2. A brain-penetrating peptide capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising a BBB-crossing domain comprising an amino acid sequence of CVGTNCY (SEQ ID NO: 2) or a functional variant thereof.

[0318] 3. The brain-penetrating peptide of paragraph 1, wherein the BBB-crossing domain is at least 13 amino acids in length.

[0319] 4. The brain-penetrating peptide of paragraph 1 or 3, wherein the BBB-crossing domain comprises 5 to 20 consecutive amino acids of one or more of SEQ ID NOs: 3 to 10, or a functional variant of one or more of SEQ ID NOs: 3 to 10.

[0320] 5. The brain-penetrating peptide of paragraph 1 or 3 or 4, wherein the BBB-crossing domain comprises a functional variant of any one of SEQ ID NO: 1 or 3 to 10, wherein the functional variant has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to any one of SEQ ID NO: 1 or 3 to 10.

[0321] 6. The brain-penetrating peptide of paragraph 2, wherein the BBB-crossing domain is at least 7 amino acids in length.

[0322] 7. The brain-penetrating peptide of paragraph 2 or 6, wherein the BBB-crossing domain comprises 5 to 10 consecutive amino acids of one or more of SEQ ID NOs: 11 to 20, 22 to 23, or a functional variant of one or more of SEQ ID NOs: 11 to 20, 22 to 23.

[0323] 8. The brain-penetrating peptide of any of paragraphs 1 to 7, wherein the amino acid sequence of the BBB-crossing domain is not CAGALCY (SEQ ID NO: 21).

[0324] 9. The brain-penetrating peptide of any one of paragraphs 1 to 8, wherein the brain-penetrating peptide selectively homes to brain parenchyma, endothelial cells, or the whole brain.

[0325] 10. The brain-penetrating peptide of paragraph 9, wherein the BBB-crossing domain is or comprises: SRRVISRAKLAAAL (SEQ ID NO: 9); SRRVISRAKLAAALE (SEQ ID NO: 3); or MLGDPILASRRVISRAKLAAALE (SEQ ID NP: 4), and

[0326] The brain-penetrating peptide selectively homes to the brain parenchymal cells.

[0327] 11. A peptide conjugate comprising

[0328] (a) the brain penetrating peptide described in any one of paragraphs 1 to 10; and

[0329] (b) cargo molecules,

[0330] wherein the cargo molecule is directly or indirectly conjugated or complexed with the brain penetrating peptide, and

[0331] wherein the cargo molecule does not cross the BBB in the absence of the brain-penetrating peptide.

[0332] 12. The peptide conjugate of paragraph 11, wherein the carrier molecule comprises one or more active agents selected from the group comprising: a therapeutic agent, a diagnostic agent, a prophylactic agent, and a nutritional agent.

[0333] 13. The peptide conjugate of paragraph 11 or 12, wherein the cargo molecule is encapsulated within or conjugated to a carrier, optionally wherein the carrier is conjugated to the brain penetrating peptide.

[0334] 14. The peptide conjugate of paragraph 13, wherein the carrier comprises a polymer particle, a lipid particle, a liposome, a gel, an inorganic particle, a viral particle, a nucleic acid nanostructure, and a virus-like particle.

[0335] 15. The peptide conjugate of paragraph 13, wherein the carrier is conjugated to the brain penetrating peptide via one or more linkers,

[0336] Optionally, wherein the one or more linkers are cleavable linkers.

[0337] 16. The peptide conjugate of any of paragraphs 12 to 15, wherein the active agent is a therapeutic agent selected from the group consisting of nucleic acids, peptides, lipids, glycolipids, glycoproteins, and small molecules.

[0338] 17. The peptide conjugate of paragraph 16, wherein the therapeutic agent is a nucleic acid selected from the group consisting of antisense molecules, aptamers, ribozymes, triplex-forming oligonucleotides, external guide sequences, RNAi, CRISPR / Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs).

[0339] 18. The peptide conjugate of paragraph 16, wherein the therapeutic agent is a small molecule.

[0340] 19. The peptide conjugate of any of paragraphs 16 to 18, wherein the therapeutic agent is selected from the group comprising an anticancer agent, an anti-inflammatory agent, and an antimicrobial agent.

[0341] 20. A pharmaceutical composition comprising the peptide conjugate of any one of paragraphs 16 to 19 and a pharmaceutically acceptable excipient for administration.

[0342] 21. The pharmaceutical composition of paragraph 20, wherein the composition is suitable for mucosal, pulmonary, intravenous or intramuscular delivery.

[0343] 22. A method of treating or preventing one or more symptoms of a disease or disorder in the brain or CNS of a subject, comprising

[0344] An effective amount of the pharmaceutical composition of paragraph 20 or 21 is administered to the subject to prevent or alleviate one or more symptoms of a disease or disorder in the subject's brain or CNS in the subject.

[0345] 23. The peptide conjugate of any of paragraphs 12 to 15, wherein the active agent is a diagnostic agent selected from the group consisting of a dye, a radionuclide, a fluorescent label, a magnetic label, and a nanoparticle.

[0346] 24. A pharmaceutical composition comprising the peptide conjugate of paragraph 23 and a pharmaceutically acceptable excipient for administration.

[0347] 25. The pharmaceutical composition of paragraph 24, wherein the composition is suitable for mucosal, pulmonary, intravenous or intramuscular delivery.

[0348] 26. Also provided is a formulation of the peptide conjugate of any one of paragraphs 11 to 19 for use in treating or preventing one or more symptoms of a disease or disorder in the brain or CNS of a subject, or a pharmaceutical composition thereof for use in treating or preventing one or more symptoms of a disease or disorder in the brain or CNS of a subject.

[0349] 27. Use of a peptide conjugate of any of paragraphs 11 to 19 for the preparation of a medicament for treating or preventing one or more symptoms of a disease or disorder in the brain or CNS of a subject, wherein the conjugate comprises one or more therapeutic agents.

[0350] 28. A method of detecting or monitoring a disease or condition in the brain or CNS of a subject, comprising

[0351] An effective amount of the pharmaceutical composition of paragraph 24 or 25 is administered to the subject to detect or monitor a disease or condition in the subject's brain or CNS in the subject.

[0352] 29. The method of any of paragraphs 22 or 28, wherein the subject is a human.

[0353] 30. The method of any of paragraphs 22 or 28 or 29, wherein the subject has or is suspected of having a disease selected from the group consisting of cancer, inflammatory diseases, neuronal disorders, HIV / AIDS, diabetes, cardiovascular diseases, infectious diseases (including viral, protozoan, bacterial diseases, and allergies), autoimmune diseases and autoimmune diseases, Alzheimer's disease, Parkinson's disease, ischemia, neurodegenerative disorders, and genetic disorders.

[0354] 31. The method of paragraph 30, wherein the disease is cancer.

[0355] 32. The method of paragraph 30, wherein the disease is Alzheimer's disease.

[0356] 33. The method of paragraph 30, wherein the disease is Parkinson's disease.

[0357] 34. The method of paragraph 30, wherein the disease is a neurodegenerative disorder.

[0358] 35. The method of any of paragraphs 22 or 28 to 34, wherein the subject has an infection.

[0359] 36. A brain-penetrating peptide conjugate capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising

[0360] (a) a BBB-crossing domain comprising the amino acid sequence RRVISRAKLAAAL (SEQ ID NO: 1) or a functional variant thereof; and

[0361] (b) an active agent selected from the group consisting of a therapeutic agent, a diagnostic agent, and a prophylactic agent;

[0362] wherein the active agent is conjugated to the BBB-passing domain, optionally wherein the active agent is conjugated via a linker.

[0363] 37. A brain-penetrating peptide conjugate capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising

[0364] (a) a BBB-crossing domain comprising the amino acid sequence CVGTNCY (SEQ ID NO: 2); and

[0365] (b) an active agent selected from the group consisting of a therapeutic agent, a diagnostic agent, and a prophylactic agent;

[0366] wherein the active agent is conjugated to the BBB-passing domain, optionally wherein the active agent is conjugated via a linker.

[0367] 38. A brain-penetrating peptide conjugate capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising

[0368] (a) a BBB-crossing domain comprising the amino acid sequence RRVISRAKLAAAL (SEQ ID NO: 1) or a functional variant thereof; and

[0369] (b) an active agent selected from the group consisting of a therapeutic agent, a diagnostic agent, and a prophylactic agent;

[0370] wherein the active agent is conjugated to the BBB-passing domain, optionally wherein the active agent is conjugated via a linker.

[0371] 39. A brain-penetrating peptide conjugate capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising

[0372] (a) a BBB-crossing domain comprising the amino acid sequence CVGTNCY (SEQ ID NO: 2); and

[0373] (b) an active agent selected from the group consisting of a therapeutic agent, a diagnostic agent, and a prophylactic agent;

[0374] wherein the active agent is conjugated to the BBB-passing domain, optionally wherein the active agent is conjugated via a linker.

[0375] 40. A brain-penetrating peptide conjugate capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising

[0376] (a) a BBB-crossing domain comprising the amino acid sequence of any one of (SEQ ID NOs: 77 to 96); and

[0377] (b) an active agent selected from the group consisting of a therapeutic agent, a diagnostic agent, and a prophylactic agent;

[0378] wherein the active agent is conjugated to the BBB-passing domain, optionally wherein the active agent is conjugated via a linker.

[0379] 41. A method of selectively delivering an active agent to one or more structures of the brain of a subject, comprising administering to the subject the composition of any one of paragraphs 38 to 40,

[0380] wherein the composition is administered to the subject by a systemic route.

[0381] 42. The method of paragraph 41, wherein the active agent comprises a therapeutic and / or prophylactic agent in an amount effective to treat or prevent one or more symptoms of a disease or disorder in the subject.

[0382] 43. The method of paragraph 42, wherein the therapeutic and / or prophylactic agent comprises one or more of the following: a protein, a carbohydrate, a lipid, a small molecule, or a nucleic acid.

[0383] 44. The method of paragraph 43, wherein the protein comprises an immunoglobulin or an antigen-binding fragment thereof.

[0384] 45. The method of any of paragraphs 42 to 44, wherein the therapeutic and / or prophylactic agent does not cross the BBB in the absence of the brain-penetrating peptide.

[0385] 46. ​​The method of any of paragraphs 42 to 45, wherein the one or more structures of the brain are selected from the group consisting of the cortex, hippocampus, and brainstem.

[0386] 47. The method of any of paragraphs 42 to 46, wherein when the therapeutic and / or prophylactic agent is administered to the subject as a conjugate with a BBB-crossing domain, its effective amount is less than the effective amount of the same therapeutic and / or prophylactic agent in the absence of the BBB-crossing domain.

[0387] 48. The method of any one of paragraphs 42 to 47, wherein the BBB-crossing domain comprises the amino acid sequence RRVISRAKLAAAL (SEQ ID NO: 1), and

[0388] wherein the active agent is selectively delivered to one or more of astrocytes, microglia, or mature neurons in the brain of the subject.

[0389] 49. The method of any one of paragraphs 42 to 47, wherein the BBB-crossing domain comprises the amino acid sequence CVGTNCY (SEQ ID NO: 2); and

[0390] wherein the active agent is selectively delivered to activated microglia in the brain of the subject.

[0391] 50. The method of paragraph 49, wherein the subject has cancer, and

[0392] wherein the active agent is selectively delivered to an area of ​​a tumor and / or brain damage within the brain of the subject.

[0393] 52. The method of paragraph 49 or 50, wherein the tumor is a glioblastoma.

[0394] 53. The method of any of paragraphs 49 to 52, wherein the active agent is a chemotherapeutic agent.

[0395] The invention will be further understood by reference to the following non-limiting examples.

[0396] Example

[0397] Example 1. Identification and development of the brain-penetrating peptide Cerepep

[0398] method

[0399] Animal procedures

[0400] Animal procedures were approved by the Animal Experimentation Committee of the Estonian Ministry of Agriculture in compliance with Estonian regulations and Directive 2010 / 63 / EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (Permits #48 and #159).

[0401] In vivo phage display

[0402] like( In vivo phage screening in male Balb / C mice was performed as described in ( et al., 2022 ) with some modifications. To reduce the background of nonspecific phage in the brain, 4.5 × 10 10 pfu of UV-inactivated control T7 phage without insert. After 30 minutes, the CX7C initial T7 phage library or phage pool recovered from the brain in the previous round of bio-panning was injected iv and circulated for 30 minutes. The mice were anesthetized and perfused intracardially with PBS. The brain was removed, homogenized in LB-NP40 (1%), and the phage in the lysate was rescued by amplification in a semi-solid medium of E. coli strain BLT5403. The peptide encoding portion of the phage genome was sequenced using Ion Torrent high-throughput sequencing (HTS). Eight mice were used in the first round of bio-panning (R1) to prevent narrow selection bottlenecks in the first round of bio-panning. Two mice were used in all subsequent rounds (R2...R5). Repeat R5, plus an additional step of separating brain CD31+ cells relative to CD31- cells by magnetic-activated cell sorting. Mouse brains were dissociated using a Dounce tissue grinder and an adult brain dissociation kit (#130-107-677), and CD31 microbeads (#130-097-418) were used to separate CD31+ cells from all other brain cells, following the manufacturer's protocol for the isolation and culture of endothelial cells from adult mouse brains. Cells were lysed in LB-NP40 (1%) and the samples were further processed following the same steps as for whole brain samples.

[0403] Bacteriophage biodistribution studies

[0404] For titer-based biodistribution studies, the nucleotide sequence encoding the target peptide is cloned into the T7 phage genomic DNA and expressed on the phage surface as a C-terminal fusion of the capsid protein. et al., 2022), cloned using complementary oligonucleotides, amplified and purified phage clones. 9 pfu of peptide-phage clones were injected iv into male Balb / C mice or 5 × 10 10 pfu of phage were injected into female Sprague-Dawley rats (n=3). After 60 minutes, the animals were anesthetized and perfused with PBS. Tissues were collected, homogenized, and the amount of phage in tissue lysates was determined by titration using E. coli strain BLT5615.

[0405] AgNP Biodistribution Study Based on Laser Ablation ICP-MS

[0406] Isotopically pure samples were prepared as described in (Braun et al., 2014; Toome et al., 2017; Pleiko et al., 2021). 107 AgNPs and 109 AgNPs were combined with biotinylated “Cerepep” peptide (biotin-Ahx-SRRVISRAKLAAAL-OH (SEQ ID NO: 9) where Ahx is aminohexanoic acid), Angiopep-2 peptide (biotin-Ahx-LGDPNSTFFYGGSRGKRNNFKTEEY-OH (SEQ ID NO: 75)), or biotin (biotin- 107 AgNP) to functionalize it. 107 AgNPs and 109 AgNPs were mixed in a 1:1 ratio and injected iv in Balb / C mice. After 3 hours of circulation time, mice were perfused intracardially with PBS. Brain and liver were collected, frozen in OCT, sectioned at 30 μm on Superfrost+ slides, and dried in a vacuum desiccator.

[0407] Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis of tissue samples was performed as follows: Agilent 8800 ICP-MS / MS was coupled to a Cetac LSX-213G2+ laser ablation unit equipped with a HelEx II ablation cell and connected using an ARIS (Aerosol Rapid Introduction System) sample introduction system. The system was adjusted using NIST 610 glass. A 65 μm square spot, a scan speed of 260 μm / sec, 20 Hz and 13.5 J / cm were used. 2 Ablation was performed with a line scan at a fluence of 100 μm. Five parallel ablations with a spacing of 65 μm were performed on each tissue. The ICP-MS was operated in single quad mode. Data collection was performed in TRA mode with a dwell time of 9.5 ms on the mass 13C and a mass 107 Ag and 109 The dwell time on Ag was 14 milliseconds, corresponding to a total duty cycle of 50 milliseconds. Data reduction was performed using Iolite v3.62. Data selection was performed using the median combined with MAD error 2SD outlier removal. Multiple parallel line grid scans were performed to generate distribution maps of whole brain sections and parts of the liver. The grid lines were directly adjacent to each other (with a 65 μm offset).

[0408] In vitro phage binding studies

[0409] To evaluate the binding of Cerepep phage, cells were cultured in 24-well plates on coverslips coated with poly-lysine until 80% to 90% confluence. Cells were incubated with Cerepep or control no-insert peptide-phage at 4°C in 1 ml of DMEM + BSA (1%) on a shaking platform at 5 × 10 7 The wells were washed 4 times with 1 ml of DMEM+BSA (1%), the cells were lysed in 1 ml of LB+NP40 and the amount of phage bound to the cells was determined by titration.

[0410] In vitro peptide binding and uptake studies

[0411] To evaluate the binding and uptake of Cerepep peptides, Neuro2A were cultured in 24-well plates on coverslips coated with polylysine until 80% to 90% confluent. FAM-Cerepep (FMA-Ahx-SRRVISRAKLAAAL-OH (SEQ ID NO: 9)) or control FAM-RPAR (FAM-RPARPAR-OH (SEQ ID NO: 74)) peptides were added to Neuro2A cells at a final concentration of 30 μM in growth medium and incubated for 1 hour at 4°C (for binding studies) or 37°C (for uptake studies). Cells were washed 4 times with growth medium and fixed in methanol. Samples were stained with anti-fluorescein antibody to enhance the FAM signal. First, the fixed cells were blocked with PBST (PBS + 0.05% Tween 20), 5% BSA, 5% goat serum at room temperature for 30 minutes, then incubated O / N at 4°C with rabbit anti-fluorescein IgG (Catalog No. A889, Thermo Fisher Scientific, MA, USA) and incubated with Alexa 647 goat anti-rabbit IgG secondary antibody for 1 hour at room temperature. The nucleus was counterstained with 1 μg / ml DAPI. The coverslips were mounted on slides with Fluoromount-G (Electron Microscopy Sciences, PA, USA), imaged using confocal microscopy (Olympus FV1200MPE, Tokyo, Japan) and analyzed using FV10-ASW4.2 software.

[0412] In vitro AgNP binding and internalization studies

[0413] To evaluate the binding and uptake of Cerepep-functionalized silver nanoparticles, Neuro2A were cultured in 24-well plates on coverslips coated with poly-lysine until 80% to 90% confluence. Biotinylated Cerepep peptide or biotin and CF555 dye as non-targeting controls were conjugated to neutravidin-AgNPs prepared as previously described (Tobi et al., 2021). Some samples were pre-incubated with 100 μM Cerepep peptide (Ac-SRRVISRAKLAAAL-OH (SEQ ID NO: 9), where Ac-acetylated) for 1 hour at 37°C to evaluate whether the binding and internalization of the particles were peptide-dependent. 10 μl (OD = 100) of CP-AgNPs or control biotin-AgNPs were added to the cells in 500 μl of culture medium and incubated at 37°C for 1 hour. In some samples, AgNPs bound to the cell surface were removed with a freshly prepared etching solution containing 10 mM Na2S2O3 and K3Fe(CN)6 in PBS, which was applied for 3 minutes. All samples were washed with DPBS to remove unbound AgNPs. Cells were fixed in methanol, stained with DAPI and analyzed by confocal microscopy as described above.

[0414] To investigate the colocalization of CP-AgNPs with endocytosis markers, Neuro2A cells were incubated with CP-AgNPs for 60 or 180 min and the samples were treated with etching solution, fixed in methanol, blocked, and stained with mouse anti-Rab5a, rat anti-Rab7a, or rabbit anti-TGN46 IgG primary antibodies and Alexa 647 goat anti-mouse, anti-rat, or anti-rabbit IgG secondary antibodies, respectively. Nuclei were stained with DAPI and the samples were analyzed by confocal microscopy.

[0415] For characterization of the CP-AgNP uptake pathway, Neuro2A cells were cultured in 6-well plates until 80% to 90% confluence and pre-incubated at 37°C for 30 minutes, and the following endocytosis inhibitors were added to the growth medium: 50 μM nystatin, 50 μM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 4 μM cytochalasin D, and 30 μM chlorpromazine. CP-AgNPs or control biotin-AgNPs were then added, and the cells were incubated at 37°C for 1 hour, treated with etching solution to remove accessible surface-bound AgNPs and subsequently washed with DPBS. The fluorescence of the labeled AgNPs was detected using a BD Accuri flow cytometer by monitoring the 555 nm channel FL2 and analyzed using FCS Express 7 software.

[0416] Proximity Mark

[0417] To evaluate the efficiency and specificity of proximity labeling, WT GBM cells were cultured in 24-well plates on coverslips coated with polylysine until 80% to 90% confluence. Cerepep and horseradish peroxidase complex (CP-HRP) were prepared by conjugating biotinylated Cerepep to streptavidin-HRP. Control biotin-HRP complexes were prepared by conjugating biotin to streptavidin-HRP. 4 μM CP-HRP or biot-HRP in DPBS was added to cells and incubated at 37°C for 1 hour. The cells were washed with DPBS. 500 μM biotin-phenol (Iris Biotech #41994-02-9) in DPBS was added to cells and incubated at 37°C for 30 minutes. To start the reaction, H2O2 was added to cells at a final concentration of 1 mM and incubated at room temperature for 2 minutes. The reaction was terminated by washing with 100U / ml catalase in DPBS. Cells were fixed in 4% PFA and biotinylated proteins were detected with streptavidin-Dylight 550 conjugate (1:500 in DPBS). Nuclei were stained with DAPI and cells were analyzed for biotinylation using confocal microscopy.

[0418] To biotinylate the receptor for the Cerepep peptide to be detected by mass spectrometry, proximity labeling was performed on WT GBM and Neuro2A cells in suspension following the same protocol as for attached cells. For all incubations of suspended cells, end-over-end rotation was used, and for washes, cells were centrifuged at 300 × g for 5 min at room temperature. 3 × 10 6 Biotinylation of cells was determined by adding streptavidin-Dylight 550 conjugate and detecting the signal by monitoring the 555 nm channel FL2 using a BD Accuri flow cytometer.

[0419] In vivo competitive screening

[0420] To determine which amino acid position is important for brain homing of Cerepep, an in vivo competitive screening approach was used. A pool of T7 peptide-phage was created in which each amino acid of the Cerepep sequence was changed one by one to alanine, and alanine to leucine. These clones, the original Cerepep peptide-phage clone, and a control no-insert phage were mixed in an equimolar ratio and 5 × 10 9 pfu of phage mixture were injected iv into male Balb / C mice (n=2). After a 1-hour circulation, animals were anesthetized and perfused with PBS. The prevalence of phage clones in the brain was determined by high-throughput sequencing.

[0421] Statistical analysis

[0422] Statistical analysis was performed using Prism 8 software. Results are expressed as mean values ​​with error bars indicating ± SD. For comparison of groups, a one-way ANOVA test was used. P values ​​were considered as follows: *p≤0.05, **p≤0.01, ***p≤0.001, and p****≤0.0001.

[0423] result

[0424] Identification of the blood-brain barrier penetrating peptide Cerepep using in vivo phage display

[0425] To identify peptides that accumulate in the mouse brain, in vivo T7 phage display was used to cross the blood-brain barrier and reach brain parenchymal cells. For round 1 biopanning, the initial CX7C T7 peptide-phage library was injected iv into mice. Phage accumulated in the brain were rescued to be used as input in subsequent rounds of biopanning. It was observed that the % accumulation of injected phage in brain tissue of rounds 1...5 of biopanning continued to increase ( Figure 1A To assess the % accumulation of injected phage in the brain over five rounds of biopanning (R1 to R5), peptide-phage were injected iv and mice were perfused with PBS for subsequent 30 min cycles. Phage rescued from the brain were re-amplified and injected for subsequent rounds of biopanning). The fifth round of biopanning was then repeated with the addition of an additional step to separate CD31+ brain endothelial cells from CD31- brain cells to detect peptides that not only accumulated in the brain but also penetrated the endothelial cell layer lining the cerebral blood vessels ( Figure 1B Total amount of phage rescued from mouse brain CD31+ and CD31- cell fractions after R5 plus an additional step to separate brain endothelial (CD31+) cells from all other brain cells (parenchymal cells; CD31-). Peptides displayed on the surface of phage rescued from brain from rounds 1 to 5 and from brain endothelial or parenchymal cell fractions were identified using Ion-Torrent HTS.

[0426] One peptide, Pep1, was the most represented peptide sequence in whole brain and in CD31+ and CD31- brain cell fractions after round 5 of biopanning ( Figure 1C ; Prevalence of Pep1 sequences in high-throughput sequencing data, shown as a percentage of all reads). Pep1 is not a CX7C peptide, but rather was generated by a frameshift mutation, resulting in a longer peptide displayed on the phage surface (Table 1A). The complete list of peptides identified from Ion Torrent high-throughput sequencing of biopanning round 5 is shown in Table 1B. Therefore, to determine the shortest sequence necessary for efficient brain homing, shorter versions of Pep1 were displayed on the phage surface (Table 1A) and tested for their accumulation in the brain ( Figure 1DCP is the shortest fragment of Pep1 peptide that retains high accumulation in the brain. 5×10 9 pfu of phage and perfused the mice with PBS after 1 hour of circulation. The amount of phage accumulated in the brain and remaining in the blood was determined by titration). The shortest peptide that still achieved high accumulation in the brain was determined to be the 14aa long peptide SRRVISRAKLAAAL (SEQ ID NO: 9), which was named "Cerepep".

[0427] Table 1A: Sequences of peptides including Pep1 (the most prevalent peptide sequence displayed on the surface of phages rescued from brain from whole brain, from CD31- and CD31+ cell fractions after R5) and shorter versions of peptides tested in vivo.

[0428]

[0429] Table 1B: Sequences of peptides identified in Ion torrent sequencing after round 5 of biopanning, ranked according to the number of reads associated with each sequence.

[0430]

[0431] Cerepep phage achieves high and selective accumulation in mouse and rat brain

[0432] To investigate brain accumulation and biodistribution of the SRRVISRAKLAAAL (SEQ ID NO: 9) (SEQ ID NO: 5) peptide in mice and rats, it was displayed on the surface of T7 phage. Peptide-phage was injected iv and mice were perfused after a 1 hour cycle, brain and control organs were collected and phage titers in each tissue were determined. Insert-free T7 phage was used as a negative control and phage displaying the BBB penetrating peptide Angiopep-2 (TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 9) was used for comparison. Cerepep phage achieved approximately 300-fold higher accumulation in mouse brain compared to control and Angiopep-2 phage ( Figure 2A ; Accumulation of peptide-phage in the brain. 5×10 9 pfu of phage and perfused the mice with PBS after a 1-hour circulation. The amount of phage accumulated in the brain was determined by titration). To evaluate the brain distribution of SRRVISRAKLAAAL (SEQ ID NO: 9), mouse brains were dissected to separate the olfactory bulb, cerebellum, brainstem, and hemispheres, and the titer in the tissues was subsequently determined. Compared to the control phage, SRRVISRAKLAAAL (SEQ ID NO: 9) had high accumulation in all brain regions tested and in the spinal cord ( Figure 2B; Distribution of peptide-phage in different regions of the central nervous system. 5×10 9 pfu of CP or no insert control phage and perfused the mice with PBS after 1 hour of circulation. The amount of phage accumulated in different regions of the central nervous system, liver and blood was determined by titration).

[0433] The brain selectivity of the SRRVISRAKLAAAL (SEQ ID NO: 9) phage after systemic administration was investigated in mice and rats by determining the titer of SRRVISRAKLAAAL (SEQ ID NO: 9) and control phage in multiple control tissues. In both mice and rats, SRRVISRAKLAAAL (SEQ ID NO: 9) showed the highest accumulation in the brain and low accumulation in all other tissues tested ( Figures 2C to 2D Biodistribution of CP peptide-phage in male and female mice - CP or no insert control phage were injected iv and mice were perfused with PBS after 1 hour circulation. The amount of phage accumulated in tissues was determined by titration).

[0434] Cerepep-functionalized silver nanoparticles homing to the mouse brain

[0435] Isotopically barcoded AgNPs coated with peptides were used to evaluate whether Cerepep (SRRVISRAKLAAAL (SEQ ID NO: 9)) could be used to increase brain homing of synthetic nanoparticles. Biotinylated Cerepep was conjugated to Neutravidin-109AgNPs (CP109AgNPs), biotin-blocked Neutravidin-107AgNPs (biot107AgNPs) were used as controls and Neutravidin-107AgNPs conjugated to biotinylated Angiopep-2 (Ang107AgNPs) were used for comparison. An equimolar mixture of 107 / 109AgNPs was injected iv in mice; after a 3-hour cycle, animals were sacrificed and AgNPs were mapped in the brain.

[0436] To visualize the silver nanoparticles functionalized with CP accumulated in the mouse brain, the distribution of isotope barcoded CP-109AgNPs and control biotin-107AgNP nanoparticles was mixed in an equimolar ratio and 200 μl of AgNPs (OD=100) was injected iv. The mice were perfused with DPBS after a 3-hour cycle. The amount of isotope barcoded targeted AgNPs and control AgNPs in the tissue was determined using LAICP-MS. In addition, CP-109AgNPs, Angiopep-2-107AgNPs and control nanoparticles were mixed in an equimolar ratio and 200 μl of AgNPs (OD=100) were injected iv. The mice were perfused with DPBS after a 3-hour cycle. The amount of isotope barcoded targeted AgNPs and control AgNPs in the tissue was determined using LAICP-MS and in the liver sections was determined using LA-ICP-MS.

[0437] The ratio of 109 / 107 AgNPs in the control organ (liver) was similar to that of particles in the input mixture (~1) ( Figure 3 ; Quantification of CP-AgNP accumulation in brain and liver was compared with Biotin-NP and Angiopep-2-AgNP).

[0438] AgNPs functionalized with SRRVISRAKLAAAL (SEQ ID NO: 9) achieved 6.72-fold higher accumulation in the brain compared to control AgNPs (95% CI 4.28 to 9.16) and 7.11-fold higher accumulation compared to Angiopep-2 functionalized AgNPs (95% CI 5.98 to 8.24). The areas positive for SRRVISRAKLAAAL (SEQ ID NO: 9) nanoparticle signals were evenly distributed between different brain regions of brain sections.

[0439] Cerepep binds to glioblastoma and neuroblastoma cells

[0440] Binding of the SRRVISRAKLAAAL (SEQ ID NO: 9) phage to a panel of different cell lines was studied in vitro. Three of the cell lines tested showed at least 50-fold higher binding of the SRRVISRAKLAAAL phage compared to the control no-insert phage ( Figure 4; Binding of CP peptide-phage to different cell lines is shown as fold relative to phage without insert. Data are shown as mean, n=2. ). The highest ratio of SRRVISRAKLAAAL (SEQ ID NO: 9) binding to control was observed in P3 stem cells (Dirkse, et al., 2019), which is a human glioblastoma cell line; followed by the mouse glioblastoma cell line WT GBM (Blouw, et al., 2003) and the commercially available mouse neuroblastoma cell line Neuro2A.

[0441] Next, the interaction of cultured Neuro2A cells with neutravidin-AgNPs labeled with NHS-CF555 dye and functionalized with biotinylated SRRVISRAKLAAAL (SEQ ID NO: 9) (CP-AgNPs) was tested. Confocal imaging showed that CP-AgNPs bound to Neuro2A cells, while control biotin-AgNPs showed only background binding; 10 μl of OD=100 CP-AgNPs or control biotin-AgNPs were added to Neuro2a cells, and such cells in some samples were pre-incubated with 100 μM free CP peptide for 1 hour at 37°C; After incubation with AgNPs for one hour at 37°C, some samples were etched to remove AgNPs bound to the cell surface. After treatment with a cell-impermeable etching solution (+Etch), there was also a CP-AgNP signal to dissolve extracellular AgNPs, indicating internalization. The binding and internalization of CP-AgNPs were abolished when cells were pre-incubated with free SRRVISRAKLAAAL (SEQ ID NO: 9), indicating that the binding and uptake of CP-AgNPs by Neuro2A cells was peptide-dependent.

[0442] The binding (at 4°C) and uptake (at 37°C) of synthetic FAM-Cerepep by Neuro2A cells were also studied, comparing it to a control FAM-RPAR peptide of similar charge. FAM-Cerepep bound to and was taken up by Neuro2A cells, while the control FAM-RPAR showed only background binding; the binding (at 4°C) and internalization (at 37°C) of the CP peptide to Neuro2A cells were visualized using cells incubated for 1 hour with 30 μM FAM-CP or the control peptide Fam-RPAR; Fam was detected with an anti-fluorescein antibody.

[0443] Cerepep endocytosis pathway

[0444] Flow cytometry was used to investigate how treatment with endocytosis inhibitors (nystatin, EIPA, cytochalasin D, and chlorpromazine) affected the uptake of CP-AgNPs in Neuro2A cells. Nystatin was used to block caveolae / lipid-mediated endocytosis, EIPA to block micropinocytosis, cytochalasin D to block actin polymerization and micropinocytosis, and chlorpromazine to block clathrin-mediated endocytosis. Nystatin was the only inhibitor that did not have a statistically significant inhibitory effect on the uptake of CP-AgNPs ( Figure 5 ; Neuro2A cells were preincubated for 30 minutes at 37°C with the following inhibitors of endocytic pathways: 50 μM nystatin, 1 mM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA); 30 μM chlorpromazine; 4 μM cytochalasin D, and untreated cells served as controls. CP-AgNPs were added to the cells and incubated for 60 minutes, and the cells were treated by etching to remove membrane-bound particles. Cells were analyzed by flow cytometry for inhibition of uptake, indicating that caveolae / lipid-mediated endocytosis did not contribute to CP-AgNP uptake by Neuro2A cells. EIPA, cytochalasin D, and chlorpromazine had similar and statistically significant inhibitory effects on the uptake of CP-AgNPs, indicating that more than one endocytic pathway is used for the uptake of CP-AgNPs by Neuro2A cells.

[0445] To map the pathway of endocytosis of CP-AgNPs, colocalization of CP-AgNPs was tracked with the following compartmental markers: early endosomal marker Rab5a, late endosomal marker Rab7a, and trans-Golgi network marker TGN46. After 60 and 180 min, CP-AgNPs showed endolysosomal uptake: colocalization with late endosomes positive for Rab7a and early endosomes positive for Rab5a.

[0446] Identification of Cerepep receptor candidates

[0447] To identify potential receptor candidates for Cerepep (SRRVISRAKLAAAL (SEQ ID NO: 9)) that facilitate blood-brain barrier penetration of the peptide, a proximity labeling method that has been optimized for this purpose (unpublished data) was used on cultured WT GBM and Neuro2A cells. CP-HPR complexes or control biotin-HRP complexes were added to the cells and cell surface proteins in the vicinity of the peptide-HRP complexes were labeled with biotin from biotin-phenol in the presence of peroxide. To determine the efficiency and specificity of this method, biotinylation of WT GBM cells was detected using a fluorescent streptavidin conjugate and confocal microscopy.

[0448] The surface of cells treated with CP-HRP was extensively biotinylated, while cells treated with control biotin-HRP complexes showed only background signal; To identify Cerepep receptor candidates, proximity ligation using Cerepep-HRP complexes and control biotin-HRP complexes was used on WT GBM or Neuro2A cells to biotinylate proteins on the cell surface in close contact with the complexes. Biotin was detected with streptavidin DyLight550. WT GBM cells were analyzed by confocal microscopy. Samples of WT GBM and Neuro2A cells were also analyzed by flow cytometry, which determined that cells treated with CP-HRP had more biotin signal than control samples treated with biotin-HRP, but the latter's signal was slightly higher than that of samples to which HRP was not added ( Figures 6A to 6B ).

[0449] The biotinylated proteins in the samples were pulled down and identified with mass spectrometry. Tables 2 and 3 contain lists of proteins identified by mass spectrometry, which have the highest spectrum counts in WT GBM and Neuro2A samples, respectively (sorted based on the spectrum counts in CP-HRP samples). Proteins with higher spectrum counts in CP-HRP samples and lower or no spectrum counts in control samples with biotin-HRP and without HRP complexes are considered to be potential receptor candidates for Cerepep (highlighted in blue). When the results of the two cell lines were compared, there was an overlap of many potential receptor candidates, but LRP1 was considered to be the most likely candidate because it was one of the top hits and has been described as a receptor for many BBB penetrating peptides including Angiopep-2 (Demeule, et al., 2008).

[0450] Cerepep binds to LRP1

[0451] Cerepep (SRRVISRAKLAAAL (SEQ ID NO: 9)) was tested for binding to human LRP1 purified from placenta using surface plasmon resonance analysis. The Cerepep-neutravidin complex produced a higher response than the LRP1-bound BBB-penetrating peptide Angiopep-2-neutravidin complex. Furthermore, as expected in the case of LRP1 ligands, the binding of the CP-neutravidin complex to LRP1 was inhibited by RAP and EDTA.

[0452] Arginine and isoleucine are essential for brain homing of Cerepep

[0453] To determine which amino acid position is important for brain homing of SRRVISRAKLAAAL (SEQ ID NO: 9), a pool of peptide-phage clones was compiled in which each amino acid position was changed to alanine (alanine to leucine) one by one. Peptide-phages were mixed in equimolar ratios, including Cerepep phage as a positive control and no insert as a negative control. The phage mixture was injected into mice and the amount of different phage clones in the brain was determined using HTS after 1 hour of circulation. The results showed that changing any of the three arginines or isoleucines to alanine eliminated the brain accumulation of phages, indicating that these amino acids are crucial for effective brain homing. Adding C-terminal alanine did not reduce brain homing of phages, indicating that Cerepep does not require a free C-terminus for effective brain homing. Surface plasmon resonance analysis using human LRP-1 immobilized on a BIAcore sensor chip was used to evaluate whether the CP-neutravidin complex interacts directly with human LRP-1. Angiopep-2-neutravidin complex was used as a positive control ( Figures 7A to 7B ).

[0454] Tables 2 and 3: Biotinylated proteins on the surface of WT GBM were pulled down and identified by mass spectrometry

[0455]

[0456] The binding of Cerepep(CP)-neutravidin complex to LRP1 is inhibited by RAP and EDTA.

[0457] Based on studies involving in vivo competitive screening assays, it was determined that arginine and isoleucine are critical for brain homing of the Cerepep (CP) peptide. Each position of the CP peptide was mutated one by one to alanine (alanine to leucine), and an in vivo competitive screening approach was used to evaluate the brain homing of each mutant peptide-phage ( Figure 7C ). A table showing the corresponding fold brain homing of different phage-peptide sequences relative to control phage is shown in Fig.7D Shown in.

[0458] Cerepep cell recruitment mechanism

[0459] To identify the receptor for the Cerepep peptide, it was shown that the binding of AgNP-Cerepep to P3 human stem cells was reduced by inhibitors of several LDL receptor (low-density lipoprotein receptor) proteins: receptor-associated protein (RAP) and anti-LRP-1 (low-density lipoprotein receptor-related protein 1, also known as alpha-2-macroglobulin receptor), apolipoprotein E receptor or cluster of differentiation (CD) 91 antibodies.

[0460] Briefly, after P3 human stem cells were exposed to AgNP-control or AgNP-Cerepep for 1 hour at 4°C, the cells were incubated with the indicated antibodies (AB) or RAP (diluted in 20% glycerol) for 1 hour at 4°C and then analyzed by FACS. Figures 18A to 18B is expressed as a multiple relative to the AgNP-Cerepep binding, where Fig.18A Incubation with anti-LRP-1 antibody is shown, while Fig.18B Incubations with RAP, LDLR antibodies, and both anti-LRP-1 and anti-LDLR antibodies are shown. The data demonstrate that anti-LRP-1 antibodies block the binding of Cerepep to its receptor.

[0461] Next, experiments were performed to assess whether and how Cerepep interacts with LRP-1 Cluster II and Cluster III.

[0462] Briefly, 50 μl of Myone Dynabeads were washed multiple times and incubated with 100 μl of 2 mM biotinylated peptide for 2 hours at room temperature. The beads were washed twice with PBS, 0.01% Tween 20-DPBS, and 0.01% PBS-BSA. The beads were then incubated overnight in PBS with 0.1% BSA with 10 nM LRP-1 cluster II or LRP-1 cluster III and Alexa Fluor 647 anti-human LRP-1 antibody (1:200) Figures 19A to 19B ).

[0463] The beads were washed several times and analyzed by FACS. The samples are as follows:

[0464] Negative control (-ve control): beads incubated with biotin (non-peptide), LRP-1 cluster III protein, and Alexa Fluor 647 anti-human antibody;

[0465] Positive control for cluster II experiments: beads incubated with biotinylated Angiopep2 peptide, LRP-1 cluster II protein, and Alexa Fluor 647 anti-human LRP-1 antibody (see Fig.19A );and

[0466] Positive control for cluster III experiments: beads incubated with biotinylated RPAR peptide, neuropilin-1b1b2 protein, anti-neuropilin-1 antibody (1:200), and Alexa Fluor 647 anti-rabbit antibody (see Fig.19B ).

[0467] 10,000 events were analyzed for each condition. The experiment was repeated three times for each condition and the scrambled sequence (SARVISRAKLARAL (SEQ ID NO: 70) was compared to Cerepep by t-test.

[0468] Cerepep-AgNPs colocalize with lysosomes

[0469] P3 stem cells were grown on coverslips and incubated with AgNP-control or AgNP-Cerepep for 1 h at 37 °C, fixed with methanol, and stained with anti-LAMP1 antibody and DAPI. Micrographs were observed under a confocal microscope with a 60× objective. Colocalization analysis of images was performed using the Colormap script ImageJ plugin. Data are available at Fig. 20 , each bar represents the average correlation index (Icorr, ImageJ output). The experiment was performed in triplicate and the error bars show the standard deviation. The data show the co-localization of AgNP-Cerepep with lysosomes.

[0470] LRP1 antibodies inhibit the binding of Cerepep in the brain.

[0471] To evaluate the ability of mAbs to inhibit Cerepep activity in the brain, 25 μg of LRP1 (N-terminal) antibody (Sigma L2295) was injected into the tail vein of mice, after a 1-hour cycle, phage was injected and 30 minutes later the animals were anesthetized and perfused. Brain and liver were collected and homogenized in LB + 1% NP40, and the phage titer in each organ was determined (see Figures 21A to 21B ). The raw data showed accumulation of phage in liver and brain ( Fig.21A ), and for brain, the fold relative to control phage is shown ( Fig. 21B ), data points are shown above the bars. The data show that LRP1 antibodies inhibit the binding of Cerepep.

[0472] FAM-Cerepep peptide crosses the blood-brain barrier.

[0473] To evaluate the ability of FAM conjugated to Cerepep to cross the BBB, 100 μl of 2 mM FAM-Cerepep or FAM scrambled control peptide (SARVISRAKLARAL (SEQ ID NO: 70)) was injected into the tail vein of mice, n = 3. After a 2.5-hour cycle, the animals were anesthetized and perfused, and the brains were collected, frozen, sectioned, fixed, permeabilized, and incubated with CD31 rat and anti-fluorescein rabbit primary antibodies for 2 hours at room temperature. After washing, the sections were incubated with Alexa Fluor 456 anti-rat and Alexa Fluor 647 anti-rabbit secondary antibodies, washed and stained with DAPI. Images were taken in 4 different brain regions (cortex, cerebellum, hippocampus, brainstem) using a confocal microscope. FAM-Cerepep can be detected in brain tissue.

[0474] FAM-Cerepep colocalizes with mature neurons.

[0475] To evaluate the ability of FAM conjugated to Cerepep to colocalize with mature neurons, 100 μl of 2 mM FAM-Cerepep or FAM scrambled (SARVISRAKLARAL (SEQ ID NO: 70)) was injected into the tail vein of mice, n=3. After a 2.5 hour cycle, the animals were anesthetized and perfused, the brains were collected, cryosectioned, fixed, permeabilized, and incubated with NeuN mouse and anti-fluorescein rabbit primary antibodies for 2 hours at room temperature. After washing, the sections were incubated with Alexa Fluor 456 anti-mouse and Alexa Fluor 647 anti-rabbit secondary antibodies, washed and stained with DAPI. Pictures were taken in 4 different brain regions (cortex, cerebellum, hippocampus, brainstem) under a confocal microscope. FAM-Cerepep associated with neurons can be detected in brain tissue. FAM-Cerepep colocalizes with astrocytes

[0476] To evaluate the ability of FAM conjugated to Cerepep to colocalize with astrocytes, 100 μl of 2 mM FAM-Cerepep or FAM scrambled (SARVISRAKLARAL (SEQ ID NO: 70)) was injected into the tail vein of mice, n=3. After a 2.5 hour cycle, the animals were anesthetized and perfused, the brains were collected, frozen, sectioned, fixed, permeabilized, and incubated with GFAP rat and anti-fluorescein rabbit primary antibodies for 2 hours at room temperature. After washing, the sections were incubated with Alexa Fluor 456 anti-rat and Alexa Fluor 647 anti-rabbit secondary antibodies, the sections were washed and stained with DAPI. Pictures were taken in 4 different brain regions (cortex, cerebellum, hippocampus, brainstem) under a confocal microscope. FAM-Cerepep associated with astrocytes can be detected in brain tissue.

[0477] FAM-Cerepep colocalizes with microglia.

[0478] To evaluate the ability of FAM conjugated to Cerepep to colocalize with microglia, 100 μl of 2 mM FAM-Cerepep or FAM scrambled (SARVISRAKLARAL (SEQ ID NO: 70)) was injected into the tail vein of mice, n=3. After a 2.5-hour cycle, the animals were anesthetized and perfused, the brains were collected, frozen, sectioned, fixed, permeabilized, and incubated with CD11b rat and anti-fluorescein rabbit primary antibodies for 2 hours at room temperature. After washing, the sections were incubated with Alexa Fluor 456 anti-rat and Alexa Fluor 647 anti-rabbit secondary antibodies, the sections were washed and stained with DAPI. Pictures were taken in 4 different brain regions (cortex, cerebellum, hippocampus, brainstem) under a confocal microscope. FAM-Cerepep associated with microglia can be detected in brain tissue.

[0479] To quantify FAM-Cerepep in different brain regions, experiments were set up to evaluate fluorescence intensity. Briefly, 100 μl of 2 mM FAM-Cerepep or FAM scrambled peptide (SARVISRAKLARAL (SEQ ID NO: 70)) was injected into the tail vein of mice, n=3. After a 2.5-hour cycle, the animals were anesthetized and perfused, and the brains were collected, cryosectioned, fixed, permeabilized, and incubated with anti-fluorescein rabbit primary antibody for 2 hours at room temperature. After washing, the sections were incubated with Alexa Fluor 647 anti-rabbit secondary antibody, washed and stained with DAPI. Pictures were taken under a confocal microscope in 4 different brain regions (cortex, cerebellum, hippocampus, brainstem), and the fluorescence intensity in each brain region was measured and quantified using ImageJ, as shown in Figure 2. Figures 22A to 22E as shown in .

[0480] HER2-FAM-Cerepep Antibody Crosses the Blood-Brain Barrier

[0481] To evaluate whether Cerepep conjugated to a cargo molecule (e.g., an antibody) crosses the blood-brain barrier, HER2-FAM scrambled (SARVISRAKLARAL (SEQ ID NO: 70)) or HER2-FAM-Cerepep antibody was injected into the tail vein of mice. After a 24-hour cycle, the animals were anesthetized and perfused, the brains were collected and divided into two hemispheres, and half of the mouse brain was cryosectioned, fixed, permeabilized, and incubated with CD31 rat and fluorescein rabbit primary antibodies for 2 hours at room temperature. After washing, the sections were incubated with Alexa Fluor 456 anti-rat and Alexa Fluor 647 anti-rabbit secondary antibodies, washed again and stained with DAPI. Pictures were taken in 3 different brain regions (cortex, hippocampus, and brainstem) under a confocal microscope. The fluorescence intensity signal from FAM was measured in each region using ImageJ, as shown in Figure 2. Fig.23 as shown in .

[0482] Cerepep library accumulates in the brain

[0483] Accumulation of the Cerepep library in mouse brain was detected in two second rounds of biopanning. A restricted Cerepep-based library containing the key residues identified in the alanine scan (XRRXIXRAXLAXXX (where x is a random amino acid; SEQ ID NO: 76)) was prepared and 5×10 9 pfu of library phage were injected into the tail vein of mice, and after 1 hour of circulation, the animals were anesthetized and perfused.

[0484] Brain, lung and liver were collected and homogenized in LB + 1% NP40. Phage titers were determined in each organ. Phage pools recovered from the brain were amplified in semi-solid medium, purified and injected into the tail vein of mice for the second round of biopanning. After rounds 1 and 2 of biopanning, raw data showed accumulation of phage in the brain ( Fig.24A ). Fig. 24BThe multiples relative to the control phage for all three organs analyzed are shown in the figure, and the data are shown on the bars. The experiments were performed in triplicate for each condition, and the error bars show the standard deviation. The data show the accumulation of the Cerepep library in brain tissue. The peptides from the restricted library screen (XRRXIXRAXLAXXX (where x is a random amino acid; SEQ ID NO: 76)) were ranked by characterization in the brain based on high-throughput DNA sequencing (ranking based on the ratio of round 2 to round 1) and weblogo was performed according to the top 20 peptides, as shown in Fig.25 as shown in .

[0485] The top 20 peptides accumulated in the brain as identified by the biopanning experiment are shown in Table 7 below.

[0486] Table 7: Top 20 peptides identified from restricted library screening

[0487]

[0488] Example 2. Identification and development of brain-penetrating peptide CVG

[0489] method

[0490] Peptides

[0491] Fluorescent peptides [labeled with 5(6)-carboxyfluorescein (FAM) and having a 6-aminohexanoic acid spacer attached to the N-terminus of the peptide] and biotinylated peptides were ordered from commercial suppliers (TAG Copenhagen, Denmark and LifeTein, USA).

[0492] Cell lines

[0493] Twenty-two different cell lines derived from human or mouse tumors or normal tissues were used in the experiments (Table 4).

[0494] Table 4. Cell lines used in in vitro and in vivo experiments.

[0495]

[0496] Peptide-phage biopanning

[0497] Competitive screening was performed with 10 previously published BBB penetration / CNS homing peptide-phages. DNA oligonucleotides encoding peptides displayed on the phage surface were purchased from Integrated DNA Technologies, Inc. (USA). The oligonucleotides were annealed and ligated into the phage genome using the T7 Select 415-1 cloning kit (Novagen) and the T7 Select system manual. Each peptide-phage was amplified individually in liquid bacterial culture medium, precipitated with PEG, recovered in PBS and titrated. The phages were mixed in equimolar ratios and purified by CsCl ultracentrifugation and dialysis. The titer of the competitive screening pool was determined and 1×10 10 After a one-hour circulation time, mice were perfused with acid buffer (500 mM NaCl, 0.1 M glycine, 1% BSA, pH 2.5) and wash buffer (1% NP40 in PBS), the collected organs were homogenized and the phage pool was amplified for high-throughput sequencing.

[0498] In vivo phage display with the CX4CYX library was performed in three rounds of selection. The phage library was injected intravenously into Balb / c mice and allowed to circulate for one hour. After circulation, the animals were anesthetized and perfused intracardially with acidic buffer and detergent buffer. Brain and control organs were collected and homogenized in 1% NP40-LB. Bound phages were rescued by amplification in E. coli and their genomic DNA was sequenced by the Ion Torrent Next Generation DNA Sequencing System.

[0499] Alanine

[0500] The phage pool for alanine-leucine scanning contained the original CAGALCY (SEQ ID NO: 21) peptide-phage and phage in which each amino acid in the CAGALCY (SEQ ID NO: 21) peptide was replaced by either alanine or leucine. The phage pool was injected iv into mice, the mice were perfused at one hour time points, the brains were collected and the phage pool from each mouse was amplified, purified and sequenced using HTS.

[0501] Phage binding assays on cell lines

[0502] To evaluate the binding of CVGTNCY (SEQ ID NO: 2) peptide-phage on the cell line, CVGTNCY (SEQ ID NO: 2) peptide-phage and control G7 phage were incubated on the attached cells (except NCH421k cells grown as speroids) in DMEM-0.5% BSA at +4°C for 1 hour. Thereafter, the cells were washed four times with DMEM-0.5% BSA and the phages were released in 1% NP40-LB to determine the titer of the samples.

[0503] Animal experiments

[0504] Animal experimental procedures were approved by the Animal Experimentation Committee of the Ministry of Agriculture of Estonia, projects #48 and #159. Balb / c mice and athymic nude mice (HD) were housed in a pathogen-free environment in the animal facility of the Institute of Biomedicine and Translational Medicine, University of Tartu (Tartu, Estonia). To induce orthotopic GBM xenografts in nude mice, 7 × 10 5 The (WT-GBM, VEGFko GBM) cells were implanted intracranially into the right striatum of the brain (coordinates: 2 mm lateral and 2 mm posterior from bregma, and 2.5 mm in depth). The intracranial tumors were grown for 6 to 7 days (WT-GBM) and 12 to 14 days (VEGFko GBM) before the experiments were performed.

[0505] Synthesis and functionalization of isotopic silver nanoparticles

[0506] Isotopically barcoded silver nanoparticles (AgNPs) were synthesized using a modified Lee and Meisel citrate method. Isotopically pure (107Ag or 109Ag) AgNO3 (50.4mg) was dissolved in 1ml of Milli-Q water (MQ, resistivity 18MΩcm) in the dark and added to 500ml of water heated at 65°C in a glass flask cleaned with Piranha solution (H2SO4 and H2O2; note: highly oxidizing acid solution). Tannic acid (1.2mg) was dissolved in 10ml of MQ and 200mg of tri-salt dihydrate of citrate was added to the solution. The mixture was added to the AgNO3 solution in the reaction vessel. The mixture was stirred vigorously at ~70°C and the reaction was allowed to proceed for 3 minutes, at which point the solution turned yellow. The flask was then transferred to a preheated hot plate, boiled for 20 minutes, and allowed to cool to room temperature. The evaporated volume was reconstituted with fresh MQ.

[0507] The particles were functionalized with neutral avidin (NA) to allow conjugation with biotinylated peptides and lipoic acid-polyethylene glycol (lk)-NH2 (PEG). The terminal amine of PEG was used for coupling of CF555-N-hydroxysuccinimide-dye (NHS-dye). Biotin-X-CVGTNCY (SEQ ID NO: 2)-OH (X, aminocaproic acid) peptide was coated on 107AgNPs, and 109AgNPs were blocked with free D-biotin. AgNPs were washed to remove free peptides by centrifugation at 7000G, decanting, and resuspending in fresh buffer (0.1M HEPES pH 7.2, 0.1M NaNO3, 0.005% Tween 20) with sonication.

[0508] In vivo biodistribution studies

[0509] CVGTNCY (SEQ ID NO: 2) peptide-phage or control phage were injected iv into mice, and after one hour of circulation, the animals were perfused with 20 ml PBS, and the organs were collected, weighed and dropped into 1% NP40-LB solution. The organs were homogenized and the titer of each tissue was determined. FAM-labeled peptides (2 mM peptide in PBS) were injected intravenously and the animals were perfused with 20 mL of PBS 3 hours later. The organs were excised, snap frozen in liquid nitrogen vapor and stored at -80°C. For confocal microscopy, the tissues were sectioned at 15 μm and stained with antibodies and DAPI.

[0510] For in vivo homing of isotope barcoded AgNPs, 107Ag and 109Ag particles were suspended in 200 μl PBS at an equimolar ratio and injected into the tail vein of Balb / c mice. After 3 hours of circulation, mice were perfused through the left ventricle of the heart with 20 mL PBS. Organs were snap-frozen in liquid nitrogen vapor and stored at -80°C until sectioning.

[0511] Laser ablation ICP-MS analysis

[0512] For the biodistribution study based on ratiometer laser ablation (LA) ICP-MS, the organs from the mice injected with Ag-NP were collected, quick-frozen, frozen and air-dried with 30 μm thick slices on Superfrost Plus slides. Using the HelEx 2 volume ablation cell on the Agilent 8800ICP-MS system, the determination of the ratio of 109Ag / 107Ag was performed in the tissue sections using the Cetac LSX-213G2+ laser ablation system (Teledyne Cetac Technologies, USA). The LA-ICP-MS settings were optimized using NIST 612 slides. The same reference sample was used to monitor the ThO / Th ratio during the analysis run, which remained below 0.3%. Monitoring 13C, 107Ag and 109Ag isotopes, the dwell times were 47.5 milliseconds, 95 milliseconds and 95 milliseconds, respectively, corresponding to a duty cycle of 0.25 seconds. 13C was used as an internal standard to account for differences in carbon content of ablated tissues.

[0513] In vitro binding of AgNPs and microscopy

[0514] WT-GBM and VEGFko cells were grown in MEM with Earl salts (Capricorn Scientific, Germany); 100 IU / ml of Pen / Strep; 1% sodium pyruvate, 0.01 M HEPES, 0.6% glucose (Applichem, USA), and 5% heat-inactivated fetal bovine serum (GE Healthcare, UK). Microscopy experiments were performed with wtAgNPs and CF555 dye. The culture medium of the cells was aspirated, the cells were washed twice with warm culture medium, and fresh culture medium was added with CVGTNCY (SEQ ID NO: 2) targeted AgNPs or biotin-AgNPs at 0.2 nM. The cells were incubated with NPs for one hour at 37°C. After incubation with AgNPs, the culture medium containing NPs was aspirated, the cells were washed, then fixed with cold methanol, and washed with PBS. The nuclei were fixed with 1 μg mL -1 The coverslips were mounted on slides with Fluoromount-G (Electron Microscopy Sciences), imaged using an Olympus FV1200MPE confocal microscope (Olympus, Germany), and the images were processed and analyzed using FV10-ASW 4.2Viewer image software (Olympus, Germany).

[0515] Immunohistochemical staining and microscopy of tissues

[0516] Cryosections (15 μm) on Superfrost Plus slides were fixed in cold methanol, washed in PBS, treated with 3% hydrogen peroxide, and blocked for 1 hour in PBS containing 0.05% Tween 20, 5% BSA, and 5% goat serum (GE Healthcare, UK). The sections were immunostained overnight at +4°C with rabbit anti-fluorescein (Catalog No. A889, Thermo Fisher Scientific, MA, USA) antibody. Immunohistochemical staining was completed with Tyramide SuperBoost kit (Invitrogen) and Pierce DAB substrate kit (Thermo Scientific). The sections were imaged with Leica AperioVERSA microscope slide scanner. Imaging was processed with Aperio Image Scope (v12.4.3.5008).

[0517] Binding studies on ELISA plates

[0518] Reelin (#8546-MR-050; R&D Systems), NRP-1b1b2 (wt and mutants), or 5% BSA were immobilized on Costar 96-well ELISA plates (#3590, Corning Life Sciences, Tewksbury, MA, USA). The multiwell plates were coated with 100 μl of 20 μg / ml recombinant protein in PBS overnight at +4°C and then blocked with 1% bovine serum albumin (BSA) in PBS overnight at +4°C. Phage (5×10 8 pfu) at +4°C overnight, and then washed 6 times with PBS containing 1% BSA and 0.1% Tween 20 (wash buffer) to remove background phage. 600 = 0.5 to recover phage and determine the titer of the sample.

[0519] result

[0520] Identification of novel CNS-homing peptides

[0521] Novel BBB-penetrating and CNS-homing peptides were identified in a stepwise process involving: phage competitive screening, alanine scanning mutagenesis, and phage display with a unique library. The first selection step included a phage competitive screen with ten previously published CNS-homing peptides (Table 5). To ensure the collection of BBB-penetrating and CNS-homing peptides, the phage competitive screening pool was injected intravenously (iv) into mice and perfusion was completed with acidic buffer and detergent buffer to remove unbound phage and block the endothelial layer of blood vessels. Based on Ion Torrent high-throughput sequencing (HTS) reads in the screen, one peptide, CAGALCY (SEQ ID NO: 21), showed the highest representation in the brain and was selected for further modification ( Fig. 8A ; An in vivo competitive phage display screen with 10 published CNS homing / BBB penetrating peptides revealed high representation of CAGALCY (SEQ ID NO: 21) in the brain). In the next step, each amino acid in the CAGALCY (SEQ ID NO: 21) peptide was replaced with either alanine or leucine to determine the essential amino acids for CNS homing. Replacement of both cysteine ​​(Cys1 and Cys6) and tyrosine (Tyr7) in the CAGALCY (SEQ ID NO: 21) peptide resulted in the most significant reduction in brain homing, indicating the importance of these amino acids for the ability of the peptide to reach the target site ( Figure 8B Alanine-leucine screening of the CAGALCY (SEQ ID NO: 21) peptide demonstrated the relevance of cysteine, glycine, and tyrosine for brain homing.

[0522] A new phage library CX4CYX was constructed based on alanine-leucine screening to find new brain homing peptides. This knowledge guides the construction of a new phage library CX4CYX (X-any amino acid), in which Cys1, Cys6 and Tyr7 are retained in their complete positions, and four amino acid variable domains are located between cysteines, and one amino acid is mutated after Tyr7. Three rounds of phage bio-panning were performed to allow enrichment of peptides in the brain. After systemic administration of the phage library (round 1) and recovery of the brain phage pool (rounds 2 and 3), acidic buffer and detergent buffer were used for perfusion again to remove non-specifically bound phages from the brain. HTS readings from rounds 1 to 3 indicate appropriate enrichment of in-frame peptides in the brain (Table 6). The first peptide in the list, CVGTNCY (SEQ ID NO: 2), showed approximately 40 times more reads in the brain in round 3 than the original GAGALCY (SEQ ID NO: 21) peptide (Table 6; Selection of identified peptides from in vivo phage display screening using the CX4CYX phage library, peptides listed according to the ratio of sequencing reads between round 3 and round 1). The four peptides that showed the highest ratio between round 3 and round 1, namely

[0523] The homing of CVGTNCY (SEQ ID NO: 2), CEGALCY (SEQ ID NO: 24), CTGSLCY (SEQ ID NO: 12) and CDGALCY (SEQ ID NO: 34)) was evaluated in an additional competitive screen ( Figure 8C In vivo competitive screening using 4 potential brain-homing peptides identified in CX4CYX phage display compared to the published original CAGALCY (SEQ ID NO: 21) peptide. Peptide CVGTNCY (SEQ ID NO: 2) demonstrated the highest characterization in the brain and was selected for detailed validation as a BBB-penetrating / CNS-homing peptide. CVGTNCY (SEQ ID NO: 2) demonstrated the highest characterization in the brain compared to the original CAGALCY (SEQ ID NO: 21) peptide and was selected for detailed characterization as a new BBB-penetrating and CNS-homing peptide.

[0524] Table 6: Selection of peptides identified from in vivo phage display screening using the CX4CYX phage library.

[0525] Peptides are listed according to the ratio of sequencing reads between round 3 and round 1.

[0526]

[0527] Biodistribution of CVGTNCY peptide in rodents

[0528] To characterize the utility of the CVGTNCY (SEQ ID NO: 2) peptide as a systemic CNS homing peptide, the biodistribution of the CVGTNCY (SEQ ID NO: 2) peptide-phage and peptide-functionalized AgNPs in rodents was analyzed. When animals were injected iv, high levels of the CVGTNCY (SEQ ID NO: 2) peptide-phage were detected in the mouse CNS and eyes compared to the control G7 phage, while in many other analyzed organs, CVGTNCY (SEQ ID NO: 2) did not show a homing trend. In the CNS, CVGTNCY (SEQ ID NO: 2) was distributed throughout the neural tissues, but showed a greater tendency to home to the hippocampus, frontal cortex, and cerebellum ( Fig. 9; Biodistribution analysis of CVGTNCY (SEQ ID NO: 2) peptide-phage in mice showed homing in the CNS; CVGTNCY (SEQ ID NO: 2) peptide-phage or control phage G7 were injected iv into mice for one hour circulation, perfused with acidic buffer and detergent buffer, and the target tissues were collected to determine the titer. The figure shows the ratio of CVGTNCY (SEQ ID NO: 2) phage counts to control G7 counts. Significantly higher accumulation of CVGTNCY (SEQ ID NO: 2) compared to control phage was observed in multiple CNS regions. In the remaining organs / tissues analyzed, no significant differences were determined between CVGTNCY (SEQ ID NO: 2) and G7 titers. Comparative biodistribution analysis between mice and rats demonstrated that in both species, CVGTNCY (SEQ ID NO: 2) preferentially homed to the CNS compared to other organs ( Fig. 10A and 10B ; CVGTNCY (SEQ ID NO: 2) peptide-phage homing to the CNS in both mice and rats. CVGTNCY (SEQ ID NO: 2) peptide-phage or control phage (G7 in mice and no insert in rats) were injected intravenously into rodents, and one hour later the animals were perfused with PBS and the tissues of interest were collected to determine the titer. The figure shows the mouse ( Fig. 10A ) and rats ( Fig. 10B ) in the CNS and control tissues relative to the ratio of CVGTNCY (SEQ ID NO: 2) phage counts in the control tissues.

[0529] Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) allows ultrasensitive detection of AgNPs in tissues of interest and has the potential to quantify the in vivo biodistribution of peptide-functionalized AgNPs. To study the distribution of CVGTNCY (SEQ ID NO: 2) functionalized AgNPs in the brain, a mixture of isotope barcoded CVGTNCY (SEQ ID NO: 2)-Ag107NPs and control-Ag109NPs was co-injected iv into Balb / c mice. The AgNPs were allowed to circulate for 3 hours, after which the animals were perfused and cryosections were prepared from snap-frozen brains and livers. LA-ICP-MS mapping of the Ag107 / Ag109 ratio in the brain showed some heterogeneity in distribution throughout the brain, meaning that some regions had Ag107 / Ag109 ratios of 30 and above. The homing of CVGTNCY (SEQ ID NO: 2)-Ag107NP was brain specific, as the Ag107 / Ag109 ratio in the control organ (liver) remained close to the input 1:1 ratio ( Fig. 10C and 10D; Ratiometric laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) spectral analysis of CVGTNCY (SEQ ID NO: 2)-AgNPs on brain sections was used to identify tissue homing in Balb / c mice injected iv with a mixture of CVGTNCY (SEQ ID NO: 2)-Ag107NPs (OD=100) and control biotin-Ag109NPs (OD=100). Mice were perfused at the 3 hour time point, and organs (brain and liver) were collected, snap-frozen, and sectioned at 30 μm. Laser ablation line scan data indicated that CVGTNCY (SEQ ID NO: 2)-Ag107NPs were distributed throughout the brain, and control-Ag109NPs showed a tendency to accumulate in the choroid plexus due to vascular leakage. The results suggest that CVGTNCY (SEQ ID NO: 2) preferentially homes to the CNS rather than other organs / tissues. CVG peptide specifically targets cortical and hippocampal neurons

[0530] To identify CVGTNCY (SEQ ID NO: 2) as a BBB-penetrating and CNS-homing peptide, the presence of CVGTNCY (SEQ ID NO: 2)-AgNPs in cleared brain was analyzed by light sheet microscopy and confocal microscopy, respectively. Mice were injected iv twice with CVGTNCY (SEQ ID NO: 2)-AgNPs or control-AgNPs 12 hours apart, and one hour before sacrificing the animals, the animals were injected iv with wheat germ agglutinin (Wheat Germ Agglutinin)-Alexa Fluor 555 conjugate to visualize blood vessels in the CNS. The animals were perfused and processed for imaging. Light sheet microscopy imaging detected CVGTNCY (SEQ ID NO: 2)-AgNPs outside of brain blood vessels, and AgNPs were present throughout the brain volume ( Fig.10E ) and especially in the white matter ( Fig.10F ) showed significantly more CVGTNCY(SEQ ID NO:2)-AgNPs compared to control-AgNPs in both domains. In conclusion, the results demonstrate that CVGTNCY(SEQ ID NO:2) crosses the BBB and homes to the brain parenchyma.

[0531] Next, colocalization analysis was performed to identify CVGTNCY (SEQ ID NO: 2) targeted cell types in the CNS. CNS sections from mice injected with CVGTNCY (SEQ ID NO: 2)-AgNPs or control-AgNPs were co-stained with cell type specific markers such as pan-neuronal NeuN, astrocyte GFAP, and oligodendrocyte Olig2. CVGTNCY(SEQ ID NO:2)-AgNPs showed colocalization with NeuN-positive cells in the CNS, in contrast, control-AgNPs showed insignificant colocalization with neurons (CVGTNCY(SEQ ID NO:2)-AgNPs colocalized with neuronal cells in the cortex and hippocampus. AgNPs functionalized with CVGTNCY(SEQ ID NO:2) (OD=100) were injected iv twice, 12 hours apart, and after perfusion of the animals, the organs were collected and processed for IF staining. Colocalization analysis of AgNPs functionalized with CVGTNCY(SEQ ID NO:2) with the pan-neuronal marker NeuN showed colocalization on confocal microscopy images. Quantification of CVGTNCY(SEQ ID NO:2)-AgNPs in NeuN-positive cells showed significant colocalization compared to control-AgNPs in hippocampal CA3, hippocampal GD, cortex, and corpus callosum; CVGTNCY(SEQ ID NO:2)-AgNPs were not detected in the cerebellum. There was significant colocalization between CVGTNCY (SEQ ID NO: 2)-AgNPs and NeuN-positive cells. Quantification of AgNPs showed significantly more CVGTNCY (SEQ ID NO: 2)-AgNPs associated with neurons in the CA3 and dentate gyrus (GD) regions of the hippocampus, as well as in the cortex and corpus callosum. Fig.11A To 11E). CVGTNCY (SEQ ID NO: 2) -AgNP also showed a trend of co-localization with cerebellar neurons, but the difference from control-AgNP was not significant. In addition, CVGTNCY (SEQ ID NO: 2) -AgNP showed association with the vascular walls of the brain parenchyma, especially co-localization with GFAP-positive astrocytes. CVGTNCY (SEQ ID NO: 2) -AgNP was detected in microglia and spinal cord neurons (motor neurons). In summary, the results confirm that CVGTNCY (SEQ ID NO: 2) is a BBB-penetrating peptide that targets astrocytes, neurons, and occasionally microglia in the CNS.

[0532] Table 5. CNS homing / BBB penetrating peptides used for in vivo competitive screening

[0533]

[0534]

[0535] The secreted extracellular matrix protein resorcin is a candidate receptor for CVG

[0536] It was planned to identify the CVGTNCY (SEQ ID NO: 2) peptide receptor using an in vitro proximity labeling approach. To complete the receptor search, the binding of the CVGTNCY (SEQ ID NO: 2) peptide-phage was tested on 22 different cell lines isolated from a variety of tumors and normal tissues. Only minimal binding of the CVGTNCY (SEQ ID NO: 2) peptide-phage was observed on all cell lines tested ( Fig.12 ; CVGTNCY(SEQ ID NO:2) peptide-phage and AgNPs functionalized with CVGTNCY(SEQ ID NO:2) showed low binding in vitro). CVGTNCY(SEQ ID NO:2) peptide-phage binding was tested on a variety of tumorigenic and non-tumorigenic cell lines. Binding efficiency was evaluated based on the titer (PFU / ml) of each sample and showed a low CVGTNCY(SEQ ID NO:2) ratio relative to control phage in the cell lines tested. This minimum binding was too low to allow proximity labeling of receptor proteins in vitro. In addition to low peptide-phage binding, no binding of CVGTNCY(SEQ ID NO:2)-AgNPs to two GBM cell lines, WT-GBM and VEGFko, was detected in vitro. In addition, when CVGTNCY(SEQ ID NO:2) peptide-phage binding was tested ex vivo on Balb / c brain homogenate, no binding was detected.

[0537] These results indicate that the CVGTNCY (SEQ ID NO: 2) receptor / binding partner is inaccessible under artificial in vitro and ex vivo conditions and can only be obtained in vivo.

[0538] Next, affinity chromatography was performed to identify the receptor for CVGTNCY (SEQ ID NO: 2), and a receptor candidate was selected, which was the secreted extracellular matrix protein reelin. The binding of CVGTNCY (SEQ ID NO: 2) to reelin was tested by a cell-free method on an ELISA plate. Recombinant human reelin (Ser1221-Gln2666; 163 kDa) was used to coat the ELISA plate and allow the CVGTNCY (SEQ ID NO: 2) peptide-phage to bind to it. After several washes, the titer of the experiment was determined to assess the binding efficiency. CVGTNCY (SEQ ID NO: 2) showed specific binding to reelin, while no binding to NRP-1 and mutant NRP1 proteins was detected ( Fig.13 ; CVGTNCY (SEQ ID NO: 2) receptor candidate).

[0539] Reticularisin was identified from affinity chromatography proteomics data. Recombinant human reticularisin (Ser1221-Gln2666; 8546-MR-050 R&D Systems) was coated onto ELISA plates, and CVGTNCY (SEQ ID NO: 2) peptide-phage was incubated with the protein to allow binding. Binding efficiency was evaluated based on the titer (PFU / ml) of each sample. Control proteins were NRP1, mutant NRP1, and BSA; the control phage was G7. CVGTNCY (SEQ ID NO: 2) showed binding to reticularisin, but not to the other tested proteins. This preliminary result suggests that reticularisin functions as a receptor for CVGTNCY (SEQ ID NO: 2).

[0540] CVG peptide targets GBM cells

[0541] Homing of CVGTNCY (SEQ ID NO: 2) was analyzed in angiogenic WT-GBM and infiltrating VEGFko GBM models. First, homing of CVGTNCY (SEQ ID NO: 2) peptide-phage was evaluated based on potency after systemic injection into glioma-bearing mice and perfusion with PBS. CVGTNCY (SEQ ID NO: 2) showed accumulation in both WT-GBM and VEGFkoGBM, being present in the respective GBM at 6-fold and 2-fold more than normal brain parenchyma ( Figures 14A to 14C ; CVGTNCY (SEQ ID NO: 2) peptide-phage was injected iv into mice bearing WT-GBM or VEGFko GBM and mice bearing no tumor. Mice were perfused at one hour time point, brain and control organs were collected and titrated; Fig.14A The figure shows the level of CVGTNCY (SEQ ID NO: 2) in GBM compared to normal brain tissue, the middle figure shows the level of control G7 phage in GBM and normal brain tissue, and the right figure shows the ratio of CVGTNCY (SEQ ID NO: 2) in GBM and normal brain relative to the control phage. At the same time, the control G7 phage also showed binding to GBM tissue in vivo like the CVGTNCY (SEQ ID NO: 2) phage, and G7 also showed a trend of binding to tumors more than to normal brain ( Fig. 14B In contrast, the ratio of CVGTNCY (SEQ ID NO: 2) to G7 was higher in normal brain parenchyma than in GBM ( Fig. 14C), indicating an enhanced permeability and retention (EPR) effect in tumors. The synthetic CVGTNCY (SEQ ID NO: 2) peptide or scrambled CVGTNCY (SEQ ID NO: 2) homing in mice bearing WT-GBM and VEGFko GBM was analyzed after iv injection and perfusion of the animals. Cryosections of glioma-bearing brains were stained with anti-FAM antibodies and a tyramide amplification kit to enhance the signal of the peptide. The CVGTNCY (SEQ ID NO: 2) peptide was detected in the brain parenchyma and tumor tissue of both GBM models. The scrambled (CVGTNCY (SEQ ID NO: 2) peptide was not detected in the brain parenchyma, but the bound (CVGTNCY (SEQ ID NO: 2) peptide was detected in GBM. The distribution of the CVGTNCY (SEQ ID NO: 2) peptide in GBM and normal brain parenchyma was examined as follows: CVGTNCY (SEQ ID NO: 2) peptide or its scrambled form (CVYTCNG (SEQ ID NO: 68)) peptide was injected iv into glioma-bearing mice, the mice were perfused after one hour of circulation and the target tissues were snap-frozen in liquid nitrogen). Immunohistochemical HRP-DAB staining was used to illustrate peptide targeting on brain cryosections. The images showed that the CVGTNCY (SEQ ID NO: 2) peptide targeted cortical neurons in the brain parenchyma and activated microglia in GBM. The scrambled peptide was shown not to target the brain parenchyma, but it targeted GBM cells. In close examination, the CVGTNCY (SEQ ID NO: 2) peptide was shown to bind to cortical neurons in normal brain and activated microglia in GBM.

[0542] CVG peptide has an adaptor protein in plasma.

[0543] It was demonstrated that CVGTNCY (SEQ ID NO: 2) peptide-phage homing to the brain parenchyma following iv injection (in vivo) Fig.15A ), in contrast, the binding of CVG (CVGTNCY; SEQ ID NO: 2) peptide-phage to brain homogenates of perfused mice was significantly impaired (ex vivo). The control peptide-phage G7 showed higher binding to ex vivo brain homogenates than to in vivo brain homogenates. This result suggests that the binding of the CVGTNCY (SEQ ID NO: 2) peptide to brain parenchymal cells depends on the presence of plasma proteins.

[0544] Next, it was shown that the binding of CVG peptide-phage to isolated brain homogenate was restored in the presence of whole blood samples. Incubation of brain homogenate with blood samples containing CVG peptide-phage significantly increased the binding of the peptide to brain parenchymal cells ( Fig. 15B ). This result indicates that CVG has adaptor proteins in plasma that direct the peptide to target brain parenchymal cells. N=3; *p<0.05.

[0545] To assess the saturation of CVG receptors in the brain, CVG peptide-streptavidin complex (SA-CVG) was injected iv in different amounts (0 μg, 10 μg, 30 μg, 100 μg). CVG peptide-phage was injected iv over 2 minutes. 10 minutes after phage injection, mice were perfused with PBS, and the brains were homogenized and titrated. Angiopep2 peptide-streptavidin complex (SA-Angiopep) and G7 phage were used as controls ( Fig.16 ).

[0546] It was shown that anti-trassin antibodies can cross the BBB after iv administration. Trassin antibodies (PA5-78413; Invitrogen) or control antibodies (made in-house) were injected iv into mice for a 3-hour cycle. After cardiac perfusion, tissues were collected and snap-frozen in isopentane. Antibodies were detected in brain tissue sections with appropriate secondary antibodies.

[0547] The observation that two different classes of resilin ligands (peptides and antibodies) exhibited increased brain tropism and BBB penetration suggests that resilin can function as a bona fide BBB shuttling protein and that different classes of systemic resilin ligands (e.g., antibodies, peptides, aptamers, LMW compounds) can be used to transport cargo across the BBB.

[0548] CVG peptide homing to glioblastoma cells in the brain

[0549] To evaluate homing of the CVG peptide in glioblastoma, CVGTNCY (SEQ ID NO: 2) peptide-phage was injected iv into mice bearing WT-GBM, VEGFko, U87MG, or NCH421k GBM and mice bearing no tumors. At the one hour time point, mice were perfused, and brain and control organs were collected and titrated. The data showed that the CVG PFU per mg GBM was greater than normal brain tissue, and the ratio of CVG to G7 control phage in GBM was lower than that in normal brain. ( Figures 17A to 17B ) The distribution of CVG peptides in GBM and brain parenchyma was also evaluated. The CVGTNCY (SEQ ID NO: 2) peptide or its scrambled form (CVYTCNG (SEQ ID NO: 68)) was injected iv into glioma-bearing mice, the mice were perfused after a 1-hour cycle and the tissues of interest were snap-frozen in liquid nitrogen. Immunohistochemical HRP-DAB staining was used to illustrate peptide targeting on brain cryosections, indicating that the CVG peptide homed to the brain parenchyma and GBM, while the scrambled peptide did not show targeting to the brain parenchyma.

[0550] CVG peptide is engulfed by activated microglia in glioblastoma.

[0551] Mice bearing WT GBM gliomas were injected iv with FAM-labeled CVG peptide for a 1 hour cycle. Mice were perfused and the tissue of interest was snap frozen in liquid nitrogen vapor. Immunofluorescence staining was performed with the following antibodies: FAM, CD68, CD11b, and CD206. The CVG peptide showed colocalization with activated microglial markers CD68, CD11b, and CD206 cells in the glioma edge and core. The scrambled CVG peptide also showed colocalization with CD68, CD11b, and CD206. This result suggests that in animals bearing gliomas, the peptide is engulfed by activated microglia.

[0552] Finally, ratiometric laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) spectral analysis of CVG (CVGTNCY; SEQ ID NO: 2) -AgNPs was performed on glioma-bearing brain sections. VEGFko, NCH421k, and U87-MG tumors were induced intracranially in nude mice. A mixture of CVGTNCY-Ag107NP (OD = 100) and control biotin-Ag109NP (OD = 100) was injected iv into tumor-bearing animals for a 3-hour cycle. Thereafter, the mice were perfused, and the organs (brain and liver) were collected, snap-frozen in nitrogen vapor, and sliced ​​at 30 μm. The results showed that the ratio of CVG-Ag107NP to control-Ag109NP in the brain parenchyma was higher than in gliomas.

[0553] References

[0554] Blouw, B, Song, H, Tihan, T, Bosze, J, Ferrara, N, Gerber, HP, Johnson, RS, and Bergers, G (2003). The hypoxic response of tumors is dependent on their microenvironment. Cancer Cell 4, 133-146.

[0555] Braun, GB et al. (2014). Etchable plasmonic nanoparticle probes to image and quantify cellular internalization. Nat Mater 13, 904-911.

[0556] Demeule,M,Régina,A,Ché,C,Poirier,J,Nguyen,T.Gabathuler,R,Castaigne,J-P,and Béliveau,R(2008).Identification and design of peptides as a new drugdelivery system for the brain.J Pharmacol Exp Ther 324,1064-1072.

[0557] Dirkse,A et al.(2019).Stem cell-associated heterogeneity inGlioblastoma results from intrinsic tumor plasticity shapcd by themicroenvironment.Nat Commun 10,1787.

[0558] Pleiko,K, K,Haugas,M,Paiste,P,Tobi,A,Kurm,K,Riekstina,U,andTeesalu,T(2021).In vivo phage display:identification of organ-spccificpeptides using deep sequencing and differential Profiling acrosstissues.Nucleic Acids Res 49,c38-e38.

[0559] K,Pleiko,K,Haugas,M,and Teesalu,T(2022).New Tools forStreamlined In Vivo Homing Peptide Identification.In:Cell PenetratingPeptides,ed.ULangel,New YorK,NY:Springer US,385-412.

[0560] Tobi,A.Willmore,AA,Kilk,K,Sidoronko,V,Braun,GB,Soomets,U,Sugahara,KN,Ruoslahti,E,and Teesalu,T(2021).Silver Nanocarriers Targeted with a CendRPeptide Potentiating the Cytotoxic Activity of an Anticancer Drug.Adv Ther 4,2000097.

[0561] Toome,K,Willmore,A-MA,Paiste,P,Tobi,A.Sugahara,KN, K,Ruoslahti,E,Braun,GB,and Teesalu,T(2017).Ratiomotric in vivo auditioning of targeted silver nanoparticles.Nanoscale 9,10094-10100.

Claims

1. A brain-penetrating peptide capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising a BBB-crossing domain comprising the amino acid sequence RRVISRAKLAAAL (SEQ ID NO: 1) or a functional variant thereof.

2. A brain-penetrating peptide capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising a BBB-crossing domain comprising an amino acid sequence of CVGTNCY (SEQ ID NO: 2) or a functional variant thereof.

3. The brain-penetrating peptide of claim 1, wherein the BBB-crossing domain is at least 13 amino acids in length.

4. The brain-penetrating peptide of claim 1 or 3, wherein the BBB-crossing domain comprises 5 to 20 consecutive amino acids of one or more of SEQ ID NOs: 3 to 10, or a functional variant of one or more of SEQ ID NOs: 3 to 10.

5. The brain-penetrating peptide of claim 1 or 3 or 4, wherein the BBB-crossing domain comprises a functional variant of any one of SEQ ID NOs: 1 or 3 to 10, the functional variant having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to any one of SEQ ID NOs: 1 or 3 to 10.

6. The brain-penetrating peptide of claim 2, wherein the BBB-crossing domain is at least 7 amino acids in length.

7. The brain-penetrating peptide of claim 2 or 6, wherein the BBB-crossing domain comprises 5 to 10 consecutive amino acids of one or more of SEQ ID NOs: 11 to 20, 22 to 23, or a functional variant of one or more of SEQ ID NOs: 11 to 20, 22 to 23.

8. The brain-penetrating peptide of any one of claims 1 to 7, wherein the amino acid sequence of the BBB-crossing domain is not CAGALCY (SEQ ID NO: 21).

9. The brain-penetrating peptide of any one of claims 1 to 8, wherein the brain-penetrating peptide selectively homes to brain parenchyma, endothelial cells, or the whole brain.

10. The brain-penetrating peptide of claim 9, wherein the BBB-crossing domain is or comprises: SRRVISRAKLAAAL (SEQ ID NO: 9); SRRVISRAKLAAALE (SEQ ID NO: 3); or MLGDPILASRRVISRAKLAAALE (SEQ ID NO: 4), and The brain-penetrating peptide selectively homes to brain parenchymal cells.

11. A peptide conjugate comprising (a) the brain-penetrating peptide of any one of claims 1 to 10; and (b) cargo molecules, wherein the cargo molecule is directly or indirectly conjugated or complexed with the brain penetrating peptide, and wherein the cargo molecule does not cross the BBB in the absence of the brain-penetrating peptide.

12. The peptide conjugate of claim 11, wherein the cargo molecule comprises one or more active agents selected from the group consisting of a therapeutic agent, a diagnostic agent, a prophylactic agent, and a nutritional agent.

13. The peptide conjugate of claim 11 or 12, wherein the cargo molecule is encapsulated in or conjugated to a carrier, optionally wherein the carrier is conjugated to the brain penetrating peptide.

14. The peptide conjugate of claim 13, wherein the carrier comprises a polymer particle, a lipid particle, a liposome, a gel, an inorganic particle, a viral particle, a nucleic acid nanostructure, and a virus-like particle.

15. The peptide conjugate of claim 13, wherein the carrier is conjugated to the brain penetrating peptide via one or more linkers, Optionally, wherein the one or more linkers are cleavable linkers.

16. The peptide conjugate of any one of claims 12 to 15, wherein the active agent is a therapeutic agent selected from the group consisting of nucleic acids, peptides, lipids, glycolipids, glycoproteins, and small molecules.

17. The peptide conjugate of claim 16, wherein the therapeutic agent is a nucleic acid selected from the group consisting of antisense molecules, aptamers, ribozymes, triplex-forming oligonucleotides, external guide sequences, RNAi, CRISPR / Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs).

18. The peptide conjugate of claim 16, wherein the therapeutic agent is a small molecule.

19. The peptide conjugate of any one of claims 16 to 18, wherein the therapeutic agent is selected from an anti-cancer agent, an anti-inflammatory agent, and an antimicrobial agent.

20. A pharmaceutical composition comprising the peptide conjugate according to any one of claims 16 to 19 and a pharmaceutically acceptable excipient for administration.

21. The pharmaceutical composition of claim 20, wherein the composition is suitable for mucosal, pulmonary, intravenous or intramuscular delivery.

22. A method of treating or preventing one or more symptoms of a disease or disorder in the brain or CNS of a subject, comprising: An effective amount of the pharmaceutical composition of claim 20 or 21 is administered to the subject to prevent or alleviate one or more symptoms of a disease or disorder in the subject's brain or CNS in the subject.

23. The peptide conjugate of any one of claims 12 to 15, wherein the active agent is a diagnostic agent selected from the group consisting of a dye, a radionuclide, a fluorescent label, a magnetic label, and a nanoparticle.

24. A pharmaceutical composition comprising the peptide conjugate according to claim 23 and a pharmaceutically acceptable excipient for administration.

25. The pharmaceutical composition of claim 24, wherein the composition is suitable for mucosal, pulmonary, intravenous or intramuscular delivery.

26. Also provided is a formulation of the peptide conjugate of any one of claims 11 to 19 for use in treating or preventing one or more symptoms of a disease or condition in the brain or CNS of a subject, or a pharmaceutical composition thereof for use in treating or preventing one or more symptoms of a disease or condition in the brain or CNS of a subject.

27. Use of a peptide conjugate as claimed in any one of claims 11 to 19 in the preparation of a medicament for treating or preventing one or more symptoms of a disease or disorder in the brain or CNS of a subject, wherein the conjugate comprises one or more therapeutic agents.

28. A method of detecting or monitoring a disease or condition in the brain or CNS of a subject, comprising: An effective amount of the pharmaceutical composition of claim 24 or 25 is administered to the subject to detect or monitor a disease or condition in the subject's brain or CNS in the subject.

29. The method of any one of claims 22 or 28, wherein the subject is a human.

30. The method of any one of claims 22 or 28 or 29, wherein the subject has or is suspected of having a disease selected from the group consisting of cancer, inflammatory diseases, neuronal disorders, HIV / AIDS, diabetes, cardiovascular disease, infectious diseases (including viral, protozoan, bacterial diseases, and allergies), autoimmune diseases and autoimmune diseases, Alzheimer's disease, Parkinson's disease, ischemia, neurodegenerative disorders, and genetic disorders.

31. The method of claim 30, wherein the disease is cancer.

32. The method of claim 30, wherein the disease is Alzheimer's disease.

33. The method of claim 30, wherein the disease is Parkinson's disease.

34. The method of claim 30, wherein the disease is a neurodegenerative disorder.

35. The method of any one of claims 22 or 28 to 34, wherein the subject has an infection.

36. A brain-penetrating peptide comprising the amino acid sequence of any one of SEQ ID NOs: 77 to 96.

37. The peptide of claim 36, further comprising a cargo molecule, wherein the cargo molecule is directly or indirectly conjugated or complexed with the brain penetrating peptide, and wherein the cargo molecule does not cross the BBB in the absence of the brain-penetrating peptide.

38. A brain-penetrating peptide conjugate capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising: (a) a BBB-crossing domain comprising the amino acid sequence RRVISRAKLAAAL (SEQ ID NO: 1) or a functional variant thereof; and (b) an active agent selected from the group consisting of a therapeutic agent, a diagnostic agent, and a prophylactic agent; wherein the active agent is conjugated to the BBB-passing domain, optionally wherein the active agent is conjugated via a linker.

39. A brain-penetrating peptide conjugate capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising: (a) a BBB-crossing domain comprising the amino acid sequence CVGTNCY (SEQ ID NO: 2); and (b) an active agent selected from the group consisting of a therapeutic agent, a diagnostic agent, and a prophylactic agent; wherein the active agent is conjugated to the BBB-passing domain, optionally wherein the active agent is conjugated via a linker.

40. A brain-penetrating peptide conjugate capable of crossing the blood-brain barrier (BBB) ​​in a subject, comprising: (a) a BBB-crossing domain comprising the amino acid sequence of any one of (SEQ ID NOs: 77 to 96); and (b) an active agent selected from the group consisting of a therapeutic agent, a diagnostic agent, and a prophylactic agent; wherein the active agent is conjugated to the BBB-passing domain, optionally wherein the active agent is conjugated via a linker.

41. A method of selectively delivering an active agent to one or more structures of the brain of a subject, comprising administering to the subject a composition of any one of claims 38 to 40, wherein the composition is administered to the subject by a systemic route.

42. The method of claim 41, wherein the active agent comprises a therapeutic and / or prophylactic agent in an amount effective to treat or prevent one or more symptoms of a disease or disorder in the subject.

43. The method of claim 42, wherein the therapeutic and / or prophylactic agent comprises one or more of the following: a protein, a carbohydrate, a lipid, a small molecule, or a nucleic acid.

44. The method of claim 43, wherein the protein comprises an immunoglobulin or an antigen-binding fragment thereof.

45. The method of any one of claims 42 to 44, wherein the therapeutic and / or prophylactic agent does not cross the BBB in the absence of the brain penetrating peptide.

46. ​​The method of any one of claims 42 to 45, wherein the one or more structures of the brain are selected from the group consisting of cortex, hippocampus, and brainstem.

47. The method of any one of claims 42 to 46, wherein when the therapeutic and / or prophylactic agent is administered to the subject as a conjugate with the BBB-crossing domain, its effective amount is less than the effective amount of the same therapeutic and / or prophylactic agent in the absence of the BBB-crossing domain.

48. The method of any one of claims 42 to 47, wherein the BBB-crossing domain comprises the amino acid sequence RRVISRAKLAAAL (SEQ ID NO: 1), and wherein the active agent is selectively delivered to one or more of astrocytes, microglia, or mature neurons in the brain of the subject.

49. The method of any one of claims 42 to 47, wherein the BBB-crossing domain comprises the amino acid sequence CVGTNCY (SEQ ID NO: 2); and wherein the active agent is selectively delivered to activated microglia in the brain of the subject.

50. The method of claim 49, wherein the subject has cancer, and wherein the active agent is selectively delivered to an area of ​​a tumor and / or brain damage within the brain of the subject.

51. The method of claim 49 or 50, wherein the tumor is a glioblastoma.

52. The method of any one of claims 49 to 51, wherein the active agent is a chemotherapeutic agent.

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