HK1227419B - Blood brain barrier shuttle - Google Patents

Description

Blood-brain barrier shuttle

Field of the Invention

The present invention relates to the field of molecular medicine and targeted delivery of therapeutic or diagnostic agents. More specifically, the present invention relates to peptides that target the cerebrospinal fluid (CSF) and brain of an organism.

background

Brain penetration and/or CSF penetration of neurological disorder drugs with low brain permeability (such as, for example, large biotherapeutic drugs or small molecule drugs) are strictly restricted by the extensive and impermeable blood-brain barrier (BBB) or blood-CSF barrier BCSFB (BCSFB) together with other cellular components of the neurovascular unit (NVU). A variety of strategies to overcome this obstacle have been tested, and one is to utilize transcytosis mediated by endogenous receptors expressed on the brain capillary endothelium. Recombinant proteins for these receptors, such as monoclonal antibodies or peptides, have been designed to allow receptor-mediated delivery of biotherapeutics and diagnostic agents to the brain. However, there have been no studies that maximize brain uptake while minimizing the missorting within brain endothelial cells (BECs) and the accumulation of certain organelles (especially those that cause biotherapeutic degradation) in BECs.

Monoclonal antibodies and other biotherapeutics have great therapeutic potential for treating pathologies in the central nervous system (CNS). However, their access to the brain is hampered by the BBB. Previous studies have shown that a very small percentage (approximately 0.1%) of IgG injected into the bloodstream is able to penetrate the CNS compartment (Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)). Due to the low concentration of antibodies in the CNS, this will inevitably limit any pharmacological effect. Cerebrospinal fluid (CSF) is in direct contact with neurons in the CNS.

Therefore, there is a need for a delivery system for drugs for neurological disorders that targets the brain of an organism.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a blood-brain barrier shuttle comprising a brain-effect fruiting body and a brain-targeting peptide, wherein the brain-targeting peptide comprises a three-amino acid peptide motif selected from the group consisting of:

Phe-Lys-Leu(FKL), Arg-Gly-Leu(RGL), Ser-Arg-Gly(SRG), Tyr-Val-Leu(YVL), Trp-Gly-Phe(WGF), Val-Leu-His(VLH), Leu- Tyr-Val(LYV), Leu-Trp-Gly(LWG), Leu-His-Ser(LHS), His-Ser-Arg(HSR), Gly-Leu-Trp(GLW), Gly-Phe-Lys(GFK), Arg-Leu- Ser(RLS), Gly-Ser-Val(GSV), Ser-Val-Ser(SVS), Leu-Gly-Ser(LGS), Val-Arg-Phe(VRF), Ser-Asn-Thr(SNT), Arg-Phe-Arg( RFR), Asn-Thr-Arg(NTR), Leu-Ser-Asn(LSN), Gly-Phe-Val(GFV), Phe-Val-Arg(FVR), Phe-Arg-Leu(FRL), Trp-Arg-Val(WRV), Phe-Ser-Leu(FSL), Val-Phe-Ser(VFS), Val-Ala-Trp(VAW), Ser-Leu-Phe(SLF), Arg-Val-Phe(RVF), Leu-Phe-Trp(LFW), Lys- Val-Ala(KVA), Phe-Trp-Lys(FWK), Ala-Trp-Arg(AWR), Val-His-Gly(VHG), Ser-Val-His(SVH), His-Gly-Val(HGV), Arg-Val- Cys(RVC), Arg-Pro-Gln(RPQ), Gln-Lys-Ile(QKI), Pro-Gln-Lys(PQK), Asn-Gly-Ala(NGA), Lys-Ile-Asn(KIN), Ile-Asn-Gly( ING), Gly-Arg-Pro(GRP), Gly-Ala-Arg(GAR), Ala-Arg-Val(ARV), Leu-Ser-Gly(LSG), Val-Asp-Ser(VDS), Ser-Val-Asp(SVD).

In a specific embodiment of the blood-brain barrier shuttle, the brain targeting peptide comprising a three-amino acid peptide motif comprises 1-25 three-amino acid peptide motifs, specifically 1-15, more specifically 1-10, even more specifically 1-5 three-amino acid peptide motifs.

In a specific embodiment of the blood-brain barrier shuttle, the brain shuttle comprises a brain effector entity, a linker, and a brain targeting peptide comprising a three-amino acid peptide motif, wherein the linker connects the effector entity to the brain targeting peptide comprising a three-amino acid peptide motif.

In a specific embodiment of the blood-brain barrier shuttle, the brain targeting peptide comprising a three-amino acid peptide motif is selected from the group consisting of Seq.Id.No.1-36, preferably Seq.Id.No.1, 6 and 8.

In a specific embodiment of the blood-brain barrier shuttle, the brain effector entity is selected from the group consisting of: a neurological disorder drug, a neurotrophic factor, a growth factor, an enzyme, a cytotoxic agent, an antibody against a brain target, a monoclonal antibody against a brain target, a peptide against a brain target.

In a specific embodiment of the blood-brain barrier shuttle, the brain target is selected from the group consisting of: β-secretase 1, Aβ, epidermal growth factor, epidermal growth factor receptor 2, Tau, phosphorylated Tau, apolipoprotein E4, α-synuclein, oligomeric fragments of α-synuclein, CD20, huntingtin, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, γ-secretase, death receptor 6, amyloid precursor protein, p75 neurotrophin receptor and caspase 6.

In a specific embodiment of the blood-brain barrier shuttle, the brain-effector fruiting entity is selected from the group consisting of a protein, a polypeptide, and a peptide.

In a specific embodiment of the blood-brain barrier shuttle, the brain effector entity comprises a full-length antibody against a brain target, preferably a full-length IgG.

In a specific embodiment of the blood-brain barrier shuttle, the effector entity is a full-length antibody against Aβ.

In a specific embodiment of the blood-brain barrier shuttle, the effector entity is a full-length antibody against phosphorylated Tau.

In a specific embodiment of the blood-brain barrier shuttle, the effector entity is a full-length antibody against alpha-synuclein.

In a second aspect, the present invention provides a pharmaceutical preparation comprising the blood-brain barrier shuttle of the present invention and a drug carrier.

Furthermore, the present invention provides the use of the brain shuttle as a drug, in particular the use of the brain shuttle for treating neurodegenerative disorders (in particular Alzheimer's disease) and for treating neuroinflammatory disorders (in particular multiple sclerosis).

Various aspects of the present invention include BBB-crossing shuttles comprising a brain-targeting peptide covalently linked to an agent to be delivered, such as a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptotic agent, an anti-angiogenic agent, a hormone, an enzyme, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, a smaller fragment of an antibody, an imaging agent, a survival factor, an anti-apoptotic agent, a hormone antagonist, or an antigen. In another aspect of the present invention, the anti-angiogenic agent can be selected from the group consisting of thrombospondin, angiostatin 5, pigment epithelium-derived factor, angiotensin, laminin peptide, fibronectin peptide, plasminogen activator inhibitor, tissue metalloproteinase inhibitor, interferon, interleukin 12, platelet factor 4, IP-10, Gro-B, thrombospondin, 2-methoxyestradiol, prolifera-related protein, carboxiamidotriazole, CMI 01, Manmastat, pentosan polysuiphate, angiopoietin 2 (Regeneron), interferon-α, herbimycin A, PNU 1451 56E, 16K prolactin fragment, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, docetaxel, polyamines, proteasome inhibitors, kinase inhibitors, signaling peptides, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline. In another aspect, the cytokine can be selected from the group consisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-α, tumor necrosis factor-α (TNF-α), or GM-CSF (granulocyte macrophage colony stimulating factor). In other embodiments of the invention, the agent can be a virus, phage, bacteria, liposomes, microparticles, magnetic beads, yeast cells, mammalian cells or cells. In certain aspects, the virus is a lentivirus, a papovavirus, a simian virus 40, a bovine papilloma virus, a polyoma virus, an adenovirus, a vaccinia virus, an adeno-associated virus (AAV), or a herpes virus. The agent can also be a eukaryotic expression vector, more preferably a gene therapy vector.

The brain targeting peptides of the present invention can be attached to a solid support, for example, an array or beads. Embodiments of the present invention can also include isolated peptide mimetics comprising a sequence that mimics a peptide motif selected from the group consisting of: Phe-Lys-Leu (FKL), Arg-Gly-Leu (RGL), Ser-Arg-Gly (SRG), Tyr-Val-Leu (YVL), Trp-Gly-Phe (WGF), Val-Leu-His (VLH), Leu-Tyr-Val (LYV), Leu-Trp-Gly (LWG), Leu-His-Ser (LHS), His-Set-Arg (HSR), Gly-Leu-Trp (GLW), Gly-Phe- Lys(GFK), Arg-Leu-Ser(RLS), Gly-Ser-Val(GSV), Ser-Val-Ser(SVS), Leu-Gly-Ser(LGS), Val-Arg-Phe(VRF), Ser-Asn-Thr(SNT), Arg-Phe- Arg(RFR), Asn-Thr-Arg(NTR), Leu-Ser-Asn(LSN), Gly-Phe-Val(GFV), Phe-Val-Arg(FVR), Phe-Arg-Leu(FRL), Trp-Arg-Val(WRV), Phe-Ser- Leu(FSL), Val-Phe-Ser(VFS), Val-Ala-Trp(VAW), Ser-Leu-Phe(SLF), Arg-Val-Phe(RVF), Leu-Phe-Trp(LFW), Lys-Val-Ala(KVA), Phe-Trp -Lys(FWK), Ala-Trp-Arg(AWR), Val-His-Gly(VHG), Ser-Val-His(SVH), His-Gly-Val(HGV), Arg-Val-Cys(RVC), Arg-Pro-Gln(RPQ), Gln-Lys -Ile (QKI), Pro-Gln-Lys (PQK), Asn-Gly-Ala (NGA), Lys-Ile-Asn (KIN), Ile-Asn-Gly (ING), Gly-Arg-Pro (GRP), Gly-Ala-Arg (GAR), Ala-Arg-Val (ARV), Leu-Ser-Gly (LSG), Val-Asp-Ser (VDS), Ser-Val-Asp (SVD), or a peptide mimetic comprising a sequence that mimics a peptide consisting of Seq. Id. No. 1 to Seq. Id. No. 36, wherein the peptide is enriched in CSF and brain.

Other embodiments include methods for targeting the delivery of an agent to the CSF and brain or its vascular system in a subject by obtaining a brain targeting peptide of the invention as described herein, or according to the methods of the invention as described herein, operably linking the peptide to the agent, and administering the peptide-linked agent to the subject. The subject can be, but is not limited to, a primate, monkey, human, mouse, dog, cat, rat, sheep, horse, cow, goat, or pig. The agent can be a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptotic agent, an anti-angiogenic agent, an enzyme, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, an antigen, a survival factor, an anti-apoptotic agent, a hormone antagonist, a virus, a phage, a bacterium, a hormone, a microparticle, a magnetic bead, a microscopic device, a yeast cell, a mammalian cell, a cell, or an expression vector.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A: Schematic depiction of in vivo phage display screening and CSF sampling. In each round of selection, phage are administered intravenously (tail vein) and then recovered from CSF and blood, amplified, pooled, and used for the next round of selection. Increased recovery of specific phage peptide clones in each subsequent round reflects the selection of peptides that preferentially target the CSF. Figure 1B: In addition, plaque forming units (pfu) based on the first round of selection illustrate the dynamics of phage in blood and CSF.

Figure 2A-D: Selection in CSF and blood after intravenous administration of a phage library. For each round of selection, all tripeptides extracted from phage isolated from CSF were pooled and sequenced. The figure shows the enrichment of certain peptide motifs in both CSF and blood. Selection was much stronger in the CSF compartment compared to the blood compartment.

Figure 3: Shows the overall in vivo selection strategy over four rounds in rats. Starting with two branches, the final fourth round of selection converges with selection in three different animals.

Figure 4: Specific phage clones in CSF and blood after four rounds of in vivo selection. This figure shows data for some peptides enriched on phage particles compared to empty phage. Many more phage clones were found in CSF compared to empty phage (based on plaque forming units (pfu)).

Figure 5: Specific phage clones bind to the brain's vasculature. Specific phage clones were administered intravenously to allow for in vivo targeting. Subsequently, phage binding specifically to brain blood vessels and capillaries was detected using immunohistochemistry. No staining was observed with empty phage, while clones Seq. Id. No. 1, Seq. Id. No. 6, and Seq. Id. No. 8, in particular, exhibited strong and specific staining of brain capillaries in the cortex.

Figure 6A-C: B: Enrichment of specific peptides linked to streptavidin (SA) in CSF. A specific peptide (Seq. Id. No. 8) (synthesized and linked to SA via N-terminal biotin) was found at higher concentrations in CSF than a control scrambled peptide. A: This also occurs when peptide Seq. Id. No. 8 is displayed on T7 phage. C: This figure also shows staining of a Seq. Id. No. 8 phage clone colonizing brain capillaries with a brain vascular marker lectin.

Figures 7A and B: Brain shuttle peptides with Seq. Id. No. 8 enhance the inhibitory activity of brain BACE1 peptides. Biotinylated BACE1 inhibitory peptides, alone (Figure 7B) or in combination with the similarly biotinylated brain shuttle peptides with Seq. Id. No. 8 (Figure 7A), pre-linked to streptavidin, were then injected intravenously (10 mg streptavidin/kg) into at least three CM cannula-treated rats. BACE1 peptide inhibitor-mediated Aβ40 reduction was measured by Aβ1-40 ELISA in blood (black triangles/circles) and CSF (grey triangles/circles) at the indicated time points. For better visualization, a dashed line at 100% was drawn in the graph. (7A) Percent reduction in blood (black triangles) and CSF (grey triangles) Aβ40 in rats injected with brain shuttles with Seq. Id. No. 8 and BACE1 inhibitor peptide pre-linked to streptavidin at a 3:1 ratio. (7B) Percent reduction of blood (black circles) and CSF (grey circles) Aβ40 in rats injected with individual BACE1 inhibitor peptides (tetramer display) pre-linked to streptavidin.

Detailed description of embodiments of the invention

definition

" Blood-brain barrier " or " BBB " refers to the physiological barrier between peripheral circulation and brain and spinal cord, and it is formed by the tight junction in the endothelial membrane of brain capillaries, thereby setting up the tight barrier that restricts molecules (even very small molecules such as urea (60 daltons)) from being transported into the brain. The BBB in the brain, the blood-spinal cord barrier in the spinal cord and the blood-retinal barrier in the retina are the connected capillary barriers in CNS, and are collectively referred to as blood-brain barrier or BBB in this article. BBB also includes blood-CSF barrier (choroid plexus), in which the barrier is made up of ependymal cells rather than capillary endothelial cells.

"Brain effector entities" refer to molecules that are transported across the BBB to the brain. Effector entities generally possess characteristic therapeutic activities that are desired to be delivered to the brain. Effector entities include neurological disorder drugs and neuroactive and cytotoxic agents such as, for example, peptides, proteins, enzymes, and antibodies, particularly monoclonal antibodies or fragments thereof directed against brain targets.

As used herein, a "brain targeting peptide" comprises at least a three amino acid peptide motif selected from the group consisting of: Phe-Lys-Leu (FKL), Arg-Gly-Leu (RGL), Ser-Arg-Gly (SRG), Tyr-Val-Leu (YVL), Trp-Gly-Phe (WGF), Val-Leu-His (VLH), Leu-Tyr-Val (LYV), Leu-Trp-Gly (LWG), Leu-His-Ser (LHS), His-Ser-Arg (HSR), Gly-Leu-Trp (GLW) , Gly-Phe-Lys(GFK), Arg-Leu-Ser(RLS), Gly-Ser-Val(GSV), Ser-Val-Ser(SVS), Leu-Gly-Ser(LGS), Val-Arg-Phe(VRF), Ser-Asn -Thr(SNT), Arg-Phe-Arg(RFR), Asn-Thr-Arg(NTR), Leu-Ser-Asn(LSN), Gly-Phe-Val(GFV), Phe-Val-Arg(FVR), Phe-Arg-Leu(FRL) ,Trp-Arg-Val(WRV),Phe-Ser-Leu(FSL),Val-Phe-Ser(VFS),Val-Ala-Trp(VAW),Ser-Leu-Phe(SLF),Arg-Val-Phe(RVF),Leu-Phe -Trp(LFW), Lys-Val-Ala(KVA), Phe-Trp-Lys(FWK), Ala-Trp-Arg(AWR), Val-His-Gly(VHG), Ser-Val-His(SVH), His-Gly-Val(HGV) , Arg-Val-Cys (RVC), Arg-Pro-Gln (RPQ), Gln-Lys-Ile (QKI), Pro-Gln-Lys (PQK), Asn-Gly-Ala (NGA), Lys-Ile-Asn (KIN), Ile-Asn-Gly (ING), Gly-Arg-Pro (GRP), Gly-Ala-Arg (GAR), Ala-Arg-Val (ARV), Leu-Ser-Gly (LSG), Val-Asp-Ser (VDS), Ser-Val-Asp (SVD). It must be understood that the three amino acid peptide motifs in the brain targeting peptides of the present invention can be sequential and/or overlapping. For example, in the case of overlapping motifs, in the brain targeting peptide, the second amino acid of a first three amino acid motif can be the first amino acid of a second three amino acid motif, and/or the third amino acid of a first amino acid motif can be the first amino acid of a third amino acid motif.

As used herein, "neurological disorder" refers to a disease or disorder that affects the CNS and/or has a cause in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, a disease or disorder of the eye, a viral or microbial infection, autoimmunity, inflammation, ischemia, a neurodegenerative disease, epileptic seizure, a behavioral disorder, and a lysosomal storage disease. For the purposes of this application, it will be understood that the CNS includes the eye, which is typically isolated from the rest of the body by the blood-retinal barrier. Specific examples of neurological diseases include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellaratrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome), syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and heterodegenerative disorders of the nervous system (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, Lafora disease, disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementias (including, but not limited to, Pick's disease and spinocerebellar ataxia), cancer (e.g., cancer of the CNS and/or brain, including brain metastases from cancer elsewhere in the body), multiple sclerosis (relapsing remitting, primary progressive, and secondary progressive forms).

A "neurological disorder drug" is a drug or therapeutic agent for treating one or more neurological diseases. Neurological disorder drugs of the present invention include, but are not limited to, small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified forms of natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small inhibitory RNAs (siRNAs) and short hairpin RNAs (shRNAs)), ribozymes, and small molecules, or active fragments of any of the foregoing. Exemplary neurological disorder drugs of the present invention are described herein and include, but are not limited to: antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules and active fragments of any of the foregoing, which themselves or specifically recognize and/or act on (i.e., inhibit, activate, or detect) CNS antigens or target molecules, such as, but not limited to, amyloid precursor protein or a portion thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophic factors. Non-limiting examples of drugs for neurological disorders and the corresponding diseases that can be treated with them: brain-derived neurotrophic factor (BDNF), chronic brain injury (neurogenesis), fibroblast growth factor 2 (FGF-2), anti-epidermal growth factor receptor brain cancer, (EGFR)-antibodies, glial cell line-derived neurotrophic factor, Parkinson's disease, (GDNF), brain-derived neurotrophic factor (BDNF), amyotrophic lateral sclerosis, depression, lysosomal enzymes, lysosomal storage diseases of the brain, ciliary neurotrophic factor (CNTF), amyotrophic lateral sclerosis, neuregulin-1, schizophrenia, anti-HER2 antibodies (e.g., trastuzumab), brain metastases from HER2-positive cancers.

An "imaging agent" is a compound that has one or more properties that allow its presence and/or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds that incorporate a labeled entity that allows detection.

"CNS antigens" or "brain targets" are antigens and/or molecules expressed in the CNS (including the brain) that can be targeted by antibodies or small molecules. Examples of such antigens and/or molecules include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, TREM2 huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), and caspase 6. In one embodiment, the antigen is BACE1.

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

As used herein, "antibody fragment" includes a portion of an intact antibody that retains the ability to bind to an antigen. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as, for example, single-chain Fab, scFv, and multispecific antibodies formed from multiple antibody fragments. The "single-chain Fab" format is described, for example, in Hust M. et al. BMC Biotechnol. 2007 Mar 8;7:14.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies comprising the group are identical and/or bind to the same epitope, except for possible variants (which may arise during the process of producing the monoclonal antibody, such variants usually existing in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). Specific examples of monoclonal antibodies herein include chimeric antibodies, humanized antibodies, and human antibodies, including antigen-binding fragments thereof. Monoclonal antibodies herein specifically include: "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America) 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World Monkeys, such as baboons, rhesus monkeys, or cynomolgus monkeys) and human constant region sequences (U.S. Patent No. 5,693,780).

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioisotopes (e.g., At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids); alkaloids) (vincristine, vinblastine, etoposide, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleases; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.

An "effective amount" of an agent, such as a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

As used herein, "linker" refers to a chemical linker or a peptide linker that covalently links the different entities of the blood-brain barrier shuttle of the present invention together. For example, the linker links the brain effector entity to the brain targeting peptide of the present invention.

A peptide linker can be used, which comprises 1-20 amino acids connected by peptide bonds. In certain embodiments, the amino acids are selected from the 20 naturally occurring amino acids. In certain other embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is a chemical linker. In certain embodiments, the linker is a single-chain peptide having an amino acid sequence of at least 25 amino acids in length, preferably 32-50 amino acids in length. In one embodiment, the linker is (GxS)n, wherein G = glycine, S = serine (x = 3, n = 8, 9 or 10 and m = 0, 1, 2 or 3) or (x = 4 and n = 6, 7 or 8 and m = 0, 1, 2 or 3), preferably, wherein x = 4, n = 6 or 7 and m = 0, 1, 2 or 3, more preferably, wherein x = 4, n = 7 and m = 2. In one embodiment, the linker is ( _G4S ) ₄ (Seq.Id.No.17). In one embodiment, the linker is (G ₄ S) ₆ G ₂ (Seq. Id. No. 13).

A variety of chemical linkers can be used for conjugation. For example, brain shuttle peptides and brain effector fruiting entities can be conjugated using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and biactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). The linker can be a "cleavable linker" to facilitate release of the effector entity after delivery to the brain. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a linker containing a disulfide bond can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020).

Covalent conjugation can be carried out directly or by a joint. In certain embodiments, direct conjugation is carried out by constructing a protein fusion (i.e., by the gene fusion of two genes encoding brain targeting peptides and effector entities and expressed as a single protein). In certain embodiments, direct conjugation is carried out by forming a covalent bond between the reactive group on one of the two parts of the brain shuttle peptide and the corresponding group on the brain effector entity or the acceptor. In certain embodiments, direct conjugation is carried out by modifying (i.e., genetic modification) one of the two molecules to be conjugated, thereby including forming a reactive group (as a non-limiting example, sulfhydryl or carboxyl) for the covalent connection with other molecules to be conjugated under appropriate conditions. As a non-limiting example, a molecule (i.e., amino acid) with a desired reactive group (i.e., cysteine residues) can be introduced, for example, introduced into the brain shuttle peptide, and formed a disulfide bond with a neurological drug. Methods for covalently conjugating nucleic acids to proteins are also known in the art (ie, photocrosslinking, see, eg, Zatsepin et al. Russ. Chem. Rev. 74:77-95 (2005)). Conjugation can also be performed using a variety of linkers. For example, the brain shuttle peptide and effector entity can be conjugated using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and biactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Peptide linkers comprising 1-20 amino acids linked by peptide bonds can also be used. In certain such embodiments, the amino acids are selected from the 20 naturally occurring amino acids. In certain other such embodiments, the one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. The linker can be a "cleavable linker," thereby facilitating release of the effector entity after delivery to the brain. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a linker containing a disulfide bond can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020).

The term "pharmaceutical formulation" refers to a preparation that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the formulation would be administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and symptom relief or improved prognosis. In certain embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

The term "phage display library" refers to a plurality of phages, wherein random heterologous peptides have been transformed into phage capsid proteins and are presented on the phage surface. In some aspects, the peptide may be restricted by the cysteine residues of the peptide. The method may further include administering the phage separated from the second subject to at least a third subject, obtaining a sample of one or more tissues or fluids (including CSF) from the third subject, and identifying the peptide sequence displayed by the phage separated from the tissue or fluid (including CSF) of the third subject. In some aspects, the administration of phage is by injection, preferably by intravenous injection. The subject can be a mammal, and in specific aspects, the mammal is a rodent, non-human primate or human. The method may further include amplifying the phage of a sample separated from a subject before being administered to another subject. Amplification may require PCR amplification of all or part of the phage nucleic acid, then the amplified fragment is cloned into the second phage, and/or phage propagation is carried out by a phage host organism, wherein the phage host organism is, for example, a bacterium that supports phage replication.

The term "in vivo cerebrospinal fluid (CSF) sampling" refers to obtaining CSF specifically from a subject. The subject can be a mammal, and in specific aspects, the mammal is a rodent, a non-human primate, or a human. The sample is obtained using a cannula inserted into the cisterna magna, as described in detail herein.

The term "simultaneously" may be used to mean that the sample is obtained within a timeframe (minutes to hours to days) that provides for sample collection from the CSF.Other embodiments of the invention include isolated peptides identified by the methods described herein.

The term "peptide motif" refers to a three amino acid peptide sequence that has the ability to target or bind to a specific tissue or receptor or has the ability to be transported across the BBB or BCSFB.

As used herein, "selective binding" in no way excludes binding to other cells or substances, but means preferential binding to a target tissue, organ, vascular system, or its receptors. Selective binding can include a 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold preference for a selected tissue/target compared to non-selected tissues/targets.

"Brain targeting peptide" as used herein is a peptide comprising at least one peptide motif as defined herein, characterized in that it is selectively localized in CSF and/or brain or its vasculature. A brain targeting peptide may comprise more than one peptide motif, and the peptide motifs may be contiguous or separated by non-motif amino acid sequences.

In some embodiments, the present invention relates to a method for the selective localization of a target peptide of a specific embodiment of the present invention. For example, selective localization can be determined by the method disclosed below, wherein the target sequence of inference is combined in the protein displayed on the outer surface of the phage. The described phage library of the targeting peptide of the multiple different amino acid sequences described in genetic modification is used to the experimenter, then the CSF or the brain that derives from one or more experimenters is gathered, and the phage that is present in the described liquid or organ or is relevant to the described liquid or organ is identified. If, compared with a control fluid or an organ, the phage expressing the targeting peptide sequence shows larger combination or location in the described liquid or an organ, then it is believed that the phage selective localization of the described expression targeting peptide sequence is in the liquid or an organ. Preferably, compared with a control fluid or an organ, the selective localization of the targeting peptide should cause the enrichment of phage or peptide in the described fluid or an organ more than twice. More preferably, compared with a control organ, in target fluid or an organ, the selective localization of the enrichment causing at least three times, four times, five times, six times, seven times, eight times, nine times, ten times or more is identified. Alternatively, when the phage recovered from the target or selected fluid or organ is injected into a second, third, fourth or more experimenter or is contacted with a second, third, fourth or more experimenter for additional screening, the phage expressing the targeting peptide sequence that selective positioning is shown preferably shows increased enrichment in the target fluid or organ compared to a control fluid or organ. Another alternative way of determining the selective positioning or binding of the phage expressing the putative target peptide preferably shows twofold, more preferably threefold or higher enrichment in the target fluid or organ compared to a control phage expressing a non-specific peptide or not genetically modified to express any putative target peptide. Another way of determining selective positioning is that the phage expressing the target peptide is blocked at least in part by a synthetic peptide comprising the target peptide sequence that is administered in conjunction with the target fluid or organ.

CSF selection

Vascular mapping by in vivo phage display reveals biochemical "addresses" that are selectively expressed within different vascular systems. This type of approach has been used to discover ligand-receptor systems that can be used to deliver agents to specific tissues (Arap et al., 1998, Pasqualini et al., 1996, Arap et al., 2002, Kolonin et al., 2001, Pasqualini et al., 2000). This screening method is based on the ability of short ligand peptides from combinatorial libraries (displayed on M13-based phage vectors) to target specific organs after systemic administration (Pasqualini et al., 2000). Peptide-targeted tissues and disease states have been separated and, in some cases, have led to the identification of corresponding vascular receptors (Arap et al., 1998, Pasqualini et al., 1996, Arap et al., 2002, Kolonin et al., 2001, Rajotte and Ruoslahti, 1999, Kolonin et al., 2002, Kolonin et al., 2004). It is noteworthy that researchers have reported the application of phage display libraries in cancer patients. A ligand motif identified as interleukin-11-like peptide and its targeting to interleukin-11 receptors are used as a potential strategy for the delivery of targeted therapeutic agents in human prostate cancer (Zurita et al., 2004). The key step in the selection of phage display random peptide libraries in vivo is the sampling strategy, that is, how to recycle phage and where to recycle phage. Preferably, for example, the in vivo selection strategy should not only be based on the combination with tissue or receptor, but also include a functional step as transport through a cell barrier. This is particularly important when the purpose is to identify a novel transport system involving the blood-brain barrier (BBB) or blood-CSF barrier (BCSFB).

In some cases, people may need to obtain highly enriched samples, which not only depends on binding, but also depends on transport by cell membrane. In these cases, typical screening methods are not optimal, and thus the methods described herein provide more effective identification methods with the targeting peptides that can be developed into the characteristics of drugs, targeting agents or diagnostic agents. The methods described herein are used for further enrichment of selected phage colonies, and based on binding and transport characteristics, different peptides are selected. The single screening in a single living subject selects a subgroup of peptides, but the colony needs to be enriched for selectivity and transport peptides. The inventor provides a method for obtaining an improved enrichment of a targeting peptide that can be used in a human subject, for example." subject" generally refers to mammals. In some preferred embodiments, the subject is a rodent, primate, monkey or human. In a more preferred embodiment, the subject is a human.

Identification of targeting peptides

The present invention includes methods for identifying one or more brain-targeting peptides or molecular targets that can be used to localize compositions in CSF, the brain, or the associated vasculature. Fluids or organs from a subject are screened for peptides having N residues, where N can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more residues, and random phage libraries are generated for several peptide motifs. In one example, different clones containing the following tripeptide motifs exhibited high frequency, selective targeting or binding, and presence in the brain in CSF:

Phe-Lys-Leu(FKL), Arg-Gly-Leu(RGL), Ser-Arg-Gly(SRG), Tyr-Val-Leu(YVL), Trp-Gly-Phe(WGF), Val-Leu-His(VLH), Leu- Tyr-Val(LYV), Leu-Trp-Gly(LWG), Leu-His-Ser(LHS), His-Ser-Arg(HSR), Gly-Leu-Trp(GLW), Gly-Phe-Lys(GFK), Arg-Leu- Ser(RLS), Gly-Ser-Val(GSV), Ser-Val-Ser(SVS), Leu-Gly-Ser(LGS), Val-Arg-Phe(VRF), Ser-Asn-Thr(SNT), Arg-Phe-Arg( RFR), Asn-Thr-Arg(NTR), Leu-Ser-Asn(LSN), Gly-Phe-Val(GFV), Phe-Val-Arg(FVR), Phe-Arg-Leu(FRL), Trp-Arg-Val(WRV), Phe-Ser-Leu(FSL), Val-Phe-Ser(VFS), Val-Ala-Trp(VAW), Ser-Leu-Phe(SLF), Arg-Val-Phe(RVF), Leu-Phe-Trp(LFW), Lys- Val-Ala(KVA), Phe-Trp-Lys(FWK), Ala-Trp-Arg(AWR), Val-His-Gly(VHG), Ser-Val-His(SVH), His-Gly-Val(HGV), Arg-Val- Cys (RVC), Arg-Pro-Gln (RPQ), Gln-Lys-Ile (QKI), Pro-Gln-Lys (PQK), Asn-Gly-Ala (NGA), Lys-Ile-Asn (KIN), Ile-Asn-Gly (ING), Gly-Arg-Pro (GRP), Gly-Ala-Arg (GAR), Ala-Arg-Val (ARV), Leu-Ser-Gly (LSG), Val-Asp-Ser (VDS), Ser-Val-Asp (SVD). Comparison of the selected peptide motifs with sequences available in online protein databases showed that multiple candidate proteins share homology or similar sequences with these peptide motifs. Mechanistic studies around these motifs continue to provide a new platform for the identification of peptides and targets for targeting CSF fluid and the brain and associated vascular systems, as well as combinations thereof. The findings will also have important clinical implications, as the newly identified motifs can serve as peptide mimetic drug leads and can be optimized to guide the delivery of different therapeutic moieties.One method involves injecting the phage library intravenously and recovering the sample a few minutes later.

" phage display library " is the collection of the phage that has been genetically modified to express one group of targeting peptides of inference on its outer surface.In a preferred embodiment, the dna sequence of the targeting peptide of encoding said inference is inserted in the gene of coding phage capsid protein in accordance with reading frame.In other preferred embodiments, the targeting peptide sequence part of said inference is all 20 kinds of amino acid whose random mixture, part is non-random thing.In certain preferred embodiments, the targeting peptide of inference of phage display library shows one or more cysteine residues in the fixed or random position in said targeting peptide sequence.For example, cysteine can be used to produce cyclic peptide.The targeting peptide of selective binding different organs, tissues or cell types can be separated (Pasqualini and Ruoslahti, 1996, Pasqualini, 1999) by " biological panning (biopanning) ".In brief, the library of the phage that will comprise the targeting peptide of inference is administered to animal, and gather the sample that comprises the organ, tissue, fluid or cell type of phage. In a preferred embodiment suitable for lytic phage, phage can be bred in vitro between multiple rounds of bio-panning in bacteria. Bacteria are lysed by phage, thereby secreting multiple copies of the phage displaying a specific insert. Phages that bind to target molecules or are transported to specific fluids or tissues can be collected from target fluids or organs and then amplified by growing them in host bacteria. If necessary, the amplified phage can be administered to the host, and samples of fluids and organs can be collected again. Multiple rounds of bio-panning can be carried out until selective binding agents and transporter colonies are obtained. The amino acid sequence of the peptide is determined by sequencing the DNA corresponding to the targeting peptide in the phage genome. The targeting peptide identified can then be produced by standard protein chemistry techniques using synthetic peptides. This method allows the targeting peptide to be circulated in the functional assay without preference, without any preconceived notions about the properties of their targets. Once a candidate target is identified as a receptor for the targeting peptide, it can be separated using standard biochemical methods. Purify and clone. In certain embodiments, a subtraction protocol can be utilized to further reduce background phage binding. The purpose of the subtraction is to remove phage from the library that bind to tissues other than the tissue of interest. In an alternative embodiment, the phage library can be pre-screened for subjects that do not have the selected fluid, tissue, or organ. Phage display technology involves genetically manipulating phages so that small peptides can be expressed on their surfaces (Smith and Scott, 1985 and 1993). The potential range of applications for this technology is quite broad, and the past decade has seen considerable progress in the construction of phage-displayed peptide libraries and the development of screening methods using libraries to isolate peptide ligands. For example, the use of peptide libraries has made it possible to characterize interaction sites and receptor-ligand binding motifs in a variety of proteins (antibodies involved in inflammatory responses or integrins that mediate cell adhesion). This method has also been used to identify novel peptide ligands as clues for the development of peptide mimetic drugs or imaging agents other than peptides (Arap et al., 1998a). Larger protein domains, such as single-chain antibodies, can also be displayed on the surface of phage particles (Arap et al., 1998a).

Selection of phage display system

In vivo selection studies previously conducted in mice preferentially utilized libraries of random peptides expressed in fUSE5 vectors as fusion proteins with gene III capsular proteins (Pasqualini and Ruoslahti, 1996). The preferred phage system for identifying transporter peptides is the T7 phage system. T7 phage particles are very small (approximately 50 nm in diameter), allowing for transport by cells using natural organelle intracellular sorting. The M13 phage system is significantly larger in size and may prevent cellular uptake and intracellular sorting. Therefore, the enrichment of target peptides in fluids and organs is prevented from being protected by cell membranes such as the blood-brain barrier (BBB) and the blood-CSF barrier (BCSFB). The number and diversity of individual clones present in a given library are important factors for successful in vivo selection. Preferably, a primary library is used, which is less likely to have an overrepresentation of defective phage clones. Preferably, a primary library with high diversity and all phage clones in equal proportion is used. The preparation of the library should be optimized to 10 ⁸ -10 ⁹ plaque forming units (pfu) / ml. In certain embodiments, a large amplification strategy is applied before each round of selection. Phage libraries displaying linear, cyclic or bicyclic peptides can be used within the scope of the present invention. However, phage libraries displaying 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more residues are preferred. Cyclic peptides are also preferred. However, due to the presence of multiple conformers with different disulfide bond arrangements, the generation of homologous synthetic peptides, although possible, can be complicated.

Targeted delivery

Peptides targeting the vasculature have been linked to cytotoxic drugs or proapoptotic peptides to generate compounds that are more potent and less toxic than the parent compound.

The present invention describes methods and compositions for selectively targeting CSF fluid and the brain. "Receptors" for targeting peptides include, but are not limited to, any molecule or macromolecular complex that binds directly or indirectly to the targeting peptide. Non-limiting examples of receptors include peptides, proteins, glycoproteins, lipoproteins, epitopes, lipids, carbohydrates, multimolecular structures, and specific conformations of one or more molecules. In preferred embodiments, a "receptor" is a naturally occurring molecule or a complex of molecules present on the surface of a cell within a target tissue or organ. Preferably, a "receptor" is a naturally occurring molecule or a complex of molecules present on or within a tissue, organ, bloodstream, or its vascular system. More preferably, a "receptor" is a transport receptor that is internalized on a cell and transported to the opposite side of the cell via a transcytosis mechanism. In certain embodiments, therapeutic agents can be attached to the targeting peptide or fusion protein, for example, for selective delivery to the CSF and brain. Suitable agents or factors for use can include any chemical compound or biological product that has a therapeutic effect on brain diseases. Chemotherapeutic agents and methods of administration, dosages, etc. are well known to those skilled in the art (e.g., see "Physicians' Desk Reference," Goodman & Oilman's "The Pharmacological Basis of Therapeutics," and "Remington's Pharmaceutical Sciences," 15th Edition, pp. 1035-1038 and 1570-1580, each of which is incorporated herein by reference in relevant part) and, in light of the disclosure herein, can be combined with the present invention. Depending on the condition of the subject being treated, some variation in dosage will need to occur. In any case, the person responsible for administration determines the appropriate dosage for the individual subject.

Pharmaceutical composition

When considering clinical applications, it may be necessary to prepare pharmaceutical compositions - expression vectors, viral stocks, proteins, antibodies and drugs - in a form suitable for the intended application. Generally, this will require preparing a composition that is substantially free of impurities that may be harmful to humans or animals. It is generally necessary to use appropriate salts and buffers to stabilize the delivery vehicle and allow it to be taken up by the target cells. When recombinant cells are introduced into a patient, a buffer is also used. The aqueous composition of the present invention can contain an effective amount of protein, peptide, fusion protein, recombinant phage and/or expression vector, which is dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other unwanted reactions when administered to an animal or human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. The use of such media and agents as pharmaceutically active substances is well known in the art. To the extent possible, its use in therapeutic compositions is considered, with the exception of any conventional media or agents that are incompatible with the protein or peptide of the present invention. Supplementary active ingredients may also be incorporated into the composition. The active compositions of the present invention may include classical pharmaceutical formulations. Administration of the compositions of the present invention may be carried out by any conventional route, as long as the target fluid, tissue, or organ is accessible via the route. This includes oral, nasal, oral, rectal, vaginal, or topical administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, or intravenous injection. The compositions are typically administered as pharmaceutically acceptable compositions (as described above). Pharmaceutical forms suitable for injectable applications include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it is easily injectable. It must be stable under the conditions of preparation and storage and must be preserved to prevent contamination by microorganisms (such as bacteria and fungi). The earner may be a solvent or dispersion medium, for example, comprising water, ethanol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. For example, by using a coating such as lecithin, in the case of a dispersant, by maintaining the required particle size and by using a surfactant, appropriate fluidity can be maintained. Prevention of microbial action can be caused by a variety of antibacterial and antifungal agents, for example, parabens, chiorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents are preferably included, for example, sugar or sodium chloride. Prolonged absorption of the injection composition can be caused by using an agent that delays absorption (for example, aluminum monostearate and gelatin) in the composition. Sterile injection solutions are prepared by combining the active compound with the required amount in a suitable solvent with the required various other ingredients listed above, followed by filtration sterilization. Typically, a dispersant is prepared by combining a variety of sterilized active ingredients into a sterile excipient comprising a basic diffusion medium and the required other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Example

The following examples are included to demonstrate preferred embodiments of the present invention. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present invention and, as such, can be considered to constitute preferred modes for its practice. However, in light of this disclosure, it will be understood by those skilled in the art that various changes can be made in the disclosed specific embodiments and still obtain the same or similar results without departing from the spirit and scope of the present invention.

Example 1: Surgical implantation of a cannula in the cisterna magna.

The cannula was inserted into the cisterna magna (CM) using a modification of the method previously described by Sarna et al. (1983) (Waring et al. 2010, 192249-253) (Sarna et al. 1983, 383-388). Anesthetized Wistar rats (200-350 g) were mounted on a stereotaxic apparatus and a midline incision was made on the shaved top of the head (all the way back to the inter-scapular region). Two holes were drilled in the top area and sealing screws were installed in the holes. Another hole was drilled at the lateral occipital crest and used to stereotactically guide the cannula into the CM. Dental cement was used around the cannula and screw to keep them in place. After the cement dried, the skin wound was sutured with 4/0 supramid yarn. When the cannula was in place, spontaneous outflow of cerebrospinal fluid (CSF) occurred. Rats were removed from the stereotaxic apparatus, received appropriate post-operative analgesia, and allowed to recover for at least one week, until no evidence of blood was observed in the CSF.All animal procedures were fully approved by the Institutional Animal Care Authority (Permission #2474).

Example 2: CSF and blood were serially collected from unanesthetized adult rats.

The conscious rat with CM cannula was gently held in the hand. The mandrain was extracted from the cannula, and 10 μL of spontaneous CSF was collected. Because the smoothness of the cannula was ultimately compromised, only clarified CSF samples were included in this study without blood contamination or signs of discoloration. 10-20 μL of blood was collected in parallel from a small incision at the end of the tail into a tube containing heparin (Sigma-Aldrich). CSF and blood were collected at different time points after intravenous injection of phage. Before collecting each CSF sample, 10-15 μL of fluid was discarded, which represents the catheter dead volume.

Example 3: T7 phage peptide library modification.

The library was constructed using the T7Select 10-3b vector as described in the T7Select system manual (Novagen) (Rosenberg et al. In Novations 6, 1-6 1996). Briefly, random 12-mer insert DNA was synthesized in the following format:

5'-GGGGATCCGAATTCT(NHH) ₁₂ TAAGCTTGCGGCCGCA-3`(Seq.Id.No.37)

←3`-TCGAACGCCGGCGT-5’(Seq.Id.No.38)

The NHH codon is used to avoid excessive representation of two stop codons and amino acids in the insert. N represents each nucleotide in an equimolar ratio of hand-mixed, and H is an equimolar ratio of adenine and cytosine nucleotides mixed manually. By continuously incubating for 3 hours with dNTPs (Novagen) and Klenow enzyme (New England Biolabs) at 37°C in Klenow buffer (New England Biolabs), the single-stranded region is converted into duplex DNA. After the reaction, the double-stranded DNA is recovered by EtOH precipitation. The resulting DNA is digested with EcoRI and HindIII restriction enzymes (both from Roche). The digested and purified (QIAquick, Qiagen) insert is then connected (T4 ligase, New England Biolabs) to a pre-digested T7 vector in frame after amino acid 348 of the capsid 10B gene. The ligation reaction was incubated at 16°C for 18 hours and then packaged in vitro. According to the instructions for use of the T7Select 10-3b cloning kit (Novagen), in vitro packaging into phage was performed, and the packaging solution was propagated once with E. coli (BLT5615, Novagen) until lysis. The lysate was centrifuged, titrated, and frozen at -80°C as a glycerol stock.

Example 4: PCR phage identification.

The variable regions of broth- or plate-grown phage were directly PCR amplified using in-house designed 454/Roche-Amplicon fusion primers. The forward fusion primer contained sequences flanking the variable region (NNK) ₁₂ (template-specific), GSFLX titanium adapter A, and a four-base library key sequence (TCAG):

5`-CCATCTCATCCCTGCGTGTCTCCGACTCAGGGAGCTGTCGTATTCCAGTC-3`(Seq.Id.No.39)

The reverse fusion primer also carries the biotin for attachment to the capture beads and the GS FLX Titanium Adapter B required for clonal amplification during emulsion PCR:

5`-Biotin-

CCTATCCCCTGTGTGCCTTGGCAGTCTCAGAACCCCTCAAGACCCGTTrA-3`(Seq.Id.No.40)

Then, according to the 454GS-FLX titanium process, the amplicon was subjected to 454/Roche pyrophosphate sequencing. For manual Sanger sequencing (Applied Biosystems Hitachi 3730 xl DNA analyzer), T7 phage DNA was PCR amplified and sequenced using the following primers:

PCR forward: 5'-AGTACGCAATGGGCCACG-3' (Seq.Id.No.41)

PCR reverse: 5'-GAGCGCATATAGTTCCTCC-3' (Seq.Id.No.42)

Sequencing forward: 5`-CAGGAGCTGTCGTATTCC-3` (Seq.Td.No.43)

Sequencing reverse: 5`-AAAAACCCCTCAAGACCCG-3` (Seq.Id.No.44)

Inserts from individual phage plaques were PCR amplified using the Roche Rapid Start DNA Polymerase Kit (following the supplier's instructions) with a hot start (95°C, 10 min) and 35 amplification cycles: 95°C for 50 sec, 50°C for 1 min, and 72°C for 1 min.

Example 5: Phage system, application and selection.

Phage from the library, wild-type phage, CSF and blood recovered phage or individual clones were propagated in Escherichia coli BL5615 in broth (TB medium) (Sigma Aldrich) or on 500 cm2 plates (ThermoScientific) at 37°C for 4 hours. Phage were extracted from the plates by rinsing the plates with Tris-EDTA buffer (Fluka Analytical) or picking plaques with a sterile pipette tip. Phage were recovered from the culture supernatant or extraction buffer by one round of polyethylene glycol precipitation (PEG8000) (Promega) and resuspended in Tris-EDTA buffer.

Before intravenous (i.v.) injection (500 μ L/ animal), endotoxin removal beads (Miltenyi Biotec) were used to remove 2-3 rounds of endotoxin from the phage of the breeding. By measuring the plaque count according to the instructions for use (T7 Select System Manual) of the supplier, the phage content in the CSF and blood samples collected at the time point shown was determined. The library purified by intravenous tail vein injection or the phage recovered by selecting the CSF of the round before injection was carried out phage selection. Then, CSF and blood samples were collected at 10min, 30min, 60min, 90min, 120min, 180min and 240min after injection. A total of four rounds of in vivo panning were carried out, wherein two selection branches were maintained, and independent analysis was performed in the first three rounds of selection. The phage inserts recovered by all CSF of the first two rounds of selection were carried out 454/Roche pyrophosphate sequencing, and the clones recovered by all CSF of the second two rounds of selection were artificially sequenced. All phage collected from the first round of selection were also subjected to 454/Roche pyrosequencing.

Example 6: Filtering and processing reading data

Roche-454 raw data were converted from binary standard flowgram format (sff, http://www.ncbi.nlm.nih.gov/Traces/trace.cgi?cmd=show&f=formats&m=doc&s=format#sff) to human-readable Pearson format (fasta, http://emboss.sourceforge.net/docs/themes/SequenceFormats.html) using vendor software ( http://454.com/my454/documentation/gs-flx system/emanuals/Part_C/wwhelp/wwhimpl/js/html/wwhelp.htm#href=PartC.1.087.html#9003035). Additional nucleotide sequence processing was performed using in-house developed C-programs and scripts (unpublished software packages) as described below.

The main data analysis consisted of a rigorous multi-step filtering step. To filter out reads that did not contain a valid 12mer insert DNA sequence, reads were sequentially aligned to the start marker defined by the nucleotide sequence GTGATGCTCGGGGATCCGAAT TCT (Seq. Id. No. 45), the stop marker TAAGCTTGCGGCCGCACTCGAGTA (Seq. Id. No. 46), and the background insert CCTGC AGGGATATCCCGGGAGCTCGTCGAC (Seq. Id. No. 47) by performing a global Needleman-Wunsch alignment (up to two mismatches allowed per alignment) (Needleman et al., 1970). Thus, reads without start or stop markers and reads containing background inserts, i.e., alignments that exceeded the allowed number of mismatches, were removed from the library. For the remaining reads, N-mer DNA sequence fragments that began with the start marker and ended before the stop marker were excised from the original read sequence and further processed (hereinafter referred to as "inserts"). When the insert is translated, the portion after the first stop codon at the 5-priming end is removed from the insert. In addition, nucleotides that produce incomplete codons at the 3-priming end are also removed. In order to exclude inserts that only contain background sequence, inserts that start with the amino acid pattern "PAG" are also removed. Peptides that are shorter than 3 amino acids in length when translated are discarded from the library. Finally, redundancy in the insert library is removed, and the frequency of each unique insert is calculated. The results of this analysis consist of a list of nucleotide sequences (inserts) and their (read) frequencies.

Example 7: Grouping of N-mer DNA inserts by sequence similarity:

In order to overcome the mistakes (for example, the problem of sequencing homopolymer fragments) of Roche-454 specific sequencing and remove more unusual redundancy (for example, due to sequencing mistakes), the N-mer inset DNA sequence (inset) filtered out before is classified into groups of similar insets (wherein allowing up to 2 mispairings) by an iterative algorithm, the iterative algorithm is as follows: insets are mainly sorted by their frequency (highest to minimum), and secondarily, in the case of equal frequency, sorted by their length (longest to shortest). As a result, the most frequent and longest insets limit the first " group ", and " group frequency " is set to be equal to the frequency of insets. Then, by pairing the Needleman-Wunsch algorithm, attempt each remaining inset in the sorted list is added to the group. If the number of mispairing, insertion or deletion is no more than threshold value 2 in the comparison, the inset is added to the group so, and therefore, the frequency of the inset is added to the total group frequency. The insets added to the group are marked when in use, and are not further processed. If an insert sequence cannot be added to an existing group, a new group with a corresponding insert frequency is generated using this insert and its label is therefore set as "used". The iteration is completed when each insert sequence is used to form a new group or can be included in an existing group. After all, the grouped inserts composed of nucleotides are translated into peptide sequences (peptide library). The result of this analysis is a list of "grouped inserts" and their corresponding frequencies, which indicate the number of reads sequenced for each insert.

Example 8: Motif generation and normalization:

Based on the list of unique peptides, the library that comprises all possible amino acid patterns is produced as described below. Length is that every possible amino acid pattern (for example " ABC ") of 3 is proposed from peptide, and joins in the universal motif library that comprises all possible patterns together with its reverse pattern (for example " CBA "). This highly redundant motif library is sorted, and redundancy is removed. Then, whether each motif in the library is present in the peptide library is detected repeatedly. If so, increase the frequency that motif exists so, and be assigned to the described motif in the motif library. These frequencies are called " motif counts ". The result that motif produces is two two-dimensional arrays that comprise all 3-amino acid patterns (motifs) and corresponding motif counts thereof, as described in detail above, and described motif counts is the measure of the sequencing reading number that causes corresponding motif when filtering, grouping and translating reading.

Finally, the motif counts for each sample were normalized using the following formula:

V _i = 10 ² n _i /∑ _j n _j

Where n _i is the number of reads containing motif i. Therefore, _vi represents the frequency of reads (or peptides) containing motif i in the sample expressed as %. P-values or unnormalized motif counts were calculated using Fisher's exact test.

Example 9: Tripeptide motif hits after 4 rounds of in vivo selection using CSF sampling in rats:

The present invention includes methods for identifying one or more targeting peptides or molecular targets that can be used to localize a composition to the CSF, brain, or associated vasculature. A random phage library is screened for X(N), where N can be 7, 8, 9, 10, 11, 12, or more residues, to generate peptide motifs. In one example, multiple clones (comprising the following tripeptide motifs: Phe-Lys-Leu (FKL), Arg-Gly-Leu (RGL), Ser-Arg-Gly (SRG), Tyr-Val-Leu (YVL), Trp-Gly-Phe (WGF), Val-Leu-His (VLH), Leu-Tyr-Val (LYV), Leu-Trp-Gly (LWG), Leu-His-Ser (LHS), His-Ser-Arg (HSR), Gly-Leu-Trp (GLW), Gly-Phe-Lys (GFK ), Arg-Leu-Ser(RLS), Gly-Ser-Val(GSV), Ser-Val-Ser(SVS), Leu-Gly-Ser(LGS), Val-Arg-Phe(VRF), Ser-Asn-Thr(SNT), Arg-Phe- Arg(RFR), Asn-Thr-Arg(NTR), Leu-Ser-Asn(LSN), Gly-Phe-Val(GFV), Phe-Val-Arg(FVR), Phe-Arg-Leu(FRL), Trp-Arg-Val(WRV), P he-Ser-Leu(FSL), Val-Phe-Ser(VFS), Val-Ala-Trp(VAW), Ser-Leu-Phe(SLF), Arg-Val-Phe(RVF), Leu-Phe-Trp(LFW), Lys-Val-Al a(KVA), Phe-Trp-Lys(FWK), Ala-Trp-Arg(AWR), Val-His-Gly(VHG), Ser-Val-His(SVH), His-Gly-Val(HGV), Arg-Val-Cys(RVC), Arg -Pro-Gln (RPQ), Gln-Lys-Ile (QKI), Pro-Gln-Lys (PQK), Asn-Gly-Ala (NGA), Lys-Ile-Asn (KIN), Ile-Asn-Gly (ING), Gly-Arg-Pro (GRP), Gly-Ala-Arg (GAR), Ala-Arg-Val (ARV), Leu-Ser-Gly (LSG), Val-Asp-Ser (VDS), Ser-Val-Asp (SVD)) show high frequency, selective binding and presence in CSF and brain.

Example 10: Peptide hits after 4 rounds of in vivo selection using CSF sampling in rats

A total of 924 sequencing runs were performed (including short reads, reads where only one species was found, and reads where no sequence was generated). The frequency of each peptide was related to the 924 total sequencing runs.

Example 11:

To investigate peptide-mediated pharmacological effects in the CNS, we conducted experiments in which we mixed a brain-shuttling peptide with Seq. Id. No. 8 with a BACE1 peptide inhibitor with streptavidin (SA), both biotinylated, at two different ratios. For one sample, we used the BACE1 peptide inhibitor alone, while for the other, we used a 1:3 ratio of the BACE1 peptide inhibitor to the brain-shuttling peptide with Seq. Id. No. 8, resulting in a conjugated SA complex fraction with a majority of the mixture of the brain-shuttling peptide with Seq. Id. No. 8 and the single BACE1 peptide inhibitor to facilitate brain delivery. Both samples were injected intravenously, and amyloid-β peptide 40 (Aβ40) levels were determined in the blood and CSF. As expected, both samples showed substantial reductions in Aβ40 levels in the blood. However, only the sample containing a mixture of a brain-shuttling peptide having Seq. Id. No. 8 and a BACE1 peptide inhibitor conjugated to SA induced a significant reduction in Aβ40 in CSF (see Figure 7A, gray line). This data suggests that the brain-shuttling peptide having Seq. Id. No. 8 is capable of transporting a large protein (SA) into the CNS and inducing pharmacological effects through the SA-conjugated BACE1 peptide inhibitor.

method

The functionality of the streptavidin-peptide-BACE1 inhibitor complex was assessed by Aβ(1-40) ELISA according to the supplier's protocol (Wako, 294-64701). Briefly, CSF samples were diluted in standard diluent (1:23) and incubated overnight at 4°C in a 96-well plate coated with the capture antibody BNT77. After five washing steps, HRP-conjugated BA27 antibody was added and incubated at 4°C for 2 hours, followed by five washing steps. Aβ(1-40) was detected by incubation in TMB solution for 30 minutes at room temperature. After color development was stopped with a stop solution, the absorbance was read at 450 nm. Prior to the Aβ(1-40) ELISA, plasma samples were subjected to solid phase extraction. Plasma was added to 0.2% DEA (Sigma) in a 96-well plate and incubated at room temperature for 30 minutes. After washing the SPE plate (Oasis, 186000679) with 100% methanol and then water, the plasma sample was added to the SPE plate and any liquid was removed. The sample was washed (5% methanol, then 30% methanol) and eluted in _NH4OH /90% methanol. After the eluate was dried at 55°C under a constant _N2 flow for 99 minutes, the sample was reconstituted in standard diluent and Aβ(1-40) was measured as described above.

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Claims (17)

1. A blood-brain barrier shuttle comprising a brain effector entity and a brain targeting peptide, wherein the brain targeting peptide comprises a three-amino acid peptide motif, wherein the brain targeting peptide comprising the three-amino acid peptide motif consists of the sequence of SEQ ID NO: 8, and the brain effector entity is a protein or peptide, wherein the brain effector entity is selected from the group consisting of: a neurological disorder drug, a growth factor, an enzyme, a cytotoxic agent, a peptide against a brain target, wherein the brain target is selected from the group consisting of: β-secretase 1, Aβ, epidermal growth factor, epidermal growth factor receptor 2, Tau, apolipoprotein E4, α-synuclein, an oligomeric fragment of α-synuclein, CD20, huntingtin, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, γ-secretase, death receptor 6, amyloid precursor protein, p75 neurotrophin receptor and caspase 6. 2 . The blood-brain barrier shuttle according to claim 1 , wherein the peptide directed against a brain target is an antibody directed against a brain target. 3 . The blood-brain barrier shuttle of claim 2 , wherein the antibody against the brain target is a monoclonal antibody against the brain target. 4. The blood-brain barrier shuttle of claim 1, wherein the brain-effecting fruiting entity is a neurotrophic factor. The blood-brain barrier shuttle of claim 1 , wherein the brain-effecting fruiting entity is a polypeptide. The blood-brain barrier shuttle of claim 1 , wherein the Tau is phosphorylated Tau. 7. The blood-brain barrier shuttle of claim 1 or 2, wherein the brain effector entity comprises a full-length antibody against a brain target. 8. The blood-brain barrier shuttle of claim 2, wherein the brain effector entity comprises full-length IgG. 9. The blood-brain barrier shuttle of claim 7, wherein the brain effector entity is a full-length antibody against Aβ. 10. The blood-brain barrier shuttle of claim 7, wherein the brain effector entity is a full-length antibody against phosphorylated Tau. 11. The blood-brain barrier shuttle of claim 7, wherein the brain effector entity is a full-length antibody against α-synuclein. 12. A pharmaceutical preparation comprising the blood-brain barrier shuttle according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier. 13. The blood-brain barrier shuttle according to any one of claims 1-2, for use as a medicament. 14. Use of the blood-brain barrier shuttle according to any one of claims 1 to 11 in the preparation of a medicament. 15. The use of claim 14, wherein the medicament is for treating a neurodegenerative disorder. 16. The use of claim 15, wherein the neurodegenerative disorder is Alzheimer's disease. 17. The blood-brain barrier shuttle of any one of claims 1-2, which transports the brain-effect fruiting entity across the blood-brain barrier.