Blood-brain barrier permeable peptide compositions comprising a VAB domain of an anti-amyloid-beta camelid single-domain heavy-chain only antibody

Abstract

Blood-brain barrier permeable peptide compositions that contain variable antigen binding domains from camelid and/or shark heavy-chain only single-domain antibodies are described. The variable antigen binding domains of the peptide compositions bind to therapeutic and diagnostic biomarkers in the central nervous system, such as the amyloid-beta peptide biomarker for Alzheimer's disease. The peptide compositions contain constant domains from human IgG, camelid IgG, and/or shark IgNAR. The peptide compositions include heavy-chain only single-domain antibodies and compositions with one or more variable antigen binding domain bound to one or more constant domains.

Claims

1. A composition comprising: a polypeptide comprising the formula R1-L2-R4-Y-R3-X-R5-L1-R2; wherein R1 comprises a variable antigen-binding domain from a camelid single-domain heavy chain antibody comprising amino acids 1-127 of the amino acid sequence of SEQ ID NO: 11 and R2 is selected from a constant domain CHL CH2 or CH3 of human IgG, a constant domain CH2 or CH3 of camelid IgG, or constant domain CHL CH2, CH3, CH4 or CH5 of shark IgNAR; wherein L1 and L2 each comprise a hinge region from a camelid single-domain heavy-chain only antibody; wherein X and Y are bifunctional linkers, selected from a maleimido-thiol conjugate and polyethylene glycol; wherein R3 comprises at least one constant domain of human IgG CH2 or CH3 domain; wherein R4 and R5 are selected from a H, a nanoparticle, a radioisotope, a fluorophore, a toxin, a biotin, a digoxigenin, an avidin, and a streptavidin; and wherein the polypeptide can cross the blood brain barrier.

2. The composition of claim 1, wherein the at least one of R4 and R5 is a biodegradable nanoparticle.

3. The composition of claim 1, wherein the nanoparticle comprises poly-butylcyanoacrylate and amino-dextran.

4. The composition of claim 1, wherein the nanoparticle has a molecular weight of about 70 kDa.

5. The composition of claim 1, wherein a hinge region corresponds to the amino acid sequence of SEQ ID NO: 13.

6. The composition of claim 1, wherein at least one of L1 and L2 is covalently conjugated to NHC(NH2.HCl)CH2CH2CH2-S-Maleimide-PEG-CONH, which in turn is conjugated to a nanoparticle.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

(2) FIG. 1 is a diagram of the tight junctions in the endothelial cell membrane that form the barrier between the blood and the brain.

(3) FIG. 2 depicts generic blood-brain permeable peptide composition structures 1 and 2 where each structure contains at least one Vab domain and each Vab domain was derived from a camelid Vab, shark V-NAR, or a combination thereof, from one or more sdAbs for an antigen.

(4) FIG. 3 is a photograph of affinity-purified A-sdAb 1a on an SDS-PAGE gel.

(5) FIG. 4 depicts the synthetic sequence of conjugating two peptides by native chemical ligation.

(6) FIG. 5 depicts the synthetic sequence of conjugating two peptides by malemido and thiol groups.

(7) FIG. 6 is a photograph of the ELISA results of A-sdAb 1a and Pierce's A-mAb binding affinity to the A-42 peptide as measured by the OD450 readings of the chromogenic yellow color generated by the reaction of the HRP secondary antibody with the TMB substrate.

(8) FIG. 7 is a photograph of the immunostaining results of immunohistochemical (IHC) staining of paraffin embedded brain tissues from the APP transgenic mouse (FIG. 7A left upper two frames) and Alzheimer's patient (FIG. 7A lower two frames); with and without primary A-sdAb (FIG. 7A frames), and staining of the same tissues with Thioflavin-S dye (FIG. 7B frames).

(9) FIG. 8 depicts the BBB permeability tests for A-sdAb 1a (FIG. 2, Structure 1, Table 1, Variant: R=1) and peptide composition 2a (single-chain of A-sdAb 1a; FIG. 2, Structure 2, Tables 2-6, variant: R1=1, R2=4, R3=2, L1=1, L2=1, R4=1, R5=1, X=1, Y=1) in mice.

(10) FIG. 9 is photographs of the immunostaining results from blood-brain barrier (BBB) permeability studies of peptide composition 2a compared to A-Mouse-IgG in the live APP transgenic mice injected in their tails with peptide composition 2a or A-Mouse-IgG.

(11) FIG. 10 is a graph of the serum retention time of the peptide composition 2a in A-mice.

(12) FIG. 11 depicts the generic synthetic sequence of synthesizing maleimido derivatized peptide composition 3 from peptide composition 2 (structure 2 in FIG. 2) and synthesizing thiolated dextran-PBCA-nanoparticles 6 from dextran 4.

(13) FIG. 12 depicts the generic conjugation, after the sequence in FIG. 11, of maleimido-peptide composition 3 to thiol-functionalized dextran-coated PBCA-nanoparticles 6, forming covalently-coated peptide composition 2-nanoparticle 7.

(14) FIG. 13 depicts the generic synthetic sequence of synthesizing maleimido derivatized-sdAb 8 from sd-Ab 1 (structure 1 in FIG. 2) and synthesizing thiolated dextran-PBCA-nanoparticles 6 from dextran 4.

(15) FIG. 14 depicts the, generic conjugation, after the sequence in FIG. 13, of maleimido-sdAb 8 to thiol-functionalized dextran-coated PBCA-nanoparticles 6, forming covalently-coated sdAb-nanoparticle 9.

(16) FIG. 15 is a structure of covalently-coated sdAb-nanoparticle 9 (structure 1 in FIG. 2) that is comprised of a maleimodio linker to form covalently-coated sdAb-nanoparticle 9 conjugated to an amino-dextran-coated-PBCA-nanoparticle 6.

(17) FIG. 16 illustrates the structures of a native candid heavy chain-only antibody (left) which is a dimer formed by intermolecular bonding of two monomeric units (right), wherein RH. Each monomer comprises the following structure: Vab-hinge region (HR)-CH2-CH3

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(18) In the following detailed description, reference is made to the accompanying drawings, which form a part thereof. The illustrative embodiments described in the detailed description, drawings and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here. The present technology is also illustrated by the examples herein, which should not be construed as limiting in any way.

(19) A. Isolation of A-Single-Domain Antibody 1A (A-sdAb 1A, Structure 1 in FIG. 2)

(20) 1. Immunization of Camelids with A1-35 Peptide

(21) All animals (llamas) were treated following NIH guidelines. First, the animals were given a complete physical examination by our veterinarian, Dr. Linda Byer, who also drew some pre-immunization blood. Immunization was then started with A.sub.1-35 synthetic peptide (200 ug) in Gerbu Pharma Adjuvant (1 ml). One month after the initial priming injection, six biweekly boosters were administered at 200 ug/injection. After the fourth booster, about 20 ml blood was drawn and serum examined for antibody titer with antigen coated 96-well ELISA plate. After immunization, 200 ml blood was drawn from the animal. Half of the blood was used to isolate single-domain A-antibody (polyclonal) with the methods below in Sections A.2-4. The second half of the blood was used to isolate peripheral blood lymphocytes (PBLs) to prepare total RNA followed by its reverse transcription to cDNA, which was then ligated into the phage vector to generate phage-display cDNA library (Section D).

(22) 2. Crude Isolation of A-sdAb 1a from Camelid Serum

(23) After immunization, 100 ml from each blood sample drawn was processed to fractionate sdAbs (MW: 90 KDa) from the classical antibodies (MW: 160 KDa). Briefly, the serum (50 ml) was concentrated on an Millipore-Amicon Ultra-15 Concentrator, molecular weight cutoff 50 KDa, by spinning the device at 4000 g, until most of the low molecular weight species passed through the membrane. The thick viscous yellow retentate (25 ml) was extracted with chloroform (325 ml) to remove fatty substances, which had contributed to the viscosity of the retentate. The resulting crude product (210 ml) was size fractionated on Superdex-200 (2.5 cm100 cm) using 1PBS as eluant. The fractions were monitored by reading OD.sub.280 on a Beckman DU-640 Spectrophotometer. After examining the fractions on a 12% SDS-PAGE gel, the fractions whose products correspond to the molecular weight of 90 KDa were pooled, concentrated and the protein concentration was measured by checking its OD.sub.280.

(24) 3. Generation of an Affinity Column for Enrichment of A-sdAb 1a

(25) 10 mg of immunogen A1-35, dissolved in 5 ml of conjugation buffer, 0.1 M NaHCO.sub.3/0.15M NaCl, pH 8.5, was conjugated with cyanogen-bromide activated Sepharose (2 gm), which had been washed with 200 ml of ice-cold 1 mM HCl. The reaction was allowed to proceed for 2 hours while the resin was allowed to gently rock on a rocker. After centrifugation, the supernatant of the reaction mixture was examined by its OD.sub.280 reading, which indicated that essentially all of the immunogen had been consumed. The resin was then washed with pH 8.5 conjugation buffer (320 ml), and then blocked with 1 M Tris.HCl, pH 8.3 (10 ml), room temperature for 2 hours. After washing the resin with 0.1 M NaHCO.sub.3/0.5M NaCl, pH 8.5, the resin was washed with 0.1 M sodium citrate (50 ml), pH 2.8 and equilibrated with 20 mM sodium phosphate buffer, pH 7.0, before using the resin for affinity purification.

(26) 4. Affinity Purification of A-sdAb 1a

(27) The crude mixture of sdAbs obtained after size fractionation on Superdex-200, which was more than 98% free of full-length conventional IgGs, was allowed to incubate with the affinity column in 1PBS, at room temperature for one hour. After one hour, the unbound material was allowed to drain through the column and the column washed with PBS until all the unbound proteins had been washed off the column. The bound A-sdAb was eluted off the column with pH 2.8 buffer (0.1 M sodium citrate, 0.2 um filtered). The eluant was adjusted to pH 7.2 by adding 1 M Tris.HCl, pH 9.0, and concentrated on Millipore-Amicon Ultra-15 concentrators (30 KDa molecular weight cutoff). The retentate was buffer exchanged to 1PBS and stored at 20 C. to obtain 1.65 mg of A-sdAb 1a (FIG. 2, Structure 1, Table 1, Variant: R=1). Its protein concentration was determined using Pierce's BCA Protein Assay Kit. The SDS-PAGE analysis of the affinity purified A-sdAb 1a is in FIG. 3. About 10 ug of the A-sdAb 1a after each step was electrophoressed on 12% SDS-PAGE gel after loading in SDS-loading buffer. The electrophoresis was performed at 100 volts for one hour, the gel was stained in 0.04% Coomassie Blue stain for 30 minutes at room temperature (RT). Coomassie blue stained SDS-PAGE (12%) protein gel of sequentially purified A-sdAb 1a, panel D: 1.sup.st and 2.sup.nd purifications were on Superdex-200; 3.sup.rd purification was done by affinity chromatography.

(28) B. Synthesis of Single-Chain A-sdAb 2a and Epitope Mapping of Peptide Composition 2a

(29) 1. Isolation of Single-Chain A-sdAb 2a from A-sdAb 1a

(30) 1.0 mg of A-sd-Ab 1a was dissolved in 400 ul of pH 7.4 PBS. To this solution was added 100 ul of 100 mM triethoxy carboxyl-phospine (TCEP) in PBS to obtain a final concentration of 20 mM. The reaction mixture was incubated at 4 C. for 12-15 hours when gel electrophoresis (10% SDS-PAGE) showed a low molecular weight species with molecular weight of 50 KDa. This product peptide composition 2a (single chain of A-sdAb 1a) was isolated by gel filtration and tested by Western and ELISA.

(31) 2. Epitope Mapping of Single-Chain A-sdAb 2a

(32) 96-Well microplates (A1-A12 through G1-12 wells) were coated in triplicate with 600 ng per well of the following synthetic amyloid-peptide segments of A1-42 peptide in Table 7.

(33) TABLE-US-00013 TABLE 7 Synthetic amyloid-peptide segments of A1-42 peptide Peptide segment Amino acid positions 1 1-16 2 5-20 3 9-24 4 13-28 5 17-32 6 21-37 7 25-41 8 29-42

(34) After coating the plate at 4 C. for 12 hours, the antigens were discarded and the wells washed with deionized water (3). The plate was blocked with 1% BSA in 50 mM Tris/150 mM NaCl, pH 7.5 for one hour. At the end of one hour, single-chain A-sdAb, 2a, 1.0 ug diluted to 2500 ul with 1% BSA/Tris buffer was added to the top row (100 ul per well in triplicates). After serial dilution all the way to 1:320000 ul, the plate was incubated with gentle shaking at room-temperature for 2 hours. At the end of 2 hour incubation, the plate was washed three times, 250 ul per well, with 0.05% tween-20/PBS. After washing, the wells were incubated with 100 ul per well of goat-anti-llama-IgG-HRP conjugate (Bethyl Labs, Texas) 1.0 ug diluted to 10 ml of 1% BSA in PBS. After one hour incubation, the plate was washed with 0.05% Tween as above. The washed well were treated with 100 ul of TMB substrate and the plate read at 370 nm. The highest antibody titer was detected with the peptide 1-16 amino acid long.

(35) Subsequently, two synthetic peptide were synthesized: the 1-8 and 9-16 peptides from the amyloid beta peptide and the above ELISA was again repeated with the plate coated with 600 ng of each of the peptide in triplicates. This time the peptide of the 9-16 amino acids gave the highest antibody titer, and no reaction took place with the sequence 1-8 mer. The epitope is between 9 to 16 amino acids with the following sequence: GYEVHHQK (SEQ ID NO: 14).

(36) C. Synthesis of Peptide Composition Structure 2 in FIG. 2 Derivatives

(37) 1. Protease Digestion of Single-Chain A-sdAb 2a to Obtain A-Vab-HR (A-Vab with L1 or L2 Linker Variant)

(38) Generation of Sepharose-Endoproteinase Glu-C Conjugate.

(39) Endoproteinase Glu-C (Worthington Biochemical Corporation), 4 mg, was conjugated to 250 mg of CNBr-activated Sepaharose (GE Healthcare, catalogue #17-0430-1) in pH 8.5 0.1 M NaHCO.sub.3/0.5M NaCl in 110 cm long spin fitted with a medium fritted disc, as described in Section A.3: Generation of an Affinity Column for Enrichment of A-sdAb 1a. After conjugation, any unbound Glu-C proteinase was removed by extensive washing of Sepharose and the column was stored in 0.1% NaN.sub.3/PBS until used. The Sepharose had swollen to about an 0.8 ml volume.

(40) Digestion of Single-Chain A-sdAb 2a and Isolation of A-Vab-HR.

(41) A-sdAb 2a (1 mg, 11 nmols) was dissolved in 1.0 ml of pH 7.5 0.1 M NaHCO.sub.3 and added to the 0.8 ml of Sepharose-Glu-C conjugate. The reaction mixture was gently rocked on a rocker for 4 hours and the contents were collected by draining the column and washing it with 4 ml of the conjugation buffer, 0.1 M NaHCO.sub.3, pH 7.5. The combined flowthrough was passed through A.sub.1-35-affinity column generated in Section A.3. After washing off the unbound material, the bound A-Vab-HR (HR=hinge region) from single-chain A-sdAb 2a was eluted with pH 2.8 0.1 M sodium citrate and the product buffer exchanged to 1PBS, pH 7.4. It was tested by ELISA.

(42) 2. Methods for Linking A-Vab-HR to Antibody Constant Domains

(43) General Method for Expression of Engineered Human Antibody Constant Domains, CH1, CH2 and CH3.

(44) Expression of engineered human constant domains CH1, CH2 and CH3 was accomplished by buying the commercially available plasmid, pFUSE-CHIg (Invitrogen: pFUSE-CHIg-hG1, pFUSE-CHIg-hG2, or pFUSE-CHIg-hG3), and using them each for transformation of E. coli strain HB2151 cells. The cultures were grown in SB media at 37 C. until an optical density of 0.7 was obtained. Expression was then induced with 1 mM IPTG (isopropyl-1-thio-b-D-galactopyranoside) at 37 C. for 15-16 hours. The bacterial cells were harvested and resuspended in a culture medium containing 10% of 50 mM Tris.HCl, 450 mM NaCl, pH 8.0. Polymyxin B sulfate (PMS) was added to the culture medium, 1:1000 volume of PMS: culture volume. After centrifuging the cell lysate at 15000 RPM for 45 minutes at 4 C., the supernatant was purified by HiTrap Ni-NTA column and tested for the respective expressed human constant domain by SDS-PAGE and Western blot.

(45) General Method for Native Chemical Ligation of A-Vab-HR to Human Constant Domain.

(46) For native chemical ligation (FIG. 4), an unprotected peptide-alpha-carboxy thioester (peptide 1) was reacted with a second peptide (peptide 2) containing an N-terminal cysteine residue to form a natural peptide linkage between A-Vab-HR and constant domain CH1 or CH2 or CH3. A-Vab-HR can be modified to be peptide 1 or peptide 2. The CH1, CH2, or CH3 domain is then modified to be the recipricol peptide 2 or peptide 1. After the reaction, the A-Vab-PEG-Human CH1, CH2, or CH3 was purified by size exclusion chromatography.

(47) General Method for Maleimido-thiol Conjugation Chemical Linkage of A-Vab-HR to Human Constant Domain.

(48) For the maleimido-thiol conjugation reaction (FIG. 5), a thiolated peptide 2 conjugates to a maleimido-derivativized peptide 1 to create aliphatic linker between A-Vab-HR and a CH1, CH2, or CH3 domain. A-Vab-HR can be modified to be peptide 1 or peptide 2. The CH1, CH2, or CH3 domain is then modified to be the recipricol peptide 2 or peptide 1. Peptide 1 was converted into a maleimido peptide by reacting in with 20-fold excess of commercial NHS-PEG-Mal (MW: 3000 Da) in pH 7.0 MOPS buffer (0.1 M MOPS/0.15 M NaCl) for one hour at room temperature. After the reaction, excess PEGreagent was removed by dialysis on Vivaspin-20 column with a MWCO: 10 KDa. To generate compatible reacting group, peptide 2 was thiolated with commercial Traut's reagent to obtain thiolated peptide 2. 1.2 molar equivalent of thiolated peptide 2 which was reacted with maleimido derivatized peptide 1 at pH 6.8 at room temperature for 2 hours. After the reaction, the A-Vab-PEG-Human CH1, CH2, or CH3 was purified by size exclusion chromatography.

(49) D. Phage-Display cDNA Library Generation of A-sdAb 1

(50) 1. Cloning of cDNA Encoding the A-sdAb 1a: mRNA Isolation and Reverse Transcription

(51) The isolation of total RNA from peripheral blood lymphocytes (PBLs) from 100 ml blood samples from immunized animals and subsequent reverse transcription to cDNA was done using commercial kits, such as PAXgene Blood RNA Tubes and Blood RNA Kit system (Qiagen, Mississauga, ON).

(52) 2. PCR Amplification of cDNA and Construction of Expression Vector

(53) Amplification of cDNA was done using PCR with primers SEQ ID NO:1 and SEQ ID NO:2. The second round of PCR amplification was done using primers with built-in restriction enzyme sites (SEQ ID NO: 3 and SEQ ID NO: 4) for insertion into pHEN4 phagemid, which was used to transform bacterial cells (WK6 E. coli). The clones were sequenced by the dideoxy sequencing method. Sequences were then translated so that they can be assigned to well defined domains of the sdAb.

(54) 3. General Method for Expression and Purification of A-sdAb 1

(55) The bacterial cells containing the proper plasmids were grown, and expression of the recombinant proteins induced with 1 mM isopropyl--D-thiogalactopyranoside (IPTG). The periplasmic proteins were extracted by osmotic shock in the presence of protease inhibitors [(4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) and leupeptin)], and recombinant protein purified by immobilized metal affinity chromatography (Ni-NTA Superflow, Qiagen). The MALDI-TOF mass spectrometry of A-sdAb 1a in sinapinic acid displayed a molecular ion at m/e 84873.5842. The molecular weight was validated by SDS-PAGE and Western blot. The purified A-sdAb 1a was further characterized by ELISA and immunohistochemical staining of tissues from transgenic mice and human patients.

(56) 4. ELISA Results for Single-Chain A-sdAb 1a

(57) FIG. 6 depicts the results of ELISA performed in Pierce's MaxiSorp plate, which had been coated with 500 ng/well of A-42 peptide at pH 9.5 overnight at room temperature. After washing the plate with water, the antigen coated wells were blocked with 1% BSA and subsequently treated with identical concentrations of A-sdAb 1a and mouse-A-mAb for the same length of time and temperature (2 hours at RT). Detection was done using HRP labeled secondary antibody and TMB as a substrate. The blue color generated by HRP reaction was quenched with 1.0 M HCl and OD450 recorded on Molecular Devices SpectraMax Plus plate reader. FIG. 6 is the plot of OD450 readings of the chromogenic yellow color generated by the reaction of the substrate with the HRP-enzyme. The single-domain antibody 1a clearly outperformed the commercial A-mouse-mAb.

(58) E. Administration and Dosage

(59) Pharmaceutical formulations of a therapeutically effective amount of a polypeptide of the invention can be administered orally or parenterally in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.

(60) Methods well known in the art for making formulations are found, for example, in Remington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Compositions intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions. The compositions may optionally contain sweetening, flavoring, coloring, perfuming, and/or preserving agents in order to provide a more palatable preparation. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient. These may include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffering agents, and/or lubricating agents (e.g., magnesium stearate) may also be used. Tablets and pills can additionally be prepared with enteric coatings.

(61) Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and soft gelatin capsules. These forms contain inert diluents commonly used in the art, such as water or an oil medium. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents.

(62) Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphthalenes, and injectable organic esters, such as ethyl oleate. Such formulations may also contain adjuvants, such as preserving, wetting, emulsifying, and dispersing agents. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for the agents of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

(63) Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions, which can be dissolved in sterile water or some other sterile injectable medium immediately before use.

(64) The amount of active polypeptide in the compositions of the invention can be varied. One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending upon a variety of factors, including the ingredient being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the subject's conditions, and the age, weight, health, and gender of the patient. In addition, the severity of the condition targeted by an agent of the invention will also have an impact on the dosage level. Generally, dosage levels of an agent of the invention of between 0.1 g/kg to 100 mg/kg of body weight are administered daily as a single dose or divided into multiple doses. Preferably, the general dosage range is between 250 g/kg to 5.0 mg/kg of body weight per day. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of the various routes of administration. For instance, oral administration generally would be expected to require higher dosage levels than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, which are well known in the art. In general, the precise therapeutically effective dosage will be determined by the attending physician in consideration of the above-identified factors.

(65) Methods for administering peptides to a subject are described, for example, in U.S. Pat. Nos. 5,830,851; 5,558,085; 5,916,582; 5,960,792; and 6,720,407, hereby incorporated by reference.

(66) F. Ex-Vivo and In-Vivo Results of Peptide Compositions 1a and 2a in Alzheimer's Disease Models

(67) 1. Detection of Amyloid Plaque in Transgenic Mouse and Human Alzheimer's Patients with Peptide Composition 2a in Ex-Vivo Experiments

(68) The specificity of A-sdAb for AP was tested by immunohistochemical (IHC) staining of paraffin embedded brain tissues from the APP transgenic mouse (FIG. 7A upper two frames) and Alzheimer's patient (FIG. 7A lower two frames), with and without primary A-sdAb. The same tissues were stained with Thioflavin-S dye (FIG. 7B). Paraffin tissues were cut in a microtome to the thickness of 5 microns, mounted on APES coated slides, dried at room temperature (RT) for 24 hours, and then deparaffinized using xylene and ethanol. Washed slides were blocked in 10% normal serum with 1% BSA (2 hours at RT), and treated with single-chain A-sdAb 2a in PBS containing 1% BSA (1:100 dilution, overnight at 4 C.). After washing slides with 0.1% Triton X-100, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide, followed by treatment with biotinylated goat-anti-llama-IgG. Detection was done with streptavidin-HRP and diaminobenzidine as HRP substrate.

(69) 2. Demonstration of BBB Permeability of A-Vab Peptide Compositions 1a and 2a in Alzheimer's-Like Transgenic Mice

(70) We tested the BBB permeability of A-sdAb 1a and single-chain A-sdAb 2a. The single-chain A-sdAb 2a had been prepared by the TCEP (triethoxy-phosphine) reduction of A-sdAb 1a above in Section B.1. To demonstrate BBB penetration, 60 ug of A-sdAb 1a or single-chain A-sdAb 2a was injected in the tail vein (FIG. 8) of the live transgenic mice (J9 strain: PDGF-APP-SW-Ind; APP: amyloid precursor protein; SW and Ind stand for Swedish and Indiana mutations in APP), according to the protocol outlined below (Table 8). The commercial mouse-A-mAb was also used for comparative purposes. Non-transgenic mice were used as negative controls.

(71) TABLE-US-00014 TABLE 8 Protocol for Demonstrating BBB Permeability Number Group Mouse Antibody Time of mice 1 APP tg mice ICBI-antibody 4 h 6 2 APP tg mice ICBI-antibody 24 h 6 3 APP tg mice Mouse-Ab-IgG 4 h 6 4 APP tg mice Mouse-Ab-IgG 24 h 6 5 Non-tg control mice ICBI-antibody 4 h 3 6 Non-tg control mice ICBI-antibody 24 h 3 7 Non-tg control mice Mouse-Ab-IgG 4 h 3 8 Non-tg control mice Mouse-Ab-IgG 24 h 3 APP = Amyloid precurson protein. Route = Tail vein, Dose = 60 ug, Treatment and duration = variable.

(72) Mice were sacrificed 4 hours and 24 hours after the injection and their brains serially sectioned. The two hemispheres were separated; the left hemisphere was rapidly snap frozen on dry ice (2 to 5 min) and stored at 80 C.; the right hemisphere was immersed in a cold 4% paraformaldehyde fixative solution.

(73) 3. Neuropathological Analysis

(74) The fixed half brain was serially sectioned sagitally with the vibratome at 40 um and stored at 20 C. in cryoprotective medium. Sections were immunostained with biotinylated anti-llama-IgG1 and detected with streptavidin-HRP using an enzyme substrate, followed by imaging with the laser confocal microscope (FIG. 9). Co-localization studies between llama IgG and A-protein were also performed by staining the tissues with Thioflavin-S dye. Digital images were analyzed with the ImageQuant program to assess numbers of lesions.

(75) 4. Results of Blood-Brain Permeability of Peptide Compositions

(76) All six transgenic mice analyzed 24 hours after a single low dose injection of 60 ug amyloid sd-antibody displayed labeling of amyloid-plaque in their central nervous system. The data shown in FIG. 9 represents data obtained only with single-chain A-sdAb 2a. Binding of peptide compositions 1a and 2a to amyloid plaque was only detected in the APP transgenic mice, not in non-transgenic mice. More importantly, A-sd-antibodies 1a and 2a labeled both the soluble/diffusible plaque and insoluble plaque, while Thioflavin-S dye labeled primarily the insoluble plaque. Soluble plaque is the one responsible for cognitive decline from Alzheimer's Disease, not the insoluble plaque, which is labeled by other neuroimaging agents such as Pittsburgh Compound B and .sup.18F-Flutemetmol. The single-chain sdAb 2a stained about 10% of all the soluble and insoluble plaque in the mouse brain, while A-sdAb 1a labeled about 3.6% of the total plaque in the same amount of time.

(77) 5. Pharmacokinetics Study of Single-Chain A-sdAb 2a for Alzheimer's Disease in Mice

(78) A pharmacokinetics (PK) study of the single-chain A-sdAb 2a was conducted in collaboration with Biotox Sciences, San Diego. In this study, three groups of mice (average weight: 25 g) were injected in the tail vein with 60 ug of single-chain A-sdAb 2a in 200 ul of PBS buffer. At a predetermined timepoint, blood was drawn from the animals the the serum was analyzed for the concentration of the single-chain A-sdAb 2a by ELISA. All three sets of animals showed identical clearance of the single-chain A-sdAb 2a from the blood (FIG. 10). FIG. 10 is a graph of the serum retention time of the single-chain A-sdAb 2a in A-mice. The X-axis represents time in hours and the Y-axis concentration of single-chain A-sdAb 2a per ml of serum. The two broken lines indicate non-linearity in the X-axis. Clearance of amyloid-plaque by binding to mouse-A-mAb and subsequent phagocytosis has been reported in the literature [Wang, Y-J, et al., Clearance of amyloid-beta in Alzheimer's disease: progress, problems and perspectives, Drug Discovery Today, 11, 931 (2006)].

(79) Although at 24 h the serum concentration of 2a in FIG. 10 dropped to about half compared to what it was at 0.5 hour, its levels stayed at 40% for 7 days, suggesting that the single-chain A-sdAb 2a has a serum life of at least 7 days and, therefore, a remarkable potential for developing diagnostic and long-acting therapeutic agents. The slow decrease in serum concentrations of the single-chain A-sdAb 2a in the first 24 h could be attributed to its binding with the amyloid-peptide.

(80) G. Synthesis of Antibody-Coated Nano-Particles with Peptide Compositions 1 and 2 from FIG. 2

(81) 1. Synthesis of Polybutylcyanoacrylate (PBCA) Nanoparticles 5

(82) To overcome the shortcomings of the prior art, this invention describes the synthesis of biodegradable polyalkylcyanoacrylate nanoparticles coated with aminated dextran and peptide compositions 1 and 2 in FIG. 2. The synthetic steps are outlined in FIGS. 11-14. To a stirring solution of aminated dextran 4 (1.0 gm) in 100 ml of 10 mM HCl (pH 2.5) was slowly added 1 ml of butylcyanoacrylate (BCA) (FIG. 11 and FIG. 13). The reaction mixture was allowed to stir at RT for 4 hours to obtain a white colloidal suspension, which was carefully neutralized with 0.1 M NaHCO.sub.3 solution to pH 7.0. This colloidal suspension was filtered through 100 um glass-fiber filter to remove large particles. The filtrate was split into 50 ml centrifuge tubes and spun at 10,000 RPM for 45 minutes. After discarding the supernatant, the particles were washed several times with deionized water, centrifuging the particles and discarding the supernatant until no more white residue was seen in the supernatant. The resulting PBCA particles, 5, were stored in 0.01% NaN.sub.3/PBS at 4 C. (FIG. 11 and FIG. 13).

(83) 2. Synthesis of Thiolated PBCA Nanoparticles 6

(84) PBCA particles 5 were washed with 50 mM MOPS buffer, pH 7.0, to remove NaN.sub.3. The particles were then treated 50 mM Traut's reagent in MOPS buffer for one hour to synthesize thiolated PBCA nanoparticles 6 (FIG. 11 and FIG. 13). The particles were then repeatedly washed to remove the unreacted Traut's reagent.

(85) 3. Synthesis of Peptide Composition Maleimido Derivatives 3 and 8

(86) Purified peptide composition 1a (1 mg, 12.5 nM) was dissolved in 50 mM MOPS buffer, pH 7.0. It was treated with NHS-PEG-Mal (MW: 3000 Da) (250 nM) at RT for 1 hour (FIG. 12). The reaction was concentrated on Amicon-Centricon Concentrators (MW Cutoff: 30 KDa) to remove hydrolyzed and unconjugated excess NHS-PEG-Mal. The purified pegylated derivative 8 was characterized by MALDI-MS and 12% SDS-PAGE gel. A similar process can be used to convert peptide composition 2 in FIG. 2 into the pegylated derivative 3 (FIG. 11).

(87) 4. Synthesis of Covalently Conjugated Peptide Composition Nanoparticles 7 and 9

(88) The conjugation of maleimido-A-sdAb 8 with thiolated PBCA nanoparticles 6 was carried out at pH 7.0 in 50 mM MOPS buffer in the presence of 5 mM EDTA for 4 hours at RT (FIG. 14). The resulting PBCA nanoparticles 9 were purified by washing off (550 ml deionized water) the unreacted maleimido antibody 3. The particles were stored in deionized water at 4 C. until used. A similar process can be used to convert pegylated derivative 3 and thiolated PBCA nanoparticles 6 into PBCA nanoparticles 7 (FIG. 12).

(89) The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods, processes and compositions within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, processes, reagents, compounds compositions or biological systems, which can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

(90) All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

(91) While certain embodiments have been illustrated and described, it should be understood that changes and modifications could be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.