Camelid single-domain antibody directed against amyloid beta and methods for producing conjugates thereof

Abstract

The present invention relates to variable domain of a camelid heavy-chain antibodies directed to amyloid β and conjugates thereof. The present invention also relates to the use of these antibody conjugates for treating or diagnosing disorders mediated by amyloid β deposits.

Claims

1. An isolated variable domain of a camelid heavy-chain antibody (VHH) directed against the fibrillar form of amyloid β, characterized in that its amino acid sequence comprises, from the N-terminus to the C-terminus, the amino acid sequence SEQ ID NO:1 (corresponding to the CDR1), the amino acid sequence SEQ ID NO:2 (corresponding to the CDR2) and the amino acid sequence SEQ ID NO:3 (corresponding to the CDR3).

2. The VHH according to claim 1, characterized in that it comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

3. A VHH derivative consisting of a polypeptide comprising the VHH of claim 1, provided that said VHH comprised in said polypeptide is able to bind the fibrillar form of amyloid β.

4. A VHH derivative characterized in that it has the amino acid sequence SEQ ID NO:8.

5. An isolated polynucleotide encoding the VHH of claim 1.

6. A recombinant expression cassette comprising the polynucleotide of claim 5 under the control of a transcriptional promoter allowing the regulation of the transcription of said polynucleotide in a host cell.

7. A recombinant vector comprising the polynucleotide of claim 5.

8. A host cell containing the recombinant expression cassette of claim 6.

9. A host cell containing the recombinant vector of claim 7.

10. A composition comprising the VHH of claim 1 linked to a substance of interest.

11. The composition of claim 10, wherein the substance of interest is selected from a peptide, an enzyme, a nucleic acid, a virus and a chemical entity.

12. The composition of claim 11, wherein the substance of interest is a NMR or MRI contrast agent selected from paramagnetic agents gadolinium (Gd), dysprosium (Dy) and manganese (Mn), and the superparamagnetic agents based on iron oxide or iron platinium, and the X-nuclei .sup.18F, .sup.13C, .sup.23Na, .sup.17O, and .sup.15N.

13. The composition of claim 10, wherein the substance of interest is selected from the group consisting of an enzyme, a fluorophore, a NMR or MRI contrast agent, a radioisotope, or a nanoparticle.

14. The composition of claim 10, wherein the substance of interest is selected from the group consisting of an analgesic compound, an anti-inflammatory compound, an antidepressant compound, a cytotoxic compound, an anticonvulsant compound or an anti-neurodegenerative compound.

15. The composition of claim 10, wherein the substance of interest is a liposome or a polymeric entity.

16. A pharmaceutical composition comprising the composition of claim 10 and a pharmaceutically acceptable carrier.

17. A kit for brain imaging, or for diagnosing or monitoring a disorder mediated by amyloid β deposits comprising a VHH of claim 1 and a diagnostic agent.

18. An in vitro or ex vivo method for diagnosing a disorder mediated by amyloid β deposits in a subject, comprising the steps of: a) contacting in vitro a biological sample from said subject with the composition of claim 13, and b) determining the presence or the absence of amyloid β deposits in said biological sample, the presence of said amyloid β deposits indicating that said subject has a disorder mediated by amyloid β deposits.

19. An in vitro or ex vivo method for monitoring the progression or regression of a disorder mediated by amyloid β deposits in a subject, comprising the steps of: a) contacting in vitro a biological sample from said subject with the composition of claim 13, b) determining the amount of fibrillar form of amyloid β in said biological sample, and c) comparing the amount determined in step (b) with the amount of fibrillar form of amyloid β previously obtained for said subject, wherein a significant increase in amount of fibrillar form of amyloid β constitutes a marker of the progression of said disorder mediated by amyloid β deposits and a significant decrease of fibrillar form of amyloid β constitutes a marker of the regression of said disorder mediated by amyloid β deposits.

20. A method for in vivo imaging amyloid β deposits in a subject comprising the steps of a) administrating a detectable quantity of the composition of claim 13 to a subject, and, b) detecting the substance of interest in said subject by an imaging method.

21. An in vitro or ex vivo method for detecting the presence or the absence of amyloid β deposits in a subject, comprising the steps of: a) contacting in vitro a biological sample from said subject with the composition of claim 13, and b) determining the presence or the absence of amyloid β deposits in said biological sample.

22. A non-site specific method for coupling a VHH of claim 1 with a substance of interest, said method comprising a conjugation step of a substance of interest with the VHH.

23. The non-site specific method of claim 22, wherein the substance of interest is a compound selected from the group consisting of a peptide, an enzyme, a nucleic acid, a virus, a fluorophore, a NMR or MRI contrast agent, a chemical entity, a radioisotope and a nanoparticle.

24. The non-site specific method of claim 23, wherein the substance of interest is a compound selected from NMR or MRI contrast agents and metallic radioisotopes.

25. The non-site specific method of claim 24 comprising the following steps: (i) the conjugation of a chelating agent activated in the form of an ester or an anhydride with lysine residues of a VHH directed against the fibrillar form of amyloid β, characterized in that its amino acid sequence comprises, from the N-terminus to the C-terminus, the amino acid sequence SEQ ID NO:1 (corresponding to the CDR1), the amino acid sequence SEQ ID NO:2 (corresponding to the CDR2) and the amino acid sequence SEQ ID NO:3 (corresponding to the CDR3), and (ii) the chelation of the ligand of step (i) with the substance of interest.

26. The non-site specific method of claim 25, wherein the substance of interest is a NMR or MRI contrast agent selected from the paramagnetic agents gadolinium (Gd), dysprosium (Dy) and manganese (Mn), and the superparamagnetic agents based on iron oxide or iron platinium, and the X-nuclei .sup.18F, .sup.13C, .sup.23Na, .sup.17O, and .sup.15N.

27. The non-site specific method of claim 25, wherein the chelating agent is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA), diethylene triamine penta-acetic acid (DTPA), 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A), nitrilotriacetic acid (NTA), D-penicillamine (Pen), 2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), 2,3-dimercaptopropanol (BAL), triethylenetetramine (Trien), the ammonium tetrathiomolybdate (TTM) anion, ethylenediaminetetraacetic acid (EDTA), 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (IB4M) or hydroxypyridinone (HOPO).

28. The non-site specific method of claim 25, wherein the substance of interest is gadolinium (Gd), and the chelating agent is DOTA.

29. The non-site specific method of claim 24 comprising the following steps: (i′) the chelation of a substance of interest with a chelating agent activated in the form of an ester or an anhydride and (ii′) the conjugation of the pre-chelated substance of interest of step (i′) with lysine residues of a VHH directed against the fibrillar form of amyloid β, characterized in that its amino acid sequence comprises, from the N-terminus to the C-terminus, the amino acid sequence SEQ ID NO:1 (corresponding to the CDR1), the amino acid sequence SEQ ID NO:2 (corresponding to the CDR2) and the amino acid sequence SEQ ID NO:3 (corresponding to the CDR3).

30. The non-site specific method of claim 29, wherein the substance of interest is a NMR or MRI contrast agent selected from the paramagnetic agents gadolinium (Gd), dysprosium (Dy) and manganese (Mn), and the superparamagnetic agents based on iron oxide or iron platinium, and the X-nuclei .sup.18F, .sup.13C, .sup.23Na, .sup.17O, and .sup.15N.

31. The non-site specific method of claim 29, wherein the chelating agent is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA), diethylene triamine penta-acetic acid (DTPA), 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A), nitrilotriacetic acid (NTA), D-penicillamine (Pen), 2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), 2,3-dimercaptopropanol (BAL), triethylenetetramine (Trien), the ammonium tetrathiomolybdate (TTM) anion, ethylenediaminetetraacetic acid (EDTA), 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (IB4M) or hydroxypyridinone (HOPO).

32. The non-site specific method of claim 29, wherein the substance of interest is gadolinium (Gd), and the chelating agent is DOTA.

Description

(1) FIG. 1 shows the immunohistochemical staining of amyloid plaques using the VHH R3VQ on human paraffin sections. 6F3D (Akiyama H. et al., 1996, Neurosci lett., 206:169-72) was used as a reference anti-Aβ antibody.

(2) FIG. 2 shows the immunohistochemical staining of amyloid β plaques using the VHH R3VQ on fresh human AD brain tissues (A and B). 4G8 (Wisniewski T. et al., 1996, B. Biochem J., 313:575-80) was used as a reference anti-Aβ antibody (C and D).

(3) FIG. 3 shows the immunohistochemical staining of amyloid plaques using the VHH R3VQ on fresh brain transgenic TauPS2APP mice tissues (A). 4G8 was used as a reference anti-Aβ antibody (B).

(4) FIG. 4 shows the Western Blot on human brain extracts and on Aβ42 revealed by VHH R3VQ (phenol red-free Ham's F12 medium (Gibco) or buffer A [PBS, pH 7.4, 0.32 M sucrose, 50 mM Hepes, 25 mM MgCl2, 0.5 mM DTT] containing protease inhibitors [200 μg/ml PMSF, 2 μg/ml pepstatin A, 4 μg/ml leupeptin, 30 μg/ml benzamidine hydrochloride]).

(5) FIG. 5A shows the immunohistochemical staining of amyloid β plaques in transgenic TauPS2APP mice paraffin embedded sections after stereotaxic injections of VHH R3VQ.

(6) FIG. 5B shows a magnification of the inset of FIG. 5A.

(7) FIG. 6A shows the immunohistochemical staining of amyloid β plaques in transgenic TauPS2APP mice paraffin embedded sections after stereotaxic injections of R3VQ-N-(DOTA/Gd).sub.1-2 2e.

(8) FIG. 6B shows a magnification of the inset of FIG. 6A.

(9) FIG. 6C shows labeling of amyloid β plaques present in the thalamus, at distance from the injection site.

(10) FIG. 6D shows a magnification of the inset of FIG. 6C.

(11) FIG. 6E shows the control performed with 4G8 antibody on the same mouse to label amyloid plaques.

(12) FIG. 7A: After soaking of a transgenic TauPS2APP mouse brain in a solution of anti-Aβ VHH-Gd 2e (R3VQ-N-(DOTA/Gd).sub.1-2 contrast agent (0.02 mg/ml, equivalent to a 0.01 mM of Gd), in vitro MR images shows hypointense spots (white arrows).

(13) FIG. 7B shows the MRI detection of amyloid plaques on the same mouse revealed by the Gd-staining.

(14) FIG. 7B shows the MRI colocalization of amyloid β plaques revealed by the Gd-staining on the same mouse (arrows).

(15) FIG. 7C shows the immunohistochemical staining of amyloid β plaques of the same mouse (arrows).

(16) FIG. 7D is a control of a transgenic TauPS2APP mouse brain soaked with a Gadolinium solution at the same concentration (0.01 mM) used with R3VQ-N-(DOTA/Gd).sub.1-2 2e.

(17) FIG. 8A: After intracerebroventricular injection, the anti-Aβ VHH-Gd 2e (R3VQ-N-(DOTA/Gd).sub.1-2; 1 μl/side at 1 μg/μl) showed hypointense spots on ex vivo MR images in the hippocampus (white arrows).

(18) FIG. 8B shows the MRI colocalization of amyloid plaques revealed by the Gd-staining on the same mouse (arrows).

(19) FIG. 8C shows the immunohistochemical staining of amyloid β plaques of the same mouse (arrows).

(20) FIG. 8D is a control with the injection in a transgenic TauPS2APP mouse of a Gadolinium solution at the same concentration (0.1 mM) used with R3VQ-N-(DOTA/Gd).sub.1-2.

(21) FIG. 9 shows the synthesis of labeled VHH (R3VQ) by non-site specific approach (A) and site specific approach (B). VHHs 1 and 3 were eluted from the affinity column in PBS/NaCl/Imidazole buffer. 1 was subjected to conjugation by a non-site-specific approach after a buffer exchange (A) and 3 was conjugated by a site-specific method (B) with (method 1) or without (method 2) a buffer exchange. The ligation sites are shown on the proteins. The labeling resulted in, respectively, polydisperse mixtures (2f shown as an example, and 2e) and chemically-defined conjugates (5 shown as an example). n′=average amount of DOTA/Gd per VHH (randomly distributed on different sites). m=exact amount of DOTA/Gd per VHH (located on a single site). The overall yield (indicated in brackets) includes all the steps from the starting protein in the affinity column elution buffer (net peptide contents). A solid-phase synthesis of the maleimido-(DOTA/Gd).sub.3 compound 4 (C) is described in FIG. 9 (C). 4 was prepared by the conventional solid-phase peptide methodology using Fmoc chemistry and HATU/DIEA as the coupling reagent. The overall yield is indicated in bracket. DOTA structural formula is shown in the inset.

(22) FIG. 10 shows (A) the amino-acid sequence alignment of anti-Aβ VHHs A7, B 10, R3VE, R3VQ and F12; CDR1, CDR2, CDR3 are underlined, (B) the amino acid sequence of R3VQ-SH 3, (C) the amino acid sequence of R3VE-SH.

(23) FIG. 11 shows the analysis and assessment of the properties of VHH R3VQ-S-(DOTA/Gd).sub.3 (compound 5 FIG. 9) obtained by site specific conjugation. (A) HPLC/MS. (B) IEF. (C) Evaluation of BBB crossing by IHC detection of VHHs in the brain after iv injection. Comparison with unconjugated protein R3VQ-SH is showed in A, B.

(24) FIG. 12 shows the MRI detection of amyloid plaques after in vitro incubation with R3VQ-S-(DOTA/Gd).sub.3. Whereas no contrast anomalies could be detected in negative control PS2APP mice brains (A), several hypointense spots were revealed after in vitro incubation of PS2APP mice brains with R3VQ-S-(DOTA/Gd).sub.3 (B, white arrows). These hypointensity were colocalized with amyloid plaques highlighted by the Gd-staining procedure used as gold standard positive control for amyloid plaques detection by MRI (C, white arrows). Experiments were realized on a 7 T spectrometer.

(25) FIG. 13 shows the ex vivo MRI detection of amyloid plaques after iv injection of R3VQ-S-(DOTA/Gd).sub.3. Mice were iv injected with PBS (negative control, A-B) or R3VQ-S-(DOTA/Gd).sub.3 at 20 mg/kg (C) or 50 mg/kg (D) and sacrificed after 5 hours. MR images were acquired at 11.7 T on extracted fixed brains. Negative controls did not display strong contrast anomalies (A and B) as compared to injected brains (C and D, white arrows). These hypointense spots were stronger and more abundant at 50 mg/kg as compared to 20 mg/kg. These hypointense spots were colocalized with amyloid plaques revealed by the positive control procedure (E and F, white arrows).

(26) FIG. 14 shows the in vitro analysis and assessment of the properties of VHH R3VQ-S-AF488 obtained by site specific conjugation. (A) HPLC/MS. (B) SDS-PAGE. (C) IEF. (D) IHC on amyloid plaques. Comparison with unconjugated protein R3VQ-SH is showed in A, B, C.

(27) FIG. 15 shows the in vivo imaging of amyloid plaques and CAA after topic brain infusion of R3VQ-S-AF488 on the cortical surface in a 2-year-old PS2APP mouse. Arrow indicates labeling of CAA. Scale bar=50 μm.

(28) FIG. 16 shows the in vivo imaging of R3VQ-S-AF 488 using two-photon microscopy. (A) In vivo imaging in the brain after iv injection of R3VQ-S-AF488 in a 2-year-old PS2APP mouse using a maximum intensity projection (MIP) reconstruction with a projected volume 360 μm deep from the surface of the cortex. T0 represents the baseline imaging before iv injection. The scale bar is 50 μm. Empty arrowheads indicate vascular Aβ and filled arrowheads indicate parenchymal Aβ deposits. (B) In vivo imaging in the brain after iv injection of R3VQ-S-AF488 in a 2-year-old PS2APP mouse 3.5 hours after injection. Empty arrowheads indicate vascular Aβ and filled arrowheads indicate parenchymal Aβ deposits. (C) Immunohistochemical staining of amyloid plaques in the PS2APP mouse that received iv injection of R3VQ-S-AF488 using anti-His mAb. Immunostaining of amyloid plaques by R3VQ was observed throughout the entire brain. (D) Comparison of immunostaining of amyloid plaques between iv 10 mg/kg and iv 50 mg/kg of R3VQ-S-AF488 in PS2APP mice showing a dose-dependent effect on IHC signal.

(29) FIG. 17 shows the in vivo imaging of R3VE using two-photon microscopy: the basic pI is crucial to allow VHH to cross the BBB. (A) IEF analysis of R3VQ and R3VE compounds. R3VE is less basic than R3VQ for both VHH and conjugate (pI around 7.5 for R3VE-S-AF488). (B) Compared to mouse receiving R3VQ-S-AF488, only cerebral amyloid angiopathy was observed in the mouse receiving intravenously R3VE-S-AF488, dose: 10 mg/kg. (C) Comparison of histological staining with anti-His mAb in mice injected iv with R3VQ-S-AF488 10 mg/kg and R3VE-S-AF488 10 mg/Kg.

(30) FIG. 18 shows the in vivo imaging in the brain after iv injection of mAb 4G8-AF488 (10 mg/Kg) in a 2-year-old PS2APP mouse. The presence of mAb 4G8 is only observed in the blood vessels. Extra-vascular signal is artefactual because it is also observed in the reference channel (red).

EXAMPLE 1

Generation of Anti-Abeta VHHs Coupled to Gadolinium Contrast Agent and their Evaluation In Vitro/In Vivo

(31) Materials and Methods

(32) 1. Production, Selection and Purification of VHH R3VQ

(33) Antigen Preparation and Induction of a Humoral Immune Response in Alpaca

(34) A-beta 42 peptide (Aβ42) (1 mg-Bachem) was dissolved in 900 μl H.sub.2O and vortexed. 100 μl PBS 10× was added and the mixture was incubated at room temperature for one month before use. 250 μl of the mixture was mixed with 250 μl of Freund complete adjuvant for the first immunization, and with 250 μl of Freund incomplete adjuvant for the following immunizations. One young adult male alpaca (Lama pacos) was immunized at days 0, 21 and 35 with 250 μg immunogen. At day 50 a serum sample was taken and the immune response monitored by ELISA using Aβ42 as antigen.

(35) Library Construction and Panning

(36) 250 ml of blood of the immunized animal was collected at day 50 and the peripheral blood lymphocytes isolated by centrifugation on a Ficoll (Pharmacia) discontinuous gradient and stored at −80° C. until further use. Total RNA and cDNA was obtained as previously described in Lafaye P. et al. (1995, Res Immunol., 146:373-382), and DNA fragments encoding VHH domains amplified by PCR using CH2FORTA4 and VHBACKA6 primers, which anneal to the 3′ and 5′ flanking region of the VH genes, respectively. The amplified product was used as template in a second round of PCR using either the primers VHBACKA4 and VHFOR36 or the primers VHBACKA4 and LHH (5′ GGACTAGTTGCGGCCGCTGGTTGTGGTTTTGGTGTCTTGGG-3′) (SEQ ID NO. 13) specific for the long hinge homodimeric antibody. The primers were complementary to the 5′ and 3′ ends of the amplified product and incorporated SfiI and NotI restriction sites at the ends of the VHH genes. The PCR products were digested and ligated into phage expression vector pHEN1. The resulting library was composed of two sub-libraries, one derived from VHH DNA-encoding genes with no hinge and the other from long hinge antibody genes. Phages were produced and isolated using both sub-libraries, and subsequently pooled.

(37) The library was panned for reactivity in parallel with a biotinylated Aβ1-42, Aβ1-40 or Aβ1-16 peptide, as previously described (Lafaye P. et al., 2009, Mol Immunol. 46:695-704). The library (10.sup.13 transducing units) was panned by incubation with each biotinylated peptide for 1 h at 37° C. under gentle agitation, then the mixture was incubated with streptavidin beads for 15′ at 37° C. A different blocking agent was used at each of the three rounds of panning: 2% skimmed milk, Licor diluted 1:4, and 4% BSA were respectively used. The concentration of biotinylated peptides used decreased at every round of panning with respectively 100 nM, 50 nM and 10 nM. Phage clones were screened by standard ELISA procedures using a HRP/anti-M13 monoclonal antibody conjugate (GE Healthcare) for detection (see below).

(38) Expression of VHHs

(39) The coding sequence of the selected nanobodies in vector pHEN1 was sub-cloned into a modified bacterial expression vector pET23 containing a 6-Histidine tag using NcoI and NotI restriction sites. Transformed E. coli BL21 (DE3) LysS cells express VHH in the cytoplasm after overnight induction with IPTG (0.5 mM) at 16° C. Purified VHHs were isolated by IMAC from cytoplasmic extracts using a HiTrap crude column charged with Ni.sup.2+ (GE Healthcare), according to the manufacturer's instructions, followed by size exclusion chromatography with a Superdex 75 column (GE Healthcare). The VHHs (in particular the R3VQ(His)-NH.sub.2; compound 1 in FIG. 9) were eluted in 50 mM sodium phosphate buffer, 300 mM NaCl and 500 mM imidazole buffer.

(40) 2. Characterization of Biochemical Properties of VHH R3VQ

(41) Immunoblots

(42) A-beta 42 peptide was resuspended in NuPAGE® LDS sample buffer (Invitrogen) containing 8M urea. Following separation by polyacrylamide gel electrophoresis (PAGE) using NuPAGE Novex 4-12% Bis-tris gel (Invitrogen), semi-dry transfer onto Hybond-C (Amersham) and western blotting were carried out using the Xcell II blot module (Invitrogen). Prior to the immunochemical reaction, membranes were blocked in a 4% skimmed milk solution. Immunoblotting of membranes was accomplished with VHH and revealed by rabbit anti-His tag (eBioscience) polyclonal antibodies followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abeam). Finally, peroxidase activity was visualized using a chemiluminescent kit (GE Healthcare).

(43) ELISA

(44) Streptavidin-coated microtiter plates (Thermo Scientific, Denmark) were coated by incubation overnight at 4° C. with 1 μg/ml of biotinylated A-beta 40 or A-beta 42 (preferably A-beta 40) diluted in PBS. Plates were washed with buffer 0.1% Tween 20 in PBS. VHH R3VQ was diluted in buffer 0.5% gelatin 0.1% Tween 20 in PBS. After 2 h incubation at 37° C., plates were washed again before adding respectively a rabbit anti-His tag polyclonal antibody (eBiosciences), followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abeam), and finally revealed by OPD (o-phenylendiamine dihydrochloride, Dako) according to manufacturer's protocol.

(45) Determination of Dissociation Constants by ELISA

(46) The binding affinity of VHHs was determined as previously described (Friguet B. et al., 1985, Immunol Methods, 77:305-19). Briefly, various concentrations of Aβ peptides (A13 fragments 1-16, 10-20, 15-25, 22-35 and 29-40) were incubated in solution overnight at 4° C. with a known quantity of VHH until equilibrium was reached. The VHH concentration used was determined by preliminary ELISA calibrations. Each mixture (100 μl) was transferred to a well of a microtiter plate previously coated with antigen and was incubated for 20 min at 4° C. The plates were washed with buffer 0.1% Tween 20 in PBS and bound VHHs were detected by adding beta-galactosidase-conjugated goat anti-rabbit Igs (Biosys, Compiegne, France) and 4-methylumbelliferyl α-D galactoside (Sigma). Fluorescence was read (Fluoroskan, Labsystem, Finland) at 460 nm, after excitation at 355 nm. KD was estimated from the slope of the regression curve obtained by plotting the reciprocal of the fraction of bound antibody versus the reciprocal of the molar concentration of antigen.

(47) Sequences Analysis

(48) VHH encoded DNAs were sequenced by GATC Biotech and sequences were treated with DNA strider.

(49) Determination of pI

(50) The pI of VHHs was determined by isoelectric focusing using IEF 2-9 Gel (Invitrogen). NEPGHE (non equilibrium pH gradient gel electrophoresis) with sample application at the anode was used because it allows optimal protein analysis in the basic range of the gel including pH 8.5 to 10.5. The protocol was detailed in SERVAGel IEF 3-10 instruction manual.

(51) 3. VHH R3VQ Coupling to MRI Contrast Agents and Characterization of the MRI Properties of the Synthesized Contrast Agents

(52) VHH R3VQ was conjugated to gadolinium (MRI contrast agent) with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) (chelating agent). Two strategies based on non-site specific and site specific coupling were used: The first strategy comprises the steps of (i) conjugation with the chelating agent DOTA to lysine residues of VHH (R3VQ-NH.sub.2 1), and (ii) subsequent chelation with a MRI contrast agent, i.e. gadolinium (Gd) (see FIG. 9A). It resulted in complex polydisperse mixture of conjugates R3VQ-N-(DOTA/Gd).sub.n 2 with randomly distributed Gd and a range of Gd:VHH stoichiometry, as shown by reverse-phase high performance liquid chromatography/mass spectrometry (RP-HPLC/MS). By varying the conditions of the DOTA conjugation step, several conjugates were prepared with different DOTA/Gd density (overall yield 60-67%). When assessed in vivo by IHC and MRI, the R3VQ-N-(DOTA/Gd).sub.n conjugate was able to recognize amyloid plaques in mouse after intra cerebro-ventricular injection. The second strategy was to use a site specific approach which involves the labeling of the VHH R3VQ with a maleimido compound (see FIG. 9B). Cys-engineered R3VQ (R3VQ-SH 3) containing from the N to the C terminus a 6-Histidine tag, a thrombin cleavage site, R3VQ VHH sequence followed by a G.sub.3S spacer and three extra amino acids CSA was cloned in vector pET23 to allow a high level of expression. The single domain products were shown to be pure to homogeneity by SDS-PAGE and by RP-HPLC/MS. The pI value of R3VQ-SH was in the range 8.5-9. A maleimido-(DOTA/Gd).sub.3 compound 4 was prepared by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. When conjugated to maleimido-(DOTA/Gd).sub.3 compound by thio-addition, R3VQ-SH was totally converted into the well-defined compound R3VQ-S-(DOTA/Gd).sub.3 5, as shown by RP-HPLC/MS, with 70% yield. The pI of R3VQ-S-(DOTA/Gd).sub.3 was slightly reduced compared to the one of the unlabeled R3VQ-SH. The binding characteristics of R3VQ-SH and R3VQ-S-(DOTA/Gd).sub.3 were determined in competitive inhibition experiments involving Aβ40 bound to the ELISA plate and soluble Aβ40. The concentration of Aβ40 giving 50% binding inhibition was calculated to be 1 μg/ml for both R3VQ-SH and R3VQ-S-(DOTA/Gd).sub.3 suggesting that the addition of DOTA/Gd does not affect the VHH binding properties. Further, following the distribution of VHH-specific immunoreactivity in transgenic B6 PS2APP mice, R3VQ-SH showed good ability to immunodetect Aβ plaques in mouse paraffin sections after antigen retrieval pretreatment.

(53) 3.1. General Synthesis Methods

(54) Unless otherwise specified, the amino-acid derivatives and the reagents are purchased from Novabiochem and Sigma-Aldrich, respectively. The concentration of the peptide and VHH solutions (net protein content) was determined by quantitative amino acid analysis (AAA) using a Beckman 6300 analyser after hydrolysis of the compounds with 6N HCl at 110° C. for 20 h. The RP-HPLC/MS analyses were performed on an Alliance 2695 system coupled to a UV detector 2487 (220 nm) and to a Q-Tofmicro™ spectrometer (Micromass) with an electrospray ionisation (positive mode) source (Waters). The samples were cooled to 4° C. on the autosampler. The linear gradient was performed with acetonitrile+0.025% formic acid (A)/water+0.04% TFA+0.05% formic acid (B) over 10 or 20 min. The column used was a XBridge™ BEH300 C18 (3.5 μm, 2.1×100 mm) (Waters) (gradient 10-100% A). The source temperature was maintained at 120° C. and the desolvation temperature at 400° C. The cone voltage was 40 V. The samples were injected at 0.4-1 mg/ml concentration in their respective buffer added with B. The expected Mr values correspond to the average mass of proteins with N-ter deleted Met and one disulfide bond. The Mr analyses were recorded on the same spectrometer in the positive mode by direct infusion (source temperature and desolvation temperature were maintained at 80° C. and 250° C., respectively). The samples were dissolved at 5 μM concentration in water/acetonitrile (1/1) with 0.1% formic acid. The purity of 4 was analyzed by RP-HPLC using an Agilent 1200 pump system with a UV detector at 220 nm. The column used was a Kromasil C18 (100 Å, 5 μm, 4.6×250 mm) (AIT) and the gradient was performed with acetonitrile (VWR) (C)/water+0.1% TFA (VWR) (D) over 20 min.

(55) 3.2. Non-Site Specific Approach

(56) The molar equivalents of all reagents are indicated relative to reactive groups (5 NH.sub.2 per R3VQ and an average of 1 DOTA/R3VQ conjugate). The overall yields (see Table 2 below) include all the synthetic steps from the starting protein 1 in the affinity column elution buffer. They were calculated by dividing the actual amount of the final products 2a-f by their expected amount (net protein contents).

(57) The R3VQ(His)-NH.sub.2 VHH 1 eluted from the affinity column was dialyzed in PBS buffer containing 300 mM NaCl (PBS/NaCl). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) (274 μg, 4 eq relative to amino groups) dissolved in PBS/NaCl (120 μl) was added to 1 (480 μl, 0.60 mg/ml) and the solution was stirred at room temperature. Aliquots (10 μl) were withdrawn every 15 min, diluted with 100 mM Tris buffer pH 7.3 (90 μl) and analyzed by HPLC/MS to monitor the reaction progress. After 3 h, the solution was cooled to 4° C. and the buffer was exchanged to 0.4M Na acetate buffer pH 5 by using Vivaspin 500 centrifugal filter device (3,000 MWCO PES) (Sartorius). The resulting DOTA-VHH conjugate (480 μl) was added with GdCl.sub.3 (149 μg, 45 eq relative to average DOTA groups) in the same buffer (5 μl). The solution was stirred at room temperature for 2.5 h. The buffer was exchanged to PBS/NaCl at 4° C. with the same Vivaspin device as above and the solution was concentrated to afford the R3VQ(His)-N-(DOTA/Gd).sub.0-2 conjugate 2f (105 μl, 1.48 mg/ml). The overall yield is 67%.

(58) The conjugate 2e was obtained using the same protocole except that DOTA-NHS was added to 1 portionwise (0.5 eq every 45 min, total of 5.5 eq relative to amino groups). The solution was stirred at room temperature for 8 h15. The overall yield is 60%.

(59) R3VQ(His)-NH.sub.2 1

(60) AAA: Ala 15.8 (16), Arg 9.1 (9), Asp+Asn 13.4 (13), Glu+Gln 16.0 (15), Gly 13.4 (14), His 5.7 (7), Ile 3.1 (3), Leu 8.4 (8), Lys 4.1 (4), Phe 4 (4), Pro 6.1 (7), Ser 11.3 (13), Thr 10.0 (11), Tyr 4.8 (5), Val 11.1 (11).

(61) MS: 15753.0996 (C.sub.681H.sub.1053N.sub.209O.sub.216S.sub.4 calcd 15752.3949)

(62) R3VQ(His)-N-(DOTA/Gd).sub.0-2 2f

(63) AAA: Ala 16.0 (16), Arg 10.0 (9), Asp+Asn 12.8 (13), Glu+Gln 15.0 (15), Gly 13.8 (14), His 6.7 (7), Ile 3.0 (3), Leu 8.2 (8), Lys 4.5 (4), Phe 4 (4), Pro 8.5 (7), Ser 10.5 (13), Thr 10.1 (11), Tyr 4.8 (5), Val 11.1 (11).

(64) MS: 16293.1328 ((DOTA/Gd).sub.1: C.sub.697H.sub.1076N.sub.213O.sub.223S.sub.4Gd calcd 16293.0263)

(65) 16833.5586 ((DOTA/Gd).sub.2: C.sub.713H.sub.1099N.sub.217O.sub.230S.sub.4Gd.sub.2 calcd 16833.6576)

(66) 3.3. Site Specific Approach

(67) Production of R3VQ-SH 3

(68) The coding sequence of a Cys-engineered VHH (R3VQ-SH 3) was cloned into a modified bacterial expression vector pET23 using NcoI and XhoI restriction sites. Transformed E. coli BL21 (DE3) pLysS cells express 3 in the cytoplasm after overnight induction with IPTG (0.5 mM) at 16° C. Purified VHHs were isolated by IMAC from cytoplasmic extracts using a HiTrap crude column charged with Ni.sup.2+ (GE Healthcare), according to manufacturer's instructions. 3 was eluted in PBS/NaCl containing 500 mM imidazole.

(69) AAA: Ala 16.1 (16), Arg 10.2 (10), Asp+Asn 13.6 (13), Glu+Gln 11.9 (11), Gly 19.1 (20), His 6.0 (7), Ile 3.1 (3), Leu 8.5 (8), Lys 2.2 (2), Phe 4 (4), Pro 4.3 (4), Ser 15.5 (18), Thr 9.5 (10), Tyr 4.8 (5), Val 12.9 (12).

(70) MS: 15723.4268 (C.sub.671H.sub.1041N.sub.213O.sub.217S.sub.5 calcd 15724.2820).

Synthesis of maleimido-(DOTA/Gd)3 4

(71) The synthesis of 4 was performed stepwise on solid-phase from Fmoc-Gly-Wang resin (143 mg, 0.093 mmol). The building blocks 1,4,7,10-tetraazacyclododecane-1,4,7-tris-tbutyl-acetate-10-(N-α-Fmoc-N-ε-acetamido-L-lysine) [Fmoc-Lys(DOTA(OtBu).sub.3))—OH] (1.1 eq) (Macrocyclics) and 6-maleimidohexanoic acid (3 eq) were incorporated manually using 2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (1.06 and 2.9 eq, respectively)/diisopropylethylamine (DIEA) (2.2 and 6 eq, respectively) as coupling reagents and dimethylformamide (DMF) (Applied Biosystems) as solvent. Fmoc-Gly-OH (3 eq) was incorporated with DIC (3 eq) in DMF. The coupling steps with the Lys, Gly and maleimido derivatives were monitored by the Kaiser test (E. Kaiser et al (1980) Anal. Biochem. 34, 595-598) and were completed in, respectively, 3 h, 2 h and 1 h. Fmoc protection was removed with 20% piperidine in DMF. After the third lysine derivative, the last coupling with 6-maleimidohexanoic acid was carried out on three-quarters of the product (0.07 mmol). The peptide-resin was suspended in 10 ml of TFA (Applied Biosystems)/water/triisopropylsilane (95/2.5/2.5 v/v/v) at 4° C. and stirred for 4 h at RT. After filtration of the resin, the solution was concentrated and the crude product precipitated with diethyl ether. After centrifugation, the pellet was dissolved in water and lyophilized to yield 119 mg of the crude DOTA-peptide which was analyzed by NMR, MS, and RP-HPLC (gradient 10-40% C, retention time 9.2 min).

(72) .sup.1H NMR (D.sub.2O): δ 6.69 (s, 2H, CH Mal), 4.18 (m, 2H, 2CH.sub.α), 4.08 (m, 1H, CH.sub.α), 3.87-3.78 (m, 6H, CH.sub.2 Gly), 3.74-3.48 (b, 24H, CH.sub.2CO DOTA), 3.34 (t, 2H, CH.sub.2 6-Mal, J.sub.5,6=0.017 Hz), 3.30-2.99 (b, 48H, CH.sub.2CH.sub.2N DOTA), 3.06 (b, 6H, CH.sub.2ε), 2.14 (m, 2H, CH.sub.2 2-Mal), 1.74-1.53 (m, 6H, CH.sub.2β), 1.49-1.34 (m, 10H, CH.sub.2δ, CH.sub.2 3-Mal, CH.sub.2 5-Mal), 1.29-1.18 (m, 6H, CH.sub.2γ), 1.16-1.06 (m, 2H, CH.sub.2 4-Mal).

(73) .sup.13C NMR (D.sub.2O): δ 177.11 (1C, CONH Mal), 175.15, 174.63, 174.36 (3C, CO Lys), 173.26 (2C, CO Mal), 172.93 (1C, COOH Gly), 171.48, 171.21 (2C, CO Gly), 163.04-162.68 (4C, CONH DOTA), 134.22 (2C, CH Mal), 120.59, 117.69, 114.79, 111.90 (TFA), 55.20-53.10 (12C, CH.sub.2CO DOTA), 54.04, 53.80, 53.47 (3C, CH.sub.α), 52.20-46.80 (24C, CH.sub.2CH.sub.2N DOTA), 42.38, 41.00 (3C, CH.sub.2 Gly), 39.10 (3C, CH.sub.2ε), 37.37 (1C, CH.sub.2 6-Mal), 35.04 (1C, CH.sub.2 2-Mal), 30.48, 30.24, 30.23 (3C, CH.sub.2β), 27.67, 27.33, 24.67 (3C, CH.sub.2 3-Mal, CH.sub.2 5-Mal, CH.sub.2δ), 25.43 (1C, CH.sub.2 4-Mal), 22.43, 22.33, 22.15 (3C, CH.sub.2γ).

(74) MS: [M+H].sup.+ 1925.9888, [M+K].sup.+ 1963.9391 (C.sub.82H.sub.136N.sub.22O.sub.31 calcd [M+H].sup.+ 1927.1195, [M+H].sup.+ 1965.2098).

(75) The DOTA-peptide intermediate (99 mg) was dissolved in 0.4M Na acetate buffer pH 5 (41 ml) and added with Gd(OAc).sub.3.xH.sub.2O (123 mg, 2 eq relative to DOTA). After stirring at 95° C. for 25 min, the solution was cooled and loaded on a C18 reverse-phase column (2 g, diameter 1.5 cm). The column was washed with four volumes of water and the product was eluted with three volumes of water/acetonitrile 1/1 affording 79 mg of product after lyophilisation. The crude DOTA/Gd peptide was purified by reverse-phase flash chromatography (30×200 mm) using a gradient with acetonitrile+0.1% TFA/buffer D over 40 min, from 5/95 to 35/65 (20 ml/min, retention time 18 min). After lyophilization of the main fraction, 61 mg of 4 were obtained with an overall yield of 44% (the overall yield includes all the synthetic steps, it was calculated on the net peptide content of the isolated product 4 based on the first Gly residue loading on the resin). 4 was analyzed by MS and RP-HPLC (gradient 5-35% C, retention time 12.2 min, purity>90%).

(76) MS: 2388.8889 (C.sub.82H.sub.127N.sub.22O.sub.31Gd.sub.3 calcd 2388.7901).

Synthesis of R3VQ-S-(DOTA/Gd)3 5

(77) The R3VQ-SH VHH 3 eluted from the affinity column was dialyzed in PBS buffer containing 300 mM NaCl (PBS/NaCl). 4 (1.35 mg, 3 eq relative to 1 thiol group per VHH) in aqueous solution (135 μl) was added to 3 (1.5 ml, 2 mg/ml in PBS/NaCl pH 6.8) and the solution was stirred at 4° C. for 3 h. The solution was then diafiltered using Vivaspin 2000 centrifugal filter device (3,000 MWCO PES) (Sartorius). Aliquots (20 μl) of 3 and 5 were diluted with buffer B (20 μl) for RP-HPLC/MS analyses. Moreover, aliquots (10 μl) of 3 and 5 were diluted in 100 mM Tris buffer pH 7.3 (90 μl) for ELISA analyses. 1 ml of 5 (2.36 mg/ml) was obtained with a yield of 70%. It was calculated by dividing the actual amount of the final product 5 by its expected amount (net protein contents).

(78) The same reaction was also performed directly in the affinity column elution buffer (PBS/NaCl containing 500 mM imidazole), and gave a 83% overall yield. The process for obtaining the R3VQ/Gd conjugates is thus improved when the conjugation is directly performed in the affinity column elution buffer (PBS/NaCl/Imidazole)): i) the number of steps is decreased to two, and ii) the overall yield is increased until 83%. This process improvement has also been validated with another VHH (data not shown).

(79) AAA: Ala 14.9 (16), Arg 10.2 (10), Asp+Asn 12.2 (13), Glu+Gln 11.1 (11), Gly 24.6 (23), His* (7), Ile 3.1 (3), Leu 8.5 (8), Lys 11.7* (5), Phe 4 (4), Pro 4.8 (4), Ser 14.9 (18), Thr 9.1 (10), Tyr 5.0 (5), Val 12.6 (12). [*His cannot be determined due to co-elution with ammonium. Lys is overestimated due to co-elution with maleimido derivative in the conditions of the analysis.]

(80) MS: 18113.7383 (C.sub.753H.sub.1168N.sub.235O.sub.248S.sub.5Gd.sub.3 calcd 18113.0720).

(81) SDS-PAGE Electrophoresis

(82) Polyacrylamide Gel electrophoresis (PAGE) was performed using NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) according to manufacturer's instructions.

(83) Determination of pI

(84) The pI of VHHs was determined by isoelectric focusing using IEF 2-9 Gel

(85) (Invitrogen). NEPGHE (non equilibrium pH gradient gel electrophoresis) with sample application at the anode was used because it allows optimal protein analysis in the basic range of the gel including pH 8.5 to 10.5. The protocol was detailed in SERVAGel IEF 3-10 instruction manual.

(86) ELISA

(87) Streptavidin-coated microtiter plates (Thermo Scientific, Denmark) were coated by incubation overnight at 4° C. with 1 μg/ml of biotinylated A-beta 40 or A-beta 42 (preferably A-beta 40) diluted in PBS. Plates were washed with buffer 0.1% Tween 20 in PBS. For the non-site specific strategy, R3VQ-NH.sub.2 1, R3VQ-N-(DOTA/Gd).sub.1-2 2e were diluted in buffer 0.5% gelatin 0.1% Tween 20 in PBS. After 2 h incubation at 37° C., plates were washed again before adding respectively a rabbit anti-His tag polyclonal antibody (eBiosciences), followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abeam), and finally revealed by OPD (o-phenylendiamine dihydrochloride, Dako) according to manufacturer's protocol. For the site specific strategy, R3VQ-SH 3 and R3VQ-S-(DOTA/Gd).sub.3 5 were diluted in the same buffer as described before, after incubation with biotinylated A-beta 40 or A-beta 42 (preferably A-beta 40), a monoclonal anti-His tag antibody (H1029—Sigma) was added, followed by peroxidase labeled goat anti-mouse antibody (ab97265-Abcam), and revealed by the same substrate.

(88) Affinity Determination

(89) The binding properties of 3 and 5 were determined by measuring the amount of soluble A-beta 40 or A-beta 42 (preferably A-beta 40) peptide able to give 50% inhibition of immobilized A-beta 40 or A-beta 42 (preferably A-beta 40) recognition. Briefly, various concentrations of A-beta 40 or A-beta 42 (preferably A-beta 40) were incubated overnight at 4° C. with a defined quantity of 3 or 5 until equilibrium was reached. The VHH concentration used has been deduced from preliminary ELISA calibrations. Each mixture (100 μl) was transferred to a well of microtiter plate previously coated with antigen and was incubated for 15 min at 4° C. After washing with PBS containing 0.1% Tween 20, unbound VHH were detected by the addition of an anti-His mAb (H1029-Sigma) followed by β-galactosidase goat anti-mouse Igs and 4-methylumbelliferyl β-D-galactoside. Fluorescence was read (Fluoroskan, Labsystem, Finland) at 460 nm, after excitation at 355 nm.

(90) Immunohistochemistry

(91) Immunohistochemistry was performed on paraffin coronal brain sections (5 μm in thickness), obtained from transgenic mouse models of amyloidosis (PS2APP mice). The sections were made with a microtome (Microm HM340E). Sections were de-paraffinized in xylene (5 min, 3 times), rehydrated through ethanol (100%×2, 90 and 70%; 5 min/step) and brought to water. They were then pretreated with 98% formic acid for 5 min and finally immersed in water for 5 min. Endogenous peroxidases were neutralized with 3% hydrogen peroxide and 20% methanol, and nonspecific binding sites were blocked with TBS-0.5% tween pH8 BSA 2% for 30 min. In the following steps, the sections were rinsed 3 times, 5 min/time in TBS-tween between steps. The sections were then incubated overnight at 4° C. with the primary antibody R3VQ-SH, diluted to 2 μg/ml in TBS-tween. Sections were then treated with mouse monoclonal anti-His-tag antibodies (H1029-Sigma) for 2 h at room temperature, and finally developed with Dako REAL™ system Peroxidase/DAB Kit (Glostrup, Denmark) according to manufacturer's protocol. After washing with water, sections were counter-stained with Harris hematoxylin and re-rinsed in water. Before being mounted, sections were dehydrated in graded ethanol solution (70, 90 and 100%) and cleared in xylene.

(92) 3.4. MRI Properties of the Synthesized Contrast Agents

(93) MRI properties of the contrast agents were assessed on a 7 T-Spectrometer (Agilent, USA) interfaced with a console running VnmrJ 2.3. The spectrometer was equipped with a rodent gradient insert of 700 mT/m. A quadrature birdcage coil (diameter: 23 mm) was used for emission and reception. The longitudinal and transverse relaxivities r1 and r2 (change in the relaxation rate per unit concentration of an agent, meaning “effectiveness” as a MRI contrast agent) were determined from linear fits of R1 (i.e. 1/T1) and R2 (i.e. 1/T2) as a function of contrast agent concentration for concentrations of 0.2, 0.15, 0.1, 0.05, 0.025, 0.01, and 0 mmol/l by using the following equations: R1(C)=R1(0)+r1×C where R1(C) is the R1 in the tubes containing the contrast agent at a concentration C, R1(0) is the R1 in the tube without the contrast agent, and C is the concentration of the contrast agent. R2(C)=R2(0)+r2×C was used to calculate r2. The samples were imaged in hematocrit tubes.

(94) T1 calculation was based on seven successive 2D multi-slice spin echo images with twenty TR values (TR=0.021, 0.04, 0.06, 0.08, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5 sec), TE=14 ms, Nex=4, FOV=10×10 mm2, Mtx=64×64, 1 slices, slice thickness=3 mm, bandwidth=50 kHz). Parametric maps of relaxation times were calculated from exponential regression curves (S=1−exp(−TR/T1)) where S is the signal intensity, TR is the repetition time and T1 is the longitudinal relaxation time (ImageJ, MRI Analysis Calculator, Karl Schmidt).

(95) T2 calculation were based on 2D multi-echo multi-slice spin echo images by using 16 Echo times (TE=10 to 160 msec); TR=3300 ms; Nex=2; bandwidth=100 kHz, FOV=10×10 mm2, Mtx=64×64, 1 slices, slice thickness=3 mm. Parametric maps of relaxation times were calculated from exponential regression curves (S=exp(−TE/T2)) where S is the signal intensity, TE is the echo time and T2 is the longitudinal relaxation time (ImageJ, MRI Analysis Calculator, Karl Schmidt).

(96) 4. In Vitro Characterization of VHH R3VQ, and VHH R3VQ Conjugates by Immunohistochemistry, Biochemistry and In-Vitro MRI

(97) Subjects

(98) Human cortical brain tissues from AD patients (Braak stage V and VI) were obtained from the NeuroCEB brain bank. This bank is associated to a brain donation program run by a consortium of patients associations (including France Alzheimer Association) and declared to the Ministry of Research and Universities, as requested by French Law. An explicit written consent was obtained for the brain donation in accordance with the French Bioethical Laws.

(99) Preclinical experiments were performed on B6TgPS2APP (Richards J. G., 2003, J Neurosci., 23:8989-9003) and APP/PS1dE9 (Garcia-Alloza M., 2006, Neurobiol Dis., 24:516-24) transgenic mice. Animal experimental procedures were performed in strict accordance with the recommendations of the EEC (86/609/EEC) and the French national committee (decree 87/848) for the care and use of laboratory animals. The animals were sacrificed using a high dose of sodium pentobarbital (100 mg/kg) and then perfusion-fixed with 10% buffered formalin. Their brains were then removed, immersed in formalin for at least 24 hours and stored at 4° C.

(100) Tissue Extracts

(101) Tissue extraction was performed according to Gong, Y. et al. (2003, Proc Natl Acad Sci USA, 100:10417-10422). Frontal cortex from AD brain (0.2 g) was homogenized in 20 volumes of phenol red-free Ham's F12 medium (Gibco) or buffer A (PBS, pH 7.4, 0.32 M sucrose, 50 mM Hepes, 25 mM MgCl2, 0.5 mM DTT) containing protease inhibitors (200 μg/ml PMSF, 2 μg/ml pepstatin A, 4 μg/ml leupeptin, 30 μg/ml benzamidine hydrochloride), and was centrifuged at 100,000×g for 1 h. The pellet was re-homogenized in 10 volumes of phenol red-free Ham's F12 medium or buffer A plus protease inhibitors and was re-centrifuged. The protein concentration of the combined supernatants was determined. An aliquot of protein was then concentrated to a volume of 60 μl or less, by using a Centricon-10 concentrator.

(102) Immunoblots

(103) Brain extracts or A-beta 42 peptide were resuspended in NuPAGE® LDS sample buffer (Invitrogen) containing 8M urea. Following separation by polyacrylamide gel electrophoresis (PAGE) using NuPAGE Novex 4-12% Bis-tris gel (Invitrogen), semi-dry transfer onto Hybond-C (Amersham) and western blotting were carried out using the Xcell II blot module (Invitrogen). Prior to the immunochemical reaction, membranes were blocked in a 4% skimmed milk solution. Immunoblotting of membranes was accomplished with VHH and revealed by rabbit anti-His tag (eBioscience) polyclonal antibodies followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abeam). Finally, peroxidase activity was visualized using a chemiluminescent kit (GE Healthcare).

(104) Immunohistochemistry

(105) Immunohistochemistry was performed on fixed tissues (paraffin-embedded or frozen sections), or alternatively on unfixed fresh tissues (frozen sections). Standard IHC protocols were applied and adapted for each tissue conditions. As most of immunostaining experiments were performed using paraffin sections, a detailed protocol for paraffin-embedded material is presented here. Immunostaining of brain tissue was performed on 4 μm thick paraffin sections. Both human and mouse tissues were used (Human AD patient, TauPS2APP mice [Grueninger F. et al., 2010, Neurobiol Dis., 37:294-306] and PS2APP transgenic mice [Richards, J. G., et al 2003, The Journal of neuroscience 23, 8989-9003]). Sections were de-paraffinized in xylene, rehydrated through ethanol (100%, 90%, and 70%), 5 min for each solution and finally brought to running tap water for 10 min. They were then incubated in 98% formic acid for 5 min, washed again under running tap water, quenched for endogenous peroxidase with 3% hydrogen peroxide and 20% methanol, and finally washed in water. Non-specific binding was blocked by incubating the sections for 30 min in 2% bovine serum albumin in TBS+0.5% Tween. Appropriate dilutions of primary antibodies (5-10 μg/ml of VHH His or Strep tag) were then applied and slices incubated overnight in a humidified chamber at room temperature. Slides were washed with TBS-Tween and incubated with secondary antibodies rabbit anti-His Tag for 1/1000 or home-made biotinylated anti-strep mAb C23-21 in TBS-Tween at room temperature for 1 h. Slides were then incubated with reagents of Dako REAL™ Detection System, Peroxidase/DAB+ according to manufacturer's instructions. Chromogenic (DAB) revelation was developed until a good signal-to-noise ratio was obtained (about 5 min). After washing with TBS-Tween, slides were counter-stained with hematoxylin. For labeling of plaques, biotinylated 4G8 (Wisniewski T et al., 1996, B. Biochem J., 313:575-80) mAb (1/10000) or 6F/3D (Akiyama H. et al., 1996, Neurosci let., 206:169-72) mAb (1/200) was used as a positive control in parallel.

(106) In vitro MRI

(107) MRI were performed on the brains of B6TgPS2APP (Richards J. G., 2003, J Neurosci., 23:8989-9003) and APP/PS1dE9 (Garcia-Alloza M., 2006, Neurobiol Dis., 24:516-24) transgenic mice (n=2, females). MRI were recorded on a 7 T-Spectrometer (Agilent, USA) interfaced with a console running VnmrJ 2.3. The spectrometer was equipped with a rodent gradient insert of 700 mT/m. A birdcage coil (RapidBiomed, GmbH, Germany) and a mouse brain surface coil (RapidBiomed GmbH, Germany) were used for emission and reception, respectively.

(108) After 4 h of membranes permeabilization in a solution of Triton 0.2% in PBS, the brain samples were soaked in a solution of phosphate buffered saline (PBS) and tested contrast agent and stored at 4° C. for at least 24 hours prior to imaging. For scanning, the brains were placed in a tight plastic tube filled with Fluorinert® (3M, Cergy-Pontoise, France), an aprotonic perfluorocarbon-based fluid that provides a black background in MR images.

(109) MR images were based on a 3D gradient-echo sequence was used (FLASH) to acquire T2*w images (TR=40 ms, TE=15 ms, FA=20°, Bw=50 kHz, Nex=16, matrix=512×512×128, FOV=13×13×13 mm.sup.2, yielding a resolution of 25×25×100 μm.sup.3 for a total Tacq of 11 h 39 min).

(110) Gd-Staining Method: A Gold Standard Method for Amyloid Plaques Detection

(111) This procedure was used to make colocalization between hypointense spots seen on MR images after the in vitro procedure, and a gold standard method revealing amyloid plaques (Petiet A. et al., 2012, Neurobiol Aging, 33:1533-44). Briefly, following in vitro experiments, the samples were soaked in a solution of PBS and 0.5 M gadoterate meglumine at a dilution of 1:200 (2.5 mM) and stored at 4° C. for at least 24 hours prior to imaging. MR images were realized in the same conditions as used for in vitro experiments.

(112) 5. In-Vivo Evaluation of VHH R3VQ, and VHH R3VQ Conjugates

(113) Subjects

(114) In vivo evaluation of VHH R3VQ, and VHH R3VQ conjugates was performed on TauPS2APP (Grueninger F. et al., 2010, Neurobiol Dis., 37:294-306) transgenic mice under the authorization previously described.

(115) In Vivo Stereotaxic Injection of VHH

(116) Stereotaxic injections were performed in TauPS2APP (n=2 female) transgenic anesthetized mice with 2 μl of VHH per injection at the rate of 0.5 μl/min. The mice were anesthetized with a mixture of isoflurane (1-2%) and air (1 L/min). They were placed on a stereotaxic frame and the skull was bilaterally perforated with a Dremel. Blunt Hamilton syringes were used to inject MR contrast agent. Each mouse received 4 injections, in the frontal cortex and the hippocampus in each hemisphere. The stereotaxic coordinates in the frontal cortex were +0.86 mm anterior from bregma, ±1.5 mm lateral from the midline, −0.65 mm ventral from dura. The stereotaxic coordinates in the hippocampus were −2.18 mm posterior from bregma, ±1.5 mm lateral from the midline, −1.8 mm ventral from dura. Two or 24 hours after the injection, mice were euthanized and perfused intracardially with 4% paraformaldehyde in PBS (pH 7.6). Brains were removed and postfixed in the same fixative overnight at 4° C. 4 μm thick paraffin sections were prepared. The presence of the VHH in cerebral tissue was detected using either of the standard immunohistochemical procedures described above.

(117) Intracarotid Perfusion of VHH in Mouse Models of Amyloidosis

(118) Intracarotid administration of VHH was performed in anesthetized TauPS2APP mice (n=2, females). The anesthesia was performed by injecting once intraperitoneally a mixture of ketamine hydrochloride (Imalgen, 50 μl of a solution diluted 1/10) and xylazine (Rompun, 50 μl of a solution diluted 1/40). The common carotid artery was exposed and cannulated with fine silicon tubing (PP25_100 FT; Portex, Ashford, UK). VHH was infused into the carotid at a constant rate using a peristaltic pump (Model PHD 2000; Harvard Apparatus, Boston, Mass., USA).

(119) Lateral Tail Vein Injection

(120) Mice were placed in an appropriate inverted beaker with hole for tail access. The animals were warmed before the injection and the tail was soaked with lukewarm water to cause vasodilatation of the vein. Injections before the MR images were performed after insertion of a catheter (27G, Microflex, Vygon, France) into the tail vein of the animals, in order to warrant the proper administration of the contrast agent. Two mg of VHH, VHH R3VQ-DOTA, or VHH R3VQ-S-(DOTA/Gd).sub.3 dissolved in 200 μl PBS was then injected into the lateral tail vein.

(121) In Vivo MRI

(122) In vivo MRI was performed on a 7 T-Spectrometer (Agilent, USA) interfaced with a console running VnmrJ 2.3 as previously described (see in vitro MRI chapter). During the MRI experiment the animals were anesthetized with a mixture of isoflurane (0.75-1.5%) and carbogen (95% O2-5% CO2) and their breathing rate was monitored. Carbogen was used to reduce the signal coming from circulating blood (Thomas et al., 2003).

(123) MR images were recorded using a high-resolution 3D-Gradient Echo sequence (29*29*117 μm.sup.3, FOV: 15*15*15 mm.sup.3, Mtx=512*512*128, TR=30 ms, TE=15 ms, flip angle=20°, Nex=1, bandwidth=25 kHz, Acquisition Time: 32 min (Petiet, A. et al., 2012, Neurobiol Aging, 33:1533-44).

(124) The T1 calculation was based on seven successive 2D multi-slice spin echo images with five TR values (TR=0.4, 0.75, 1.5, 2.5, and 5 s, TE=14 ms, Nex=1, FOV=25×25 mm2, Mtx=128×128, 6 slices, slice thickness=1 mm, bandwidth=50 kHz). Parametric maps of relaxation times were calculated from exponential regression curves (S=1−exp(−TR/T1)) where S is the signal intensity, TR is the repetition time and T1 is the longitudinal relaxation time (ImageJ, MRI Analysis Calculator, Karl Schmidt). Relaxation times were measured from cortical regions in the frontal part of the brain.

(125) Ex Vivo MRI

(126) 6 h after in vivo intracerebroventricular injections of VHH-S-(DOTA/Gd).sub.3 (1 μg/side), mice were perfused (PFA 4%) and their brains were extracted prior to ex vivo MR images. For each procedure, controls were performed by using an equivalent solution of Gd (i.e: 0.1 mM).

(127) Results

(128) 1. Library Construction, and Selection of Specific Anti-Aβ VHH

(129) VHHs were amplified by PCR and cloned in vector pHEN1. Subsequent transformations yielded a library of about 10.sup.8 clones. VHHs displaying the best affinity were selected by phage display through 3 palming cycles with biotinylated Aβ1-42, Aβ1-40 or Aβ1-16 peptides. 46 individual clones were tested by ELISA from Aβ42 panning, 192 clones from Aβ40 and 192 clones from Aβ16. 46/46, 110/192 and 163/192 were found positive from Aβ42, Aβ40 and Aβ16 pannings, respectively. These positive clones were sequenced and 45, 65 and 118 VHH sequences were respectively identified. Finally 3 families of VHH were selected (A7/B10, F12 and R3VQ) (see Table 1 below). A7/B10 VHHs were found 11, 64 and 117 times after pannings against, respectively, biotinylated Aβ1-42, Aβ1-40 or Aβ1-16. VHH R3VE/Q were found respectively 34 times and once after panning against biotinylated Aβ1-42 and Aβ1-40 while VHH F12 were found once after Aβ16 panning.

(130) These VHHs were subcloned in vector pET23 or in vector pASK IBA2 to allow a high level of expression of VHH with, respectively, a His-tag or a Streptavidin-tag. Yields of 1-2 mg/I of bacterial culture were obtained. The single domain products were shown to be pure to homogeneity by SDS-PAGE (data not shown); their pI values were above 8.5. Subsequent experiments were performed with the two constructs.

(131) R3VQ is kept at 4° C. or for long term storage at −20° C. with glycerol. It is not stable frozen without glycerol.

(132) DLS experiments showed that R3VQ is monomeric and not aggregated after purification.

(133) Amino-acid sequence alignment of anti-Aβ VHHs A7, B10, R3VE, R3VQ and F12 is shown in FIG. 10.

(134) 2. Recognition of Amyloid Plaques by VHH R3VQ

(135) Immunoreactivity of VHH R3VQ for Aβ and Amyloid Lesions

(136) It was examined the distribution of VHH-specific immunoreactivity in human AD brains and transgenic TauPS2APP mice. R3VQ showed good ability to immunodetect Aβ plaques and cerebral amyloid angiopathy (CAA) in human paraffin sections after antigen retrieval pretreatment (FIG. 1). No labelling was observed with wild type mice. Paralleling result on paraffin-embedded tissues, it was showed that Aβ immunodetection using R3VQ can be readily obtained on free-floating vibratome sections (data not shown) and, more importantly, on fresh tissues from AD human brains and from mouse brain sections without the use of any antigen retrieval pre-treatment (FIG. 2-3). Noticeably the strong background signal and low signal/noise ratio observed with free-floating sections obtained on a freezing microtome, precluded the use of this material for R3VQ IHC. Amyloid plaques immunolabelling was undetectable for VHH A7/B10 and F12 and no longer experiments were performed with these VHHs.

(137) To confirm the immunoreactivity of VHH R3VQ on brain tissues, western-blot immunoassays were performed on brain extracts obtained from AD patients. Four principal bands, corresponding to Aβ oligomers between 40 and 55 kDa, were immunodetected with VHH R3VQ. In parallel R3VQ recognized three bands with Aβ42 peptide between 6 and 17 kDa corresponding to monomers, dimers and trimers (FIG. 4).

(138) R3VQ Recognizes the Central Region of Aβ42

(139) VHH R3VQ was shown to be specific for fibrillar synthetic Aβ42 peptide in inhibition assays using Aβ42. VHH R3VQ had a KD of 17 nM. To further determine the epitope recognized by VHH R3VQ, the concentration required for 50% inhibition (IC50) was determined for Aβ40 and for peptides corresponding to different Aβ fragments (1-16, 10-20, 15-25, 22-35 and 29-40). VHH R3VQ did neither recognize fragments 1-16 nor 29-40. The IC50 of VHH R3VQ for Aβ 16-35 was 16 nM, suggesting that VHH R3VQ recognizes an epitope located in the central part of Aβ42. R3VQ did not recognize APP by flow cytometry using the H4 stable clone provided by Roche (data not shown).

(140) VHH R3VQ Labels Amyloid Plaques In Vivo after Stereotaxic Injection

(141) Two μg of VHH R3VQ were injected stereotaxically into the hippocampus or the cortex of the left hemisphere of the mouse brain (2 mice). 2 h or 24 h after the injection, the animals were sacrificed and brain sections were collected Immunostaining of amyloid plaques was observed indicating that VHH R3VQ labeled fibrillar Aβ in vivo. Moreover, it was noticed a brown halo in the cortex indicating the diffusion of VHH R3VQ into brain tissues (FIGS. 5A and 5B).

(142) VHH R3VQ Crosses the BBB In Vivo

(143) VHH R3VQ was tested in vivo for its ability to cross the BBB. Four mgs of VHH was injected via the left carotid artery over a period of 60 min. Following the injection, the diffusion of VHH into cerebral tissues was allowed for 1 hour before the mice were euthanized and perfused with fixative. Immunostaining of amyloid plaques was observed in cortex, hippocampus and thalamus. This staining was faint in the right hemisphere contralateral to the injected carotid.

(144) The experiments showed that unlabeled R3VQ 1) was able to cross BBB following slow intra-carotid infusion, 2) specifically recognized amyloid plaques in vivo, and 3) is a good candidate for MRI studies.

(145) Comparison Between VHH R3VQ and Known VHHs Directed Against Amyloid β

(146) Table 1 below summarizes the labeling of VHHs directed against amyloid obtained by immunization of an alpaca with the peptide Aβ42, the peptide Aβ1-10 coupled to the ovalbumin or fibrillar form of Aβ42. The selections of the VHHs have been carried out by phage display, except for VHHs R1.3, R1.5 and R3.3 which have been selected by ribosome display.

(147) TABLE-US-00001 TABLE 1 Labeling of VHHs directed against amyloid β obtained by immunization of an alpaca with the peptide Aβ42, the peptide Aβ1-10 coupled to the ovalbumin or fibrillar form of Aβ42. VHHs L1-3, L35, 61-3, V31-1 are disclosed in Lafaye P. et al., 2009, Molecular Immunology, 49: 695-704. The other VHHs were obtained according to the method disclosed above. Labeling VHHs Amyloid Amyloid Amyloid clones Immunogens Selection angiopathy (CAA) oligomers plaques Aβ42 oligomer Aβ42 coated tubes L1-3 0 0 0 L35 0 0 0 61-3 0 0 0 V31-1 0 ++ 0 Aβ1-10 coated tubes L3 0 0 0 L4 0 0 0 L7 0 0 0 V2 0 0 0 V17 0 0 V11 0 0 0 OVA Aβ1-10 Aβ1-16 coated tubes NN1 ++ 0 0 NN3 ++ 0 + NN4 Fibrillar Aβ42 Aβ42 coated tube 2D5 0 0 0 3F7 0 0 0 2A5 0 0 0 3B4 0 0 0 3B10 0 0 0 3H7 0 0 0 Fibrillar Aβ42 Ribosome display + Aβ42 coated tubes R1.3 ++ 0 0 R1.5 0 0 0 R2.3 ++ 0 0 R3.3 ++ 0 + Fibrillar Aβ42 Biotinylated Aβ40 or biotinylated Aβ16 biotinyle + magnetic beads B10 0 0 0 A7 0 0 0 F12 0 0 0 Fibrillar Aβ42 Biotinylated Aβ42 + magnetic beads R3VE ++ 0 +++ R3VQ Control AcM +++ +++ 6F/3D

(148) The results show that among the 27 VHHs tested only VHH R3VE/Q is able to label amyloid angiopathy and amyloid plaques but not amyloid oligomers.

(149) 3. Antibody Coupling to MRI Contrast Agent

(150) Non-Site Specific Approach:

(151) Conjugation was performed with NHS-activated DOTA to VHH lysine residues. Subsequent chelation with Gd has resulted in conjugates with variable ratios of DOTA/Gd on the protein (Table 2). Monitoring of both steps by HPLC/MS has allowed to optimize the process. Nearly complete chelation could be achieved at room temperature.

(152) Two initial conjugates (2a and b) have showed no Aβ binding activity due to instability during storage at −20° C. without glycerol. By varying experimental conditions, four new conjugates have been prepared in 0.5-1 mg scale with a good recovery (64-74%). The first series (2c and d) has a higher DOTA/Gd density than the second series (2e and f). Compared to the original VHH 1 Aβ recognition in ELISA, the binding of 2c and 2d is low (˜10%) while the binding of 2e and 2f is hardly affected (50 to 100%). This difference might be due to the overall lower density of DOTA/Gd and/or inherent stability of the VHH.

(153) TABLE-US-00002 TABLE 2 Characteristics of R3VQ(His)-N-(DOTA/Gd) conjugates Average Average Overall ELISA vs Experimental conditions DOTA/ Gd/ yield original Compound Initial storage Conjugation.sup.a Chelation.sup.a protein.sup.b protein.sup.b (%) VHH 2a −20° C. 12 × 2 eq, 12 h 20° C., (3) 4 5 (6) (3) 4 (5) 67 − 2 h 30 2b −20° C. 12 × 2 eq, 12 h 60° C., (3) 4 (5) (3) 4 (5) 65 − 15 min 2c −20° C. + 12 × 2 eq, 12 h 20° C., (1) 2 (3) (1) 2 (3) nd + glycerol 2 h 30 2d −20° C. + 12 × 2 eq, 12 h 60° C., (1) 2 (3) (1) 2 (3) nd + glycerol 20 min 2e  +4° C. 11 × 0.5 eq, 20° C., (0) 1 2 (3) (0) 1 2 (3) 60 ++ 8 h 15 2 h 30 2f  +4° C. 4 eq, 3 h 20° C., (0) 1 (2) (0) 1 (2) 67 ++ 2 h 30 .sup.aThe reactions are performed at room temperature. .sup.bDetermined by MS. The minor compounds are in brackets.

(154) Site Specific Approach:

(155) This strategy involves the labeling of the Cys-engineered R3VQ VHH (R3VQ-SH 3) (SEQ ID NO 8) with a maleimido-(DOTA/Gd).sub.3 compound 4 (see FIG. 9B).

(156) DOTA is represented as the monoacyl moiety. The overall yield is indicated in brackets.

(157) When conjugated to 4 by thioaddition, 3 was totally converted into the well-defined compound R3VQ-S-(DOTA/Gd).sub.3 5, as shown by RP-HPLC/MS, with 79% yield. The pI of 5 was slightly reduced compared to the one of the unlabeled R3VQ-SH. The binding characteristics of R3VQ-SH and R3VQ-S-(DOTA/Gd).sub.3 were determined in competitive inhibition experiments involving Aβ40 bound to the ELISA plate and soluble Aβ40. The concentration of Aβ40 giving 50% binding inhibition was calculated to be 1 μg/ml for both R3VQ-SH and R3VQ-S-(DOTA/Gd).sub.3 suggesting that the addition of DOTA/Gd does not affect the VHH binding properties. Further, following the distribution of VHH-specific immunoreactivity in transgenic B6.PS2APP mice, R3VQ-SH showed good ability to immunodetect Aβ plaques in mouse paraffin sections after antigen retrieval pretreatment.

(158) R3VQ-SH was constructed with a C-terminal Cys residue for coupling to Maleimido-DOTA/Gd (FIG. 9). 15 mg of purified protein is expressed per L of culture. However several constructs have been realized before and are summarized below: Strep-R3VQSSfree-Cys-Thr-His containing from the Cter to the Nter a strep tag, VHH R3VQ with Cys mutated to Val and Ser, a Cys, a thrombin cleavage site and a his tag. This protein was expressed at very low yield Gig protein/1). Tag-R3VQSSfree-Cys and His Tag-R3VQSSfree-Cys containing from the Cter to the Nter a tag (either Strep or His tag), VHH R3VQ with Cys mutated to Val and Ser and a Cys; Both proteins were expressed at very low yield (μg protein/1). Tag-R3VQSSfree-Cys-Ser-Ala containing from the Cter to the Nter a tag (either Strep or His tag), VHH R3VQ with Cys mutated to Val and Ser, a Cys, a Ser and an Ala. These proteins were expressed at very low yield (μg protein/I). Tag-Thr R3VQSSfree-Cys-Ser-Ala containing from the Cter to the Nter a tag (either Strep or His tag), a thrombin cleavage site, VHH R3VQ with Cys mutated to Val and Ser, a Cys, a Ser and an Ala. These proteins were expressed at very low yield (μg protein/1).

(159) 4. Detection of Amyloid Plaques with VHH R3VQ Conjugated with Gd Contrast Agent

(160) R3VQ-N-(DOTA/Gd).sub.(1-2) Labels Amyloid Plaques In Vivo after Stereotaxic Injection

(161) Two μg of VHH R3VQ-N-(DOTA/Gd).sub.1-2 (2e) were injected stereotaxically into the hippocampus or the cortex of the left hemisphere of the mouse brain (2 mice). 4 h after the injection, the animals were sacrificed and brain sections were collected. Immunostaining of amyloid plaques was observed indicating that R3VQ-N-(DOTA/Gd).sub.n′ labeled fibrillar Aβ in vivo (FIGS. 6A and 6B). FIGS. 6C and 6D shows labeling of amyloid β plaques present in the thalamus, at distance from the injection site. A control was performed with 4G8 antibody on the same mouse to label amyloid plaques (FIG. 6E).

(162) In-Vitro Imaging

(163) Imaging of brains of TauPS2APP mice soaked in a solution of R3VQ-N-(DOTA/Gd).sub.1-2 (2e contrast agent at a final concentration of 0.02 mg/ml, equivalent to a 0.01 mM of Gd) revaled several hypointense spots (n=2; FIG. 7A, arrows) that could not be detected in the brains of control mice images in the same condition (data not shown) that could be colocalized with amyloid plaques revealed by the Gd-staining method (FIG. 7B, arrows). IHC confirmed the large diffusion of VHH-DOTA/Gd and the labeling of amyloid plaques in the same area (FIG. 7C, arrows), even if the distortion induced by the paraffin procedure did not allow point-to-point registration between MRI and IHC. Moreover, no hypointense spots could be detected in the brains of control mice images in the same condition (n=2; data not shown) or in the brains of TauPS2APP mice soaked in Gadolinium solution at the same concentration (i.e., 0.01 mM; FIG. 7D).

(164) Ex-Vivo Imaging after Intracerebral, Intracarotid or IV Injection

(165) After intracerebroventricular injection, the anti-Aβ VHH-Gd 2e (R3VQ-N-(DOTA/Gd).sub.1-2) showed hypointense spots on ex vivo images in the hippocampus (FIG. 8A, arrows) that correspond to amyloid plaques as confirmed by Gd-staining on the same mouse (FIG. 8B, arrows). IHC confirmed the labeling of amyloid plaques in the same area (FIG. 8C, arrows), even if the distortion induced by the paraffin procedure did not allow point-to-point registration between MRI and IHC. Moreover, no hypointense spots could be detected with the injection in a transgenic TauPS2APP mouse of a Gadolinium solution at the same concentration (0.1 mM) used with R3VQ-N-(DOTA/Gd).sub.1-2.

(166) Evaluation of R3VQ-S-(DOTA/Gd).sub.3 by In Vitro MRI

(167) R3VQ-S-(DOTA/Gd).sub.3 was synthesized by site specific approach as described above. The HPLC/MS, pI, and IHC assays confirm the biochemical properties (i.e. purity by HPLC/MS, pI, and IHC reactivity against Aβ) of R3VQ-S-(DOTA/Gd).sub.3 (see FIG. 11).

(168) The potential of R3VQ-S-(DOTA/Gd).sub.3 to induce MR contrast modification was evaluated after in vitro incubation of brains from PS2APP mice (Richards, J. G., et al., 2003, The Journal of neuroscience: the official journal of the Society for Neuroscience 23, 8989-9003) (n=2) with R3VQ-S-(DOTA/Gd).sub.3 at 0.1 mg/ml as described above. Images acquired at 7 T revealed hypointense spots in the cortex as compared to brains of PS2APP mice under negative control condition (FIGS. 12A and B). To confirm the nature of these hypointense spots as amyloid plaques, brains were submitted to a Gd-staining procedure used as the gold-standard method for MRI detection of amyloid plaques (FIG. 12C). Analyses of images obtained after the Gd-staining procedures allowed the co-registration of the amyloid plaques detected by Gd-staining with the hypointense spots revealed by the R3VQ-S-(DOTA/Gd).sub.3 (FIG. 12, white arrows). These results suggest that R3VQ-S-(DOTA/Gd).sub.3 passively diffuses in postmortem tissues and targets amyloid deposits allowing their detection by in vitro MRI.

(169) Evaluation of R3VQ-S-(DOTA/Gd).sub.3 by Ex-Vivo MRI after Peripheral (Intravenous) Injections

(170) R3VQ-S-(DOTA/Gd).sub.3 ability to reveal amyloid plaques by MRI was then investigated after intravenous injection in the tail vein (20 mg/kg and 50 mg/kg) of 18-month-old PS2APP mice as described above. The MR images acquired at 11.7 T of the PS2APP mice injected intravenously with R3VQ-S-(DOTA/Gd).sub.3 were acquired after brain extraction 5 hours following injection. Contrary to MR images of the control condition (PS2APP mice injected with PBS) (FIGS. 13A and B), images obtained on ex vivo brains that received intravenous injection of R3VQ-S-(DOTA/Gd).sub.3 showed numerous hypointense spots (FIGS. 13C and D). These hypointense spots were co-registered with contrast anomalies corresponding to amyloid plaques as detected with a gold-standard Gd-staining procedure (FIGS. 13E and F). These spots were more intense with the 50 mg/kg dose suggesting, according to the two-photons results, that R3VQ brain penetration and its potency to label brain AB lesions were dose-dependent.

EXAMPLE 2

Generation of Anti-Abeta VHHs Coupled to a Fluorophore Agent and its Evaluation In Vitro/In Vivo

(171) 1. Materials and Methods

(172) Unless explicitly mentioned hereafter the materials and methods are the same as described for Example 1.

(173) 2. In Vivo Targeting of Aβ-Positive Lesions

(174) Conjugation of R3VQ-SH with AF488 Fluorophore

(175) In order to realize the coupling between R3VQ VHH and a fluorophore, a site specific conjugation was implemented as described above. Briefly, an additional cysteine residue was inserted in the C terminal part of the sequence of R3VQ (referred to as R3VQ-SH), thus allowing a C terminal thio-addition of a maleimido-Alexa Fluor® 488 (AF488). By this way, it was obtained a well defined conjugate, referred to as R3VQ-S-AF488, with a single AF488 on the VHH (FIG. 14).

(176) SDS-PAGE and HPLC/MS analyses showed the expected molecular weight (increase of 698 Da) corresponding to the addition of AF488 to the molecule. These data confirm the labeling and the purity of the conjugate (FIGS. 14A and B).

(177) The isoelectric point (pI) of R3VQ-SH and R3VQ-S-AF488 were analyzed by NEPHGE using IEF 3-10 gel. The pI of R3VQ-SH was between 8.5 and 9.5 (FIG. 14C), similar to R3VQ's pI. The addition of AF488 to R3VQ-SH slightly decreased its pI, which was around 8.3, however, still basic (FIG. 14C). The hydrodynamic radius (R.sub.H) of R3VQ-SH and R3VQ-S-AF488 were measured by DLS. The size distribution over time showed an average R.sub.H of 2.66±0.0788 nm for R3VQ-SH and an average R.sub.H of 2.34±0.106 nm for R3VQ-S-AF488, which suggested that both of them were in monomeric form in solution. Immunostaining of amyloid plaques by R3VQ-S-AF488 was confirmed in vitro by IHC using brain slices from PS2APP mice (FIG. 14D).

(178) Diffusion of R3VQ-S-AF488 after Topic Brain Infusion (Two-Photon Imaging)

(179) Craniotomy was performed on a PS2APP mouse to obtain a skull window over the right posterior cortex. 15 μg (10 μl) of R3VQ-S-AF488 was applied directly onto the exposed brain after peeling off the dura. R3VQ-S-AF488 diffusion was followed up for about 2 hours by two-photon microscopy. Typical specific staining of amyloid plaques and CAA was detected in brain parenchyma, suggesting that R3VQ-S-AF488 was able to diffuse and detect in vivo extracellular and vascular AB-positive lesions after pericortical infusion (FIG. 15).

(180) Diffusion of R3VQ-S-AF488 after Intravenous Injection (Two-Photon Imaging)

(181) The integrity of the blood-brain barrier (BBB) of the tested mice was first checked by MRI (see control experiments below) to ensure absence of leakages that may artificially favor brain penetration of the intravenous (iv) injected VHHs.

(182) A 50-mg/kg dose of R3VQ-S-AF488 was injected in the tail vein of one mouse. The conjugate extravasation and staining in the brain was recorded for 3.5 hours post injection using two-photon microscopy on brain window (z=from the surface up to 360 μm deep). FIG. 16A displays in vivo imaging reconstruction (Maximum Intensity Projection—MIP) of R3VQ-S-AF488 over time up to 30 min in the same region. Few seconds after iv injection, strong staining of arborescent vessels was observed and declined dramatically 20 min later with only few capillary vessels remaining stained. This suggested a short half-life of conjugated VHH in the circulation (10-20 min). Shortly after injection a green fluorescent “cloud” formed and spread in the parenchymal space, presenting similarities with the spherical diffusion observed following stereotaxic injection of R3VQ (see above). 30 min after iv injection, amyloid plaques began to be visualized. Vascular Aβ (CAA) was also observed. The absence of signal in the red channel demonstrated that the fluorescent signal was specific (data not shown) and not due to general autofluorescence. Further imaging showed that in vivo staining of Aβ deposition in plaques and vessels remained up to 3.5 hours after injection (FIG. 16B), suggesting a brain half-life of R3VQ-S-AF488 extending over several hours. Four hours after the intravenous injection of R3VQ-S-AF488, the brain was harvested and 5 μm-thick paraffin sections were prepared. IHC was then performed with anti-His mAb to confirm the diffusion and labeling of amyloid plaques by R3VQ-S-AF488. Immunostaining of amyloid plaques by R3VQ-S-AF488 was observed throughout the entire brain with an accompanying brown background which could correspond to the diffusion halo of the VHH (FIG. 16C). Additional experiments were performed with a lower dose of R3VQ-S-AF488 (10 mg/kg) and in vivo detection of Aβ deposition was observed but with decreased intensity. These results suggested that R3VQ brain penetration and its potency to label brain Aβ lesions were dose-dependent, which was confirmed by IHC (FIG. 16D).

(183) Basic pI of VHH is a Key Factor for its Ability to Transmigrate Across the BBB

(184) Maleimido-AF488 conjugated R3VE was also prepared, whose pI was around 7.5 (FIGS. 17A and B) (VHH R3VE was described above). A 10 mg/kg dose of R3VE-S-AF488 was intravenously injected in a PS2APP mouse. 45 minutes after injection, only cerebral amyloid angiopathy was observed without labeling of amyloid plaques (FIG. 17C). 4 hours after the intravenous injection of R3VE-S-AF488, the brain was harvested and 5 μm-thick paraffin sections were prepared. IHC was then performed with anti-His mAb to detect the presence of intrinsic R3VE-S-AF488 in the brain (FIG. 17D). Compared with the result obtained with R3VQ-S-AF488 using the same dose (see above and FIG. 16D), only a very limited labeling of amyloid plaques was observed throughout the entire brain, suggesting that the positive electric charges present on the surface of VHHs play a role for brain penetration of these antibodies across the BBB.

(185) Control Experiments

(186) Evaluation in Amyloid Free Mouse

(187) R3VQ-S-AF488 was intravenously injected in a wild type, amyloid-free, C57BL/6 mouse. No specific in vivo staining in the brain parenchyma was observed using two-photon microscopy assay (data not shown).

(188) Comparison with Conventional IgG Antibody

(189) Injection of mAb 4G8-AF488 iv in a PS2APP mouse only allowed to detect CAA by two-photon imaging but not amyloid plaques indicating no significant extravasation of this standard anti-AB immunoglobulin (FIG. 18).

(190) Assessment of Blood-Brain Barrier Integrity in Mice Used for Two Photon Imaging

(191) BBB permeability of the PS2APP mice (2-year-old) used for two-photon experiments was tested using DOTAREM iv injection (0.2 ml Gd 500 mM). This MRI contrast agent is unable to cross the BBB with the exception of pathological conditions leading to local leakages of the barrier. This MRI exam, used also in human, shows an increase of signal in areas where the BBB is disrupted (V. M. Runge et al., American Journal of Roentgenology, 1994, 162, 431-435; M. A. Ibrahim et al., Investigative radiology, 1998, 33, 153-162). The absence of signal modification in the tested mice suggested the integrity of their BBB. Two other age-matched PS2APP mice were also MRI-assessed using the same method and no disruption of the BBB was observed (data not shown).