NEW ANTI TAU SVQIVYKPV EPITOPE SINGLE DOMAIN ANTIBODY

Abstract

The invention relates to generation, optimization and characterisation of VHH targeted against Tau MTBD (microtubule-binding domain) with high affinity, obtained by screening from a naïve synthetic library. The inventors optimized version of a lead VHH which is able to inhibit Tau aggregation in vitro and in HEK 293 aggregation-reporting cellular model, providing a new tool in Tau immunotherapies. Accordingly the invention relates to new VHH antibody that specifically binds with high affinity Tau species, especially the epitope region involved in Tau aggregation. Moreover, the inventors found that immunization with the optimized version of this lead VHH prevented the formation of neurofibrillary tangles induced by injection of extracellular h-AD in mouse model. Thus, these specific antibodies can be used for the therapy of tauopathy disorders such as Progressive supranuclear palsy (PSP).

Claims

1. An isolated anti-Tau single domain antibody, wherein said isolated anti-Tau single domain antibody binds to an epitope comprising residues SVQIVYKPV (SEQ ID NO:1) of the Tau protein with a KD of 150 nM or less, 80 nM or less, or 50 nM or less.

2. The isolated anti-Tau single domain antibody according to claim 1 wherein the isolated anti-Tau single domain antibody comprises a variable heavy chain having at least 70% of identity with sequence set forth as SEQ ID NO:2.

3. The isolated anti-Tau single domain antibody according to claim 2, wherein said isolated anti-Tau single domain antibody comprises a variable heavy chain (VH) having an amino acid sequence as set forth as SEQ ID NO:2

4. The isolated anti-Tau single domain antibody according to claim 1 which is a humanized single domain antibody.

5. The isolated anti-Tau single domain antibody according to claim 1 wherein said isolated anti-Tau single domain antibody has a heavy chain comprising i) a VH-CDR1 having at least 7, 6, 5, 4, 3, 2, or 1 conservative substitutions within the VH-CDR1 of single domain antibody Z70 (SEQ ID No 3), ii) a VH-CDR2 having at least having at least 7, 6, 5, 4, 3, 2, or 1 conservative substitutions within the VH-CDR2 of single domain antibody Z70 (SEQ ID No 4) and iii) a VH-CDR3 having at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative substitutions within the VH-CDR3 of single domain antibody Z70 (SEQ ID No 5).

6. The isolated anti-Tau single domain antibody according to claim 5, wherein said isolated anti-Tau single domain antibody comprises a CDR1 having a sequence set forth as SEQ ID NO: 3, a CDR2 having a sequence set forth as SEQ ID NO:4 and a CDR3 having a sequence set forth as SEQ ID NO:5.

7. The isolated anti-Tau single domain antibody according to claim 1, which comprises a heavy chain framework region sharing a homology or identity of 80% or more with a framework region having an amino acid sequence consisting of SEQ ID NOs: 8 to 11.

8. The isolated anti-Tau single domain antibody according to claim 1 wherein said isolated anti-Tau single domain antibody is able to reduce the level of pathological aggregation of Tau protein.

9. An isolated anti-Tau single domain antibody, comprising a heavy chain having the following sequences: TABLE-US-00016 VH FR1: (SEQ ID NO: 21) M-A-E-V-Q-L-Q-A-S-G-G-V-F-V-Q- S-G-G-S-L-R-L-Xaa1-C-A-A-S-G wherein Xaa1 is Serine (S) or Cysteine (C); TABLE-US-00017 VH-CDR1: (SEQ ID NO: 22) A-T-S-Xaa2-F-D-G wherein Xaa2 is Threonine (T) or Cysteine (C); TABLE-US-00018 VH-FR2: (SEQ ID NO: 23) M-G-W-F-R-Q-A-P-G-K-E-Xaa3-E-F-V-S-A-I-S wherein Xaa3 is Arginine (R) or Lysine (K); TABLE-US-00019 VH-CDR2: (SEQ ID NO: 24) Y-Xaa4-Q-G-S-Y-T wherein Xaa4 is Glutamic acid (E) or Glycine (G); TABLE-US-00020 VH-FR3: (SEQ ID NO: 25) Y-Y-A-D-S-V-K-G-R-F-T-I-S-R-D-N-S-K-N-M-V-Y-L- Q-M-N-S-L-Xaa5-A-E-D-T-A-Xaa6-Y-Y-C-A wherein Xaa5 is Arginine (R) or Glycine (G) and Xaa6 is Threonine (T)) or Serine (S); TABLE-US-00021 VH-CDR3: (SEQ ID NO: 26) Xaa7-A-Y-E-G-D-L-Y-A-F-D-S wherein Xaa7 is Proline (P) or Serine (S); and TABLE-US-00022 VH-FR4: (SEQ ID NO: 27) Y-Xaa8-Xaa9-Q-G-T-Q-V-T-V-S-S- wherein Xaa8 is Glycine (G) or Glutamic acid (E) and Xaa9 is Glycine (G) or Glutamic acid (E).

10. The isolated anti-Tau single domain antibody according to claim 9, wherein Xaa5 is Arginine (R); Xaa7 is Proline (P) and Xaa8 is Glycine (G).

11. A nucleic acid sequence encoding the isolated anti-Tau single domain antibody according to claim 1.

12. A vector comprising a nucleic acid sequence according to claim 11.

13. An in vitro method for detecting human Tau peptide in a biological sample, comprising contacting the biological sample with the anti-Tau single domain antibody according to claim 1 under conditions under conditions that permit formation of an immune complex between the human Tau peptide and the anti-Tau single domain antibody, and detecting and/or measuring the immune complex that is formed.

14. (canceled)

15. A pharmaceutical composition comprising the isolated anti-Tau single domain antibody according to claim 1, or a vector according to claim 12.

16. A method of treating a tauopathy in a subject in need thereof, comprising, administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 15.

17. The method of claim 16, wherein the tauopathy is selected from the group consisting of Alzheimer's Disease Down syndrome; Guam parkinsonism dementia complex; Dementia pugilistica; myotonic dystrophies; Niemann-Pick disease type C; Pick disease; argyrophilic grain disease; Fronto-temporal dementia; Cortico-basal degeneration; Pallido-ponto-nigral degeneration; Progressive supranuclear palsy; and a Prion disorder.

18. The method according to claim 17, wherein the tauopathy is Progressive supranuclear palsy (PSP).

19. (canceled)

20. The method of claim 17, wherein the Prion disorder is Gerstmann-Sträussler-Scheinker disease with tangles.

Description

FIGURES

[0140] FIG. 1. VHH E4-1 binds to the MTBR of Tau. A. Overlay of two-dimensional .sup.1H, .sup.15N HSQC spectra of Tau (black) with Tau mixed with non-labelled VHH E4-1 (overlayed in gray) (n=1). B. Same spectra enlargements showing broadened resonances corresponding to residues implicated in the interaction. C. Normalized intensities I/I0 of corresponding resonances in the two-dimensional spectra of Tau with equimolar quantity of VHH E4-1 (I) or free in solution (TO) for residues along the Tau sequence. Overlapping resonances are not considered (x-axis is not scaled). A double-arrow indicates the region containing the corresponding major broadened resonances, which was mapped to the R2-R3 repeats in the MTBD.

[0141] FIG. 2 VHH Z70 is optimized for intracellular activity and has a better affinity for Tau than VHH E4-1 A. Results from yeast two-hybrid. A growth test on non-selective medium (labeled growth, lacking only leucine and tryptophane) or on selective medium (labeled interaction, lacking leucine, tryptophane and histidine) was performed with dilutions 1/10, 1/100, 1/1000 and 1/10000 (top to bottom) of the diploid yeast culture expressing both bait and prey constructs. Positive and negative controls of interaction consist respectively in Smad/Smurf interaction.sup.54 and Tau alone (empty vector). VHH E4-1 did not interact with Tau in yeast whereas VHH Z70 did. B. Structure of the VHHs and sequence alignment between VHH E4-1 and VHH Z70 resulting in 4 mutations in the framework domain: G12V, P16S, T81M and W114G. C, D. Sensorgrams (reference subtracted data) of single cycle kinetics analysis performed on immobilized biotinylated Tau, with five injections of VHH at 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, and 2 μM. C of VHH E4-1 and D of VHH Z70 (n=1). Dissociation equilibrium constant Kd were calculated from the ratio of off-rate and on-rate kinetic constants koff/kon. E. Sensorgram (reference subtracted data) of single-cycle kinetics analysis performed on immobilized VHH Z70 on a CMS chip, with five injections of SUMO-Tau peptide Tau[273-318] (n=1). kon, Koff and KD are included in the table 3. Red line corresponds to the fit curve, black line to the measurement.

[0142] FIG. 3. VHH E4-1 and VHH Z70 inhibit in vitro Tau aggregation of Tau (10 μM) in the absence of heparin (black curve), in the presence of heparin and of increasing concentration of A. VHH F8-2 B. VHH E4-1 and C. VHH Z70 (0, 1, 2,5, 5 and 10 μM) followed by Thioflavin T fluorescence at 490 nm (n=3). Error bars: standard deviation.

[0143] FIG. 4. VHH Z70 mutants inhibit in vitro Tau aggregation. Aggregation of Tau (10 μM) in the absence of heparin (negative control), in the presence of heparin and of VHH mutants (ratio 1 Tau/0.2 VHH) followed by Thioflavin T fluorescence at 490 nm. (n=3): Mean value is presented

[0144] FIG. 5. VHH Z70 and optimized Z70 blocks intracellular aggregation of Tau MTBD in HEK 293 Tau RD P301S FRET Biosensor cells. A-D. Analysis of Tau seeding in HEK 293 Tau RD P301S FRET Biosensor cells A) by confocal microscopy for cells transfected with vehicle (HEPES buffer) B) by flow cytometry with a FRET-gate for cells transfected with vehicle C) by confocal microscopy for cells transfected with MTBD seeds. Positive cells that have incorporated MTBD seeds show yellow dots corresponding to FRET signal D) by flow cytometry with a FRET-gate for cells transfected with MTBD seeds. E. Percentage of FRET positive cells determined from FACS data for cells transfected as in B, D or transfected with mCherry-VHH F8-2 followed by MTBD seeds. F-I Analysis of Tau seeding in HEK 293 Tau RD P301S FRET Biosensor cells F-G by confocal microscopy for cells transfected with F) mCherry-VHH F8-2 or G) with mCherry VHH F8-2 followed by MTBD seeds. Cells transfected with mCherry-VHH have a red color, FRET is visualized as yellow dots H-I by flow cytometry with a mCherry FRET gate for cells transfected with H) mCherry-VHH F8-2 followed by MTBD seeds (n=3) or I) mCherry-VHH Z70 followed by MTBD seeds (n=3). J. Percentage of mCherry-gated FRET positive cells, determined from FACS data for cells transfected with mCherry-VHH, as stated on x-axis, followed by MTBD seeds (at minimum n=3). A significant decrease of FRET signal, reporting a decrease intracellular MTBD aggregation, is observed in the presence of VHH Z70 and mutated Z70. Error bars: standard deviations.

[0145] FIG. 6. Activity of VHH Z70 in ThyTau30 mouse model. A data point corresponds to the quantification for one hemisphere of the immunoreactive signal summed over 5 slices (at specific Bregma conserved for all animals). Results are presented as percentage of marker occupancy corresponding to the ratio of immunoreactive area normalized to the area of the region of interest. Lentiviral expression of VHH Z70 (Z70) or a VHH recognizing GFP (GREN), stereotaxic injection of AD brain lysates (AD) or PBS (PBS). Error bars correspond to the S.E.M.

[0146]

TABLE-US-00012 TABLE 3 VHH Kon 1/M.s Koff 1/s KD (nM) E4-1 4982 0.0017 345 Z70 18100 0.0026 147

EXAMPLE 1: (SELECTION OF VHHS TARGETING TAU)

[0147] Materials and Methods

[0148] Screening and Selection of VHHs Directed Against Tau Protein

[0149] Recombinant Tau protein was biotinylated using EZ-Link™ Sulfo-NHS-Biotin (Thermo Fisher Scientific) using manufacturer conditions except for a two-fold molecular excess of Sulfo-NHS-Biotin. The unreacted Sulfo-NHS-Biotin was eliminated using Prepacked Columns Sepadextran™ 25 Medium SC (Proteigene). The Nali-H1 library of VHHs was screened against the recombinant biotinylated-Tau as described previously.sup.44. Briefly, biotinylated-Tau protein was bound to Dynabeads™ M-280 Streptavidin (Invitrogen) at each round of selection, at a concentration gradually decreased: 100 nM in round1, 50 nM in round 2 and 10 nM in round3. Biotinylated-Tau binding was verified by Western Blot using Streptavidin Protein, HRP (Thermo Fisher Scientific). 3×10.sup.11 phages of the Nali-H1 library were used in the first round of selection. After the third round, 186 clones were randomly picked and tested in non-absorbed Phage ELISA assay using avidin-plates and biotinylated-Tau Antigen (5 μg/ml) for cross-validation.sup.45.

[0150] Production and Purification of VHHs

[0151] Competent Escherichia coli BL21 (DE3) bacterial cells were transformed with the various PHEN2-VHH constructs. Recombinant E. coli cells produced proteins targeted to the periplasm after induction by 1 mM IPTG (isopropylthiogalactoside). Production was pursued for 4 hours at 28° C. before centrifugation to collect the cell pellet. Pellet was suspended in 200 mM Tris-HCl, 500 mM sucrose, 0.5 mM EDTA, pH 8 and incubated 30 min on ice. 50 mM Tris-HCl, 125 mM sucrose, 0.125 mM EDTA, pH 8 and complete protease inhibitor (Roche) were then added to the cells suspension and incubation continued 30 min on ice. After centrifugation, the supernatant, corresponding to the periplasmic extract, was recovered. The VHHs were purified by immobilized-metal affinity chromatography (HisTrap HP, 1 mL, GE healthcare) followed by size exclusion chromatography (Hiload 16/60, Superdex 75, prep grade, GE healthcare) in NMR buffer (50 mM NaPi pH 6.7, 30 mM NaCl, 2.5 mM EDTA, 1 mM DTT).

[0152] Production and Purification of Labelled .sup.15N Tau 2N4R, .sup.15N Tau 2N3R and .sup.15N Tau MTBD

[0153] pET15b-Tau recombinant T7lac expression plasmid was transformed into competent E. coli BL21 (DE3) bacterial cells. A small scale culture was grown in LB medium at 37° C. and was added at 1:10 V/V to 1 L of a modified M9 medium containing MEM vitamin mix 1× (Sigma-Aldrich), 4 g of glucose, 1 g of .sup.15N—NH4Cl (Sigma-Aldrich), 0.5 g of .sup.15N-enriched ISOGROW (Sigma-Aldrich), 0.1 mM CaCl2 and 2 mM MgSO4. Recombinant .sup.15N Tau production was induced with 0.5 mM IPTG when the culture reached an optical density at 600 nm of 0.8. Proteins were first purified by heating the bacterial extract, obtained in 50 mM phosphate buffer pH 6.5, 2.5 mM EDTA and supplemented with complete protease inhibitors cocktail (Sigma-Aldrich), 15 min at 75° C. The resulting supernatant was next passed on a cation exchange chromatography column (Hitrap SP sepharose FF, 5 mL, GE healthcare) with 50 mM sodium phosphate buffer (NaPi) pH 6.5 and eluted with a NaCl gradient. Tau proteins were buffer-exchanged against 50 mM ammonium bicarbonate (Hiload 16/60 desalting column, GE Healthcare) for lyophilization. The same protocol was used to produce and purify Tau 2N3R isoform and Tau[245-368] (designated MTBD, also called K18 fragment). Detailed procedure can be found in.sup.46.

[0154] Production and Purification of SUMO-Tau Peptides

[0155] cDNA encoding Tau[273-318] peptide, was amplified from Tau 2N4R cDNA by PCR. cDNA was cloned by a ligation independent protocol into vector pETNKI-HisSUMO3-LIC as described in.sup.47. Tau peptide was expressed as N-terminal SUMO protein fusion with a N-terminal HisTag. His-SUMO-Tau peptide was purified by affinity chromatography on Ni-NTA resin followed by size exclusion chromatography (Hiload 16/60, Superdex 75, prep grade, GE healthcare) in SPR buffer (HBS-EP+, GE Healthcare).

[0156] Nuclear Magnetic Resonance Spectroscopy Experiments

[0157] Analysis of the .sup.15N Tau/VHH interactions were performed at 298K on a Bruker 900 MHz spectrometer equipped with cryogenic probe. TMSP (trimethyl silyl propionate) was used as internal reference. Lyophilized .sup.15N Tau were diluted in a buffer containing 50 mM NaPi, 30 mM NaCl, 2.5 mM EDTA, 1 mM DTT, and 10% D20, pH 6.7 and mixed with VHH at 100 μM final concentration for each protein. 200 μL of each mix in 3 mm tubes were sufficient to obtain the 2D .sup.1H, .sup.15N HSQC spectra. .sup.1H, .sup.15N HSQC were acquired with 3072 and 416 points in the direct and indirect dimensions, respectively for 12.6 and 25 ppm, in the .sup.1H and .sup.15N dimensions, respectively, with 32 scans. Data were processed with Bruker Topspin and analyzed with Sparky (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco).

[0158] Optimization of VHH E4-1 for Intracellular Expression

[0159] VHH E4-1 cDNA was amplified from E4-1-pHEN2 plasmid using Taq polymerase with 14 mM MgCl2 and 0.2 mM MnCl2 and a modified nucleotide pool according to.sup.48. The amplified cDNAs were transformed in yeast Y187 strain, together with a digested empty derivative of pGADGH vector.sup.49, allowing recombination by gap repair in the vector. The VHH cDNAs are expressed as preys, with an N-terminal Gal4-activation domain fusion (E4-1-Gal4AD). A library of 2.1 million clones was obtained, collected and aliquoted. Tau variant 0N4R isoform (NM_016834.4) was expressed as bait with an N-terminal fusion with lexA (Tau-LexA) from pB29 vector, which is derived from the original pBTM116.sup.50. The library was screened at saturation, with 20 million tested diploids, using cell-to-cell mating protocol.sup.51. A single clone was selected, named VHH Z70. A one-to-one mating assay was used to test for interaction using a mating protocol with L40DGal4 (mata) transformed with the bait and Y187 (mata) yeast strains transformed with the prey.sup.51. The interaction pairs were tested in triplicate on selective media by streak.

[0160] Tau Fragment Library Construction

[0161] Tau cDNA (NM_016834.4) was amplified from Tau-LexA bait vector. 5 μg of the PCR product was subjected to Fragmentase® treatment (New England Biolab, NEB) until a smear of fragments was detected around 400-500 pb by agarose gel electrophoresis. The DNA fragments were purified by phenol/chloroform extraction and ethanol precipitation. The DNA fragments were next subjected to end repair (NEB) and dA-tailing adaptation, using Blunt/TA ligase master mix with NEBNext® Adaptor hairpin loop (NEB), followed by AMPure XP bead (Beckman Coulter) purification. After USER® enzyme digestion (NEB), DNA fragments were amplified with 15 cycles of PCR using NEBNext® Q5® Hot Start HiFi PCR Master Mix (NEB), which allowed to add Gap Repair recombination sequences for the cloning in Gal4-AD prey plasmid pP7. The library comprised 50000 independent clones.

[0162] Tau Fragment Library Screening

[0163] The coding sequence for VHH Z70 was PCR-amplified and cloned into pB27 as a C-terminal fusion to LexA (LexA-VHHZ70). The construct was used to produce a bait to screen the Tau fragments library constructed into pP7. pB27 and pP7 derived from the original pBTM116.sup.50 and pGADGH.sup.49 plasmids, respectively. The Tau fragment library was screened using a mating approach with YHGX13 (Y187 ade2-101::loxP-kanMX-loxP, mata) and L40DGal4 (mata) yeast strains.sup.51. 90 His+ colonies corresponding to 267.103 tested diploids were selected on a medium lacking tryptophan, leucine and histidine. The prey fragments of the positive clones were amplified by PCR and sequenced at their 5′ and 3′ junctions.

[0164] Surface Plasmon Resonance Experiments

[0165] Affinity measurements were performed on a BIAcore T200 optical biosensor instrument (GE Healthcare). Recombinant Tau proteins were biotinylated with 5 molar excess of NHS-biotin conjugates (Thermofisher) during 4 hours at 4° C. Capture of biotinylated Tau was performed on a streptavidin SA sensorchip in HBS-EP+ buffer (GE Healthcare). One flow cell was used as a reference to evaluate nonspecific binding and provide background correction. Biotinylated-Tau was injected at a flow-rate of 30 μL/min, until the total amount of captured Tau reached 500 resonance units (RUs). VHHs were injected sequentially with increasing concentrations ranging between 0.125 and 2 μM in a single cycle, with regeneration (3 successive washes of 1M NaCl) between each VHH. On the other hand, VHH Z70 was immobilized on a CMS chip in HBS-EP+ buffer (GE Healthcare) and increasing concentrations, ranging between 0.125 and 2 μM of the SUMO-Tau peptide, were successively injected. Single-Cycle Kinetics (SCK) analysis.sup.52 was performed to determine association kon and dissociation Koff rate constants by curve fitting of the sensorgrams using the 1:1 Langmuir model of interaction of the BIAevaluation sotware 2.0 (GE Healthcare). Dissociation equilibrium constants (KD) was calculated as kon/Koff.

[0166] Results

[0167] Identification of a Synthetic VHH Directed Against Tau Microtubule-Binding Domain

[0168] A synthetic phage-display library of humanized llama single-domain antibody (Moutel et al., 2016) was screened against a preparation of biotinylated recombinant full-length Tau protein, corresponding to its longest isoform (Tau 2N4R, designated as Tau). After validation with non-absorbed phage ELISA, 20 clones were selected from the screen for further analysis. We used nuclear magnetic resonance (NMR) spectroscopy to identify the epitope site recognized by each of the validated VHHs, based on resonance perturbation mapping in .sup.1H, .sup.15N HSQC spectra of .sup.15N-Tau. Interaction was visualized as a perturbation of resonance that can be a modification of the chemical shift value or of the peak intensity when comparing spectra of Tau alone in solution or in the presence of a VHH. As most of the resonances from the .sup.1H, .sup.15N spectrum of Tau have been assigned.sup.28,29, each perturbation can be linked to a specific amino acid residue in Tau sequence. One VHH, named VHH E4-1, affected resonances in Tau spectrum corresponding to residues in the MTBD (FIG. 1). In the spectrum of Tau in the presence of VHH E4-1, a number of resonances are broadened beyond detection compared to the Tau control spectrum (FIG. 1a). Intensity ratios of corresponding resonances in these two spectra, plotted along the Tau sequence, allowed the identification of the Tau MTBD domain as the target of VHH E4-1 interaction (FIG. 1b). The epitope mapping was refined using a Tau fragment that corresponded to the isolated MTBD. The smaller size of this Tau fragment resulted in less resonance overlap in the corresponding Tau[245-368].sup.1H, .sup.15N spectrum and made identification of the binding site easier. The affected resonances corresponded to amino acid residues located in a stretch expanding from residue V275 to K317 (data not shown). VHH E4-1 thus bound within the R2-R3 repeats of the MTBD.

[0169] Optimization of Lead VHH E4-1 into Variant VHH Z70

[0170] An important property of a VHH is its capacity to be produced and to recognize its targets in the cytoplasmic environment, inside the cells. However, VHHs might not all be efficient once expressed in a cell, due to improper folding and/or poor stability. Indeed, VHH E4-1 proved to be a poor binder of Tau when using Yeast 2-Hybrid to test its intracellular binding capacity.sup.30,31 (FIG. 2a). VHH E4-1 was thus next submitted to a round of optimization, using yeast two-hybrid system, to maximize its capacity to recognize its target when expressed in a cellular environment. First, we built a cDNA mutant library by random mutagenesis, targeting the whole sequence of VHH E4-1 to produce a variety of VHH preys (C-terminal Gal4-activation domain fusion) against the Tau bait (N-terminal LexA fusion). The library was transformed in yeast and screen by cell-to-cell mating to get positive colonies in conditions corresponding to undetected VHH E4-1-Tau interaction (FIG. 2a). An optimized mutant, named VHH Z70, was selected, resulting from 4 mutations G12V, P16S, T81M and W114G located in the framework domains, outside the recognition loops or CDR (FIG. 2a-b), suggesting that the epitope recognized by this mutant is unaltered. Conservation of the epitope was confirmed by resonance perturbation mapping, using labelled MTBD in the same manner as for the lead VHH E4-1. Interaction of VHH E4-1 and VHH Z70 with Tau were further characterized using surface plasmon resonance spectroscopy (SPR) with biotinylated-Tau immobilized at the surface of a streptavidin-functionalized chip. The assay provided the kinetic parameters of the interaction, characterized by dissociation constants Kd of 345 nM for VHH E4-1 (FIG. 2c) and of 147 nM for mutant VHH Z70 (FIG. 2d). VHH Z70, optimized for intracellular activity, had a better affinity for its target than VHH E4-1, the major optimization concerning the association constant (kon). SPR was additionally performed with VHH Z70 immobilized on the chips. A fusion protein corresponding to a SUMO domain fused at its C-terminus to a Tau peptide [273-318], corresponding to the identified VHH binding site, was injected into the flux. VHH Z70 interacted with the fused peptide with a Kd of 85 nM (FIG. 2e), confirming that the region 275 to 317 in Tau sequence was sufficient for VHH-Z70 binding.

[0171] Identification of the Minimal Tau Epitope Recognized by VHH Z70

[0172] The binding site identified by NMR for both lead VHH E4-1 and optimized VHH Z70 was larger than expected for an epitope, about 40 contiguous amino-acid residues showing strong reduction of their resonance intensities (275 to 317). However, the NMR epitope mapping by resonance intensity decrease does not allow identification of the residues in a direct interaction. The decrease in resonance intensity can result from local immobilization of the disordered protein due to the binding, decreasing local tumbling and increasing relaxation. Accordingly, the Tau domain involved in the VHH interaction, which contained the PHF6 and PHF6*, was described as presenting local extended secondary structure.sup.29 and thus represented a relatively rigid stretch that could explain the extended region of immobilization upon binding. Alternatively, decrease resonance intensity can be due to chemical exchange between bound and unbound states that can result in line broadening, depending on the affinity and chemical shift change resulting from the interaction. In this case, the observed binding in the repeat region of Tau, given the level of sequence redundancy, could correspond to binding to R2 or R3 repeats, even if one is a secondary site of low affinity. To lift the ambiguity, and determine the minimal epitope that VHH Z70 can recognize, an epitope mapping was performed using yeast two-hybrid (267.103 tested interactions) with a library of Tau fragments as preys (GAL4 activation domain-Tau_fragments), and VHH Z70 as bait (LexA-VHH fusion). 90 positive clones were selected from a small-scale cell-to-cell mating screen. Comparison of the Tau prey fragment sequences corresponding to these 90 interactions identified peptide .sub.305SVQIVYKPV.sub.313 (SEQ ID No 1) as the minimal recognition sequence of Tau that VHH Z70 can bind. The sequence is localized in the R3 repeat of the MTBD domain and contains the PHF6 peptide VQIVYK (SEQ ID No 19). We next used Tau2N3R isoform, which lacks the R2 repeat and so does not contain the PHF6* peptide, to confirm that the R3 repeat, containing the PHF6 peptide, was sufficient for the interaction. As observed in the resonance intensity profile, the interaction of VHH Z70 with Tau2N3R is maintained, and the most affected resonances in the Tau spectrum corresponded to the PHF6 residues in the R3 repeats.

EXAMPLE 2: (VARIANTS OF Z70)

[0173] Materiel and Methods

[0174] Optimization of VHH Z70

[0175] VHH Z70 was amplified from pHEN2 plasmid using Taq polymerase with 14 mM MgCl2 and 0.2 mM MnCl2 and a modified nucleotide pool according to.sup.48. The amplified cDNAs were transformed in yeast Y187 strain, together with a digested empty derivative of pGADGH vector 49, allowing recombination by gap repair in the vector. The VHH cDNAs are expressed as preys, with a C-terminal Gal4-activation domain fusion (Gal4AD-Z70). A library of 2.1 million clones was obtained, collected and aliquoted. Tau variant 0N4R isoform (NM_016834.4) was expressed as bait with an N-terminal fusion with lexA (Tau-LexA) from pB29 vector, which is derived from the original pBTM116.sup.50. The library was screened with 0.5 mM 3-aminotriazol at saturation, with 40 million tested diploids, using cell-to-cell mating protocol.sup.51. Most redundant mutants with 1 to 4 mutations were selected for further analysis.

[0176] Surface Plasmon Resonance Experiments

[0177] Affinity measurements were performed on a BIAcore T200 optical biosensor instrument (GE Healthcare). Recombinant Tau proteins were biotinylated with 5 molar excess of NHS-biotin conjugates (Thermofisher) during 4 hours at 4° C. Capture of biotinylated Tau was performed on a streptavidin SA sensorchip in HBS-EP+ buffer (GE Healthcare). One flow cell was used as a reference to evaluate nonspecific binding and provide background correction. Biotinylated-Tau was injected at a flow-rate of 30 μL/min, until the total amount of captured Tau reached 500 resonance units (RUs). VHHs were injected sequentially with increasing concentrations ranging between 0.125 and 2 μM in a single cycle, with regeneration (3 successive washes of 1M NaCl) between each VHH. Single-Cycle Kinetics (SCK) analysis.sup.52 was performed to determine association kon and dissociation Koff rate constants by curve fitting of the sensorgrams using the 1:1 Langmuir model of interaction of the BIAevaluation sotware 2.0 (GE Healthcare). Dissociation equilibrium constants (Kd) was calculated as kon/Koff.

[0178] Results

[0179] Optimization of VHH Z70

[0180] A mutant library of VHH Z70 has been screened for mutants displaying a stronger affinity in yeast two-hybrid system as described above. 8 mutants were selected and their affinity have been further characterized using SPR experiments. All the selected mutants displayed an enhanced affinity toward Tau protein (cf table 4), the best having a KD of 22.6 nM meaning an improvement of more than 6 times.

TABLE-US-00013 TABLE 4 VHH kon 1/M.s Koff 1/s KD (nM) Z70 1.81E+04 0.002671 147 Mut1 1.07E+05 0.0024213 22.6 Mut3 2.60E+04 0.0020972 80.5 Mut5 1.09E+04 0.0008825 80.8 Mut9 4.01E+04 0.0020485 51 Mut12 3.39E+04 0.0020714 61.1 Mut14 1.88E+04 0.0013966 74.2 Mut15 2.13E+04 0.0020344 95.6 Mut20 2.12E+04 0.0008902 41.8

EXAMPLE 3: (INHIBITION OF TAU AGGREGATION)

[0181] Materiel and Methods

[0182] In Vitro Kinetic Aggregation Assays

[0183] Tau 2N4R aggregation assays were performed with 10 μM Tau and with increasing concentrations of VHHs (between 0 and 10 μM) in buffer containing 50 mM IVIES pH 6.9, 3 mM NaCl, 2.5 mM EDTA, 0.33 mM freshly prepared DTT, 2.5 mM heparin H3 (Sigma-Aldrich) and 50 μM Thioflavin T (Sigma-Aldrich), at 37° C. Experiments were reproduced 3 times in triplicates for each condition. The resulting fluorescence of Thioflavin T was recorded every 5 min/cycle within 200 cycles using PHERAstar plate-reader. The different measures were normalized in % of fluorescence, 100% being defined as the maximum value reached in the positive Tau control, in each experiment.

[0184] Transmission Electron Microscopy

[0185] The same samples from the aggregation assays were recovered and a 10 μl sample of each Tau:VHH ratio 1:1 condition was loaded on a formvar/carbon-coated grid (for 5 min and rinsed twice with water). After drying, the grids were stained with 1% uranyl acetate for 1 min. Tau fibrils were observed under a transmission electron microscope (EM 902 Zeiss).

[0186] Aggregation Seeding Assays in HEK293 Reporter Cell-Line

[0187] Stable HEK293 Tau RD P301S FRET Biosensor cells (ATCC CRL-3275) were plated at a density of 100 k cells/well in 24-well plates. For confocal analysis, cells were plated on poly-D-lysine and laminin coated slides at a density of 100 k cells/well in 24-well plates. At 60% confluency, cells were first transfected with the various pmCherry-N1 plasmid constructs allowing expression of the mCherry-VHHs. Transfection complexes were obtained by mixing 500 ng of plasmid diluted in 40 μL of opti-MEM medium, which include 18.5 μL (46.25% v/v) of opti-MEM medium+1.5 μL (3.75% v/v) Lipofectamine 2000 (Invitrogen). Resulting liposomes were incubated at room temperature for 20 min before addition to the cells. Cells were incubated for 24 hours with the liposomes and 1 mL/well of high glucose DMEM medium (ATCC) with Fetal Bovine Serum 1% (Life technologies). The transfection efficiency was estimated to reach about 46%, for all mCherry-VHHs plasmids. Eight μM of recombinant MTBD seeds were prepared in vitro, in the presence of 8 μM heparin, as described.sup.32. Cells were then treated with MTBD seeds (10 nM/well) in the presence of transfection reagents forming liposomes as here above described.

[0188] Confocal Analysis

[0189] Cells were first washed twice with PBS and fixed in 4% paraformaldehyde for 20 min and next washed 3 times with 50 mM NH4Cl in PBS. Glass slides were mounted with DAKO mounting medium (Agilent). Fluorescence imaging acquisitions were performed using an inverted confocal microscope (LSM 710, Zeiss, Jena, Germany) with a 40-times oil-immersion lens (NA 1.3 with an optical resolution of 176 nm). CFP, YFP and FRET, and mCherry fluorescence were imaged using UV, Argon 458/514 nm, DPSS 561 nm and Helium/Neon 633 nm lasers, respectively. A focal plane was collected for each specimen. Images were processed with ZEN software.

[0190] FRET Flow Cytometry

[0191] Cells were recovered with trypsin 0.05% and fixed in 2% paraformaldehyde for 10 min, then suspended in PBS. Flow cytometry was performed on an ARIA SORP BD (Biosciences). To measure CFP emission fluorescence and FRET, cells were excited with a 405 nm laser. The fluorescence was captured with either a 466/40 or a 529/30 nm filter, respectively. To measure YFP fluorescence, a 488 nm laser was used for excitation and emission fluorescence was captured with a 529/30 nm filter. mCherry cells were excited with a 561 nm laser and fluorescence was captured with a 610/20 nm filter. To selectively detect and quantify FRET, gating was used as described.sup.32, 53. 3 independent experiments were done in triplicate or quadruplicate, with at least 10,000 cells per replicate analyzed.

[0192] Statistical Analysis

[0193] Experiments were performed at least in triplicate and obtained from three independent experiments. Statistical analyses were performed using the Mann-Whitney U-Test to determine the p-value.

[0194] Results

[0195] Inhibition of In Vitro Tau Aggregation

[0196] VHHs E4-1 and VHH Z70 recognizing Tau peptide PHF6, known to nucleate the aggregation and to form the core of Tau fibers, were assayed for their capacity to interfere with Tau in vitro aggregation. The assays were carried out with Tau recombinant protein in the presence of heparin, using thioflavin T as a dye whose fluorescence is increased in presence of aggregates (FIG. 3). Negative and positive controls consisted in Tau without or with heparin, respectively. An additional control was performed in the presence of VHH F8-2, a VHH issued from the initial phage-library screen, which targeted Tau C-terminal domain. At 10 μM of Tau, the observed amount of aggregates was maximal (defined as 100%) for the positive control after 8 h of incubation at 37° C., while no fluorescence change was detected for the negative control (FIG. 3A-C). At equimolar concentration of Tau:VHH F8-2, the fluorescence signal reached 91.2% (±3.8%, standard deviation), showing that VHH F8-2 did not affect the aggregation of Tau (FIG. 3A). In contrast, at a molar ratio of 1:0.25 Tau: VHH E4-1, the maximal fluorescence signal reached 86.9% (±2.4%). Additionally, about 3.8 h were needed to gain 50% of maximal signal, compare to 2.5 h for the positive control, showing a slower aggregation kinetic in the presence of VHH E4-1 (FIG. 3B). At a 1:1 Tau:VHH E4-1 molar ratio, the fluorescence signal only reached 58.3% (±3.9%) and more than 12.8 h were necessary to gain 50% of maximal signal (FIG. 3B). VHH Z70 had an even stronger inhibition effect on the aggregation of Tau than the lead VHH E4-1. At a 1:1 Tau:VHH Z70 molar ratio, the maximal fluorescence signal barely reached above the negative control level, at 4.1% (±0.1%) (FIG. 3C). The link between the thioflavin T fluorescence measurements in our assays and the formation of Tau aggregates at the end point of each aggregation assay was confirmed by Transmission Electron Microscopy (TEM) imaging that allowed direct visualization of typical Tau fibers, whether present (data not shown). Large amounts of fibrils were observed for Tau in the presence of heparin only or in the additional presence of VHH F8-2, but shorter filaments with VHH E4-1 and practically none with VHH Z70. In conclusion, lead VHH E4-1 and its optimized variant VHH Z70 had both the capacity to inhibit the aggregation of Tau in vitro and their relative activity is related to their affinity for Tau.

[0197] Inhibition of In Vitro Tau Aggregation by Optimized VHH Z70 Mutants

[0198] VHH Z70 and the derived mutants recognizing Tau peptide PHF6, known to nucleate the aggregation and to form the core of Tau fibers, were assayed for their capacity to interfere with Tau in vitro aggregation. The assays were carried out with Tau recombinant protein in the presence of heparin, using thioflavin T as a dye whose fluorescence is increased in presence of aggregates (FIG. 3). Negative and positive controls consisted in Tau without or with heparin, respectively. Using 10 μM of Tau, the observed amount of aggregates was maximal (defined as 100%) for the positive control after 18 h of incubation at 37° C., while no fluorescence change was detected for the negative control.

[0199] Mut3, Mut5, Mut14 and Mut20 displayed inhibition ability similar to that of VHH Z70 while Mut1 and Mut12 displayed an even better inhibition of Tau aggregation in this assay. Mut1 and Mut12 are able to significantly inhibit Tau aggregation using a ratio of 0.2 VHH to 1 Tau (FIG. 4).

[0200] In conclusion, VHH Z70 and the tested derived mutants had the capacity to inhibit the aggregation of Tau in vitro using low ratio of VHH/Tau (FIG. 4).

[0201] Inhibition of Tau Seeding in HEK293 Tau Repeat Domain (RD) P301S FRET Biosensor Aggregation Reporter Cells

[0202] The capacity of VHH E4-1 and VHH Z70 to block the intracellular aggregation in the HEK293 Tau RD P301S FRET Biosensor reporter cell line model was next investigated. This cell line constitutively expresses Tau RD (MTBD), with a P301S mutation, fused to either CFP (Cyan Fluorescent Protein) or YFP (Yellow Fluorescent Protein) that together generate a FRET (Forster Resonance Energy Transfer) signal upon MTBD-P301S aggregation.sup.32. For cells treated with HEPES buffer only, FRET signal is detected neither by confocal microscopy nor by flow cytometry (FIG. 5A-B). The intracellular aggregation of MTBD-P301S protein is induced by treating the cells with Tau seeds, the MTBD fragment in vitro aggregated in HEPES buffer with heparin associated to liposomes to help cell penetration.sup.32, leading to a FRET signal (yellow fluorescence by confocal and 16%±0.8% FRET gated positive cells, FIG. 5C-E). In addition, mCherry-VHH F8-2 was transfected and served as negative control since its binding is outside the MTBD. Similarly to the previous experiment in the absence of any VHH, 15.4% (±1% s.e.m) FRET-gated positive cells were visualized after Tau seeding (FIG. 5E-H), providing a reference for 100% seeding. To evaluate the efficiency of VHHs E4-1 and Z70 to inhibit aggregation in this model, plasmids that expressed each VHH fused to mCherry protein were transfected one day prior to addition of the Tau seeds. With a mCherry gate to detect FRET signal selectively in mCherry-VHH positive cells, the percentage of FRET positive cells transfected by mCherry VHHs E4-1 and Z70 were compared to the cells transfected by mCherry VHH F8-2, which did not affect the seeding in cells (FIG. 5E). The FRET signal reduction for mCherry-VHH E4-1 positive cells was not significant, with a percentage of FRET positive cells decreasing to 15.2±1.6%, compare to 16.5±4.8% FRET signal for mCherry-VHH F8-2 negative control (7.8% seeding inhibition, FIG. 5H-J). Conversely, mCherry-VHH Z70 clearly affected the intracellular aggregation of MTBD-P301S, as the observed FRET signal for the corresponding transfected cells was significantly decreased to 9.9±2.9% (40% seeding inhibition, FIG. 5H-J).

[0203] mCherry-VHH Mut1, Mut3 and Mut12 affected the intracellular aggregation of MTBD-P301S similarly to VHHZ70, as the observed FRET signals for the corresponding transfected cells were significantly (p<0.001) decreased to 8.2 (±1.9, standard deviation), 8.2±1.7, 8.5±1.6 (50,50,48% seeding inhibition, FIGS. 4H, 4I and 4J). mCherry-VHH Mut5, Mut14 and Mut20 inhibited the intracellular aggregation of MTBD-P301S but were less efficient compare to VHH Z70, as the observed FRET signals for the corresponding transfected cells were decreased to 12.1±3.1, 12.7±5.4 and 11.2±4.8 (27,23,32% seeding inhibition, FIG. 5H-J).

[0204] From all that measurements, we concluded that the amount of intracellular aggregates of MTBDP301S Tau was reduced by more than 40% in the presence of the mCherry-VHH Z70, showing the efficiency of VHH Z70 to block Tau seeding in this cellular model. Similarly, the mutated VHH Z70 (VHH Mut) showed a range of inhibition efficiency in the same cellular model, ranging from about 25% inhibition to 50% inhibition, depending on the specific mutation(s) of VHH Z70 (FIG. 5J). The poorer seeding inhibition capacity of VHH-E4-1 is likely due to its poor intracellular activity compared to VHH Z70 (FIG. 2).

EXAMPLE 4: ACTIVITY OF VHH Z70 IN THYTAU30 MOUSE MODEL

[0205] Materiel and Methods

[0206] 3-month-old Tg30tau mice, expressing human 1N4RTau transgene mutated as P301SG272V under the control of Thy1.2 promoter.sup.55, were injected in hippocampus of both hemispheres with lentiviral vectors expressing VHH Z70 or a VHH directed against the green fluorescent protein. 2 weeks later, these mice were submitted to stereotaxic injections of AD human brain homogenate (2 μl, 5.5 μg/μl) or PBS (2 μl) in the hippocampus CA1 region of both hemispheres, as previously described in detail.sup.56. The combination resulted in four groups of 3 mice per group. The mice were sacrificed after a month delay from the injection of the brain extract. Brains were collected, fixed and sliced. Cryostat section slices were next used for immunohistochemistry. Brain slices were incubated with the primary antibody AT8. Labelling was amplified by incubation with an antimouse biotinylated IgG (1:400 in PBS-0.2% Triton™ X-100, Vector) followed by the application of the avidin-biotin-HRP complexe (ABC kit, 1:400 in PBS, Vector) prior to addition of diaminobenzidine tetrahydrochloride (DAB, Vector) in Tris-HCl 0.1 mol/l, pH 7.6, containing H2O2 for visualization. Mounted brain sections were analysed using stereology software (Mercator image analysis system; Explora Nova, La Rochelle, France). The CA1 region of the hippocampus was chosen as quantification zone, at 5 specific bregma locations situated between between 1.7 and 3.7 to remain at close distance to the stereotaxic injection site. The 5 selected locations were conserved for all mice to ensure accurate comparison of the four groups. The quantification was performed for these 5 slides per mouse, at a specific threshold presenting a minimum background. The quantification corresponds to the sum of the detected signal in the 5 slices.

[0207] Results

[0208] The model consisting of the injection of AD brain-derived material into the hippocampus of Tg30tau mice was previously described.sup.56 and shown to induce development of tauopathy (FIG. 6; GREN+AD circle). At 3-month old age, corresponding to the time of injection, background tau pathology is already present although to a lesser extent compare to injection with seeds (FIG. 6; GREN+PBS triangle). The appearance of tau pathology was evaluated 1 month post-injection by immunohistochemistry with the monoclonal AT8 antibody. For the immunization studies, VHH Z70 and control VHH directed against GFP (not present in mouse brains) were used.

[0209] In h-AD injected Tg30tau mice treated with VHH Z70, the AT8 detected in the CA1 fields was on average lower compare to Tg30tau mice treated with the negative-control VHH directed against GFP. The decrease in the average AT8 labeling detected in the CA1 fields in 4.5 month of age also showed the positive effect of VHH Z70 on decreasing the background pathology detected at this age (FIG. 6). These results suggested that immunization with VHH Z70 prevented the formation of neurofibrillary tangles induced by injection of extracellular h-AD or spontaneously developing in Tg30Tau.

TABLE-US-00014 TABLE 5 Useful amino acid sequences for practicing the invention SEQ ID NO amino acid sequence 1 (Tau epitope) SVQIVYKPV 2: VH of Z70 MAEVQLQASGGVFVQSGGSLRLSCAASGATSTFDG antibody MGWFRQAPGKEREFVSAISYEQGSYTYYADSVKGR FTISRDNSKNMVYLQMNSLRAEDTATYYCAPAYEG DLYAFDSYGGQGTQVTVSS 3: VH-CDR1 of Z70 ATSTFDG 4: VH-CDR2 of Z70 YEQGSYT 5: VH-CDR3 of Z70 PAYEGDLYAFDS 6: VH FR1 of Z70 MAEVQLQASGGVFVQSGGSLRLSCAASG 7: VH-FR2 of Z70 MGWFRQAPGKEREFVSAIS 8: VH-FR3 of Z70 YYADSVKGRFTISRDNSKNMVYLQMNSLRAEDTA TYYCA 9: VH-FR4 of Z70 YGGQGTQVTVSS 10: VH Mut1 MAEVQLQASGGVFVQSGGSLRLSCAASGATSTFDG MGWFRQAPGKEREFVSAISYEQGSYTYYADSVKGR FTISRDNSKNMVYLQMNSLRAEDTATYYCAPAYEG DLYAFDSYGEQGTQVTVSS 11: VH Mut3 MAEVQLQASGGVFVQSGGSLRLSCAASGATSTFDG MGWFRQAPGKEKEFVSAISYEQGSYTYYADSVKG RFTISRDNSKNMVYLQMNSLRAEDTATYYCAPAYE GDLYAFDSYGGQGTQVTVSS 12: VH Mut5 MAEVQLQASGGVFVQSGGSLRLSCAASGATSTFDG MGWFRQAPGKEREFVSAISYEQGSYTYYADSVKGR FTISRDNSKNMVYLQMNSLRAEDTASYYCAPAYEG DLYAFDSYGGQGTQVTVSS 13: VH Mut9 MAEVQLQASGGVFVQSGGSLRLSCAASGATSTFDG MGWFRQAPGKEREFVSAISYEQGSYTYYADSVKGR FTISRDNSKNMVYLQMNSLRAEDTATYYCASAYEG DLYAFDSYGEQGTQVTVSS 14: VH Mut12 MAEVQLQASGGVFVQSGGSLRLCCAASGATSTFDG MGWFRQAPGKEREFVSAISYEQGSYTYYADSVKGR FTISRDNSKNMVYLQMNSLRAEDTATYYCAPAYEG DLYAFDSYGEQGTQVTVSS 15: VH Mut14 MAEVQLQASGGVFVQSGGSLRLSCAASGATSIFDG MGWFRQAPGKEREFVSAISYGQGSYTYYADSVKG RFTISRDNSKNMVYLQMNSLRAEDTATYYCAPAYE GDLYAFDSYGEQGTQVTVSS 16: VH Mut15 MAEVQLQASGGVFVQSGGSLRLSCAASGATSTFDG MGWFRQAPGKEREFVSAISYEQGSYTYYADSVKGR FTISRDNSKNMVYLQMNSLGAEDTATYYCASAYEG DLYAFDSYEGQGTQVTVSS 17: VH Mut20 MAEVQLQASGGVFVQSGGSLRLSCAASGATSTFDG MGWFRQAPGKEKEFVSAISYEQGSYTYYADSVKG RFTISRDNSKNMVYLQMNSLRAEDTATYYCAPAYE GDLYAFDSYGEQGTQVTVSS 18: Tau [273-318] GKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQI VYKPVDLSKV 19: PHF6 peptide VQIVYK 20: VH of Abx M-A-E-V-Q-L-Q-A-S-G-G-V-F-V-Q-S-G-G-S-L-R-L- Xaa1-C-A-A-S-G-A-T-S-Xaa2-F-D-G M-G-W-F-R-Q- A-P-G-K-E-Xaa3-E-F-V-S-A-I-S-Y-Xaa4-Q-G-S-Y-T-Y- Y-A-D-S-V-K-G-R-F-T-I-S-R-D-N-S-K-N-M-V-Y-L-Q- M-N-S-L-Xaa5-A-E-D-T-A--Xaa6-Y-Y-C-A-Xaa7-A-Y- E-G-D-L-Y-A-F-D-S-Y-Xaa8-Xaa9-Q-G-T-Q-V-T-V-S- S- 21: VH FR1 of Abx M-A-E-V-Q-L-Q-A-S-G-G-V-F-V-Q-S-G-G-S-L-R-L- Xaa1-C-A-A-S-G 22: VH CDR1 of Abx A-T-S-Xaa2-F-D-G 23: VH FR2 of Abx M-G-W-F-R-Q-A-P-G-K-E-Xaa3-E-F-V-S-A-I-S 24: VH CDR2 of Abx Y-Xaa4-Q-G-S-Y-T 25: VH FR3 of Abx Y-Y-A-D-S-V-K-G-R-F-T-I-S-R-D-N-S-K-N-M-V-Y-L- Q-M-N-S-L-Xaa5-A-E-D-T-A-Xaa6-Y-Y-C-A 26: VH CDR3 of Abx Xaa7-A-Y-E-G-D-L-Y-A-F-D-S 27: VH FR4 of Abx Y-Xaa8-Xaa9-Q-G-T-Q-V-T-V-S-S- 28: VH of E4-1 MAEVQLQASGGGFVQPGGSLRLSCAASGATSTFDG antibody MGWFRQAPGKEREFVSAISYEQGSYTYYADSVKGR (FIG. 2B) FTISRDNSKNTVYLQMNSLRAEDTATYYCAPAYEG DLYAFDSYWGQGTQVTVSSAA 29: Minibody Z70 MYRMQLLSCIALSLALVTNSISAMAEVQLQASGGV FVQSGGSLRLSCAASGATSTFDGMGWFRQAPGKER EFVSAISYEQGSYTYYADSVKGRFTISRDNSKNMVY LQMNSLRAEDTATYYCAPAYEGDLYAFDSYGGQG TQVTVSSAAARSPPLKECPPCAAPDLLGGPSVFIFPP KIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWM SGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYV LPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGR TEQNYKNTATVLDSDGSYFMYSKLRVQKSTWE RGSLFACSVVHEGLHNHLTTKTISRSLGK 30: VH of Z70 MAEVQLQASGGVFVQSGGSLRLSCAASGATSTFDG antibody MGWFRQAPGKEREFVSAISYEQGSYTYYADSVKGR (FIG. 2B) FTISRDNSKNMVYLQMNSLRAEDTATYYCAPAYEG DLYAFDSYGGQGTQVTVSSAA

TABLE-US-00015 TABLE 6 nucleotide sequences for practicing the invention SEQ ID NO nucleotide sequence 31: VH of Z70 atggcggaagtgcagctgcaggcttccgggggagtatttgtgcagtcgggggg antibody gtcattgcgactgagctgcgccgcatccggagcaacttcaacatttgacggtatg ggctggtttcgtcaggcccctggcaaggagagagagttcgtttccgccatctccta cgaacaagggtcgtatacatactacgctgacagcgtaaagggaagatttacaatt agccgggataactccaaaaacatggtctatctccagatgaacagcctcagggcc gaggacacagctacgtattactgtgcacctgcatatgagggtgacctgtatgcattt gactcgtacgggggacaggggacgcaggtaactgtgagtagc 32: plasmid sequence cgaaggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcc with Z70 minibody cacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctaga gaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctt tttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatct ctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtc gcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggta agtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctac ctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctt tgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgcctacctga gatcaccggcgaaggagggccaccatgtacaggatgcaactcctgtcttgcattg cactaagtcttgcacttgtcacgaattcgatatcggccatggcggaagtgcagctg caggcttccgggggagtatttgtgcagtcgggggggtcattgcgactgagctgc gccgcatccggagcaacttcaacatttgacggtatgggctggtttcgtcaggccc ctggcaaggagagagagttcgtttccgccatctcctacgaacaagggtcgtatac atactacgctgacagcgtaaagggaagatttacaattagccgggataactccaaa aacatggtctatctccagatgaacagcctcagggccgaggacacagctacgtatt actgtgcacctgcatatgagggtgacctgtatgcatttgactcgtacgggggaca ggggacgcaggtaactgtgagtagcgcggccgctagatctcctccactcaaaga gtgtcccccatgcgcagctccagacctcttgggtggaccatccgtcttcatcttccc tccaaagatcaaggatgtactcatgatctccctgagccctatggtcacatgtgtggt ggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaa cgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtac tctccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaa ggagttcaaatgcaaggtcaacaacagagccctcccatcccccatcgagaaaac catctcaaaacccagagggccagtaagagctccacaggtatatgtcttgcctcca ccagcagaagagatgactaagaaagagttcagtctgacctgcatgatcacaggc ttcttacctgccgaaattgctgtggactggaccagcaatgggcgtacagagcaaa actacaagaacaccgcaacagtcctggactctgatggttcttacttcatgtacagc aagctcagagtacaaaagagcacttgggaaagaggaagtcttttcgcctgctcag tggtccacgagggtctgcacaatcaccttacgactaagaccatctcccggtctctg ggtaaatgagctagctggccagacatgataagatacattgatgagtttggacaaa ccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgct ttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcatttt atgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctac aaatgtggtatggaattaattctaaaatacagcatagcaaaactttaacctccaaatc aagcctctacttgaatccttttctgagggatgaataaggcataggcatcaggggct gttgccaatgtgcattagctgtttgcagcctcaccttctttcatggagtttaagatata gtgtattttcccaaggtttgaactagctcttcatttctttatgttttaaatgcactgacct cccacattccctttttagtaaaatattcagaaataatttaaatacatcattgcaatgaa aataaatgttttttattaggcagaatccagatgctcaaggcccttcataatatccccc agtttagtagttggacttagggaacaaaggaacctttaatagaaattggacagcaa gaaagcgagcttctagcttatcctcagtcctgctcctctgccacaaagtgcacgca gttgccggccgggtcgcgcagggcgaactcccgcccccacggctgctcgccg atctcggtcatggccggcccggaggcgtcccggaagttcgtggacacgacctcc gaccactcggcgtacagctcgtccaggccgcgcacccacacccaggccaggg tgttgtccggcaccacctggtcctggaccgcgctgatgaacagggtcacgtcgtc ccggaccacaccggcgaagtcgtcctccacgaagtcccgggagaacccgagc cggtcggtccagaactcgaccgctccggcgacgtcgcgcgcggtgagcaccg gaacggcactggtcaacttggccatgatggctcctcctgtcaggagaggaaaga gaagaaggttagtacaattgctatagtgagttgtattatactatgcagatatactatg ccaatgattaattgtcaaactagggctgcagggttcatagtgccacttttcctgcact gccccatctcctgcccaccctttcccaggcatagacagtcagtgacttaccaaact cacaggagggagaaggcagaagcttgagacagacccgcgggaccgccgaac tgcgaggggacgtggctagggcggcttcttttatggtgcgccggccctcggagg cagggcgctcggggaggcctagcggccaatctgcggtggcaggaggcgggg ccgaaggccgtgcctgaccaatccggagcacataggagtctcagccccccgcc ccaaagcaaggggaagtcacgcgcctgtagcgccagcgtgttgtgaaatgggg gcttgggggggttggggccctgactagtcaaaacaaactcccattgacgtcaatg gggtggagacttggaaatccccgtgagtcaaaccgctatccacgcccattgatgt actgccaaaaccgcatcatcatggtaatagcgatgactaatacgtagatgtactgc caagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggccatt taccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtactg ccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccct attggcgttactatgggaacatacgtcattattgacgtcaatgggcgggggtcgtt gggcggtcagccaggcgggccatttaccgtaagttatgtaacgcctgcaggttaa ttaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggcc gcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcg acgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtt tccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggat acctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgta ggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccc cccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaaccc ggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcag agcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggc tacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcgga aaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtt tttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctt tgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattt tggtcatggctagttaattaacatttaaatcagcggccggccgcaataaaatatcttt attttcattacatctgtgtgttggttttttgtgtgaatcgtaactaacatacgctctccat caaaacaaaacgaaacaaaacaaactagcaaaataggctgtccccagtgcaagt gcaggtgccagaacatttctctat

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