Patent Application
20100266576
The invention relates to the use of camelid single-domain antibodies for detecting an oligomeric form of the amyloid β peptide 42 and their applications.
Alzheimer's disease (AD) is a progressive, irreversible brain disorder with no known cause or cure. Extracellular fibrillar deposits and senile plaques are prominent and universal AD features. The major component of these aggregates is a water-soluble 40 or 42 amino acid polypeptide called Amyloid β (Aβ). However, the initial focus on the water-insoluble fibrillar amyloid as the central structure in AD pathology has evolved during the last 10 years. This was due to several outstanding discoveries such as the finding of a water-soluble fraction of oligomeric Aβ in the human brain (Kuo et al., 1996). These isolated soluble oligomers were toxic to neurons in culture. The presence and toxicity of oligomeric Aβ was then confirmed and the name of ADDLs (Aβ-derived diffusible ligands) was proposed for these structures (Lambert et al., 1998). Depending on conditions, ADDL compositions can contain predominantly trimers-hexamers, with larger structures of up to 24-mers. ADDLs show important regionally selective neurotoxicity, sparing neurons in the cerebellum while selectively killing neurons in hippocampal CA1 region and entorhinal cortex (Klein et al., 2001). Moreover, oligomers are able to inhibit hippocampal long-term potentiation (LTP) in rats in vivo (Walsh et al., 2002) and in hippocampal slices (Wang et al., 2002; Wang et al., 2004). Recently, it has been showed that cognitive deficits are directly attributable to low amounts of soluble oligomeric forms of Amyloid β; trimers and at a lesser extent, dimers and tetramers being particularly active (Cleary et al., 2005; Townsend et al., 2006).
Different models have been proposed for the formation of Aβ fibrils. A simple model proposed by Bitan et al. (2003) illustrates how Aβ42 may assemble. Monomers rapidly oligomerize into paranuclei that in turn associate to form large oligomers and protofibrils. Monomers, paranuclei (pentamers, hexamers), and oligomers are predominantly unstructured, but do contain some β-strand and α-helix elements. Protofibril formation involves substantial conformational rearrangements, during which unstructured, α-helix and β-strand elements transform into predominantly β sheet/β-turn structures. The final step in the pathway is protofibril maturation into fibrils, a process that appears to be irreversible, at least kinetically (Lomakin et al., 1997).
The need for accurate diagnosis of disorder mediated by amyloid β peptide oligomers such as AD or the Down syndrome is increasingly important as therapeutics become available in some cases. Therefore, a reliable assay for Aβ oligomers would be extremely helpful. However these assays are impaired because monoclonal and polyclonal antibodies, generated by immunization with Aβ oligomers, raised against oligomers also recognize fibrils (Lacor et al., 2004; Lambert et al., 2001; Lambert et al., 2007). These antibodies show, in AD brain sections, the expected AD type immunoreactivity and also a pericellular immunoreactivity (Lambert et al., 2007; Lacor et al., 2004; Gong et al., 2003). A monoclonal antibody raised against oligomers was also obtained by Lee et al. (2006). It recognizes oligomers, fibrils and mature senile plaques on AD brain sections. Fayed et al. (2003) prepared polyclonal rabbit antibodies that specifically recognize a conformation specific of Aβ high-MW oligomers distinct from that of soluble monomers, low-MW oligomers, and fibrils. A way to discriminate between the different conformations of Aβ is to use alternative “binders” which could recognize non conventional epitopes.
A significant proportion of camelid antibodies are single-domain antibodies, which interact with the antigen via a single heavy-chain binding domain devoid of light chain. This domain is referred to as “VHH” or “VHH antibody”. Recombinant VHH is the minimal-sized, intact antigen-binding domain. The absence of VL domain allows the VHHs to attain a higher structural flexibility than that of VH domains associated with VLs. Furthermore, the complementarity determining regions (CDRs) of VHHs, and especially CDR3, are statistically longer than those of conventional VH-VL antibodies (Muyldermans et al., 2001). Small size and increased plasticity appear to endow VHHs with unique potentialities: for instance, several VHHs are capable of inhibiting enzymatic activity by interacting with the active site cavity of enzymes such as α-amylase, carbonic anhydrase and hen egg lysozyme (Desmyter et al., 1996; Desmyter et al., 2002; Transue et al., 1998; Lauwereys et al., 1998). These features may allow camelid VHHs to recognize other unique epitopes that are poorly immunogenic for conventional antibodies.
International Application No. WO 2004/044204 describes variable fragments of camelid single-chain antibodies (VHH antibodies) capable to specifically bind the amyloid β peptide 42. Among these VHH antibodies, one particular antibody, referred to as VHH V31-1, has been shown to specifically recognize the carboxy terminal end of Aβ42 peptide (Aβ42) in its fibrillar form and intraneuronal Aβ42 deposits.
The Inventors have now found that contrary to what was described in said International Application No. WO 2004/044204, VHH V31-1 antibody does not recognize Aβ42 in its water-insoluble fibrillar form but specifically recognizes water-soluble low-molecular oligomers of Aβ42.
Furthermore the Inventors have also found that unexpectedly said VHH V31-1 antibody inhibits fibrillogenesis, i.e. Aβ fibril formation, in spite of the fact that it specifically binds the C-terminus of Aβ42. Indeed, previous studies have indicated that antibodies specific of regions 1-6 (Solomon, 2002; Legleiter et al., 2004) and 17-20 (Liu et al., 2004; Legleiter et al., 2004) of Aβ42 inhibited in vitro aggregation and cytotoxicity of Aβ, while antibodies specific of the C-terminus did not inhibit aggregation (Bard et al., 2000; Liu et al., 2004). The regions 17-20 and the C-terminus play critical roles in the aggregation (Ma and Nussinov, 2002; Balbach et al., 2000; Halverson et al., 1990; Jarrett et al., 1993 and Hilbich et al., 1991), which may explain why antibodies specific of the central region of Aβ inhibited aggregation. However there is no clear explanation why previously reported C-terminus specific antibodies had no effect on the aggregation of Aβ.
Therefore, in a first aspect, the present invention relates to the use of a VHH antibody of camelid binding an epitope located at the C-terminal end of the amyloid β peptide 42, for detecting in vitro in an appropriate biological sample or tissue, or in vivo in an organ (for instance the brain), an oligomeric form of the amyloid β peptide 42.
A VHH antibody of camelid (camel, dromedary, llama, alpaca, . . . ) refers usually to a variable fragment of a camelid single-chain antibody (See Nguyen et al., 2001; Muyldermans, 2001), and also comprises according to the present invention:
In a preferred embodiment, the VHH antibody of the present invention is a variable fragment of a camelid single-chain antibody having the amino acid sequence of SEQ ID NO: 1. This VHH antibody has been described in International Application No. WO 2004/044204. A host cell expressing VHH V31-1 is available at the Collection Nationale de Cultures de Microorganismes (CNCM), 28 rue du Dr. Roux, 75724 Paris Cedex 15, France, under the number I-2936.
As used herein, an “amyloid β peptide” refers to a peptide generated from amyloid precursor protein (APP) by β- and γ-secretase-mediated cleavage. Preferably, the amyloid β peptide is the amyloid β peptide 42 (Aβ42) having the amino acid sequence of SEQ ID NO: 2.
As used herein, the term “oligomer” refers typically to the initially formed, metastable multimer in an amyloid formation reaction (Kodali et al., 2007). Some protofibrils might also be considered oligomers.
In a preferred embodiment of the invention, the oligomeric form (oligomer) of said amyloid β peptide 42 is selected from the group consisting of a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, preferably a dimer, trimer, tetramer or dodecamer (12-mer), and more preferably trimer and tetramer.
The characterization of the binding of a camelid VHH antibody according to the present invention to an epitope located at the C-terminal end of the amyloid β peptide 42 can be performed by an ELISA-based binding assay as described in Example 2 below. Advantageously, the dissociation constant of said VHH antibody is about or less than 2. 10−8 M. Dissociation constant measurements may be made using methods known to those skilled in the art, including using the method described in Friguet et al. (1985).
The term “C-terminal end of the amyloid β peptide 42” as used herein, refers to the amino acids 25 to 42, particularly to the amino acids 29 to 42 of the amyloid β peptide of SEQ ID NO:2.
In a preferred embodiment of the invention, the epitope consists of a peptide selected from the group consisting of the peptide Aβ 29-40 having the amino acid sequence of SEQ ID NO: 9, the peptide Aβ 33-42 having the amino acid sequence of SEQ ID NO: 10 and the peptide Aβ 35-42 having the amino acid sequence of SEQ ID NO: 11 in the annexed listing of sequences.
The term “detecting” means assessing the presence or absence of an oligomeric form of the amyloid β peptide 42 as defined here above, in vitro or ex vivo in an appropriate biological sample or tissue (obtained for example by brain biopsy) or in vivo in an organ, for instance the brain. This process involves the ability of said VHH antibody (of SEQ ID NO: 1) to bind an oligomeric form of the amyloid β peptide 42.
The detection can be performed by any method known in the art in vitro in an appropriate biological sample or tissue or in vivo in an organ (for instance the brain). These methods include an appropriate immunochemical technique such as:
By way of example, this may be accomplished by linking to said VHH antibody a detectable label that can be visualized or measured, or by using ligands (e.g., an antibody linked to a detectable label) that specifically bind to said VHH antibody.
The level of binding is preferably detected quantitatively.
Advantageously, the detection of the presence of an oligomeric form of the amyloid β peptide 42 as described above is a prognostic marker of appearance of a disorder mediated by amyloid β peptide oligomers, preferably a neurodegenerative disorder such as Alzheimer's disease or the Down syndrome.
In a preferred embodiment of the invention, the detectable label is selected from the group consisting of:
In a second aspect, the present invention provides a method for determining in vitro in an appropriate biological sample or a tissue, or in vivo in an organ (for instance the brain), an early-stage of a disorder mediated by amyloid β peptide oligomers, preferably by Aβ42 oligomers, (for instance a neurodegenerative disorder such as Alzheimer's disease or the Down Syndrome), comprising a step of detecting, in a subject (a mammal, preferably a human), an oligomeric form of an amyloid β peptide 42 as defined here above, by contacting said appropriate biological sample, tissue or organ with a VHH antibody as defined above, preferably the VHH antibody of SEQ ID NO: 1, preferably labelled with a detectable label as defined above.
According to another aspect, the present invention relates to a method of diagnostic of a disorder mediated by amyloid β peptide oligomers, preferably by Aβ42 oligomers as defined above, in a subject (a mammal, preferably a human) comprising the steps of:
a) contacting an appropriate biological sample, tissue or organ, with a VHH antibody of camelid binding an epitope located at the C-terminal end of an amyloid β peptide 42 as defined above, preferably the VHH antibody having the amino acid sequence of SEQ ID NO: 1, and
b) detecting the binding of said VHH antibody to said biological sample, tissue or organ, a binding constituting a marker of the presence of said disorder (for instance Alzheimer's disease or the Down Syndrome).
According to the invention, said contacting step may be performed in vitro, ex vivo or in vivo.
When the contacting step is performed in vivo, the organ is preferably the brain.
The present invention also relates to a method of diagnostic of a disorder mediated by amyloid β peptide oligomers, preferably by Aβ42 oligomers, in a subject (a mammal, preferably a human) comprising the steps of:
a) contacting in vitro or ex vivo an appropriate biological sample or tissue, or in vivo (for instance the brain), with a VHH antibody of camelid binding an epitope located at the C-terminal end of an amyloid β peptide 42 as defined above, preferably the VHH antibody having the amino acid sequence of SEQ ID NO: 1,
b) determining the amount of amyloid β peptide 42 oligomers in said biological sample, tissue or brain, and
c) comparing the amount determined in step (b) with a standard, a difference in amount constituting a marker of the presence of said disorder (for instance Alzheimer's disease or Down Syndrome).
As used herein, the term “standard” refers to the amount of amyloid β peptide 42 oligomers in said appropriate biological or in the brain, which has been determined in a large population of subjects not suffering from a disorder mediated by amyloid β peptide oligomers.
In another aspect, the present invention relates to the use of a VHH antibody of camelid binding an epitope located at the C-terminal end of an amyloid β peptide 42 as defined above, preferably the VHH antibody of camelid having the amino acid sequence of SEQ ID NO: 1 for the preparation of a medicament for treating or preventing a disorder mediated by amyloid β peptide oligomers, preferably by amyloid β peptide 42 oligomers.
The term “treating” includes the administration of said VHH antibody to a patient who has said disorder, a symptom of said disorder or a predisposition toward said disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptoms of the disorder, or the predisposition toward disorder.
The term “preventing” means that the progression of said disorder is reduced and/or eliminated, or that the onset of said disorder is delayed or eliminated.
In a preferred embodiment, said medicament is administered to a subject so as to inhibit the formation of amyloid β peptide fibrils, preferably Aβ42 fibrils, and/or to slow down the progression of said disorder.
In a particular embodiment of the present invention, the medicament can be administered to a subject (a mammal or a human) either directly into the brain or by injection, preferably by intravenous, intraperitoneal, intramuscular or subcutaneous injection. Indeed, the Inventors have shown that the said VHH antibody is capable of transmigrate across a blood-brain barrier model.
In another preferred embodiment, the disorder is a neurodegenerative disorder such as Alzheimer's disease or the Down syndrome.
All amyloid fibrils share common features, including a high content of β-sheet in a classical <<cross-β>> pattern, a fibrillar morphology in electron microscopy, and the ability to bind and alter the spectroscopic properties of heterochromatic dyes Congo red and Thioflavin T (Sumner Makin and Serpell, 2005; Westermark, 2005).
Then, the present invention also relates to a method of monitoring the therapeutic effect of a VHH antibody of camelid binding an epitope located at the C-terminal end of an amyloid β peptide 42 as defined above, preferably the VHH antibody of camelid having the amino acid sequence of SEQ ID NO: 1 on the regression of a disorder mediated by amyloid β peptide oligomers in a subject comprising the steps of:
a) contacting in vitro or ex vivo an appropriate biological sample with a compound binding β-sheet amyloid structures such as amyloid fibrils, preferably Congo Red (sodium salt of benzidinediazo-bis-1-naphtylamine-4-sulfonic acid) or thioflavin T (ThT),
b) determining the amount of β-sheet amyloid structures,
c) comparing the amount so determined with amounts previously obtained for the subject, a decrease in amount constituting a marker of the regression of said disorder.
In addition to the preceding features, the invention further comprises other features which will emerge from the following description, which refers to examples illustrating the present invention, as well as to the appended figures.
The following examples illustrate the invention but in no way limit it.
Materials
Aβ42 (SEQ ID NO: 2) and the different Aβ42 fragments (1-11, 10-20, 15-25, 22-35, 29-40 and 33-42 fragments of SEQ ID NO: 2, respectively SEQ ID NO: 5, 6, 7, 8, 9 and 10) used were purchased from Bachem. The monoclonal anti-body (mAb) 6F/3D anti-Ab 8-17 (Dako) recognizes synthetic amyloid peptides by dot- and western-blotting. It also specifically stains all type of amyloid deposits in AD brains.
Subjects
Human cortical brain tissue was obtained from the Hôpital Pitié-La Salpetrière, Paris, France. Postmortem brain tissue was examined from representative neurologically normal controls and AD patient (staged Braak-VI, according to Braak and Braak, 1991).
VHH Antibodies and Expression Thereof in a pET System
VHH V31-1 (SEQ ID NO: 1); a host cell expressing VHH V31-1 is available at the Collection Nationale de Cultures de Microorganismes (CNCM), 28 rue du Dr. Roux, 75724 Paris Cedex 15, France, under the number I-2936; it was filed on Sep. 20, 2002;
VHH L1-3 (SEQ ID NO: 3); a host cell expressing VHH L1-3 is available at the CNCM, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France, under the number I-2934; it was filed on Sep. 20, 2002;
VHH L35 (SEQ ID NO: 4); a host cell expressing VHH L35 is available at the CNCM, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France, under the number I-2935; it was filed on Sep. 20, 2002;
VHH 61-3 (SEQ ID NO: 12); a host cell expressing VHH 61-3 is available at the CNCM, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France, under the number I-2933; it was filed on Sep. 20, 2002.
The coding sequences of VHH V31-1, VHH L1-3, VHH L35 and VHH 61-3 antibodies inserted in vector pHEN1, described in International Application No. WO 2004/044204, were subcloned in vector pET22 using the NcoI and NotI restriction sites according to the manufacturer's instructions (Novagen, Darmstadt, Germany). Transformed E. coli BL 21 (DE3) cells expressed VHH antibodies in the periplasm after induction by IPTG 1 mM for 3 hours at 20° C. Periplasmic extracts were obtained by spheroplasting cells, suspended in 50 mM sodium phosphate buffer pH 8 containing 20% sucrose and 1 mM EDTA, and hydrolysing the peptidoglycan with 5 mg/ml lysozyme for 20 min at 4° C., in the presence of protease inhibitors (Complete™, Boehringer Mannheim, Germany). The suspension was then centrifuged 2 mM at 10,000 rpm. The supernatant corresponding to the periplasmic extract was kept at 4° C. Purified VHH antibodies were obtained by IMAC using a chelating agarose column charged with Ni2+ (Superflow Ni-NTA, Qiagen Ltd, UK) according to manufacturer's instructions. The protein content was measured using the Bradford reagent. The purity of the final preparation was evaluated by SDS-PAGE with Coomassie staining and by Western blot.
ELISA
A modified version of a standard ELISA was used to test for the presence of VHH antibodies in culture supernatants. Microtiter plates (Nunc, Denmark) were coated by incubation overnight at 4° C. with 1 μg/ml of antigen diluted in PBS. Plates were washed four times with buffer A (0.1% Tween 20 in PBS), and VHHs were diluted in buffer B (0.5% gelatin in buffer A). The plates were incubated for 2 hours at 37° C. and washed again, before adding a horseradish peroxidase-labeled rabbit anti-c-myc (A14) (Santa Cruz, Calif., USA) or with a rabbit anti-His tag antibody (Santa Cruz, Calif., USA). Then, the plates were washed with buffer A, and freshly prepared 0.2% orthophenylenediamine (Dakopatts A/S, Glostrup, Denmark), 0.03% H2O2 in 0.1 M citrate buffer, pH 5.2, were added to each well. The peroxidase reaction was stopped by adding 3 M HCl, and the optical density was measured at 490 nm.
Determination of Dissociation Constants by ELISA
Binding affinity of VHH antibodies was determined as described in Friguet et al., (1985). Various concentrations of Aβ peptides were incubated in solution overnight at 4° C. with a known quantity of VHH antibody until equilibrium was reached. The VHH antibody 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 A and free VHH antibodies were detected by adding β-galactosidase-conjugated goat anti-rabbit Igs (Biosys, Compiegne, France) and 4-methylumbelliferyl β-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.
Western Blot
Aβ peptides were suspended in PBS. To an aliquot (5 μl), an equal volume of gel loading buffer was added and then treated at 100° C. for 5 min. 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 either VHH antibody or mAb, and respectively revealed by peroxidase-labeled rabbit anti-c-myc (A14) anti-bodies (Santa Cruz, Calif., USA), or rabbit anti-His tag (Santa Cruz, Calif., USA) followed by peroxidase labeled goat anti-rabbit immunoglobulins. Finally, peroxidase activity was visualized using a chemiluminescent kit (Amersham).
Dot-Blot
Frozen brain samples were homogenized (1:10 (w/v)) in the Laemmli sample buffer containing 0.25% (W/v) dithiothreitol (DTT) and heat-treated for 10 min. Each sample was then centrifuged at 100,000 g for 60 min. Resulting pellets were resuspended in 500 μl of 100% formic acid and left under agitation at room temperature for 3 hours. Fractions were then centrifuged for 1 hr at 14000×g (Permanne et al., 1995). The supernatants were removed and 2 μl of a serial dilution (from ½ to 1/164) were dot-blotted onto Hybond-C nitrocellulose membranes (Amersham). Membranes were blocked and processed for immunorevelation as described for the western blot. Dot-blots were digitized on a Gel-doc (Biorad) at a resolution of 72 dots/inch and saved as 8-bit gray scale PICT files (256 shades of gray). Intensities of immunoreactivity were calculated for each dot with Quantity one software (Biorad).
Preparation of Aβ42 Monomers and Protofibrils
Samples of 1 mg of Aβ42 powder were dissolved in 500 μl of hexafluoroisopropanol (Sigma), gently stirred at 4° C. for 7 days, sonicated for 10 min using a Branson ultrasonic bath sonicator and then centrifuged for 10 min at 16000×g. Aliquots containing 50 μg of peptides each were lyophilised and stored at −20° C. Aliquots were dissolved in double distilled water or in PBS pH=7.4 and incubated at 37° C. until use.
Dynamic Light Scattering (DLS)
DLS (also termed quasi-elastic light scattering) was used to measure the average diffusion coefficient distribution of Aβ particles of the particles. Each particle present in the sample is characterized by its hydrodynamic radius (RH), corresponding to the radius of a sphere with a diffusion coefficient equal to that measured. RH measurements were made at 25° C. with a DynaPro MS800 instrument (Protein solution-Wyatt) equipped with a gallium aluminium arsenide 825 nm laser. An aliquot of Aβ42 peptide was diluted to 0.20 μM in double-distilled water and incubated at 37° C. Samples (110 μl) were placed directly into a 3 mm optical pathlength quartz cuvette (Hellma), and the total light scattering intensity at a 90° angle was collected using a 10-s averaging acquisition time. Particle translational diffusion coefficients (DT) were calculated from autocorrelated light intensity data (usually 30-40 points) and converted to RH with the Stokes-Einstein equation. A distribution plot of intensity versus RH was calculated using the Sedfit 9.3 analysis software (www.analyticalultracentrifugation.com), and intensity-weighted mean RH values were obtained from each subpeak.
Thioflavin T (ThT) Fluorescence Assay
Fluorescence emission of ThT is shifted when it binds to β-sheet aggregate structures such as amyloid fibrils (LeVine, 1993). An aliquot of Aβ42 peptide was diluted to 20 μM in double-distilled water or in PBS pH 7.4 and incubated at 37° C. Aβ42 aggregation was measured by periodically removing 30 μl aliquots from the incubation samples and adding them to 2 ml of 5 μM ThT solution (50 mM phosphate buffer, pH=6.5). Fluorescence intensity was monitored at an excitation wavelength of 450 nm and an emission wavelength of 482 nm on a spectrofluorometer using 1 cm light-path quartz cuvettes with both excitation and emission bandwidths of 5 nm. Readings were the results of an average of three values after substracting the fluorescence contribution of free ThT. Each experiment was performed in duplicate.
Statistical Analysis
A two-tailed Student's t-test was used for comparison of the fibrillogenesis of Aβ42 in the presence or absence of antibody fragments. P values <0.05 were considered as statistically different.
Immunocytochemistry
Immunostaining of brain tissue was performed on 7 μM thick paraffin sections. Sections were de-paraffinized in xylene, rehydrated through ethanol
(100%, 96%, and 90%) and finally brought to water. They were incubated in 90% formic acid, washed again in water, quenched for endogenous peroxidase with 3% hydrogen peroxyde and 20% methanol, and finally washed in water. Non-specific binding was blocked by incubating the sections for 10 minutes in 2% bovine serum albumin in TBS+0.5% Tween. Appropriate dilutions of primary antibodies were then applied overnight in a humidified chamber at room temperature (typically 1 μg/ml for VHH, and 1:200 for 6F/3D mAb). Slides were washed with TBS-Tween and incubated with secondary antibodies (rabbit anti-His Tag or biotinylated anti-mouse immunoglobulins) in TBS-Tween at room temperature for 2 hours. Slides were then incubated with either peroxidase goat anti-rabbit immunoglobulins or streptavidine-peroxidase fusions, and developed with diaminobenzidine (DAB) for 2 minutes. After washing with TBS-Tween, slides were counter-stained with hematoxylin and eosin.
Amyloid Extraction
Human cortical brain tissue was obtained from the GTE Neuro-CEB of Hopital de la Pitié-Salpétrière, Paris, France. Amyloid extraction was performed according to Delacourte et al., 2002. Briefly, a total of 50 mg of brain tissue was homogenized in 500 μl of pure formic acid. One hundred μl were evaporated under nitrogen, solubilized in 100 μl of the LDS sample buffer (NuPage, Invitrogen) containing 2% β-mercaptoethanol and boiled 10 minutes before electrophoresis.
1. Characterization of the Epitopes Recognized by VHH V31-1
International Application No. WO 2004/044204 discloses that VHH V31-1 recognizes the carboxy terminal end of Aβ42. To further characterize the epitope recognized by VHH V31-1, the KD was determined for peptides corresponding to different Aβ fragments (1-11, 10-20, 15-25, 22-35, 29-40 and 33-42). VHH V31-1 did not recognize the fragments 1-10, 10-20, 15-25 and 22-35. The KD of VHH V31-1 for Aβ 29-40 and Aβ 33-42 was in the same order of magnitude, respectively 2(±0.8). 10−8 M for Aβ 29-40 and 2.10−8 M for Aβ 33-42, suggesting that VHH V31-1 recognizes an epitope located at the C-terminal end of Aβ42.
2. VHH V31-1 Recognizes Preferentially the Oligomeric Form of Aβ42
To further characterize VHH V31-1, its binding was tested by immunoblotting on Aβ. Aβ42 was incubated for several days at 37° C. and an aliquot was removed at days 0, 1, 3 and 7. Standard Aβ42 preparation, i.e., <<aging>> by incubation of high concentrations of Aβ42 at 37° C. for several days, lead to mixed fractions. After SDS denaturation, such preparations showed not only gel-excluded Aβ-reactive material (i.e., SDS-insoluble fibrils), but also Aβ42 monomer and oligomers (Hartley et al., 1999). VHH V31-1 labelled the 4 kDa band corresponding to the monomer, the 8-16 kDa bands corresponding to dimers, trimers and tetramers, and the 50-80 KDa bands corresponding to 12-mers, but not the additional band at the very top of the gel, suggesting that V31-1 recognizes specifically Aβ42 in its oligomeric form, but not its fibrillar form (
DLS was then used to study the evolution of the size distribution of Aβ42 particles along the oligomerization process. Aβ42 peptide was dissolved in hexafluoroisopropanol (HFIP), sonicated and then centrifuged to completely remove associated peptides. Lyophilized aliquots were kept at −20° C., resuspended in double-distilled water and analyzed by DLS at different incubation times. At T-1 h, the size distribution for Aβ42 showed a single peak with a weight-average hydrodynamic radius (RH) of approximately 40 nm (
The kinetics of fibril formation was monitored using a ThT assay (Table I below). ThT is a fluorescent dye that specifically binds to fibrillar structures. Aβ42 was resuspended in water and aliquots were periodically removed and added to a ThT solution. A time-dependent increase was observed, suggesting that progressive formation of amyloid fibrils was taking place.
The binding of V31-1 to the different fractions of Aβ42 was analysed in ELISA (
Dot-blot immunoassays were performed on Aβ42 incubated for more than 20 days at 37° C., composed mostly of fibrils. A strong Aβ42 immunoreactivity was obtained with mAb 6F/3D. Conversely, Aβ42 immunoreactivity was weak with VHH V31-1 (
3. VHH V31-1 Recognizes Intraneuronal Aβ42 Oligomers but not Amyloid Plaques
The distribution of VHH-specific immunoreactivity in human AD brains was examined. Stained AD brain tissue slices revealed significant intraneuronal immunoreactivity for VHH V31-1, while that of VHH 61-3 was very faint and that of VHH L1-3 undetectable (
To confirm the immunoreactivity of VHH V31-1 on brain tissues, dot-blot immunoassays were performed on formic acid fractions obtained from an AD patient and from a control. A strong Aβ42 immunoreactivity was obtained in the AD patient fraction. Conversely, Aβ42 immunoreactivity was weak for the control patient (
4. VHH V31-1 Inhibits the Formation of Fibrils
To investigate the effects of the VHH antibodies on fibril formation, conditions were selected (lyophilized Aβ42 is resuspended in water then diluted in PBS at the working concentration and sonicated) under which both Aβ42 is able to aggregate in vitro and the VHH antibody can bind tightly to Aβ42. Similar experiments were performed with an irrelevant VHH antibody (L35; SEQ ID NO: 4; described in International Application No. WO 2004/044204) as a negative control. A striking feature of the VHH antibodies is their capacity to withstand prolonged incubation at 37° C. (Arbabi Ghahroudi et al., 1997).
In the absence of VHH V31-1, the DLS data showed that Aβ42 readily aggregated with the formation of 2 peaks at day 1, one around 30 nm and the other around 100-150 nm. Then as early as day 2, a new peak centred around 300 nm could be detected, which then evolved to 600-700 nm at day 7 (Table II).
The aggregation of Aβ42 therefore appears to be similar when it is diluted in water (
No significant decrease of fluorescence was observed when Aβ42 is co-incubated with an equimolar concentration of VHH L35.
Yan and Wang (2006) have recently shown that the C terminus of Aβ42 is more rigid than that of Aβ40 and is likely preordered for its β-conformation in fibrils and oligomers, therefore acting as an internal seed for aggregation. This notion is supported by recent bioinformatic studies of Aβ42 showing that the C-terminal six-residues of Aβ42 have the best match for the NNQQNY conformation in the crystal structure of fibrils (Thompson et al., 2006; Nelson et al., 2005). It can be postulated that VHH V31-1 may recognize this seed in oligomers and preclude the formation of aggregates.
5. VHH V31-1 Recognizes A042 Trimers and Tetramers in Human Cortical Brain Tissue
The distribution of VHH-specific immunoreactivity in human cortical brain tissue was examined. Two main bands at 16 and 20 KDa were immunodetected in the cortical brain formic acid fractions with VHH V31-1 corresponding to Aβ42 trimers and tetramers (
Immortalized human brain endothelial cells hCMEC/D3 have been previously described in detail in Weksler et al. (2005). Cell viability in the presence of VHH antibodies (VHH V31-1 and L1-3) was assessed by MTT assay as described in Weksler et al. (2005).
The permeability of hCMEC/D3 cell monolayers to VHH antibodies was measured on transwell polycarbonate insert filters (pore size 3 μm, Corning, Brumath, France) as described in Weksler et al. (2005). hCMEC/D3 cells were seeded on the filters at a confluent density of 2×105 cells/cm2 in EGM-2 medium.
Transport studies were performed 3 days post-seeding as described in Weksler et al. (2005). Experiments were initiated by adding VHH antibodies to the upper chamber containing either collagen, coated inserts without cells, hCMEC/D3 cells or hCMEC/D3 cells pre-exposed to various pharmacological modulators for 30 min. Transport studies were conducted at 37° C. The lower chamber was sampled at various time intervals (10, 30 and 60 min) and the presence of VHH antibodies was determined by ELISA and Western Blot (see results).
Results
| Number | Date | Country | Kind |
|---|---|---|---|
| 07290812.2 | Jun 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2008/002671 | 6/26/2008 | WO | 00 | 3/29/2010 |
